Studies on Free Amino Acids of Aphid <i>Schlechtendalia chinensis</i> and the Host Plant <i>Rhus chinensis</i>

WANG Chao; CHEN Xiao-ming; YANG Zi-xiang; CHEN Hang; SHAO Shu-xia; WU Hai-xia

doi:10.13275/j.cnki.lykxyj.2018.03.015

Volume 31 Issue 3

Jul. 2019

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Studies on Free Amino Acids of Aphid Schlechtendalia chinensis and the Host Plant Rhus chinensis

1.
Research Institute of Resources Insects, Chinese Academy of Forestry, Key Laboratory of Cultivating and Utilization of Resources Insects, State Forestry Administration, Kunming 650224, Yunnan, China
2.
Southwest Forestry University, Kunming 650224, Yunnan, China

Corresponding author: CHEN Xiao-ming, cafcxm@139.com ;
Received Date: 2017-12-18

Abstract

Objective to illustrate the nutrition relationship of free amino acids in aphid Schlechtendalia chinensis, horn gall and host plant Rhus chinensis during their cooperative growth. Method The contents of free amino acids in aphid, horn gall, leaf with gall, leaf without gall and CK (leaf on the tree without aphid) were detected by automatic amino acid analyzer Biochrom30+, and the total amount of free amino acids (FAA), the content of essential amino acids (EAA), the ratio of EAA/TAA and several free amino acids in the early, middle and later stages of gall growth were analyzed by variance analysis. Result All samples in different stages contained the same components, including 32 free amino acids (17 protein amino acids and 15 non-protein amino acids) and 2 other components (no ammonia). The concentration of free amino acids (FAA) from big to small was aphid (5.569) > horn gall (2.122) > leaf with gall (0.560) > leaf not gall (0.537) > CK (-0.114). The amount of EAA in aphids increased gradually with the generation increasing. The variation range of EAA/TAA from big to small was CK (1.73%)>leaf with gall (0.52%) = leaf without gall (0.52%)>horn gall (0.43%) >aphid (0.02%). And the EAA/TAA of aphids was stable at 27.91 + 0.01%. It indicated that the parasitation of aphid may decrease the variation range of EAA/TAA in the tree significantly. The contents of arginine and phenylalanine in aphids increased by 2.93 times during the growth of horn gall, and the content of proline in plant tissues also increased, and its increasing range from big to small was horn gall (0.028) > leaf with gall (0.003) > leaf without gall (-0.001) > CK (-0.018). Conclusion The aphid parasitation cannot change the amino acid components in leaves of R. chinensis, but increase the concentrations of free amino acids. The increasing trend of total free amino acids in R. chinensis is almost consistent with the increasing trend of the aphid individual number and horn gall volume during the gall growth process and that may reflect the balance between the aphid feeding and plant defense.
- Schlechtendalia chinensis,
- horn gall,
- Rhus chinensis,
- free amino acid

