Cytochrome P450 Monooxygenases CYP6AY3 and CYP6CW1 Regulate Rice Black-Streaked Dwarf Virus Replication in Laodelphax striatellus (Fallén)

The small brown planthopper, Laodelphax striatellus (Fallén), is an important agricultural pest that causes significant losses by sucking and transmitting multiple plant viruses, such as rice black-streaked dwarf virus (RBSDV). Insecticides are commonly used to control planthoppers and cause the induction or overexpression of cytochrome P450 monooxygenases (P450s) from the CYP3 and CYP4 clades after insecticide application. However, little is known about the roles of insecticides and P450s in the regulation of viral replication in insects. In this study, RBSDV-infected L. striatellus were injected with imidacloprid, deltamethrin, pymetrozine, and buprofezin, respectively. The insecticide treatments caused a significant decrease in RBSDV abundance in L. striatellus. Treatment of piperonyl butoxide (PBO), an effective inhibitor of P450s, significantly increased the RBSDV abundance in L. striatellus. Fourteen P450 candidate genes in the CYP3 clade and 21 in the CYP4 clade were systematically identified in L. striatellus, and their expression patterns were analyzed under RBSDV infection, in different tissues, and at different developmental stages. Among the thirty-five P450 genes, the expression level of CYP6CW1 was the highest, while CYP6AY3 was the lowest after RBSDV infection. Knockdown of CYP6CW1 and CYP6AY3 significantly increased the virus abundance and promoted virus replication in L. striatellus. Overall, our data reveal that CYP6CW1 and CYP6AY3 play a critical role in the regulation of virus replication in L. striatellus.     

淡色库蚊细胞色素P450抗性相关基因克隆与初步鉴定

[目的 ]探讨淡色库蚊细胞色素P45 0与溴氰菊酯抗药性的关系。 [方法 ]采用一对昆虫细胞色素P45 0简并引物 ,以反转录 聚合酶链反应从淡色库蚊扩增特异片段 ,T/A直接克隆法筛选阳性克隆 ,对新序列进行cDNA芯片和逆Northern分析。 [结果 ]从淡色库蚊对溴氰菊酯敏感品系和抗性品系获得 112个阳性克隆 ,其中 2 4个阳性克隆测序后显示为细胞色素P45 0新序列 ,由GenBank登录上网 ;经国际细胞色素P45 0命名委员会鉴定分别属CYP4家族CYP4C、CYP4D、CYP4H和CYP4J等 4个亚家族 ;2 4个CYP4cDNA片段中 ,来自敏感品系的 2个片段(NYDS3和NYDS5 )和来自抗性品系的 4个片段 (NYDR6、NYDR9、NYDR15和NYDR17)在两品系间存在差别 ,cDNA芯片信号亮度值均是抗性品系大于敏感品系 ,倍数在 3 .1～ 9.7范围内 ;NYDR17仅与抗性探针杂交 ;逆Northern再鉴定 ,获得了与cDNA芯片一致的结果。 [结论 ]淡色库蚊CYP4与溴氰菊酯抗性相关 ;在淡色库蚊溴氰菊酯抗性机理中 ,可能存在P45 0基因点突变而导致的特异表达。     

嗜人按蚊溴氰菊酯抗性分子机理研究Ⅲ嗜人按蚊P450基因mRNA荧光半定量RT-PCR分析

目的探讨嗜人按蚊CYP6、CYP4基因与溴氰菊酯抗性的关系。方法采用荧光定量 RT-PCR方法,对嗜人按蚊体内CYP6和CYP4基因的mRNA进行半定量检测分析。结果嗜人按蚊抗性品系中CYP6基因mRNA的含量约为敏感品系的1．39倍。抗性品系中CYP4基因mRNA 的含量约为敏感品系的3．63倍。结论嗜人按蚊抗性品系体内的CYP6和CYP4的mRNA量高于其敏感品系,提示嗜人按蚊溴氰菊酯产生抗性机理可能与其细胞色素P450表达量增高有关。     

