二化螟滞育生物钟蛋白TIME-EA4基因的克隆及时空和温度诱导表达分析

【目的】生物测时蛋白TIME-EA4是昆虫特有的一种滞育生物钟蛋白。本文旨在克隆二化螟Chilo suppressalis TIME-EA4基因,研究其在二化螟不同发育时期和不同组织中的时空表达模式及在不同田间种群和不同温度诱导下的滞育和非滞育种群中的表达水平。【方法】通过RACE技术克隆二化螟TIME-EA4基因全长c DNA序列,利用荧光定量PCR的方法检测TIME-EA4基因在二化螟不同发育时期、6龄幼虫不同组织中的表达模式,及在不同季节采集的田间种群和不同温度处理1h后的滞育和非滞育种群6龄幼虫中的表达量变化。【结果】从二化螟中克隆获得TIME-EA4的c DNA序列(Gen Bank登录号:KU356855),全长821 bp,开放阅读框516 bp,编码172个氨基酸;TIME-EA4基因在二化螟不同发育时期和不同组织中均有表达,且在早期蛹、雌雄成虫以及6龄幼虫的头部、脂肪体和中肠中表达水平较高,同时在滞育幼虫组织中的表达高于非滞育幼虫。此外,TIME-EA4基因在滞育种群中的表达量是非滞育种群的3倍左右,同时在非滞育种群中,该基因的表达明显受10℃及以下低温的诱导,且在10℃时表达量最高,而在滞育种群中该基因的表达受温度影响不明显。【结论】结果说明TIME-EA4基因与二化螟的抗低温胁迫以及低温滞育相关。本研究为TIME-EA4在农业害虫滞育的分子作用机制研究奠定了基础。     

CO_2倍增和转Bt水稻对二化螟幼虫的生理影响

通过开顶式气室研究了CO2浓度倍增(750vs.375μL/L)对取食转Bt水稻及其对照亲本秀水11的二化螟Chilo suppressalis(Walker)幼虫体内代谢酶乙酰胆碱酯酶(AChE)和保护酶(超氧化物歧化酶SOD、过氧化物酶POD和过氧化氢酶CAT)活力的影响,以期明确该类害虫对大气CO2浓度升高及转Bt水稻的生理响应。研究结果表明:(1)AChE受CO2浓度水平影响显著,与对照CO2处理相比,高CO2条件下该酶活力显著降低;(2)高CO2水平及水稻中Bt毒素蛋白的存在均会导致二化螟幼虫体内POD活力显著降低,SOD初期活力显著升高,但不能维持较长时间,CAT活力表现为先升后降的特点;(3)当受到水稻品种、CO2浓度水平和取食时间的综合影响时,以上代谢酶和保护酶活力会发生显著变化。短时间内二化螟幼虫可通过体内以上生理代谢调节减缓受CO2浓度升高和水稻中Bt毒素蛋白的不利影响,但长时间环境胁迫下这种应对不利环境的响应机制将会失去作用,最终导致种群适合度的降低。     

Biochemical properties of different entomopathogenic fungi and their virulence against Chilo suppressalis (Lepidoptera: Crambidae) larvae

