【作者】 黄琼；

【导师】 周定刚；

【作者基本信息】 四川农业大学 ， 特种经济动物饲养， 2010， 博士

【摘要】 昆虫被誉为当今地球上尚未被充分利用的最大生物资源之一。我国昆虫种类繁多、资源丰富,合理开发利用我国的昆虫资源,无疑会带来良好的经济、社会和生态效益。黄粉虫作为我国传统的饲用和食用昆虫之一,其食物来源广泛、生活力强、易于人工饲养、世代周期短、饲养成本低、营养丰富,长期以来被广泛用作饲养畜禽和其它一些特种经济动物的饲料(或饵料),现已广泛应用于农业、畜牧业、食品和医疗保健方面,具有极高的开发利用价值和广阔的市场前景。近年来,我国黄粉虫养殖规模日益扩大,山东、河北等地已形成以黄粉虫养殖和综合开发利用为核心的产业链,而种虫质量退化已成为制约黄粉虫产业进一步持续健康发展的重要瓶颈。围绕这一严重制约黄粉虫产业发展的瓶颈问题,本文对黄粉虫的品系选育及2种不同品系黄粉虫的生长发育性状、繁殖能力、抗逆性、营养价值及主要逆境协迫相关同工酶进行了较为系统地研究,同时也对这2种品系黄粉虫主要逆境协迫相关基因的克隆与表达进行了初步研究。其主要研究结果及结论如下：1.经过连续12代的自然选育,获得了遗传性状稳定的黄、黑2种色型黄粉虫。2.黑色型黄粉虫比黄色型黄粉虫发育更快、更整齐。黑色型黄粉虫幼虫共历经12-15龄,黄色型黄粉虫幼虫共历经12-17龄,但这2种色型黄粉虫幼虫所历经的虫龄数均以14龄居多。黄、黑2色型黄粉虫幼虫虫龄数为14的比例分别为27%和53%；其中,黑色型黄粉虫幼虫历期明显短于黄色型幼虫,虫龄数按14计,黄、黑2色型黄粉虫的幼虫历期分别为154.3±7.9d和134.1±3.2d。此外,这2种色型黄粉虫卵和蛹的历期分别约为7d和10d；它们的孵化率、化蛹率分别约为83%和97%,羽化率均为81%以上。3. 2种色型黄粉虫同日龄幼虫的存活率、体重及体重增长率均差异不显著；但黄色型黄粉虫幼虫对饲料的平均利用效率明显高于同日龄黑色型幼虫。其中,黄色型黄粉虫幼虫的平均饲料消化率、转化率和利用率(%)分别为66.5±0.1、52.3±1.2和33.6±0.7,而黑色型黄粉虫幼虫的平均饲料消化率、转化率和利用率(%)则分别为62.1±0.2、47.8±0.2和29.1±0.2。4.常规饲养条件下,2种色型黄粉虫成虫的雌、雄性比无显著差异,但黄色型雌成虫的累积产卵量明显高于黑色型雌成虫。并且,相同试验条件下,2种色型黄粉虫雌成虫的初始产卵日龄、产卵历期和产卵量日变化规律也基本一致。常规饲养条件下,2种色型黄粉虫雌成虫的初始产卵日龄为4日龄,产卵高峰在羽化后的第14-44d；黄、黑2色型黄粉虫的累积产卵量分别为625.5±25.5粒／雌和529.9±17.4粒/雌。5. 2种色型黄粉虫的耐热、耐寒性差异明显。耐热性试验表明,黑色型黄粉虫幼虫、蛹及成虫对试验高温(45℃)的耐受性明显高于同日龄的黄色型幼虫、蛹和成虫；同时,耐寒性试验表明,黑色型黄粉虫幼虫和蛹对试验低温(-25℃)的耐受性也明显高于同日龄黄色型幼虫和蛹,但相同日龄的2种色型黄粉虫成虫对试验低温(-25℃)的耐受性差异不显著。6. 2种色型黄粉虫抗药性差异显著。本文以不同浓度梯度的苦参碱杀虫剂为供试药剂,对黄、黑2色型黄粉虫幼虫和成虫的抗药性进行了研究,结果表明,黑色型黄粉虫幼虫和成虫对苦参碱的耐受性明显高于同日龄的黄色型幼虫和成虫。在处理后72h,苦参碱对黄、黑2色型黄粉虫60日龄幼虫的毒杀中浓度(LC50)分别为107.63mg.ML-1和178.63mg.ML-1；对黄、黑2色型黄粉虫7日龄成虫的毒杀中浓度(LC50)分别为95.89mg.ML-1和162.54mg.ML-1.7. 2种色型黄粉虫的抗病性也差异明显。本文以脂多糖(LPS)作为供试致病因子,对黄、黑2色型黄粉虫的抗病性进行了初步研究,结果表明,注射5μL相同浓度(0.5-2.0mg·ML-1)的LPS溶液后,黑色型黄粉虫幼虫、蛹和成虫的存活率明显高于同日龄的黄色型幼虫、蛹和成虫。由此可见,黑色型黄粉虫对LPS的耐受性明显高于黄色型黄粉虫。8.营养成分分析表明,2种色型黄粉虫的营养成分丰富,具有极高的营养和开发利用价值。2种色型黄粉虫幼虫、蛹和成虫的蛋白质含量约为干重的48%-63%,其中含有18种氨基酸,必需氨基酸含量约占总氨基酸的48%-49%；同时,还富含油酸、亚油酸、亚麻酸等多种不饱和脂肪酸及K、Na、Ca、Mg、Fe、Cu、Mn、Zn、Se等多种矿物质和微量元素。此外,2种色型黄粉虫的营养组成还各具特点：黄色型黄粉虫幼虫和成虫的粗蛋白、总氨基酸及必需氨基酸含量明显高于黑色型幼虫和成虫,而黑色型黄粉虫幼虫、蛹和成虫的粗脂肪含量又明显高于黄色型幼虫、蛹和成虫。9. 2种色型黄粉虫主要逆境协迫相关同工酶电泳结果表明,黄、黑2色型黄粉虫同一发育阶段的同工酶酶谱具有相似相异性。除SOD同工酶外,这2种色型黄粉虫其余同工酶POD、EST、COD、MPO和DPO)的酶谱均存在差异,其中尤以它们的EST、POD和MPO同工酶酶谱差异最明显,可考虑作为黄粉虫种下分类的工具酶。10. 2种色型黄粉虫主要逆境协迫相关同工酶酶活检测表明,在常规饲养条件下,黑色型黄粉虫的多数供试同工酶活性均大于同日龄的黄色型黄粉虫；同时,在试验低温(-25℃)、高温(45℃)或杀虫剂(苦参碱)协迫下,2种色型黄粉虫的多数保护酶和解毒酶均被抑制,而它们的防御酶却被激活。此外,在上述逆境因子协迫下,相同日龄的黄、黑2色型黄粉虫,它们的同一种保护酶(或解毒酶)活性抑制率及同一种防御酶活性增长率均有所不同。11.黄、黑2色型黄粉虫逆境协迫相关基因核心片段克隆与序列分析表明,2种色型黄粉虫间,细胞色素氧化酶亚基Ⅰ(COI)、酚氧化酶原(PPO)和抗冻蛋白(AFP)基因的序列同源性都很高,但它们的这3个基因序列中仍存在一定的碱基差异。12.黄、黑2色型黄粉虫幼虫的热休克蛋白(HSP)基因和抗冻蛋白(AFP)基因表达研究表明,在试验低温(或高温)协迫下,黑色型黄粉虫幼虫的AFPmRNA(或HSPmRNA)表达量均显著提高,而同日龄的黄色型黄粉虫幼虫的AFPmRNA(或HSPmRNA)表达量却无显著增加；同时,与同日龄黄色型黄粉虫幼虫相比较,黑色型黄粉虫幼虫的AFP(或HSP)对试验低温(或试验高温)表现出更强的应答。更多还原

【Abstract】 Insects are the largest biological resources today on the earth which haven’t yet been fully utilized. Considering the fact that there are a great variety of insects and rich in natural resources of insects in China, it will undoubtedly produce good benefits on economy, society and ecology with a rational development and utilization of our country’s insects resources. Tenebrio molitor is one of traditional feed and edibile insects in China. Not only there are large quantities of food sources to raise T. molitor, but also it has a short generation cycle and strong living ability. In addition, it is rich in nutritional ingredients, easy to raise and characterized by low feeding cost. It had been only used as feed stuff or bait for livestock, poultry and other special economic animals in