Impact of Urban Agriculture on the Species Distribution and Insecticide Resistance Profile of <i>Anopheles gambiae s.s.</i>and <i>Anopheles coluzzii</i>in Accra Metropolis, Ghana

Malaria incidence in urban areas has generally been low compared to rural areas but recent data indicate that urban malaria remains a public health problem. It is therefore important to understand the factors that promote urban malaria to help formulate future vector control strategies. This study compared Anopheles gambiae s.l. (A. gambiae s.l.) species composition, distribution and insecticide resistance mechanisms between vegetable and non-vegetable growing areas in Accra Metropolis. Four sites were selected within the city of Accra which comprised of two vegetable-growing and two non-vegetable growing areas. WHO susceptibility tests were carried out on adults A. gambiae s.l. reared from larvae collected from the sites. Five insecticides were tested and the A. gambiae complex, resistance genotypes and enzyme activities of each population were characterized. All A. gambiae s.l. populations tested were resistant to all the insecticides, but relatively lower mortalities were observed in the vegetable growing areas. The mortality against 0.05% deltamethrin was 2.6% (Opeibea) and 12.5% (Korle-Bu) for the vegetable growing areas and 36.2% (Achimota) and 38.9% (Mataheko) in the non-vegetable growing areas. Anopheles gambiae s.s. (95% of Opeibea population) and Anopheles coluzzii, (98% of Korle-Bu population) were the dominant species in the vegetable growing areas. The voltage-gated sodium channel (Vgsc-1014F) frequencies of all the populations were similar but the acetylcholinesterase (ace-1) frequencies were significantly lower (p A. gambiae s.l. from Opeibea than from the other areas. The contribution of urban agriculture in the development of insecticide resistance needs to be considered in the formulation of future vector control strategies alongside other domestic usages.

Observation of granulations in the basal body of ovarioles and follicular dilatations for the determination of physiological age of Anopheles gambiae s.s.

Objective:To explore ovariole basal body granulations and follicular dilatations for determining physiological age inAnopheles gambiaes.s.(An.gambiaes.s.).Methods:Mosquitoes were collected by using window trap catch and identified morphologically.For the first lot ofmosquitoes,they were dissected,and ovary was left in distilled water for reading ovarian tracheoles and the second was cut and transferred to another blade in a physiological liquid for verification of ovariole basal body granulations.The same approach was followed with the second lot of mosquitoes where follicular dilatations were found after classic dilaceration of ovaries were transferred into physiological liquid.The other body parts of mosquitoes were used to identify the species of theAn.gambiaes.s.complex by PCR.Results:Among the 123An.gambiae s.s.of the first lot,the method of Detinova determined the age of 89 mosquitoes versus 114 for the observation of granulations(P>0.05).Among the 112An.gambiae s.s.of the second lot,the method of Detinova determined the age of 84 mosquitoes versus 93 for the observation of follicular dilatations(P>0.05).Unlike the method of Detinova,observation of follicular dilatations and basal body granulations of ovarioles were possible beyond the stage II Christophers.Conclusions:Overall,the observation of follicular dilatations and ovariole basal body granulations are reliable for the determination of the physiological age inAn.gambiaes.s.Furthermore,these two methods can be used beyond the stage II.

Larvicidal efficacy of monoterpenes against the larvae of Anopheles gambiae

Objective:To evaluate the larvicidal efficacy of eight volatile components of essential oils against 3rd instar larvae of Anopheles gambiae s.s.Methods:Larvicidal effects of each compound were evaluated in both laboratory and semi-field trials.Stock solution was prepared and serial dilutions were made in six concentrations for each compound.A total of 20 larvae were exposed to larvicides for each replicate and monitored at intervals of 12,24,48 and 72 h.Larvae monitoring was done on basis of dead and live larvae in all intervals.Results:All assayed compounds were larvicides and presented varying degrees of larval toxicity,with LC50 values ranging from 1.28 to 1 938.92 mg/L depending on the treatment time(12,24,48 or 72 h).(-)-Perillyl alcohol presented the strongest larvicidal activity towards Anopheles gambiae larvae,with LC_(50) values of 73.60,18.36,1.72 and1.28 mg/L after 12,24,48 and 72 h of exposure,respectively.The next strongest were(-)-isopulegol(LC_(50)= 135.10,49.39,34.39 and 20.22 mg/L) and(-)-carvone epoxide(LC_(50)= 168.86,124.74,80.84 and 23.46 mg/L).After 12,24 and 48 h of treatment,hydroxydihydrocarvone was the least toxic compound,with LC_(50) values of 1 938.92,1 172.18 and 401.03 mg/L,respectively.Conclusions:The data obtained in this study suggest that all evaluated monoterpenes,especially(-)-perillyl alcohol,have remarkable larvicidal effects and may be considered as potential sources for the development of suitable natural larvicides for mosquito management programs.Further small-scale field trials should be conducted.

