感染布氏白僵菌后油松毛虫血淋巴中海藻糖酶活性、海藻糖和葡萄糖含量的变化

为了揭示病原真菌白僵菌Beauveria侵染昆虫过程中如何利用虫体内糖类物质作为自身营养, 本研究测定了布氏白僵菌Beauveria brongniartii (Sacc.) Petch （2382菌株）感染油松毛虫Dendrolimus tabulaeformis Tsai et Liu幼虫后, 虫体血淋巴中酸性海藻糖降解酶活性及海藻糖和葡萄糖含量的变化。油松毛虫4龄幼虫感染菌株孢子悬浮液后, 血淋巴中酸性海藻糖降解酶的活性明显高于对照组, 感染后第3天酶活性达到最大值（0.2786 U/mg）, 此后第4-6 天酶活性逐渐降低; 染菌后的6 d中, 血淋巴中海藻糖含量显著低于对照组, 同样在感染后第4天其含量逐渐降低, 第6天时降到最低值。相比之下, 处理组血淋巴中的葡萄糖含量显著高于对照组; 处理组其含量在第1-3天内呈现快速升高趋势, 在第3天达到最大值（7.7615 mmol/L）, 然后逐渐降低。结果说明, 白僵菌侵入昆虫血淋巴后, 菌株代谢产生酸性海藻糖降解酶, 将血淋巴中的海藻糖水解成为葡萄糖, 然后为真菌利用, 破坏了虫体内的血糖平衡, 这是一个相互连接的生理代谢和生化反应过程。     

金龟子绿僵菌对斜纹夜蛾幼虫的生防效果、抗氧化酶活性和肠道细菌群落的影响

【目的】测定金龟子绿僵菌(Metarhizium anisopliae)对斜纹夜蛾(Spodoptera litura) 2龄幼虫的毒力,研究金龟子绿僵菌侵染后寄主体内抗氧化酶活性和肠道内细菌群落的变化,探讨斜纹夜蛾对金龟子绿僵菌侵染的防御机制。【方法】采用浸渍法测定不同浓度金龟子绿僵菌对斜纹夜蛾2龄幼虫的毒力;应用IlluminaMiSeq高通量测序技术测定肠道细菌群落。【结果】不同浓度的孢悬液对斜纹夜蛾2龄幼虫均有一定的毒力,处理7 d时半致死浓度(LC_(50))为3.944 107个孢子/mL;浓度为1.0×10~9个孢子/mL时,半致死时间最短(LT_(50))为4.6 d,校正后的死亡率为81.03%。处理后未致死的斜纹夜蛾幼虫体内抗氧化酶活性显著高于对照组。处理后致死的斜纹夜蛾幼虫肠道细菌群落多样性显著高于对照组;且处理后致死的斜纹夜蛾幼虫肠道细菌群落组成与对照组差异显著。【结论】金龟子绿僵菌对斜纹夜蛾幼虫的致死率和致死效率与金龟子绿僵菌的浓度呈正相关;斜纹夜蛾幼虫体内的抗氧化酶可能在抵抗金龟子绿僵菌侵染的过程中起重要作用。金龟子绿僵菌的侵染会导致斜纹夜蛾幼虫肠道细菌群落多样性升高和组成发生变化,Enterococcus、Escherichia和Pseudomonas等属可能是影响斜纹夜蛾幼虫抵抗金龟子绿僵菌侵染致死的重要因素。     

绿僵菌与3种杀虫剂混配对樟巢螟的协同作用

采用定量喷雾法测定了绿僵菌、白僵菌以及3种杀虫剂对樟巢螟Orthaga achatina低龄幼虫的室内毒力,并测定了金龟子绿僵菌Ma09与3种杀虫剂混配后的协同作用。结果显示：4种昆虫病原菌菌株均对樟巢螟2~3龄幼虫具有较好的致死效果,用绿僵菌和白僵菌孢悬液处理后,死亡高峰期在3~4 d;白僵菌GDLY9的防治效果最好,处理浓度为1×109孢子/mL下LT50和LT90分别为3.01、3.95 d,金龟子绿僵菌Ma09、3297效果相当,黄绿绿僵菌Mf985的LT50相对低一些,但是LT90优于金龟子绿僵菌Ma09和3297。金龟子绿僵菌Ma09与苯氧威、除虫脲和短稳杆菌混配后均表现出增效作用,协同指数分别为20.43、27.53和33.13。     

