Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet,Brassica species,and protease inhibitor

The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one‐dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease‐encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin‐like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin‐like gene McSP34. The expression of the trypsin‐like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources. Published 2010 Wiley Periodicals, Inc.     

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism

The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography‐tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α‐amylase) or other reactions (β‐1,3‐glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C‐Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase‐2 (McCHS‐2), chitinase (McCHI), and β‐N‐acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS‐2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS‐2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.     

甘蓝夜蛾几丁质酶基因cDNA序列的克隆与序列分析

昆虫几丁质酶在害虫生物防治中具有很大的发展潜力。以甘蓝夜蛾Mamestra brassicae L.预蛹期幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),扩增得到其几丁质酶的cDNA序列。该序列含有2826个碱基,包括1个1689个碱基的开放阅读框,预测编码1个含562个氨基酸的多肽,分子量约为62.6kDa,等电点为5.30。推导得到的氨基酸序列含有2个N-位糖基化位点,22个O-位糖基化位点,氨基酸序列与其他昆虫,尤其是鳞翅目昆虫的几丁质酶高度同源。获得的甘蓝夜蛾几丁质酶基因cDNA序列已经登录GenBank并获得登录号FJ436415。     

苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶（serine protease, SP）基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP（GenBank登录号：JX866720）,该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP（GenBank登录号：CAA72962）的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.     

甘蓝夜蛾和八字地老虎肌动蛋白基因的克隆与序列分析

肌动蛋白是细胞骨架微丝的主要组成成分,在肌肉收缩、细胞骨架形成、细胞移动等方面起重要作用。以鳞翅目夜蛾科昆虫甘蓝夜蛾Mamestra brassicae L.和八字地老虎Agrotis c-nigrum 3龄幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到2种昆虫的肌动蛋白的cDNA序列,甘蓝夜蛾肌动蛋白的cDNA序列含有1441个碱基,而八字地老虎肌动蛋白的cDNA序列含有1411个碱基。2种昆虫的该基因的cDNA序列均包括1个1131个碱基的开放阅读框,编码1个含376个氨基酸的蛋白。甘蓝夜蛾肌动蛋白分子量约为41.8kDa;八字地老虎肌动蛋白分子量约为41.9kDa。Prosite软件分析结果表明,甘蓝夜蛾和八字地老虎肌动蛋白氨基酸序列中存在3个肌动蛋白特征片段。GenBank数据库搜索及序列比对结果表明,甘蓝夜蛾肌动蛋白属于肌肉特异型肌动蛋白,八字地老虎肌动蛋白属于细胞质特异型肌动蛋白。2个基因的cDNA序列已经登录GenBank并获得登录号,甘蓝夜蛾肌动蛋白cDNA序列登录号为EU035314,八字地老虎肌动蛋白cDNA序列登录号为EU035315。利用RT-PCR技术在八字地老虎4龄、5龄、6龄幼虫、蛹期4个不同发育阶段和6龄期的肠道、体壁、脂肪体3种不同组织中都检测到了肌动蛋白基因在mRNA水平的表达。     

Evaluation of the Palaearctic Eurithia consobrina (Diptera: Tachinidae) as a Potential Biocontrol Agent for Mamestra configurata (Lepidoptera: Noctuidae) in Canada

The evaluation of Eurithia consobrina (Meigen) as a candidate for introduction against bertha armyworm, Mamestra configurata (Walker) (Lepidoptera: Noctuidae), in western Canada included studies of its host range, distribution, synchronization with host, constancy, abundance and life history in Europe. In addition, its diapause induction and its coldhardiness were compared with those of the target host M. configurata and its native tachinid parasitoid, Athrycia cinerea (Coq.). E. consobrina was found to fit criteria for a successful biocontrol agent reasonably well. It also had the potential to fill a largely unoccupied niche in the parasitoid complex of M. configurata. E. consobrina has a facultative diapause of the long-day type, similar to A. cinerea, but is less sensitive to diapause-inducing conditions. E. consobrina is at least as coldhardy as A. cinerea and more coldhardy than M. configurata. Laboratory exposures to temperatures of 0 C for 140 days apparently selected for greater coldhardiness, and later generations survived exposures of 140 days at -15 C without significant mortality. Adult E. consobrina were released at three locations in Manitoba in 1986 and 1987, but establishment has not been confirmed.     