We determined the virulence of Beauveria bassiana, Metarhizium anisopliae, Isaria fumosoroseus and Lecanicilium lecanii against larvae of Chilo suppressalis Walker by bioassay and evaluated several enzymatic and non-enzymatic components. LC₅₀ values of the entomopathogenic fungi revealed 90, 32, 45,000, 4600, 42,000 and 1,540,000 spores/larva for isolates BB1–BB3 of B. bassiana, I. fumosoroseus, M. anisopliae and L. lecanii, respectively. Isolate BB3 and I. fumosoroseus had the highest amounts of total protein and hydrophobin and isolates BB3 and M. anisopliae showed the highest activities of lipases and chitinases. In case of proteases, the highest activities were observed for Pr1 of BB1 and Pr2 of L. lecanii. The highest general esterase activities were obtained in I. fumosoroseus and BB1 when 1-naphtyl acetate and 2-naphtyl acetate were used as substrates. The highest activity of glutathione S-transferase (GST) was observed in I. fumosoroseus by using both reagents but BB1 demonstrated the highest activities of alkaline phosphatase and acid phosphatase. Clustering of the fungi using biochemical enzymes revealed BB2 and BB3 as a separate group of entomopathogenic fungi. In another group, I. fumosoroseus and L. lecanii had the most similarity and were separated from BB1 and M. anisopliae. The fungi exhibited different virulence on larvae of C. suppressalis by producing adhering protein and extracellular enzymes. Overall, results of the bioassays and clustering based on enzymatic activities revealed that isolate BB2 was the most effective fungus against larvae of C. suppressalis.     

二化螟水稻种群与茭白种群光周期反应的比较

本文报道了用水稻和茭白分别饲养二化螟水稻种群和茭白种群的光周期反应。光周期反应曲线显示,用水稻饲养茭白种群或用茭白饲养水稻种群,无论是在短光照还是在长光照条件下,绝大多数幼虫被诱导进入滞育,丧失了各自原有的光周期反应特性,表明这两个种群已分化到仅适应其本身寄主的程度。用水稻饲养的茭白种群仅有5%～6%的个体化蛹,且其幼虫期较用茭白饲养的延长了15～18天;而用茭白饲养的水稻种群有30%～40%的个体化蛹,其幼虫期与水稻饲养的仅相差3～8天。茭白种群用水稻饲养时32日龄幼虫体重仅为茭白饲养的53.1%,而水稻种群用水稻饲养时32日龄幼虫体重为茭白饲养的79.5%。这些结果表明,茭白种群不适应取食水稻,而水稻种群对取食茭白则有一定的适应能力。根据这些结果,我们认为：（1）这两个种群已出现种下分化的迹象;（2）茭白种植不会对水稻田二化螟的发生产生大的影响。     

温度胁迫对二化螟滞育幼虫生理指标的影响

为从生理生化水平上探讨二化螟滞育幼虫应对温度胁迫的生理机制,分别对系列温度胁迫(STS)和梯度温度胁迫(GTS)处理后的幼虫水、脂质、总糖、小分子碳水化合物含量及超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性进行了测定.结果表明:随着温度的降低,两种处理二化螟滞育幼虫虫体含水量趋于减少,且0℃以下时GTS处理下降幅度较STS处理显著;两种处理脂质含量均逐步下降且二者间无显著差异;两种处理总糖含量分别先减后增和持续下降,均检测出4种小分子碳水化合物,其中STS处理葡萄糖、甘油和果糖含量先增后减,海藻糖含量变化与此相反,而GTS处理海藻糖含量先减后增,葡萄糖和甘油含量呈相反变化,果糖含量无变化;14～-14℃范围内STS处理SOD和POD活性较GTS处理低,CAT活性相反.二化螟滞育幼虫生理指标的变化反映了其应对不同温度胁迫的生理响应.     

Influence of soil moisture on supercooling capacity and associated physiological parameters of overwintering larvae of rice stem borer

Influence of soil moisture on the supercooling capacity and associated physiological parameters of overwintering larvae of the rice stem borer Chilo suppressalis was examined by exposing larvae to soil moistures of 25, 50, 75 and 100% of saturated soil water content (SSWC) at ambient temperature for 30 d from December 2007 to January 2008 in Beijing, China. At the end of the exposure, supercooling points (SCPs) varied significantly among the treatments, the lowest being in the larvae exposed to soil moisture of 25% SSWC. Fresh weight was significantly higher in the larvae exposed to soil moisture of 100% SSWC than in those kept at 25 and 50% SSWC. Dry weight and body water content (% fresh weight) were not different among the treatments. Glucose and trehalose contents were markedly lower, and glycerol content was significantly higher in the larvae confined to soil moisture of 25% SSWC than in those exposed to the other soil moisture treatments. It is suggested that variation in body water content (% fresh weight) contributes to the differences in SCPs of the overwintering C. suppressalis larvae in all treatments, but the influence of soil moisture treatments on supercooling capacity are caused through changes in glycerol content.     

