【作者】 吴杰；

【导师】 胡福良；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 熊蜂是一类多食性的社会性昆虫,是多种虫媒植物,特别是豆科、茄科及一些濒危植物的重要传粉者。熊蜂与蜜蜂同属于一个科,都以花蜜和花粉为主要食物,但熊蜂授粉特性比蜜蜂优越,它们更适应低温、低光照和高湿环境；此外,熊蜂还具有声震授粉特性。20世纪80年代末,欧洲一些国家的野生熊蜂的人工周年繁育技术获得突破,使得熊蜂的人工授粉应用取得重大进展。目前,地熊蜂(Bombus terrestris)已应用于多种温室作物的授粉,取得良好的经济效益和生态效益,成为欧洲一些国家优良的授粉蜂种。由于商业利益,国外熊蜂繁育公司对熊蜂的人工繁育技术进行严格保密,使得进口熊蜂成本特别高,另外进口熊蜂可能会对本土熊蜂资源造成严重危害。因此,90年代末,我国科学家也开始了熊蜂的人工繁育和授粉应用技术研究。目前,已从本国熊蜂种类中筛选出6种具有授粉应用价值的蜂种,小峰熊蜂即是其中的一个优良授粉蜂种。本论文通过采取实地调查、分子生物学技术、实验室饲养及温室实验等技术手段,研究了小峰熊蜂的地理分布、形态学特征、系统发育地位、生物学特性、温室传粉生态学等内容,旨在为我国熊蜂资源的保护及合理的人工利用研究提供科学依据。本文主要研究内容和结果如下：1.通过实地调查及查阅文献和中国科学院动物所馆藏标本,研究了小峰熊蜂的地理分布；并根据实地调查记录的数据,了解了小峰熊蜂采访植物的种类。结果表明,小峰熊蜂属于古北区、东洋区和日本区蜂种,分布于中国、朝鲜、日本和俄罗斯,在我国主要分布于华北地区、东北地区和青藏高原东部,在海拔50-3500m之间都有分布,其中,尤以海拔1000-2500m之间分布最为丰富。小峰熊蜂采访植物种类涉及到11个科28个种,偏向于采访菊科、豆科、蔷薇科和唇形科植物。2.利用细胞色素氧化酶Ⅰ基因(COI基因)序列对9个亚属13种熊蜂的系统发育进行预测,结果表明,该基因序列能够反应亚属级的系统进化关系,在Bombus s.str亚属内B.hypocrita和B.patagiatus这两种蜂分化最晚,而B.ignitus分化最早。3.实地调查结果显示,华北地区小峰熊蜂的活动规律为：3月中下旬蜂王开始出蛰,5月上旬第一批工蜂就会羽化出房,7月群势达高峰,雄蜂和子代蜂王羽化出房,蜂王出房后1个星期左右开始交配,9月下旬至10月上旬老蜂王、工蜂和雄蜂逐渐解体消亡,10月上旬蜂王进入休眠状态。4.比较了不同的花粉(新鲜杏花粉、干杏花粉、新鲜油菜花粉及新鲜杏花粉和新鲜油菜花粉的混合花粉)对实验室饲养条件下野生蜂王产卵率、第1只工蜂出房时间、蜂王产卵前期时间、蜂群成群前期时间、子代蜂王数量的影响。结果显示,4种花粉(组合)对野生蜂王产卵率和第一只工蜂的出房时间影响差异不显著,但对蜂王的产卵前期时间、蜂群成群前期时间、子代蜂王数量影响差异显著。其中,新鲜杏花粉、干杏花粉和混合花粉有利于缩短蜂群成群前期时间,提高成群率；油菜花粉不利于蜂群饲养开始阶段,但其与杏花粉混和利于子代蜂王的产生。5.比较了不同饲料(5种花粉和2种糖类组合饲料及不添加花粉的2种糖类饲料,共12组)对无王小峰熊蜂工蜂群生长发育的影响。结果表明,只饲喂糖类食物时,工蜂寿命显著短于有花粉的饲料组,而且工蜂不产卵。含有花粉的各饲料组之间工蜂寿命存在显著差异,饲喂玉米花粉的工蜂寿命小于其他4种花粉组。在工蜂产卵前期时间和卵杯数量方面,10种含有花粉的饲料组之间差异不显著；但在产卵量、幼虫拖出数、雄蜂出房时间和子代雄蜂数量等方面,各饲料组之间差异较大。白砂糖和杏花粉组的产卵量最高；杏花粉组和茶花粉组的幼虫拖出数量显著低于其它花粉组;油菜花粉组和杏花粉组的雄蜂出房时间显著短于其它花粉组;杏花粉组的子代雄蜂数量显著高于其它饲料组。6.研究了4种常见的设施作物农药(莫比朗、清源保、世高、绿乳铜)对3种熊蜂[小峰熊蜂、密林熊蜂(B. patagiatus)和火红熊蜂(B. pyrosoma)]的毒性。结果显示,4种农药对小峰熊蜂的致死率显著低于对密林熊蜂和火红熊蜂的致死率,对密林熊蜂和火红熊蜂致死率则没有显著差异。莫比朗对小峰熊蜂的内吸毒性LD50(0.0028μg.a.i/蜂)显著高于对火红熊蜂(0.0023μg.a.i/蜂)和密林熊蜂(0.0021μg.a.i/蜂)的内吸毒性LD50。莫比朗对熊蜂的致死率显著高于其他3种农药和空白对照组,清源保对熊蜂的致死率显著高于空白对照组,但是世高和绿乳铜对熊蜂的致死率与空白组相比没有显著差异。本研究结果表明,3种试验蜂种中,小峰熊蜂对4种农药的毒性敏感性最低,同时本研究结果认为设施农业中,当应用熊蜂进行授粉时,可施用低毒农药如世高和绿乳铜代替毒性大的农药。7.对温室杏园小峰熊蜂的活动变化及其影响因子进行观测。结果表明,整个花期熊蜂的采集活动比较活跃,小峰熊蜂日采集活动呈现两个明显的高峰期,分别在9：30～11：00和13：30～15：30。熊蜂采集活动变化与湿度、花蜜浓度呈负相关,而与温度、光强、花蜜重量、花粉量呈正相关,但只有与湿度、花蜜量、花粉量的相关性达到显著水平。多重逐步回归分析表明湿度和花粉量是影响熊蜂采集活动变化的主要因子,具有显著的直接效应,而其它影响因子起间接效应。8.比较了小峰熊蜂和意大利蜜蜂(Apis mellifera ligustica)在温室授粉时的传粉行为,及不同传粉方式花粉活力及柱头上花粉量的差异。结果表明,意大利蜜蜂和小峰熊蜂采集行为存在很大的差异,小峰熊蜂出巢温度和工作温度都低于意大利蜜蜂,意大利蜜蜂访花速度比小峰熊蜂快。经小峰熊蜂、意大利蜜蜂和人工传粉三种授粉方式传粉的柱头上花粉数量有显著差异,其中,小峰熊蜂传粉后柱头花粉量最多,意大利蜜蜂授粉次之,人工授粉最少。意大利蜜蜂、小峰熊蜂和人工采集的花粉活力分别为79.95%、53.25%和33.30%。蜂类采集的花粉活力显著大于人工采集的花粉活力；蜜蜂采集的花粉活力大于熊蜂采集的花粉活力,这可能是蜜蜂的进化程度较高,其对花粉识别能力较强的结果。综上所述,本论文研究通过连续3年对我国小峰熊蜂的分布进行调查、查阅相关文献和馆藏标本,了解了小峰熊蜂的地理分布、形态学特征、分布海拔高度、年生活史及采访植物种类；同时,利用条形码序列COI基因片段对9个亚属13种熊蜂的系统发育进行预测,清楚地显示了小峰熊蜂的系统发育地位。本论文还对人工饲养小峰熊蜂饲料进行了研究,明确了2种花粉对小峰熊蜂蜂群生长发育的影响；并对5种花粉和2种糖类组合饲料对工蜂生长发育影响进行了研究,为合理组配小峰熊蜂饲料奠定了理论依据；对小峰熊蜂温室授粉生态学进行了研究,探索了熊蜂对4种温室常用农药的敏感性,为利用熊蜂为温室农作物授粉奠定了基础。更多还原

