Cloning and overexpression of CYP6F1, a cytochrome P450 gene, from deltamethrin-resistant Culex pipiens pallens

CYP6F1 (GenBank/EMBL accession No. AY662654), a novel gene with a complete encoding sequence in the cytochrome P450 family 6, was cloned and sequenced from deltamethrin-resistant 4th instar larvae of Culex pipiens pallens. The cDNA sequence of CYP6F1 has an open reading frame of 1527 bp, which encodes a putative protein of 508 amino acid residues. The deduced amino acid sequence of CYP6F1 indicated that the encoded P450 has conserved domains of a putative membrane-anchoring signal,putative reductase-binding sites, a typical heme-binding site, an ETLR motif and substrate recognition sites.Semi-quantitative RT-PCR analysis indicated that the CYP6F1 gene was expressed to a greater extent in the deltamethrin-resistant strain than in the susceptible strain of Cx. pipiens pallens. The expression levels of the CYP6F1 gene in the deltamethrin-resistant 1 st, 2nd, 3rd, 4th instar larvae and adult female mosquitoes differed, with highest expression levels in the 4th instar larvae. In addition, the CYP6F1 gene was stably expressed in mosquito C6/36 cells, and the expected 61.2 kDa band was identified by Western blotting. The cells transfected with CYP6F1 had an increased resistance to deltamethrin as compared with control cells.These results indicate that CYP6F1 is expressed at higher levels in the deltamethrin-resistant strain, and may confer some insecticide resistance in Cx. pipiens pallens.     

Serine proteinase over-expression in relation to deltamethrin resistance in Culex pipiens pallens

Two serine proteinase genes were isolated from Culex pipiens pallens as significantly up-regulated genes in a deltamethrin-resistant strain through a combination of suppression substractive hybridization and gene expression profiling by macroarrays. These two genes were found to be expressed at least threefold higher in the resistant strain than in the susceptible one. By using rapid amplification of cDNA ends to screen the constructed cDNA library, we cloned these two sequences. There were 909 bp with an open reading frame of 786 bp in the sequence of trypsin cDNA (GenBank/NCBI AF468495), the deduced protein had 261 amino acids, which was most similar to the trypsin gene of Anopheles gambiae. There were 992 bp with an open reading frame of 816 bp in the chymotrypsin cDNA (GenBank/NCBI AY034060), and its deduced amino acid sequence had 271 amino acids, which was most similar to the chymotrypsin-like protein from Aedes aegypti. The two genes were stably expressed in mosquito C6/36 cells, and the expected 29 and 30 kDa bands were shown with Western blot, respectively. In these cells, after deltamethrin treatment, they had protective effects on the viability. The results indicate that trypsin and chymotrypsin were more highly expressed in the deltamethrin-resistant strain, and was related to insecticide resistance in mosquitoes, Cx. pipiens pallens.     

The role of miR-2∼13∼71 cluster in resistance to deltamethrin in Culex pipiens pallens

Excessive and continuous application of deltamethrin has resulted in the development of deltamethrin resistance among mosquitoes, which becomes a major obstacle for mosquito control. In a previous study, differentially expressed miRNAs between deltamethrin-susceptible (DS) strain and deltamethrin-resistant (DR) strain using illumina sequencing in Culex pipiens pallens were identified. In this study, we applied RNAi and the Centers for Disease Control and Prevention (CDC) bottle bioassay to investigate the relationship between miR-2∼13∼71 cluster (miR-2, miR-13 and miR-71) and deltamethrin resistance. We used quantitative real-time PCR (qRT-PCR) to measure expression levels of miR-2∼13∼71 clusters. MiR-2∼13∼71 cluster was down regulated in adult female mosquitoes from the DR strain and played important roles in deltamethrin resistance through regulating target genes, CYP9J35 and CYP325BG3. Knocking down CYP9J35 and CYP325BG3 resulted in decreased mortality of DR mosquitoes. This study provides the first evidence that miRNA clusters are associated with deltamethrin resistance in mosquitoes. Moreover, we investigated the regulatory networks formed between miR-2∼13∼71 cluster and its target genes, which provide a better understanding of the mechanism involved in deltamethrin resistance.     

淡色库蚊酯酶等位基因及其在自然种群中的频率分布

酯酶基因扩增所产生的酯酶活性升高是库蚊Culex pipiens对有机磷杀虫剂抗性的主要机理之一。采用分子杂交技术和限制性酶切片段长度多态性(RFLP)分析,已鉴定出多种酯酶等位基因类型。该文通过酯酶基因特异性片段的PCR扩增及扩增片段的酶切片段分析,对淡色库蚊Culex pipiens pallens四种有机磷抗性品系的酯酶等位基因进行分型,并测定分析自然种群中不同酶型的频率分布。研究结果表明：PCR分型方法具有快速、准确的特点。不同的有机磷杀虫剂对酯酶等位基因具有明显的选择作用。双硫磷品系为B₁型;毒死蜱和敌百虫品系为B₂型;马拉硫磷品系为B₁型和B₁/B₂杂合型。不同地区采集的种群表现出不同的酶型频率分布。该文就杀虫剂对酯酶等位基因选择作用及自然种群的酶型频率分布进行了讨论。     

