用核糖体DNA鉴别PCR区分中华按蚊和嗜人按蚊

目的：建立鉴别中华按蚊和嗜人按蚊的ＰＣＲ检测技术，并对现场标本进行检测。方法：根据中华按蚊和嗜人按蚊的ｒＤＮＡ ＩＴＳ２序列差异，设计种特异引物，用ＰＣＲ技术扩增种特异ＤＮＡ片段（中华按蚊４２５ｂｐ，嗜人按蚊２５３ｂｐ），根据扩增片段的长度对两种按蚊进行鉴别。结果：应用该法检测中华按蚊和嗜人按蚊的３个实验室品系共７０例，经卵鉴定的嗜人按蚊野外标本２０例，均显示单一的种特异扩增带。凉山按蚊、贵阳按蚊     

中华按蚊和嗜人按蚊三种同工酶的比较研究

<正> 近年来,同工酶电泳技术日益为国内外学者所重视,并用同工酶作为遗传标志进行物种鉴定,探讨自然种群基因结构的变化及种内、种间的亲缘关系。本文用垂直平板型聚丙烯酰胺凝胶电泳(PAGE)对闽北种群数量大、传播疟疾和马来丝虫病能力     

嗜人按蚊rDNA-ITS2基因的克隆鉴定及SNP分析

目的克隆和分析嗜人按蚊核糖体DNA（rDNA）第2内转录间隔区（ITS2）序列,研究嗜人按蚊rDNA-ITS2序列的种属特异性及不同个体rDNA-ITS2序列的单核苷酸多态性（Single Nuclear Polymorphism,SNP）。方法用DNA提取试剂盒从嗜人按蚊中提取DNA摸板,利用蚊虫5．8s和28S序列的保守性设计特异引物进行聚合酶链反应以扩增嗜人按蚊rDNA-ITS2基因,扩增产物经回收纯化后连接到TA载体,经amp^＋LB固体平板筛选的阳性克隆。经amp^＋LB液体培养基培养后进行PCR鉴定、EcoRI酶切鉴定和序列分析。结果经PCR反应扩增出全长556bp的嗜人按蚊rDNA-ITS2基因。纯化后重组克隆经PCB鉴定可重现556bp的特异性条带,其酶切产物亦与目的基因PCR产物位置相同。嗜人按蚊6个克隆的同一性为99．6％,其rDNA-ITS2基因单核苷酸存在颠换和插入,在556的范围内有一个A→T,一个G→T的颠换;一个T的插入。结论嗜人按蚊rDNA-ITS2序列在种间高度保守,但不同个体间也存在一定的SNP多态性。     

中华按蚊与嗜人按蚊生物学实验研究

在实验室内对中华按蚊和嗜人按蚊的主要繁殖率指标以及雄蚊、未吸血雌蚊和饱血蚊体重等进行实验研究,在温度26±1℃,温度80±10％的实验室内,中华按蚊的产卵率、平均每蚊首次产卵数、孵化率、化蛹率、成成蚊率分别为79.31％、127.29±47.50、86.56％、87.61％、95.89％、89.93％:而嗜人按蚊则分别为75％、114.71±47.37、75.06％、82.42％、94.44％、95.55％:中华按蚊雄蚊、未吸血蚊、饱血蚊体重、平均吸血量分别为1.12±0.24、1.44±0.32、3.94±0.892.50mg:而嗜人按蚊则分别为0.93±0.24、1.06±0.25、2.93±0.55、1.847mg。经统计学分析,它们的产卵率、平均产卵数和羽化率没有显著性差别,而孵化率、化蛹率、成成蚊率、蚊体重(包括雄蚊、未吸血蚊和饱血蚊)等则有非常显著性差别。中华按蚊羽化为成虫的数量与工龄幼虫之比(S值)为0.7562:而嗜人按蚊的S值则为0.7434     

广东省嗜人按蚊与中华按蚊疟疾传播强度的比较

目的比较广东省嗜人按蚊与中华按蚊疟疾传播强度，为控制疟疾爆发流行提供科学依据。方法采用按蚊密度调查等方法在广东省珠海市和惠州市进行媒介能量的调查和研究。结果广东省珠海市嗜人按蚊的媒介能量为5．863，中华按蚊为0．663；广东省惠州市嗜人按蚊的媒介能量为2．770，中华按蚊为0．249。结论嗜人按蚊的媒介能量是中华按蚊的8～11倍，说明嗜人按蚊在广东的传疟作用较中华按蚊更为重要，占主要地位。     

海南省嗜人按蚊地理分布调查

1992和1993年的6～9月，在海南省19个市、县，22个乡（镇）、农（林）场选89个居民点开展嗜人按蚊地理分布调查。结果除了在1990年8月曾发现嗜人按蚊的文昌县铺前镇南北沟一个点捕获嗜人按蚊30只（人房5只，牛诱25只）外，其余88个点，均未发现嗜人按蚊，表明该种蚊在海南省的分布很局限。     