绿僵菌产海藻糖水解酶培养条件研究

丝状真菌绿僵菌能产生一系列二糖水解酶,其中包括海藻糖水解酶。这些酶在绿僵菌对昆虫的致病过程中起着重要的作用。本文研究了不同碳源、氮源对金龟子绿僵菌Metarhizium anisopliae var. acridum菌株CQMa102产生与分解昆虫血淋巴中海藻糖等二糖相关的海藻糖水解酶活性的影响。结果表明:分别以葡萄糖、麦芽糖、蔗糖、山梨醇和可溶性淀粉为碳源,金龟子绿僵菌均可产生海藻糖水解酶,但最佳碳源是可溶性淀粉,因为由其诱导产生的海藻糖水解酶具有最高的总活性和比活性以及更多的同工酶,山梨醇次之。硝态氮(NaNO₃)作为唯一氮源时,几乎检测不出海藻糖水解酶活性,而铵态氮((NH₄)₂SO₄)或NaNO₃和有机氮(蛋白胨和酵母浸膏)混合氮源作氮源时,海藻糖水解酶活性都很高。在绿僵菌菌丝提取液和滤液的海藻糖水解酶活性比较中发现:CQMa102在多数碳源的培养基中产生的海藻糖水解酶主要分泌到培养基中,仅有少数结合在细胞壁上。     

金龟子绿僵菌及其粗毒素对樟巢螟幼虫的致病性

【目的】为了筛选感染樟巢螟Orthaga achatina幼虫的高致病力金龟子绿僵菌Metarhizium anisopliae菌株,并研究绿僵菌粗毒素对幼虫的毒力和取食粗毒素后幼虫的血淋巴细胞免疫反应。【方法】以死亡率 时间几率值法和TDM模型分析绿僵菌及其粗毒素对樟巢螟幼虫的致病力,并显微观察处理幼虫的血淋巴细胞的变化。【结果】绿僵菌菌株Ma1291-2对樟巢螟幼虫有较强的致病力,以浓度(1.0±0.5)×108个孢子/mL的孢子悬液接菌11 d后,幼虫的校正死亡率和僵虫率分别为99.8%±2.6%和86.9%±1.3%,LT₅₀为6.29 d。各菌株产粗毒素水平与其对幼虫的校正死亡率和LT₅₀呈显著相关。通过时间-剂量-死亡率模型参数估算,Ma1291-2菌株及其粗毒素对幼虫致死效应较强的时间段分别为接菌后6-7 d和3-4.5 d。幼虫取食绿僵菌粗毒素后2 d,总血细胞、浆血细胞、珠血细胞和类绛血细胞浓度上升到最高值,而后下降;粒血细胞浓度2 d后开始极显著上升,第3天达到最高值后急速下降;原血细胞前3 d未发现有明显的数量变化,第4天显著下降。幼虫取食粗毒素3~4 d后,浆血细胞和粒血细胞有破裂,珠血细胞和类绛血细胞有黑化现象,原血细胞病态变化不明显。【结论】樟巢螟幼虫取食绿僵菌粗毒素后2-3 d,幼虫血淋巴细胞对粗毒素的免疫反应最强烈,且粗毒素对血细胞有毒害和破坏作用。本研究为该害虫的生物防治提供了一定的理论与应用基础。     