The molecular mechanism for DDT detoxification in Anopheles gambiae: A molecular docking study

The epsilon class glutathione-S-transferase of Anopheles gambiae, agGSTe2, is capable of metabolizing DDT. A molecular docking analysis of DDT with agGSTe2 support an E2 elimination mechanism wherein the glutathione sulfur serves as the base to convert DDT to DDE.

Efficacy of the Microbial Larvicide VectoMax^®G against <i>Anopheles gambiae</i>s.l. and <i>Culex</i>spp. Larvae under Laboratory and Open Field Trial Experiments in the City of Yaoundé, Cameroon

Background: With the rapid expansion of insecticide resistance limiting the effectiveness of insecticide-based vector control interventions, integrated control strategies associating larviciding could be appropriate to improve current control efforts. The present experimental study assesses laboratory and field efficacy of the larvicide VectoMax®G on Anopheline and Culicine larval stages in Yaoundé. Methods: The effect of the larvicide VectoMax®G, a combination of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs), on larval development was assessed during both laboratory and open field trial experiments. Laboratory experiments permitted the evaluation of five different concentrations with four replicates/experiments. Laboratory experiments were conducted with Anopheles coluzzii “Ngousso” and Culex quinquefasciatus laboratory strains. Open field trials were conducted using sixteen plastic containers with a diameter of 0.31 m buried in an array of four rows with 4 containers each. Distance between rows and between containers in a row was 1 meter. This experiment permitted to test the effect of the microbial larvicide VectoMax®G under operational application conditions on field mosquito populations. Results: The time to induce 100% mortality after exposure to serial concentrations of the larvicide varied according to the dose from 4 - 12 hours for An. coluzzii and 6 - 9 hours for Cx. quinquefasciatus in laboratory experiments. Measurements of the residual activity indicated that all VectoMax®G concentrations were still active after 35 days and killed 86% - 100% of larvae. Lethal dose of VectoMax®G killing 50% of larvae was estimated at 5.24 × 10-8 mg/m2 for An. coluzzii and 1.25 × 10-8 mg/m2 for Cx. quinquefasciatus. The lethal concentration inducing 95% mortality was estimated at 3.13 × 10-7 mg/m2 for An. coluzzii and 2.5 × 10-8 mg/m2 for Cx. quinquefasciatus. Open field trials tests indicated that sub-lethal concentrations of VectoMax®G successfully killed 100% An. gambiae s.l. larvae within 24 hours, while with Culex spp. larvae, 100% mortality was recorded after 48 hours post-treatment. Natural recolonization of water containers by larvae was recorded between 3 and 6 days respectively after the treatment with sublethal doses. Late instar larvae were recorded 5 and 6 days after treatment. When the jars were treated with reference dosage or supra doses of VectoMax®G, recolonization of water containers was observed six days after treatments. No pupae of both species were found 6 and 7 days post-treatment. Conclusions: The study indicated high efficacy of the microbial larvicide VectoMax®G against Anopheline and Culex larvae. Microbial larvicides such as VectoMax®G could be appropriate for controlling mosquito population particularly in areas experiencing high insecticide resistance or outdoor biting mosquitoes.

