取食转Bt基因抗虫玉米后亚洲玉米螟幼虫中肠的组织病理变化

利用透射显微镜（TEM）观察亚洲玉米螟Ostrinia furnacalis (Guenée)幼虫取食了表达Cry1Ab杀虫蛋白的转Bt基因玉米心叶组织后中肠的组织病理变化, 以探讨转Bt基因玉米对亚洲玉米螟的致病机理, 为其合理、安全和持续利用提供理论依据。结果表明：亚洲玉米螟取食Bt玉米后中肠细胞及其细胞器发生了明显的病变。取食Bt玉米12 h后中肠细胞开始病变, 首先微绒毛脱落、内质网开始肿胀, 24 h后内质网肿胀、增多, 杯状细胞杯腔增大, 48 h后微绒毛大量脱落, 细胞开始空泡化, 随着取食时间的增加, 细胞空泡化程度加剧, 在感染前期细胞间的病变程度差异较大。微绒毛脱落、内质网肿胀断裂是在多数取食Bt玉米的亚洲玉米螟中肠细胞发生的普遍病变。由此表明, 人工修饰的Cry1Ab基因导入到玉米染色体组中所表达的杀虫蛋白可使玉米螟幼虫中肠细胞发生病变, 最终导致其死亡。     

转cry1Ab基因水稻对二化螟幼虫血细胞的影响

研究了转cry1Ab基因水稻“克螟稻1号”对二化螟Chilo suppressalis幼虫细胞免疫系统的影响。结果表明,转cry1Ab基因水稻对二化螟幼虫的血细胞影响明显,取食转cry1Ab基因水稻后,二化螟幼虫各类血细胞都明显低于取食非转基因水稻“秀水11”的对照组（原血细胞和囊血细胞在取食初期例外）,随取食时间延长,各类血细胞数量及血细胞总数均呈递减的趋势。从各类血细胞所占血细胞总数的百分比来看,原血细胞在取食36 h后锐减,而浆血细胞和粒血细胞则比例增加,其余珠血细胞、囊血细胞的变化不明显。另外,血细胞还出现空泡化、肿胀等病态变化,致使血细胞快速破裂。由此推测转cry1Ab基因水稻自身表达的毒蛋白能严重干扰靶标昆虫二化螟幼虫的细胞免疫系统。     

转Bt基因水稻对稻纵卷叶螟幼虫体内三种保护酶活性的影响

用转Cry1Ab/Cry1Ac基因水稻的叶片饲喂稻纵卷叶螟Cnaphalocrocis medinalis (Guenée)幼虫,采用酶活力测定方法研究了转基因水稻对稻纵卷叶螟幼虫体内3种保护酶（SOD,CAT和POD）活性的影响。结果表明：取食转基因水稻叶片4 h后,幼虫体内SOD酶活性比对照提高了91.5%,与对照有显著差异;36 h后活性达到最大值,但与对照差异不显著。取食转基因水稻叶片24 h后,幼虫体内CAT酶活性达到最大值且高于对照,但与对照差异不显著;48 h后酶活性受到显著抑制,与对照差异显著。取食转基因水稻叶片12 h后,幼虫体内POD酶活性达到最大值,与对照差异不显著;48 h后酶活性逐渐下降达到最小值,比对照下降68.05%。实验同时测定了幼虫体内及其粪便中Bt毒蛋白含量的变化,结果表明取食转基因水稻叶片12 h后,随着大量取食进入体内的毒蛋白随粪便排出,幼虫体内的毒蛋白含量一直低于粪便中的含量,且在24 h时两者差异达到最大。由于Bt毒蛋白在幼虫体内的积累,扰乱了稻纵卷叶螟幼虫体内SOD,CAT和POD 3种保护酶的动态平衡,使虫体内自由基的清除遇到障碍,从而对其产生毒害作用。     

