【作者】 何超；

【导师】 花蕾；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2013， 博士

【摘要】 梨小食心虫Grapholita molesta (Busck)是一种重要的蛀果害虫,食性杂,寄主范围广。近年来,由于农业产业结构调整及气候变化等原因,致使该虫的危害逐年加重,甚至在一些地区或不同年份,该虫在果园的种群数量及危害程度已经成为果品生产中的主要害虫,给果品生产造成巨大的经济损失。在我国西北地区,梨小食心虫在自然条件下,老熟幼虫的滞育长达5个月左右,越冬幼虫能否顺利越冬是影响翌年种群数量的一个重要因素。目前对梨小食心虫的滞育的研究报道较少,仅从滞育的生态学方面进行了一些初步的研究。为完善对梨小食心虫滞育特性及其影响因子作用的认识,进一步探明滞育形成对该虫种群动态的调节作用,提高梨小食心虫预测预报和综合治理的水平,本文对梨小食心虫滞育的生理及生态机制进行了深入研究,主要研究结果如下：1.室内对梨小食心虫滞育诱导的光周期反应、临界光周期、敏感光照虫期及温周期反应进行了研究。在生态光周期范围内,梨小食心虫属短日照滞育型昆虫。在20℃下,当光周期为8L：16D,10L：14D和12L：12D时,梨小食心虫的滞育率均达95%以上,滞育诱导的临界光周期为13.75L：10.25D；在24℃下,当光周期为10L：14D和12L：12D时,滞育率均达95%以上,诱导滞育的临界光周期为13.68L：10.32D。在幼虫期,分别给不同日龄幼虫以12L：12D和16L：8D光照处理后,在不同发育时间内幼虫对滞育诱导光周期反应均显示出一定程度的敏感性,无明显的临界时间点,但仍以第5～8日龄幼虫接受滞育诱导光周期反应更为敏感；连续接受短光照的时间越长,滞育率越高。温度不是影响梨小食心虫滞育诱导的关键因素,但能调节光周期对滞育诱导的作用效果；在10-25℃的中位温度下,较低的温度能促进滞育的形成,而5℃的低温和30℃的高温在一定程度上抑制短光照对滞育的诱导作用,且温度只在暗期发挥作用。2.对梨小食心虫滞育诱导的光周期钟机制进行了研究。24h昼夜循环光周期反应表明,在20℃,梨小食心虫滞育诱导的临界暗长约为10.25h,在24℃,约为10.32h；12～16h的暗期可诱导最高滞育的发生。非24h昼夜循环光周期反应表明,不同光期的滞育诱导反应曲线明显不同,但均在暗期为12h时滞育发生率最高,暗期长度在该虫滞育的诱导中发挥重要作用,但光照长度能明显调节暗期对滞育诱导的作用效果。暗期干扰试验表明,1h光脉冲干扰光周期10L：14D、11L：13D和13L：11D的滞育发生率明显低于干扰光周期12L：12D,且均表现为在暗期的中间对光脉冲最敏感,光脉冲的干扰效果与所干扰的光周期密切相关。在Nanda-Hamner和Bunsow试验光周期反应中,二者滞育发生率普遍较低,在24h内均未显示滞育诱导的节律性波动。这些结果表明控制梨小食心虫滞育诱导的光周期钟是基于一个非昼夜节律的沙漏钟模型或阻尼振荡器模型。3.在4℃、10℃和24℃三种温度下对梨小食心虫的滞育解除进行了研究。滞育幼虫在4和10℃下处理120天,可完全解除滞育,10℃比4℃更有利于滞育的解除；而24℃下未见滞育幼虫化蛹；表明低温对梨小食心虫的滞育解除有重要作用,滞育幼虫在一定低温条件下,经历一定的时期,才能解除滞育。4.用蒽酮比色法、乙酰丙酮比色法和考马斯亮蓝G-250染色法分别对梨小食心虫滞育过程中体内总糖、糖原、海藻糖、甘油和蛋白质等能源物质的代谢特征进行了研究。在低温条件下,滞育幼虫体内糖原含量明显减少,且不同温度条件下糖原的代谢速率不同,4℃下幼虫体内糖原利用率高于10℃；总糖含量的变化趋势与糖原相似：海藻糖含量在4℃、10℃和24℃2下均升高,且保持较高水平；甘油含量在4℃下明显增加,出现3个峰值,在10℃和24℃下,甘油含量并没有显著增加,表明只有达到一定的低温,甘油的合成才能够被诱导；甘油是梨小食心虫的主要抗冻物质；蛋白质含量在3种温度下均升高,但在4℃和10℃下变化差异不显著,在24℃下差异显著。滞育期间,这些代谢物质的变化受滞育发育及温度影响,其含量的动态变化可以反映梨小食心虫耐寒性强弱。5.用3,5-二硝基水杨酸法、双试剂法及代谢酶活力测试盒分别测定了梨小食心虫滞育过程中海藻糖酶(TRE)、山梨醇脱氢酶(SDH)及己糖激酶(HK)、丙酮酸激酶(PK)和碱性磷酸酶(ALP)的活力变化。在低温条件下,滞育幼虫体内各酶活力明显不同,TRE活力在滞育初期显著提高,而后随滞育时间的延长而逐渐降低；SDH活力总体变化较为平缓,变化量较小；HK、PK和ALP活力在滞育前期均显著降低,且随后一直保持在较低水平。滞育期间,5种代谢酶的活力均与滞育发育及温度有关；通过相关代谢酶的调节,梨小食心虫的代谢速率维持在较低水平,提高了虫体的耐寒能力。6.用H2O2含量测试盒及保护酶活力测试盒分别测定了梨小食心虫滞育过程中过氧化氢(H2O2)含量及超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活力变化。在低温条件下,滞育幼虫体内H2O2的含量明显增加,SOD和CAT的活力在滞育初期均呈下降趋势,之后显著升高,POD活力在滞育初期减少,且随后一直维持在较低水平。滞育期间,H2O2的含量和3种保护酶的活力受滞育发育及温度影响,其变化与虫体的抗寒有着较密切关系。7.用高效液相色谱法、紫外比色法和碘-淀粉比色法分别测定梨小食心虫滞育过程中多胺含量及其代谢酶活力动态。在低温条件下,滞育幼虫体内腐胺(Put)含量在滞育初期略有减少,之后迅速升高：亚精胺(Spd)含量在滞育后明显升高,且一直保持在较高水平；4℃下Put和Spd含量明显高于10℃；Spm含量在滞育前期下降,之后显著升高。鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(SAMDC)活力均表现出不同程度的升高,且ODC活力大于SAMDC活力,表明鸟氨酸途径是该虫滞育过程中多胺合成的主要途径；多胺氧化酶(PAO)活力在滞育过程中也升高,在4℃时活力变化存在显著差异,在10℃和24℃时不存在显著差异。滞育期间,多胺代谢与滞育发育及环境温度有关；低温下多胺的积累有利于提高梨小食心虫滞育幼虫的抗寒能力。更多还原

【Abstract】 The oriental fruit moth, Grapholita molesta (Busck), is a key pest of stone fruit, its feeding habits is miscellaneous, and it has a broad range of host plant. In recent years, due to the agricultural structure adjusted and the climate changed, resulting in most large-scale infestations of this pest had occurred throughout the growing season in many regions every year. In some regions, this pest had become a major pest according to the populations and harmful degree, it has caused serious economic losses. In the Northwest of China, this pest overwintered as mature diapausing larvae for about five months under natural conditions. Whether the diapausing larvae can successfully overwinter, affects the population dynamics of this species in the following year. Studies on the diapause ecology of oriental fruit moth have been documented. However, very little work has been devoted to investigate the physiological and biochemical mechanisms of diapause in this species. To improve understanding of the diapause characteristics and its surroundings impact factor in this species, further to know the regulatory effect of diapause on the population dynamics of this pest, provide novel targets