快速检测蚊虫乙酰胆碱酯酶活性的方法

本研究采用微板法,根据乙酰硫代胆碱-二硫双硝基苯甲酸法的原理,检测了敏感品系和抗残杀威抗性品系淡色库故CulexpipienspallensCoguillett）单个蚊虫的乙酰胆碱酯酶（AchE）的活性。给果表明：敏感品系和抗性品系蚊虫的AchE活性有较大差别,敏感品系蚊虫的AchE可被一定量的杀虫剂抑制,而抗性品系的AchE则不被抑制,因此该方法能够区分敏感蚊虫和抗性蚊虫,用于抗性测定,具有简单易行,检测快速等优点。     

淡色库蚊细胞色素P450基因研究

采用一对昆虫细胞色素P450简并引物,以反转录-聚合酶链反应从淡色库蚊对溴氰菊酯敏感品系和抗性品系成虫RNA扩增到约485 bp和510 bp两个片段,将这两个片段与PinPoint^TMXa-1 T质粒重组,然后克隆至大肠杆菌JM109菌株,筛选获得68个阳性克隆;其中24个阳性克隆测序后与GenBank资料对照,显示为细胞色素P450新序列;分子系统学研究显示,24个新基因（等位基因）分别属CYP4家族CYP4C、CYP4D、CYP4H和CYP4J等4个亚家族,其已由细胞色素P450命名委员会命名和GenBank登录上网;其中CYP4C23可能是一个假基因,CYP4H13具有一段58个碱基长度的内含子,CYP4J4V1在近3′端具有一个终止密码子TAG.     

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。     

Detection and widespread distribution of sodium channel alleles characteristic of insecticide resistance in Culex pipiens complex mosquitoes in China

Culex pipiens complex mosquitoes are widely distributed throughout China and are known to be important disease vectors. Two pyrethroid resistance associated mutations have been identified in Cx. pipiens complex (Diptera: Culicidae), but there is little information on the diversity and distribution of kdr alleles in pyrethroid resistance in Cx. pipiens complex mosquitoes in China. In the present study, we report on a modified three tube allele-specific (AS)-PCR method for detecting the 1014F and 1014S alleles. The new technique was applied to identify the distribution of the two alleles in natural Cx. pipiens complex populations in China. The results confirmed that the new method is both sensitive and specific. The 1014F allele was found in all 14 of the field populations tested (frequency ranged from 6.8 to 76.2%) and the 1014S allele was found in almost two-thirds (frequency from 2.4 to 28.6%), indicating that the genotypes known to be associated with pyrethroid resistance are widespread in China. The resistance-associated alleles were more common in southern Chinese sampling sites than in northern sites. The coexistence of the two resistant mutations in individual mosquitoes was also observed in five of the field populations. Two alternative mutations within the L1014 codon were identified in Culex pipiens molestus Forskal, 1775, including a non-synonymous mutation resulting in a 1014C substitution.     

Molecular Ecology of Pyrethroid Knockdown Resistance in Culex pipiens pallens Mosquitoes

Pyrethroid insecticides have been extensively used in China and worldwide for public health pest control. Accurate resistance monitoring is essential to guide the rational use of insecticides and resistance management. Here we examined the nucleotide diversity of the para-sodium channel gene, which confers knockdown resistance (kdr) in Culex pipiens pallens mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations. We developed and validated allele-specific PCR and the real-time TaqMan methods for resistance diagnosis. The real-time TaqMan method is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity. Significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014S mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance monitoring in natural Cx. pipiens pallens populations in the East China region. The laboratory selection experiment found that L1014F mutation frequency, but not L1014S mutation, responded to deltamethrin selection, suggesting that the L1014F mutation is the key mutation conferring resistance to deltamethrin. High L1014F mutation frequency detected in six populations of Cx. pipens pallens suggests high prevalence of pyrethroid resistance in Eastern China, calling for further surveys to map the resistance in China and for investigating alternative mosquito control strategies.     

Genetic differentiation of Culex pipiens (Diptera: Culicidae) in China

The population genetic structures of Culex pipiens Linnaeus were evaluated in China over a 2000 km transect that encompasses the two subspecies, C. p. pallens and C. p. quinquefasciatus. Four polymorphic allozyme loci were investigated in 1376 mosquitoes sampled from 20 populations across four provinces. These loci were not statistically dependent with no apparent heterozygote deficit or excess. On a regional scale (intra-province), a low (Fst=0.007-0.016) and significant genetic differentiation was found, with no clear geographical pattern. On a wider scale (inter-province), the genetic differentiation was higher (Fst=0.059), and an isolation by distance emerged. The results are compared with previous population genetic surveys of this mosquito species in different geographic areas over the world. The overall pattern suggests that Culex pipiens requires considerable distance (500-1000 km) to show isolation by distance, irrespective of the subspecies (C. p. pipiens, C. p. quinquefasciatus and C. p. pallens) or the geographic location.     