the past, and nowadays it has been widely applied into agriculture, animal husbandry, food and medical care. So it has a high value for development and utilization and has a bright market future. Recently, the scale of the industry in breeding T. molitor has been increasing gradually and a new industry chain has emerged, in such provinces as Shangdong and Hebei, the core of which is raising and comprehensive utilization of T. molitor. However the quality of the varieties of T. molitor is degenerating, which has become the bottleneck restricting the further sustained and healthy development of the industry. Around the problem, the selection of varieties of T. molitor was studied. The growth and development traits, the reproductive capacity, the resistance to stress and the nutritional value of 2 kinds of color patterns of T. molitor were determined. The main stress-related isozymes of 2 kinds of color patterns of T. molitor were studied systematically. Furthermore, the preliminary researches on cloning and expression of the main stress-related genes of the 2 kinds of color patterns of T. molitor were carried out, too. The main results and conclusions were given as follows:1. The yellow and black color pattern of T. molitor with good genetic stability were obtained through natural selection of 12 continual generations.2. The black color pattern of T. molitor grew faster and more orderly than the the yellow color pattern. The black-color-pattern larvae of T. molitor went through 12-15 instars in all, while the yellow-color-pattern larvae went through 12-17 instars. But most of the yellow and black color pattern of larvae went through 14 instars. There were 27% larvae with 14 instars in the yellow-color-pattern and 53% in the black-color-patern. And the developmental duration of the black-color-pattern larvae was remarkably shorter than that of the yellow-color-pattern larvae. If taking the larvae with 14 instars into consideration, the developmental durations of the yellow and black color patterns of larvae were 154.3±7.9d and 134.1±3.2d respectively. In addition, the eggs and pupae developmental durations of the 2 kinds of color patterns of T. molitor were about 7d and 10d respectively. The hatching rate and pupation rate of the 2 kinds of color patterns of T. molitor were approximately 83% and 97%. The emergence rate of theirs were above 81%.3. There were no significant differences in each of the weight, growth rate and survival rate between the same days old larvae of 2 kinds of color patterns of T. molitor. But the yellow-color-pattern larvae had a significantly higher average food utilization efficiency than the black-color-pattern larvae of the same days. The average approximate digestibility, efficiency of converting digesting food and efficiency of converting ingested food(%) of the yellow-color-pattern larvae of T. molitor were 66.5±0.1,52.3±1.2 and 33.6±0.7 respectively, while those of the black-color-pattern larvae of the same days were 62.1±0.2,47.8±0.2 and 29.1±0.2 respectively.4. Under routine breeding conditions, the cumulative fecundity of the yellow-color-pattern female adults of T. molitor was obviously higher than that of the black-color-pattern female adults. But there was no significant difference in the sex ratio of the adults between the 2 kinds of color patterns of T. molitor. Under the same test conditions, the age at first egg, durations of oviposition and the daily variation