【Abstract】 Bumblebee is a polyphagous social insect. It is an important pollinator for many entomophilous pollination plants, especially for Leguminosae, Solanaceae and endangerous plant pollination. Bumblebee and honeybee belong to the same family (Hymenoptera, Apidae), and both feed on neatar and pollen. However, Bumblebee has the advantage over honeybee with that it can pollinate flowers at lower temperature, lower light intensity, and higher humidity. More importantly, bumblebee can provide the buzz-pollination that is crucial for crops like tomatoes.In the end of 1980s, techiques for year-round rearing of bumblebee were successfully created, with which the use of bumblebee for greenhouse pollination comes true. In Europe Bombus terrestris has been imported by many countries for greenhouse crop pollination. It is an excellent pollinating species for many crops such as tomatoes, eggplants. Bumblebee pollination brings significant economic, social and ecological benefits.Bumblebee producers have developed their specific rearing systems, which are kept as primarily secrets. As a result, the cost to import bumblebee colonies for crop pollination is extremely high. What is more important is that the exotic species may lead to severe negative effects on native species. Therefore, the techniques for artificially rearing bumblebees and for their pollination application have been researched in China since the end of 1990s. Now, six species of bumblebees have been selected for pollination application from native species. B. hypocrita is one of the excellent species.In the present research, the distribution, morhpology, phylogeny, biology and pollination ecology of B. hypocrita were studied, with methods combing field survey, molecular biology, laboratory rearing, greenhouse experiments, etc.. The aim of the research was to provide a theoretical basis for protecting bumblebee resources as well as the application of bumblebee pollination. The main content and results are listed as follows.1. The distribution of B. hypocrita was studied by field survey, investigation on the specimens collected by Institute of Zoology, Chinese Academy of Sciences and on the data reported in references. The results showed that B. hypocrita belongs to the Oriental, Japanese, Palaearctic Regions species, distributed in China, Korea, Japan and Russia. In China, it was mainly distributed in the east of Tibetan Plateau, Northern China and Northeastern China, with elevation between 50 m to 3500 m above sea level, in which the elevation 1000 m to 2500 m above see level had more richful ditribution. B. hypocrita foraged on plants of 28 species, which were classified into 11 families, with preference on Composite, Rosaceae, Leguminosae and Labiatae family。2. The barcoding sequence of COI gene was used to analyse the phylogeny of 13 species bumblebees belonging to 9 subgenuses. The results showed that the sequence of COI gene can reflect evolution relations of species in the same subgenus. The subgenus of Bombus s. str is monophyletic, in which B. ignitus diverged earliest and B. hypocrita and B. patagiatus diverged latest.3. The annual activity of B. hypocrita in North China was studied by field survey. In the middle of March, queens emerge from their hibernacula and search for suitable nest sites. Once a suitable site is located, the queen constructs her brood nest and deposits eggs. The first worker emerges in the first ten-days period of May. The population of the colony reaches the peak in July, after which queens and males are produced. Young queens mate about one