褐飞虱过氧化物酶基因NlPOD1的克隆、鉴定及功能分析

【目的】探明褐飞虱Nilaparvata lugens过氧化物酶基因NlPOD1的分子特性、表达模式和生物学功能。【方法】基于褐飞虱转录组数据,利用PCR技术克隆获得NlPOD1基因全长cDNA序列;利用生物信息学手段分析其序列特征;通过qRT-PCR技术检测NlPOD1在褐飞虱不同发育时期(卵、1-5龄若虫和初羽化雌雄成虫)和5龄若虫不同组织(头部、脂肪体、血淋巴和肠道)中的表达水平,以及不同微生物(大肠杆菌Escherichia coli、金黄色葡萄球菌Staphylococcus aureus和金龟子绿僵菌Metarhizium anisopliae)（1×10⁷/mL）注射感染褐飞虱5龄若虫不同时间后的诱导表达模式;利用RNAi技术沉默褐飞虱 5 龄若虫NlPOD1基因,并通过生物测定分析NlPOD1基因沉默和接种金龟子绿僵菌(1×108/mL)后褐飞虱的存活率及致死中时(LT₅₀)。【结果】获得褐飞虱NlPOD1全长 cDNA 序列(GenBank登录号: MZ682107),其开放阅读框长2 049 bp,编码682个氨基酸,含有一个典型的动物亚铁血红素过氧化物酶结构域(An_peroxidasedomain),且N端包含一段由29个氨基酸残基组成的信号肽。系统发育分析表明,NlPOD1与半翅目其他昆虫的POD亲缘关系较近,其中与茶翅蝽Halyomorpha halys POD亲缘关系最近。发育表达谱结果表明,NlPOD1在褐飞虱不同发育阶段均有表达,其中卵期表达量最低,5龄若虫中表达量最高;组织表达谱结果表明,NlPOD1在褐飞虱5龄若虫不同组织中均有表达,且肠道和血淋巴中的表达量显著高于头部和脂肪体中的;微生物诱导表达模式表明,与注射PBS的对照组相比,大肠杆菌诱导48 h内各时间点NlPOD1在褐飞虱5龄若虫中的表达量显著上调,而在金黄色葡萄球菌和金龟子绿僵菌诱导下,NlPOD1表达量均呈现先上调后回稳的态势。RNAi分析结果显示,显微注射dsNlPOD1可显著抑制NlPOD1的表达水平。通过RNAi抑制NlPOD1表达后褐飞虱5龄若虫存活率不变,但注射dsNlPOD1联合金龟子绿僵菌感染对褐飞虱5龄若虫的LT₅₀(4.5 d)显著低于注射dsGFP联合金龟子绿僵菌感染的对照组的LT₅₀(5.4 d),说明沉默NlPOD1后褐飞虱对金龟子绿僵菌侵染的抵御能力显著降低。【结论】NlPOD1在褐飞虱病原防御过程中发挥重要作用,可作为开发RNAi和病原真菌联合介导的褐飞虱防治技术中的一个潜在靶标。     

实时荧光定量PCR检测感病蝗虫体内绿僵菌rDNA基因

根据绿僵菌23SrDNA的转录间隔区(ITS)和5.8S的基因序列,设计检测感病蝗虫体内绿僵菌菌体DNA分子的特异引物,通过优化感病蝗虫菌体DNA快速制备方法,建立了基于荧光定量PCR的定量检测体系。该检测方法专一、灵敏,检测下限为10pg/μl绿僵菌,并且能从刚侵染48h的东亚飞蝗血淋巴中,早期定量检测到在虫体血淋巴中增殖的绿僵菌的rDNA。     

从青杨天牛分离的几种致病真菌

从青杨天牛虫尸上分离出枝顶孢霉、球孢白僵菌、金龟子绿僵菌、轮状镰刀菌和尖孢镰刀菌5种病原真菌,其中枝顶孢霉为国内新记录。利用青杨天牛蛹对五种致病真菌进行了生物测定,5种病原菌的致病力顺序为:金龟子绿僵菌>球孢白僵菌>枝顶孢霉>尖孢镰刀菌>轮状镰刀菌。青杨天牛不同虫期对金龟子绿僵菌的易感性顺序为:1龄幼虫>蛹>老熟幼虫。利用金龟子绿僵菌和枝顶孢霉菌液田间防治青杨天牛幼虫,死亡率分别为95.56％及48.72％。     

