截形叶螨对哒螨灵、阿维菌素和阿维·哒螨灵的抗性选育和抗性稳定性研究

【目的】明确截形叶螨Tetranychus truncatus Ehara对哒螨灵、阿维菌素和阿维·哒螨灵3种田间常用药剂产生抗性的速率和稳定性,为叶螨的抗性综合治理提供一定的理论依据。【方法】采用室内生测法,对截形叶螨进行药剂的抗性筛选、衰退和再恢复规律研究。【结果】经过连续30代的药剂汰选,截形叶螨对哒螨灵、阿维菌素和阿维·哒螨灵3种药剂产生了不同程度的抗药性,抗性指数分别达到197.50、19.56和12.57;停止喷药后,其抗性都有所下降,其中截形叶螨对哒螨灵的抗性最不稳定,培育至30代后,抗性衰退率达到63.54%,对阿维菌素的抗性较为稳定,抗性衰退率为23.30%;再次恢复用药后,截形叶螨对哒螨灵、阿维菌素和阿维哒螨灵抗性再度回升,以抗哒螨灵品系的抗性恢复最快,药剂汰选30代后,增长率达到了58.47%,阿维·哒螨灵次之(增长率为38.67%),抗阿维菌素的品系抗性恢复最慢,增长率仅为22.86%。【结论】截形叶螨对哒螨灵抗性不稳定,停止用药后,敏感性易恢复,对阿维菌素和阿维·哒螨灵的抗性较稳定,一旦抗性产生不易衰退,故田间应用时应交替轮换用药。     

柑橘全爪螨田间种群敏感性测定及三种主要解毒酶活性比较

为明确重庆地区柑橘全爪螨Panonychus citri(McGregor)对常用杀螨剂的抗性水平,本研究采用阿维菌素、哒螨灵、三唑锡、螺螨酯4种不同类型杀螨剂对柑橘全爪螨重庆北碚种群、璧山种群、武隆种群和忠县种群进行了田间敏感性测定。结果表明,柑橘全爪螨4个种群对三唑锡表现最不敏感,致死中浓度LC50在209.9～370.9mg/L之间。璧山种群对阿维菌素敏感性最高,武隆种群和忠县种群对阿维菌素的相对抗性分别达12倍和11倍。哒螨灵监测结果表明,北碚种群的抗性水平显著高于其他3个种群。而北碚种群对螺螨酯的LC50仅为1.2mg/L,显著低于其他种群。柑橘全爪螨4个种群解毒酶活性研究发现,解毒酶活性的高低与不同种群抗性水平之间并没有明显相关性,这可能同各地区施药背景不同、综合防治措施不同、各杀螨剂作用机理不同、不同种群体内代谢抗性及靶标抗性水平差异有关。     

基于转录组的苹小卷叶蛾杀虫剂靶标及解毒代谢相关基因分析

【目的】建立苹小卷叶蛾Adoxophyes orana转录组数据库,挖掘杀虫剂靶标及解毒代谢相关基因。【方法】采用Illumina HiSeq~(TM) 2000高通量测序技术对苹小卷叶蛾进行转录组测序,挖掘并分析杀虫剂靶标基因;利用qPCR检测6个杀虫剂靶标基因在苹小卷叶蛾卵、幼虫、蛹和成虫各不同发育阶段的表达;挖掘并分析苹小卷叶蛾转录组中解毒代谢相关基因的代谢通路及进化关系。【结果】通过组装有效序列共获得48 610条unigene(GenBank登录号:GGMW00000000)。挖掘鉴定到155个杀虫剂靶标unigene;qPCR结果显示,1个蜕皮激素受体(ecdysone receptor, ECR)、2个乙酰胆碱酯酶(acetylcholinesterase, AChE)、1个氯离子通道蛋白(chloride channel, CLC)、1个几丁质酶(chitinase, CS)和1个鱼尼丁受体(ryanodine receptor, RyR)基因在苹小卷叶蛾不同发育阶段均存在表达差异。挖掘鉴定到69个羧酸酯酶(carboxylesterase, CarE)unigene、66个谷胱甘肽S-转移酶(glutathion S-transferase, GST)unigene和205个细胞色素P450(cytochrome P450)unigene等解毒代谢相关基因,共鉴定20个CarE unigene, 32个GST unigene和30个P450 unigene与有毒物质代谢相关的通路有关。基于氨基酸序列对具有完整ORF的unigene聚类分析结果显示:12个CarEs中9个为G类,即鳞翅目保幼激素类;分别有10个AoGSTs属于Delta和Epsilon亚家族;18个P450全部聚到CYP3集团。【结论】该研究有助于苹小卷叶蛾杀虫剂靶标基因的挖掘及抗药性的研究。     

