Uptake and persistence of ingested antibody in the mosquito Anopheles stephensi

A sensitive ELISA was developed to monitor the persistence of a specific antibody, rabbit anti-BSA, in the bloodmeal, haemolymph and tissues of the mosquito Anopheles stephensi Liston. Different concentrations of anti-BSA were fed to female mosquitoes in sheep blood, via a membrane-feeder, and it was found that antibody persisted in the gut as the bloodmeal was digested: concentrations present at 24 h were directly related to those fed. Homogenates of mosquito bodies, from which the intact guts had been removed, were always antibody-positive up to 9 days post-feeding, indicating that undigested antibody had passed through the gut wall into the haemocoele. Haemolymph was extracted from mosquitoes at different times post-feeding, using a microcapillary and manipulator, and antibody was detected in several of the assays. The level of antibodies in the haemolymph 24 h post-feeding was less than half of the level in mosquito heads, indicating removal of antibodies from the haemolymph, perhaps by binding onto haemocoelic tissues. The relevance of these results to the ingestion, survival and fate of antibody against malaria sporozoites is discussed.     

Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum

Variation in susceptibility of the vector Anopheles stephensi Liston to the human malaria parasite Plasmodium falciparum (Welch) was demonstrated using twelve strains of mosquitoes and one strain of parasites cultured in vitro. The Beech strain of An. stephensi exhibited greatest natural refractoriness, but with high intrapopulation variability. By selection for the required characteristic, two refractory lines of the Punjab strain and one highly susceptible line of the Sind strain were obtained. The median number of oocysts in the two refractory lines was less than 4% of that in the unselected line, whilst the highly susceptible line yielded about twice as many oocysts as the unselected line. Selection progressed more by keeping the descendants of individual females separate and selecting between them (individual selection) rather than pooling the progeny of all selected mosquitoes (mass selection). Using the former procedure many lines were lost due to inbreeding depression, but the outcome was more successful.     

Innate immunity against malaria parasites in Anopheles gambiae

Malaria continues to exert a huge toll in the world today, causing approximately 400 million cases and killing between 1-2 million people annually. Most of the malaria burden is borne by countries in Africa. For this reason, the major vector for malaria in this continent, Anopheles gambiae, is under intense study. With the completion of the draft sequence of this important vector, efforts are underway to develop novel control strategies. One promising area is to harness the power of the innate immunity of this mosquito species to block the transmission of the malaria parasites. Recent studies have demonstrated that Toll and Imd signaling pathways and other immunity-related genes （encoding proteins possibly function in recognition or as effector molecules） play significant roles in two different arms of innate immunity： level of infection intensity and melanization of Plasmodium oocysts. The challenges in the future are to understand how the functions of these different genes are coordinated in defense against malaria parasites, and if different arms of innate immunity are cross-regulated or coordinated.     

Comparative fecundity and associated factors for two sibling species of the Anopheles gambiaecomplex occurring sympatrically in The Gambia

Abstract. For two sibling species of mosquitoes belonging to the Anopheles gambiae complex of malaria vectors, the effects of body size (wing length) and bloodmeal size (haematin excretion) on fecundity of wild females were investigated in The Gambia, West Africa. Freshly blood-fed individuals from sympatric populations of An.arabiensis and An.gambiae sensu stricto were sampled by collection at 07.00–09.00 hours from within bednets during July/August 1993, at the beginning of the rainy season. The possible confounding effect of infection with Plasmodium parasites was removed by eliminating infected mosquitoes from the study samples. An.arabiensis females comprised 75% of the An.gambiae sensu law population and were significantly larger (greater mean wing length) than those of An.gambiae s.s. mosquitoes. Mean egg production per female (for the subsequent gonotrophic cycle, excluding pre-gravids) for the two species was not significantly different, though the relationship between wing length and egg production showed An.gambiae s.s. to be more fecund than the An.arabiensis of the same size. Pre-gravid An.gambiae s.s. had consumed significandy smaller bloodmeals than gravid females but the mean wing length of these two gonotrophic categories was not significantly different. In contrast, An.arabiensis pre-gravids were smaller and had consumed smaller bloodmeals than the gravids.     

