高温对家蚕三品系血淋巴中糖水平的影响（英文）

家蚕Bombyx mori的两个二化性品系热耐受型NB4D2和热敏感型CSR2均适合于温带气候,而多化性的PM（Pure Mysore) 品系适合于热带气候,将这3种品系5龄幼虫分别置于32℃和36℃的高温下,观察高温对其5龄幼虫至蛹期血淋巴中糖含量及海藻糖酶活性的影响。结果表明： PM幼虫和蛹的死亡率均小于NB4D2和CSR2。在蜕皮期间血淋巴海藻糖水平较高,而葡萄糖水平及海藻糖酶活性较低。32℃和36℃的高温下,幼虫蜕皮期间血淋巴中糖含量及海藻糖酶活性仅在其各自的水平上表现为小幅度的增加。蜕皮后幼虫血淋巴中海藻糖含量显著下降,而葡萄糖含量和海藻糖酶活性显著上升。在较高温度下,蜕皮后幼虫血淋巴中海藻糖含量下降幅度更大,而葡萄糖含量及海藻糖酶活性上升水平也更加显著。25±1℃下取食幼虫血淋巴中葡萄糖含量显著下降,海藻糖含量显著上升;3℃和36℃下PM 和NB4D2取食幼虫血淋巴葡萄糖和海藻糖含量以及海藻糖酶活性增加,而CSR2均减少或降低。吐丝幼虫血淋巴中葡萄糖含量及海藻糖酶活性显著下降,海藻糖小幅度下降。而在较高温度下,耐热型PM 和NB4D2吐丝家蚕血淋巴糖含量含量和海藻糖酶活性明显增加,而热敏感型CSR2的则明显下降。这3种品系蛹发育期的血淋巴糖含量及海藻糖酶活性均下降。在两较高温度下,PM蛹期血淋巴糖和海藻糖酶活性增加,而NB4D2 36℃时增加幅度小于32℃时。对于CSR2,32℃时观察到其血淋巴葡萄糖含量增加,但当环境温度增加到36℃时其血淋巴葡萄糖含量降至正常水平下。然而,当CSR2的蛹置于32℃和36℃时血淋巴海藻糖含量及其酶活性下降,且36℃时下降幅度更大。因此,桑蚕对高温的适应取决于家蚕的品系及发育阶段,并可通过其血淋巴糖及海藻糖酶活性水平进行验证。     

马尾松毛虫感染绿僵菌后某些生化指标的变化

3～ 4龄马尾松毛虫Dendrolimuspunctatus幼虫被金龟子绿僵菌Metarhiziumanisopliae感染后 1～ 4d,血淋巴中血细胞总数和可溶性蛋白浓度均显著高于同期未感染的幼虫。利用聚丙烯酰胺凝胶电泳技术 ,分析了感染后 3 ,4,6d幼虫体壁组织中可溶性蛋白和过氧化物酶 ,以及血淋巴中过氧化物酶的变化。结果显示 ,金龟子绿僵菌的侵染对马尾松毛虫幼虫体壁组织蛋白质代谢产生影响 ,过氧化物酶活性有减弱趋势     

几种植物源化合物对棉铃虫海藻糖酶活性及相关碳水化合物含量的影响

碳水化合物对昆虫的能量代谢和物质合成具有重要的作用。本研究选用2种一般性生物碱（氢溴酸东莨菪碱和烟碱）以及2种β-葡萄糖苷类化合物（七叶灵和皂角苷）, 研究其在不同浓度下对棉铃虫Helicoverpa armigera (Hübner)幼虫体内海藻糖酶活性及相关碳水化合物代谢的影响。结果表明: 用饲喂法处理3龄幼虫96 h后, 皂角苷对棉铃虫幼虫的活体抑制效果明显, 且随添加物浓度增高, 棉铃虫死亡率上升, 10, 20, 40 g/L浓度下棉铃虫的均重分别是0.194, 0.089和0.034 g, 分别为对照的86.99%, 39.91%和15.24%。对海藻糖酶活性及其相关代谢酶的测定结果表明, 2种苷类化合物显著抑制中肠海藻糖酶活性, 饲喂40 g/L皂角苷的试虫中肠海藻糖酶比活力仅是对照组的54.21%; 饲喂30 g/L七叶灵的试虫中肠海藻糖酶比活力为对照组的83.73%。而2种生物碱类化合物显著抑制血淋巴和脂肪体中海藻糖酶活性, 20 g/L氢溴酸东莨菪碱对棉铃虫血淋巴和脂肪体组织的海藻糖酶活性抑制率分别为7.24%和71.43%; 而20 g/L烟碱对试虫血淋巴和脂肪体组织的海藻糖酶活性抑制率为26.29%和33.44%。用氢溴酸东莨菪碱、 烟碱和七叶灵处理试虫后, 血淋巴海藻糖含量都有所增高。4种化合物能够导致试虫糖原磷酸化酶活性变化, 其中, 皂角苷在中肠和脂肪体表现为显著抑制作用, 而随外源化合物浓度变化, 糖原含量和糖原磷酸化酶活性表现为此消彼长关系。饲喂4种植物源化合物的试虫血淋巴中葡萄糖浓度变化和其海藻糖变化一致。本研究证明β-葡萄糖苷类化合物是海藻糖酶抑制剂, 在作为先导化合物进行农药创制开发方面具有重要意义。     

