Existence of Two Acetylcholinesterases in the Mosquito Culex pipiens (Diptera: Culicidae)

Abstract: Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens . The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 × 10⁻⁴ M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 × 10⁻⁴ M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 × 10⁻⁴ M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.     

一个致倦库蚊杀虫剂敏感品系的筛选

化学防治是控制蚊虫传播疾病的主要方法, 抗性监测表明我国蚊虫已对有机磷、 有机氯、 氨基甲酸酯和拟除虫菊酯类杀虫剂产生了不同程度的抗性。蚊虫抗药性的分子机制主要包括靶标抗性和三大解毒酶家族带来的代谢抗性。筛选对杀虫剂敏感的品系是抗性监测和抗性机理研究必不可少的材料。本研究通过从一个致倦库蚊Culex pipiens quinquefasciatus野生种群筛选无乙酰胆碱酯酶G119S突变且具有低活性羧酸酯酶、 P450单加氧酶和谷胱甘肽-S-转移酶的单雌系, 建立了一个对杀虫剂敏感的致倦库蚊品系。该品系的羧酸酯酶活性是敏感品系S-lab的2.5倍, P450单加氧酶和谷胱甘肽-S-转移酶的活性与S-lab相当。生物测定表明, 与S-lab相比, 该品系对有机磷杀虫剂有低于2倍的抗性, 对氨基甲酸酯和拟除虫菊酯类杀虫剂没有抗性, 可以作为相对敏感品系用于抗性监测。     

Effects of blood meal source on the reproduction of Culex pipiens quinquefasciatus (Diptera: Culicidae)

Culex pipiens quinquefasciatus were fed blood meals from a live chicken (LC), chicken blood in Alsever's (AC) solution, defibrinated bovine blood (DB), or bovine blood in citrate (CB) and incubated at 28° C. The effects of different blood meal sources were evaluated with respect to rates of blood feeding and reproduction (i.e., fecundity and fertility) over two gonotrophic cycles. Mosquitoes that fed on the first blood meal were subjected to a second blood meal as follows (first blood meal / second blood meal): LC/LC, LC/DB, DB/DB, CB/CB, AC/AC. Fecundity and fertility of Cx. p. quinquefasciatus were significantly (P < 0.05) greater in mosquitoes fed LC blood; however, fecundity and fertility in different treatment groups varied by gonotrophic cycle. These results contribute to our understanding of the impact of blood meal source on feeding and reproduction in Cx. p. quinquefasciatus. The potential impacts of blood meal source on virus transmission experiments are discussed.     

致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用

为探讨致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用,通过病毒分离、逆转录聚合酶链反应、透射电镜等技术进行了相关研究。结果表明：吸食感染性血液后,登革Ⅱ型病毒能侵染白纹伊蚊中肠上皮细胞并大量复制,但不能侵染致倦库蚊中肠上皮细胞。以上研究证明致倦库蚊对登革Ⅱ型病毒存在中肠感染屏障。     

Comparison of chlorpyrifos resistance in Culex pipiens pipiens (Diptera: Culicidae) collected from Northern and Southern Tunisia

In this study, we investigated resistance to the organophosphates chlorpyrifos in Tunisian populations of Culex pipiens pipiens. Three field populations were collected from Northern and central Tunisia between 2003 and 2005 and used for the bioassays tests. Our results registered moderate and high levels of resistance to chlorpyrifos which ranged from 33.8 to 111. The chlorpyrifos resistant populations were highly resistant to propoxur indicated an insensitive acetylcholinesterase 1 (AChE 1). The highest frequency of AChE 1 resistant phenotypes (64%) was recorded in the most resistant population (sample # 1). Bioassays conducted in the presence of synergists showed that not esterases were involved as the resistance mechanism to chlorpyrifos. However, CYP450 was partly involved in the resistance of the most resistant sample (# 1). Starch electrophoresis showed that three esterases were present in studied samples: A2‐B2, A4‐B4 and B12. Results are discussed in relation to the selection pressure caused by insecticide treatments.     