斑蝥素对粘虫和小菜蛾幼虫中肠组织的影响

粘虫 Mythimna separata (Walker) 和小菜蛾Plutella xylostella L.的幼虫经斑蝥素处理后,中肠组织发生明显的病变。粘虫幼虫中肠柱状细胞微绒毛脱落,杯状细胞变大,质膜消失;线粒体不规则肿胀,内嵴排列紊乱、溶解、断裂;粗面内质网扩张明显,核糖体脱落;细胞核肿胀,染色质浓缩,偏移。小菜蛾幼虫中肠组织病变更为明显。表明斑蝥素主要作用于中肠的膜系统,为进一步探讨其毒理机制提供了理论依据。     

不同饲料对稻纵卷叶螟取食代谢及消化酶活性影响

【目的】研究稻纵卷叶螟Cnaphalocrocismedialis对水稻、玉米和人工饲料的取食代谢效应以及取食后幼虫生长发育、物质积累和中肠消化酶活性变化。【方法】在实验室条件下以水稻叶片、玉米叶片及人工饲料饲养稻纵卷叶螟,观察生长发育状况,取特定龄期幼虫测定消化代谢、营养物质积累及中肠消化酶活性。【结果】取食水稻的稻纵卷叶螟蛹重最高、历期最短,与玉米和人工饲料差异显著,取食人工饲料幼虫历期最长。取食不同饲料后单雌产卵量、产卵历期以及成虫寿命之间无显著差异;取食水稻的食物转化率显著高于玉米和人工饲料,取食人工饲料近似消化率最高;取食水稻和玉米的幼虫体内脂肪积累量显著高于人工饲料,取食人工饲料幼虫体内蛋白质和可溶性糖含量明显增加,显著高于水稻和玉米。人工饲料的幼虫体内总蛋白酶、淀粉酶、脂肪酶三种消化酶活性显著高于水稻和玉米。【结论】不同饲料饲养对稻纵卷叶螟生长发育及营养物质累积均有影响,而稻纵卷叶螟幼虫可能通过调节取食量及消化酶活性的方式保证对食料中营养物质摄取。     

水稻品种对稻纵卷叶螟抗性的物理及生化机制

稻纵卷叶螟Cnaphalocrocis medinalis连年大发生,为在其治理中培育和推广抗虫品种,本文应用扫描电镜和生化测定研究了5个常用水稻栽培品种扬辐粳8号、扬稻6号、扬粳9538、淮稻9号和宁梗1号叶片中的硅含量、硅颗粒大小、蜡质含量等物理指标和一些生化指标与抗虫性的关系,并比较了稻纵卷叶螟幼虫危害不同水稻品种后其体内生化反应的差异。结果表明抗虫品种宁梗1号、淮稻9号叶片表面的硅含量显著高于感虫品种,硅颗粒大小在品种间无显著差异。宁粳1号叶片蜡质含量显著高于其他品种。由此表明叶片表面硅和蜡质含量与抗性有关。稻纵卷叶螟幼虫取食后,抗虫品种体内的过氧化物酶（POD）、超氧化物岐化酶（SOD）活性显著高于感虫品种,但丙二醛(MDA)含量低于感虫品种。稻纵卷叶螟幼虫取食不同品种后,其体内一些酶的活性明显不同,取食抗虫品种的幼虫体内POD和谷胱甘肽-S-转移酶(GST)活性显著高于取食其他品种的幼虫,SOD活性显著低于取食感虫品种的幼虫, 表明抗虫品种对稻纵卷叶螟幼虫有毒害作用。     

