不同方法处理的家蝇蛹对蝇蛹金小蜂繁殖的影响

通过适当方法处理寄主并长期保存,是大量繁殖寄生蜂的重要途径.蝇蛹金小蜂是多种有害蝇类的蛹期重要寄生蜂,在生物防治上具有重要价值.本文探讨了蝇蛹金小蜂对-20℃冷冻、6℃冷藏和二氧化碳窒息处理1、3和30 d的家蝇蛹以及热处死和热处死后冷藏保存30 d家蝇蛹的寄生能力.结果表明:蝇蛹金小蜂可以利用上述蝇蛹,且其后代在胫节长度上均与源自新鲜蛹的寄生蜂胫节长度无显著差异;但除冷冻方法外,寄生蜂后代产量均随寄主保存时间的延长而降低.在保存30 d的前提下,冷冻方法保存的寄主上寄生蜂后代最多.表明在大量繁殖蝇蛹金小蜂时,可以利用冷冻等方法对寄主进行处理并保存.     

家蝇蛹和果蝇蛹繁育的蝇蛹金小蜂对果蝇蛹的寄生比较

【目的】蝇蛹金小蜂Pachycrepoideus vindemmiae(Rondani)是杨梅园等果园果蝇类害虫蛹期常见寄生蜂种类,在对果蝇类害虫的生物防治上具有重要价值。本文旨在探讨使用家蝇蝇蛹为替代寄主繁育蝇蛹金小蜂的方法。【方法】探讨分别以家蝇蛹和果蝇蛹繁育的蝇蛹金小蜂对家蝇和果蝇蝇蛹的选择性,并比较了在两种寄主上繁育的蝇蛹金小蜂在大小、寿命、产卵期、后代产量和性比等方面的差异。【结果】结果表明与果蝇蛹相比,家蝇蛹明显较大,在家蝇蛹上发育的蝇蛹金小蜂后代个体也明显较大;家蝇蛹和果蝇蛹发育的寄生蜂雌蜂寿命为(13.4±4.11)和(3.94±2.49)d、产卵期分别为(11.4±4.11)和(3.13±2.42)d、单头雌蜂后代雌蜂数量分别为(34.31±31.83)和(7.88±3.58)头,在家蝇蛹上繁育的寄生蜂明显具有较长的寿命和产卵期、更多的雌雄蜂后代数量;在对家蝇蛹和果蝇蛹的选择上,繁育自家蝇和果蝇的蝇蛹金小蜂雌蜂选择频率的差异不大。【结论】利用家蝇蛹繁殖的蝇蛹金小蜂在寄生果蝇蛹时具有更大优势,在繁殖蝇蛹金小蜂控制杨梅园等果蝇的为害时,可以选择家蝇蛹作为替代寄主。     

家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase, PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。     

管氏肿腿蜂毒液丝氨酸蛋白酶同源物SgSPH对寄主血淋巴酚氧化酶活性的影响

[目的]本研究旨在通过克隆表达管氏肿腿蜂Scleroderma guani毒液丝氨酸蛋白酶同源物(serine protease homologue,SPH)基因SgSPH,探索其编码的毒液蛋白对寄主血淋巴酚氧化酶活性的影响.[方法]利用RT-PCR技术克隆管氏肿腿蜂毒液SgSPH基因的开放阅读框(ORF),采用生物信...     

蝇蛹金小蜂对桔小实蝇蛹的寄生功能反应

在温度25±1℃、相对湿度70±5%、光照周期（L∶D）14 h∶10 h条件下,研究了蝇蛹金小蜂对桔小实蝇蛹的寄生功能反应及干扰效应。结果表明,寄主密度、寄主日龄均影响寄生蜂的寄生效能。蝇蛹金小蜂对桔小实蝇2日龄（N=2）、4日龄（N=4）蛹的寄生功能反应均符合HollingⅡ模型,其方程分别为Na= 0.4494N2/ (1+ 0.0294N2)、Na= 0.5586N4/ (1+ 0.0253N4)。24h内单头雌成蜂最多可寄生2日龄、4日龄蝇蛹数量分别为15.29、22.08头。自身密度对蝇蛹金小蜂寄生产生一定的干扰效应,其干扰效应符合Hassell－Varley模型（a= 0.0719P－0.2526）,表明蝇蛹金小蜂雌蜂的发现域随着自身密度的增加而逐渐变小,雌蜂个体间干扰效应降低了寄生效能。     