Effect of body size on energy reserves in Culex pipiens pallens females (Diptera: Culicidae)

We examined the relationship between body size and energy reserves of female Culex pipiens pallens under laboratory conditions. Small and large‐sized adult mosquitoes were obtained from larvae reared at different densities. There was a significantly positive relationship between pupal body weight and adult wing length depending on body sizes. Studies showed large females contained significantly higher contents of glycogen and lipid but lower protein stores in comparison to small females. Large sized mosquitoes accumulated higher percentages of glycogen (21.2% vs 15.9%) and lipid (45.8% vs 30.7%) but lower levels of protein (33.0% vs 53.4%) than small females. This result suggests that larval‐derived glycogen and lipid stores may be important in determining the body size of female Cx. pipiens pallens.     

Larvicidal potency of selected xerophytic plant extracts on Culex pipiens (Diptera: Culicidae)

Chemical insecticides released into the environment may have adverse biological effects. Therefore, there is a need for ecofriendly insecticides for mosquito control. Xerophytic plant extracts that may provide more ecofriendly active component were evaluated against Culex pipiens 4th instars. Plant extracts prepared using different solvents with a Soxhlet apparatus and different concentrations were tested against Culex pipiens larvae. The effects were observed at 24 h and 72 h intervals and LD₅₀ and LD₉₀ values determined. Chloroform (CHCl₃) and ethyl acetate (EtOAc) extracts of Althaea ludwigii were the most effective against Cx. pipiens 4^th instars, but were highly dependent on extract concentrations and exposure time. Results suggest that A. ludwigii extracts contain bioactive compounds, such as phenols and saponins, that may provide effective Cx. pipiens larval control. However, the extract was found to be toxic to zebrafish larvae, and may be toxic to other aquatic fauna. Further studies to determine the active components and toxicity to other fauna are needed.     

致倦库蚊主要组织结构的石蜡切片观察

【目的】蚊虫是传播人类多种疾病的重要媒介害虫, 对其组织形态学的认识是开展众多领域研究的基础。本文通过研究致倦库蚊Culex pipiens quinquefasciatus成虫组织结构及形态, 为媒介蚊虫的抗药性研究及有效防治提供基础材料。【方法】采用改进的石蜡切片法和HE染色, 结合活体内脏器官解剖及光学显微镜观察, 从形态学和组织学水平对致倦库蚊组织结构做详尽展示。【结果】获得结构完整、 染色清晰、 定位准确的消化排泄系统、 生殖系统、 神经系统、 呼吸系统等HE染色石蜡切片。【结论】探讨了改进制片和染色过程中一些步骤及注意事项。研究结果为利用原位杂交、 免疫组化等方法研究蚊虫体内抗药性基因的准确定位及基因功能分析提供了可靠的基础。     

41种植物精油对淡色库蚊的熏蒸活性

为发现具有较高杀蚊活性的精油, 推动植物精油类卫生杀虫剂的创制, 本研究采用三角瓶熏蒸法测定了17科41种植物精油对淡色库蚊Culex pipiens pallens雌成蚊的熏蒸活性, 并进一步采用密闭圆筒法验证其杀虫毒力大小。三角瓶熏蒸法测定表明, 在12 μL/L浓度下, 留兰香油（spearmint oil）、 薰衣草油（lavender oil）、 山苍子油（Litsea cubeba oil）等26种精油均有较强的熏蒸作用, KT₅₀小于15 min。该26种精油用密闭圆筒熏蒸法进一步测定表明, 在浓度为10 μL/L时, 冬青油（wintergreen oil）、 艾叶油（blumea oil）、 薄荷油（peppermint oil）、 桉叶油（Eucalyptus robusta oil）和蓝桉油（E. globulus oil）等植物精油熏蒸活性较强, 其KT₅₀分别为16.91, 21.20, 22.57, 18.43和19.48 min。结果证明冬青油、 艾叶油、 薄荷油、 桉叶油和蓝桉油等5种精油对淡色库蚊具有较强的熏蒸活性, 具备作为淡色库蚊防控剂开发的潜力, 值得进一步研究。     

