Responses of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens mosquitoes (Diptera: Culicidae) to cool and humid refugium conditions

Like all terrestrial arthropods, mosquitoes must cope with the threat of desiccation. To gain insight into their survival strategies, we recorded the behavioral responses of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens offered zones of different microclimatic conditions in laboratory cages. The cooled refugium was at 25.6 degrees C, 86% RH and the control was at 28.5 degrees C, 75% RH, i.e., a difference in saturation deficit of 3.9 mm Hg between the two zones. We show that newly-emerged adults, with no access to water or sugar, prefer the cooler and more humid refugium with a saturation deficit half that in the control and where the mosquitoes could reduce their metabolic rate. This response is delayed in Ae. aegypti, perhaps because the energy reserves accumulated as larvae are higher in this species. This study shows that mosquitoes under stress can use their thermohygroreceptor cells to guide them to locations that facilitate survival.     

Susceptibility of Anopheles messeae Fall. and Culex pipiens pipiens L. Larvae to entomopathogenic Fungi Metarhizium

The mortality rate of Anopheles messeae Fll. and Culex pipiens pipiens L. mosquito larvae infected with entopathogenic fungi Metarhizium brunneum and M. robertsii has been studied in laboratory conditions. It was shown that C. pipiens larvae are more susceptible to a water suspension of fungal conidia treatment while A. messeae larvae are more susceptible to treatment with dry conodia. The mortality of mosquito larvae infected with the most virulent strain and dose of 2 × 10⁶ conidia per cm² was 87.5–92.5%. Prospects for the use of fungi in the biocontrol of bloodsucking mosquitoes are discussed.     

Identification and biochemical characterization of the Anopheles gambiae 3-hydroxykynurenine transaminase

Spontaneous oxidation of 3-hydroxykynureine (3-HK), a metabolic intermediate of the tryptophan degradation pathway, elicits a remarkable oxidative stress response in animal tissues. In the yellow fever mosquito Aedes aegypti the excess of this toxic metabolic intermediate is efficiently removed by a specific 3-HK transaminase, which converts 3-HK into the more stable compound xanthurenic acid. In anopheline mosquitoes transmitting malaria, xanthurenic acid plays an important role in Plasmodium gametocyte maturation and fertility. Using the sequence information provided by the Anopheles gambiae genome and available ESTs, we adopted a PCR-based approach to isolate a 3-HK transaminase coding sequence from the main human malaria vector A. gambiae. Tissue and developmental expression analysis revealed an almost ubiquitary profile, which is in agreement with the physiological role of the enzyme in mosquito development and 3-HK detoxification. A high yield procedure for the expression and purification of a fully active recombinant version of the protein has been developed. Recombinant A. gambiae 3-HK transaminase is a dimeric pyridoxal 5'-phosphate dependent enzyme, showing an optimum pH of 7.8 and a comparable catalytic efficiency for both 3-HK and its immediate catabolic precursor kynurenine. This study may be useful for the identification of 3-HK transaminase inhibitors of potential interest as malaria transmission-blocking drugs or effective insecticides.     

Comparative responses of ovipositing Anopheles gambiae and Culex quinquefasciatus females to the presence of Culex egg rafts and larvae

Field observations have demonstrated that gravid Anopheles gambiae Giles s.s. (Diptera: Culicidae) are selective in their choice of oviposition sites. For example, immature stages of An. gambiae s.s. are rarely found in water that contains Culex quinquefasciatus Say immatures. The possibility that this may, in part at least, reflect a response by ovipositing An. gambiae s.s. females to volatile signals associated with Culex juveniles was evaluated by testing the response of An. gambiae s.s. females to varying densities of Cx. quinquefasciatus egg rafts and/or larvae in oviposition choice assays. For comparison, the oviposition choices of Cx. quinquefasciatus to conspecific egg rafts and/or larvae were similarly assayed. At a low density of Cx. quinquefasciatus egg rafts (1-15 egg rafts/100 mL water), An. gambiae s.s. females laid more eggs in the treatment water than in the control, with a maximum of twice as many in the treatment water at 5 egg rafts/100 mL water. At higher egg raft densities and in all treatments that included Cx. quinquefasciatus larvae, oviposition decreased significantly in the treatment dishes in a density-dependent manner. As previous studies have indicated, ovipositing Cx. quinquefasciatus females were attracted to and laid egg rafts in dishes containing conspecific egg rafts and, interestingly, also in dishes containing larvae.     

