Diet factors responsible for the change of the glucose oxidase activity in labial salivary glands of Helicoverpa armigera

We investigated the change of the glucose oxidase (GOX) activity in labial salivary glands of Helicoverpa armigera larvae fed with the artificial diet or host plant tobacco and the major factors responsible for such a change. Throughout larval development, the labial salivary GOX activities in caterpillars reared on the artificial diet were remarkably higher than those fed with the plant. After fifth-instar plant-fed caterpillars were transferred to the artificial diet, their labial salivary GOX activity increased quickly, which was closely correlated with the time spent feeding on the artificial diet. The total sugar content of the artificial diet was 68 times higher than that of the tobacco leaves. We hypothesized that sugars and secondary metabolites are the possible causes of induction of GOX activity. When fifth-instar caterpillars were fed with tobacco leaves coated with glucose or sucrose, their labial salivary GOX activity was significantly higher than those fed with leaves without sugar coating. Following native PAGE, 1 single band of the labial salivary GOX was observed in all the caterpillars fed with different diets, implying that only the activity of the isoenzyme was changed in response to different diets. Furthermore, the labial salivary GOX activity was determined after caterpillars were fed with artificial diets containing chlorogenic acid, rutin, and quercetin. The results showed that all these phenolic compounds had no effect on the GOX activity. We conclude that sugar in diets was a major factor influencing the labial salivary GOX activity of the larvae. Arch. Insect Biochem. Physiol. 2008.     

Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants

The insect salivary enzyme glucose oxidase (GOX) can inhibit wound-inducible nicotine production in tobacco, Nicotiana tabacum. We examined whether salivary gland extracts of Helicoverpa zea lacking active GOX could still suppress nicotine in tobacco, Nicotiana tabacum, and whether GOX could suppress wound-inducible defenses of another Solanaceous plant, tomato Lycopersicon esculentum. Tobacco leaves were wounded with a cork borer and treated with water, salivary gland extracts with active GOX (SxG), or salivary gland extracts with inactive GOX (SxI). After three days, leaves treated with SxG had significantly less nicotine than all other wounded treatments. Neonates that fed on the terminal leaves of tobacco plants treated with SxG had significantly higher survival than neonates that fed on leaves treated with either SxI or water. This evidence supports the assertion that GOX is the salivary factor responsible for the suppression of tobacco plant nicotine production by H. zea saliva. Results for the NahG tobacco plants, which lack salicylic acid (SA) due to a transgene for bacterial SA hydroxylase, indicate that suppression of nicotine by GOX does not require SA. However, tobacco leaves that were wounded and treated with SxG had significantly higher levels of the SA-mediated PR-1a protein than leaves treated with SxI or water. Leaves of tomato plants wounded with scissors and then treated with SxG had trypsin inhibitor levels that were moderately lower than plants wounded and treated with purified GOX, water, or SxI. However, all the wounded tomato leaves irrespective of treatment resulted in lower caterpillar growth rates than the non-wounded tomato leaves. Glucose oxidase is the first insect salivary enzyme shown to suppress wound-inducible herbivore defenses of plants.     

苜蓿夜蛾葡萄糖氧化酶cDNA的克隆、表达及特性研究

【目的】从苜蓿夜蛾Heliothis viriplaca体内克隆葡萄糖氧化酶(Glucose oxidase,GOX)基因cDNA序列,并进行原核表达及活性测定。同时对GOX在不同组织的特异表达及对葡萄糖诱导反应的特性进行研究。【方法】以苜蓿夜蛾为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术,扩增苜蓿夜蛾GOX基因全长cDNA序列。利用原核表达载体p ET-21b在大肠杆菌中表达苜蓿夜蛾的GOX基因,并用Ni-NTA亲和层析柱将带His-tag的目的蛋白进行纯化,再利用梯度透析法进行复性,以葡萄糖为底物进行酶的活性测定。同时利用Real-time PCR分析GOX的组织特异性表达和对葡萄糖的诱导反应。【结果】该cDNA序列有2 154个碱基,开放阅读框1 824个碱基,编码607个氨基酸组成的多肽,分子量为67.04 ku,多肽的等电点为5.13。该序列命名为Hv GOX,在Gen Bank的登录号为KT907054。序列分析表明,Hv GOX的氨基酸序列与其他昆虫的GOX氨基酸序列高度同源。该基因在大肠杆菌表达系统中成功地进行了诱导表达,表达出与预测的蛋白分子量相符的融合蛋白。由原核表达载体表达后的蛋白经过变性、纯化和复性后有活性。Real-time PCR结果表明,该基因在苜蓿夜蛾的不同组织均有m RNA水平的特异表达,其中在唾腺中的表达量最高;同时用0.01%、0.1%、1%和10%葡萄糖溶液浸泡的大豆叶喂食的幼虫,幼虫体内的GOX的表达均被诱导,且在10%的浓度时诱导表达量最高。【结论】本研究在苜蓿夜蛾体内获得一个新的葡萄糖氧化酶基因,该结果为进一步研究葡萄糖氧化酶在昆虫体内的生物功能奠定基础。     

