苹果蠹蛾气味结合蛋白基因CpomOBP20的克隆及表达分析

[目的] 通过克隆苹果蠹蛾气味结合蛋白CpomOBP20基因cDNA序列,分析其序列特征和表达谱,旨在更好地了解OBP基因在苹果蠹蛾生命活动过程中的作用,为该害虫的绿色防控提供理论支撑。[方法] 采用RT-PCR法扩增苹果蠹蛾气味结合蛋白CpomOBP20基因cDNA序列,并使用生物信息学软件对其核苷酸和氨基酸序列进行分析;基于qPCR技术分析CpomOBP20基因在苹果蠹蛾4龄幼虫不同组织(头、血淋巴、表皮、脂肪体、中肠、马氏管和唾液腺)以及雌雄成虫不同末端组织(头、触角、下唇须、喙、足和翅)中的表达情况,利用分子对接研究了CpomOBP20与3种保幼激素的结合能力。[结果] 苹果蠹蛾气味结合蛋白CpomOBP20基因的开放阅读框长459 bp,共编码152个氨基酸,等电点为6.30,蛋白分子质量为16.264 ku,N末端具有20个氨基酸组成的信号肽序列,蛋白质序列中具有6个保守的半胱氨酸残基,属于Classical OBP。序列分析表明,CpomOBP20的氨基酸序列与小菜蛾OBP (XP_011557123.1)的一致性最高,在亲缘关系上更加接近。qPCR结果表明,CpomOBP20基因在苹果蠹蛾4龄幼虫以及雌雄成虫不同组织中均有表达,其中在4龄幼虫的血淋巴中表达量最高,在雌雄成虫表达量最高的分别是翅和足,其次是头部。分子对接结果表明,CpomOBP20与3种保幼激素均具有较好的结合能力,可能参与保幼激素的结合与转运。[结论] 本研究明确了CpomOBP20的核苷酸和氨基酸序列的组成及编码蛋白的理化性质,并推测CpomOBP20的作用可能不仅局限于嗅觉识别,在非嗅觉器官中也可能起着重要的生理作用,为今后更深入地探究气味结合蛋白在苹果蠹蛾生命活动中的作用机理提供数据支撑。     

Effect of plant architecture and hail nets on temperature of codling moth habitats in apple orchards

Plant architecture of apple trees in commercial orchards was rapidly changed from traditional tall trees to dwarf trees to optimize yield and fruit quality. Additionally, hail nets are widely used to prevent yield loss by hail. These changes are expected to considerably influence the orchard microclimate and thus the developmental rates of pest insects in apple. However, these relationships have not yet been fully elucidated. The present study was conducted over the seasonal cycle to investigate the influence of plant architecture and hail nets on the habitat temperatures of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), in apple, Malus domestica Borkh. (Rosaceae). Within the canopies, leaf area index (LAI) and global site factor (GSF) were quantified using hemispherical photography. Temperature was analysed for the main habitats of the different codling moth stages, i.e., air within the canopy, bark of tree stems, and apple fruit. In dwarf trees, LAI was lower, leading to a higher GSF than in tall trees. Hail nets did not influence LAI and GSF. Results for dwarf trees compare as follows with those for tall trees: Average air temperatures within the canopy were 0.7 °C higher during daytime, whereas 0.4 °C lower at night. Mean surface temperatures of bark were 0.9 °C higher on sunny and 0.4 °C on overcast days. Mean surface temperatures of apple fruits were 1.8–2.7 °C higher on sunny days, but 0.6 °C cooler on overcast days. The effect of hail nets was confined to a reduction of the air temperature within the canopy by approximately 0.2–0.8 °C. Bark and apple surface temperatures were not significantly affected. Based on the temperature differences in the habitats considered, the calculated development of the codling moth in dwarf trees was on average 3 days faster than in tall trees. The calculations imply a negligible effect of hail nets on codling moth development.     