淡色库蚊中抗性相关羧酸酯酶的纯化及其生化性质

在库蚊Culex pipiens品系中,非专一性酯酶活性的升高是对有机磷杀虫剂产生抗性的重要机理之一。应用SDS/PAGE,比较淡色库蚊Culex pipiens pallens抗敌百虫品系(RD)、敏感型品系(S)和抗苄呋菊酯品系(PY)中可溶性总蛋白质带型,显示RD中含有一条特异蛋白带,其它两个品系中未检出。在RD成虫匀浆液总蛋白中含量高达2.1%。分子量测定为66 kD。应用柱层析法分离得到了较纯的纯品。以α-NA为底物测得K_m=64.1 mmol/L,V_max=249.8 mmol/(L·mg·min)。与羧酸酯酶相比较：其K_m值小于已报道的抗性品系及非抗性品系A-酯酶和B-酯酶。V_max值比已报道抗性品系A-酯酶低,比B-酯酶高。较高浓度的敌百虫并不能抑制其酶活,属于A-酯酶。在昆虫体内可能主要通过结合隔离作用(sequestration)提高昆虫对有机磷的耐受性,对有机磷杀虫剂水解作用的可能性也不能排除。     

Reproductive physiology of Anopheles gambiae and Anopheles atroparvus.

When exposed to a human host, Anopheles gambiae started probing 4 h post-eclosion, but 95% successfully blood-fed by 16-20 h with maximal blood volumes of 5- 10 microl per female. When fed sugar, the 95% feeding was not observed until 36-40 h post-eclosion; sugar meals appeared to interfere with blood meals. Similarly in An. atroparvus, maximum volumes were 10 microl when starved but only 6 microl when fed sugar. This species did not bite before 2 d, and 95% biting was by 4 d. Given single blood meals to water-kept An. gambiae, a threshold body size for oogenesis was detected. With wing lengths below 2.8 mm, eggs never matured, but when sugar-fed, females of all sizes matured eggs including the synthesis of maternal deposits. Although sugar feeding interfered with blood feeding, more lipid was transferred to the yolk. In water-kept An. atroparvus only 5% of the females produced eggs. When sugar-fed for 4 d, all females matured eggs, so in this species sugar feeding appeared to be essential for oogenesis. An. gambiae always took multiple blood meals, tested at any time after the first ones, leading to 120 mature eggs/female. Yolk composition was 3.9 mcal protein and 3.8 mcal lipid/oocyte when kept on water, but 2.8 meal protein and 4.3 mcal lipid/oocyte with intermittent sugar meals, thus marking a surprising flexibility in synthesis of yolk protein and lipid that strongly depends on additional carbohydrates sources. Only 80% of water-fed An. atroparvus re-fed 2 d after a first blood meal with small females taking three blood meals but they still showed reduced fecundity. Only the large water-fed females matured eggs, with blood volumes higher than 9-12 microl. When fed sugar, the blood meal input was reduced, but oogenesis was possible, whereas water-fed females required three blood meals to reach the caloric level comparable to pre-feeding sugar-fed females. Water-fedAn. gambiae could survive on daily blood meals alone, but survival was further extended by intermittent sugar meals. When offered a blood donor daily, there was a behavioral difference. Females maintained alone showed a more or less regular 3 d feeding and oviposition activity, while females kept in groups fed daily followed a daily oviposition pattern, suggesting gonotrophic discordance.     