转cry1Ab/1Ac,cry1C,cry2A基因水稻对田间稻纵卷叶螟及其捕食类天敌的影响

在转Bt基因水稻商业化之前需要评价其对靶标害虫及其天敌的影响.本研究连续2年在3个地点调查了3种转Bt水稻材料和非转基因水稻田间稻纵卷叶螟为害情况,稻纵卷叶螟及其4种捕食类天敌的种群密度、相对丰富度和种群动态.结果表明,水稻材料对卷叶率和稻纵卷叶螟幼虫种群密度有显著影响.非转基因水稻田间卷叶率显著高于3种转Bt基因水稻田.在调查期间,不同地点3种非转基因水稻田间稻纵卷叶螟幼虫的种群密度以及它在植食类功能团内的相对丰富度均显著高于3种转Bt基因水稻田.水稻材料、水稻材料×调查日期、水稻材料×调查年份、水稻材料×调查日期×调查年份对稻纵卷叶螟幼虫的种群动态均有显著影响.但是,3种转Bt基因水稻材料和非转基因水稻田间4种捕食类天敌的相对丰富度、种群密度以及种群动态基本没有显著差异.从上述结果可推论,转Bt基因水稻中的Bt蛋白能明显抑制稻纵卷叶螟的发生,但对4种捕食类天敌没有显著影响.     

Cry1Ab蛋白对稻纵卷叶螟幼虫体内三种保护酶活性的影响

室内采用酶活力测定方法研究转B t基因水稻表达的Cry1Ab蛋白对稻纵卷叶螟幼虫体内3种保护酶(AChE、SOD和CAT)活性的影响。结果表明,用转B t基因水稻处理稻纵卷叶螟幼虫,饲喂4 h、36 h后,其体内AChE活力分别比对照增加了52.09%、128.51%,并且都极显著(P<0.01)高于对照组;取食转基因水稻叶片4 h后,幼虫体内SOD酶活性比对照提高了91.5%,与对照有显著差异;36 h后活性达到最大值,但与对照差异不显著。取食转基因水稻叶片24 h后,幼虫体内CAT酶活性达到最大值且高于对照,但与对照差异不显著;48 h后酶活性受到显著抑制,与对照差异显著。同时,测定了幼虫体内及其粪便中Cry1Ab蛋白含量的变化,结果表明,取食转基因水稻叶片12 h后,进入体内的Cry1Ab蛋白随粪便排出,幼虫体内的Cry1Ab蛋白含量一直低于粪便中的含量,且在24 h时两者差异达到最大。由于Cry1Ab蛋白在幼虫体内的积累,扰乱了稻纵卷叶螟幼虫体内AChE、SOD和C AT保护酶的动态平衡,使虫体内自由基的清除遇到障碍,从而对其产生毒害作用。     

水稻施用硅肥对稻纵卷叶螟幼虫保护酶和解毒酶活性的影响

【目的】研究施硅水稻对稻纵卷叶螟Cnaphalocrocis medinalis(Guenée)幼虫保护酶(过氧化氢酶CAT、过氧化物酶POD、超氧化物歧化酶SOD)和解毒酶(谷胱甘肽-S-转移酶GST、乙酰胆碱酯酶ACh E)活性的影响,为探明硅增强水稻抗稻纵卷叶螟的机理和稻纵卷叶螟的综合治理提供依据。【方法】采用感虫水稻品种TN1,设置2种施硅水平,即0.32 g Si/kg土壤(Si+)和不施硅(Si-),测定取食水稻24、48、72、96 h后3龄幼虫体内CAT、POD、SOD、GST、ACh E活性的动态变化。【结果】幼虫取食Si+水稻前期(24~48 h),CAT、POD、SOD活性呈上升趋势,并在48 h时达到最高值;取食后期(48~96 h),CAT、POD、SOD活性下降并在96 h时显著低于Si-处理。在Si-处理中,幼虫CAT和POD活性"先降低、再升高",SOD活性呈一直增大的趋势。取食Si+水稻幼虫GST活性始终显著高于Si-处理,而ACh E活性均低于Si-水稻,并在72 h和96 h时显著低于Si-水稻。处理间幼虫存活率存在显著差异,Si+水稻上1龄和3龄幼虫的存活率显著低于Si-水稻。这些结果说明,硅处理可能使稻纵卷叶螟幼虫产生应激反应引起保护酶活性在短时间内增大,但取食一段时间后保护酶活性下降。ACh E活性受到抑制,会引起昆虫神经传导的异常反应,造成昆虫死亡。【结论】硅可能通过参与植物的生理代谢过程,诱导植物增加次生代谢物的合成和积累,引起稻纵卷叶螟保护酶和解毒酶的活性变化,可能会影响稻纵卷叶螟的存活。     