嗜人按蚊溴氰菊酯抗性分子机理研究：Ⅲ嗜人按蚊基P450因mRNA荧光半定量RT-PCR分析

目的 探讨嗜人按蚊CYP6、CYP4基因与溴氰菊酯抗性的关系。方法 采用荧光定量RT—PCR方法，对嗜人按蚊体内CYP6和CYP4基因的mRNA进行半定量检测分析。结果 嗜人按蚊抗性品系中CYP6基因mRNA的含量约为敏感品系的1．39倍。抗性品系中CYP4基因mRNA的含量约为敏感品系的3．63倍。结论 嗜人按蚊抗性品系体内的CYP6和CYP4的mRNA量高于其敏感品系，提示嗜人按蚊溴氰菊酯产生抗性机理可能与其细胞色素P450表达量增高有关。     

Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions

Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b₅ knockout mouse livers to validate P450 activation and establish the role for b₅ in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or “pyrethrome.” Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450–insecticide interactions and aiding the development of unique tools for disease control.Pyrethroids are synthetic analogs of pyrethrins, botanical chemicals derived from chrysanthemum flowers (1). They are highly potent insecticides with low mammalian toxicity that are used worldwide in ∼3,500 registered products in agricultural, medicinal, veterinary, and public health sectors. Importantly, they are the only class of insecticide recommended for insecticide-treated nets for malaria control. More than 254 million insecticide-treated nets were distributed across Africa between 2008–2010 (2). Similar to antibiotics, pyrethroids are critical for controlling a diverse spectrum of diseases. Unfortunately, similar to antibiotics, such intense exposure affects health and drives the rapid evolution of insecticide resistance (3).Pyrethroids are structurally highly diverse (4) but share a common architecture comprising a cyclopropane acid group coupled to an alcohol moiety, as exemplified by deltamethrin (Fig. 1A). Traditionally, they are divided into two classes (type 1 and type 2), depending on the absence (type 1) or presence (type 2) of an α-cyano group (Fig. 1B). Pyrethroids work by blocking the voltage-gated sodium channels, causing paralysis in arthropods, and resulting in death (3). Resistance is commonly associated with target site modification or metabolic resistance, in which increased rates of biotransformation, generally by P450s, esterases, and GSTs, reduce toxic potency (3).Open in a separate windowFig. 1.Conversion of deltamethrin into PyABPs. (A) Structure of deltamethrin with constituent acid and alcohol moieties. Primary sites of P450 hydroxylation are indicated by bold arrows at the 2′ and 4′ positions, and minor routes of hydroxylation are indicated with open arrows (1). (B) Conversion of deltamethrin to a PyABPP involves the addition of an alkyne warhead and a clickable handle. The structures of the general probe and the PyABPs synthesized are illustrated. Alkyne warhead groups were located in the 2′ or 4′ positions, whereas alkyne click handles replaced the cyano group (type 1) or terminal bromides (type 2). The general P450 probe 2-EN is boxed parallel to its type 1 pyrethroid analog, P2.Although the toxicity and metabolism of pyrethroids in mammals and insects have been extensively characterized (1), the role of specific enzymes and pathways involved in pyrethroid clearance is unclear. In insects, P450s are key enzymes involved with metabolic degradation, with constitutive overexpression of specific P450s leading to pyrethroid resistance (5, 6). Similarly, in mammals, the toxic potency of pyrethroids is inversely related to their rates of metabolic elimination (7), with both P450 oxidation and carboxyl esterase-mediated hydrolysis playing major roles. Humans have 57 P450 genes, rodents ∼80 P450 genes, and insects up to ∼200 P450 genes (8). Where genome information exists, genetic and microarray-based studies of pyrethroid-resistant versus susceptible populations have been used to identify P450s potentially capable of pyrethroid metabolism (3, 5). However, relatively few P450s have been functionally validated through recombinant P450 expression. Thus, probes able to identify pyrethroid-metabolizing enzymes directly would aid our understanding of the fundamental processes of insecticide–organism interactions, expanding our understanding of the risks of pyrethroid exposure to mammals and the enzymatic mechanisms of metabolism and resistance used by insects.Activity-based protein profiling (ABPP) has been described for the functional profiling of P450s (9, 10). The activity-based probes (ABPs) work in a mechanism-dependent manner to covalently label P450s, whereby the labeling events are detectable by adding a fluorescent reporter group via copper-catalyzed azide-alkyne cycloaddition (“click chemistry”) onto the probe−P450 adducts (9, 10). Furthermore, affinity tags can also be incorporated to pull down and identify probe–P450 adducts. The major advantage of ABPPs is their ability to directly assess enzyme activity. In this article, we have designed and synthesized a group of seven pyrethroid mimetic ABPs (PyABPs) on the basis of the deltamethrin scaffold (Fig. 1B) for the targeted identification of pyrethroid-metabolizing P450s in highly divergent organisms. We have investigated their reactivity profiles against pyrethroid metabolizing and nonmetabolizing recombinant mosquito P450s and mouse and rat liver microsomes. We show that PyABPs can be used to reveal pyrethroid structure–activity relationships, and they also have been used to identify pyrethroid-reactive P450s and related detoxification enzymes in rat liver microsomes, demonstrating their potential for directly assessing pyrethroid-metabolizing enzyme activity.     