Responses of Anopheles gambiae complex mosquitoes to the use of untreated bednets in The Gambia

Population dynamics of the Anopheles gambiae complex of malaria vector mosquitoes were studied in four small hamlets in The Gambia. Bednets were used to reduce man/vector contact in two of the hamlets. High densities of An. gambiae, sensu lato, were present for only 3-8 weeks during the rainy season, depending on the position of the hamlet within the study area. The proportions of blood-fed mosquitoes caught indoors (83.0%) and existing from houses (11.6%) were lower in hamlets where bednets were used than in hamlets without (96.5% and 33.1% respectively). Fewer of the blood-fed mosquitoes had fed on man in houses where people slept under bednets (68.2%) than in those without (81.5%). However, the average number of infective bites received by children was still greater than one a year in hamlets where bednets were used. Consequently bednets are considered unlikely to be an effective malaria control measure so long as they are untreated with insecticide.     

Selection of Anopheles dirus for refractoriness and susceptibility to Plasmodium yoelii nigeriensis

Two lines of the Oriental malaria vector mosquito Anopheles dirus species A (Diptera: Culicidae), one fully refractory and one fully susceptible to Plasmodium yoelii nigeriensis (an African rodent malaria parasite), were established after 17 generations of mass selection, followed by single female selection for one or two generations. Prior to selection, the stock colony of An. dirus was 17% refractory. Both lines of An. dirus produced abundant ookinetes that started to invade the midgut within 24h post-infection, as seen in histological sections. In most of the refractory mosquitoes, oocysts stopped development <12 h post-invasion, indicating a rapid defence mechanism. Dead P. y. nigeriensis parasites were apparently localized as small melanized spots (2-5 microm) seen in wet preparations of mosquito midguts dissected 5-7 days post infective bloodmeal. In some refractory An. dirus females, apart from the spots, a small number of totally encapsulated oocysts (c. 10 microm) were also present. These larger melanized parasites predominated in a few females: they appeared 2-3 days post-infection as a secondary delayed defence mechanism. The progeny of reciprocal matings between susceptible and refractory lines had approximately 50% susceptibility. Backcrosses of F1 hybrids with susceptible or refractory lines increased or decreased the susceptibility of backcross progeny accordingly. Overall, these results suggest polygenic control of susceptibility to P. y. nigeriensis infection. The refractory line of An. dirus showed normal susceptibility to natural infections of the human malarias P. falciparum and P. vivax from local patients.     

Pteridine fluorescence for age determination of Anopheles mosquitoes

The age structure of mosquito populations is of great relevance to understanding the dynamics of disease transmission and in monitoring the success of control operations. Unfortunately, the ovarian dissection methods currently available for determining the age of adult mosquitoes are technically difficult, slow and may be of limited value, because the proportion of diagnostic ovarioles in the ovary declines with age. By means of reversed-phase HPLC this study investigated the malaria vectors Anopheles gambiae and An. stephensi to see if changes in fluorescent pteridine pigments, which have been used in other insects to determine the age of field-caught individuals, may be useful for age determination in mosquitoes. Whole body fluorescence was inversely proportional to age (P < 0.001, r2 > 91%) up to 30 days postemergence, with the regression values: y = 40580-706x for An. gambiae, and y = 52896-681x for An. stephensi. In both species the main pteridines were 6-biopterin, pterin-6-carboxylic acid and an unidentified fluorescent compound. An. gambiae had only 50-70% as much fluorescence as An. stephensi, and fluorescent compounds were relatively more concentrated in the head than in the thorax (ratios 1:0.8 An. gambiae; 1:0.5 An. stephensi). The results of this laboratory study are encouraging. It seems feasible that this simpler and faster technique of fluorescence quantification could yield results of equivalent accuracy to the interpretation of ovarian dissection. A double-blind field trial comparing the accuracy of this technique to marked, released and recaptured mosquitoes is required to test the usefulness of the pteridine method in the field.     