Influence of dietary nutritional composition on caterpillar salivary enzyme activity

Caterpillars are faced with nutritional challenges when feeding on plants. In addition to harmful secondary metabolites and protein- and water-limitations, tissues may be carbohydrate-rich which may attenuate optimal caterpillar performance. Therefore, caterpillars have multiple strategies to cope with surplus carbohydrates. In this study, we raise the possibility of a pre-ingestive mechanism to metabolically deal with excess dietary sugars. Many Noctuid caterpillars secrete the labial salivary enzyme glucose oxidase (GOX), which oxidizes glucose to hydrogen peroxide and gluconate, a nutritionally unavailable carbohydrate to the insect. Beet armyworm, Spodoptera exigua, larvae were restricted to diets varying in protein to digestible carbohydrate (P:C) ratio (42p:21c; 33p:30c; 21p:42c) and total nutrient concentration (42% and 63%). High mortality and longer developmental time were observed when caterpillars were reared on the C-biased, P-poor diet (21p:42c). As the carbohydrate content of the diet increased, caterpillars egested excess glucose and a diet-dependent difference in assimilated carbohydrates and pupal biomass was not observed, even though caterpillars restricted to the C-biased diet (21p:42c) accumulated greater pupal lipid reserves. Larval labial salivary GOX activity was also diet-dependent and gluconate, the product of GOX activity, was detected in the frass. Unexpectedly, GOX activity was strongly and positively correlated with dietary protein content.     

The host plant as a factor in the synthesis and secretion of salivary glucose oxidase in larval Helicoverpa zea

We investigated the effect of the host plant on the synthesis and secretion of the elicitor glucose oxidase in the salivary glands of larval Helicoverpa zea. Glucose oxidase catalyses the oxidation of d-glucose to produce d-gluconic acid and hydrogen peroxide. Previous studies have found that the product hydrogen peroxide is primarily responsible for suppressing the wound-inducible defenses of the host plant. Using an antibody specific for glucose oxidase, we determined the effect of the host plant on the rate of secretion of glucose oxidase. Larval H. zea secrete microgram amounts of the enzyme glucose oxidase from their principal salivary glands, the labial glands. Larvae reared on different host plants produce varying amounts of glucose oxidase in their labial glands. We used a tissue printing procedure with our antibody to determine if larvae secrete glucose oxidase directly at the feeding or wound sites. Significant amounts of the enzyme are deposited at the feeding site, although some is deposited outside the feeding margins.     

棉铃虫幼虫唾液腺cDNA文库的构建及EST分析

棉铃虫Helicoverpa armigera (Hübner)幼虫唾液中的各种酶类及各种生化组分在棉铃虫与植物相互作用及协同进化中起到重要作用; 唾液腺是棉铃虫唾液成分的合成器官。本研究通过构建棉铃虫幼虫唾液腺全长cDNA文库, 测序得到1 502条EST序列, 聚类分析后获得821个unigenes, 为筛选棉铃虫与寄主互作信号因子提供基因信息资源。使用Blast2 GO软件对821个unigenes进行了比对和功能注释, 初步获得棉铃虫幼虫唾液腺中mRNA的构成特征。结果显示, 在棉铃虫唾液腺ESTs文库中, 鉴定得到脂类相关消化酶基因17个, 糖类相关消化酶基因5个, 半胱氨酸蛋白酶基因1个, 丝氨酸蛋白酶基因20个（其中16个为新发现）, 提示唾液腺的主要功能是分泌消化酶进行预消化; 还发现在棉铃虫幼虫唾液腺中存在表皮蛋白、 气味结合蛋白和化学感受蛋白基因。结果为研究棉铃虫预消化系统打下基础。     

抑制葡萄糖氧化酶基因降低黏虫幼虫对苏云金芽孢杆菌侵染的抗性(英文)

