【作者】 卢芙萍；

【导师】 符悦冠；

【作者基本信息】 海南大学 ， 作物害虫学， 2012， 博士

【摘要】 木薯单爪螨(Mononychellus mcgregori),是新入侵我国的重要危险性害螨,主要危害木薯叶片,导致叶片褪绿黄化,枝条干枯,严重时整株死亡,导致减产20～40%。该螨2008年首次在我国海南儋州发现,目前已在海南、广西、广东以及云南部分地区大量发生危害。其自2008年入侵我国热区后,种群数量在迅速增加,虽然这与缺乏有效天敌以及无其他相同生态位的螨类与其竞争等因素有关,但对入侵环境的适应性是木薯单爪螨种群快速扩张的前提。连续跟踪调查表明,该螨2008年较难采集到,至2010年年底已成片发生,且其发生期从一开始的仅在冬、春季发生已发展到目前的全年发生,因此,该螨已在逐渐对我国热区气候环境产生适应。外来物种对温度胁迫耐受力的高低对其入侵新环境后的扩散以及种群扩张具有重要影响。高温适应性的生理生化及分子机制对揭示外来物种种群扩张、种间替代和种群生态预测均有重要的理论和实践意义。木薯单爪螨原发生地为南美洲,作为一种热带土著害螨,对高温的适应性是其发生为害的重要基础,也是该螨入侵我国热区后,可用于预测其分布、扩散和成灾机制的最基础资料,对其高温适应性研究可为进一步探讨木薯单爪螨入侵我国后的生态适应性、种群扩张机制以及有效控制措施的制定奠定基础。因此,本研究在确定了适宜木薯单爪螨生长发育与繁殖的温度、湿度和光照条件,以及木薯品种后,采用人工气候箱室内饲养、分光光度计终点测定法及荧光定量PCR (RTFQPCR)方法探讨了高温(30℃、33℃、36℃、39℃和42℃)对木薯单爪螨发育与繁殖、保护酶(PPO、POD、AsA-POD、CAT和SOD)活性及其基因相对表达量以及热激蛋白Hsp70基因相对表达量的影响,旨在从生物学、生理学及分子生物学层面上初步阐明木薯单爪螨的高温适应性及其机制,为进一步探讨木薯单爪螨高温适应性的分子及其生态适应性机理提供理论依据和前期工作基础。主要研究结果如下：1.木薯单爪螨发育与繁殖的适宜温度为24～27℃,适宜湿度为70%～90%,适宜光照为12～14h,适宜木薯品种(系)为CM1210-10。在此适宜条件下,木薯单爪螨的发育历期为,卵期4.17±0.29d,幼螨期2.67±0.29d,前若螨期2.17±0.29d,后若螨期2.33±0.29d,卵到成螨期11.34±0.29d,后代孵化率为100%,平均每雌产卵量为49.00±1.00粒,后代性比为89.36%±1.53,成螨寿命为19.00±0.50d。2.高温对木薯单爪螨的发育与繁殖存在显著影响。42℃为木薯单爪螨发育与繁殖的极限温度,在42℃高温条件下,木薯单爪螨的卵不能孵化,不能完成后续发育；在30℃～39℃高温条件下,木薯单爪螨的发育历期和雌成螨寿命随着温度的升高逐渐缩短,发育历期由25℃(对照)时的11.34d缩短为39℃高温时的9.83d,成螨寿命由19.00d缩短为3.00d；后代产卵量随着温度升高而显著减少,由49.00粒减少为7.00粒；在30℃～33℃范围内,木薯单爪螨的卵均可完全孵化,但在36℃和39℃高温下,木薯单爪螨的后代卵孵化率极显著下降,仅分别为32.05%和13.42%；在30℃和33℃下,木薯单爪螨F1代雌性百分率分别为76.67%和76.40%,显著低于其它温度；36℃和39℃下木薯单爪螨F1代雌性百分率无显著变化。3.卵、幼螨、前若螨、后若螨和成螨受高温胁迫不同时间后均显著影响木薯单爪螨的生长发育与繁殖。各龄螨经高温胁迫不同时间后置于室温发育,其随后各龄期的发育历期随着胁迫温度的升高和胁迫时间的延长而显著延长,卵孵化率、后代雌性百分率与后代雌成螨寿命均显著下降；随着龄期的增加,木薯单爪螨对高温的耐受性有所增强,但前若螨对高温的耐受能力稍大于后若螨,其在经36℃及以上高温胁迫不同时间后,无论在后代产卵量上还是卵的孵化率上均高于后若螨。各龄螨对高温的耐受性大小表现为成螨>前若螨>后若螨>幼螨>卵。4.高温胁迫显著影响木薯单爪螨多酚氧化酶(PPO)、过氧化物酶(POD)、抗坏血酸过氧化物酶(AsA-POD)和过氧化氢酶(CAT)活性。木薯单爪螨各龄螨经高温胁迫不同时间后,体内PPO、POD、AsA-POD和CAT活性发生了不同程度的变化。高温胁迫后,前若螨和成螨PPO、POD和AsA-POD活性以及成螨CAT活性在1h时达最高,随后随处理时间的延长,活性逐渐降低,但前若螨经42℃极端高温胁迫1h后体内CAT活性降为最低。而胁迫幼螨和后若螨不同时间后,其体内PPO、POD、AsA-POD和SOD活性与对照均无显著差异,但经极端高温胁迫1h后幼螨体内CAT活性显著升高,而后若螨CAT活性却显著降低。各龄螨经高温胁迫后,SOD活性与对照相比无显著变化。5.高温胁迫1h是木薯单爪螨的最敏感时间。木薯单爪螨各龄螨经高温胁迫1h后,不但其产卵量、后代孵化率均显著降低,且体内酶活性发生了显著变化,尤其是42℃极端高温处理1h。经42℃高温处理各龄螨1h后平均每雌产卵量降低为幼螨9.00粒,前若螨18.67粒,后若螨11.33粒,成螨21.00粒；后代卵孵化率降低为幼螨30.82%,前若螨57.33%,后若螨54.11%,成螨61.19%。PPO、POD、AsA-POD和CAT活性分别为对照的0.92、5.85、2.9和1.56倍(幼螨),1.91、1.00、1.00和0.14倍(前若螨),0.92、0.99、1.00和0.17倍(后若螨),1.81、12.52、9.00和1.75(成螨)。6.极端高温胁迫1h后,木薯单爪螨成螨PPO、POD、AsA-POD、CAT和SOD基因的相对表达量显著升高,分别高达对照的5.19、4.38、152.23、321.42和3.41倍,且与其酶活性变化趋势存在一致性,酶活性值分别升高为对照的1.81、12.52、9.00、1.75和1.14倍。保护酶POD、AsA-POD、CAT和SOD可能在木薯单爪螨应对高温胁迫中具有协同防止氧自由基毒害及提高对高温胁迫的抵抗作用,PPO基因表达和酶活性的升高可能在提高木薯单爪螨对高温的免疫力中发挥着一定作用。7.极端高温胁迫1h后,木薯单爪螨成螨热激蛋白基因Hsp70的相对表达量显著升高,为对照的56.85倍。说明热激蛋白Hsp70可能在木薯单爪螨高温适应性中具有重要作用,其基因表达量的升高可能与木薯单爪螨的高温适应性有关。更多还原

【Abstract】 Cassava green mite Mononychellus mcgregori is a newly invasive pest mite of cassava in China. It mainly harms the green components of buds, young leaves and stems of cassava, with damaged leaves deformed by yellow blot, which results in leaf chlorosis, dried branches and even plant death in serious cases, a40～60%reduction in production in the case of serious damage. It was firstly reported in Danzhou, Hainan province, China in2008and has since become more widespread. It is known to occur currently in important cassava-planting areas such as Guangxi, Guangdong and Yunnan where it causes significant damage to cassava. Since it’s invasive in tropical area of China, the populations have increasing quickly. The adaptation to its invasive environment are the fundamental for the population expansion of M. mcgregori, although the population growth have something to do with the deficiency of natural enemies and that it dose not have other pest mite species with the same ecological niche to compete with. The continually survey showed that, the green mite were very difficult to collected in2008, but in2010, the mites developed to vast areas of land and occurrence not only