【作者】 李晓娟；

【导师】 骆有庆； 贺伟；

【作者基本信息】 北京林业大学 ， 森林保护， 2010， 博士

【摘要】 光肩星天牛(Anoplophora glabripennis Motschulsky)属鞘翅目,天牛科,沟胫天牛亚科,星天牛属,是我国重要林木蛀干害虫。在我国广泛分布,主要危害杨属(Populus spp.)、柳属(Salix spp.)、榆属(Ulmus spp.)和槭属(Acer spp.)的树木,对我国林业造成巨大的损失。国内外众多学者对光肩星天牛寄主选择以及光肩星天牛纤维素酶均有大量的研究,但光肩星天牛体内纤维素酶与寄主选择的关系尚未明确。为了明确光肩星天牛体内纤维酶与寄主选择的关系,本文选取四种不同寄主来源(新疆杨、箭杆杨、合作杨和漳河柳)光肩星天牛幼虫和成虫、取食不同树种(臭椿、毛白杨、合作杨、旱柳和复叶槭)光肩星天牛成虫、取食原寄主树种与非原寄主树种光肩星天牛成虫、不同虫态光肩星天牛、不同龄期光肩星天牛幼虫等为研究对象,采用纤维素酶活性测定、纤维素酶同工酶检测、肠道组织结构观察比较等研究方法。研究结果如下：(1)以新疆杨、箭杆杨、合作杨和漳河柳四种寄主来源光肩星天牛成虫和幼虫为研究对象,测定其肠道内三类纤维素酶活性,结果表明,不同寄主来源光肩星天牛雌与雄成虫之间的Cx酶、C1酶以及β-萄糖苷酶活性均无显著性差异,同种寄主来源光肩星天牛雄成虫的Cx酶活性显著高于雌成虫,同种寄主来源光肩星天牛雌雄成虫的C1酶和β-萄糖苷酶活性无显著性差异；不同寄主来源光肩星天牛幼虫Cx酶、C1酶以及β-萄糖苷酶活性均无显著性差异。以聚丙烯酰胺凝胶电泳法检测上述四种寄主来源光肩星天牛成虫和幼虫Cx酶同工酶,结果显示,不同寄主来源幼虫、雌成虫以及雄成虫的Cx酶同工酶带型和迁移距离无明显差异,同种寄主来源雌雄成虫之间亦无明显差异。在本研究结果中,三类纤维素酶活性和同工酶谱具有高度的一致性,分别显示出不同寄主来源光肩星天牛成虫纤维素酶在酶活性和Cx酶同工酶方面是没有明显差异的,这表明,光肩星天牛纤维素酶与幼虫寄主树种之间无明显的关联性。(2)取合作杨来源光肩星天牛成虫,分别饲以臭椿、毛白杨、合作杨、旱柳和复叶槭五种不同树种的新鲜枝条,3d后检测其纤维素酶活性,研究结果为,取食复叶槭雌成虫Cx酶活性显著高于雄成虫,取食其它树种雌雄成虫Cx酶活性无显著性差异,对照雄成虫(饥饿3d)Cx酶活性显著高于雌成虫；取食各树种雌雄成虫之间C1酶活性均无显著性差异；取食复叶槭雌成虫β-葡萄糖苷酶活性显著高于雄成虫,取食其它树种雌雄成虫β-葡萄糖苷酶活性无显著性差异。取食复叶槭雌成虫Cx酶活性显著高于取食臭椿与对照雌成虫,与取食其它树种无显著性差异,取食旱柳、合作杨雌成虫Cx酶活性显著高于对照,与取食其它树种无显著性差异；取食各树种雄成虫Cx酶活性均显著低于对照,取食复叶槭、合作杨和臭椿雄成虫Cx酶活性无显著性差异,取食旱柳、毛白杨、合作杨和臭椿雄成虫Cx酶活性无显著性差异。取食各树种雌成虫以及雄成虫之间C1酶活性和β-葡萄糖苷酶活性均无显著性差异。纤维素酶作为一种诱导性酶,本研究发现,光肩星天牛成虫取食与否以及取食材料的不同都对其纤维素酶活性大小有很大影响,尤其是对于Cx酶。由此可见,光肩星天牛纤维素酶与成虫寄主树种选择之间有一定的关联性。(3)取旱柳和合作杨两种寄主来源光肩星天牛成虫,分别饲以旱柳、合作杨和复叶槭三种寄主树种的新鲜枝条,3d后检测其纤维素酶活性,结果为,旱柳和合作杨来源光肩星天牛取食三种寄主树种光肩星天牛雌雄成虫之间Cx酶和β-葡萄糖苷酶活性均无显著性差异,旱柳和合作杨来源光肩星天牛雌成虫以及雄成虫之间Cx酶活性和β-葡萄糖苷酶活性均无显著性差异,除了合作杨来源光肩星天牛成虫取食旱柳雄成虫β-葡萄糖苷酶活性显著低于取食合作杨和复叶槭雄成虫。两种寄主来源光肩星天牛,取食原寄主与其它寄主树种之后,雌成虫之间和雄成虫之间纤维素酶活性都无明显差异,除了合作杨来源雄成虫取食复叶槭和合作杨之后,表现出比取食旱柳高的β-葡萄糖苷酶活性。这是光肩星天牛成虫对原寄主树种的“记忆效应”,在纤维素酶活性方面的体现,还是由天牛本身取食状态所导致,有待于进一步研究。(4)检测光肩星天牛蛹和未出树体成虫体内纤维素酶活性,结果显示,光肩星天牛蛹体内各纤维素酶活性均极其微弱,在0.06μmol(葡萄糖)·g-1(FW)·h-1以内,雌雄蛹之间Cx酶、β-葡萄糖苷酶和C1酶活性均无显著性差异；未出树体光肩星天牛雌雄成虫之间Cx酶、β-葡萄糖苷酶和C1酶活性均无显著性差异。树体内光肩星天牛成虫体内纤维素酶活性已较蛹体内有很多增加,但仍然没有达到从羽化孔出树体后光肩星天牛成虫体内纤维素酶活性的水平,研究中也发现树体内光肩星天牛成虫纤维素酶活性大小波动比较大,尤其是Cx酶和β-葡萄糖苷酶,这表明光肩星天牛羽化之后,体内纤维素酶的功能以及分布是一个不断完善的过程。(5)取食寄主树种与取食人工饲料光肩星天牛幼龄幼虫纤维素酶活性均显著高于老熟幼虫,这有可能是由于老熟幼虫相对取食量降低而导致,也有可能是由于随着天牛幼虫的生长,其体内的纤维素酶活性到达一个稳定的水平,但体重却不断增加,从而使相对酶活性降低。(6)解剖光肩星天牛幼虫以及雌雄成虫,取出肠道并区别前中后肠,分别测定其纤维素酶活性,结果显示,光肩星天牛幼虫90.4%的Cx酶活性和93.4%的β-萄糖苷酶活性分布在中肠；光肩星天牛雌成虫65.4%的Cx酶活性和94.2%的β-萄糖苷酶分布在中肠,31.5%的Cx酶活性分布在后肠；光肩星天牛雄成虫89.0%的Cx酶活性和92.0%的β-萄糖苷酶活性分布在中肠；在光肩星天牛雌成虫后肠组织结构中观察到有囊状结构。这使得作者对光肩星天牛体内纤维素酶的来源,有了一个新的推测,即光肩星天牛幼虫和雄成虫体内的纤维素酶均由自身分泌,而光肩星天牛雌成虫对天然纤维素的消化,有后肠共生微生物的参与。对于这一推测的验证则需要进一步的研究。更多还原

【Abstract】 The Asian Longhorn Beetle (ALB), Anoplophora glabripennis (Motschulsky) (Coleoptera:Cerambycid ae:Lamiinae:Lamiini) is one of the most important forest pests and widely distribute in China, and caused great losses to the forest. Poplars (Populus spp.), willows (Salix spp.), maples (Acer spp.) and elm (Ulmus spp) are the primary host tree species.Because of the serious threat posed by A. glabripennis, a large amount of research of cellulase and host selection has focused on this species in recent years, but the relationship of cellulase and host selection is unclear.To investigate the relationship between cellulase activity and host selection, A. glabripennis larvae and adults from four different host tree species (Populus alba L. var. bolleana Bunge; P. nigra var. thevestina (Dode)Bean; P. simonii Carriere×P. alba var. pyramidlis cv. opera Hsu., and Salix matsudana f. lobato-glandulosa Fang & Liu), adults fed on five different tree species (Ailanthus altissima Swingle, P. tomentosa Carriere, P. simonii Carriere×P. alba var. pyramidlis cv. opera Hsu., S. matsudana Koidz, and Acer negundo L.), adults fed on original and non-original host tree species, insect in different state and larvae in different instars were selected.The enzyme activities of endoglucanase, exoglucanase and (3-glucosidase in the intestines of the insects were measured, the endoglucanase isozymes were detected with a CMC-incorporated polyacrylamide gel, and the intestinal tissue paraffin section of A. glabripennis were also been observed. The results show that:(1) Adults and larvae from four different host tree species (P. alba L. var. bolleana Bunge; P. nigra var. thevestina (Dode)Bean; P. simonii Carriere×P. alba var. pyramidlis cv. opera Hsu., and S. matsudana f. lobato-glandulosa Fang & Liu) were chosen, and three kinds of cellulase activities in the intestines of the insects were measured.The results were:there were no significant differences in the activities of endoglucanase, exoglucanase andβ-glucosidase in males and females emerging from different host tree species. When adults emerged from the same host tree species, there were no significant differences in the activities of exoglucanase andβ-glucosidase between males and females, but the activity of endoglucanase in males was clearly higher than that in females. There were no significant differences in endoglucanase, exoglucanase andβ-glucosidase activities between larvae from different host tree species.The endoglucanase isozymes were detected with a CMC-incorporated polyacrylamide gel, the patterns and migration of endoglucanase isozymes of males and females emerging from different host tree species are clearly not different, neither are they different when emerging from the same hosts. No difference in larvae from different host tree species.From the results, it can be concluded that there exists strong consistency between the enzyme activities of cellulase and isozymes patterns. There were no significant differences between the cellulase activities in A. glabripennis from different host tree species, as well as the endoglucanase isozymes patterns.This show that, there is no obvious relevance between cellulase and the larvae host tree species in A. glabripennis.(2) Adults were emerging from opera poplar, fed them with fresh branches of A. altissima Swingle, P. tomentosa Carriere, P. simonii Carriere×P. alba var. pyramidlis cv. opera Hsu., S. matsudana Koidz, and A. negundo L. respectively for three days.Cellulase activities were measured. The results were:Significant differences were found in the activities of endoglucanase between male and female fed on A. negundo L. and control, low activity of endoglucanase was occurred in both males and females fed on A. altissima Swingle. There were no significant differences in exoglucanase activities between males and females. Significant differences were noted inβ-glucosidase activity between male and female fed on A. negundo L., but there were no significant differences between male and female fed on other trees.As a kind of induced enzymes, this study found that, the kind of trees which the adults fed on has great effect on the activity of cellulase in adults, especially for endoglucanase. Thus, there is some relevance between cellulase and host tree selection in A. glabripennis adults.