Fauna and some biological characteristics of Anopheles mosquitoes(Diptera:Culicidae) in Kalaleh County,Golestan Province,northeast of Iran

Objective:To determine fauna and some ecological aspects of Anopheles mosquitoes in northeast of Iran.Methods:In this descriptive study,3 villages in Kalaleh County were selected in different geographical zones.Anopheles mosquitoes were collected biweekly from May to October using standard dipping method for larvae,and hand catch,total catch,artificial pit shelter as well as night-biting collections on human and animal baits for adults.Results:Totally 399 larvae and 2 602 adults of Anopheles mosquitoes were collected and identified as 2 species:Anopheles superpictus s.l.(An.superpictus s.l.) and Anopheles maculipennis s.l.The dominant species was An.superpictus s.l.(92.1%).Activity of these mosquitoes found to be started from middle of May and extended till September with two peaks of activity in July and August.Conclusions:An.superpictus s.l.as one of the main malaria vectors in Iran as well as some other parts of the world is the dominant species in the study area.This species has high potential for transmission and possibility of establishing a transmission cycle with low abundance.Other species,Anopheles maculipennis s.l.also has introduced as a malaria vector in northern parts of Iran.As this Anopheles is a complex species,genetic studies are recommended to determine the members of this complex in the study area.

应用mtDNA-COⅠ基因序列研究我国大劣按蚊A、D群体的遗传差异

目的应用线粒体DNA细胞色素氧化酶亚单位Ⅰ(mtDNA-COⅠ)基因序列特征阐明我国大劣按蚊A、D群体的遗传差异和分化水平。方法研究样本包括大劣按蚊A海南群体(n=13),大劣按蚊D云南江城群体(n=17)和勐腊群体(n=17),所有样本均以rDNA-ITS2序列差异为依据,进行分子鉴别确认;扩增和测定mtDNA-COⅠ基因片段,用MEGA(Version2.1)、TCS(Version1.12)、ARLEQUIN(Version2.0)等软件对基因序列进行生物信息学分析。结果分析mtDNA-COⅠ基因中长度为959bp的片段,显示大劣按蚊A的单倍型共3个,大劣按蚊D的单倍型共6个,均匀分布于3个群体。勐腊群体内错配平均数(7.4412)明显大于江城群体(1.2794)和海南群体(1.0513),表明勐腊群体内个体间分化程度最大。分步AMOVA的计算结果显示群体间基因交流有限(Fst=0.7999),群体间变异(79.99%)明显大于群体内变异(20.01%)。结论我国大劣按蚊A、D群体之间的遗传差异较小,个体间的分化水平较高。

微小按蚊不同发育期蛋白质和同工酶的研究

用聚丙烯酰胺凝胶等电点聚焦电泳法,比较研究了微小按蚊(广义)卵、二龄和四龄幼虫、蛹及雌性成虫蛋白质和酯酶同工酶电泳图谱。微小按蚊(广义)不同发育期蛋白质等电点pH值,卵、幼虫、蛹和成虫显带区在pH4.1～9.5,其中在酸性区显带最多,碱性次之;酯酶同工酶等电点pH值显带区在pH4.2～7.8,其中酸性区显带最多,中性次之。并对微小按蚊复合体作了讨论。

中华按蚊谷胱甘肽-S-转移酶E6(AsGSTE6)的表达纯化与结晶

【目的】对中华按蚊Anopheles sinensis谷胱甘肽-S-转移酶E6(As GSTE6)进行初步晶体学研究,为深入研究其空间结构奠定基础。【方法】利用生物信息学软件,首先对中华按蚊As GSTE6进行生物信息学预测和分析;然后进行分子克隆,构建重组子p ET28a-As GSTe6;利用大肠杆菌Escherichia coli原核表达系统进行诱导表达;采用Ni-NTA金属螯合层析和葡聚糖凝胶层析的方法对重组蛋白进行纯化;通过葡聚糖凝胶层析和化学交联的结果分析蛋白的聚合状态;运用坐滴蒸气扩散法对重组蛋白进行结晶筛选。【结果】生物信息学分析结果表明,As GSTE6分子量为25.28 k D,无信号肽和跨膜区,是亲水性蛋白;三级结构预测结果显示,As GSTE6主要由一个小的βαβαββα折叠模式的N端结构域和一个大的全螺旋的C端结构域组成。多重序列比对结果表明,在不同昆虫中GSTE6具有高度保守性。分子克隆得到中华按蚊As GSTE6的编码基因As GSTe6序列,大小为672 bp。在大肠杆菌中As GSTE6主要在上清中有大量表达,为可溶性表达;通过镍柱亲和层析和凝胶过滤层析纯化出了高纯度且稳定的As GSTE6重组蛋白;凝胶过滤层析结合化学交联的结果证实体外纯化的融合蛋白As GSTE6主要呈二聚体状态;通过晶体筛选获得了As GSTE6的晶体。【结论】运用结晶学的方法获得了As GSTE6的晶体,这为后续解析As GSTE6的三维结构奠定了基础。