白僵菌的调查和鉴定

白僵菌[Beauveria bassiana (Bals.)Vuill.]是一种重要的虫生真菌,我国广泛用于防治玉米螟和松毛虫等农、林害虫。但关于自然界昆虫白僵病的发生特点和寄主范围等在国内报道很少,对于白僵菌的分类也比较混乱,有人认为B.globulifera应区别于白僵菌(B.bassiana)作为一个独立的种存在,但有人则认为B.globulifera应是白僵菌(B.bassiana)的异名,不能作为一个独立的种。作者在虫生真菌的采集调查中,从1962年冬至1965年夏共分离到白僵菌属真菌55个菌株。根据对这些菌株的研究,同意后一种的看法。 现将调查和鉴定结果报道如下。     

粘虫的血糖代谢

本文通过纸上层析和化学方法,研究了粘虫在蛹化前后各虫期血淋巴液中的总糖、海藻糖、葡萄糖和血淋巴的还原值,并与蓖麻蚕进行了比较。此外,还测定了血淋巴海藻糖酶的活性变化。结果表明:1)在蛹化前后血淋巴液中的总糖与海藻糖,自末龄幼虫进食以后开始大量积累,到幼虫老熟时达最高。在幼虫蜕皮和停食化蛹过程,血糖含量明显地减少。雌体含量均高于雄体。血淋巴液中的糖量变化与粘虫的生长发育有密切关系。2)和蓖麻蚕相似,在蜕皮和停食以后的化蛹过程,血淋巴中的还原值显著增加,这种增加主要是由于葡萄糖、核糖以及其它还原物质的出现或数量的增加所引起。3)末龄幼虫血淋巴液中的总糖及海藻糖的百分比含量,均比同虫期的蓖麻蚕的含量低2—5倍。粘虫幼虫血淋巴中的海藻糖量只占总糖的66.3%—94%,而蓖麻蚕则为98%—100%。4)幼虫血淋巴中的海藻糖酶活力与家蚕、蓖麻蚕的相同,均在进食期间无活力表现或活力极低;在幼虫蜕皮和化蛹过程则明显出现活力。5)血淋巴液中的糖量变化与海藻糖酶活力变化有明显的负相关。文中还分析和讨论了粘虫血糖代谢的特点以及各种糖类物质在生长发育中的作用。     

油松毛虫受球孢白僵菌感染的组织病理学变化

为了研究昆虫病原真菌对松毛虫的致病机理, 提供北方地区松毛虫生物防治的科学依据, 本研究采用球孢白僵菌Beauveria bassiana (Bals.) Vuillemin 菌株1573感染油松毛虫Dendrolimus tabulaeformis Tsai et Liu, 通过扫描电镜和石蜡切片光学显微镜观察技术, 研究了菌株的感染过程和虫体的组织病理学变化。结果表明, 该病原真菌通过穿透表皮入侵油松毛虫, 染菌后24 h, 观察到分生孢子附着于头部的颅顶, 单眼、触角和口器的基部, 在胸、腹部附着于毛簇、毛瘤、棘状突和节间褶。染菌后36 h, 孢子萌发长出菌丝, 在入侵部位, 菌丝的端部特化成附着胞和侵入钉。染菌后48 h, 菌丝依靠机械力和胞外酶的作用穿透表皮, 虫体表皮出现了裂痕和黑化。染菌后72 h, 菌丝已进入体腔, 感染血淋巴、脂肪体、肌肉、消化道、丝腺和神经组织, 并利用血液和内部组织器官作为营养大量繁殖, 此时, 虫体发涨, 表皮变暗。染菌后96 h, 菌丝占据了松毛虫的血腔, 内部的组织结构被完全瓦解, 松毛虫死亡。最后, 菌丝冲破体壁, 在尸体表面长出新的分生孢子。这些结果说明, 球孢白僵菌B. bassiana菌株1573是一种油松毛虫的高致病性菌株, 可以引起虫体的一系列组织病理变化而致其死亡。     