(3) Adults were emerging from P. simonii Carriere×P. alba var. pyramidlis cv. opera Hsu. and S. matsudana Koidz, fed them with fresh branches of P. simonii Carriere×P. alba var. pyramidlis cv. opera Hsu., S. matsudana Koidz and A. negundo L. respectively for three days.Cellulase activities were measured. The results were:In A. glabripennis adults from S. matsudana Koidz fed on different host tree species, there are no significant differences in the enzyme activities of endoglucanase between male and female fed on three host trees, neither inβ-glucosidase. No significant differences in the enzyme activities of endoglucanase andβ-glucosidase was found between male or female fed on three host tree species. In A. glabripennis adults from P. simonii Carriere×P. alba var. pyramidlis cv. opera Hsu. fed on different host tree species, there are no significant differences in the enzyme activities of endoglucanase between male and female fed on three host trees, neither in P-glucosidase. No significant differences in the enzyme activities of endoglucanase and P-glucosidase was found between male or female fed on three host tree species, except the endoglucanase activity in male fed on S. matsudana Koidz is lower than that fed on A. negundo L. and P. simonii Carriere×P. alba var. pyramidlis cv. opera Hsu..(4) In the research, extremely weak cellulase activity was detected in A. glabripennis pupae, not more than 0.06μmol (glucose)·g-1 (FW)·h-1, no significant differences in the enzyme activities of endoglucanase, exoglucanase and P-glucosidase between male pupae and female pupae. In the inner adults, no significant differences in the enzyme activities of endoglucanase, exoglucanase andβ-glucosidase between male and female.There was a great fluctuation in the cellulase activity, this may show that the cellulase system in A. glabripennis adults is gradually perfect after adult emergence till coming out of the emergence hole. (5) Larvae fed on host tree species under natural growth situation had a significant reduction in both endoglucanase andβ-glucosidase activities. The activities of endoglucanase in the first and second instars are significantly higher than the followed three instars, and the activities ofβ-glucosidase in the former three instars are significantly higher than the followed two instars. This trend also occurred in the larvae fed on artificial diet. The low cellulase activity of matured larvae may caused by low relative comsumption rate or stable cellulase activity level with increasing weight.(6) Intestine of A. glabripennis larvae, female and male were tookout, foregut, midgut and hindgut were distinguished and the cellulase activity was measured respectively, the results show that:90.4% endoglucanase activity and 93.4%β-glucosidase activity of larvae were distributed in midgut.65.4% endoglucanase activity and 94.2%β-glucosidase activity of female were distributed in midgut, and 31.5 %endoglucanase activity distributed in hindgut.89.0%endoglucanase activity and 92.0%β-glucosidase activity of male were distributed in midgut. Cystic structure was discovered in the intestinal tissue paraffin section of female A. glabripennis. This makes cellulase source in A. glabripennis, with a new assumption that cellulase in A. glabripennis larvae and male adult was secreted by itself, and A. glabripennis adult female digest natural cellulose with the participation of symbiotic microorganism in there hindgut. For verification of this speculation requires further study.更多还原