Improved insecticidal activity of a genetically modified baculovirus expressing the immunosuppressive CrV1 protein from a polydnavirus against Spodoptera exigua

Recombinant baculoviruses could be used as biological insecticides through the introduction and expression of exogenous genes (such as those coding for proteins) that interfere with metabolism, metamorphosis (toxins, hormones, and enzymes), and immune system of the insects. The CrV1 secreted protein of Cotesia rubecula polydnavirus (PDV) is responsible for the actin depolymerisation in haemocytes and the abolishment of immune functions such as phagocytosis and cell spreading, thus allowing the successful embryonic development of the parasitoid wasp. CrV1 cDNA was cloned into C6 strain of Autographa californica multiple nucleopolyhedrovirus (AcMNPV-C6-CrV1) under p10 promoter to construct a recombinant virus. The recombinant virus was then tested against the insect pest Spodoptera exigua. The recombinant virus expressing CrV1 protein showed significantly lower LC₅₀ and shorter LT₅₀ as compared with the AcMNPV-C6 wild-type virus. The potential of recombinant baculoviruses expressing PDV genes in relation to their virulence is discussed.     

海木提取物对菜青虫幼虫的生物活性

测试了海木提取物对菜青虫幼虫的拒食活性,并对拒食活性高的部分进行了乙酰胆碱酯酶活性、蛋白水解酶活性等测定。海木的氯仿部分表现出明显的拒食活性(拒食率97.7%),同时还能抑制菜青虫的乙酰胆碱酯酶活性,提高蛋白水解酶活性,降低菜青虫体内蛋白质含量。     

银杏外种皮提取液对菜粉蝶幼虫的毒力研究

用银杏外种皮水煮提取液进行室内菜粉蝶毒力实验和田间药效测定试验。结果表明,相当于0.75g/ml的银杏外种皮提取液对菜粉蝶3龄幼虫室内浸虫试验,幼虫死亡率为76.6%,浸叶饲喂健虫的幼虫死亡率为63.3%。0.15g/ml银杏外种皮提取液对菜粉蝶3龄幼虫室内浸虫试验,幼虫死亡率为53.3%,浸叶饲喂健虫死亡率为43.3%。0.75g/ml的银杏外种皮提取液对菜粉蝶5龄幼虫室内浸虫试验,幼虫死亡率仅为33.3%。0.75g/ml银杏外种皮提取液对菜粉蝶3龄幼虫田间药效测定,其幼虫死亡率为64%,校正防治效果为63.6%。     

红楝提取物对菜青虫幼虫的生物活性

对红楝的乙酸乙酯抽提部分进行了粗分并对各部分进行了拒食活性测试,并对活性较高的部分进行了乙酰胆碱酯酶活性、蛋白酶活性、蛋白质含量等测定。编号为6的部分拒食率最高为60．9％。在对其进行进一步研究中发现红楝对幕青虫的神经系统影响较明显,而胃毒毒性却不十分理想。     

竹提取物对棉铃虫幼虫及菜青虫的拒食活性

研究结果表明 ,质量浓度为 1 0g L的 1 0种供试竹提取物对棉Helicoverpaarmigera铃虫均具有较强的拒食作用 ,1 2h拒食率最高达 95 . 0 3 % ,最低为 74. 0 7% ,其中毛金竹 (Phyllostachysnigravar.henonis)、白纹短穗竹 (Brachystachyumalbostriatum)提取物对棉铃虫 3龄幼虫的AFC50 (拒食中浓度 )分别为 2 . 0g L、2. 7g L ,但 2 4h、48h拒食率均明显下降。对菜青虫 3龄pierisrapae幼虫的拒食效果以凤凰竹 (Bambusamultiplex)提取物最好 ,2 4h拒食率为 71. 69% ;对 4龄菜青虫的拒食效果以毛金竹提取物最好 ,拒食率为71 . 45 % ,其中毛金竹、白纹短穗竹 2种竹提取物对菜青虫 3龄幼虫的AFC50 分别为 2 .68g L和 3 . 3 7g L。研究结果对于开发环境友好农药以及充分利用竹类资源均具有重要意义。     

假臭草对菜青虫杀虫活性的研究

测定了假臭草根、茎、叶的甲醇提取液对菜青虫的生物活性。结果表明假臭草根、茎、叶的甲醇提取液对菜青虫无触杀活性;假臭草提取液对菜青虫的拒食活性强弱依次为叶茎根,在浓度为1 g/mL时拒食率分别为36.51%、63.34%和79.33%;假臭草茎和叶的甲醇提取液对菜青虫有一定的胃毒作用,在浓度为1 g/mL时,假臭草叶和茎甲醇提取液对菜青虫的胃毒活性分别为66.67%和33.33%。     

