【作者】 查玉平；

【导师】 雷朝亮； 陈其才；

【作者基本信息】 华中农业大学 ， 动物学， 2008， 博士

【摘要】 棉铃虫Helicoverpa armigera(H(u丨¨)ber),属于夜蛾科(Noctuidae),是我国乃至世界上的重要农业害虫。由于它具有寄主范围广、繁殖潜能大、种群能远距离迁移和对环境适应能力强等特点,条件适宜时常大面积爆发成灾,造成多种经济作物的严重损失。棉铃虫和其他夜蛾一样具有听器,位于后胸两侧,并且最佳调谐频率范围是15-30 kHz。当棉铃虫成虫飞翔时可以发出主频在20-40 kHz范围内的超声脉冲。为了详细地了解棉铃虫的听觉机理,为治理棉铃虫提供听觉方面相关的理论基础和依据。本文主要研究了棉铃虫对非蝙蝠特征的超声波生理生化反应以及棉铃虫侦测超声波的分子基础。主要结果如下:1.超声波对棉铃虫生殖及生长发育的影响无论是有或无超声波辐照的棉铃虫雌成虫在前4天的产卵数是逐渐增加的,而后的几天产卵量逐渐下降。暴露于超声波辐照下的棉铃虫雌成虫产卵集中在开始产卵的第3天至第6天,对照组的棉铃虫雌成虫产卵高峰要比前者延长一天。超声波辐照的棉铃虫雌成虫产卵量要高于对照组的,且达到了显著差异水平。测试组的棉铃虫雌成虫产卵量比对照组的增加了54.5%。对照组的棉铃虫雌成虫所产的卵孵化率为61.5%,而测试组的为67.6%,两者之间无显著差异。测试组的雌蛾具有1.43个精包,要多于对照组的1.28个。暴露于超声波辐照下的幼虫的生长情况和对照组幼虫的生长情况无显著差异。测试组的幼虫化蛹率要低于对照组的,前者为77.7%,而后者为92.2%,两者之间达到了显著差异水平。暴露于超声波辐照下的蛹羽化率要低于无超声波辐照蛹的羽化率,但是没有达到显著差异水平。2.超声波对棉铃虫乙酰胆碱酯酶(AChE)活性的影响长时间暴露于超声波影响下,棉铃虫的AChE活性异常升高。处理12 h测试组的棉铃虫AChE活性增长最大,达到了显著水平,是对照组活性的2.61倍。其次是处理6 h测试组的棉铃虫AChE活性增长,达到了显著水平,比对照组酶活增长了149%。处理时间最长的24 h测试组的棉铃虫AChE活性增长幅度很小,同对照组相比仅仅增长了16%。在短时间超声波刺激试验中,超声波对棉铃虫成虫的AChE活性影响显著。棉铃虫雌成虫在暴露于超声波20 min时,AChE活性变化最大,与对照组相比增长了74%,达到统计学显著水平。其他测试组的AChE活性变化不显著,仅在对照组AChE活性的86%-103%之间变动。与对棉铃虫成虫的影响不同的是,超声波对棉铃虫蛹的AChE活性主要起到抑制作用;当在30 min时棉铃虫蛹的AChE活性达到最低为0.085 U/mg prot,是对照组的56%,差异显著。其次在50 min时,棉铃虫蛹的AChE活性为0.0947 U/mg prot,是对照组的63%,差异显著。当超声波处理棉铃虫幼虫达20 min时,有一个显著的抑制作用,AChE活性仅为0.018 U/mg prot,是对照组活性的15%。然而在40 min和60 min时超声波又刺激棉铃虫幼虫AChE活性显著增强,分别为0.285 U/mg prot和0.2553 U/mg prot,是对照组活性的2.3倍和2.1倍。3.超声波对棉铃虫抗氧化酶活性的影响当棉铃虫长时间暴露于超声波辐照下,其超氧化物歧化酶(SOD)活性没有发生显著的变化。长时间暴露于超声波辐照下,棉铃虫雌成虫的过氧化氢酶(CAT)活性变化达到了显著水平。当棉铃虫雌成虫被超声波辐照6 h时CAT活性显著增加,是对照组的2.2倍。超声波辐照棉铃虫雌成虫12 h也使得CAT活性显著增加,比对照组增长了38.5%。经过24 h的超声波辐照,棉铃虫雌成虫CAT活性反而开始下降,并达到了统计学显著差异水平。长时间暴露于超声波辐照下,棉铃虫的过氧化物酶(POD)活性也发生了显著变化。超声波辐照6 h后,棉铃虫POD活性显著增加,为对照组的124.3%。棉铃虫经过超声波辐照12 h,POD活性同对照组相比有所增加,虽然增长幅度小于6 h超声波辐照,但仍然达到了极显著差异水平。经过24 h的超声波辐照,棉铃虫POD活性同CAT活性变化一样,反而开始下降,但没有达到了统计学显著差异水平。当短时间暴露于超声波辐照下,棉铃虫成虫、蛹和幼虫的SOD和CAT以及棉铃虫蛹短时间POD活性均没有发生显著变化。当棉铃虫雌成虫短时间暴露于超声波辐照下,其POD活性变化达到了显著差异水平。在超声波辐照20 min和40 min时,棉铃虫雌成虫POD活性达到波峰,其中40 min时棉铃虫雌成虫POD活性与对照组相比达到了极显著差异。短时间的超声波辐照对于棉铃虫幼虫的POD活性具有极显著的影响。除了10 min和60 min是略高于对照组外,其他处理时间长度的超声波辐照使棉铃虫幼虫的POD活性显著增强。4.棉铃虫Nanchung(Nan)基因全长cDNA克隆及序列分析利用RT-PCR和RACE的方法从棉铃虫中得到Nan基因全长cDNA序列。该序列蛋白编码区长1020 bp。由该序列推定的蛋白序列由339个氨基酸残基组成。利用DNAStar软件分析,推测蛋白的分子量39.191 kDa,等电点为6.76。对氨基酸序列进行疏水性分析,说明其氨基酸序列具有亲水性。对蛋白的二级结构进行预测,结果表明该蛋白的二级结构主要为α-螺旋结构。利用生物软件DNAStar分析以下无脊椎动物间的演化关系,结果发现:棉铃虫与家蚕的遗传距离最近,其次是膜翅目的意大利蜜蜂和丽蝇蛹集金小蜂、鞘翅目的赤拟谷盗、双翅目的黑腹果蝇、冈比亚按蚊、致倦库蚊和埃及伊蚊,最后是外群秀丽隐杆线虫。5.棉铃虫Inactive(Iav)基因部分cDNA克隆及序列分析利用RT-PCR和RACE的方法从棉铃虫中得到Iav基因部分cDNA序列。该序列蛋白编码区长1209 bp。由该序列推定的蛋白序列由402个氨基酸残基组成。利用DNAStar软件分析,推测蛋白的分子量45.132 kDa,等电点为6.65。对氨基酸序列进行疏水性分析,疏水性平均值为-1.75,说明其氨基酸序列具有亲水性。对蛋白的二级结构进行预测,结果表明该蛋白的二级结构主要为α-螺旋结构。利用生物软件DNAStar分析以下无脊椎动物间的演化关系,结果发现同Nan基因进化树类似:棉铃虫与家蚕的遗传距离最近,其次是蜜蜂,接着是蚊和果蝇,最后是外类群线虫。更多还原