单宁酸对淡色库蚊抗氰戊菊酯品系和敏感品系幼虫生长发育的影响

单宁酸是一种植物次生代谢物, 为探索其用于蚊幼虫防治的可能性,在室内测定了其对淡色库蚊Culex pipiens pallens抗氰戊菊酯品系和敏感品系1～4龄幼虫的毒性,并观察了其对存活幼虫生长发育的影响。结果表明,淡色库蚊敏感品系幼虫对单宁酸的敏感性比抗氰戊菊酯品系的要高,1～4龄幼虫分别高6.4、4.9、4.7和2.0倍。4个龄期幼虫中,无论是敏感品系还是抗氰戊菊酯品系,均是1龄幼虫对单宁酸的敏感性最高,3龄幼虫最低。在1 000 mg/L单宁酸持续作用下,敏感品系和抗氰戊菊酯品系各龄幼虫的存活率,均随处理时间延长而降低。与对照相比,饲养在100 mg/L～500 mg/L单宁酸溶液中的存活幼虫发育历期延长,敏感品系和抗氰戊菊酯品系发育历期分别延长了34.5～38.3 h和59.2～93.4 h。其中,125 mg/L浓度处理的敏感品系1～4龄幼虫,其发育历期与对照的差异达到了显著水平(P<0.05);抗性品系则在250 mg/L作用下也达到了差异显著水平(P<0.05)。但100～250 mg/L单宁酸处理淡色库蚊抗氰戊菊酯品系和敏感品系1龄幼虫,对其存活幼虫的化蛹率、羽化率和成虫性比均无显著影响。表明单宁酸对淡色库蚊幼虫的影响主要是延迟其生长发育,且影响程度与蚊虫对氰戊菊酯的敏感性有关。     

Two Bacillus sphaericus binary toxins share the midgut receptor binding site: implications for resistance of Culex pipiens complex (Diptera: Culicidae) larvae

This work demonstrates that Bin1 and Bin2 toxins, produced by Bacillus sphaericus strains IAB59 and 2362, respectively, share a binding site in midgut brush border membranes (BBMF) from Culex pipiens complex larvae. However, a colony selected with strain IAB59, displaying a resistance ratio of only 42-fold to IAB59, but a 162,000-fold resistance to strain 2362, was found to miss receptors for Bin2 in the BBMF. This correlates with results showing that Bin1, produced in strain IAB59, failed to bind specifically to BBMF from other colony highly resistant to strain 2362. Data indicate the loss of the BBMF bound receptor as a general mechanism of resistance to binary toxins in mosquito.     

不同地理株尖音库蚊复组雄蚊尾器的光学镜和扫描电镜观察

本研究对烟台、济南、徐州、镇江、杭州、福州及厦门等不同纬度地区尖音库蚊复组的雄蚊尾器在光学镜下进行了观察。结果表明:在杭州以南地区,致倦库蚊为本复组的唯一成员,属南方组;而徐州及镇江地区的本复组成员中,尚包括淡色库蚊及中间型,与武汉地区的类似,统属中介组。济南以北地区的本复组蚊虫,仅有淡色库蚊,属于北方组。 通过光学显微镜和扫描电镜观察,进一步揭示了本复组雄蚊尾器的形态随纬度发生递交的现象;阳茎侧板中叶的形态特征是我国古北界与东洋界交错过渡地带本复组蚊虫的可靠鉴别依据。在过渡地带,所谓纯的淡色库蚊或致倦库蚊是很难得到的,这一假想在蚊虫抗性试验中具有实际意义。在扫描电镜下,还发现了在光学显微镜下未见过的一些特征,它们在分类学上的地位及其生理意义有待进一步探讨。但是,尾器的感觉毛数量多、分布广,可能在完成交配行为中具有重要意义。     