利用SAS中非线性混和效应模型分析城市中阿拉伯按蚊水体滋生环境的时空变异系数

目前对于时间序列分析中出现的空间自相关现象，已经有很多的的文献详述了如何对这种现象进行识别与建模分析。但是，对于撒哈拉以南非洲地区疟疾的主要媒介-阿拉伯按蚊幼虫滋生地的地理空间相关性方面的研究还没研究报道。在空间回归分析中，空间相关性主要通过空间滤波方法完成，该滤波方法通过分析时间序列中随机误差的分布特征。运用基于地理连接矩阵的非参数特征向量分解空间滤波方法，以寻求能鉴别阿拉伯按蚊幼虫滋生水生环境的预测因子。首先，选择肯尼亚的两个城市，Kisumu和Malindi，进行了蚊虫滋生水体的现场采样工作。然后获取快鸟（QuickBird）卫星数据；并利用该数据对冈比亚按蚊的滋生水体进行遥感影像分析。把这些水体的地理空间信息数据输入SAS／GIS模型中，进行单因素分析、相关分析、数据分布分析、以及全局空间自相关统计。然后，利用空间协方差参数生成局部空间自相关指数（如：Moran’s指数）。并由该模型生成了选择性特征向量的粗子集，此粗子集可以被用来研究这些样方变量间的空间互作效应。最后，利用贝叶斯系数估计法定义矩阵中各参数的期望先验参数值，得知水体环境的深度是决定该水体能否滋生阿拉伯按蚊的关键预测性因素，并且深度与其滋生可能性呈明显的正相关关系；由转移空间连接矩阵生成的特征向量的特殊子集可以获知由阿拉伯按蚊水生环境预测因子以及所建立的空间回归模型中空间误差的相关性。

河南省部分地区溴氰菊酯抗性中华按蚊代谢酶活性的调查研究

目的了解河南省部分地区溴氰菊酯抗性中华按蚊的代谢酶活性,探讨中华按蚊对溴氰菊酯的代谢抗性机制。方法 2010年8月在河南省桐柏、淮滨县和永城市采集中华按蚊样本,子1代成蚊用接触筒内成蚊药膜滤纸接触法,按照WHO标准,区分溴氰菊酯抗性与敏感样本,并用微量滴定板法测定抗性样本的非特异性酯酶、谷胱甘肽S转移酶(GST)和P450单加氧酶活性。以实验室敏感品系蚊为对照。结果淮滨、永城和桐柏三地中华按蚊抗性样本的A、B型非特异性酯酶活性均高于对照组(t=2.41,3.30,10.31,7.67,7.90,11.17,均P<0.05);永城和淮滨地区中华按蚊抗性样本GST活性高于对照组的敏感品系(t=3.687,2.484,均P<0.05);淮滨、永城及桐柏三地的中华按蚊抗性样本和敏感品系的P450单加氧酶活性的差异均无统计学意义(t=-1.489,0.397,-0.413,均P>0.05)。结论永城、淮滨两个地区中华按蚊的溴氰菊酯抗性与酯酶和GST活性增高有关;桐柏地区中华按蚊的溴氰菊酯抗性与酯酶活性增高有关。