Effect of radiation on fecundity and fertility of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) from South Africa

Codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) is the key pest of pome fruit in South Africa, and it’s control in apple and pear orchards relies on the application of insecticides and pheromone‐mediated mating disruption. Development of resistance to insecticides and placement of restrictions on the use of certain insecticides has made control of codling moth in South Africa increasingly problematic. The use of the sterile insect technique (SIT) as a control tactic for codling moth is under investigation as a potential addition to the current control strategy. We investigated the radiosensitivity of a laboratory strain of codling moth that was established from moths collected from commercial and organic orchards in the Western Cape, South Africa. Fecundity and fertility of this strain following radiation were consistent with values for the codling moth strain in the Canadian rearing facility in British Columbia. For both strains, the female codling moth was considerably more radiosensitive than the male. At a radiation dose of 100 Gy or higher, treated females were 100% sterile. The fertility of the South African strain was higher (86.3%) than for the Canadian strain (71.9%). This difference in fertility between the two strains was maintained when the dose of radiation was 100 Gy. However, the level of fertility was very similar between the two strains for doses ≥150 Gy. Therefore, based upon previously published work and the data from this study, an operational dose of 150 Gy is recommended for future codling moth SIT programmes in South Africa.     

Post-application of anti-desiccant agents improves efficacy of entomopathogenic nematodes in formulated host cadavers or aqueous suspension against diapausing codling moth larvae (Lepidoptera: Tortricidae)

Codling moth (CM), Cydia pomonella (L.) is the most serious pest of apple and other pome fruit worldwide. In temperate climates, diapausing cocooned larvae make up 100% of the population. Control of this stage would reduce or eliminate damage by first generation CM in late spring and early summer. Entomopathogenic nematodes (EPNs) are good candidates for control of CM in the cryptic habitats where the larvae overwinter. The two predominant limiting factors for EPNs are adequate moisture and temperatures below 15°C. Formulation that maintains moisture and enables survival of EPN infective juveniles (IJs) until they can infect overwintering larvae would significantly improve their utility for protection of apple, pear and walnut. In laboratory studies conducted in moist mulch (consisting of apple and conifer wood), Galleria mellonella (L.) larvae infected with Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), or Heterorhabditis bacteriophora Poinar and coated with starch and clay, produced mean mortalities of 42, 88, and 24%, respectively in CM larvae. Mulched field plots treated with formulated S. carpocapsae- or S. feltiae-infected G. mellonella larvae, then followed by an application of wood flour foam as an anti-desiccant, resulted in 56 and 86% mortality, respectively. Comparative tests of aqueous suspensions of S. carpocapsae IJs applied to cardboard bands on apple tree trunks followed by water, fire retardant gel or foam resulted in 11, 35, and 85% respective mortalities. Identical tests with S. feltiae resulted in 20, 19, and 97% respective mortalities. Our research with cadaver formulations of EPNs in mulch and aqueous suspensions on tree trunks combined with anti-desiccant agents, demonstrated significant improvement in larvicidal activity for diapausing cocooned CM larvae.     

Structured populations of the oriental fruit moth in an agricultural ecosystem

Intercontinental trade has led to multiple introductions of invasive pest species at a global scale. Molecular analyses of the structure of populations support the understanding of ecological strategies and evolutionary patterns that promote successful biological invasions. The oriental fruit moth, Grapholita (=Cydia) molesta, is a cosmopolitan and economically destructive pest of stone and pome fruits, expanding its distribution range concomitantly with global climate warming. We used ten newly developed polymorphic microsatellite markers to examine the genetic structure of G. molesta populations in an agricultural ecosystem in the Emilia‐Romagna region of northern Italy. Larvae collected in eight sampling sites were assigned to a mosaic of five populations with significant intra‐regional structure. Inferred measures of gene flow within populations implicated both active dispersal, and passive dispersal associated with accidental anthropogenic displacements. Small effective population sizes, coupled with high inbreeding levels, highlighted the effect of orchard management practices on the observed patterns of genetic variation within the sampling sites. Isolation by distance did not appear to play a major role at the spatial scale considered. Our results provide new insights into the population genetics and dynamics of an invasive pest species at a regional scale.     

Effect of monosodium glutamate on apple leaf consumption by codling moth larvae

We have demonstrated that larvae of codling moth, Cydia pomonella (L) can successfully complete their first instar when fed apple leaves instead of fruit. Larvae fed leaves after hatching maximized their feeding intensity (about 320 g/larva/day) on day 2. Weight gain revealed a stereotypic sigmoid pattern that peaked on day 3. Although the maximum body weight of larvae fed leaves was 70–85% less than for larvae maintained on apples or on artificial diet, 100% of larvae fed leaves molted to the second instar 3–5 days after hatching. Our investigation revealed a diurnal pattern of leaf ingestion, and neonates' feeding intensity decreased significantly during the scotophase. We also demonstrated that monosodium glutamate (MSG) increased feeding on leaves by codling moth larvae. Depending on the duration of the bioassay, and larval age at time of initial exposure, 0.05 mg/ml and 0.1 mg/ml MSG increased apple leaf consumption by 25–60% over leaves alone. The effect of monosodium glutamate was best demonstrated during the first day following hatching. Exposure to MSG also accelerated molting to the second instar. Larvae exposed to MSG initiated consumption of leaf tissue significantly earlier than control neonates. The feeding stimulatory effect of MSG was not observed if exposure to this chemical was delayed until 3–4 h after hatch.The addition of feeding stimulants to pesticides that act via the alimentary tract may reduce the amount of active ingredients needed to maintain the efficacy of these formulations. Here, we postulate that first instar codling moth larvae are potential targets for treatment with pesticide formulations enhanced with monosodium glutamate.     

