黑肩绿盲蝽Cyrtorhinusl ividipennis Reuter（半翅目、盲蝽科）是广泛分布于稻田的一种重要的捕食性天敌,主要捕食褐飞虱和稻叶蝉的卵或若虫,该天敌对有效控制稻飞虱的危害发挥重要的作用。在稻田生态系统中营造益于黑肩绿盲蝽种群增长的自然环境,对于褐飞虱的防治具有重要的实践意义。以前研究表明黑肩绿盲蝽取食水稻花穗（水稻茎秆中含褐飞虱卵）比仅取食褐飞虱卵（水稻茎秆中含褐飞虱卵）产更多的卵。基于转录组数据分析表明,与仅取食褐飞虱卵（水稻茎秆中含褐飞虱卵）相比,黑肩绿盲蝽取食水稻花穗（水稻茎秆中含褐飞虱卵）后激活InR/FoxO通路,且E-cadherin/A rmadillo表达量显著上升。我们进一步又测定了水稻花穗、5%蜂蜜和水稻茎秆内多种糖含量,发现水稻花穗和5%蜂蜜中的葡萄糖和海藻糖含量显著高于水稻植物。因此,我们推测InR/FoxO通路响应糖变化介导E-cadherin/Armadillo调控黑肩绿盲蝽雌虫生殖的机制。该研究结果将揭示营养因子糖类在黑肩绿盲蝽生殖中的作用,同时也将初步阐明了稻田旁边种植花穗植物有利于害虫天敌种群增长的作用机理。本论文主要研究结果如下:1、InR/FoxO通路响应糖变化介导E-cadherin/Armadillo表达与蛋白互作采用液相色谱法测定了水稻花穗、5%蜂蜜和水稻茎秆中11种植物体内常见且易被昆虫吸收的单糖、双糖和三糖的含量,结果表明水稻花穗和5%蜂蜜中葡萄糖和海藻糖显著高于水稻茎秆,其中水稻花穗和5%蜂蜜溶液中葡萄糖含量与水稻茎秆内的含量相比分别高出了 70%和191%,水稻花穗和5%蜂蜜溶液中海藻糖含量与水稻茎秆内的含量相比分别高出了 93%和480%。与取食含褐飞虱卵的水稻茎秆相比,黑肩绿盲蝽取食含褐飞虱卵的水稻花穗或含褐飞虱卵的水稻茎杆+5%蜂蜜诱导InR/FoxO通路相关基因和E-cadherin/Armadillo上调表达,E-cadherin/Armadillo蛋白表达量也显著增加,Vg表达上调和Vg蛋白表达量也增加。与注射ddH2O对照相比,注射外源0.3 mM葡萄糖显著诱导InR/FoxO通路相关基因表达上调和E-cadherin/Armadillo表达上调,并且E-cadherin/Armadillo蛋白表达量也上调,Vg表达上调和Vg蛋白表达量也增加,但注射海藻糖无影响。注射外源0.2 mM二氧双胍（metformin）处理显著抑制InR/FoxO通路相关基因表达和E-cadherin/Armadillo表达量下降,并且E-cadherin/Armadillo蛋白表达量也下降,Vg表达下降和Vg蛋白表达量也下降。沉默InR/FoxO通路相关基因后显著影响了 InR/FoxO通路转导,且E-cadherin/Armadillo基因和蛋白水平表达量均显著下降。dsE-cadherin 或 dsArmadillo 处理仅导致 E-cadherin/Armadillo 同时下降,对InR/FoxO通路相关基因表达水平无影响。通过酵母双杂和GST pull down实验证明E-cadherin和Armadillo蛋白存在相互作用。该结果阐明黑肩绿盲蝽InR/FoxO通路响应糖变化介导E-cadherin/Armadillo表达与蛋白互作。2、E-cadherin/Armadillo调控黑肩绿盲蝽雌虫生殖E-cadherin/Armadillo时空表达特征分析表明,E-cadherin和Armadilo在卵巢中表达最高,在胸部、中肠和脂肪组织中也有一定的表达;在胚胎时期和成虫时期表达最高,且在整个发育阶段都有不同程度的表达。与dsGFP处理的对照相比,dsRNAs（dsE-cadherin 或 dsArmadillo 或 dsE-cadherin/Armadillo）处理显著降低了黑肩绿盲蝽卵巢和脂肪体内可溶性蛋白质含量,Vg表达和Vg蛋白的合成,但对雌虫寿命无显著性影响,同时雌虫的平均产卵显著下降了约50%。与dsGFP处理的对照相比,dsRNAs处理显著抑制黑肩绿盲蝽雌虫卵巢发育和卵巢小管对Vg的吸收及胚胎的发育,同时降低了黑肩绿盲蝽子代（F1）种群数量、卵孵化率和种群增长指数。该结果表明E-cadherin/Armadillo影响黑肩绿盲蝽生殖。综上所述,该研究结果揭示了营养因子葡萄糖诱导InR/FoxO通路介导E-cadherin/Armadillo调控黑肩绿盲蝽雌虫生殖的作用机理,为进一步优化黑肩绿盲蝽纯人工饲料的配比和室内人工大规模饲养黑肩绿盲蝽提供理论基础,同时也初步阐明了稻田旁边种植花穗植物更有利于害虫天敌种群增长的作用机理,为害虫绿色防控提供新的策略。

