【作者】 王洪亮；

【导师】 仵均祥；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2007， 博士

【摘要】 滞育是大多数昆虫长期进化选择的结果,是适应不良环境的重要生理生态对策之一,是种群能够持续繁衍和发展的一种特殊表现形式。麦红吸浆虫是典型的滞育型昆虫,具有滞育率高、滞育能力强、高度延长滞育和2次滞育的特点,特有的滞育多态现象也是引起间歇性、局域性暴发成灾的主要原因之一。麦红吸浆虫广泛分布于我国小麦主产区,在我国历史上曾多次发生。近年来,麦红吸浆虫在历史上的老灾区猖獗成灾的势头得到了有效遏制,但适生区却进一步扩大,并在河北、北京等地频发成灾,危害呈加重趋势。本研究从山梨醇和几丁质含量、调控糖醇化合物代谢的海藻糖酶和山梨醇脱氢酶活性、3种保护酶的活性、调节几丁质含量的几丁质酶活性和不同滞育状态基因组DNA的多态性、滞育基因的克隆等方面,研究了麦红吸浆虫的滞育机理,对阐明小麦吸浆虫的成灾规律、准确测报及大面积的综合治理具有重要的意义。1.滞育麦红吸浆虫山梨醇含量的变化麦红吸浆虫从脱离麦穗进入土中滞育到当年10月中旬,一直未检测到山梨醇。至10月下旬,幼虫体内的山梨醇才被检测到,但含量很低。此后,随着滞育时间的延长和温度降低,山梨醇的含量快速增加。表明山梨醇高的含量可以作为衡量麦红吸浆虫越冬滞育状态的指标。同期滞育裸露幼虫的山梨醇含量较结茧幼虫略高,但二者之间无明显差异;2年滞育的麦红吸浆虫较当年滞育的同期阶段高。2.滞育麦红吸浆虫海藻糖酶和山梨醇脱氢酶活性的变化海藻糖酶活性在滞育年周期中呈现出低-高-低-高的变化趋势,表明海藻糖酶活性的高低与麦红吸浆虫不同滞育阶段的能量转换有密切关系;同期滞育裸露幼虫海藻糖酶活性较结茧幼虫稍高,但变化趋势相同;2年滞育的麦红吸浆虫海藻糖酶活性较当年滞育的略低,不同滞育年限麦红吸浆虫海藻糖酶活性无明显差异。不同滞育时期麦红吸浆虫从脱离麦穗到当年11月以前,一直未检测到山梨醇脱氢酶的活性。从翌年元月上旬起,检测到山梨醇脱氢酶的活性。从翌年2月底至4月中旬,山梨醇脱氢酶的活性急剧增加。因此,高的山梨醇脱氢酶活性可作为麦红吸浆虫打破滞育的标志。同期滞育裸露幼虫的山梨醇脱氢酶活性较结茧幼虫稍高,但二者之间差异不显著;2年滞育麦红吸浆虫的山梨醇脱氢酶活性仍能被检测到,但维持在一个较低水平。3.滞育麦红吸浆虫保护酶系的活性变化特点不同滞育时期麦红吸浆虫之间的SOD、POD和CAT活性存在着显著的差异性。SOD活性在滞育年周期中呈现出低-略高-低-高的变化规律,SOD活性高低与麦红吸浆虫滞育状态密切相关,可作为衡量麦红吸浆虫滞育状态的指标;而POD和CAT活性在在滞育年周期中呈现出高-低-高的变化规律,表明在滞育期间,POD和CAT对清除滞育麦红吸浆虫体内的自由基起到了一定的作用。麦红吸浆虫越冬滞育阶段比越夏滞育阶段的CAT活性高,表明了麦红吸浆虫滞育期间CAT活性与温度有关。落土滞育前麦穗幼虫的3种保护酶活性均为最高。在入土到翌年破土而出的整个滞育期间,活性都受到不同程度的抑制,均呈下降趋势。同期滞育的裸露幼虫SOD、CAT和POD酶活性较结茧幼虫略高,但二者的酶活性没有显著的差异性。当年滞育的麦红吸浆虫SOD、POD和CAT活性较2年滞育的高,不同滞育年限之间活性有明显差异性。4.滞育麦红吸浆虫的几丁质酶活性和几丁质含量变化特点在不同滞育时期麦红吸浆虫之间的几丁质酶活性存在着明显的差异性。从入土到翌年2月底,在滞育的裸露幼虫体内一直未检测到几丁质酶的活性,仅在翌年4月上旬测出微弱的活性。而在4月中下旬又未能测到酶活性;圆茧幼虫在当年的9月中旬就检测到几丁质酶的活性,随后很快就又测不到酶活性,直到翌年2月下旬才再一次测到酶活性。之后,在4月上旬,酶活性快速上升,4月中下旬又开始下降;这表明在圆茧幼虫体内,几丁质酶表现出持续高活性的时间较短。同期滞育的裸露幼虫和圆茧幼虫的几丁质酶活性有显著差异性。除未检测出活性的阶段外,圆茧幼虫体内的几丁质酶活性远高于裸露幼虫;2年滞育的麦红吸浆虫几丁质酶活性较当年滞育的高。在不同滞育年限之间,裸露幼虫的几丁质酶活性无明显差异,而在圆茧幼虫中却存在着明显的差异性。滞育裸露幼虫体内几丁质含量在滞育年周期中变化幅度较小,不存在显著差异性。而滞育圆茧幼虫的几丁质含量却有着明显的差异性。从麦穗幼虫转变为圆茧幼虫,几丁质含量急剧升高,但在翌年2月下旬即将打破滞育时又快速下降,表明几丁质也是一种能量贮存形式,与滞育期间的能量转换有关;在滞育年周期中,圆茧幼虫的几丁质含量远高于裸露幼虫,两者之间的差异显著;2年滞育的几丁质含量较同期当年滞育的高,但二者之间差异不明显。5.滞育麦红吸浆虫DNA的多态性应用5个重复序列引物,通过PCR扩增试验,研究了麦红吸浆虫DNA的多态性。结果表明:(1)5个重复序列引物共扩增了104条核酸带,分子量介于202-1163bp之间;(2)不同滞育群体的麦红吸浆虫的平均相似性指数范围在0.573-0.936之间,在种群3和种群4之间,最大的平均相似性指数是0.936,在群体1、3、4、5和群体6、7、8之间平均相似性指数较高(均超过0.81);(3)不同滞育状态麦红吸浆虫群体的遗传变异大部分来自群体间,产生于群体间的变异为62.41%,来自群体内的变异为37.59%。6.单头麦红吸浆虫RNA的提取和滞育基因的克隆首次从单头麦红吸浆虫中抽提出RNA。28S和18S条带清晰,无拖尾,麦穗幼虫的28S和18S条带亮且宽,而进入滞育状态的圆茧幼虫RNA条带较细并且发暗。表明处于生长发育状态的幼虫,代谢速度较快,转录的RNA的量较多。但以基因组DNA和cDNA为模板,均未扩出麦红吸浆虫的DH基因。表明麦红吸浆虫的DH基因可能与其它昆虫的的同源性较差,根据目前已知其他昆虫滞育基因序列设计的引物很难扩增出麦红吸浆虫的滞育基因。更多还原

【Abstract】 The wheat blossom midge (WBM), Sitodiplosis mosellana (Gehin) is one of most damaged pests distributing in the main wheat-growing regions of China and other areas of world. Many times of outbrokes and plagues caused great losses to wheat production have been recorded in history., Outbreaks and plagues of WBM have been controlled in broader area in China during recently past decade years, but WBM displayed a trend to extend their habitats at certain wheat-growing areas such as Hebei, Beijing, Shandong and has a potential threat to local wheat yield. Diapause, as a result of evolution and adapting selection of most insects fitting to natural environment changes, is one of the most important ecological and physiological strategies. It is also a special survival form which makes