【Abstract】 The cotton bollworm,Helicoverpa armigera(H(u|¨)bner)(Lepidoptera:Noctuidae) is a videspread polyphagous pest using more than 60 crops as host plants and has high mobility and fecundity.Like other noctuid moths,H.armigera has tympanal ears on the lateroposterior edge of the metathoracic segment,which can detect the echolocation calls of insectivorous bats and have an auditory best frequency at 15-30 kHz with high sensitivity.The male and female of H.armigera are both able to produce ultrasonic signals in the range of 20-40 kHz while flying.In the present study,in order to know that the mechanism of H.armigera hearing,effects of ultrasound from a commercial device, LHC20,on fecundity of adults and development of larvae H.armigera were investigated in laboratory.And Changes in the activities of acetylcholinesterase(ACHE),superoxide dismutase(SOD),catalase(CAT),and peroxidase(POD) were measured in H.armigera after ultrasound stress.And H.armigera Nan and Iav genes were cloned and sequenced. The major results are summarized as following:1.Physiological effects of ultrasound on H.armigeraThe number of eggs laid by females of H.armigera in the absence and presence of ultrasound increased gradually with adult age up to 4 days,and then decreased gradually over the next 6 days.Females exposed to ultrasound from the LHC20 device laid most of their eggs between the third and sixth day,whereas those in the control treatment did between the third and seventh day.Ultrasound from devices significantly increased the number of eggs laid by H.armigera.The number of eggs laid by females was increased by 54.5%when exposed to ultrasound from the LHC20 device,respectively,compared to the control treatment.Viability of eggs laid by females in the control treatment was 61.51%,but those exposed to ultrasound from the LHC20 device was 67.64%.However, this difference was not significant.Across all three tests,each female,on an average,had 1.43 spermatophores in the presence of ultrasound and 1.28 spermatophores in the absence of ultrasound.This difference was significant.The weight per larva in separate test containers with ultrasound was not significantly than the weight per larva in separate test containers without ultrasound.The larva of H. armigera in the absence and presence of ultrasound had the similar daily development pattern.The pupation rate in separate test containers with ultrasound was significantly than the pupation rate in separate test containers without ultrasound.The eclosion rate in plastic containers with ultrasound was not significantly than the eclosion rate in plastic containers without ultrasound.2.Effect of ultrasound on acetylcholinesterase activity in H.armigeraThe AChE activities were significantly elevated compared with the control,after exposure to ultrasound stress for 6 h or 12 h.The maximum AChE activity was 0.434 U/mg prot after 12 h ultrasound stress exposure,and was about 2.6-fold the control.The activity of AChE was only 0.291 U/mg prot after 24 h ultrasound stress exposure,a total increase of 16%.The results of the ultrasounds