褐飞虱Nilparvata lugens（Stal）,半翅目、飞虱科（Hemiptera:Delphacidae）,是一种专食性水稻重大害虫。以前研究表明药剂诱导褐飞虱雄虫的交配因子经交配传递使雌虫产生显著的交配后效应,但交配后效应的作用途径及分子机制不清楚。基于羽化后2 d交配与未交配褐飞虱雌虫的数字基因表达谱分析,发现交配后雌虫体内adenylyl cyclase 9（NlAC9）与 cAMP-dependent protein kinase（PKA）type Ⅰ regulatory subunit（NlPKAR）显著上调,且均属于cAMP/PKA信号通路;进一步研究结果表明NlAC9、NlPKAR与褐飞虱雌虫产卵和种群增长有关,且发现交配后雌虫卵巢内章鱼胺（OA）含量显著增加。OA是昆虫体内主要生物胺,具有神经递质、神经激素和神经调质三重作用,调控昆虫交配、排卵、产卵等多种行为和生理过程。因此,本研究提出“OA响应交配激活cAMP/PKA通路调控褐飞虱生殖”的假设。本论文主要研究结果如下:1、OA响应雄虫交配激活cAMP/PKA信号通路的特征（1）采用双抗体夹心法（E1ISA）检测发现交配后雌虫卵巢内OA含量显著增加,雌虫体内酪胺-β-羟化酶（TβH,OA重要的合成酶）、腺苷酸环化酶（AC）、cAMP依赖性蛋白激酶A（PKA）酶活、OA和cAMP含量也显著增加;实时荧光定量（qRT-PCR）结果表明交配后雌虫体内NlTβH和NlOctβ2R表达量上调,cAMP-PKA信号通路主要基因NlAC9、NlPKAR、NlCRREB1和NlCBP表达量也上调;蛋白免疫印迹（western blot）分析发现 cAMP-response element-binding protein 1（CREB1）的磷酸化（p-CREB1）水平显著增加。（2）未交配雌虫外源注射OA后cAMP含量、AC、PKA酶活、CREB1磷酸化水平、NlOctβ2R、NlAC9、NlPKAR、NlPKAc、NlCREB1表达与对照相比均有部分上升。上述结果初步证明OA响应交配激活下游cAMP/PKA通路。2、OA在褐飞虱生殖中的功能（1）OA/cAMP/PKA信号通路相关基因的时空表达特征分析:研究结果表明各基因在褐飞虱雌虫的成虫阶段均有较高表达;且在头部、脂肪体、卵巢组织中有特异性表达,暗示OA偶联cAMP/PKA通路在雌虫卵巢和脂肪体中发挥作用。（2）沉默NlTβH对雌虫生理生化和生殖力的影响:与dsGFP对照相比,NlTβH表达及OA含量显著下降,TA（tyramine,合成OA前体）含量上升;cAMP/PKA信号通路主要基因表达下调、p-CREB1蛋白水平和cAMP含量减少;AC和PKA酶活性降低;雌虫体重、脂肪体和卵巢可溶性蛋白含量、NlJHAMT、Met、Vg、VgR表达、JH Ⅲ滴度、Vg蛋白水平等均显著减少;雌虫产卵量、产卵历期及寿命显著降低。补偿JH后（dsTβH+methoprene）NlTβH、NlJHAMT、Met、Vg表达量及JHⅢ滴度、Vg蛋白水平显著上升;补救OA后（dsTβH+OA）NlTβH、Vg、VgR表达、Vg蛋白水平、OA含量、JH Ⅲ滴度显著上升。以上结果表明沉默NlTβH影响OA及JH合成进而调节雌虫生殖生殖,暗示OA与JH的协同作用。（3）OA与JH互作关系:virgin+OA处理显著增加NlJHAMT、Met的表达及JH Ⅲ滴度,表明OA影响JH合成;virgin+methoprene（保幼激素类似物）处理后NlTβH表达量、OA含量也显著增加,表明JH影响OA合成。（4）沉默OA/cAMP/PKA通路关键基因(TβH、Octβ2R、ACH、PKAR和CR显著抑制雌虫卵巢正常发育及卵巢小管内Vg的吸收积累;dsTβH,dsOctβ2R和dsCREB1处理后F1代种群数量、孵化率、种群趋势增长指数（population growth index,PGI）均显著降低。以上结果表明OA偶联cAMP/PKA通路与JH协同调控褐飞虱雌虫生殖。综上所述,本论文:（1）解析OA响应交配的特征;（2）阐明OA的生理功能;（3）揭示OA协同JH对褐飞虱生殖的调控。

褐飞虱（Nilaparvata lugens Stal）,半翅目、飞虱科（Hemiptera:Delphacidae）具有繁殖速度快、生命周期短以及环境适应性强等特点,主要栖息于水稻茎秆刺吸汁液,造成植株生长缓慢、分蘖延迟、产量下降,严重时可导致叶片干枯、植株死亡,是我国和许多亚洲国家水稻上的重要害虫。三氟苯嘧啶是杜邦公司研发的新型介离子类杀虫剂,具有高效、持效、用量少和对环境友好等特点,主要用于防治稻飞虱等害虫。有研究表明三氟苯嘧啶作为水稻种衣剂可以长效防治褐飞虱种群,但其长效作用机理尚不清楚。本文研究了 4种不同浓度22.5（低）、45.0（中）、67.5（中高）和90.0 g a.i./ha（高）的三氟苯嘧啶拌种处理对室内和田间水稻生理生化指标的影响以及对褐飞虱取食行为、生殖与田间种群的影响,进一步阐明三氟苯嘧啶作为种衣剂对褐飞虱具有长效防治的作用机理,为褐飞虱的防治提供新策略。主要研究结果如下:（1）三氟苯嘧啶拌种处理对室内水稻生理生化及褐飞虱取食行为和生殖的影响研究结果表明,三氟苯嘧啶拌种处理与对照相比,水稻发芽率和出苗率无显著差异。