insects to be sustainable under bad environmental conditions for reproduction and development of their population. The wheat blossom midge, Sitodiplosis mosellana (Gehin) is one of typical diapause insects with high diapause ratio, powerful diapause potential, extremely prolonged diapause time and re-diapause. It is reported that diapause polymorphism of WBM is one of key factors affecting population fluctuation with occasional, regional and uneven dumpling characteristics in the filed.In this paper, sorbitol content, activities of trehalases, sorbitol dehydrogenase (SDH) and 3 kinds of protective enzymes, SOD, POD, CAT were determined and DNA polymorphism was studied with 5 repeat sequence primers during various diapause stages of WBM. The main results are as follows.1. Contents of sorbitol in S. mosellana were undetected in larvae collected from wheat heads or from soil before mid-October of the same year. It could be detected with a lower level in larvae from on 25th October of the same year. Sorbitol contents were increased rapidly as diapause time becom longer and temperature gradually decreased afterward. This change suggested that large amounts of sorbitol were accumulated in order to cope with cold in winter. High contents of sorbitol were regarded as index of WBM in over-wintering stage. No significant difference of sorbitol contents was found between non-cocooned larvae and cocooned larvae with the same diapause stage, but the non-cocooned larvae had a lightly high sorbitol content than the cocooned larvae. Sorbitol contents of WBM larvae with 2-year diapause period were also detectable and more than that of larvae with 1-year diapause period at the same phase.2. Activities of trehalase and SDH in WBM larvae were measured during its various diapause stages. Results showed that activity of trehalase in grown larvae collected from wheat heads was higher than that of larvae from soil. It decreased quickly during initial period of larvae entering soil. Uplifted in mid-September. Dropped again in late October of the same year and increased steeply in late February of the next year in turn. There was a close relationship between high activity of trehalase and energy transition for WBM larvae in different diapause stage. Activity of SDH in WBM larvae was not detectable from wheat mature to November. But it increased sharply from January to April. High SDH activiy was regarded as a index for WBM that was in breaking diapause stage. Activities of both trehalase and SDH in Non-cocooned larvae were higher than that of cocooned larva in the same diapause stage. An identical change trend was seen on activity of trehalase and SDH. No significant difference of activity in either trehalase or SDH was found between one-year and two-year diapause larvae.3. Activities of SOD, CAT and POD in WBM larvae were measured and result showed that there exited a remarkable difference of SOD activities in WBM larvae with the different diapause stage. SOD activities were reduced gradually after grown larvae fell into soil from wheat ears. There were not significant differences of SOD activities between no-cocooned larvae and cocooned larvae with the same diapause stage. A similar change trend of SOD activities was seen in both