Impact of permethrin-treated bednets on malaria transmission by the Anopheles gambiae complex in The Gambia

Malaria vector mosquitoes belonging to the Anopheles gambiae complex were studied in four hamlets in The Gambia. All inhabitants were given bednets treated either with a placebo (milk) in two hamlets or with the pyrethroid insecticide permethrin (500 mg/m2) in two other hamlets. Malaria transmission occurred mainly during a few weeks of the rainy season, in September and October 1987. The indoor resting densities of mosquitoes in permethrin-treated hamlets were reduced, and we estimated over 90% reduction in biting on man by An. gambiae Giles sensu stricto in these hamlets. No mosquitoes were found under permethrin-treated bednets compared with eighty-one recovered from placebo-treated bednets. Mosquitoes exited more readily from rooms where permethrin-treated bednets were used than from rooms with placebo-treated nets. The annual mean probability that a child would receive an infective bite was estimated to be 0.09 in hamlets with insecticide-treated bednets, compared with 1.9 where placebo-treated bednets were used. Permethrin-treated bednets are therefore recommended as a means of effectively reducing the risk of exposure to malaria transmission, particularly in areas of low seasonal transmission.     

Isolation and characterization of polymorphic microsatellite markers from Asian malaria mosquito Anopheles sinensis (Diptera: Culicidae)

Microsatellite-containing region were isolated and characterized in Anopheles sinensis, a primary vector of malaria parasites in Asia. An enrichment protocol yielded 252 microsatellite sequences. We designed primers to amplify 20 unique microsatellites, 14 of which amplify cleanly and were polymorphic. A survey of 24 individuals showed that 12 loci are highly variable with the number of alleles ranging from two to 11, and expected heterozygosity ranging from 0.116 to 0.903. These markers will be useful for population genetic studies and genome mapping in A. sinensis.     

斯氏按蚊肽聚糖识别蛋白基因PGRP-LC1的克隆及功能分析

天生免疫系统是昆虫抵御外界病原入侵的主要方式。目前研究发现, Imd信号通路与按蚊感染柏氏疟原虫Plasmodium berghei的强度密切相关, 而PGRP-LC1是Imd信号通路最上游的受体之一。为了研究斯氏按蚊Anopheles stephensi肽聚糖识别蛋白PGRP-LC1, 采用RT-PCR并结合RACE技术克隆斯氏按蚊PGRP-LC1基因, 通过序列比较分析, 得到两条cDNA序列, 其开放阅读框分别为1 365 bp和1 290 bp, 3′非编码区为320 bp, 5′非编码区为240 bp。将两条cDNA分别命名为AsPGRP-LC1a（GenBank注册号 GU214232）和AsPGRP-LC1b（GenBank注册号GU214233）。AsPGRP-LC1a编码454个氨基酸, 分子量约为49.07 kDa;AsPGRP-LC1b编码429个氨基酸, 分子量约为46.3 kDa。AsPGRP-LC1b比AsPGRP-LC1a少一个长度为75 bp的外显子, 该外显子在冈比亚按蚊Anopheles gambiae PGRP-LC1基因的某些可变剪切形式中也有发现。分别将两个斯氏按蚊PGRP-LC1基因在冈比亚按蚊细胞系L3-5和斯氏按蚊细胞系MSQ43中过量表达, 通过双荧光素酶检测系统检测抗菌肽的表达情况, 结果显示克隆得到的PGRP-LC1基因在两种细胞系中均能够启动Imd信号通路, 为进一步研究斯氏按蚊的Imd信号通路提供了依据。