水稻茎秆内三氟苯嘧啶残留量分析表明,水稻播种后71d～115d,水稻茎秆内残留量保持一个相对稳定的水平（大约0.04 mg.kg-1）。稻谷内三氟苯嘧啶残留量大约为0.015 mg.kg-1,显著低于残留标准1.0 mg.kg-1。4种不同浓度的三氟苯嘧啶拌种处理显著影响水稻茎秆内与褐飞虱抗性有关的抗性物质及蔗糖和游离氨基酸含量。与对照相比,4种浓度的三氟苯嘧啶拌种处理后水稻茎秆内草酸含量增加了 69%～267%,类黄酮含量增加了 63%～434%;中浓度及以上浓度的三氟苯嘧啶拌种处理后水稻茎秆内总酚含量提高了 79%～159%,胼胝质含量上升139%～301%;4种浓度三氟苯嘧啶拌种处理后水稻茎秆内游离氨基酸含量降低了 33%～71%。然而,中浓度及以上浓度的三氟苯嘧啶拌种处理后水稻茎秆内蔗糖含量增加了 85%～275%。褐飞虱取食选择性数据分析表明,褐飞虱更倾向于取食未处理的水稻。刺探电位技术（Electrical penetration graph,EPG）分析4种不同浓度的三氟苯嘧啶拌种处理对褐飞虱雌虫取食行为的影响,与对照相比,中浓度及以上浓度的三氟苯嘧啶拌种处理后褐飞虱雌虫非刺探时间（np波持续时间）增加了 56%～80%,刺探次数（N1波出现次数）减少了 20%～44%,韧皮部取食时间（N4波持续时间）减少了 24%～71%,N4波出现次数减少了 27%～60%。与对照相比,中高浓度和高浓度的三氟苯嘧啶拌种处理后导致褐飞虱因取食受阻使其蜜露排泄量减少了 19%～23%,中浓度及以上浓度的三氟苯嘧啶拌种处理后导致褐飞虱雌成虫脂肪体和卵巢可溶性蛋白分别减少了 38%～71%和21%～44%,保幼激素滴度降低了 14%～29%,蜕皮激素滴度降低了 16%～37%,Vg表达量下降了 46%～62%（中浓度处理除外）,JHAMT表达量下降了 23%～54%。与对照相比,中高浓度和高浓度的三氟苯嘧啶拌种处理后,褐飞虱雌成虫产卵量减少了 61%～68%,产卵历期缩短了 53%～62%,雌成虫寿命减少了 51%～63%,卵孵化率下降了 51%～61%;中浓度及以上浓度的三氟苯嘧啶拌种处理后子代数量与对照相比减少了 22%～88%。Western blot分析表明,三氟苯嘧啶拌种处理抑制褐飞虱雌虫脂肪体内Vg蛋白的合成。卵巢免疫荧光分析表明,褐飞虱取食中浓度及以上浓度的三氟苯嘧啶拌种处理的水稻后显著影响了褐飞虱雌虫卵巢的发育、卵的成熟和卵巢小管对Vg吸收。（2）三氟苯嘧啶拌种处理对田间水稻生理生化及褐飞虱取食行为和田间种群的影响水稻茎秆内三氟苯嘧啶残留量分析表明,田间水稻播种后82d～130d,水稻茎秆内残留量保持一个相对稳定的水平（大约0.03 mg.kg-1）。4种不同浓度的三氟苯嘧啶拌种处理影响了田间水稻茎秆内与褐飞虱抗性有关次生代谢物及蔗糖和游离氨基酸的含量。研究结果表明,4种不同浓度三氟苯嘧啶拌种处理水稻后,与对照相比,播种后60d水稻茎秆内草酸含量升高了 36%～174%,类黄酮含量升高了 58%～290%,总酚含量升高了 89%～262%,胼胝质含量上升了 89%～256%,游离氨基酸含量减少了 23%～69%,蔗糖含量升高了 37%～343%;播种后90d水稻茎秆内草酸含量升高了 37%～185%,类黄酮含量升高了 82%～264%,总酚含量增加了 82%～264%,胼胝质含量增加了 61%～230%,游离氨基酸含量减少了 21%～68%,蔗糖含量升高了 29%～288%。二因素分析表明,4种浓度三氟苯嘧啶拌种处理后60d和90d水稻体内次生代谢物和游离氨基酸含量无显著性差异。水稻茎秆蔗糖浓度与三氟苯嘧啶拌种处理浓度因素和处理天数因素上均有显著差异,且处理浓度和处理天数存在显著的互作效应。EPG数据分析表明,与对照相比,4种浓度的三氟苯嘧啶拌种后,褐飞虱雌虫非刺探时间（np波持续时间）增加了 66%～198%,刺探次数（N1波出现次数）减少了37%～62%,韧皮部取食时间（N4波持续时间）减少了 24%～71%,N4波出现次数减少了 3 4%～49%（低浓度处理除外）。田间褐飞虱百丛虫口数调查结果表明,4种不同浓度的三氟苯嘧啶拌种处理后,2017年水稻播种后56 d～133 d,机插秧田间褐飞虱防治效果达80%以上,2018年水稻播种后56d～119d,机插秧田间褐飞虱防治效果达93%以上,2019年水稻播种后62 d～110 d,机插秧田间褐飞虱防治效果达83%以上,2020年水稻播种后61 d～130 d,机插秧田间褐飞虱防治效果达93%以上。综上所述,我们推测三氟苯嘧啶作为种衣剂对褐飞虱具有长效防治的原因可能是三氟苯嘧啶毒性与水稻体内的次生代谢物质变化的协同作用。首先,三氟苯嘧啶拌种处理后,水稻生长前期茎秆内三氟苯嘧啶残留量高于稻飞虱的亚致死剂量,对稻飞虱产生一定的毒害作用,从而可以有效控制稻飞虱种群;其次,后期水稻体内的三氟苯嘧啶的残留量低于稻飞虱亚致死剂量,可能由于三氟苯嘧啶拌种处理改变了水稻茎秆内与稻飞虱抗性相关的次生代谢物含量,从而抑制稻飞虱的取食行为及影响稻飞虱体内的生化参数和生殖参数,降低稻飞虱生殖,导致田间稻飞虱种群数量减少。