References

[1]	Stern D L. Phylogenetic evidence that aphids, rather than plants, determine gall morphology[J]. Proceedings of the Royal Society of London, 1995, 260(1357):85-89. doi: 10.1098/rspb.1995.0063
[2]	Stone G N, Schönrogge K. The adaptive significance of insect gall morphology[J]. Trends in Ecology & Evolution, 2003, 18(10):512-522.
[3]	Mattson J, Mattson Jr. Herbivory in relation to plant nitrogen content[J]. Annual Review of Ecology & Systematics, 1980, 11(1):119-161.
[4]	Karley A J, Douglas A E, Parker W E. Amino acid composition and nutritional quality of potato leaf phloem sap for aphids[J]. Journal of Experimental Biology, 2002, 205(Pt19):3009-3018.
[5]	朱玉永, 陈立, 王俊刚.豌豆蚜取食对蚕豆韧皮部及自身氨基酸含量的影响[J].新疆农业科学, 2014, 51(7):1284-1291.
[6]	邵淑霞.角倍蚜瘿内世代种群动态及寄主植物对蚜虫刺激的光合和脱落酸响应[D].北京: 中国林业科学研究院, 2013.
[7]	刘平.角倍蚜的生态适应性研究[D].北京: 中国林业科学研究院, 2014.
[8]	国家化肥质量监督检验中心.水溶肥料游离氨基酸含量的测定[S].农业标准, 2010.
[9]	许再福.普通昆虫学[M].北京:科学出版社, 2009.
[10]	Chen J, Zhang Y, Wang C, et al. Proline induces calcium-mediated oxidative burst and salicylic acid signaling[J]. Amino Acids, 2011, 40(5):1473-1484. doi: 10.1007/s00726-010-0757-2
[11]	马双敏, 虞泓, 李晨程, 等.植物虫瘿[J].昆虫知识, 2008, 45(2):330-335. doi: 10.3969/j.issn.0452-8255.2008.02.037
[12]	王光钺.栗瘿蜂致瘿生物学及其对寄主植物生长发育的影响[D].临安: 浙江农林大学, 2010.
[13]	Allison S D, Schultz J C. Biochemical responses of chestnut oak to a galling cynipid[J]. Journal of Chemical Ecology, 2005, 31(1):151-166. doi: 10.1007/s10886-005-0981-5
[14]	Petersen M K, Sandstrom J P. Outcome of indirect competition between two aphid species mediated by responses in their common host plant[J]. Functional Ecology, 2001, 15(4):525-534. doi: 10.1046/j.0269-8463.2001.00552.x
[15]	Koyama Y, Yao I, Akimoto S. Aphid galls accumulate high concentrations of amino acids:a support for the nutrition hypothesis for gall formation[J]. Entomologia Experimentalis Et Applicata, 2004, 113(1):35-44. doi: 10.1111/eea.2004.113.issue-1
[16]	Dsouza M R, Ravishankar B E. Nutritional sink formation in galls of Ficus glomerata Roxb. (Moraceae) by the insect Pauropsylla depressa (Psyllidae, Hemiptera)[J]. Tropical Ecology, 2014, 55(1):129-136.
[17]	Larson K C, Whitham T G. Manipulation of food resources by a gall-forming aphid:the physiology of sink-source interactions[J]. Oecologia, 1991, 88(1):15-21. doi: 10.1007/BF00328398
[18]	王小艺, 杨忠岐, 魏可, 等.昆虫翅型分化的表型可塑性机制[J].生态学报, 2015, 35(12):3988-3999.
[19]	Braendle C, Davis G K, Brisson J A, et al. Wing dimorphism in aphids[J]. Heredity, 2006, 97(3):192-199. doi: 10.1038/sj.hdy.6800863

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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角倍蚜(Schlechtendalia chinensis Bell)在盐肤木(Rhus chinensis Mill)上形成的虫瘿称为角倍，而角倍是五倍子中分布最广泛、产量最大的种类，其主要成分单宁酸，广泛应用于化工、医药、食品和轻工等行业，是我国传统的林特产品。致瘿昆虫的寄生会对寄主植物生理代谢产生影响，虫瘿是植物发育过程被昆虫改变的结果^[1-2]。氨基酸是生命营养物质蛋白质合成的必要底物，生物体内以游离状态存在的氨基酸被称为游离氨基酸。游离氨基酸是蚜虫生长发育所必需氮素的唯一来源，并以氮素为基础合成自身所需的各种物质^[3-5]，也是蚜虫与植物之间相互联系的纽带，对其营养和生长发育的调控具有重要作用。精氨酸是昆虫肌肉磷酸精氨酸的合成前体物质，苯丙氨酸是昆虫体壁暗化和硬化作用的酚、醌的来源物质^[6]；脯氨酸作为植物体内一种广泛存在的渗透调节物质，在植物响应逆境胁迫及生长发育过程中发挥多种保护功能^[7]。角倍蚜虫瘿的形成和生长是如何影响寄主盐肤木营养生理代谢的，迄今未见报道。因此，测定和分析角倍蚜、角倍和盐肤木内的游离氨基酸含量变化，有助于深入理解角倍蚜取食攻击与盐肤木防御之间的平衡关系，明确虫瘿生长和营养物质的源库转化关系。