in spring and winter, but also in summer and autumn. Therefore, the cassava green mite M. mcgregori has gradually developed adaptation to the climate of tropical area of China.Whether a alien species can survive the temperature stress after its invasive had important influence on its dispersal in the new environment as well as its population expansion. The physiological, biochemical as well as the molecular mechanism for an alien species to adapt the high temperature have important significance in theory and practice to reveal the population expansion, population replacement as well as the population prediction. The origin M. mcgregori is South America and as a tropical native mite, the adaptation to high temperature was the significant foundation for its occurrence and damage and was also the most fundamental material for us to predict the distribution, disperse as well as the population occurrence mechanism of the pest mite after its invasion in China. The study on the adaptation to high temperature of M. mcgregori can lay the groundwork for investigate further the ecological adaptation, population expansion mechanism as well as the forming of its effective control and management measures.Therefore, to understand more of the mechanisms behind the invasion and spread of M. mcgregori in China, as well as the adaptation of this mite to a tropical environment, the mainly ecological factors affected the development and reproduction of the mite were firstly studied, then the effects of high temperature exposure on the development and reproduction, protective enzyme activities and the relative expression of protective enzyme genes as well as the heat shock protein70(Hsp70) gene via spectrophotometry method and realtime fluorescence quantitative PCR(RTFQ PCR) were also studied, so that the thermostability as well as its mechanisms were revealed based on the biology, physiology as well as the molecular. The results will provide a theoretical basis and precondition for further research on the molecular mechanisms of the thermostability and ecological adaptability of M. mcgregori.The main results are as follows:1. The suitable temperature, humidity, photoperiod and host for the development and reproduction of M. mcgregori were24～27℃,70%～90%,12～14h and the local cassava cultivars "Bread", respectively. Under the above suitable conditions, the development duration of M. mcgregori were as follows:eggs durations was4.17±0.29d, larvae duration was2.67±0.29d, protonymphs duration was2.17±0.29d, detonymph duration was2.33±0.29d and eggs to adults duration was11.34±0.29d. The eggs hatchability was100%, the fecundity of per female was9.00±1.00, the female percentage was89.36%±1.53%and the female adult mite lifespan was19.00±0.50d.2. High temperature significantly affected the development and the reproduction of M. mcgregori.42℃was the extremely high temperature for the development and the reproduction of M. mcgregori and under the temperature the eggs could not hatch and not fullfill their subsequent development. With the increasing of the temperature from30℃to39℃, the development duration and the female adult mite lifespan decreased significantly. The development duration decreased fromll.34d (25℃) to9.83d (39℃), the female adult mite lifespan decreased from19.00d (25℃) to3.00d (39℃) and the fecundity of per offspring female decreased significantly, from49.00(25℃) to7.00(39℃). The eggs could completely hatch when temperature increase from30℃to33℃. but the hatchability significantly decreased at36℃with32.05%and39℃with13.42%, respectively. At24℃, the female percentage was the highest with89.36%, but significantly decreased30℃with76.67%and33℃with76.40%, respectively. In addition, there was no difference in the female percentage at36℃and39℃.3. Exposure of eggs, larvae, protonymphs, deutonymphs and adults to high temperature significantly affected the