The brown planthopper (BPH), Nilaparvata lugens (Stal) is a resurgent pest of rice crops throughout Asia. We recently discovered that octopamine (OA) and OA2B2 operate in the BPH mating system, where it mediates a wide range of molecular, physiological and behavioural changes. Here, we report on outcomes of experiments designed to test the hypothesis that OA/OA2B2 signalling mediates responses to three abiotic stressors, starvation, high temperature (37 degrees C), and induced oxidative stress. We found per os RNAi-mediated OA2B2 silencing led to significantly decreased survival, measured in days, following exposure to each of these stressors. We selected a biologically costly process, reproductive biology, as a biotic stressor. Silencing of OA2B2 led to decreased total protein content in ovaries and fat bodies, downregulated expression of vitellogenin (Vg) and Vg receptor (VgR), inhibited fat body Vg protein synthesis, shortened the oviposition period, prolonged the preoviposition period, reduced the number of laid eggs, body weight and female longevity. In addition, the silencing treatments also led to inhibited ovarian development, and ovarian Vg uptake, reduced numbers of egg masses and offspring and lower hatching rates and population growth index. These data support our hypothesis that OA2B2 acts in mediating BPH resistance to biotic and abiotic stressors.

Mating triggers physiological and behavioral changes in female insects. In many species, females experience postmating behavioral and physiological changes that define a post-mated state. These changes are comprised of several conditions, including long-term refractoriness to re-mating and increased production and laying of eggs. Here, we report that mating led to several changes in brown planthopper (BPH) females, including increased octopamine (OA), cAMP concentrations, and activities of several enzymes. Mating also led to changes in the expression of several genes acting in female physiology, including those in the cAMP/PKA signal transduction pathway. OA injections into virgin females led to