黄氏枝膜叶蜂生物学特性及金龟子绿僵菌对其幼虫的致病力

【目的】黄氏枝膜叶蜂Cladiucha huangbki是新发现的叶蜂科(Tenthredinidae)枝膜叶蜂属Cladiucha种类,以幼虫取食危害乳源木莲Manglietia yuyuanensis叶片,虫口密度大, 严重影响乳源木莲的生长。本研究旨在明确该虫的生物学特性,并筛选出对该虫具有高致病力的金龟子绿僵菌Metarhizium anisopliae菌株,为高效治理该虫提供理论基础和生物防治资源。【方法】以林间调查和室内饲养观察相结合的方式,观察黄氏枝膜叶蜂的各发育阶段形态特征和生物学特性(寄主植物、分布和生活史);采用孢子悬浮液喷雾法筛选高致病力金龟子绿僵菌菌株。【结果】林间和室内观察结果表明,黄氏枝膜叶蜂幼虫取食木兰科木莲属植物,在福建永安市一年发生1代,以老熟幼虫(预蛹)在土室中越夏、越冬;翌年3月上旬化蛹,3月下旬成虫羽化,4月上旬为雌虫产卵高峰期,4月中旬幼虫出现,4月下旬至5月上旬为幼虫危害高峰期,5月中旬老熟幼虫开始入土做土室,入土深度30~150 mm。室内金龟子绿僵菌不同菌株对黄氏枝膜叶蜂幼虫致病力测定结果表明,以1×10⁷孢子/mL悬浮液喷雾11 d后,不同菌株对其幼虫的校正死亡率均超过85%,其中MaQZ-01, MaYTTR-04-05, Ma3和MaFZ-13 4个菌株感染的叶蜂幼虫校正死亡率为100%。菌株MaFZ-08感染引起的黄氏枝膜叶蜂僵虫率最高,为60%;其次是MaYTTR-04-05,引起的僵虫率为50%。金龟子绿僵菌各菌株对黄氏枝膜叶蜂幼虫的致死中时间(LT₅₀)相差不大,在6 d左右,其中MaYTTR-04-05菌株的LT₅₀最短,为5.090 d。【结论】黄氏枝膜叶蜂在福建永安一年发生1代,5月中、下旬老熟幼虫入土做土室化为预蛹越夏越冬。本研究筛选出对黄氏枝膜叶蜂幼虫致病力强的金龟子绿僵菌MaYTTR-04-05菌株,在其防治上有良好的应用前景。     

家蝇幼虫血淋巴蛋白组及血淋巴中热稳定蛋白研究

目的 初步研究家蝇幼虫血淋巴蛋白组学特征及其中的热稳定蛋白.方法 使用破壁法提取三龄幼虫血淋巴原液,设置全血淋巴组、沸水浴处理组,采用不同pH范围双向电泳胶条,分析血淋巴中蛋白的pH、分子量分布特点,采用二级质谱对蛋白点进行分子鉴定.结果 家蝇幼虫血淋巴中蛋白的分子量主要分布在10 kDa～66kDa之间,采用pH3～10的IPG胶,检测到286个蛋白点,采用pH4～7的IPG胶,测到601个蛋白点;血淋巴经沸水浴处理后,检测到41个蛋白点,分子量低于30 kDa,比未处理组减少了93.1％,对其中11个蛋白点进行质谱分析,鉴定出4个功能蛋白,包括存储蛋白、气味结合蛋白、铁蛋白重链样蛋白和铁蛋白2轻链蛋白同系物.结论 双向电泳能很好地分析家蝇幼虫血淋巴中的蛋白,血淋巴中的蛋白等电点主要分布在pH4～7之间;血淋巴经沸水浴处理后可去掉大量变性蛋白,其上清中可能包含耐热性较好的功能蛋白.     

家蚕幼虫血细胞密度变化成因及血细胞密度与耐高温性的关系

[目的]血细胞是昆虫血淋巴免疫的主导者.调查家蚕Bombyx mori幼虫血细胞密度变化和成因、血细胞密度与家蚕抗性的关系,是研究家蚕血细胞相关的免疫调控和抗性育种的重要组成.[方法]用细胞计数板统计家蚕品种大造不同龄期(4龄第1-4天、5龄第1-8天和上蔟期)幼虫10 μL血淋巴中的血细胞数目并计算血细胞密度,利用I...     

烟青虫核型多角体病毒的复制和染病后血淋巴蛋白的变化

用烟青虫(Hiliothis assulta)核型多角体病毒感染5龄初烟青虫幼虫,以染病后24、48·,72、96/120小时分别测定了虫体血淋巴蛋白浓度的变化。幼虫染病后24小时血淋巴蛋白浓度要高于同期对照组,72小时后染病幼虫血淋巴蛋白浓度较对照急剧下降。在染病及对照血淋巴样品中电泳分析(PAGE)三种蛋白(普通蛋白、糖蛋白和脂蛋白)均可分别染出22条、3条和3条带。分析结果表明,在虫体正常生长代谢过程中发生变化的主要蛋白可能大多为糖脂复合蛋白,但病毒的侵染能抑制这些变化的产生。电镜观察染病毒后幼虫的中肠组织和气管上皮组织,发现中肠染病轻微,不形成多角体。气管上皮细胞感染情况表明其属于对NPV感染较为敏感的组织之一,并在虫体染病后的病毒二次感染上可能起着重要作用。     