寄主转换对稻纵卷叶螟寄主种群适合度及生命表参数的影响

【目的】稻纵卷叶螟Cnaphalocrocis medinalis是水稻的迁飞性害虫之一,同时也能在玉米上完成世代。本研究旨在探究寄主转换对稻纵卷叶螟寄主种群适合度的影响。【方法】稻纵卷叶螟玉米种群和水稻种群分别取食原寄主和转换寄主,形成取食玉米的稻纵卷叶螟玉米种群、取食水稻的稻纵卷叶螟玉米种群、取食玉米的稻纵卷叶螟水稻种群和取食水稻的稻纵卷叶螟水稻种群4个处理组合,构建这些处理组合的稻纵卷叶螟寄主种群年龄 龄期两性生命表,测定其适合度和生命表参数。【结果】稻纵卷叶螟幼期发育历期以取食水稻的稻纵卷叶螟水稻种群最长(29.2 d),以取食水稻的稻纵卷叶螟玉米种群最短(24.8 d);适合度指标(蛹重、化蛹率、羽化率、产卵量)、生命表参数(存活率、最大繁殖力、最高繁殖值、幼期寿命期望值)、种群参数(内禀增长率、周限增长率、净增殖率)均在两种群取食玉米时最大,在取食水稻的稻纵卷叶螟玉米种群中最小(除蛹重外),而且取食玉米的稻纵卷叶螟水稻种群又往往优于取食水稻的稻纵卷叶螟水稻种群。【结论】稻纵卷叶螟玉米种群在寄主转换后适合度和种群增长显著下降,而稻纵卷叶螟水稻种群在寄主转换后适合度和种群增长有所上升,表明玉米比水稻更有利于提高稻纵卷叶螟的适合度和种群增长,这一与自然条件下稻纵卷叶螟极少在玉米上发生为害相悖的现象值得深入研究。     

苦皮藤素V对东方粘虫中肠细胞及其消化酶活性的影响

苦皮藤素V是从杀虫植物苦皮藤Celastrus angulatus Max.根皮中分离的一种对昆虫具有毒杀活性的新化合物。该文通过电镜观察和生化分析研究了其对东方粘虫Mythimnaseparata(walker)幼虫中肠组织及中肠主要消化酶活性的影响。电镜观察发现,中毒试虫的中肠细胞及其细胞器发生明显病变：柱状细胞顶膜微绒毛零乱、减少;线粒体肿胀,出现空白亮区,双层膜不完整;细胞质密度降低,细胞器排列紊乱;内质网池扩张,囊泡化,粗面内质网减少;杯状细胞杯腔变大,微绒毛减少。消化酶活性测定结果表明,中毒试虫中肠的蛋白酶、淀粉酶及脂肪酶的活性和正常虫相比,无显著变化。因此认为,苦皮藤素V主要作用于中肠细胞的质膜及其内膜系统。     

Changes in the structure of Nippostrongylus brasiliensis intestinal cells during development from the free-living to the parasitic stages

The ultrastructure of Nippostrongylus brasiliensis intestinal cells was examined in free-living, feeding second-stage larvae, infective, nonfeeding third-stage larvae, and parasitic, feeding third-stage larvae. The intestinal cells of second-stage larvae were characterized by a well-developed microvillar border, large numbers of ribosomes, Golgi complexes, rough endoplasmic reticulum, and nuclei with prominent nucleoli. The intestinal cells of infective, third-stage larvae had very few microvilli and the cells were extremely narrow. Few ribosomes, Golgi complexes, and little rough endoplasmic reticulum were present. Nuclei did not contain nucleoli. When worms were introduced into an in vitro culture system, development of intestinal cells began. By 36 hr, microvilli were well differentiated and the cysoplasm contained numerous ribosomes and Golgi complexes, and rough endoplasmic reticulum, mitochondria, and nucleoli were prominent. These morphological changes were related to changes in the physiology of Nippostrongylus brasiliensis which occur during development from a free-living to parasitic form.     