no-cocooned larvae and cocooned larvae. Change gradient of SOD activities of WBM larvae with one-year diapause was higher than that of WBM larvae with two-year diapause.Activities of POD and CAT in mature larvae living on the wheat head were higher than that of diapause larvae in the soil. They dropped quickly after larvae entered into soil from wheat heads. There was not a significant difference of SOD, POD and CAT activity between no-cocooned larvae and cocooned larvae at the same diapause stage. Activities of SOD, POD and CAT in WBM larvae with one-year diapause were slightly higher than that in WBM larvae with two-year diapause, there was a obvious differencc between lavae with one-year diapause and with two-year diapause.4 Chitinase activities and chitin contents were measured in larvae of the wheat midge during mature and diapause stage. The result indicated that there exited remarkable difference for chitinase activities of S. mosellana at the different diapause stage. The chitinase activitives in the cocooned larvae were higher than that of no-cocooned larvae. Chitinase activities of WBM larvae with one-year diapause were slightly higher than that in WBM larvae with two-year diapause. There existed significant differences for chitinase activities of the cocooned larvae of S. mosellana between in the one-year diapause period and in the two-year diapause period, but not existed in the no-cocooned larvae.Chitin contents of no-cocooned larvae in the different diapause stage changed slightly, not existed remarkable differences among them. But the circumstances of the cocooned larvae were opposite to no-cocooned larvae, changed steeply. Chitin contents of cocooned larvae were higher than that of no-cocooned larvae. No obvious difference of were found for chitin contents between in the one-year diapause period and in the two-year diapause period.5 The DNA polymorphism of WBM at different diapause stage was analyzed with 5 repeat sequence primers by the PCR technique. Results showed that (1) five primers generated 104 DNA bands with molecular weights from 202 to 1163bp; (2) Average similarity index of WBM at different diapause stage was in 0.573-0.936; (3) Genetic variation of WBM at different diapause stage was mainly produced from intergroups; (4) different diapause populations of the WBM may be divided into three main categories on the basis of genetic distances. The paper is the first report of the quantified genetic diversity among different diapause population of the wheat blossom midges.6. RNA was extracted from single larva of WBM firstly. An effective method of RNA extracting from the single WBM was proposed. 28S and 18S bands were all clear and not tail phenomena. The two bands of the wheat-ear larvae were lighter and wider than that of cocooned larvae, which suggested slower metabolic activities and lower quantities of RNA transcribe in the diapause larvae. Clone of diapause hormone gene of WBM was experimently carried out from genomic DNA and cDNA, but failed to get any result. There maybe has a lower homology of the diapause hormone gene between WBM and other insects. So it was difficult to design primers according to current DH gene sequence from other insects so as to amplify DH gene of WBM.更多还原