Resistance of Australian Helicoverpa armigera to fenvalerate is due to the chimeric P450 enzyme CYP337B3

Worldwide, increasing numbers of insects have evolved resistance to a wide range of pesticides, which hampers their control in the field and, therefore, threatens agriculture. Members of the carboxylesterase and cytochrome P450 monooxygenase superfamilies are prominent candidates to confer metabolic resistance to pyrethroid insecticides. Both carboxylesterases and P450 enzymes have been shown to be involved in pyrethroid resistance in Australian Helicoverpa armigera, the noctuid species possessing by far the most reported resistance cases worldwide. However, specific enzymes responsible for pyrethroid resistance in field populations of this species have not yet been identified. Here, we show that the resistance toward fenvalerate in an Australian strain of H. armigera is due to a unique P450 enzyme, CYP337B3, which arose from unequal crossing-over between two parental P450 genes, resulting in a chimeric enzyme. CYP337B3 is capable of metabolizing fenvalerate into 4′-hydroxyfenvalerate, which exhibits no toxic effect on susceptible larvae; enzymes from the parental P450 genes showed no detectable fenvalerate metabolism. Furthermore, a polymorphic H. armigera strain could be bred into a susceptible line possessing the parental genes CYP337B1 and CYP337B2 and a resistant line possessing only CYP337B3. The exclusive presence of CYP337B3 in resistant insects of this strain confers a 42-fold resistance to fenvalerate. Thus, in addition to previously documented genetic mechanisms of resistance, recombination can also generate selectively advantageous variants, such as this chimeric P450 enzyme with an altered substrate specificity leading to a potent resistance mechanism.     

The Anopheles gambiae detoxification chip: a highly specific microarray to study metabolic-based insecticide resistance in malaria vectors

Metabolic pathways play an important role in insecticide resistance, but the full spectra of the genes involved in resistance has not been established. We constructed a microarray containing unique fragments from 230 Anopheles gambiae genes putatively involved in insecticide metabolism [cytochrome P450s (P450s), GSTs, and carboxylesterases and redox genes, partners of the P450 oxidative metabolic complex, and various controls]. We used this detox chip to monitor the expression of the detoxifying genes in insecticide resistant and susceptible An. gambiae laboratory strains. Five genes were strongly up-regulated in the dichlorodiphenyltrichloroethane-resistant strain ZAN/U. These genes included the GST GSTE2, which has previously been implicated in dichlorodiphenyltrichloroethane resistance, two P450s, and two peroxidase genes. GSTE2 was also elevated in the pyrethroid-resistant RSP strain. In addition, the P450 CYP325A3, belonging to a class not previously associated with insecticide resistance, was expressed at statistically higher levels in this strain. The applications of this detox chip and its potential contribution to malaria vector insecticide resistance management programs are discussed.     

Insecticide resistance of Triatoma infestans (Hemiptera,Reduviidae) vector of Chagas disease in Bolivia

Objective To define the insecticide resistance status of Triatoma infestans to deltamethrin (pyrethroid), malathion (organophosphate) and bendiocarb (carbamate) in Bolivia. Methods Fifty populations of T. infestans were sampled in Bolivian human dwellings. Quantal response data were obtained by topical applications of 0.2 μl of insecticide–acetone solutions on nymphs N1 of the F1 generations. For most populations, dose–mortality relationships and resistance ratios (RR) were analysed. Discriminating concentrations were established for each insecticide with a susceptible reference strain and used on the other field populations. A tarsal‐contact diagnostic test using insecticide impregnated papers was designed to rapidly identify deltamethrin‐resistant populations in the field. Results Discriminating concentrations for topical applications were 5, 70 and 120 ng active ingredient per insect for deltamethrin, bendiocarb and malathion, respectively. The diagnostic concentration for deltamethrin was 0.30% for the 1‐h exposure by tarsal contact. All populations sampled in human dwellings exhibited significant levels of resistance to deltamethrin, from 6 to 491 and varied among regions. Resistant populations did not recover complete susceptibility to deltamethrin when the synergist piperonyl butoxide (PBO) was used. None of the sampled populations exhibited significant resistance to bendiocarb (all RR₅₀ < 1.8) or malathion (all RR₅₀ < 2.2). Conclusion In Bolivia, most ‘domestic’T. infestans populations are resistant to deltamethrin. Because insecticide vector control is the only selection pressure, resistance likely originates from it. Switching from pyrethroids to organophosphates or carbamates could be a short‐term solution to control this vector, but other alternative integrated control strategies should also be considered in the long term.     