week after emergence. In the end of September and the first ten-days period of October, the colony dwindles and callapse; newly mated queens start to hibernate over the winter.4. The effects of different kinds of pollen on the development of B. hypocrita colonies were studied. The results showed that there was no significant difference on the effects of different kinds of pollen on the proportion of egg-laying queens, or on the timing of the first worker emergence. However, different pollen diets did have significantly different effects on the time that queens took to start to oviposit, the success rate for colony founding, and the number of young queens produced. All three diets consisted of apricot pollen were better than fresh rape pollen for colony initiation and queen production. Fresh and dried apricot pollen had similar effects on colony development rates, but fresh pollen was better for producing more young queens. Overall, the highest numbers of young queens were produced with the mixture of fresh apricot and rape pollens. Therefore, we recommended feeding queen bumble bees with fresh apricot pollen in the initial stage of colony founding and then changing diet to mixed pollen.5. The effect of the different diets on the reproductive success of B. hypocrita workers was investigated by feeding queenless micro-colonies workers with combination of sugar or honey with six kinds of pollen. The results showed that the workers just fed with sugar did not lay eggs and had significantly lower longevity. Feeding different kinds of pollen also caused significant difference on the longevity of workers. The longevity of workers fed on corn pollen was significantly lower than those of workers fed on other pollens. Feeding different kinds of pollen did not have significant difference on the effects on worker’s pre-oviposition time and number of egg cups, but had significant difference on the effects on the numbers of eggs, larval ejection, duration of male development and the number of progeny. Among all the treatments, sucrose and apricot pollen treatment delivered the highest number of eggs, apricot and tea pollen treatments caused lower number of larval ejection, rape and apricot pollen treatments resulted in shorter male development time, and apricot pollen treatment contributed to higher number of progeny. It was concluded that the worker colony development was significant affected by different feeds. So, we should provide with different food for bumblebee colony at different development phases.6. The effects of four pesticides (Mosplian, Kingbo, Score and Lvrtong), applied commonly in greenhouses in China, on three bumblebee species (B. hypocrita, B. ignitus and B. patagiatus) were studied. The results showed that the mortality of B. hypocrita after contacting the four pesticides was significantly lower than that of B. patagiatus and B. ignitus, but there was no significant difference between the mortality of B. patagiatus and B. ignitus. The oral toxicity LD50 value of Mosplian to B. hypocrita (0.0028μg.a.i./bee) was significantly higher than that to B. ignitus (0.0023μg.a.i./bee) and B. patagiatus (0.0021μg.a.i./bee). As long as the three species were concerned, it