瓜实蝇2种蛹寄生蜂生防潜能比较

[目的] 比较研究蝇蛹俑小蜂与蝇蛹金小蜂对瓜实蝇的控制潜能。[方法] 采用非选择性实验测定蝇蛹俑小蜂与蝇蛹金小蜂对瓜实蝇蛹的寄生效能、繁殖能力,并研究覆土厚度对2种寄生蜂寄生效能的影响。[结果] 2种寄生蜂对瓜实蝇的寄生率无显著性差异,但蝇蛹金小蜂的平均单雌产后代数比蝇蛹俑小蜂的多,分别为30和23头。土壤厚度显著影响2种寄生蜂的寄生效能,随着土壤厚度的增加,2种寄生蜂对瓜实蝇蛹的寄生率均显著下降,但蝇蛹金小蜂的寄生率下降更为迅速;蝇蛹俑小蜂最深可寄生8 cm土壤下的瓜实蝇蛹,而蝇蛹金小蜂在土壤厚度达到3 cm时就不能完成寄生。[结论] 蝇蛹俑小蜂较蝇蛹金小蜂更适合应用于瓜实蝇蛹的生物防治。     

腰带长体茧蜂寄生对亚洲玉米螟幼虫体内酚氧化酶活性的影响

通过对被腰带长体茧蜂Macrocentrus cingulum Brischke寄生的5龄亚洲玉米螟Ostrinia furnacalis Guenée幼虫体内不同组织中酚氧化酶活性的测定,采用体外注射腰带长体茧蜂雌性成蜂的萼液成分、毒液成分、萼液与毒液混合物的方法,研究了寄生蜂各种主要生理因子对寄主血清中酚氧化酶活性的影响。结果表明： 寄生蜂寄生可明显抑制寄主体内的酚氧化酶活性,减少黑色素产生;被寄生组FITC标记的血细胞阳性百分率低于未被寄生组,差异极显著( P<0.01）;萼液成分可明显地抑制亚洲玉米螟幼虫血清中酚氧化酶的活性 （P<0.01）;萼液与毒液混合物对酚氧化酶活性也有明显抑制作用（P<0.01）。研究认为寄生蜂产卵时注入的萼液、毒液可对寄主昆虫酚氧化酶活性产生明显的抑制作用,其中萼液是抑制寄主免疫能力的主要因素。     

蝇蛹金小蜂对家蝇蛹的寄生策略

蝇蛹金小蜂Pachycrepoideus vindemmiae Rondani是家蝇Musca domestica蛹期常见寄生蜂种类。本文探讨蝇蛹金小蜂对寄主日龄的选择策略以及该寄生蜂的寿命、产卵历期和后代数量等规律。结果表明寄生蜂可利用各日龄的蝇蛹,寄生高龄期蝇蛹时,寄生蜂后代产量显著降低,既未出蜂也未出蝇的死亡蝇蛹比例显著增加;寄生蜂寿命为(11.89±6.99)d,产卵历期为(9.58±6.67)d,单个雌蜂后代产量为(33.74±18.08)头,雄性后代的发育历期显著短于雌性后代,随着寄生蜂产卵历期的延长,寄生蜂后代产量下降,雄性后代比例增加。     