本研究从角倍核心分布区的盐肤木林中取样，在角倍蚜虫瘿生长的初期(无翅干雌一代)、中期(无翅干雌二代)和后期(有翅干雌即秋迁蚜)，分别采集有虫瘿盐肤木上的角倍蚜、虫瘿、有虫瘿叶、无虫瘿叶和无虫瘿盐肤木上的叶片(CK)样品，采用氨基酸全自动分析仪Biochrom30+测定角倍蚜、角倍(虫瘿)、有虫瘿叶、无虫瘿叶及未结角倍的盐肤木叶片(CK)游离氨基酸种类和含量，分析虫瘿生长过程中昆虫必需氨基酸总量(EAA)、检出总量(TAA)及二者的比值(EAA/TAA)等，并对昆虫精氨酸和苯丙氨酸以及与植物逆境生理密切相关的游离脯氨酸进行分析。研究结果有助于理解角倍蚜与寄主盐肤木间的营养物质源库的转化和平衡关系，也可为角倍蚜翅型分化机理的研究提供参考。

1. 材料与方法

1.1. 试验材料

试验材料采自云南省昭通市盐津县牛寨村试验基地(104°39′ E，28°10′ N，海拔860 m)的3年生盐肤木林，为结倍树上的角倍蚜、角倍、有倍叶、无倍叶及对照树的无倍叶(CK)，选择叶片时，保持叶位一致，生长势相同，无病虫害。在林间采集新鲜样本，立即放置于液氮罐保存，带回实验室内处理和制备，采集时间和特征见表 1。

角倍生长时期 Gall growth stage	采集时间(月-日) Collection date	角倍大小 Gall size /cm³	世代 Generation	蚜虫特征 Aphid characteristic	蚜虫数量/个 Aphidnumber	蚜虫不同虫型百分比 Percentage of different type of aphid/%
初期Early stagd	07-09	0.69±0.30 f	无翅干雌一代	无翅、浅黄色	18.00±2.16 f	100
	07-27	1.06±0.30 e	无翅干雌一代	无翅、浅黄色	94.00±4.97 e	≥95
中期Middle stage	08-18	2.47±0.11 d	无翅干雌二代	无翅、深褐色	568.33±27.21 d	≥80
	09-14	15.38±0.99 c	无翅干雌二代	无翅、深褐色	7 668.33±387.63 c	≥95
后期Late stage	09-29	53.68±5.81 ab	有翅干雌(秋迁蚜)	有翅、黑色	12 973.67±210.37 b	≥80
	10-11	57.07±3.26 ab	有翅干雌(秋迁蚜)	有翅、黑色	19 372.67±325.43 a	≥95
注：角倍蚜的生活周期包括干母、无翅干雌一代、无翅干雌二代和有翅干雌(秋迁蚜)等^[6]，其中，干母和干雌在封闭的瘿内生长和繁殖，个体数量可达数千至上万头^[7]。表中小写字母表示不同样本间的差异显著性，同一列中不同字母表示差异显著(P＜0.05)。 Note: The life cycle of S. chinensis include fundatrix, first generation of wingless fundatrigenia, second generation of wingless fundatrigenia, and alate fundatrigenia (autumnmigrant aphid). The fundatrix and fundatrigenia grow and reproduce in closed gall, and its individual may increased to thousands. Note: lower-case letters represent significant difference among different samples (vertical comparison). The different letter in the same column means significant difference (P＜0.05).

Table 1. Horn gall collection date and aphid sample characteristics

1.2. 仪器与试剂

仪器：烘箱XMTD-8222(上海精宏)、全自动氨基酸分析仪Biochrom30+(Biochrom)、程序真空干燥箱VOS-300VD(EYELA)、旋转蒸发仪N-1000V-W(东京理化)、真空泵MHY-24595(北京美华仪)、水解管(硬质玻璃管35 mL)、离心机(10 000 r·min^-1以上)、0.45 μm微孔滤膜及过滤器。

试剂：柠檬酸钠、盐酸、盐酸溶液、氢氧化钠溶液、磺基水杨酸液、EDTA-Na溶液、柠檬酸钠缓冲液、34种氨基酸混合标准品(laborservice onken 5.403.151)。