不同地理种群尖音库蚊复组抗性动态和遗传多样性

通过生物测定、蛋白质电泳和等位酶分析等方法对5个不同地区的尖音库蚊复组蚊虫Culex pipiens complex的抗性水平、种群中非特异性酯酶基因表型分布和种群遗传多样性进行了研究。不同地理种群的抗性检测结果表明：5个种群分别对敌敌畏、对硫磷、氯菊酯和溴氰菊酯的抗性较高,对残杀威、巴沙和胺菊酯的抗性较低;朝阳种群对敌敌畏抗性最高（55.7倍）,武汉种群次之;佛山种群对氯菊酯和溴氰菊酯的抗性比率高达123倍和23.9倍。酯酶电泳结果显示：5个种群间酯酶多态性存在差异,广州和佛山两个库蚊种群酯酶表型多态性最高,有B1,A2-B2,A8-B8,A9-B9,B10和A11-B11等6种酯酶表型,提示高活性酯酶是主要的抗性机制。群体遗传学研究表明：每位点平均等位基因数（A）为2.76,平均多态位点百分率（P）为64.45％,平均预期杂合度（He）为0.1943,种群间遗传分化系数（Fst）值为0.10,平均基因流(Nm)＝2.57,说明5个种群有较丰富的遗传多样性,种群内遗传多样性高于种群之间。据此推测,种群间可以通过迁徙等方式进行基因交流,使得遗传结构、抗性水平朝一致性方向变化。本研究对我国尖音库蚊复组蚊虫的综合治理有一定指导意义。     

Repellency of inseminated Culex pipiens (Diptera: Culicidae) females to various wing-beat sound frequencies in modulated intensities in a flight chamber

Wing‐beat sound frequencies at 370, 440, 493 and 554 Hz were evaluated for female mosquito repellency. One hundred inseminated females of Culex pipiens were introduced into a 30 cm × 30 cm × 3 m screened cage with an electronic device (cellular phone) emitting selected frequencies positioned at one end of the chamber. The mosquitoes were counted in areas of 0.0–0.5, 0.5–1.0, 1.0–2.0 and 2.0–3.0 m from the cellular phone. In a second test, 50 Cx. pipiens were released in the screened cage with a shaved rat as bait placed beside the cellular phone (370 Hz) at the center of the chamber. Statistical analysis (anova ) showed that significantly fewer (P < 0.05) mosquitoes rested near the cellular phone at 370 Hz, but not at the other frequencies. Similarly, there was significant reduction (mean 33.0%) in the number of mosquitoes taking a blood meal on the shaved rat when the cellular phone was turned on (P < 0.01).     

Repellent efficacy of wood vinegar against Culex pipiens pallens and Aedes togoi (Diptera: Culicidae) under laboratory and semi-field conditions

The repellent efficacy of wood vinegar was assessed against mosquitoes under laboratory conditions at 1, 5, 10, 20, 40, 60 and 80% concentrations. The study evaluated whether wood vinegar is able to repel Culex pipiens pallens Coquillet and Aedes togoi (Theobald) from the human body and if so at what concentrations. The tests were conducted using the arm-in-cage method in 80 × 40 × 40 cm screened mosquito cages. The data were analyzed and compared with those of N,N-Diethyl-3-methylbenzamide (deet) at 10.3% concentration. The results showed that wood vinegar provided mosquito repellence of varying degree depending on the concentration used. The observed repellence averaged from as low as 39.6% at 5.0% concentration to as high as 100% at 80% concentration against Ae. togoi. Repellence against Cx. pipiens pallens was high being 90.3% at 20% concentration, 92.2% at 40% concentration, 93.9% at 60% concentration and 100% at 80% concentration. The duration of protection time tests showed that the 40% and 60% concentrations of the wood vinegar give protection from landing of Ae. togoi for a period of up to 7 h, though the lower concentration gave lower protection after the first five hours. The results indicated that wood vinegar has mosquito repellent characteristics that tend to vary with the concentration used and the species of mosquitoes. Wood vinegar in this case was very effective in repelling Cx. pipiens pallens, even at lower concentrations while higher concentrations were required to repel Ae. togoi.     