两种金小蜂毒液对菜粉蝶蛹血细胞延展、存活及包囊作用的影响

活体微注射测定结果表明,将0.5毒囊当量（venom reservoir equivalent, VRE）的蝶蛹金小蜂毒液注射于其寄主菜粉蝶蛹体内,注射后4～24 h寄主浆血细胞和颗粒血细胞的延展、存活和对Sephadex A50微珠的包囊能力显著下降;以0.002～0.02 VRE/μL的该蜂毒液处理其离体寄主血细胞均能产生同样的生理效应。该毒液抑制90%菜粉蝶蛹浆血细胞和颗粒血细胞延展的浓度各为0.00076 VRE/μL和0.00804 VRE/μL。可见,蝶蛹金小蜂毒液能显著抑制其寄主血细胞的延展和包囊作用,并导致血细胞的死亡。然而,同样条件下丽蝇蛹集金小蜂毒液对其非自然寄主菜粉蝶蛹的血细胞延展、存活和包囊作用则无任何效应。可见,寄生蜂毒液的生理作用具有明显的寄主特异性。     

一种水稻蛋白酶抑制剂基因的克隆及其结构分析

参照水稻蛋白酶抑制剂部分氨基酸序列 ,利用水稻偏爱密码子设计引物 ,经 PCR扩增 ,从我国水稻 (Oryza sativa)品种“中花 8号”中克隆到一个长 40 8bp的基因。序列测定和分析表明 ,克隆到的是一个未见报道的新的水稻蛋白酶抑制剂基因 ,该基因编码了一个由 1 33个氨基酸组成 ,具有重复双功能结构域和以抑制胰蛋白酶为主的活性中心的包曼 -伯克 (Bowman- Birk)型蛋白酶抑制剂 ,该基因推导的氨基酸序列与大麦、小麦、豆类等的某些蛋白酶抑制剂的氨基酸序列具有较高的同源性 ,与该家族的水稻的一种胰蛋白酶抑制剂氨基酸全序列同源性高达 75%。     

Negative regulation of prophenoloxidase (proPO) activation by a clip-domain serine proteinase homolog (SPH) from endoparasitoid venom

Most parasitic wasps inject maternal factors into the host hemocoel to suppress the host immune system and ensure successful development of their progeny. Melanization is one of the insect defence mechanisms against intruding pathogens or parasites. We previously isolated from the venom of Cotesia rubecula a 50 kDa protein that blocked melanization in the hemolymph of its host, Pieris rapae [Insect Biochem. Mol. Biol. 33 (2003) 1017]. This protein, designated Vn50, is a serine proteinase homolog (SPH) containing an amino-terminal clip domain. In this work, we demonstrated that recombinant Vn50 bound P. rapae hemolymph components that were recognized by antisera to Tenebrio molitor prophenoloxidase (proPO) and Manduca sexta proPO-activating proteinase (PAP). Vn50 is stable in the host hemolymph-it remained intact for at least 72 h after parasitization. Using M. sexta as a model system, we found that Vn50 efficiently down-regulated proPO activation mediated by M. sexta PAP-1, SPH-1, and SPH-2. Vn50 did not inhibit active phenoloxidase (PO) or PAP-1, but it significantly reduced the proteolysis of proPO. If recombinant Vn50 binds P. rapae proPO and PAP (as suggested by the antibody reactions), it is likely that the molecular interactions among M. sexta proPO, PAP-1, and SPHs were impaired by this venom protein. A similar strategy might be employed by C. rubecula to negatively impact the proPO activation reaction in its natural host.     

The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. The molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel. In the presence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions. The PPO was shown to be a glycoprotein and its isoelectric point was 6.2. The amino-acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sexta. The activation of the PPO with chymotrypsin was investigated and two proteins of 67 and 50 kDa were found to be products of the proteolytic cleavage. The N-terminus of the G. mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited significant homology with highly conserved sequences found in arthopod hemocyanins and insect storage proteins, which indicates that the PPO belongs to this family.     

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.     