桔全爪螨对阿维菌素和甲氰菊酯的抗性现实遗传力及风险评估

在实验室抗性选育的基础上,应用数量遗传学中的域性状分析法,研究了桔全爪螨北碚种群对阿维菌素和甲氰菊酯2种杀螨剂的抗性现实遗传力,并对2种药剂在不同杀死率下抗性发展的速率进行了预测.结果表明:用阿维菌素和甲氰菊酯分别不连续汰选11及16代后,桔全爪螨对两者的抗性分别为3.8和29.9倍,抗性现实遗传力分别为0.0475和0.1544.在室内选择条件下,杀死率为50%～90%时,要获得10倍抗性,甲氰菊酯仅需要7～16代,阿维菌素则需要12～26代.而在田间选择情况下,2种药剂都将需要更长的时间.抗性筛选结果表明,生物源农药阿维菌素的抗性风险明显低于菊酯类农药甲氰菊酯.试验结果可为桔全爪螨抗性治理提供参考.     

食料对南亚果实蝇解毒酶活力的影响

昆虫解毒酶是一类异质酶系, 对分解大量的内源或外源有毒物质、维持正常生理代谢起着重要作用。本文采用生物化学的方法测定了5种寄主果实对南亚果实蝇Bactrocera tau Walker 3个虫态体内总蛋白含量和5种解毒酶的活力。双因子方差分析显示, 南亚果实蝇种群取食黄瓜Cucumis sativus L.、南瓜Cucurbita moschala L.、丝瓜Luffa cylindrical L.、冬瓜Benincasa hispida (Thunb.) Coqn.和苦瓜Momordica charantia L.后, 体内蛋白含量和解毒酶活性均存在显著差异。以丝瓜为食料时, 南亚果实蝇体内蛋白含量较高; 而以黄瓜和冬瓜为食料时蛋白含量则相对较低。在以上5种寄主果实间, 南亚果实蝇的羧酸酯酶（CarE）活性在黄瓜和南瓜上较高, 细胞色素P450 O-脱甲基和谷胱甘肽S-转移酶（GST）活性在苦瓜上较高, 酸性磷酸酯酶（ACP）和碱性磷酸酯酶（ALP）活性却分别在黄瓜和南瓜上较低。在幼虫、蛹和成虫3个虫态间, 解毒酶活性亦存在显著差异, 成虫具有较高的CarE活性;幼虫具有较高的细胞色素P450 O-脱甲基, GST和ALP活性,但具有较低的ACP活性;除ACP外,蛹期解毒酶活性均较低。据以上结果可以推测,南亚果实蝇解毒酶活力受寄主果实种类以及该种群本身发育阶段的影响。     