of the fecundity of the yellow-color-pattern female adults were all basically the same as those of the black-color-pattern female adults. The statistical analyses showed that, under routine breeding conditions, the cumulative fecundity of the female adults of the yellow and black color pattern of T. molitor were 625.5±25.5eggs/female and 529.9±17.4eggs/female respectively. The female adults of the 2 kinds of color patterns were both 4 days after emergence when they were at first egg, and their oviposition peaks were both between the 14th and 44th day after emergence.5. There were significant differences both in heat resistance and cold resistance between the 2 kinds color patterns of T. molitor. The heat-resistance tests showed that the tolerance of the black-color-pattern larvae, pupae and adults to the high temperature (45℃) was stronger than that of the yellow-color-pattern larvae, pupae and adults of the same days. At the same time, the cold-resistance tests showed that the tolerance of the black-color-pattern larvae and pupae to the low temperature (-25℃) was stronger than that of the yellow-color-pattern larvae and pupae of the same days. But There was no significant difference between the tolerance of the adults of the 2 kinds of color patterns of T. molitor to the low temperature (-25℃).6. There was significant difference in the resistance to insecticides between the 2 kinds of color patterns of T. molitor. In the thesis, the Matrine pesticides of different concentration gradients were used to test the insecticides resistance of the larvae and adults of 2 kinds of color patterns of T. molitor. The results showed that the tolerance of the black-color-pattern larvae and adults to the Matrine pesticides was stronger than that of yellow-color-pattern larvae and adults of the same days. The LC50 values of Matrine insecticides against the 60-days-old larvae of the yellow and black color pattern were107.63mg·ML-1(72h) and 178.63 mg·ML-1(72h) respectively, while the LC50 values of Matrine pesticides against the 7-days-old adults of the yellow and black color pattern were 95.89 mg·ML-1(72h) and 162.54 mg·ML-1(72h) respectively.7. There was significant difference in the resistance to the pathogenic fator between the 2 kinds of color patterns of T. molitor, too. In this thesis, lipopolysaccharide (LPS) was used as the pathogenic fator to study the disease resistance of the 2 kinds of color patterns of T. molitor. The results showed that, after injecting 5μL LPS (0.5-2.0 mg·ML-1) solutions of the same concentrations, the survival rates of the black-color-pattern larvae, pupae and adults were significantly higher than those of the yellow-color-pattern larvae, pupae and adults of the same days. It was thus evident that the tolerance of black color pattern of T. molitor to LPS was stronger than that of the yellow-color-pattern.8. The analysis of nutritional components showed that the 2 kinds of color patterns of T. molitor have the high nutritional and utilization value since they are rich in nutrients. The protein contents of the larvae, pupae and adults of the 2 kinds of color patterns were about 48%-63% of their dry matter. They contained 18 kinds of amino acids, of which the essential amino acids occupied 48%-49%. Meanwhile, they are also rich in oleic acid, linoleic acid, linolenic acid and other unsaturated fatty acids and K, Na, Ca, Mg, Fe, Cu, Mn, Zn, Se and other minerals and trace elements. In