嗜人按蚊化学感受器的超微结构和功能研究

采用扫描电镜、电生理方法和缩腿试验对嗜人按蚊研究，发现其唇瓣上的感受器为味觉感受器，足Ｉ跗节上的感毛有嗅觉功能和味觉功能，尾感器上有嗅觉功能。     

内陆嗜人按蚊与海岛嗜人按蚊幼虫对盐水耐受性比较

目的了解内陆嗜人按蚊与海岛嗜人按蚊对盐水的耐受能力有无差异。方法将四川、辽宁嗜人按蚊和珠海横琴岛嗜人按蚊幼虫分别放入6个不同浓度的盐水中孵化，观测不同浓度盐水中蚊卵孵化率及幼虫发育情况。结果3种嗜人按蚊在不同浓度盐水中均可孵化；随着盐浓度的增高，幼虫死亡率上升，蛹化率下降；1％以下盐浓度3种嗜人按蚊幼虫均可发育至蛹期，并能正常羽化为成蚊；按蚊在1．5％盐浓度水体中均不能由卵发育至成蚊。结论海岛嗜人按蚊与内陆嗜人按蚊幼虫对盐的耐受性无差别，都能适应一定盐度和对盐的耐受程度，其幼虫均具有较强的渗透调节机制和适应能力。     

中华按蚊多态微卫星DNA位点的筛选和特征研究

目的:对中华按蚊多态的微卫星DNA位点进行分离和筛选,并阐明其特征.方法:应用中华按蚊基因组DNA的酶切片段与生物素标记的寡核苷酸探针杂交,亲合素富集和超滤离心浓缩目的片段,扩增放大,克隆并测序,构建中华按蚊微卫星DNA库.在其中挑选合适的微卫星位点,建立扩增体系.应用中华按蚊现场样本扩增不同的微卫星位点,聚丙烯酰胺凝胶电泳筛选具有多态性的微卫星位点.结果:构建的中华按蚊微卫星DNA库中包含252条序列,GenBank注册登记号为EF620047～EF620298.其中,双核苷酸重复最为常见,三核苷酸重复次之,多核苷酸重复少见;含(CA)和(GT)重复的序列最多,重复次数最多可达56次;完整序列占35.3%,非完整序列占20.2%,复合序列占18.7%,其余为非典型序列.设计了22对引物扩增不同的微卫星位点,其中20个位点产生特异的PCR产物,PAGE结果显示其中15个位点具有多态性.结论:获得了中华按蚊新的多态微卫星位点共15个,为中华按蚊群体遗传和其他相关研究提供了分子标志.     

Sequence differences of rDNA-ITS2 and species-diagnostic PCR assay of Anopheles sinensis and Anopheles anthropophagus from China

AnophelessinensisandAn.anthropophagusareconsideredimportantvectorsofmalariaandfilariasisinChina.Itisoftendifficulttodistinguishbetweenthetwospeciesofadultmosquitoesbecauseoftheirmorphologicalsimilarity,andidentificationalwaysreliesonthecharacteristicsoftheegg.Severaltechniques,suchasallozyme,chromosome,restrictionfragmentlengthpolymorphism(RFLP)andDNAprobehavebeentriedforidentifyingthespecies,butthesemethodssufferedsomelimitationsforfielduse[IJ.RibosomalDNA(rDNA)asanattractivetargetform…     

益母草类中药原植物的核核糖体内转录间隔区序列分析

目的分析益母草属4种植物nrDNAITS序列,探讨其在益母草药材DNA分子鉴别中的作用。方法对4种益母草属植物分别进行ITS序列的PCR扩增和测序,并运用CLUSTRALX和MEGA软件进行分析。结果测得4种植物的ITS序列,包括ITS1、5.8S和ITS2全长序列以及18S、26S部分序列;益母草及其易混淆的3种植物ITS1、ITS2序列的差异性分别为7.2%～18.8%和14.2%～27%。根据ITS序列特征所构建的系统树与传统分类有所不同。结论ITS序列特征可作为益母草种间鉴别的有效分子标记。     