1.3. 样品预处理和制备

植物叶片组织鲜样105℃杀青，85℃烘干，固体样经四分法缩分后，迅速研磨，过0.5 mm孔径筛，混合均匀，置于洁净、干燥的容器中保存。称取样品3.000 g，加入150 mL去离子水，于95℃水浴锅中加热溶解，在提取过程中不断振荡，直至液面无漂浮物时停止加热。将提取液置于250 mL容量瓶中定容，放置过夜。吸取上清液2 mL于10 mL离心管中，加入2 mL磺基水杨酸溶液，混匀，放置1 h。加入1 mL EDTA-Na和1 mL 0.06 mol·L^-1的盐酸溶液，混匀，用0.45 μm微孔滤膜过滤。用移液枪吸取滤液1 mL于2 mL离心管中，于45℃下真空干燥56 h。残留物用1 mL、pH值2.2的柠檬酸钠缓冲液溶解后，超声震荡15 min，用一次性注射器吸取1 mL，0.45 μm微孔滤膜过滤到测定样品瓶中待检。

昆虫角倍蚜鲜样用玻璃匀浆器在冰水条件下匀浆，将匀浆液转入1.5 mL Eppendorf管内进行低温离心，15 000 r·min^-1，15 min；取0.6 mL上清液，再加入0.4 mL、10%磺基水杨酸溶液，再离心10 min，15 000 r·min^-1，取上清液保存在-4℃冰箱待检。

1.4. 测试方法和条件

采用全自动氨基酸分析仪Biochrom30+(Biochrom)(Lithium column锂柱，Column# B-5219, Column# H-0037)参照《水溶肥料游离氨基酸含量测定方法》NY/T 1975-2010 ^[8]对样品进行外标法测定，柱温53℃，泵流速25 mL·h^-1，泵压力90 kg·cm^-2，分析时间120 min，进样体积20 μL，检测波长570 nm、440 nm。每一个样平行测定3次，取平均值。单位为质量百分数。

1.5. 数据分析

采用单因素(One-way ANOVA)方差分析对角倍蚜、角倍、有倍叶、无倍叶及对照的氨基酸质量分数(%)进行分析；所有数据分析均在SPSS 17.0统计软件中完成，显著度水平为P＜0.05。

3. 讨论

虫瘿是某些昆虫通过产卵或取食刺激，诱导寄主植物细胞异常增生而形成的具有特定结构的增生组织^{[2, 11-12]}，致瘿昆虫在瘿内生活和繁殖，从瘿壁获取营养^[13]。有研究表明，致瘿昆虫可以影响寄主植物体内游离氨基酸的组成及含量^{[4, 14-15]}，虫瘿内可溶性糖、可溶性蛋白质和游离氨基酸等营养物质的含量高于正常组织^[16]，某些致瘿蚜虫通过操纵虫瘿从周围组织调取营养^[17]，满足自身的生长需求。角倍是一种典型的蚜虫虫瘿，本研究中虽然角倍蚜的取食和致瘿没有改变寄主盐肤木叶片的游离氨基酸组成，但却显著改变了植株不同部位的游离氨基酸含量，盐肤木各组织中的游离氨基酸总量变化大小倍数从大到小依次为角倍(2.122)>有倍叶(0.560)>无倍叶(0.537)>CK(-0.114)，其中，角倍的游离氨基酸含量约为对照叶片的20倍，约为邻近有倍叶或无倍叶的4倍，而且这种氨基酸含量的升高与瘿内蚜虫个体数量和虫瘿体积的增加趋势一致，虫瘿和邻近叶片的游离氨基酸含量快速增加反映了盐肤木的抗虫能力随蚜虫数量的增加而增强。

昆虫翅型分化，其遗传因素和环境因素均对表型变异起作用^[18]，产生无翅蚜是为了种群繁殖最大化，有翅蚜是为了适应种群扩散^[19]。在角倍生长后期，虽然各样品游离氨基酸总量从大到小均为角倍蚜>角倍>有倍叶>无倍叶>CK，但角倍蚜与有倍叶、无倍叶的游离氨基酸总量呈上升趋势，而角倍与对照呈下降趋势。这种变化可能与蚜虫的翅型变化和即将到来的迁飞有关。