截形叶螨抗哒螨灵品系和敏感品系体内解毒酶活性的变化

【目的】通过对截形叶螨 Tetranychus truncatus Ehara抗哒螨灵品系体内解毒酶活性分析和增效剂与哒螨灵混用的增效作用测定,明确截形叶螨对哒螨灵的抗性动态及抗性机理,获得抗性治理的途径。【方法】采用室内生测法培育截形叶螨抗哒螨灵品系,微量滴度酶标板测定抗性和敏感品系体内解毒酶比活性、米氏常数（K_m）及最大反应速度（V_max）,再用增效醚（PBO）、顺丁烯二酸二乙酯（DEM）和磷酸三甲苯酯（TPP）进行增效作用测定。【结果】室内筛选的截形叶螨对哒螨灵产生了抗性,筛选至49代,抗性倍数达到955.25;PBO,TPP和DEM对哒螨灵药剂有不同程度的增效作用,相对增效系数分别为95.97%, 85.14%和97.37%;抗哒螨灵品系体内的羧酸酯酶（CarE）、谷胱甘肽转移酶（GSTs）、多功能氧化酶（MFO）活性较敏感品系显著性提高（P＜0.05）,酸性磷酸酯酶（ACP）和碱性磷酸酯酶（ALP）的活性与敏感品系差异不大(P>0.05);抗性品系中的CarE,GSTs和MFO 3种解毒酶的米氏常数（K_m）下降,最大反应速度（V_max）高于敏感品系。【结论】截形叶螨对哒螨灵产生抗性可能与其体内CarE,GSTs和MFO 3种解毒酶与底物的亲和力提高和代谢能力增强有关;3种增效剂(PBO, TPP和 DEM)与哒螨灵混用能提高对截形叶螨的毒杀效果。     

亚致死剂量阿维菌素和氟虫睛处理小菜蛾对寄生蜂菜蛾绒茧蜂生长发育的影响

分别在小菜蛾体内的菜蛾绒茧蜂处于卵期、早期幼虫和中期幼虫时,饲喂小菜蛾2龄幼虫亚致死剂量（＝LC₁₀）的阿维菌素和氟虫睛,研究上述杀虫剂处理对寄主体内菜蛾绒茧蜂结茧率和羽化率的影响。结果表明： 在菜蛾绒茧蜂处于卵期、早期幼虫和中期幼虫时,饲喂小菜蛾LC₁₀剂量阿维菌素处理的菜叶后,菜蛾绒茧蜂的结茧率分别下降26.6%,22.8%和5.8%,饲喂小菜蛾LC₁₀剂量氟虫睛处理的菜叶后,菜蛾绒茧蜂的结茧率分别下降76.9%,42.5%和18.5%。上述阿维菌素处理对菜蛾绒茧蜂成虫羽化率影响不显著,但上述氟虫睛处理可显著抑制菜蛾绒茧蜂成虫羽化率,在菜蛾绒茧蜂处于卵期、早期幼虫和中期幼虫时,饲喂小菜蛾LC₁₀ 剂量氟虫睛处理的菜叶可导致菜蛾绒茧蜂成虫羽化率分别下降53.1%,36.1%和47.8%。结果显示,即便是对寄主小菜蛾幼虫很低的剂量（LC₁₀剂量）也会显著危害小菜蛾幼虫体内的菜蛾绒茧蜂的生长发育。此外,饲喂小菜蛾幼虫亚致死剂量杀虫剂对菜蛾绒茧蜂生长发育的影响与杀虫剂种类及蛾绒茧蜂发育阶段有关。     

牛蒡L1-2组分对桔全爪螨的毒性和几种代谢酶的作用

【目的】探讨杀螨植物牛蒡Arctium lappa L.提取物中主要杀螨成分L1-2的杀螨作用机理。【方法】采用叶片浸渍法处理桔全爪螨Panonychus citri雌成螨后,测定了静止期、兴奋期、痉挛期、麻痹期、复苏和死亡期5个中毒阶段试虫体内几种代谢酶的活性。【结果】L1-2组分在静止期和复苏期对羧酸酯酶（carboxylesterase, CarE）具有一定的抑制作用,在其他时期均激活CarE活性。除了静止期外,在其他时期均能激活乙酰胆碱酯酶（acetylcholinesterase , AChE）和谷胱甘肽转移酶（glutathione S-transferases, GSTs）的活性,在痉挛期和麻痹期活性增强,随后在麻痹期和复苏期降低。【结论】L1-2组分对CarE的抑制与其毒杀活性有关,而中毒试虫的复苏可能与AChE和GSTs有关。该组分可在较长时期内影响桔全爪螨的神经传导及消化和生殖系统,具有潜在的应用研究价值。     

