杜仲梦尼夜蛾幼虫肠道可培养细菌的组成及其功能

doi:10.11707/j.1001-7488.20201113

摘要/Abstract

摘要：

目的: 研究杜仲梦尼夜蛾幼虫肠道可培养细菌的种类组成及其功能，揭示其对宿主昆虫生长发育、食物消化和营养利用方面的影响。方法: 采用传统的微生物分离培养方法和16S rRNA序列分析进行幼虫肠道细菌的分离和鉴定，采用二硝基水杨酸法(DNS法)结合高效液相色谱法(HPLC)研究肠道菌对生物大分子(纤维素、果胶和淀粉)及杜仲叶成分的降解作用，最后通过抗生素处理，研究肠道菌对幼虫生长发育和营养指标的影响。结果: 利用传统微生物分离培养方法从幼虫肠道分离鉴定出10株不同的细菌，DNA序列显示它们分别为变形菌门、厚壁菌门和放线菌门的10个属的细菌，包括短波单胞菌属、肠球菌属、假单胞菌属、鞘氨醇单胞菌属、克吕沃尔菌属、不动杆菌属、莫拉菌属、土壤杆菌属、微杆菌属和节杆菌属。这些菌株对生物大分子(纤维素、果胶和淀粉)均有不同程度的降解作用，并影响杜仲叶中物质的变化。饲喂抗生素(1 μg·mL^-1氯霉素)消除肠道菌后可影响幼虫的生长发育，显著降低幼虫的化蛹率和低龄幼虫的取食和消化能力。结论: 从杜仲梦尼夜蛾幼虫肠道分离获得10株不同的细菌，它们在杜仲梦尼夜蛾幼虫的生长发育、食物消化、营养物质利用等方面发挥着重要作用。初步确定了杜仲梦尼夜蛾幼虫能够取食和利用含胶的杜仲叶片，为自身生长发育提供营养物质，与其肠道微生物有着密不可分的关系。

关键词: 杜仲梦尼夜蛾, 幼虫肠道细菌, 可培养微生物分离, 鳞翅目

Abstract:

Objective: Gut microbiota can help host insects to degrade indigestible ingredients and harmful substance in food. The ability to digest and utilize the leaves of Eucomnia ulmoides in Orthosia songi (Lepidoptera: Noctuidae) larvae may be related with gut microbiota. Therefore,this study aims to investigate the gut bacterial composition of larvae in O. songi,so as to reveal the effects of intestinal bacteria on the growth and development,food digestion,nutrition utilization of host insects. Method: The traditional methods of microbial isolation and culture and the sequencing of 16S rRNA gene were used to isolate and identify the gut bacteria from the larvae. The dinitrosalicylic acid (DNS) and HPLC methods were used to verify the degradation of biomacromolecules (cellulose,pectin and starch) and chemical substances in leaves of Eucommia ulmoides by gut bacteria. Then the effects of gut bacteria on the growth and nutritional utilization of larvae were investigated by feeding 1 μg·mL^-1 chloramphenicol,an antibiotic. Result: Ten different bacterial strains were isolated from the larval guts by traditional microbial isolation and culture methods,and their DNA sequences showed that they belonged to ten genera in Proteobacteria,Firmicutes and Actinobacteria,including Brevundimonas,Enterococcus,Pseudomonas,Sphingomonas,Kluyvera,Acinetobacter,Moraxinella,Agrobacterium,Microbacterium, and Arthrobacter. These strains were able to cause degradation to biomacromolecules (cellulose,pectin and starch) in varying degrees,and affect the changes of some substances in Eucommia ulmoides leaves. The feeding test with 1μg/mL chloramphenicol affected the growth and development of larvae,and significantly reduced the pupation rate of larvae and the feeding and digestive ability of the younger larvae. Conclusion: Ten bacterial strains were isolated from the larval guts of O. songi,and they play important roles in the growth and development,food digestion and nutrient utilization of O. songi larvae.

Key words: Orthosia songi, gut bacteria of larvae, isolation of culturable microbe, Lepidoptera

中图分类号:

S718.83

郭彤彤,朱铭强,李晋鹏,郭新荣,贺虹. 杜仲梦尼夜蛾幼虫肠道可培养细菌的组成及其功能[J]. 林业科学, 2020, 56(11): 124-133.

Tongtong Guo,Mingqiang Zhu,Jinpeng Li,Xinrong Guo,Hong He. Composition and Functions of Cultural Bacteria in the Larval Guts of Orthosia songi(Lepidoptera: Noctuidae)[J]. Scientia Silvae Sinicae, 2020, 56(11): 124-133.