4. 结论

(1) 角倍蚜取食不会改变寄主盐肤木叶片中游离氨基酸组分，但可使游离氨基酸总量增加。

(2) 在角倍生长过程中，盐肤木植株游离氨基酸总量与角倍蚜个体数量和角倍体积的增加趋势基本一致，反映了角倍蚜取食与盐肤木防御之间的平衡。

Reference (19)

[1]	Stern D L. Phylogenetic evidence that aphids, rather than plants, determine gall morphology[J]. Proceedings of the Royal Society of London, 1995, 260(1357): 85-89. doi: 10.1098/rspb.1995.0063
[2]	Stone G N, Schönrogge K. The adaptive significance of insect gall morphology[J]. Trends in Ecology & Evolution, 2003, 18(10): 512-522.
[3]	Mattson J, Mattson Jr. Herbivory in relation to plant nitrogen content[J]. Annual Review of Ecology & Systematics, 1980, 11(1): 119-161.
[4]	Karley A J, Douglas A E, Parker W E. Amino acid composition and nutritional quality of potato leaf phloem sap for aphids[J]. Journal of Experimental Biology, 2002, 205(Pt19): 3009-3018.
[5]	朱玉永, 陈立, 王俊刚. 豌豆蚜取食对蚕豆韧皮部及自身氨基酸含量的影响[J]. 新疆农业科学, 2014, 51(7): 1284-1291.
[6]	邵淑霞.角倍蚜瘿内世代种群动态及寄主植物对蚜虫刺激的光合和脱落酸响应[D].北京: 中国林业科学研究院, 2013.
[7]	刘平.角倍蚜的生态适应性研究[D].北京: 中国林业科学研究院, 2014.
[8]	国家化肥质量监督检验中心.水溶肥料游离氨基酸含量的测定[S].农业标准, 2010.
[9]	许再福.普通昆虫学[M].北京:科学出版社, 2009.
[10]	Chen J, Zhang Y, Wang C. Proline induces calcium-mediated oxidative burst and salicylic acid signaling[J]. Amino Acids, 2011, 40(5): 1473-1484. doi: 10.1007/s00726-010-0757-2
[11]	马双敏, 虞泓, 李晨程. 植物虫瘿[J]. 昆虫知识, 2008, 45(2): 330-335. doi: 10.3969/j.issn.0452-8255.2008.02.037
[12]	王光钺.栗瘿蜂致瘿生物学及其对寄主植物生长发育的影响[D].临安: 浙江农林大学, 2010.
[13]	Allison S D, Schultz J C. Biochemical responses of chestnut oak to a galling cynipid[J]. Journal of Chemical Ecology, 2005, 31(1): 151-166. doi: 10.1007/s10886-005-0981-5
[14]	Petersen M K, Sandstrom J P. Outcome of indirect competition between two aphid species mediated by responses in their common host plant[J]. Functional Ecology, 2001, 15(4): 525-534. doi: 10.1046/j.0269-8463.2001.00552.x
[15]	Koyama Y, Yao I, Akimoto S. Aphid galls accumulate high concentrations of amino acids:a support for the nutrition hypothesis for gall formation[J]. Entomologia Experimentalis Et Applicata, 2004, 113(1): 35-44. doi: 10.1111/eea.2004.113.issue-1
[16]	Dsouza M R, Ravishankar B E. Nutritional sink formation in galls of Ficus glomerata Roxb. (Moraceae) by the insect Pauropsylla depressa (Psyllidae, Hemiptera)[J]. Tropical Ecology, 2014, 55(1): 129-136.
[17]	Larson K C, Whitham T G. Manipulation of food resources by a gall-forming aphid:the physiology of sink-source interactions[J]. Oecologia, 1991, 88(1): 15-21. doi: 10.1007/BF00328398
[18]	王小艺, 杨忠岐, 魏可. 昆虫翅型分化的表型可塑性机制[J]. 生态学报, 2015, 35(12): 3988-3999.
[19]	Braendle C, Davis G K, Brisson J A. Wing dimorphism in aphids[J]. Heredity, 2006, 97(3): 192-199. doi: 10.1038/sj.hdy.6800863