白僵菌孢子和提取毒素对亚洲玉米螟血淋巴蛋白质的影响

以白僵菌孢子和提取毒素感染亚洲玉米螟Ostrinta furnacalis幼虫,引起血淋巴蛋白质含量降低,蛋白质凝胶电泳区带减少。无论是孢子感染或经饲喂和注入虫体毒素,试虫血淋巴中皆有不受毒作用影响的稳定性蛋白质存在。还可看出,培养液提取毒素对试虫血淋巴蛋白质的影响大于由菌粉提取的毒素。此外证实了提取毒素经贮存7年仍有毒效。     

蓖麻蚕在变态期间代谢作用的研究——Ⅱ.海藻糖酶的性质及其在代谢中的作用

本文以蓖麻蚕蛹化前后各虫期为材料,通过对不同组织中海藻糖酶活力及其糖含量的测定,结果发现了:1)血淋巴海藻糖酶活力仅在眠期蜕皮过程显现,其它虫期该酶不表现活力;2)海藻糖酶抑制物仅存在于血淋巴中,抑制物的存在虽然使海藻糖酶经常处于不活动状态,但后者却具有一定的潜在活力;3)饥饿能促使对海藻糖酶抑制的解除,随着酶活力的显现,此血糖的含量水平不断下降;4)消化道中海藻糖酶的分布依次为中肠>后肠>前肠,酶活力随幼虫进食而增加,吐丝以后显著下降;5)血淋巴和脂肪体中糖代谢与海藻糖酶活力变化之间存在一定的联系。文中明确了不同组织中海藻糖酶活力变化在昆虫糖代谢中的作用,并讨论了它的生理意义。     

淡足侧沟茧蜂寄生对甜菜夜蛾幼虫血淋巴保护酶和免疫酶的影响

[目的]为探讨淡足侧沟茧蜂Microplitis pallidipes Szepligeti调控寄主甜菜夜蛾Spodoptera exigua(H(u|¨)bner)的生理机制。[方法]测定了淡足侧沟茧蜂寄生甜菜夜蛾后,寄主幼虫血淋巴中保护酶(SOD、CAT及POD)和免疫酶(PO和LSZ)活性的变化。[结果]寄生后连续5 d观察时间内,从寄生后第1天开始,寄主幼虫血淋巴中SOD活性就高于未寄生幼虫(对照),且在寄生后第4~5天达到显著水平;被寄生幼虫血淋巴中CAT活性始终高于对照幼虫,且在寄生后第1、第4~5天达到显著水平;同样,甜菜夜蛾被寄生后,其血淋巴中POD活性明显高于对照幼虫,且在寄生后第1~2、第5天达显著水平;寄生后第1天,寄生与对照组酚氧化酶活性差异不显著,但寄生后第2~5天均达到显著水平;较对照组,寄生后第1~2天,被寄生幼虫溶菌酶活性增加但无显著差异,而寄生后3~4d溶菌酶活性显著降低。[结论]本研究揭示,淡足侧沟茧蜂寄生刺激了甜菜夜蛾幼虫体内的应激反应,诱导寄主体内SOD、CAT和POD活性明显上升,阻止寄生蜂成功寄生;同时,寄生蜂寄生诱导了寄主免疫酶(PO、LSZ)活性的变化,免疫酶活性的变化是寄主免疫系统对寄生蜂寄生的一种应急免疫反应。     