development and reproduction of M. mcgregori. With the increase of the temperature and exposure time, the development duration significantly prolonged, but the eggs hatchability, female percentage and longevity of female adults were all significantly decreased. The tolerance of M. mcgregori to high temperature was some improved with the increased of instars, however, the tolerance of pronymphs were some higher than deutonymphs, for that not only the fecundity but also the offspring hatchability of the exposed protonymphs were higher than deutonymphs. The tolerance of all the development stage to high temperature were ranked in the sequence:adults> protonymphs> deutonymphs> larvae> eggs. However, they usually recovered more slowly after the high temperature exposure termination.4. Exposure of eggs, larvae, protonymphs, deutonymphs and adults to high temperature for different time significantly affected the activities of polyphenol oxidase (PPO), peroxidase (POD), ascorbate peroxidase (AsA-POD) and catalase (CAT) in M. mcgregori. Activities of PPO, POD, AsA-POD and CAT in protonymphs and adults some varied with the increase of temperatures and exposure time, and were the highest at1h-exposure, and then decreased except in larvae in which the CAT activity decreased to the lowest after exposed to the extremely high temperature (42℃) for1h. Howerve, there were no significant difference in changes of activities of PPO, POD, AsA-POD and superoxide dismutase(SOD) in larvae and deutonymphs.Howerer, the activities of CAT increased significantly in larvae and decreased in detonymphs after exposed to the extremely high temperature (42℃) for1h. No significantly changes were observed in SOD activities after all the development stages were exposed to high temperature for different time.5. Exposure to high temperatures for1hour was the most sensitive time for M. mcgregori. The fecundity and the offspring eggs hatchability decreased significantly and the the activities of PPO, POD, AsA-POD and CAT in protonymphs and adults changed significantly after exposure to high temperatures for1h. After exposure to extremely high temperatures of42℃for1h especially, the fecundity per female decreased to9.00for larvae,18.67for protonymphs,11.33for deutonymphs and21.00for adults; the offspring hatchability decreased to30.82%for larvae,57.33%for protonymphs,54.11%for deutonymphs and61.19%for adults; and the activities of PPO, POD, AsA-POD and CAT were0.92,5.85,2.9and1.56times (larvae),1.91,1.00,1.00and0.14times (protonymphs),0.92,0.99,1.00and0.17times (deutonymphs),1.81,12.52,9.00and1.75(adults).6. The relative expression of PPO, POD, AsA-POD, CAT and SOD gene via RTFQ PCR were increased in adults after they were exposed to extremely high temperature (42℃) for1h, which increased5.19,4.38,152.23,321.42and3.41times that of the control, respectively, and were in accord with their enzyme activities. These results suggested that protective enzymes of POD, AsA-POD, CAT and SOD were associate with the thermostability of M. mcgregori and they might have synergistic effect on protect the mite against toxic oxygen intermediates and perhaps were associate with the resistance of high temperature in M. mcgregori.The increasing of PPO activities as well as its gene expression were associate with the immunity improvement of the mite to high temperature stress. These results provide a theoretical basis and precondition for further research on the molecular mechanisms of the thermostability and ecological adaptability of M. mcgregori.7. The relative expression of heat shock protein70(Hsp70) gene via realtime fluorescence quantitative PCR (RTFQ PCR) were increased to56.85times that of the control after exposure adults to extremely high temperature (42℃) for1h. It suggested that the elevated expression of HSP70gene was related to the adaptation of M. mcgregori to high temperature.更多还原