similar changes. RNAi silencing of the gene encoding tyramine beta-hydroxylase, involved in the final step in OA synthesis, led to decreased expression of these genes, and reduced the cAMP/PKA signaling. At the whole-organism level, the RNAi treatments led to reduced fecundity, body weights, and longevity. RNAi silencing of genes acting in OA signaling led to truncated ovarian development, egg maturation, and ovarian vitellogenin (Vg) uptake. The impact of these decreases is also registered at the population level, seen as decreased population growth. We infer that OA signaling modulates the postmating state in female BPH and possibly other hemipterans.

In Asian rice systems, Cyrtorhinus lividipennis Reuter is an important predator that preys on rice planthopper eggs and young nymphs, as a primary food source. Alanine aminotransferase (ALT) acts in many physiological and biochemical processes in insects. We cloned the full-length complementary DNA of C. lividipennis ClALT. Expression analysis showed higher expression in the fat body and midgut compared to other tissues. It is expressed in all C. lividipennis developmental stages and at least four organs. Silencing of ClALT by RNA interference significantly decreased the ClALT enzyme activity and ClALT expression compared to dsGFP-treated controls at 2 days after emergence (DAE). Silencing of ClALT influenced free hemolymph amino acid compositions, resulting in a reduction of Aspartic acid (Asp) and Alanine (Ala) proportions, and increased Cysteine (Cys) and Valine (Val) proportions in females at 2 DAE. dsClALT treatments led to decreased soluble total protein concentrations in ovary and fat body, and to lower reduced vitellogenin (Vg) expression, body weight, and the numbers of laid eggs. The double-stranded RNA viruse treatments also led to prolonged preoviposition periods and hindered ovarian development. Western blot analysis indicated that silencing ClALT also led to reduced fat body Vg protein abundance at 2 DAE. These data support our hypothesis that ClALT influences amino acid metabolism and fecundity in C. lividipennis.