几种杀虫真菌对二化螟幼虫的毒力测定与高毒力菌株的分子鉴定

【目的】测定杀虫真菌对二化螟Chilo suppressalis(Walker)幼虫的室内毒力,明确高毒力菌株的种类,为应用杀虫真菌防治二化螟提供理论基础。【方法】采用喷雾法测定10株杀虫真菌在同种浓度、湿度和温度下对二化螟幼虫的室内毒力,选择毒力最强的菌株,利用扫描电镜观察杀虫真菌分生孢子对二化螟的侵染过程;根据该菌株的生物学特性,利用白僵菌Beauveria sp.18S r DNA ITS序列进行PCR扩增、琼脂糖凝胶电泳检测和序列分析,并在核酸序列数据库中进行同源性序列对比。【结果】真菌YQ147、QING、Bbr14、Bbr84在浓度为1.0×108孢子/m L下均对二化螟幼虫具有较强的致病力,其中真菌YQ147在处理后第10天对二化螟累计校正死亡率达到78.4%,LT50为5.8 d,分子鉴定的结果显示该菌为白僵菌属球孢白僵菌Beauveria bassiana。【结论】明确了球孢白僵菌对二化螟的室内毒力最强,这为利用其对二化螟进行生物防治提供了基础。     

Identification of an extracellular acid trehalase and its gene involved in fungal pathogenesis of Metarizium anisopliae

Trehalose is the main sugar in the haemolymph of insects and is a key nutrient source for an insect pathogenic fungus. Secretion of trehalose-hydrolysing enzymes may be a prerequisite for successful exploitation of this resource by the pathogen. An acid trehalase [EC 3.2.1.28] was purified to homogeneity from a culture of a locust-specific pathogen, Metarhizium anisopliae, and its properties were characterized. The gene (ATM1) of this acid trehalase was also isolated. The pure enzyme can efficiently hydrolyze haemolymph trehalose into glucose in vitro. The new acid trehalase appearing in the haemolymph of Locusta migratoria infected with M. anisopliae had the same pI and substrate specificity as the purified fungal acid trehalase, and the concentration of trehalose in the haemolymph decreased sharply after infection. RT-PCR also revealed the ATM1 gene's expression in the haemolymph of the infected insects. Our results indicated that the acid trehalase may serve as an "energy scavenger" and deplete blood trehalose during fungal pathogenesis.     

Modulation of trehalase activity in two insect cell lines by virus infection and trehalose

Trehalase (EC 3.2.1.28), an important glycosidase involved in regulating trehalose levels and metabolic energy in insects, was measured in cell lines from fall army worm, Spodoptera frugiperda and salt marsh caterpillar, Estigmene acrea, treated with either glucose or trehalose in the presence or absence of Tipula Iridescent Virus (TIV), a cytoplasmic deoxyribovirus. In medium containing 15-35 mM trehalose, both of these cells increased their trehalase activities by 4.5 to 8x the basal levels from cells in glucose medium. Trehalase activity was rapidly reduced after cells were exposed to TIV. Maximum loss in activity (70-90%), occurring about the same time as peak viral DNA synthesis, was significantly delayed when cells were pre-incubated with 30 mM trehalose. These experiments demonstrate the potential utility of trehalase as a marker for monitoring stresses induced by viral infection and changes in nutrition.     

Involvement of adipokinetic hormone in the homeostatic control of hemolymph trehalose concentration in the larvae of Bombyx mori

Prior to wandering, 5th instar larvae of the silkworm, Bombyx mori, maintain constant hemolymph titers of trehalose. Head ligation of day 3, 5th instar larvae significantly decreased the hemolymph trehalose concentrations, but the concentrations did not decrease in starved larvae. After being diluted by replacement of larval hemolymph with insect Ringer's solution, the trehalose concentrations recovered the initial levels in 90 min in the non-ligated larvae, while they were not restored in 90 min in the neck-ligated larvae. These results suggest that a head factor(s) with hypertrehalosemic activity is involved in the homeostatic control of hemolymph trehalose concentration. When adipokinetic hormone (AKH) was injected into neck-ligated larvae, the trehalose concentrations increased in 2 h and decreased thereafter. Repeated injections of AKH every 4 h maintained the concentrations for 12 h. These findings suggest that AKH induces a hypertrehalosemic response and is involved in the homeostasis of hemolymph trehalose concentration in the larval feeding period.     