can be concluded that B. hypocrita was least susceptible to the four pesticides. The mortality of each bumblebee species after contact with Mosplian was significantly higher than that for the other three pesticides and the control group. The mortality of bumblebee in Kingbo group was significantly higher than that of control group, but Score and Lvrtong exposed groups showed no significant increase in mortality when compared to the control group. It can therefore be concluded that the pesticides differ in their negative influences on bumblebees, and that Mosplian is the most harmful.7. Foraging activities of B. hypocrita and their influence factors were studied. The results showed that bumblebee foraging activity was lively during the all flowering period. The daily foraging activity of bumblebee appears two peaks. The first peak was at 9:30～11:00 and the second at 13:30～15:30. Foraging activities of B. hypocrita was negatively correlated with humidity and nectar concentration and positively with temperature, light intensity, nectar weight and pollen quantity. Only humidity, nectar weight and pollen quantity reached significant difference level. Multiple selection regression analysis revealed that humidity and pollen quantity were direct factors and others were indirect.8. Foraging behavior of B. hypocrita and Apis mellifera was significantly different. The foraging temperature of B. hypocrita was lower than that of A. mellifera. However, the foraging frequence of A. mellifera was faster than that of B. hypocrita. The amount of pollen grain on the stigma was significantly different among three pollination methods. The order of the amount of pollen grain was as follows:B. hypocrita> A. mellifera> manual pollintion. The pollen activity collected by B. hypocrita, A. mellifera and hand were 79.95%,53.25% and 33.30%, respectively. The activity of pollen collected by A. mellifera was higher than that collectey by B. hypocrita. The reason for this could be that honeybee was more advanced than bumblebee in the aspect of evolution thus was able to recognize pollen with higher activity.To sum up, a complete distribution map of B. hypocrita was drawn according to the data collected from field survey in the last 3 years, the analysis of the specimens stored in Institute of Zoology, Chinese Academy of Sciences and data reported in references. The geographical distribution, morhpology, distribution elevation, annual activity and kinds of foraging plant of B. hypocrita were also studied by field survey. The phylogeny of B. hypocrita was predicted by analysis on the sequence of COI gene, which showed the evolution position of B. hypocrita in Bombus s. str clearly. Moreover, the effects of diets on colony development and reproductive success of queenless workers were also studied, which provided important informations on artificial rearing of B. hypocrita in laboratory. At last, the sensitivity of B. hypocrite, B. patagiatus and B. pyrosoma to four pesticides commonly used in greenhouse was compared and the pollination ecology of B. hypocrita was studied. The results have provided a theoretical basis for protecting bumblebee resource and artificial pollination application.更多还原