昆虫的耐寒性及其影响因素

昆虫是变温动物,种群的繁衍面临如何安全度过漫长而寒冷的冬季的挑战.为了安全越冬,昆虫必须适应冬季的低温,增强耐寒能力是昆虫的一种重要的季节适应机制.近年来,关于昆虫耐寒性的研究不断开展,研究内容涉及影响耐寒性的因素、耐寒性的机制等方面.影响昆虫耐寒性的因素包括环境因素,如气候的季节变化、纬度或海拔的差异等,以及昆虫自身的发育阶段、滞育发生等.     

Glycerol and glucose accumulation during diapause in a tropical beetle

Abstract. Carbohydrate changes were measured during the 10-month adult diapause in the neotropical beetle, Stenotarsus rotundus Arrow (Endomychidae, Coleoptera) to test the hypothesis that metabolic suppression causes the accumulation of polyols in the absence of the need for cold tolerance. Glycerol and glucose both accumulate during the first 3 months from June to September, but decline in October, accumulate again in December-January and finally decline after January and stay low until the adults leave their diapause site in April. Any adaptive significance for this pattern is unknown but its coincidence with previously measured metabolic suppression suggests that glycerol and glucose accumulate as a result of metabolic adjustments during diapause. The relevance of these results to the evolution of polyol accumulation for cryoprotection in temperate insects in discussed.     

Chill injury in the eggs of the migratory locust, Locusta migratoria （Orthoptera： Acrididae）： the time-temperature relationship with high-temperature interruption

Abstract Mortality of the overwintering egg of the migratory locust, Locusta migratoria L. , was attributed to chill injury because of its occurrence well above the egg's super cooling point. In this study, two parameters, upper limit of chill injury zone (ULCIZ) and sum of the injurious temperature (SIT), were used to examine the locust egg's cold hardiness. The value of ULCIZ for the locust egg is 1.06 ± 0.54°C, and the SIT is -329.7 (hour · degree). The superoxide dismutase (SOD) and catalase (CAT) activities changed dramatically after cold stress, indicating that oxygen and hydroxide free radicals are probably efficiently detoxified at low temperatures. It was suggested that the nature of chill injury in locust egg might be a complex of metabolic disorder and a non-proportional decrease in enzymatic reaction and transports, because the LDH activity at low temperature increased significantly and the ATPase activity decreased with prolonged duration of exposure to low temperatures. The results from high temperature interruption revealed that the high temperature intervals significantly increased the survival of locust eggs.     

一种采集水稻二化螟卵(块)的高效简便新方法

比较了二化螟Chilosuppressalis卵块的塑料袋收集法和稻株法。结果表明 ,塑料袋法和稻株法收集的卵粒总数及卵孵化率都没有显著的差异 ,均为 2 79粒卵 雌蛾和 95 %。塑料袋法收集的卵块数较稻株法的多 ,但小卵块比例较高 ( <1 0 0粒卵 卵块 )。还讨论了采用塑料袋法收集二化螟及其它昆虫卵的优点。     