香芹酮和薄荷醇手性异构体对淡色库蚊熏蒸及驱避活性

【目的】比较香芹酮和薄荷醇各手性异构体对淡色库蚊Culex pipiens pallens成蚊的熏蒸及驱避活性,为其应用提供理论依据。【方法】以3日龄淡色库蚊雌成蚊为供试昆虫,采用三角瓶熏蒸法比较测定香芹酮和薄荷醇的5种手性异构体的击倒与熏蒸致死活性;用Y-型嗅觉仪法比较测定驱避作用;将活性较好单体制成电热蚊香片,用方箱法进一步评价药效。【结果】5种异构体对淡色库蚊均具有一定的击倒和致死作用,其中L-香芹酮的击倒速度最快,KT50为9.75 min;D-香芹酮的致死作用最强,LC50为0.26μL/L。L-香芹酮和D-香芹酮对淡色库蚊均具较好的驱避作用,在0.5μL剂量下,处理30 min后的驱避率仍达100%。方箱法实验表明,以200μL的L-香芹酮和D-香芹酮为有效成分的电热蚊香片加热1 h后,对淡色库蚊雌成蚊的KT50分别为13.70和17.90 min。【结论】香芹酮对淡色库蚊雌成蚊的熏杀活性优于薄荷醇,且不同异构体的活性存在显著差异,L-香芹酮具备被开发为植物源卫生害虫防控剂的潜力。     

Influence of light and habitat on predation of Culex quinquefasciatus (Diptera: Culicidae) larvae by the waterbugs (Hemiptera: Heteroptera)

The influence of light and habitat structure on the predation of Culex quinquefasciatus larvae by the common heteropteran water bug, Diplonychus (=Sphaerodema) annulatus, D. rusticus and Anisops bouvieri was assessed in the laboratory. It was revealed that water bugs predate more in presence of light than in dark conditions. While A. bouvieri consumed more prey in structured conditions, D. annulatus and D. rusticus consumed more prey in open conditions. The selection of prey size as well as the respective numbers varied between predators and treatments significantly. Prey vulnerability (PV), an indicator of predatory efficiency, was highest for D. annulatus, moderate for D. rusticus and low for A. bouvieri. Prey consumption and PV values under different treatment conditions indicate that for belostomatid water bugs D. annulatus and D. rusticus, the order of prey consumption under different habitat and light/dark combinations is light open > dark open > light vegetated > dark vegetated. In the case of the backswimmer, A. bouvieri, the order of prey consumption appeared to be light structured > dark structured > light open > dark open. These findings were consistent with resource‐partitioning by water bug species sharing the same guild. If the observed results are extended to natural settings the efficiency of these predators in controlling mosquito populations will vary with the structural complexity of habitats and the intensity of light.     

冬青油与肉桂油对淡色库蚊的生物活性

为进一步明确冬青油（wintergreen oil）与肉桂油（cinnamon oil）对蚊虫的生物活性, 本研究采用浸液法、 “Y”型嗅觉仪法和密闭三角瓶熏蒸法, 分别测定了两种精油对淡色库蚊Culex pipiens pallens幼虫的毒杀作用以及对成虫的驱避和熏杀活性。结果显示： 肉桂油和冬青油对淡色库蚊幼虫均具有较强的毒杀作用, 处理24 h的LC₅₀分别为71.87 mg/L和 102.83 mg/L。在0.5 μL的供试剂量下, 冬青油与肉桂油在20 min内对淡色库蚊的驱避率均在80%以上, 显示了较好的驱蚊活性。冬青油对淡色库蚊具有明显的熏蒸击倒作用, 12 μL/L浓度下的KT₅₀为3.97 min; 而肉桂油则具有较好的熏蒸致死活性, 熏蒸5 h的LC₅₀ 为0.31 μL/L。冬青油与肉桂油按1∶1体积比混配后对淡色库蚊表现出较好的击倒和致死效果。冬青油和肉桂油既对淡色库蚊幼虫具有较强的毒杀作用, 又对淡色库蚊雌成虫表现出较好的驱避和熏杀活性, 具有开发成为植物源蚊虫防控剂的潜力。     