Biology of three species of Agistemus (Acari: Stigmaeidae): life table parameters using eggs of Panonychus citri or pollen of Malephora crocea as food

The biology and life table parameters of Agistemus industani Gonzalez, A. cyprius Gonzalez, and A. floridanus Gonzalez (Acari: Stigmaeidae) were studied under laboratory conditions using two food sources: Panonychus citri (McGregor) eggs or ice plant, Malephora crocea (Jacquin) Schwantes pollen at 25 degrees C. The larval, protonymph, deutonymph, and adult stages of A. industani fed on citrus red mite eggs. All active stages of A. industani, except the larva, fed on all P. citri stages and the larval stage could not feed on P. citri adults. All immature stages of A. industani fed on M. crocea pollen. Agistemus cyprius larvae fed on P. citri eggs and larvae or ice plant pollen. The nymphal stages fed on P. citri eggs, larvae, and protonymphs but not deutonymphs or adults while A. cyprius deutonymphs and adults fed on all P. citri stages. Adult and nymphal stages of A. cyprius fed on ice plant pollen and successfully completed their development while A. floridanus did not. Agistemus floridanus larvae fed only on P. citri eggs, while the other stages fed on P. citri eggs, larvae, and protonymphs. The developmental times from egg to adult for A. industani and A. cyprius when fed M. crocea pollen were 11.3 and 13.4 days, respectively. Agistemus floridanus was unable to complete its life cycle on a diet of only M. crocea pollen. Agistemus industani, A. cyprius, and A. floridanus completed development from egg to adult in 11.7, 13.8, and 10.8 days, respectively, when fed P. citri eggs. The intrinsic rate of increase (r(m)) values for A. cyprius and A. industani were 0.0311 and 0.1201 per day on the pollen diet. The net reproductive rate (Ro) was 3.58 for A. cyprius and 10.07 for A. industani with generation times (T) of 45.2 and 35.1 days, respectively, on the ice plant pollen diet. The r(m) values for A. cyprius, A. floridanus, and A. industani on the P. citri egg only diet were: 0.0562, 0.1001, and 0.1031 per day, respectively. The Ro values for each species fed P. citri eggs only were: 6.36, 7.90, and 18.70 for A. cyprius, A. floridanus, and A. industani and the generation times (T) for each of the three species were: 35.2, 29.9 and 37.8 days, respectively.     