Inhibition of fungal growth in thermoregulating locusts,Locusta migratoria,infected by the fungus Metarhizium anisopliae var acridum

The locust, Locusta migratoria, has the capacity to develop a behavioural fever which reduces fungal infection by Metarhizium anisopliae var acridum. We investigated hemocyte and blastospore kinetics in infected insects under conditions that did or did not allow thermoregulation. Hemocyte concentrations were severely reduced in inoculated insects that did not thermoregulate but remained similar to those of controls in inoculated insects that were allowed to thermoregulate. Reductions in hemocyte counts were accompanied by an increase in the concentration of blastospores. In non-thermoregulating insects, circulating blastospores were first observed two days post-inoculation and had heavily colonized the hemolymph by day 5; in contrast, no blastospores were recovered from hemolymph of inoculated-thermoregulating insects. We used fluorescein isothiocyanate (FITC)-labelled silica beads to examine in vivo phagocytosis in thermoregulating and non-thermoregulating locusts. In the absence of fungus, a greater proportion of beads were engulfed by hemocytes in thermoregulating than in non-thermoregulating locusts early (4 and 24h) after bead injection, but the proportions were similar thereafter. In infected locusts, phagocytosis in non-thermoregulating insects was progressively impaired; such impairment, however, was not observed in challenged, thermoregulating insects. Our results suggest that thermoregulation helped keep fungal growth in check, apparently through the maintenance of hemocyte population levels and the direct inhibition of blastospore propagation by elevated temperatures.     

Effects of infection by larvae of Angiostrongylus cantonensis (Nematoda, Metastrongylidae) on the metabolism of the experimental intermediate host Biomphalaria glabrata

The effect of infection by Angiostrongylus cantonensis on the activity of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the concentration of total proteins, uric acid and urea in the hemolymph of Biomphalaria glabrata were investigated. The snails were dissected after 1, 2 and 3 weeks of infection to collect the hemolymph. The infection by A. cantonensis induced severe changes in the host snail's metabolism, triggering physiological mechanisms to minimize the deleterious effects caused by the larvae. There was a significant decrease in the concentration of total proteins in the infected snails, which occurred gradually as the infection advanced. This change was accompanied by an increase in the concentrations of urea and a decrease in the levels of uric acid in the hemolymph, suggesting that in this model the infection induces proteolysis and inversion of the excretion pattern of the infected snails. Besides this, variations in the activities of the aminotransferases were observed, with significantly higher levels in the infected groups than in the control group. These results indicate an increase in the protein metabolism of the infected snails, since there was an increase in nitrogen catabolites such as urea.     

The entomopathogenic fungus Nomuraea rileyi impairs cellular immunity of its host Helicoverpa armigera

In this study, we investigated the effect of the entomopathogenic fungus Nomuraea rileyi on Helicoverpa armigera cellular immune responses. Nomuraea rileyi infection had no effect on total hemocyte count (THC), but impaired hemocyte‐mediated phagocytosis, nodulation, and encapsulation responses. Nomuraea rileyi infection led to a significant reduction in hemocyte spreading. An in vitro assay revealed that plasma from N. rileyi infected H. armigera larvae suppressed the spreading ability of hemocytes from naïve larvae. We infer that N. rileyi suppresses the cellular immune response of its host, possibly by secreting exogenous, cytotoxic compounds into the host's hemolymph.     