Induction of volatile emissions from ripening apple fruits infested with Cydia pomonella and the attraction of adult females

We studied the effect of herbivory by Cydia pomonella L. (Lepidoptera: Tortricidae) first instar larvae on the volatile emissions from immature apples collected at the end of August. Volatiles from infested, artificially damaged, and healthy fruit were collected using solid-phase microextraction and analysed using combined gas chromatography mass spectrometry. The results show that neonate C. pomonella larvae induce the release of E--ocimene from immature fruit compared to healthy or artificially damaged fruit. Seven further compounds, a terpene, an alcohol, four aldehydes, and a ketone, were consistently found in all treatments. There were no significant differences amongst these latter compounds, although the amount of the terpene E,E--farnesene was three to five times higher from the infested as compared to healthy fruits. Headspace volatiles were also collected on Tenax to test for behavioural responses to the volatiles. Volatile collections from infested fruit attracted mated adult females in a Y-tube olfactometer, whereas no preference was evinced for volatiles from healthy or artificially damaged fruits when compared to a solvent control.     

Characterisation of Cydia pomonella granulovirus from codling moths in a laboratory colony and in orchards of British Columbia*

Sequence analyses indicate that a granulovirus isolated from a laboratory colony of codling moth in British Columbia, Canada is identical or very similar to the Mexican isolate of Cydia pomonella granulovirus (CpGV-M). In addition to codling moth, the vims was found to cause mortality of secondary leafroller pests of North American orchards, including the fruit tree leafroller and the obliquebanded leafroller. A survey in apple growing regions of British Columbia indicates that 23% of the wild population of codling moth larvae carry CpGV.     

Interference with the mate-finding communication system of the Chinese tortrix Cydia trasias (Lep., Olethreutidae) using synthetic sex pheromones

Abstract: Interference with the mate-finding communication system of Cydia trasias (Meyrick) using sex pheromone dispensers (60% E8,E10-dodecadienyl acetate and 40% E8,E10-dodecadien-1-ol, 0.5 mg/dispenser) was investigated in three plots of the Chinese scholar-tree, Sophora japonica L., in Beijing, China, in 2000. Treatments were evaluated by pheromone-baited traps, caged virgin females, pupating and hibernating populations on tree trunks, and assessment of larval damage in petioles and seed pods. Six to eight natural rubber septum dispensers were placed in each tree at the pheromone-treated plots at three separate periods: at the beginning of overwintering generation flight, the first- and the second-generation flights. Application of pheromone dispensers reduced the incidence of mating of virgin females in treated plots relative to those in the control plots. Pheromone-baited trap catches were lowered on average by 96%, suggesting a high level of disruption. Average larval density on tree trunks was significantly lower in the pheromone-treated plots than in the untreated control plots. The mean percentage of Chinese scholar-tree petioles infested with first- and second-generation larvae, and seed pods infested with third-generation larvae was significantly greater in untreated control plots than in pheromone-treatment plots. These results suggest that disruption of pheromone-based communication in C. trasias is prospective, with synthetic sex pheromone on trees in urban environments.     

Olfactory orientation of the truffle beetle, Leiodes cinnamomea

Although the truffle beetle, Leiodes cinnamomea, inflicts substantial damage to the ripe stage of fruiting bodies of the economically important black truffle (Tuber melanosporum), it is not attracted by ripe truffle odours. Rather, male beetles are attracted to infested truffles only in the presence of female beetles, suggesting that the former employ a pheromone to locate truffles over short distances. In contrast, female beetles show no attraction to infested or uninfested truffles, suggesting that they employ other cues, possibly linked to odours emitted by truffles prior to the ripe stage. We hypothesize that the chemical composition of truffle volatiles changes over the life of the truffle fruiting body, being attractive to insects early on and to mammals just prior to decomposition.