细胞色素P450介导的昆虫抗药性研究进展

昆虫细胞色素P450在昆虫抗药性、解毒代谢等方面具有重大研究价值。本文概述了昆虫细胞色素P450在昆虫抗药性中所起的作用及其目前的一些研究进展,从分子学角度分析了昆虫细胞色素P450介导昆虫抗药性的分子机制。     

Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid‐resistant Aedes aegypti?

Background Pyrethroid resistance can be considered the main threat to the continued control of many mosquito vectors of disease. Piperonyl butoxide (PBO) has been used as a synergist to help increase the efficacy of certain insecticides. This enhancement stems from its ability to inhibit two major metabolic enzyme systems, P450s and non‐specific esterases, and to enhance cuticular penetration of the insecticide. Objective To compare the mortality of a characterized resistant Aedes aegypti strain, Nha Trang, from Vietnam and the susceptible laboratory strain Bora Bora on netting with the pyrethroid deltamethrin (DM) alone and in combination with PBO. Methods Resistance mechanisms were characterized using molecular and bioassay techniques; standard PCR was used to test for the kdr target site mutation. Potential genes conferring metabolic resistance to DM were identified with microarray analysis using the Ae. aegypti‘detox chip’. These data were analysed alongside results from WHO susceptibility tests. P450, CYP9J32, was significantly overexpressed in the DM‐resistant strain compared with the susceptible Bora Bora strain. Another five genes involved with oxidative stress responses in mosquitoes were also significantly overexpressed. The Nha Trang strain was homozygous for two kdr mutations. WHO cone bioassays were used to investigate mortality with incorporated DM‐treated nets with and without PBO. PBO used in combination with DM resulted in higher mortality than DM alone. Conclusion Synergists may have an important role to play in the future design of vector control products in an era when alternatives to pyrethroids are scarce.     

Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome P450s: Evidence for involvement in host-plant allelochemical resistance

Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3′ RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (CYP28) from two species of isoquinoline alkaloid-resistant Drosophila and the cosmopolitan species Drosophila hydei. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous in vitro metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect–host-plant relationships through xenobiotic detoxification.     

白纹伊蚊细胞色素P450 CYP6N3基因的原核表达及鉴定

目的　对白纹伊蚊细胞色素P4 5 0CYP6N3基因进行原核表达 ,以获得高效表达蛋白。方法　根据CYP6N3基因的全长cRNA为模板进行RT -PCR。产物经T -A克隆测序鉴定后 ,亚克隆入原核融合表达载体 pGEX - 4T - 1(含有编码 2 6KDaGST的基因序列 )中 ,在大肠杆菌BL2 1(DE3)中进行原核表达。将细菌总蛋白进行SDS聚丙烯酰胺凝胶电泳分析 ,通过Westernblot分析鉴定目的蛋白的位置 ,并运用核酸蛋白分析仪扫描凝胶以确定表达产物的含量。结果　获得了高效表达的融合蛋白GST -CYP6N3,表达量占菌体总蛋白的 37 4 9%。结论　本实验成功地异源表达了白纹伊蚊CYP6N3基因 ,为体外重建细胞色素P4 5 0CYP6N3单加氧酶系 ,了解CYP6N3基因结构功能关系奠定基础     

Differences in the expression of xenobiotic-metabolizing enzymes between islets derived from the ventral and dorsal anlage of the pancreas