小地老虎几丁质酶基因的克隆与序列分析

利用昆虫几丁质酶对几丁质的调控作用破坏几丁质新陈代谢的平衡来防治害虫, 在生物防治策略中具有很大的发展潜力。从处于预蛹期的小地老虎Agrotls ipsilon (Hufnagel)体中肠内提取总的RNA, 经反转录, 利用cDNA末端快速扩增技术（RACE）获得了几丁质酶基因的cDNA序列。该基因序列已经登录GenBank并获得登录号为EU035316。该序列长度为2 823个碱基, 含有一个1 674个碱基的开放读码框。开放读码框编码558个氨基酸残基, 预测的分子量为62.5 kDa, 等电点5.12。推导得到的氨基酸序列含有2个N-位糖基化位点,20个O-位糖基化位点, 含有2个几丁质酶所具有的保守序列：N-端的催化区和C-端的几丁质结合区。氨基酸序列与其他昆虫, 特别是鳞翅目昆虫的几丁质酶高度同源。     

New chemical attractants for trapping Lacanobia subjuncta, Mamestra configurata, and Xestia c-nigrum (Lepidoptera: Noctuidae)

Significant numbers of 3 pest species of noctuid moths were captured in traps baited with acetic acid, 3-methyl-1-butanol, and 3-methyl-1-pentanol. These were Lacanobia subjuncta (Grote & Robinson); Mamestra configurata Walker, bertha armyworm; and Xestia c-nigrum (L.), spotted cutworm. The combination of acetic acid and 3-methyl-1-butanol was superior to the individual chemicals in attracting all 3 species, whereas the combination of acetic acid and 3-methyl-1-pentanol was superior to the individual chemicals in attracting X. c-nigrum. For the 3 species of moths, numbers captured were similar in traps baited with the combination of acetic acid and 3-methyl-1-butanol or acetic acid and 3-methyl-1-pentanol. Traps baited with these attractants captured both males and females at a ratio near 1:1.     

Aestival dormancy in the cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae)

Summary A highly specific recognition system, capable of foreseeing and distinguishing between two critical points in time, exists in Mamestra brassicae (Lepidoptera: Noctuidae). Both points in time, the onset of a drought period and the end of the growth season, require different growth patterns of the pupae. In order to minimize the likelihood of weather-induced mortality and to maximize fitness, individuals of M. brassicae must enter aestival dormancy or hibernal diapause, respectively, before the onset of drought or frost. This study is primarily concerned with aestival dormancy. Normally, the pupal period of dormancy-free developing individuals amounts to approximately 20 to 30 days. A modified pupal period of approximately 35 to 80 days is defined as aestival dormancy. The onset of aestival dormancy is triggered by day lengths exceeding an innate individaul-specific threshold. The results reported in this paper indicate that the photoperiodic response curve represents largely the genetic variability within a population with respect to the thresholds triggering aestival dormancy. This variability in thresholds is considered to reflect the frequency of correlation of a distinct day length with a certain onset of drought period in the past. Furthermore, the innate thresholds are characterized by a temperature dependent norm of reaction. Our results also indicate, that a strong genetical component is involved in variability of duration of the pupal period. This variability in duration of aestivation reflects the frequency of drought periods of a certain length in the past. The adaptive significance of both the variation in day length thresholds and duration of aestival dormancy is discussed with respect to the number of generations per season, and the synchronization of the individual life cycles with the seasonal changing environmental conditions.This study is dedicated to Prof. H.J. Müller, Jena, for his 75th anniversaryThis research was supported by the Deutsche Forschungsgemeinschaft (Sa 259/3-1)     

Development of spermiduct and seminal vesicle during pharate adult of the cabbage armyworm,Mamestra brassicae, (Lepidoptera; Noctuidae)

Summary

Development of the upper vas deferens and seminal vesicle, and the ecdysteroid titers of the cabbage armyworm (Mamestra brassicae) during pharate adult stage were investigated to provide evidence of endogenous control of the sperm movement through the reproductive tract. Apparently, development of the upper vas deferens is initiated when ecdysteroid titers increase after pupation and continues until eclosion. Sperm movement correlates with the decrease of ecdysteroid titers. When ecdysteroid titers remained at low levels, sperm release from the testis was observed one day before eclosion.     