Surface properties of Culex pipiens pipiens eggs and the behaviour of the female during egg-raft assembly

ABSTRACT. Eggs of the mosquito Culex pipiens pipiens L. float vertically because a flexible corolla at the anterior (lower) pole has a hydrophilic lower surface and a hydrofuge upper surface. The corolla assumes different configurations in the egg raft; the surface forces operating on eggs are briefly analysed. The outer surface of the chorion is hydrofuge, but in eggs floating on water there is a jacket of water surrounding the egg in the spaces between the tubercles of the exochorion, below the meshwork connecting them. The humidity over natural water can be low enough to cause continuous evaporation from the jacket; water is replenished through openings within the lower surface of the corolla. The disposition of the corolla prevents the uptake of materials confined to the water surface outside, but the mechanism could concentrate solutes in the jacket, with possible deleterious consequences. Eggs are joined together by inter-digitation of the regularly spaced tubercles. The behaviour by which the female assembles the egg raft is described in detail, explaining the form of the raft and the patterns in which eggs are packed.     

埃及伊蚊、冈比亚按蚊和致倦库蚊polyUBQ基因的电子克隆及其序列分析

针对具有选择性蛋白质降解功能的泛素在调控昆虫生长发育过程中的重要作用,探讨埃及伊蚊、冈比亚按蚊和致倦库蚊基因组中polyUBQ基因的有关生物信息。采用电子克隆的方法钓取3种蚊虫基因组中polyUBQ基因序列,分析其特征、分子进化关系、遗传多态性和密码子偏爱性。结果显示,成功获取埃及伊蚊、冈比亚按蚊和致倦库蚊polyUBQ基因序列,命名为Aa-polyUBQ(GenBank登录号:AAGE02005963)、Ag-polyUBQ(ABKP02009650)和Cq-polyUBQ(AAWU01023041),分别编码1 065个、218个和533个氨基酸残基,各具14个、3个和7个泛素单体,Aa-polyUBQ、Ag-polyUBQ和Cq-polyUBQ蛋白二级结构主要元件是延伸带和无规则卷曲,Leu、Ile和Lys是构成3种蛋白的主要氨基酸,亚细胞主要定位于细胞质和细胞核,无前导肽、信号肽和跨膜结构域,除Ag-polyUBQ蛋白外均呈碱性;3种基因序列的同源性较高(83.8%-88.2%)且遗传距离较近(0.129-0.187);检出135个多态位点,共生成3个单倍型,单倍型多样性(Hd=1.000)、平均核苷...     

Combined pyrethroid and carbamate 'two-in-one' treated mosquito nets: field efficacy against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus

A new approach is proposed in the treatment of mosquito nets, using a 'two-in-one' combination of pyrethroid and non-pyrethroid insecticides applied to different parts of bednets. The objectives are mainly to overcome certain limitations of pyrethroid-impregnated bednets currently recommended for malaria control purposes. Apart from developing alternatives to pyrethroid dependency, we sought to counteract pyrethroid irritant effects on mosquitoes (excito-repellency) and resistance to pyrethroids. The idea takes advantage of the presumed host-seeking behaviour of mosquitoes confronted by a net draped over a bed, whereby the mosquito may explore the net from the top downwards. Thus, nets could be more effective if treated on the upper part with residual non-irritant insecticide (carbamate or organophosphate) and with a pyrethroid on the lower part. Sequential exposure to different insecticides with distinct modes of action is equivalent to the use of a mixture as a potential method of managing insecticide resistance. We also intended to improve the control of nuisance mosquitoes, especially Culex quinquefasciatus Say (Diptera: Culicidae) that often survive pyrethroids, in order to encourage public compliance with use of insecticide-treated nets (ITNs). Polyester bednets were pretreated with residual pyrethroid (bifenthrin 50 mg/m2 or deltamethrin 25 mg/m2) on the lower half and with carbamate (carbosulfan 300 mg/m2) on the upper half to minimize contact with net users. Unreplicated examples of these 'two-in-one' treated nets were field-tested against wild mosquitoes, in comparison with an untreated net and bednets treated with each insecticide alone, including PermaNet wash-resistant formulation of deltamethrin 50 mg/m2. Overnight tests involved volunteers sleeping under the experimental bednets in verandah-trap huts at Yaokofikro, near Bouaké in C te d'Ivoire, where the main malaria vector Anopheles gambiae Giles, as well as Culex quinquefasciatus Say, are highly resistant to pyrethroids. Efficacy of these ITNs was assessed in the huts by four entomological criteria: deterrency and induced exophily (effects on hut entry and exit), blood-feeding and mortality rates (immediate and delayed). Overall, the best impact was achieved by the bednet treated with carbosulfan alone, followed by 'two-in-one' treatments with carbosulfan plus pyrethroid. Blood-feeding rates were 13% An. gambiae and 17% Cx. quinquefasciatus in huts with untreated nets, but only 3% with carbosulfan ITNs, 7-11% with combined ITN treatment, 6-8% An. gambiae and 12-14% Cx. quinquefasciatus with pyrethroid alone. Mosquitoes that entered the huts were killed sooner by nets with combined treatment than by pyrethroid alone. Mortality-rates in response to ITNs with carbosulfan (alone or combined with pyrethroid) were significantly greater for Cx. quinquefasciatus, but not for An. gambiae, compared to ITNs with only pyrethroid. About 20% of sleepers reported potential side-effects (headache and/or sneezing) from use of ITN treated with carbosulfan alone. Further development of this new 'two-in-one' ITN concept requires a range of investigations (choice of effective products, cost-benefit analysis, safety, etc.) leading to factory production of wash-resistant insecticidal nets treated with complementary insecticides.     

Viability of Culex pipiens pipiens eggs affected by nutrition and aposymbiosis

Raisins were a better source of carbohydrate than sucrose for reproduction by autogenous Culex pipiens. A blood meal increased the number of eggs per raft from 49 autogenously to 114. Eggs of aposymbiotic females produced autogenously did not hatch, but 34% of the eggs were viable if the mosquitoes fed on chickens. With repeated blood meals the number of eggs per raft and the rate of embryonation and hatching declined in each successive gonotrophic cycle. In about $\text{1}\text{4}$ of the unhatched eggs of normal females there were no fully developed embryos, while many more of the unhatched eggs of aposymbiotic females contained no evidence of embryonic development. After the fifth blood meal, neither normal nor aposymbiotic insects oviposited. The ovaries of the nulliparous females contained approximately 10% of the potential number of mature terminal oocytes. Proximally in the ovarioles there were dilatations and coiled tracheoles indicating egg resorption. There were fewer parous follicles in aposymbiotic than in normal females. Larval rearing water, i.e., infusion in which larvae had been reared, was more attractive than fresh infusion for oviposition by normal, autogenous mosquitoes. The degree of embryonation of the eggs was lower and the hatching success rate was poorer in fresh infusion than in larval rearing water.     

The hemolymph proteome of Anopheles gambiae

We used two-dimensional SDS-PAGE and microsequencing or peptide mass fingerprinting to identify major proteins in the hemolymph of Anopheles gambiae. We found approximately 280 protein spots in hemolymph and identified 28 spots, representing 26 individual proteins. Most of these proteins have known or predicted functions in immunity, iron transport, or lipid biology. Many of the proteins have been found in hemolymph in other insects but one protein is novel: a new member of the ML family (involved in lipid recognition). Three of the identified proteins increased in spot intensity or appeared de novo following bacterial injection: a phenoloxidase, and two chitinase-like proteins. A subset of proteins decreased following bacterial injections: these included the light and heavy chains of ferritin. Several proteins appeared in hemolymph following any wound or injection. Most of these are metabolic enzymes lacking signal peptides that are likely to be released as a result of damage to muscles and other tissues by injury. The map will provide a useful tool for examining changes in hemolymph proteins following blood feeding and infection by parasites.