BACKGROUND Triflumezopyrim (TFM), a novel mesoionic insecticide, has high efficiency at a low dosage, and is mainly used to control hopper species. A previous study demonstrated that seed dressing with TFM effectively controlled rice planthopper populations in mechanically transplanted rice fields; however, mode of action for control was unclear.RESULTS The study shows that seed dressing with TFM resulted in elevated levels of oxalic acid, flavonoids, phenolic substances, callose and other compounds associated with Nilaparvata lugens resistance in rice plants, and low TFM residue content in rice plant stem and grain. Host choice behavioral experiments showed that N. lugens females prefer feeding on untreated rice plants. Electrical penetration graph (EPG) data showed that seed dressing with TFM at medium and high doses significantly prolonged the non-probing period and inhibited phloem ingestion in N. lugens females. These changes led to a significant decrease in female secretion of honeydew, expression of genes encoding vitellogenin and juvenile hormone acid methyltransferase, body weight and longevity, and significantly influenced several physiological parameters resulting in impaired oocyte growth, fecundity and population. Field survey data showed that seed dressing with TFM was efficacious and relatively durable in protecting rice plants from infestation by planthoppers.CONCLUSION This study revealed that seed dressing with TFM enhances rice plant resistance to N. lugens by limiting phloem ingestion and increasing the N. lugens non-probing period; this leads to reduced fecundity of females and lowers N. lugens numbers in the field.

褐飞虱(Nilaparvata lugens)是中国长江流域及华南和西南广大稻区的重要害虫，也是一种典型的药剂诱导再猖獗。以前研究表明药剂诱导雄虫的生殖效应经交配传导刺激雌虫生殖，但其作用机制不清楚。我们研究结果表明交配后的雌虫卵巢OA含量显著增加,TβH、AC和PKA酶活性、OA和cAMP含量显著增加,TβH和Octβ2R表达水平上调,cAMP/PKA通路关键基因AC9、PKAR、CREB1和CBP表达水平也上调。通过蛋白印迹分析发现CREB1的磷酸化水平增加。未交配的褐飞虱处女雌注射OA后，其体内cAMP含量增加,AC和PKA酶活性增加,CREB1磷酸化水平,cAMP/PKA通路关键基因表达水平显著增加。沉默褐飞虱雌虫体内TβH后其体内OA含量下降,cAMP/PKA信号通路主要相关基因表达下降,CREB1磷酸化水平下降,AC和PKA酶活性降低，雌虫脂肪体和卵巢内蛋白质含量下降、雌虫体重下降;JHAMT、Met、Vg和VgR表达水平也下降,JHIII滴度和Vg蛋白含量均显著下降，导致雌虫卵巢量、产巢历期及雌虫寿命缩短。进一步研究表明，外源OA处理未交配的雌虫后与注射PBS对照相比,JHAMT、Met表达水平增加,JHIII滴度增加，表明OA影响JH合成；进一步通过保幼激素类似物methoprene补偿实验，结果表明TβH 表达量上调,OA含量也显著增加，表明JH影响OA合成。沉默OA/cAMP/PKA通路相关基因后显著抑制褐飞虱雌虫卵巢发育及卵巢小管内Vg的吸收和积累,dsTβH,dsOctβ2R和dsCREB1处理后F1代种群数量、孵化率、种群趋势增长指数均显著降低。本研究结果解析了OA响应交配介导cAMP/PKA通路协同JH调节雌虫生殖的机制。该研究结果揭示了雌虫响应雄虫交配产生交配后效应的可能作用机理，为褐飞虱的防治提供新的途径。