南昌国境口岸中华按蚊氟氯氰菊酯抗药性及其机制研究

目的了解南昌昌北国际机场中华按蚊的氟氯氰菊酯抗药性,初步探索其产生机制。方法应用WHO成蚊生测法检测当地中华按蚊抗药性水平,将生测过程中即将死亡蚊虫归为敏感蚊,存活蚊虫归为抗性蚊,检测并比较两组蚊虫P450单加氧酶、谷胱甘肽巯基转移酶(GST)活性;同时记录每只敏感蚊生测死亡时间,对生测死亡时间与P450单加氧酶、GST活性进行相关分析。结果当地中华按蚊生测死亡率为59.5%;抗性蚊、敏感蚊、实验室敏感蚊的P450单加氧酶活性差异具有统计学意义(F=151.89,P<0.01),其中抗性蚊>敏感蚊>实验室敏感蚊;但三者GST活性差异无统计学意义(F=0.72,P>0.05);蚊虫生测死亡时间与P450单加氧酶活性呈正相关性,相关系数为0.88,回归方程为y=79.479+1.512x;生测死亡时间与GST活性不具有相关性。结论南昌昌北国际机场中华按蚊对氟氯氰菊酯已产生抗药性,P450单加氧酶活性增强可能是其抗药性产生的机制之一。

我国多斑按蚊复合体mtDNA-COⅠ基因序列特征与系统发育学研究

目的测序并比较多斑按蚊复合体5成员种(伪威氏按蚊、多斑按蚊、威氏按蚊、达罗毗按蚊及塞沃按蚊)mtD-NA-COⅠ基因序列差异,并探讨其系统进化关系。方法多斑按蚊复合体经形态学和ITS2分子鉴定种型后,PCR扩增多斑按蚊复合体mtDNA-COⅠ基因序列,采用Chromas软件核对,必要时手动调整,采用Bioedit软件进行比对分析,采用Mega3.0软件构建系统进化树。结果 PCR扩增mtDNA-COⅠ基因序列全长约645～660bp,平均G+C含量为29.1%,A+T含量为70.9%。5种不同算法计算的5成员种成对种间距离有较小偏差,但种间遗传距离范围基本趋于一致,以塞沃按蚊、达罗毗按蚊及多斑按蚊亲缘关系最近,其后依次为威氏按蚊和伪威氏按蚊。结论根据mtDNA-COⅠ基因序列,采用不同方法构建的多斑按蚊复合体系统进化树结果基本一致,该基因序列适用于多斑按蚊复合体分析系统学分析。

库态按蚊（昆虫纲：双翅目）的研究现状：从生态学研究到其传疟作用

库态按蚊是一个由五种亲缘种型组成的按蚊复合体，分别被命名为A、B、C、D和E型，其中除B型以外，其他各型均属于传疟媒介。库态按蚊广泛分布于埃塞俄比亚及其以东的亚洲地区，包括印度和中国南部等。在某些南亚国家，库态按蚊既是当地的优势按蚊种，也是主要的传疟媒介之一，对当地疟疾的流行起着非常重要的作用。通过多线染色体技术和PCR能鉴定出按蚊复合体中的所有成员。该文对库态按蚊的生态学、传疟作用及其相关问题的研究文献进行综合的评述，主要包括：（1）最近所涉及的库态按蚊传疟媒介领域的研究，（2）库态按蚊的地理分布、生态学和库态按蚊幼虫及成虫的生态特征研究，以及每个种型的媒介能量等，（3）有关库态按蚊的媒介控制方面的研究进展，（4）对今后库态按蚊研究的展望。

中华按蚊GSTO1基因的鉴定及生物信息学分析

谷胱甘肽S-转移酶(GST)是有机体内一类具有解毒和抗氧化等多功能的超家族蛋白酶。在昆虫中GST主要分为七大家族,Omega家族(GSTO)为研究较少的家族。在本研究中,通过Blast方法搜索中华按蚊(Anopheles sinensis)转录组数据,得到中华按蚊GSTO1cDNA序列,全长1 059bp,其中阅读框ORF长744bp,编码248个氨基酸。推测该基因编码蛋白的分子量和等电点分别为28.5kD和6.92。分析发现该蛋白不存在跨膜区和信号肽,亚细胞定位预测显示该蛋白质位于细胞质中。同源性分析表明GSTO1蛋白与达氏按蚊(Anopheles darlingi)氨基酸序列相似性最高。本研究进一步丰富了GST蛋白的基础数据,为进一步研究昆虫GSTO蛋白的功能奠定了理论基础。