Ecdysteroid levels/profiles of the parasitoid wasp,Diapetimorpha introita,reared on its host,Spodoptera frugiperda and on an artificial diet

Diapetimorpha introita is an ichneumonid ectoparasitoid of the fall armyworm, Spodoptera frugiperda. Since it has been reported that D. introita wasps reared on an artificial diet exhibit a significantly lower percentage of adult eclosion and fecundity than host-reared wasps, this study was undertaken to elucidate the factors responsible for the reduced viability observed in diet-reared wasps. A system of markers has been devised to track the development (from the initiation of cocooning through adult eclosion) of D. introita. Although wasps reared on artificial diet developed more slowly than did those reared on host pupae, both diet- and host-reared wasps passed through the same stages of development - the eyes enlarged and moved backward, the gut was purged and upon ecdysis the exarate pupa emerged. The thorax was the first to darken, followed by the head and then the abdomen. Pharate pupal formation occurred before gut purge. Two peaks of hemolymph ecdysteroids were observed, one in wasps in which gut purge was almost complete and the second in day-2 exarate pupae. Ecdysone and 20-hydroxyecdysone were the major ecdysteroids present in hemolymph sampled at these times. Small quantities of 20,26-dihydroxyecdysone, polar ecdysteroids and/or possibly 26-hydroxyecdysone were also present. In six stages of development, hemolymph ecdysteroid titers were significantly higher in host-reared than in diet-reared wasps (Eye 1, Eye 2, Gut Purge 2, Pharate Pupa, Head/Thorax Dark, and Abdomen Dark). Relatively high percentages of mortality were observed in diet-reared wasps in four of these stages and in two others which occurred in close proximity to one of the stages, the Abdomen Dark stage. Thus, insufficient ecdysteroid in the hemolymph may be responsible, in part, for the relatively high percentage of mortality that occurred in wasps reared on an artificial diet.     

Chymotrypsin inhibitors in mosquitoes: Activity profile during development and after blood feeding

Chymotrypsin and trypsin inhibitors persist throughout all developmental instars of Aedes aegypti. After a blood meal, inhibitor activity against chymotrypsin was more than double that of sugar-fed females, but only weak activity was detected in midguts where proteinase inhibitors has been thought to regulate proteinases during blood digestion. A fourfold increase in the ratio of abdominal/thoracic inhibitor activity after the blood meal strongly suggested that fat body, or other abdominal tissues, represent the major source of inhibitor. Chymotrypsin inhibitor activity was deposited in maturing oocytes. Similar results were obtained with blood-fed Anopheles albimanus. Chymotrypsin inhibitor was active against different mosquito proteinases and against bovine α-chymotrypsin and trypsin, but not against subtilisin, pancreatic elastase, or fungal proteases; chymotrypsin inhibitors did not interfere with bacterial growth. The hypothesis on the regulation of blood digestion through the action of proteinase inhibitors during the gonotrophic cycle was abandoned and its involvement in the phenoloxidase cascade in the mosquito egg chorion is suggested instead. Arch. Insect Biochem. Physiol. 36:315–333, 1997. © 1997 Wiley-Liss, Inc.     

Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.     

一种新的螯虾丝氨酸蛋白酶抑制物基因的克隆表达及其活性分析

根据本课题组从克氏原螯虾中新发现的丝氨酸蛋白酶抑制物的基因序列(GenBank登录号CD644775)设计一对引物,应用逆转录-聚合酶链式反应(RT-PCR)技术,从螯虾血淋巴细胞中扩增出丝氨酸蛋白酶抑制物基因PCI188,将其连入原核表达载体pET-32a,转化至大肠杆菌Rosetta株和BL21株中进行蛋白表达,结果该蛋白只在前者表达。表达产物用免疫转印检测,出现50kD的特异性条带,与螯虾PCI188基因编码的蛋白大小相符。将融合蛋白纯化后免疫新西兰兔,用免疫血清与螯虾血淋巴作用后测定酚氧化酶活力,结果显示,酚氧化酶活力有所升高,从而首次证实螯虾PCI188编码的蛋白对丝氨酸蛋白酶有抑制作用。