for pest forecasting and integrated control. In this paper, the ecological, physiological adaptation mechanisms of diapause of this pest were conducted in-depth in experimental conditions. The main results were summarized as follows.1. The photoperiod response type, critical photoperiod, sensitive stage of diapause induction and the effect of temperature on diapause induction in oriental fruit moth were determined in the laboratory. In ecological light cycle, this pest was a typical short-day diapause type. At20℃, when the photoperiod was8L:16D,10L:14D and12L:12D, the diapause rate>95%, the critical photoperiod was13.75L:10.25D. At24℃, when the photoperiod was10L:14D and12L:12D, diapause rate>95%, the critical photoperiod was13.68L:10.32D. During the larval stage, the age in different day of larvae were treatmented under12L:12D and16L:8D light respectively, all results showed a certain degree of sensitivity to diapause inducting photoperiod, although there was no obvious critical point in time, but still the age in5-8th day of larvae was more sensitive to diapause inducing photoperiod. The larvae continoued in the short day length was longer, the rate of diapause was higher. Temperature was not a key influential factor in diapause induction, but could adjust the effect of short photoperiod on diapause induction. At the mediam temperature of10-25℃, low temperature could promote diapause induction. The low temperature of5℃and the high temperature of30℃, to some extent, inhibited diapause induction of short photoperiod. Temperature played an important role only in the dark period.2. Photoperiodic clock mechanism of diapause induction in oriental fruit moh was investigated. The photoperiodic response under24h light-dark cycles showed that the critical night length was10.25h at20℃and10.32h at24℃, high incidences of diapause were at intermediate scotophases of12-16h. Under non24h light-dark cycles, the response curves of diapause induction were significantly different in different photophase, but the length of12h in scotophase all resulted in the highest rate of diapause. The length of scotophase played an essential role in diapause induction, but the length of photophase could adjust the diapause inducting effect of scotophase. In night interruption experiments showed that the percentage of diapause was significantly lower in the scotophase of10L:14D,11L:13D and13L:11D which were interrupted by1h light pulse than that of12L:12D, the most highly photo-sensitive position occurred in the middle of the dark period, and interruption effects had a relationship with photoperiod. Nanda-Hamner and Bunsow experiments showed that the percentage of diapause were all significantly lower and the diapause induction curve did not show rhythmic fluctuations within24h in this species. These results suggested photoperiodic clock in this pest showed an hourglass-liker response model or a rapidly damping oscillator model.3. At4℃,10℃and24℃, diapause termination of oriental fruit moth were systematically investigated. Diapause larvae could be terminated by exposing them to low temperature of4℃and10℃for120days, it seemed to be more conducive to complete diapause development at the medium-low temperature of10℃compared to the low temperature of4℃. Diapause larvae could not be terminated at24℃until day120. The result indicated that low temperature had a significantly effect on diapause termination. Diapause larvae only in low temperature, experienced a period could be terminated.4. The anthrone colorimetry, acetylacetone colorimetry and coomassie brilliant blue G-250dying were used to analyze the dynamic metabolic processes of total sugars, trehalose, glycogen, glycerol and protein in diapausing larvae oriental fruit moth. At low temperatures, glycogen content decreased significantly, its metabolic rate was different under different temperature, and