广州地区荷木夜间树干液流补水的影响因子及其对蒸腾的贡献

明确树木夜间水分补充现象有助于提高总蒸腾量和冠层气孔导度估算的精确度,进一步认识冠层蒸腾与树干液流之间存在的时滞关系.本研究采用热消散探针法测定了广州地区的荷木树干液流密度,同步监测了主要的环境因子,从不同时间尺度分析了树干夜间液流的水分补充现象.结果表明：与白天相比,荷木夜间液流密度较小,旱季变化幅度比湿季大;夜间水分补充的时间段主要在前半夜(18:00-22:00);年内各季节夜间水分补充量之间没有显著差异,与环境因子之间的偏相关关系不显著,但与胸径、树高、冠幅、树干生物量、冠层生物量的回归曲线拟合很好,表明树形特征和生物量能更好地解释夜间补水的变化;各季节夜间水分补充量对总蒸腾量的贡献有显著差异,旱季明显高于湿季.     

Preliminary studies on levels of sensitivity to phosphoric esters in the Italian population of the Culex pipiens complex

A preliminary survey of the susceptibility level to organophosphate (OP) insecticides in six populations of Culex pipiens from Central Italy has been carried out. The correlation between OP resistance and highly active esterase electromorphs has also been investigated. The bioassay and electrophoresis results show an association between OP resistance and the highly active esterase allele Est-3A and confirm what it was found in populations of C. pipiens from Southern France. Est-3A appears to be a reliable indicator of OP resistance in mosquitoes of italian C. pipiens.     

Resistance of field-collected populations of Culex pipiens pallens (Diptera: Culicidae) to insecticides in the Republic of Korea

Toxicities of 10 insecticides were examined against late third instars of Culex pipiens pallens, the northern house mosquito, using a direct-contact mortality bioassay. Several strains of mosquitoes were tested (insecticide-susceptible KS-CP strain and five geospatially distant field-collected strains (DG-CP, US-CP, BS-CP, GS-CP, and SG-CP)) and identified by polymerase chain reaction. Marked regional variations of insecticide susceptibility were observed. Extremely high to low levels of resistance were measured: bifenthrin, resistance ratio (RR) = 1-521; β-cyfluthrin, RR = 16-397; α-cypermethrin, RR = 9-343; deltamethrin, RR = 1-40; etofenprox, RR = 2-42; permethrin, RR = 3-46; chlorpyrifos, RR = 2-675; fenitrothion, RR = 0.5-364; and fenthion, RR = 2-360. All strains were susceptible to one or more of the insecticides examined. These results indicate that careful selection and rotational use of these insecticides may result in continued satisfactory control against field populations of northern house mosquitoes.     

Comparison of Anopheles gambiae and Culex pipiens acetycholinesterase 1 biochemical properties

Selection of insensitive acetycholinesterase 1 (AChE1) has occurred in several mosquito species controlled with carbamate (CX) and organophosphate (OP) insecticides. In case of pyrethroid resistance, these insecticides represent an alternative for disease vector control program. Their heavy use in agriculture has selected resistant populations of Anopheles gambiae in West Africa. The evolution of resistance has to be studied to prevent, or at least slow down, the spread of resistant mosquito in wild populations. An. gambiae shares the same resistance mechanism to CX and OP insecticides as Culex pipiens, which was attributed to the G119S substitution in the AChE1 enzyme. By comparing resistant AChE1 from both species, we show here that similar resistance levels are obtained toward 10 insecticides of both classes. Moreover, similar AChE1 activity levels are recorded between either susceptible or resistant mosquitoes of both species. Enzymes belonging to both species seem thus to share identical properties. Consequently, we hypothesize that fitness cost associated with AChE1 insensitivity in C. pipiens mosquitoes should be similar in An. gambiae and thus be used in strategies to control resistant populations where malaria is prevalent.     

Monitoring of Insecticide Resistance in Field-Collected Populations of Culex pipiens pallens (Diptera: Culicidae)

A susceptible strain and six field populations of the northern house mosquito, Culex pipiens pallens Coquillett, were collected in Seoul, Paju, Daejeon, Heongseong, Jinhae, and Buan, and tested for their susceptibility to seven insecticides by topical application. In tests with female Cx. pipiens pallens, marked regional variations of insecticide susceptibility were observed. Five field-collected populations of Cx. pipiens pallens, except for the Seoul colony, exhibitedhightoveryhighlevelsofresistanceto cyfluthrin [resistance ratio (RR), 96–554] and deltamethrin (RR, 192–959). Low to moderate levels of resistance were observed in bendiocarb (RR, 14–46), chlorpyrifos [RR, 7–38; exceptions: Buan (RR, 63) and Jinhae (RR, 80) colonies], λ-cyhalothrin (RR, 5–23), S-bioallethrin (RR, 1–47), and permethrin [RR, 2–27; exception: Jinhae colony (RR, 247)]. These results indicate that careful selection and rotational use of these insecticides should result in continued satisfactory control against field populations of Cx. pipiens pallens.