【目的】本研究旨在探讨葡萄糖氧化酶(glucose oxidase, GOX)在黏虫Mythimna separata发育、消化和免疫防御中的作用。【方法】采用转录组测序技术克隆了黏虫GOX基因的cDNA序列。qRT-PCR检测该GOX基因在黏虫不同发育阶段(1-6龄幼虫、蛹和1日龄成虫)和4龄第1天幼虫不同组织(前肠、中肠、后肠、下唇腺、马氏管、脂肪体和表皮)、以及取食用不同浓度(0.01%, 0.1%, 1%和10%)葡萄糖溶液浸泡10 s的玉米叶片及饥饿和再取食条件下的4龄第1天幼虫中的特异性表达模式。通过RNAi和生物测定研究该GOX基因在黏虫对苏云金芽孢杆菌Bacillus thuringiensis抗性中的功能。【结果】克隆获得了一条新的长2 187 bp的黏虫GOX基因MsGOX cDNA序列(GenBank登录号: KY348779),其开放阅读框长1 821 bp,编码606个氨基酸,预测分子量为66.4 kD。发育表达谱结果表明MsGOX在黏虫不同发育阶段表达量不同,在4龄幼虫期表达量最高;组织表达谱结果表明MsGOX在黏虫4龄第1天幼虫各组织中均有表达,在下唇腺中表达量最高。饲喂幼虫不同浓度葡萄糖对MsGOX转录的诱导效果不同,在葡萄糖浓度为10%时,该基因的表达水平达到最高。随着饥饿时间的延长,4龄第1天幼虫中MsGOX的表达水平逐渐升高,并在饥饿24 h时达到高峰,饥饿后再用玉米叶片饲喂幼虫,MsGOX的表达水平逐渐回升。注射dsMsGOX 48 h后,MsGOX的表达水平较对照(注射dsEGFP)降低了88.3%; 与同一时间点的对照相比,注射dsMsGOX后48和72 h幼虫 的体重、体长和消化系数均显著降低,苏云金芽孢杆菌侵染48和72 h提高了幼虫的校正死亡率。【结论】MsGOX可能参与了黏虫中肠的消化和抗菌,研究结果为进一步研究黏虫MsGOX的功能并提出新的黏虫防治策略提供了依据。     

Evidence that caterpillar labial saliva suppresses infectivity of potential bacterial pathogens

Salivary enzyme, glucose oxidase (GOX) from the caterpillar Helicoverpa zea, catalyzes the conversion of glucose to gluconic acid and hydrogen peroxide. Because hydrogen peroxide has well-known antimicrobial properties, we examined whether caterpillar labial saliva could reduce the infectivity of bacterial pathogens. We examined the effects of caterpillar saliva on the growth of two bacteria species Serratia marcescens and Pseudomonas aeruginosa. Wells formed in LB agar contained a solution of salivary gland extract (Sx) and glucose, GOX and glucose, Sx only, GOX only, or glucose only. After 18 h of incubation, the diameter of cleared bacteria was measured. Wells treated with only GOX, Sx, or glucose showed no measurable area of clearing, while wells treated with GOX with glucose or Sx with glucose had considerable clearing. To determine if saliva could provide protection to caterpillars in vivo, a surgery was performed on caterpillars that prevented the secretion of labial saliva. Caterpillars were fed a diet containing either no added bacteria or treated with high levels of S. marcescens or P. aeruginosa. Caterpillars that could not secrete saliva had significantly higher levels of mortality when feeding on diet treated with either bacterium than caterpillars that could secrete saliva when feeding on equal levels of bacteria-treated diet. Our evidence demonstrates for the first time that insect saliva in situ can provide protection against bacterial pathogens and that the salivary enzyme GOX appears to provide the antimicrobial properties.     

Brachymeria lasus: effects of nutrient level on in vitro larval growth of a chalcid insect parasite

Exponential weight gain equations and growth rates were determined during larval development of Brachymeria lasus reared aseptically on chemically defined media with varying amino acid and glucose levels. The rate of weight gain increased from 0 to 2% dietary glucose and mean growth rate increased from 160 to 260 micrograms/mg x day. Similar results were observed for larvae reared on media containing 1 to 4% free amino acids. At higher nutrient levels, growth rate and rate of weight gain declined and maximal growth occurred at dietary osmolalities of 500-700 mOsm/kg. A further experiment utilizing nutritionally inert L-glucose suggested that this osmolality range does not reflect the optimal for growth but rather the point at which the detrimental effects of dietary osmotic pressure exceed the nutritive benefit of the dietary ingredients.     