Sympatric occurrence of Culex pipiens (Diptera,Culicidae) biotypes pipiens,molestus and their hybrids in Portugal,Western Europe: feeding patterns and habitat determinants

Culex (Culex) pipiens (Diptera: Culicidae) has two recognized biotypes, pipiens and molestus, which differ in physiology and behaviour; this difference may influence vectorial capacity for West Nile virus (WNV). Our goal was first to determine the presence of Cx. pipiens populations in 31 locations in Portugal and to subsequently analyse their host‐feeding preferences and habitat determinants. Molecular identification of Cx. pipiens forms and their hybrids was performed in 97 females; bloodmeal sources were identified in 59 engorged specimens. Overall, 61.9% of specimens were identified as Cx. pipiens f. pipiens, 20.6% as Cx. pipiens f. molestus, and 17.5% as hybrid forms. Culex pipiens f. pipiens fed preferentially on birds, and Cx. pipiens f. molestus on humans. Hybrid forms fed mostly on birds, but human bloodmeals were common. With reference to habitat, Cx. pipiens f. pipiens and hybrid forms were positively correlated with peri‐urban habitats. Our results confirm the sympatric presence of different Cx. pipiens biotypes in 14 of the 31 locations studied. Peri‐urban areas were a common habitat of all biotypes and may represent zones of hybridization. The feeding preferences and sympatric distribution of the Cx. pipiens biotypes observed in Portugal favour the epizootic circulation of WNV and the occurrence of disease outbreaks of WNV.     

Inhibition of the growth and development of mosquito larvae of Culex pipiens L. (Diptera: Culicidae) treated with extract from flower of Matricaria chamomilla (Asteraceae)

Mosquitoes can transfer many adverse diseases to human and animals therefore, there is a need to fight their spread. Among promising larvicidal sources is the use of plant extracts, which will play an important role in the future. This study was conducted to assess the larvicidal activity of Ferula hermonis Boiss,Achillea millefolium, Salvia officnalis, Psidium guaja and Matricaria chamomilla extract against Culex pipiens. The plant materials were extracted with hexane, dichloromethane, ethyl acetate, and methanol using a Soxhlet extractor. The extracts were evaluated for larvicidal activity against Cx. pipiens the mortality was monitored after 24 and 48 h of exposure. None of the extracts tested showed larvicidal acitivties except M. chamomilla. The ethyl acetate extract showed the most promising larvicidal activity with LC₅₀ values of 287.1 and 209.4 ppm after 24 and 48 h of exposure, respectively. Treatment of the eggs with different concentrations of the active extract decreased the hatchability of the eggs dose dependently from 95 to 86.49%. Similarly, the pupal duration increased in treated groups. The larval period lasted for 12 d, whereas that of the control group lasted for 10 d. Furthermore, the pupal period lasted 3 d (control 2 d) in treated groups. The data also revealed a significant decrease in the growth index in treated groups (0.00–7.53) than that of the control (8.5). The GC–MS analysis revealed the presence of 1.6.10‐dodecatriene, 7,11‐dimethyl‐3‐methylene (89.68%), 1,6‐cyclodecadiene, 1‐methyl‐5‐methylene‐8‐(1‐methylethyl)‐, [S‐(E,E)] (6.34%), 2H‐pyran ?3‐ol (4.04%), and 2H‐1‐benzopyran‐2‐one (0.079%).