甜菜夜蛾微孢子虫研究:Ⅲ.超微结构与致病机理

从甜菜夜蛾幼虫体内首次分离到一种侵染寄主脂肪体、马氏管和中肠的微孢子虫,该微孢子虫对甜菜夜蛾、菜青虫和棉铃虫幼虫有较强的致病力,并可经卵垂直传播;水平传播主要借助于病虫的粪便及虫尸。用电镜观察了该微孢子虫感染甜菜夜蛾幼虫后的超微结构变化。结果表明：寄主细胞被感染后,细胞核膨大并变形,由正常的圆球形被挤压成长条状,但核膜及核周间隙没有发生变化;线粒体体积变小,嵴增宽,嵴的排列方向发生改变,双层膜部分     

中华硬蜱叮咬不同免疫力新西兰兔后中肠上皮组织的形态动态变化

通过光镜和电镜观察了中华硬蜱Ixodes sinensis叮咬初次和再次感染宿主新西兰兔后不同时间（叮咬后24 h、48 h、72 h以及第 5天、第8天）中肠上皮组织的形态学动态变化。结果显示： 中华硬蜱叮咬前中肠上皮主要由替代细胞和少量体积较大的消化细胞构成;替代细胞数量多、体积小、呈圆形、胞质染色浅 。中华硬蜱叮咬初次感染宿主后,消化细胞随叮咬时间延长而增多增大,微绒毛较密集,排列整齐,胞质内细胞器丰富,各单位膜结构清晰,并出现顶端小管、小泡、大量脂滴和高铁血红素颗粒;近基膜的细胞膜内褶形成发达的基底迷路系统。中华硬蜱叮咬再次感染宿主后,中肠可发生一系列明显的病理变化,中肠基膜出现变薄、松散和断裂现象,消化细胞破裂、空泡化,消化细胞数量减少;消化细胞微绒毛减少、变短、排列不整,线粒体肿大,体嵴减少、变短甚至髓样变,粗面内质网扩张,脂粒及高铁血红素颗粒减少,细胞膜吞饮、吞噬现象减弱,消化细胞内结构紊乱和破坏。该研究结果提示初次叮咬导致了宿主的免疫抗性,再次叮咬后蜱中肠是宿主免疫力的主要作用部位。     

A new enveloped helical virus from the blue crab,Callinectes sapidus

Quite different ultrastructural changes were observed in the columnar cell and the goblet cell of the silkworm midgut after administration of the crystalline toxin of Bacillus thuringiensis. Shortly after the ingestion of the toxin, the deep infoldings of the basal cell membrane of some columnar cells became very irregular in shape and the mitochondria near the basal region were transformed into a condensed form. A few goblet cells showed relatively high electron density in the cytoplasm. The earliest pathological changes were slight and located in a region lying between the first and second thirds of the midgut. With the passage of time, they spread anteriorly and posteriorly to include the entire anterior two thirds of the midgut and became more profound. The cytoplasm of columnar cells became very electron transparent. Most mitochondria were transformed into a condensed form and the endoplasmic reticulum assumed a vacuole-like configuration. The basal infoldings of the cell membrane almost disappeared. On the other hand, the cytoplasm of the goblet cells became very electron dense and granular. The clear basal infoldings of the cell membrane were enlarged making a striking contrast with the dense cytoplasm. However, the mitochondria and the endoplasmic reticulum did not show any pathological deformation.     

Toxicological assessment of pollen from different bt rice lines on Bombyx mori (Lepidoptera: Bombyxidae)

The relative toxicity of Bt rice pollen to domestic silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), was assessed by a leaf-dip bioassay under laboratory conditions. Silkworm first instars were sensitive to pollen from Bt rice lines, B1 and KMD1, but were not sensitive to pollen from Bt rice line TT9-3. First instars were 1.34-2.12 times more sensitive to B1 pollen than older instars. Bioassays of subacute toxicity under a worst-case scenario suggested that continuous exposure to a sublethal dose of B1 pollen or equivalent doses of non-Bt rice pollen affected silkworm survival and development. Young larvae were more affected by continuous exposure to Bt pollen than older larvae but less affected by non-Bt pollen. Ultrastructural observations showed that Cry proteins associated with Bt pollen were released into the larval lumen and resulted in pathological midgut changes and negative impacts on silkworm survival and development. However, considering that the sublethal dose of Bt pollen (LC15) used in this study is equivalent to the highest detected density of rice pollen on mulberry leaf under field conditions and that the likelihood of such high density of rice pollen occurring in the fields is extremely low, we suggest that the risk of Bt rice pollen on silkworm rearing is negligible.     