Trehalose-hydrolysing enzymes of Metarhizium anisopliae and their role in pathogenesis of the tobacco hornworm,Manduca sexta

Trehalose is the main haemolymph sugar in most insects including the tobacco hornworm, Manduca sexta, and is potentially a prime target for an invading pathogenic fungus. There was considerably more trehalose-hydrolysing activity in the haemolymph of caterpillars infected with Metarhizium anisopliae than in controls. This appeared to be due primarily to additional isoforms; one of which could also hydrolyse maltose and was designated an alpha-glucosidase. A comparable isoform was identified in in vitro culture of the fungus, supporting a fungal origin for the in vivo enzyme. The in vitro fungal enzyme, alpha-glucosidase-1 (alpha-gluc-1), was purified to homogeneity and partially characterised. A study with the trehalase inhibitor trehazolin and C14 trehalose suggested that extracellular hydrolysis is important for fungal mobilisation of trehalose. Haemolymph glucose increases significantly during mycosis of tobacco hornworm larvae by M. anisopliae, consistent with the hydrolysis of trehalose by extracellular fungal enzymes. The implications for the host insect are discussed.     

α-Glucose-1-phosphate formation by a novel trehalose phosphorylase from Flammulina velutipes

A novel type of trehalose phosphorylase was found in a basidiomycete. Flammulina velutipes . The enzyme catalyzes both the reversible phosphorolysis of trehalose to form α-glucose 1-phosphate and glucose and also the synthesis of trehalose. Comparison of the specific activity of trehalose phosphorylase with that of trehalase suggested that the function of the former enzyme was more important in the fruit-bodies of this fungus.     

Fate and effects of the trehalase inhibitor trehazolin in the migratory locust (Locusta migratoria)

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 μg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.     

Determination of trehalose in biological samples by a simple and stable trehalase preparation

A three step purification procedure for trehalase from Saccharomyces cerevisiae with a recovery of 76% of the original activity is presented. The enzyme was activated by a heat shock treatment prior to homogenization of the cells. A mutant strain deleted in SUC genes was used to avoid contamination by invertase. The lyophylized enzyme was stable for, at least, 5 months and could be used to determine trehalose in the range 25 to 500 nmol. The preparation was free of inspecific phosphatases allowing for trehalose determinations in yeast cell free extracts and in insect hemolymph.     

沉默类胰岛素多肽(Ilp)基因对褐飞虱翅和卵巢发育及海藻糖代谢的影响

【目的】类胰岛素多肽(insulin-like peptide, Ilp)位于胰岛素信号通路最上游,其在糖类物质调控中发挥关键作用。本研究则旨在探究Ilp在褐飞虱Nilaparvata lugens海藻糖代谢平衡的调控作用。【方法】以褐飞虱5龄若虫为实验材料,采用RNAi技术干扰Ilps的表达,观察RNAi后褐飞虱的表型以及雌成虫的卵巢发育。RNAi 48 h与72 h后,采用生化方法测定褐飞虱5龄若虫体内海藻糖、糖原和葡萄糖含量以及海藻糖酶活性变化;采用qPCR检测海藻糖酶基因(TRE1-1, TRE1-2和TRE2)和海藻糖合成酶基因(TPS1和TPS2)的表达量变化。【结果】dsRNA可有效抑制Ilps的表达,导致褐飞虱出现异常翅型,且注射dsIlp3以及dsIlp1+dsIlp2+dsIlp3+dsIlp4发现褐飞虱2日龄雌成虫卵巢发育不完全。分别注射dsIlp1-4和dsIlp1+dsIlp2+dsIlp3+dsIlp4后48 h均能显著提高葡萄糖含量;分别注射dsIlp2, dsIlp3及dsIlp4后48 h显著提高了糖原含量;分别注射dsIlp3和dsIlp4后48 h能够显著提高海藻糖含量,而分别抑制Ilp2和Ilp4 72 h后海藻糖含量显著下降,但注射dsIlp1+dsIlp2+dsIlp3+dsIlp4后48和72 h海藻糖含量都显著上升。注射dsIlp1+dsIlp2+dsIlp3+dsIlp4后72 h可溶性海藻糖酶活性均显著上升,膜结合型海藻糖酶活性则在分别注射dsIlp3, dsIlp4和dsIlp1+dsIlp2+dsIlp3+dsIlp4后72 h显著下降;分别注射dsIlp1, dsIlp2和dsIlp4后TRE1-1, TRE1-2, TRE2, TPS1和TPS2的表达量显著下降。【结论】沉默Ilp基因对褐飞虱的发育以及繁殖有一定的阻碍作用,且能够提高褐飞虱体内葡萄糖与糖原的含量,下调海藻糖酶与海藻糖合成酶基因表达量,提高可溶性海藻糖酶的活性,进而调控海藻糖的代谢。