addition, the nutritional composition of the 2 kinds of color patterns of T. molitor was characterized by that the yellow-color-pattern larvae and adults had the higher contents of crude protein, total amino acids and essential amino acids, whereas the black-color-pattern larvae, pupae and adults possesed a higher content of crude fat.9. The results of the main stress-related isozymes electrophoresis of the 2 kinds of color patterns of T. molitor showed that there were some similarities and differences in the isozymes zymograms between the yellow and black color pattern of T. molitor at the same developmental stages. Except there was no clear difference in the SOD isozyme zymograms between the 2 kinds of color patterns of T. molitor, there were obvious differeces in all the other isozymes zymograms,inciuding POD, EST, COD, MPO and DPO, between the yellow and black color pattern of T. molitor. Especially, there were so significant differences in each of EST, POD and MPO isozymes zymograms between the 2 kinds of color patterns of T. molitor that they might be considered as tool enzymes to classify the different varieties of the species.10. The determination of the main stress-related isozymes activities of the 2 kinds of color patterns of T. molitor of the same days showed that the activities of the most tested isozymes of the black color pattern were higher than those of the yellow color pattern under the routine rearing conditions. At the same time, Under each stress of the cold (-25℃), heat (45℃) and insecticides (Matrine), the protective enzymes and detoxification enzymes were mostly inhibited, whereas the defensive enzymes were activated. In addition, there were some differences both in the inhibition ratios of the same kind of protective and detoxification enzymes activities between the yellow and black color pattern of T. molitor under the above stress. And there were some differences in the growth rates of the same kind of defensive enzymes between the 2 kinds of color patterns of T. molitor under the above stress, too.11. The cloning and sequence analysis of the core fragments of some stress-related genes of the 2 kinds of color patterns of T. molitor showed that there was a high nucleotide sequence homology in each of the cytochrome oxidase subunit I(COI) cDNA, prophenoloxidase (PPO) cDNA and antifreeze protein(AFP) cDNA between the 2 kinds of color patterns of T. molitor. But there were still a few different nucleotides in each of the above core fragments between the yellow and black color pattern of T. molitor.12. The studies on the expression of HSP and AFP genes of the 2 kinds of color patterns of T. molitor larvae showed that, under the cold stress, the AFPmRNA expression of the black-color-pattern larvae had a significant up-regulation, whereas there was no visible increase in the AFPmRNA production of the-yellow-color-pattern larvae. At the same time, under the heat stress, the HSPmRNA expression of the black-color-pattern larvae had a significant up-regulation, whereas there was no visible increase in the HSPmRNA production of the yellow-color-pattern larvae. In addition, the response of the black-color-pattern larvae AFP to the testing cold-stress was significantly higher than that of the yellow-color-pattern larvae of the same days, and the response of the black-color-pattern larvae HSP to the testing heat-stress was obviously higher than that of the yellow-color-pattern larvae, too.更多还原