意大利蝗卵发育过程中血蓝蛋白基因表达分析

【目的】意大利蝗Calliptamus italicus是新疆草原的主要优势危害种,以卵在土壤中越冬。呼吸代谢可反映蝗卵的生理状态,呼吸蛋白对于呼吸系统不完善的蝗卵尤为重要。本研究旨在明确意大利蝗卵发育过程中血蓝蛋白基因的表达情况。【方法】采用实时荧光定量PCR方法检测不同发育阶段的蝗卵以及1龄蝗蝻的血蓝蛋白2个亚基基因Hc1和Hc2的表达量。【结果】根据解剖形态观察,将意大利蝗越冬卵的整个发育过程分为10个阶段,包括9个卵发育阶段(C-Ⅰ-C-Ⅹ)和1龄蝗蝻阶段(C-Ⅹ)。Hc1和Hc2在越冬蝗卵各发育阶段以及1龄蝗蝻中均有表达。其中,在蝗卵早期发育阶段(C-Ⅰ, C-Ⅱ和C-Ⅲ),Hc1表达量逐渐增加,C-Ⅲ阶段表达量显著高于C-Ⅰ和C-Ⅱ阶段;滞育阶段(C-Ⅳ, C-Ⅴ和C-Ⅵ),胚胎发育停滞,Hc1表达量较C-Ⅲ,C-Ⅶ和C-Ⅷ阶段低;滞育后发育阶段(C-Ⅶ和C-Ⅷ),蝗卵解除滞育,快速发育,Hc1表达量较早期发育阶段和滞育阶段高,其中,C-Ⅷ阶段Hc1表达量最高(212.3156±10.5470),显著高于其他所有阶段;1龄蝗蝻(C-Ⅹ)的Hc1表达量最低,为0.4017±0.1010。Hc2表达量在C-Ⅴ阶段最高(679.7511±54.5719),显著高于其他所有阶段;除C-Ⅴ阶段外,其他各阶段之间Hc2表达量差异均不显著。Hc1在蝗卵滞育后阶段高表达,而Hc2在蝗卵滞育阶段高表达。【结论】血蓝蛋白亚基基因Hc1和Hc2在整个意大利蝗卵发育过程均有表达,且具有阶段特异性。Hc1与Hc2协同作用为蝗卵发育供氧,其中,Hc1主要负责蝗卵滞育后发育期间的氧气运载,而Hc2主要维持滞育期间的氧气运载,且载氧效率较低。研究结果可为进一步探讨意大利蝗卵的抗逆机制提供科学依据。     

基于沟眶象属两近缘种不同虫态转录组的气味受体基因鉴定及表达分析

[目的]基于沟眶象属Eucryptorrhynchus两近缘种沟眶象E.scrobiculatus和臭椿沟眶象E.brandti不同虫态的转录组数据进行气味受体(odorant receptor,OR)基因的鉴定及表达特征分析,补充两种象甲的气味受体信息,为之后的功能研究提供理论依据.[方法]从沟眶象(幼虫、蛹和成虫)...     

灰飞虱唾液腺三大解毒酶家族的转录组分析

灰飞虱Laodelphax striatellus （Fallén）是危害多种禾本科经济作物的重要刺吸式害虫。唾液腺对刺吸式口器昆虫取食植物尤其重要, 其分泌的唾液可以帮助刺吸式口器昆虫刺穿植物、 消化食物、 解毒植物的次生物质。细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）、 谷胱甘肽S 转移酶（glutathione S transferase, GST）和羧酸酯酶（carboxylesterase, CarE）是昆虫主要的解毒酶系。为了分析解毒酶基因在灰飞虱唾液腺中的表达谱, 本研究对灰飞虱成虫唾液腺进行转录组测序、 重头组装和注释, 并与豌豆蚜Acyrthosiphon pisum和西方蜜蜂Apis mellifera的同源蛋白进行系统发育分析。发现有9个谷胱甘肽S 转移酶（glutathione S transferase, GST）基因、 22个羧酸酯酶（carboxylesterase, CarE）基因和39个细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）基因在灰飞虱唾液腺中表达。通过对同源蛋白进行系统发育分析, 发现灰飞虱唾液腺大部分的CarE是参与消化/解毒和激素/信息素的加工, 而参与神经/发育的CarE很少; 灰飞虱唾液腺表达的P450基因远远少于豌豆蚜和西方蜜蜂基因组的P450基因数, 且只有CYP6和CYP4家族的成员; GST家族在3种昆虫的保守性最高。研究结果为灰飞虱对寄主植物和杀虫剂的适应性研究奠定了基础。     