utilization rate was higher at4℃than10℃. The change of total sugar content was fairly similar with glycogen. Trehalose content increased significantly during diapause development at4℃and10℃, as well as at24℃and maintained at a higher level. Glycerol level was significantly increased at4℃and showed three peaks, while at10℃and 24℃, glycerol content did not significantly accumulate, it showed that only reached at determinate low temperature, the synthesis of glycerol could be induced. Glycerol is mainly cryoprotectants of oriental fruit moth during diapause period. Protein content increased during diapause development at4℃、10℃and24℃, changed insignificantly at4℃and10℃and significantly at24℃. During diapause period, the changes of the content of those test chemical substances were significantly affected by diapause development and temperature and could reflect the cold-resistance of overwintering larvae of oriental fruit moth.5. The activities of several metabolic enzymes including trehalase (TRE), sorbitol dehydrogenase (SDH), hexokinase (HK), pyruvate kinase (PK) and alkaline phosphatase (ALP) in diapausing larvae oriental fruit moth were measured using3,5-dinitrosalicylic acid colorimetry, dual reagent method and metabolic enzyme activity testing kits. The activities of these metabolic enzymes were significantly different during diapause development at low temperatures. Initially TRE activity significantly increased, then gradually decreased with the prolongation of the time of diapause. SDH activity changed more gently and was no larger fluctuation. HK, PK and ALP activities significantly reduced during the early phase, and then fluctuated at a lower level during the late phase. During diapause period, diapause intensity and temperature had significantly affected on these metabolic enzymes. The metabolic rate remained at low level through the regulation of metabolic enzymes could improve the ability of cold hardiness of oriental fruit moth.6. The content of hydrogen peroxide (H2O2) and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) in diapausing larvae of oriental fruit moth were determined by using the protective enzyme testing kits. At low temperatures, H2O2content was significantly increased, the activities of SOD and CAT decreased during the early phase of diapause, then significantly consistent increased. POD activities decreased in the early stage of diapause, and maintained at low level. During diapause period, H2O2content and protective enzymes activities were significantly affected by temperature and diapause intensity, and had a relationship with cold hardiness.7. The dynamic of polyamine content and its metabolic enzyme activities of oriental fruit moth were examined by high performance liquid chromatography, UV colorimetry and iodine-starch colorimetry. At low temperature, putrescine (Put) content slightly decreased in the early period of diapause, then rapidly rised, spermidine (Spd) content increased and maintained high level during the whole diapause development, Put and Spd content was significantly higher at4℃than that of at10℃, spermine (Spm) content decreased in the early phase of diapause, then significantly increased in the late phase of diapause. The activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) increased significantly, ODC activities were higher than SAMDC, indicating the biosynthetic pathway of ornithine is the main way of polyamine metabolism in oriental fruit moth during diapause development. The activities of polyamine oxidase (PAO) also increased during diapause development, and were significantly differences at4℃, but no significantly differences at10℃and24℃. During diapause period, polyamine metabolism was significantly affected by diapause development and temperature, and the polyamine accumulation could help to improve diapause larvae of hardiness at low temperature.更多还原