艾蒿提取物对菜青虫的生物活性

采用微波辅助提取、溶剂浸提 ,以正己烷、乙醇、正己烷 +乙醇 (体积比 1∶1 )为溶剂从艾蒿ArtemisiaargyiL啨ｖｌ.etVant茎叶得到了提取物。另外 ,还用水蒸气蒸馏法获得了精油。生物活性试验表明 ,它们对菜青虫PierisrapaeL .具有较强的拒食活性和一定的触杀活性。其中 ,正己烷为溶剂的微波辅助提取物的拒食活性和触杀活性最强。     

14种菊科植物提取物对菜青虫的杀虫活性

用胜红蓟、黄花蒿、青蒿、艾蒿、鬼针草、三叶鬼针草、鱼眼草、小鱼眼草、紫茎泽兰、千里光、希签草、万寿菊、肿柄菊、苍耳等１４种菊科植物的石油醚、氯仿、丙酮、乙醇、甲醇和水提取物对菜青虫５龄幼虫的生测结果表明,黄花蒿、青蒿、艾蒿、鱼眼草、小鱼眼草、紫茎泽兰、万寿菊等７种菊科植物甲醇和乙醇提取物具有较高的杀虫活性。此结果为进一步从菊科植物中提取,分离和筛选高活性杀虫活性成分奠定了基础。     

决明子胰蛋白酶抑制剂COTI对菜青虫生长抑制作用的研究

从决明子中分离得到胰蛋白酶抑制剂COTI,利用其缓冲液浸泡油菜叶片饲喂菜青虫(Pieris rapae L)幼虫后,幼虫体重明显下降,分析显示菜青虫中肠提取物中蛋白酶活性受到COTI的抑制,表明决明子胰蛋白酶抑制剂对菜青虫的生长具有明显的抑制作用。     

Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host,Pieris rapae

Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host (Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of approximately 14 and approximately 17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts.     

三种植物提取物对菜青虫拒食活性的研究

本文测定了胡椒?葱?川楝子3种植物的石油醚和丙酮提取物对菜青虫3龄幼虫的拒食作用,结果表明,3种供试植物提取物5~40 mg/mL浓度处理24 h对菜青虫幼虫均有一定拒食作用,拒食率达到31.8%~99.8%。其中胡椒的石油醚提取液拒食作用最大,浓度为40 mg/mL的处理效果最好,拒食率达99.8%。总体上看,3种供试植物的石油醚提取物拒食作用大于丙酮提取物,24 h的拒食效果的优于48 h。     

菜青虫不同虫态及虫龄的多酚氧化酶性质比较

初步比较了菜青虫Pieris rapae (L.)不同虫态及虫龄的多酚氧化酶(polyphenol oxidase)的性质。结果表明,不同虫态及虫龄的多酚氧化酶活力有很大的不同,其中以5龄幼虫酶活力最高,蛹酶活力最低。酶活力大小依次为： 5龄幼虫> 预蛹>4龄幼虫>3龄幼虫>蛹。以邻苯二酚为底物,研究了pH对多酚氧化酶活力的影响,结果表明酶催化底物氧化均有一个最适pH区域,不同虫态及虫龄的多酚氧化酶最适pH基本相同,其值为7.0±0.2 。不同虫态及虫龄的多酚氧化酶催化底物氧化的最适温度有很大的差异。3龄、4龄、5龄幼虫、预蛹和蛹的多酚氧化酶活力的最适温度分别为36.0±0.5℃、38.5±1.0℃、43.0±1.0℃、45.5±1.0℃和50.0±1.5℃,说明蛹期的多酚氧化酶活力的最适温度较高。进一步比较催化底物氧化反应的活化能,测得3龄、4龄、5龄幼虫、预蛹和蛹的多酚氧化酶活化能分别为：4 3.10±0.28、36.50±0.27、25.79±0.32、30.10±0.21和58.88±0.39 kJ/mol 。通过测定不同虫态及虫龄的多酚氧化酶催化邻苯二酚和L-多巴氧化反应的动力学特征参数,比较了不同虫期的酶对底物的亲和力。     

Identification of highly pathogenic Beauveria bassiana strain against Pieris rapae larvae

Seven different strains of Beauveria bassiana were used in a bioassay on Pieris rapae larvae. The results showed that an B. bassiana strain showed relatively high pathogenicity towards P. rapae larvae. The adjusted mortality rate was 92.86 %, and the infection rate was 85.71 % in 10 days post inoculation. Molecular identification was performed to identify the unknown strain. Internal Transcribed Spacer sequence analysis showed that the polymerase chain reaction amplicon length of the unknown strain of Beauveria sp. was 573 bp, and sequence similarity to the known B. bassiana sequences in the NCBI database was 99 %. The B. bassiana strain was named Bb01. The changes of proteins and PPO of P. rapae larvae infected by B. bassiana Bb01 strain at different times was determined. The activity of PPO increased in 1–6 d and decreased in 7 d again after inoculation. The B. bassiana invaded into the insect body affected the balance of the proteins and PPO.     