Background/Aims: Chronic pancreatitis and pancreatic cancer have been linked to the exposure of environmental chemicals (xenobiotics), which generally require metabolic activation to highly reactive toxic or carcinogenic intermediates. The primary enzyme system involved is made up of numerous cytochrome P450 mono-oxygenases (CYP). Glutathione S-transferases (GST) belong to the enzyme systems that catalyze the conjugation of the reactive intermediates produced by CYPs to less toxic or readily excretable metabolites. Because the majority of chronic pancreatitis and pancreatic cancers develop in the organ's head, we compared the expression of selected CYP and GST enzymes between the tissues deriving from the ventral anlage (head) and dorsal anlage (corpus, tail). Methods: A total of 20 normal pancreatic tissue specimen from organ donors and early autopsy cases were processed immunohistochemically by using antibodies to CYP 1A1, 1A2, 2B6, 2C8/9/19, 2D6, 2E1, 3A1, 3A2 and 3A4, GST-α, GST-μ and GST-π, and the NADPH cytochrome P450 oxido-reductase (NA-OR), the specificity of which has been verified in our previous study by Western blot and RT-PCR analyses. Results: In all pancreatic regions, most of the enzymes were expressed in islet cells. However, more islets in the head region expressed CYP 2B6, 2C8/9/19, 2E1 and the NA-OR, than those in the body and tail. Moreover, the expression of CYP 2B6 and 2E1 was restricted to the pancreatic polypeptide (PP) cells, and the concentration of CYP 3A1 and 3A4 was stronger in PP cells than in other islet cells. On the other hand, GST-μ and GST-π were expressed primarily in islet cells of the body and tail. Conclusion: The greater content of xenobiotic-metabolizing and carcinogen-activating CYP enzymes and a lower expression of detoxifying GST enzymes in the head of the pancreas could be one reason for the greater susceptibility of this region for inflammatory and malignant diseases.     

A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis

A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b₅, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This ω-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.     

Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT

One of the challenges faced in malarial control is the acquisition of insecticide resistance that has developed in mosquitoes that are vectors for this disease. Anopheles gambiae, which has been the major mosquito vector of the malaria parasite Plasmodium falciparum in Africa, has over the years developed resistance to insecticides including dieldrin, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pyrethroids. Previous microarray studies using fragments of 230 An. gambiae genes identified five P450 loci, including CYP4C27, CYP4H15, CYP6Z1, CYP6Z2, and CYP12F1, that showed significantly higher expression in the DDT-resistant ZAN/U strain compared with the DDT-susceptible Kisumu strain. To predict whether either of the CYP6Z1 and CYP6Z2 proteins might potentially metabolize DDT, we generated and compared molecular models of these two proteins with and without DDT docked in their catalytic sites. This comparison indicated that, although these two CYP6Z proteins share high sequence identity, their metabolic profiles were likely to differ dramatically from the larger catalytic site of CYP6Z1, potentially involved in DDT metabolism, and the more constrained catalytic site of CYP6Z2, not likely to metabolize DDT. Heterologous expressions of these proteins have corroborated these predictions: only CYP6Z1 is capable of metabolizing DDT. Overlays of these models indicate that slight differences in the backbone of SRS1 and variations of side chains in SRS2 and SRS4 account for the significant differences in their catalytic site volumes and DDT-metabolic capacities. These data identify CYP6Z1 as one important target for inhibitor design aimed at inactivating insecticide-metabolizing P450s in natural populations of this malarial mosquito.     

Irritability of malaria vector, Anopheles sacharovi to different insecticides in a malaria-prone area

ObjectiveTo determine the susceptibility and irritability level of malaria vector Anopheles sacharovi (An. sachrovi) to different insecticides in a malaria-prone area.MethodsSusceptibility and irritability levels of field collected strain of An. sacharovi to WHO standard papers of DDT 4%, dieldrin 0.4%, malathion 5%, fenitrothion 1%, permethrin 0.75%, and deltamethrin 0.05% were determined in East Azerbaijan of Iran during reemerging of malaria as described by WHO.ResultsResults showed that at the diagnostic dose of insecticides this species exhibited resistance to DDT, tolerant to dieldrin and but somehow susceptible to fenitrothion, malathion, permethrin and deltamethrin. The results of irritability of this species to DDT, lambdacyhalothrin, permethrin cyfluthrin and deltamethrin revealed that DDT had had the most and deltamethrin the least irritancy effect. The average number of take offs/fly/minutes for DDT was 0.8±0.2. The order of irritability for permethrin, lambdacyhalothrin, cyfluthrin and deltamethrin were 0.7±0.2, 0.5±0.2, 0.5±0.3, and 0.2±0.1, respectively.ConclusionsResults of this study reveals the responsiveness of the main malaria vector to different insecticides. This phenomenon is depending on several factors such as type and background of insecticide used previously, insecticide properties, and physiology of the species. Careful monitoring of insecticide resistance and irritability level of species could provide a clue for appropriate selection of insecticide for malaria control.     