Isolation and characterisation of a cytochrome b5 cDNA clone from Helicoverpa armigera (Hubner): possible involvement of cytochrome b5 in cytochrome P450 CYP6B7 activity towards pyrethroids

A cDNA clone specific for cytochrome b5 was isolated from Helicoverpa armigera. This sequence corresponded to a mRNA of an estimated 544 nucleotides in length excluding the poly A tail. The mRNA contained an open reading frame of 381 nucleotides encoding a protein of 127 amino acid residues with a molecular weight of 14,564 Daltons. The encoded protein sequence showed 51% protein sequence identity with cytochrome b5 from M. domestica and 36-37% identity with mammalian and avian cytochrome b5 sequences. Northern analysis of larval RNA using this cDNA as probe, revealed that cytochrome b5 mRNA expression is tissue specific with the mRNAs being expressed in abundance in the midguts of larvae, at a lower level in fatbody but is not detectable in larval integument. During normal development this mRNA was undetectable in eggs but was present at similar levels from first to fifth instar larvae. The mRNA was expressed at very low levels in pupae and adult moths. The cytochrome b5 mRNA was found to be inducible by treatment with the monoterpene, a-pinene, and to be over-expressed in some individuals of a pyrethroid resistant population of H. armigera. The induction and over-expression patterns were identical to the cytochrome P450, CYP6B7 mRNA. The present data suggests that cytochrome b5 may be involved in CYP6B7 mediated pyrethroid resistance in H. armigera.     

Higher accumulation of proteinase inhibitors in flowers than leaves and fruits as a possible basis for differential feeding preference of Helicoverpa armigera on tomato (Lycopersicon esculentum Mill, Cv. Dhanashree)

Tomato (Lycopersicon esculentum, Mill; cultivar- Dhanashree) proteinase inhibitors (PIs) were tested for their trypsin inhibitory (TI) and Helicoverpa armigera gut proteinases inhibitory (HGPI) activity in different organs of the tomato plants. Analysis of TI and HGPI distribution in various parts of the plant showed that flowers accumulated about 300 and 1000 times higher levels of TI while 700 and 400 times higher levels of HGPI as compared to those in leaves and fruits, respectively. Field observation that H. armigera larvae infest leaves and fruits but not the flowers could be at least partially attributed to the protective role-played by the higher levels of PIs in the flower tissue. Tomato PIs inhibited about 50-80% HGP activity of H. armigera larvae feeding on various host plants including tomato, of larvae exposed to non-host plant PIs and of various larval instars. Tomato PIs were found to be highly stable to insect proteinases wherein incubation of inhibitor with HGP even for 3h at optimum conditions did not affect inhibitory activity. Bioassay using H. armigera larvae fed on artificial diet containing tomato PIs revealed adverse effect on larval growth, pupae development, adult formation and fecundity.     

不同食料植物对棉铃虫生长发育和繁殖的影响

报道了棉花、烟草、番茄和辣椒4种植物对棉铃虫Helicoverpa armigera生长发育和繁殖的影响。棉铃虫成虫喜欢在番茄上产卵,在辣椒上的着卵量最少。初孵幼虫喜选食嫩棉叶,选食辣椒嫩叶的虫数最少。4组幼虫取食嫩叶时的平均相对生长速率都有显著差异,顺序为棉叶组>烟叶组>番茄叶组>辣椒叶组。幼虫存活率以取食棉花时最高,取食番茄时最低。棉花组成虫的产卵量最高,烟草组的产卵量最低。取食棉花的棉铃虫种群增长的速度约为取食番茄时的14倍。6龄幼虫能有效利用和转化棉铃、烟草蒴果、辣椒果实,而对番茄果实的利用和转化效率较低。棉铃虫可分别以这4种植物的不同器官为食完成世代循环。其中,棉花是最适宜的寄主,辣椒和番茄是较不适宜的寄主。     

棉铃虫HaKettin1基因的全长cDNA克隆、序列分析和表达谱

【目的】为了克隆棉铃虫Helicoverpa armigera编码肌肉蛋白Kettin基因的全长cDNA序列以及鉴定该基因在棉铃虫发育周期内的表达模式。【方法】利用兼并引物,通过分段RT-PCR和5′-和3′-RACE的方法克隆全长cDNA序列。利用半定量RT-PCR进行表达谱分析。【结果】编码棉铃虫Kettin蛋白的基因HaKettin1全长cDNA序列为13 805 bp,包含一个13 365 bp的开放阅读框,编码4 454个氨基酸,蛋白分子量约为504.3 kD。组织表达结果显示HaKettin1基因在棉铃虫的整个生育周期都有表达,幼虫期的表达尤为显著。【结论】HaKettin1与家蚕的Kettin蛋白具有90％的同源性,表明鳞翅目昆虫的Kettin蛋白之间具有很高的保守性。表达谱结果显示HaKettin1基因在棉铃虫的整个发育过程中都发挥重要作用。