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菌株编号 Strain code	Blast结果中一致性最高的菌株(GenBank登录号) (一致性) BLAST with the highest identity (GenBank accession No.) (Identity)	序列登录号 Accession No.
LT1	肠球菌属Enterococcus sp. (KJ124182.1)(99%)	MK571186
NT2	土壤杆菌属Agrobacterium sp. (KT230833.1)(99%)	MK571187
NA3	不动杆菌属Acinetobacter sp. (KY910080.1)(99%)	MK571188
NT4	莫拉菌属Moraxella sp.(FJ380954.1)(99%)	MK571189
NA5	假单胞菌属Pseudomonas sp. (JX675235.1)(99%)	MK571190
NA6	克吕沃尔菌属Kluyvera sp. (GQ915083.1)(99%)	MK571191
NT7	短波单胞菌属Brevundimonas sp. (KF735808.1)(99%)	MK571192
NA8	鞘氨醇单胞菌属Sphingomonas sp. (EU130999.1)(99%)	MK571193
NA9	节杆菌属Arthrobacter sp.(KU060821.1)(99%)	MK571194
NT10	微杆菌属Microbacterium sp. (EU558969.1)(99%)	MK571195

幼虫龄期 Larval instars	发育历期 Developmental duration/d		头宽 Head width/mm		体长 Body length/mm		体质量 Body mass/mg
幼虫龄期 Larval instars	抗生素处理 Antibiotics treatment	CK	抗生素处理 Antibiotics treatment	CK	抗生素处理 Antibiotics treatment	CK	抗生素处理 Antibiotics treatment	CK
1龄 1st instar	4.10±0.84a	3.93±0.87a	0.4±0.11a	0.42±0.12a	2.94±0.44a	3.52±0.37a	0.48±0.13a	0.58±0.18a
2龄 2nd instar	2.87±0.94a	2.73±0.74a	0.57±0.12a	0.66±0.29a	4.88±1.08a	5.74±1.05a	1.28±0.40a	2.24±0.44a
3龄 3rd instar	2.70±0.70a	2.57±0.73a	0.56±0.06a	0.87±0.06a	6.87±0.97a	7.48±0.66a	8.10±4.41a	9.07±3.03a
4龄 4th instar	3.80±0.76a	3.57±0.78a	1.03±0.08a	1.13±0.09a	11.00±1.35a	11.05±1.18a	24.85±3.55a	25.94±5.38a
5龄 5th instar	3.80±0.85a	3.47±0.71a	1.70±0.14a	1.74±0.16a	14.93±2.28a	15.09±1.83a	78.79±35.36a	83.07±31.95a
6龄 6th instar	5.60±0.86a	5.43±1.22a	2.47±0.05a	2.48±0.05a	21.92±2.46a	22.24±1.76a	180.02±44.06a	190.9±35.16a

幼虫龄期 Larval instars	幼虫相对生长率RGR		食物利用率ECI		食物转化率ECD		消耗指数CI		近似消耗率AD
幼虫龄期 Larval instars	抗生素处理 Antibiotics treatment	CK	抗生素处理 Antibiotics treatment	CK	抗生素处理 Antibiotics treatment	CK	抗生素处理 Antibiotics treatment	CK	抗生素处理 Antibiotics treatment	CK
1龄 1st instar	0.34±0.05b	0.49±0.07a	0.08±0.05b	0.21±0.02a	0.08±0.02b	0.42±0.01a	4.45±2.29a	4.4±2.23a	0.94±0.25a	0.91±0.27a
2龄 2nd instar	0.17±0.07b	0.57±0.07a	0.07±0.07b	0.31±0.01a	0.12±0.04b	0.58±0.07a	2.44±1.25a	1.86±0.65a	0.58±0.12a	0.53±0.13a
3龄 3rd instar	0.15±0.04b	0.48±0.02a	0.15±0.00b	0.47±0.04a	0.25±0.02b	0.83±0.25a	1.03±0.45a	1.02±0.47a	0.59±0.25a	0.57±0.25a
4龄 4th instar	0.07±0.01b	0.19±0.02a	0.08±0.01b	0.2±0.01a	0.19±0.00b	0.49±0.03a	0.88±0.40a	0.86±0.38a	0.44±0.00a	0.42±0.00a
5龄 5th instar	0.11±0.01a	0.13±0.01a	0.1±0.01a	0.16±0.00a	0.18±0.02b	0.29±0.05a	1.06±0.55a	0.8±0.34a	0.59±0.00a	0.57±0.03a
6龄 6th instar	0.04±0.01a	0.09±0.01a	0.04±0.00a	0.09±0.01a	0.26±0.01a	0.43±0.01a	1.18±0.35a	0.97±0.44a	0.24±0.00a	0.21±0.01a

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