中华按蚊代谢抗性相关基因多态性分析

目的 测定中华按蚊现场样本的宏基因组,分析代谢解毒酶相关基因的多态性。方法 在山东济宁曹县和北湖区用人帐诱捕采集按蚊,基于形态和分子特征鉴定确认蚊种,进行宏基因组Illumina测序。从NCBI数据库下载属于CYP450s、GSTs和Coe基因家族的中华按蚊8个代谢解毒酶基因序列。应用DNASTAR将宏基因组测序结果与代谢解毒酶相关基因序列进行对比,在SeqMan Pro软件上分别进行读取和SNP分析,根据开放阅读框确定SNP的位置,并判别其为同义突变SNP或非同义突变SNP。结果 分子鉴定的山东济宁曹县和北湖区中华按蚊样本分别为31只和12只,分为2组进行宏基因组测序。Illumina测序每组样本产生的reads数近200 Mb,平均长度101 bp,GC含量平均为41%（曹县）和42%（北湖区）,mapping到8个代谢解毒酶基因序列上的reads数为391~2 810条,每个位点的平均覆盖度的范围为23.72~144.93。本研究中华按蚊8个代谢抗性基因共有135个SNP位点,其中109个导致同义突变,占80.74%,26个非同义突变SNP位点（19.26%）;每Kb的SNPs位点数为8.73~29.94;非同义突变SNPs在密码子的位置主要是第1位,占50.00%（13/26）,第2位和第3位分别占26.92%（7/26）和23.08%（6/26）。结论 Illumina测序获得中华按蚊宏基因组序列中代谢抗性相关基因具有相当程度的多态性,可为进一步研究中华按蚊的抗性分子机制提供重要数据。     

赫坎按蚊种团中5种4株的数值分类

本文报告应用系统聚类分析和主成分分析法对赫坎按蚊种团中的中华按蚊4个地理株以及嗜人按蚊、贵阳按蚊、昆明按蚊和凉山按蚊进行分析的结果。并对分析的具体步骤作了简单介绍。     

Larvicidal and repellent potential of Moringa oleifera against malarial vector,Anopheles stephensi Liston (Insecta: Diptera: Culicidae)

Objective

To evaluate the larvicidal and pupicidal potential of the methanolic extracts from Moringa oleifera (M. oleifera) plant seeds against malarial vector Anopheles stephensi (A. stephensi) mosquitoes at different concentrations (20, 40, 60, 80 and 100 ppm).

Methods

M. oleifera was collected from the area of around Bharathiar University, Coimbatore. The dried plant materials were powdered by an electrical blender. From each sample, 100 g of the plant material were extracted with 300 mL of methanol for 8 h in a Soxhlet apparatus. The extracts were evaporated to dryness in rotary vacuum evaporator to yield 122 mg and 110 mg of dark greenish material (residue) from Arcang amara and Ocimum basilicum, respectively. One gram of the each plant residue was dissolved separately in 100 mL of acetone (stock solution) from which different concentrations, i.e., 20, 40, 60, 80 and 100 ppm were prepared.

Results

Larvicidal activity of M. oleifera exhibited in the first to fourth instar larvae of the A. stephensi, and the LC₅₀ and LC₉₀ values were 57.79 ppm and 125.93 ppm for the first instar, 63.90 ppm and 133.07 ppm for the second instar, 72.45 ppm and 139.82 ppm for the third instar, 78.93 ppm and 143.20 ppm for the fourth instar, respectively. During the pupal stage the methanolic extract of M. oleifera showed that the LC₅₀ and LC₉₀ values were 67.77 ppm and 141.00 ppm, respectively.

Conclusions

The present study indicates that the phytochemicals derived from M. oleifera seeds extracts are effective mosquito vector control agents and the plant extracts may be used for further integrated pest management programs.     

Ovicidal and repellent activities of botanical extracts against Culex quinquefasciatus,Aedes aegypti and Anopheles stephensi (Diptera: Culicidae)

Objective

To determine the ovicidal and repellent activities of methanol leaf extract of Ervatamia coronaria (E. coronaria) and Caesalpinia pulcherrima (C. pulcherrima) against Culex quinquefasciatus (Cx. quinquefasciatus), Aedes aegypti (Ae. aegypti) and Anopheles stephensi (An. stephensi).

Methods

The ovicidal activity was determined against three mosquito species at various concentrations ranging from 50-450 ppm under the laboratory conditions. The hatch rates were assessed 48 h after treatment. The repellent efficacy was determined against three mosquito species at three concentrations viz., 1.0, 2.5 and 5.0 mg/cm² under the laboratory conditions.

Results

The crude extract of E. coronaria exerted zero hatchability (100% mortality) at 250, 200 and 150 ppm for Cx. quinquefasciatus, Ae. aegypti and An. stephensi, respectively. The crude extract of C. pulcherrima exerted zero hatchability (100% mortality) at 375, 300 and 225 ppm for Cx. quinquefasciatus, Ae. aegypti and An. Stephensi, respectively. The methanol extract of E. coronaria found to be more repellenct than C. pulcherrima extract. A higher concentration of 5.0 mg/cm² provided 100% protection up to 150, 180 and 210 min against Cx. quinquefasciatus, Ae. aegypti and An. stephensi, respectively. The results clearly showed that repellent activity was dose dependent.

Conclusions

From the results it can be concluded the crude extracts of E. coronaria and C. pulcherrima are an excellent potential for controlling Cx. quinquefasciatus, Ae. aegypti and An. stephensi mosquitoes.