Enzymatic properties of phenoloxidase from Pieris rapae (Lepidoptera) larvae

The kinetic parameters of partially purified phenoloxidase (PO, EC. 1.14.18.1) from the 5th instar larvae of Pieris rapae (Lepidoptera) were determined, using L‐3, 4‐dihydroxyphenylalanine (L‐DOPA) as substrate. The optimal pH and temperature of the enzyme for the oxidation of L‐DOPA were determined to be at pH 7.0 and at 42°C, respectively. The enzyme was stable between pH 6.5 and 7.4 and at temperatures lower than 37°C. At pH 6.8 and 37°C, the Michaelis constant (K_m) and maximal velocity (V_m) of the enzyme for the oxidation of L‐DOPA were determined to be 0.80 μmol/L and 1.84 μmol/ L/min, respectively. Tetra‐hexylresorcinol and 4‐dodecylresorcinol effectively inhibited activity of phenoloxidase and this inhibition was reversible and competitive, with the IC₅₀ of 1.50 and 1.12 μmol/L, respectively. The inhibition constants were estimated to be 0.50 and 0.47 μmol/L, respectively.     

茶皂素对菜青虫的拒食作用方式及机制

采用注射、熏蒸、点滴、饲喂、内吸等方法测定了茶皂素对菜粉蝶Ｐｉｅｒｉｓ　ｒａｐａｅ　Ｌ．５龄幼虫的拒食活性。结果表明,茶皂素对５龄菜青虫具有很强的饲喂拒食活性,２４小时选择性拒食中浓度ＡＦＣ_(５０)为４０．８８ｍｇ／Ｌ,非选择性拒食中浓度为３２１．９２ｍｇ／Ｌ;并具有一定的内吸拒食作用,尤其是根部施药法内吸拒食活性较明显。茶皂素对菜青虫无显著的注射、熏蒸、点滴拒食作用。     

Cloning and expression profiles of tumor suppressor QM homologue in response to granulovirus in Pieris rapae

The tumor suppressor, QM, has been cloned and characterized from various model organisms such as human, plant and invertebrates. Yet, it has not been seriously investigated for its role in conjunction with antiviral mechanisms involving innate insect immunity. From the expressed sequence tag (ESTs) project, conducted with larval cDNA library of cabbage butterfly, Pieris rapae, a partial fragment (718 bp) of QM homologue, termed PrQM containing 660 bp long open reading frame (ORF) encoding protein of 219 amino acids was identified. In silico analysis of PrQM ORF revealed the presence of ribosomal protein L10a/L10e type domain. Phylogenetic analysis of the P. rapae QM‐like protein showed high amino acid sequence similarity with other PrQM polypeptides identified from Heliothis virescenes (95%), Plutella rapae (92%), Bombyx mori (92%), Drosophila melanogaster (89%), and Polyrhachis vicina (85%). The butterfly QM has the closest phylogenetic relationship to a moth (Hv) QM homologue. Further investigations revealed the expression of PrQM at all developmental stages, with pronounced presence at the egg stage. In addition, spatial pattern analysis indicated its high expression in the head, salivary gland, integument and fat body with visible presence in Malpighian tubule and gut. Time course expression studies conducted after immune‐challenge with lipoteichoic acid (LTA) showed the induction of PrQM mRNA at 12 h and 24 h after challenge and also in response to granulovirus (GV). Results of this investigation therefore suggest possible role of QM‐like proteins from Pieris rapae to be involved in innate antiviral immune responses. Further elucidation on the precise function of PrQM during antiviral immune responses by using RNA interference remains a viable research front.     

拟青霉菌代谢产物对菜粉蝶幼虫的生物活性

淡紫拟青霉(Paecilomyces lilacinus)甲醇萃取物柱层析活性馏分DM6和拟青霉(Paeecilomyces sp.)菌株X1甲醇萃取物柱层析活性馏分X1M5对菜粉蝶Pieris rapae(L.)4龄幼虫具有较强的选择性和非选择性拒食作用,处理后24h选择性AFC50分别为5.8652mg/mL和3.5239mg/mL,非选择性AFC50分别为9.0095mg/mL和14.0234mg/mL。饲喂法、注射法、点滴法测定结果表明,DM6和X1M5对菜粉蝶4龄幼虫具有较强的毒杀活性,其中注射法处理后72h,DM6和X1M5对菜粉蝶4龄幼虫的LD50分别为0.1846μg/头和0.6784μg/头。DM6和X1M5以5.0mg/mL饲喂法处理菜粉蝶3龄幼虫后,试虫生长发育受到强烈的抑制作用,发育抑制率分别为73.34%和63.30%。这些结果对于进一步研究拟青霉菌杀虫活性成分具有重要参考意义。