CHANGES IN THE HEMOLYMPH PROTEIN PROFILES IN Galleria mellonella INFECTED WITH Bacillus thuringiensis INVOLVE APOLIPOPHORIN III. THE EFFECT OF HEAT SHOCK

This report concerns the effect of heat shock on host–pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis. We show enhanced activity against Gram‐positive bacteria in the hemolymph of larvae pre‐exposed to heat shock before infection with B. thuringiensis. Heat shock influenced the protein pattern in the hemolymph of infected larvae: more peptides with a molecular weight below 10 kDa were detected in comparison with nonshocked animals. Additionally, we noticed that the amount of apolipophorin III (apoLp‐III) in the hemolymph decreased transiently following infection, which was considerably higher in larvae pre‐exposed to heat shock. On the other hand, its expression in the fat body showed a consequent infection‐induced decline, observed equally in shocked and nonshocked animals. This suggests that the amount of apoLp‐III in the hemolymph of G. mellonella larvae is regulated at multiple levels. We also report that this protein is more resistant to degradation in the hemolymph of larvae pre‐exposed to heat shock in comparison to nonshocked larvae. Two‐dimensional analysis revealed the presence of three isoforms of apoLp‐III, all susceptible to proteolytic degradation. However, one of them was the most abundant, both in the protease‐treated and untreated hemolymph. Taking into consideration that, in general, apoLp‐III has a stimulative effect on different immune‐related hemolymph proteins and peptides, the reported findings bring us closer to understanding the effect of heat shock on the resistance of G. mellonella to infection.     

Pathogenicity of a new China variety of Metarhizium anisopliae (M. Anisopliae var. Dcjhyium) to subterranean termite Odontotermes formosanus

The pathogenicity of a new China variety of Metarhizium anisopliae (M. anisopliae var. dcjhyium) against the subterranean termite Odontotermes formosanus and the effect of the fungal fermented solution on hemolymph intracellular calcium were studied in laboratory. Conidia from the M. anisopliae var. dcjhyium were highly virulent for O. formosanus causing approximately 100% mortality 3 days post-inoculation in the concentration of 3x10(8) conidia/ml. The conidial clumps with conidial chains distributed on the cadavers of termite. When the termite was treated with 3x10(5) conidia/ml for 2 days, two constitutive proteins (91 and 105kDa) disappeared and a new specific protein (40kDa) appeared in the hemolymph of survivors relative to the controls. Hemolymph cells treated by the fungal fermented solution had a significantly higher level of intracellular calcium than controls 30min after treatment (x1.7). When the termite O. formosanus was infected by the entomopathogenic fungus M. anisopliae var. dcjhyium, hyphae invaded the integument and body cavity of the termite; well-developed muscles and fat tissue in the thorax of termite were decomposed and absorbed by hyphae, and formed the net structure; Hyphae seriously destroyed hemolymph, various tissues, pipelines and produced large number of conidia in the body of termite.     

Inhibition of hemocyte microaggregation reactions in Rhodnius prolixus larvae orally infected with Trypanosoma rangeli

Hemocoelic inoculation of epimastigotes of Trypanosoma rangeli strain H14 into 5th-instar larvae of Rhodnius prolixus previously fed on blood containing the same parasites, showed reduced number of hemocyte microaggregates in the hemolymph, enhanced number of flagellates in the hemolymph as well as increased mortality of these insects. All these effects were counteracted by combined inoculation of R. prolixus with T. rangeli and arachidonic acid. In vitro assays using hemolymph taken from insects previously fed on blood containing parasites showed that hemocyte microaggregation reactions were also attenuated when T. rangeli is used as inducer of the reaction, and that simultaneous applying T. rangeli with arachidonic counteracted the hemocyte microaggregation inhibition. We suggest that arachidonic acid pathway can be a mediator of hemocyte microaggregation reactions in the hemolymph of insects inoculated with T. rangeli, and that oral infection with this protozoan inhibits the release of arachidonic acid.     

二化螟幼虫被二化螟绒茧蜂寄生后血淋巴的生理生化变化

二化螟Chila suppressalis幼虫被二化螟绒茧蜂Apanteles chilonis产卵寄生后,血细胞含量增加,在被产卵寄生后的第6—10天,血细胞数最比对照幼虫增加了1.53—2.79倍,但血细胞形成被囊的能力很弱,对于蜂卵和蜂幼虫形成被囊的百分率分别为0.34％和0.62％。绒茧蜂寄生后,对二化螟幼虫生理生化有一定的影响,在寄生前期和后期,寄主幼虫血淋巴蛋白质浓度显著下降,在寄生后第2天,血淋巴游离氨基酸总浓度明显上升,以后随蜂幼虫发育而逐渐下降。在16种氨基酸中,对苏、丝、赖、精氨酸的影响最大。此外,本文对二化螟绒茧蜂的营养生理和寄生后调节寄主的能力进行了探讨。     

Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella.

Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.