二化螟对杀螟硫磷产生抗性的机理

本文就二化螟Ckilo suppressalis Walker对杀螟硫磷抗性发生的机理进行了较为全面的研究。活体增效作用研究表明,脱叶磷(DEF)和氧化胡椒基丁醚(PBO)对抗性种群可分别增效1.3和34.8倍。这表明了多功能氧化酶(MFO)活性增强,而似乎与水解酶的活性无关。对寓体解毒酶的进一步研究表明,抗性和敏感种群的非特异性酯酶活力无明显差异,但羧酸酮酶(CarE,加入10^-4mol/L的毒扁豆碱)和MFO的活力在抗性种群中有所增高。R种群幼虫CarE活力分别是s的2.42和2.92倍(以a-和β-乙酸萘酯为底物);R种群幼虫MFO的O-脱甲基作用的活力为s幼虫的1.43倍(以对硝基茴香醚为底物);R幼虫乙酰胆碱酯酶(AChE)对乙酰胆碱的活力是s的1.3倍,两者相差不大。但从米氏常数K_m)和最大反应速度(V_max)看,R幼虫是s幼虫的1.9和1.6倍;以对氧磷为抑制剂的R幼虫150是s幼虫的2倍。这些表明了R和s的AChE是不同的。研究结果还表明,二化螟对杀螟硫磷抗性的机理至少包括：(1)CarE活性的增高;(2)MFO的O-脱甲基活力的增高和(3)AChE敏感性的降低等。     

二化螟对杀螟硫磷产生抗性的机理

本文就二化螟Ckilo suppressalis Walker对杀螟硫磷抗性发生的机理进行了较为全面的研究。活体增效作用研究表明,脱叶磷(DEF)和氧化胡椒基丁醚(PBO)对抗性种群可分别增效1.3和34.8倍。这表明了多功能氧化酶(MFO)活性增强,而似乎与水解酶的活性无关。对寓体解毒酶的进一步研究表明,抗性和敏感种群的非特异性酯酶活力无明显差异,但羧酸酮酶(CarE,加入10^-4mol/L的毒扁豆碱)和MFO的活力在抗性种群中有所增高。R种群幼虫CarE活力分别是s的2.42和2.92倍(以a-和β-乙酸萘酯为底物);R种群幼虫MFO的O-脱甲基作用的活力为s幼虫的1.43倍(以对硝基茴香醚为底物);R幼虫乙酰胆碱酯酶(AChE)对乙酰胆碱的活力是s的1.3倍,两者相差不大。但从米氏常数K_m)和最大反应速度(V_max)看,R幼虫是s幼虫的1.9和1.6倍;以对氧磷为抑制剂的R幼虫150是s幼虫的2倍。这些表明了R和s的AChE是不同的。研究结果还表明,二化螟对杀螟硫磷抗性的机理至少包括：(1)CarE活性的增高;(2)MFO的O-脱甲基活力的增高和(3)AChE敏感性的降低等。     

抗二化螟的水稻品种筛选

【目的】筛选抗二化螟Chilo suppressalis水稻品种资源,明确其抗虫机理,是农业防治的基础。【方法】采用室内人工接虫为害和大田自然受害相结合的方法,鉴定了不同的水稻品种抗性;同时通过目测不同水稻植株外部形态特征、在网罩内供试品种上接入成虫后2 d的产卵量、幼虫孵化后2 d观察稻株内二化螟的存活率,探讨抗虫品种的抗虫机理。【结果】从344个水稻品种资源和当家品种中筛选出"碑田倒"和"银间杂糯"2个抗虫品种,以及"农飞"和"三香糯"等65个中抗品种,进一步对鉴定出的2个抗虫品种和65个中抗品种进行大田验证,发现符合率达88.06%;其中,抗虫品种"碑田倒"和"银间杂糯"稻株叶舌、叶耳紧贴,叶鞘紧包,脉间距较窄,其植株上的产卵量和幼虫存活率显著少于感虫品种"香稻"、"赣南早"、"三粒寸"、"赣优晚"及中抗品种"赛湖早"、"TKM6"。【结论】在籼稻、粳稻和糯稻中均可筛选出抗虫和中抗品种。其中,糯稻中的抗虫品种和中抗品种较多,占糯稻品种的33.3%;粳稻品种次之,中抗品种占20%,籼稻品种较少,为18.83%。其抗虫机理主要是稻株外部形态特征不利于二化螟蚁螟侵入、成虫产卵量少和幼虫生长发育不良、存活率低。