取食Cry2Ab蛋白后棉铃虫幼虫中肠组织的病理变化

为了明确Cry2Ab杀虫蛋白的作用机制, 利用透射电镜观察了棉铃虫Helicoverpa armigera (Hübner)3龄末幼虫取食含Cry2Ab蛋白（8 μg/g）饲料后中肠的组织病理变化, 并与分别取食含Cry1Ac蛋白（0.97 μg/g）饲料和正常饲料的棉铃虫进行了比较。结果表明： 棉铃 虫取食Cry2Ab蛋白后中肠细胞及其细胞器均发生了明显的病变, 主要表现为： 中肠杯状细胞的杯腔肿胀或拉长, 部分柱状细胞被杯状细 胞挤压出来, 微绒毛脱落, 细胞核皱缩, 质膜和核膜不清晰, 染色质凝聚, 线粒体拉伸变形, 内质网肿胀断裂; 并且随着取食时间 的延长病变越来越明显。与取食Cry1Ac蛋白的棉铃虫相比, Cry2Ab引起棉铃虫中肠组织发生病变的速度较慢。本研究可为Cry2Ab作为转基 因棉花的重要杀虫蛋白在将来更好地发挥作用提供理论依据。     

Digestive cells in the midgut of Triatoma vitticeps (Stal, 1859) in different starvation periods

Triatoma vitticeps (Stal, 1859) is a hematophagous Hemiptera that, although being considered wild, can be found in households, being a potential Chagas’ disease vector. This work describes the histology and ultrastructure of the midgut of T. vitticeps under different starvation periods. Fifteen adults of both sexes starved for 3, 7, 20 and 25 days were studied. In general, digestive cells had apical microvilli, basal plasma membrane infoldings and central nucleus. The perimicrovillar membrane was found in all insects examined. Digestive cells of anterior midgut had lipid droplets, glycogen granules, developed basal labyrinth associated with mitochondria suggesting their role in nutrient storage and in fluid and ion transport. The cells of median and posterior regions of the midgut were rich in rough endoplasmic reticulum, lysosomes, vesicles and granules with different electron-densities. Moreover, cells of the posterior portion of the midgut had hemozoyn granules and mitochondria in the apical cytoplasm close to microvilli, suggesting their role in blood digestion and active nutrient absorption. The midgut of T. vitticeps showed differences in digestive cells associated with the time after feeding, and the increase of vesicles amount in long starvation periods, which suggests enzyme storage, which is readily used after a blood meal.     

Midgut ultrastructure of Culex tarsalis (Diptera:Culcidae) before and after a bloodmeal

The midgut ultrastructure of raisin- and blood-fed female mosquitoes, Culex tarsalis Coquillett, was examined. The raisin-fed midgut is characterized by: (1) large nuclei, (2) small mitochondria, (3) short segments of rough endoplasmic reticulum, (4) rough endoplasmic reticular vesicles in the posterior midgut only, and (5) increased autophagic activity with age. Blood feeding elicits drastic changes in midgut epithelial structures: (1) nuclei are smaller, (2) mitochondria are much enlarged, (3) rough endoplasmic reticular vesicles disappear, (4) rough endoplasmic reticular whorls appear, (5) residual lyosomal figures are abundant, and (6) an intercellular accumulation of an electron-opaque material is noted. The significance of rough endoplasmic reticular whorls and vesicles in bloodmeal digestion is discussed. In addition, the concept of a functional host ‘gut barrier’ to infection by pathogens is examined as related to a possible by-pass mechanism.