with different lengths of time have significant but different effects on acetylcholinesterase activities in different developmental stages of H. armigera.The enzyme activities were increased in 20 min ultrasound-stress groups in adults,and decreased in 30 and 50 min groups in pupa,whereas they were reduced in 20 min groups and enhanced in 40 and 60 min groups significantly in larvae.3.Effect of ultrasound on the activity of antioxidant enzymes in H.armigeraThe SOD activities did not change significantly compared with the control after ultrasound exposure for different lengths of time,and they were between 94%and 106% of the control value.The ultrasound stress induced change in CAT activity related to the different durations.The CAT activity increase significantly at 6 h and 12 h ultrasound stress exposure,and the activity of CAT was about 2.2- and 1.4-fold the control, respectively.However,the CAT activity was significantly declined under 24 h ultrasound stress exposure,and it was only 68%of the control value.The increase was statistically significant under 6 h and 12 h ultrasound stress exposure,and the activities of POD both were 1.2-fold the control value.No significant change in POD activity was observed at 24 h of ultrasound stress exposure,and the POD activity was 98%of the control value.In adult,the activities of SOD and CAT changed irregularly but not significantly compared with the control after ultrasound exposure.However,the SOD and CAT activities declined after ultrasound exposure in larvae,and the SOD activity increased after ultrasound exposure in pupae.A significant increase in POD activity in adult H. armigera was observed at 40 min ultrasound exposure.And the POD activity in larvae H. armigera increased significantly after 20 min,30 min,40 min,and 50 min ultrasound exposed,respectively.4.Cloning and sequence of cDNA of Nanchung gene from H.armigeraThe cDNA of Nan gene was cloned from black porgy ovary by means of reverse transcriptase and polymerase chain reaction(RT-PCR) and 5’ and 3’-rapid amplification of cDNA ends(RACE) analyses.The cDNA is 1020 bp in length and encodes a putative protein of 339 amino acids(m.w.39.191 kD,pI 6.76).The comparison of Nan amino acid sequence with those of nearly of all the other known members of insects showed that Bombyx mori shared the highest identity with H.armigera.5.Cloning and sequence of cDNA of Inactive gene from H.armigeraThe cDNA of Iav gene was cloned from black porgy ovary by means of reverse transcriptase and polymerase chain reaction(RT-PCR) and 5’ and 3’-rapid amplification of cDNA ends(RACE) analyses.The cDNA is 1209 bp in length and encodes a putative protein of 402 amino acids(m.w.45.132 kD,pI 6.65).The comparison of Nan amino acid sequence with those of nearly of all the other known members of insects showed that Bombyx mori shared the highest identity with H.armigera.更多还原