中华按蚊细胞色素P450基因表达特征分析

目的 探讨6种溴氰菊酯抗性候选细胞色素P450（CYP）基因（CYP6M3、CYP6Y1、CYP6P5、CYP4H14、CYP4G17、CYP12F16）在中华按蚊体内的表达特征。方法 收集中华按蚊不同发育时期（卵、幼虫、蛹、雌性成蚊和雄性成蚊）和组织（唾液腺、马氏管、中肠、卵巢以及脂肪体）样本,以及雌性成蚊在暴露于不同溴氰菊酯剂量（0、1.25、3.75、6.25、12.5 μg/瓶）和时间（0,5、15、30、60 min）后的样本.提取总RNA,利用反转录实时定量PCR（qPCR）技术分析CYP6M3、CYP6Y1、CYP6P5、CYP4H14、CYP4G17、CYP12F16基因在中华按蚊不同发育时期、组织以及不同溴氰菊酯接触剂量和时间下的相对表达量。结果 CYP6M3与CYP6Y1基因在雄性中华按蚊成蚊体内的表达量最高,CYP6M3基因在雄性成蚊体内的表达量是雌性成蚊的35.1倍,CYP6Y1基因在雄性成蚊体内的表达量是雌性成蚊的61.4倍;CYP4H14基因在幼虫期表达量最低,且在雌性成蚊体内表达量是四龄幼虫体内表达量的22.5倍。候选CYP基因在中华按蚊不同组织内的表达量差异具有统计学意义,CYP6M3基因在马氏管内的表达量是在卵巢中的38.9倍,CYP6Y1基因在脂肪体内的表达量是在卵巢中的9.1倍,CYP6P5基因在中肠内的表达量是在卵巢中的30.3倍,CYP4G17基因在脂肪体内的表达量是在卵巢中的4.6倍,CYP12F16基因在马氏管内的表达量是在卵巢中的4.4倍。接触不同溴氰菊酯剂量和时间对候选CYP基因的表达水平表现出一定的诱导效应,影响候选CYP基因在中华按蚊体内的表达。结论 候选CYP基因在不同发育期中华按蚊体内和不同组织中差异表达,暴露于不同溴氰菊酯剂量和时间影响CYP基因在中华按蚊体内表达。     

Yearly changes of insecticide susceptiblity and possible insecticide resistance mechanisms of Anopheles maculipennis Meigen (Diptera: Culicidae) in Turkey

To evaluate the adulticide susceptibility and yearly changes of Anopheles maculipennis Meigen (Diptera: Culicidae) in Thrace, five mosquito populations were evaluated against the resistance status of four different adulticides. Three biochemical resistance mechanisms and yearly changes of activities were investigated. All the strains were highly resistant to DDT, and all the strains were placed in the resistance surveillance category for malathion, permethrin and deltamethrin in 2007. Although DDT mortality rates had increased from 2007 to 2008 except in the Seremkoy strain, malathion, permethrin and deltamethrin mortality rates have decreased in all of the tested strains. High rates of increase were determined for nonspecific esterases (NSEs) activity by using the substrate p-NPA and these results showed correlation with malathion mortality rates. All the strains showed high level of glutathione S transferases (GSTs), and their activity level had significantly increased from 2007 to 2008. Different insecticide susceptibility statuses were observed between localities, and high DDT resistance was observed although DDT was banned in the 1980s. Biochemical assay results suggest that NSEs and GSTs could play a role insecticide resistance in all tested strains. Malathion susceptibility has decreased in all the tested strains and NSE's activity is possibly the main enzymatic mechanism related to the insecticide resistance. DDT resistance is at a high degree in all the strains and GST's activity is probably related to this situation. GST's activity could play an important role for permethrin and deltamethrin susceptibility but needs to be confirmed for molecular studies. Our results provide important data on insecticide susceptibility and change over time for the Anopheles maculipennis populations in Turkey.