类别 Category	氨基酸总量Total content of free amino acids/%
	初期Early stage		中期Middle stage		后期Late stage
	07-09	07-27	08-18	09-14	09-29	10-11
角倍蚜S.chinensis	2.895±0.001 fB	2.904±0.002 eC	4.796±0.002 dA	5.874±0.002 cA	6.344±0.004 bA	8.464±0.005 aA
角倍Horn gall	2.139±0.001 fE	2.373±0.001 eE	3.098±0.001 dC	3.512±0.003 cB	4.478±0.002 aB	4.261±0.003 bB
有倍叶Leaf with gall	2.985±0.001 fA	3.280±0.002 cA	3.230±0.001 dB	3.063±0.001 eC	3.335±0.002 bC	3.545±0.000 aC
无倍叶Leaf without gall	2.787±0.002 fD	2.916±0.002 eB	2.928±0.001 dE	2.984±0.003 cD	3.223±0.003 bD	3.324±0.001 aD
CK	2.836±0.002 bC	2.877±0.002 aD	2.960±0.002 cD	2.605±0.002 fE	2.762±0.002 eE	2.722±0.002 fE
注：同一行中小写字母不同表示差异显著(横向比较)(P＜0.05)，同一列中大写字母不同表示差异显著(纵向比较)(P＜0.05)，下同。 Note: Different lower-case letter at the same lines means significant difference (horizontal comparison) (P＜0.05); Different capital letter means significant difference (vertical comparison) (P＜0.05). The same below.

类别 Category	10种昆虫必须氨基酸总量Total content of 10 essential amino acids/%
	初期Early stage		中期Middle stage		后期Late stage
	07-09	07-27	08-18	09-14	09-29	10-11
角倍蚜S.chinensis	1.027±0.000 fB	1.030±0.000 eC	1.701±0.001 dA	2.080±0.000 cA	2.248±0.001 bA	3.001±0.002 aA
角倍Horn gall	0.759±0.000 fE	0.842±0.001 eE	1.098±0.000 dC	1.245±0.001 cB	1.588±0.001 aB	1.510±0.001 bB
有倍叶Leaf with gall	1.058±0.000 fA	1.162±0.000 cA	1.145±0.001 dB	1.086±0.000 eC	1.182±0.004 bC	1.259±0.001 aC
无倍叶Leaf without gall	0.989±0.001 fD	1.033±0.001 eB	1.038±0.000 dE	1.058±0.001 cD	1.142±0.001 bD	1.184±0.001 aD
CK	1.006±0.001 cC	1.020±0.000 bD	1.049±0.000 aD	0.923±0.001 fE	0.961±0.001 eE	0.976±0.000 dE

类别Category		游离酸含量The content of several free amino acids/%
		初期Early stage		中期Middle stage		后期Late stage
		07-09	07-27	08-18	09-14	09-29	10-11
角倍蚜 S. chinensis	精氨酸Arg	0.120±0.000f	0.121±0.000e	0.199±0.000d	0.243±0.001c	0.263±0.001b	0.351±0.001a
角倍蚜 S. chinensis	苯丙氨酸Phe	0.114±0.000ef	0.114±0.000ef	0.189±0.000d	0.231±0.001c	0.250±0.000b	0.334±0.001a
角倍Horn gall	脯氨酸Pro	0.027±0.001fD	0.030±0.001eD	0.038±0.001dB	0.044±0.001cA	0.056±0.001aA	0.055±0.000bA
有倍叶Leaf with gall		0.038±0.001efA	0.041±0.001bcdA	0.041±0.001bcdA	0.038±0.001efBC	0.044±0.001aB	0.041±0.001bcdB
无倍叶Leaf without gall		0.035±0.001efBC	0.037±0.001bcdBC	0.037±0.001cdCD	0.038±0.001bBC	0.042±0.001aC	0.034±0.001efC
CK		0.036±0.001bcBC	0.036±0.001bcBC	0.037±0.001aCD	0.033±0.001eD	0.035±0.001dcD	0.018±0.001fD

Studies on Free Amino Acids of Aphid Schlechtendalia chinensis and the Host Plant Rhus chinensis