二斑叶螨对七种杀螨剂的抗药性测定及其机理研究

室内测定了相对敏感种群(S)和抗性种群(R)对常用7种杀螨剂的敏感性,并测定了羧酸酯酶、谷胱甘肽S-转移酶和乙酰胆碱酯酶3种酶的比活力。结果表明:二斑叶螨Tetranychus urticae Koch R种群已对甲氰菊酯和哒螨灵产生了抗性,抗性倍数分别为5.45和105.47。其中,甲氰菊酯对雌成螨的毒力最低(>3000mg/L),已远远超过田间推荐剂量,不宜继续使用。酶活测定结果表明:谷胱甘肽S-转移酶解毒活性的提高是二斑叶螨对甲氰菊酯产生抗性的原因之一;二斑叶螨对哒螨灵抗性的增强可能与羧酸酯酶有关。     

Identification of three protein disulfide isomerase members from Haemaphysalis longicornis tick

Three genes encoding putative protein disulfide isomerase (PDI) were isolated from the Haemaphysalis longicornis EST database and designed as HlPDI-1, HlPDI-2, and HlPDI-3. All three PDI genes contain two typical PDI active sites CXXC and encode putative 435, 499, and 488 amino acids, respectively. The recombinant proteins expressed in Escherichia coli all show PDI activities, and the activities were inhibited by a PDI-specific inhibitor, zinc bacitracin. Western blot analysis and real-time PCR revealed that three HlPDIs were present in all the developmental stages of the tick as well as in the midgut, salivary glands, ovary, hemolymph, and fatbody of adult female ticks, but the three genes were expressed at the highest level in the egg stage. HlPDI-1 is expressed primarily in the ovary and secondarily in the salivary glands. HlPDI-2 and HlPDI-3 are expressed primarily in the salivary gland, suggesting that the PDI genes are important for tick biology, especially for egg development, and that they play distinct roles in different tissues. Blood feeding induced significantly increased expression of HlPDI-1 and HlPDI-3 in both partially fed nymphs and adults. Babesia gibsoni-infected larval ticks expressed HlPDI-1 and HlPDI-3 2.0 and 4.0 times higher than uninfected normal larval ticks, respectively. The results indicate that HlPDI-1 and HlPDI-3 might be involved in tick blood feeding and Babesia parasite infection in ticks.     

Residual Toxicity of Avermectin b1 and Pyridaben to Eight Commercially Produced Beneficial Arthropod Species Used for Control of Greenhouse Pests

Residual toxicities of avermectin b1 and pyridaben for 2- to 28-day exposure periods were assessed in laboratory and greenhouse trials for eight species of beneficial arthropods that are commercially produced for greenhouse pest management. In laboratory trials, Amblyseius degenerans Berlese, Aphidius colemani Viereck, Aphidoletes aphidimyza (Rondani), Dacnusa sibirica Telenga, Encarsia formosa (Gahan), and Orius insidiosus (Say) showed high mortality (>85%) when exposed to ≤6-day residues of both acaricides. Lower toxicities were observed for pyridaben to the predatory mites Amblyseius cucumeris (Oudermans) and Phytoseiulus persimilis Athias-Henroit and for avermectin b1 to A. cucumeris after exposure to ≤6-day residues. In greenhouse trials, pyridaben showed significantly higher residual toxicity to all beneficial species than avermectin b1. Pyridaben had high residual toxicity (40–60% mortality) to E. formosa, A. colemani, A. aphidimyza, A. degenerans, and P. persimilis 6 days after treatment. Residual toxicity of pyridaben to D. sibirica, A. cucumeris, and O. insidiosus decreased to a low level (<15% mortality) after 6 days. Avermectin b1 was slightly toxic or nontoxic to the predaceous mites A. cucumeris, A. degenerans, and P. persimilis. Toxicity of avermectin b1 to E. formosa, A. colemani, D. sibirica, and O. insidiosus rapidly decreased to <25% mortality 6 days after application. Based on the results of the greenhouse trials, avermectin b1 was considered suitable for use with predacious mites and could be combined in integrated pest management (IPM) programs with other beneficial species after residual toxicity is taken into consideration. Pyridaben can also be combined in IPM programs with A. cucumeris, O. insidiosus, and D. sibirica after a 6-day residual period.