Responses of Mamestra brassicae (Lepidoptera: Noctuidae) to crowding: interactions with disease resistance,colour phase and growth

This study examines phenotypic plasticity in relation to rearing density in larvae of the moth, Mamestra brassicae. Larval phase, growth rate, weight at moulting and susceptibility to disease were quantified when reared at five densities. Larvae develop more quickly, but attain a smaller size and are more susceptible to disease, when reared at high than at intermediate densities. They also exhibit a higher degree of melanisation than larvae reared at intermediate densities, or singly. A review of the literature suggests that a switch to a rapidly developing dark phase at high densities is a widespread phenomenon within the Lepidoptera. Rapid development at the expense of attaining a large size, and increased melanisation, are interpreted as adaptive responses to reach pupation before food supplies are depleted, as is likely when larval density is high. High susceptibility to viral infection at high density may be a result of physiological stress associated with rapid development, or due to a shift in allocation of resources from resistance to development: larvae that developed quickly were more susceptible to infection. Larvae reared singly appeared to be less fit than larvae reared at intermediate densities: they exhibited many of the characteristics of larvae reared at high density, particularly low weight, a right-hand skew in their weight frequency distribution, and high susceptibility to disease. I hypothesise that expression of resistance may be phenotypically plastic with regard to environment. Contact with other larvae may, up to a point, stimulate both growth and resistance to infection, for the risk of infection will increase with the density of conspecifics.     

12a-羟基鱼藤酮对斜纹夜蛾生殖力的影响及其作用机理

为探讨12a-羟基鱼藤酮对斜纹夜蛾Prodenia litura (Fabricius)生殖力的影响及其作用机理, 本文测定了该虫取食12a-羟基鱼藤酮后的产卵量。在用高效液相色谱仪确定其卵巢组织中存在12a-羟基鱼藤酮后, 通过MTT法测定该化合物对斜纹夜蛾卵巢细胞（PL细胞）的细胞毒性,并利用流式细胞仪检测该化合物对PL细胞细胞周期、膜电位、胞内钙离子浓度、线粒体膜电位的影响。结果表明:斜纹夜蛾幼虫取食12a-羟基鱼藤酮后产卵量下降80％以上, 并从卵巢组织中检测出该化合物。12a-羟基鱼藤酮对PL细胞增殖具有抑制活性, 其IC₅₀为6.6 mg/L。该化合物将PL细胞周期阻滞于S期, 导致PL细胞膜电位、线粒体膜电位和细胞内游离钙离子浓度均显著升高。该化合物导致斜纹夜蛾繁殖力降低。12a-羟基鱼藤酮将PL细胞增殖阻滞于细胞周期的S期。同时, 该化合物对卵巢细胞具有较弱的毒杀活性, 导致一些卵巢细胞死亡。由于上述原因, 卵巢细胞数量逐渐减少而导致卵巢管萎缩。卵巢管的萎缩使卵巢管内的卵母细胞不能发育成卵细胞, 故其生殖力下降。     

The effects of photoperiod and temperature on calling behaviour and egg development of the bertha armyworm,Mamestra configurata (Lepidoptera: Noctuidae)

The effects of photoperiod and temperature on calling behaviour and the effects of temperature on the relationship between egg development and calling of virgin females of Mamestra configurata Walker were examined at 3 photoperiodic regimes and seven constant temperatures. Photoperiod affected the diel periodicity of calling and length of the daily calling period. The mean onset calling times were similar if the preceding scotophase(s) was 6–10 h long, but the onset of calling was delayed by 1–5 h if the preceding scotophase(s) was 12–18 h long. Long-term, constant temperature (from emergence until death) affected 4 aspects of calling: age at first calling, diel periodicity, length of the daily calling period, and percentage of females calling. Short-term temperature changes (during a single scotophase) affected calling in 3 ways: diel periodicity, length of the daily calling period, and percentage of females calling. The optimal temperature range for calling was, at least, 10–25°C; the upper limit and threshold were near 35 and < 5°C. The time of first calling was synchronized closely with the appearance of the first chorionated eggs in the ovaries at 10–35°C. Egg development and calling were adversely affected at 30 and 35°C. The physiological and ecological significance of the data are discussed.