亚致死剂量呋虫胺对意大利蜜蜂采集蜂免疫解毒相关基因表达和酶活性的影响

doi:10.3969/j.issn.1004-1524.2023.03.10

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (3): 575-581.DOI: 10.3969/j.issn.1004-1524.2023.03.10

亚致死剂量呋虫胺对意大利蜜蜂采集蜂免疫解毒相关基因表达和酶活性的影响

张宇昊¹(), 马卫华², 刘晋佳¹, 马秀梅¹, 姜玉锁¹^,^*()

1.山西农业大学动物科学学院,山西太谷 030801
2.山西农业大学园艺学院,山西太谷 030801

收稿日期:2021-09-06 出版日期:2023-03-25 发布日期:2023-04-07
通讯作者: ^*姜玉锁,E-mail:jiangys-001@163.com
作者简介:张宇昊(1996—),男,浙江义乌人,硕士研究生,研究方向为动物遗传育种与繁殖。E-mail:zhangyuhao1005@126.com
基金资助:
现代农业蜂产业技术体系建设专项资金(CARS-45-KXJ5)

Effects of sublethal doses of dinotefuran on genes expression and enzymes activities related to immune and detoxification in foragers of Apis mellifera ligustica

ZHANG Yuhao¹(), MA Weihua², LIU Jinjia¹, MA Xiumei¹, JIANG Yusuo¹^,^*()

1. College of Animal Sciences, Shanxi Agricultural University, Taigu 030801, Shanxi, China
2. College of Horticulture, Shanxi Agricultural University, Taigu 030801, Shanxi, China

Received:2021-09-06 Online:2023-03-25 Published:2023-04-07

摘要/Abstract

摘要：

为确定呋虫胺对意大利蜜蜂Apis mellifera ligustica采集蜂的亚致死浓度,探究亚致死剂量呋虫胺对意大利蜜蜂采集蜂免疫解毒相关基因的表达及酶活性的影响,本研究采用饲喂法测定呋虫胺对意大利蜜蜂采集蜂的毒力曲线。应用qRT-PCR技术研究LC₂₀和LC₅₀浓度呋虫胺对蜜蜂Abaecin,Apidaecins,Defensin-1,Hymenoptaecin,AChE-2,PPO,GST及Cyp450 mRNA相对表达量的影响。测定蜜蜂体内多酚氧化酶、乙酰胆碱酯酶、超氧化物歧化酶、谷胱甘肽S-转移酶和过氧化氢酶活性的变化。结果表明,呋虫胺胁迫意大利蜜蜂采集蜂的LC₂₀和LC₅₀为0.458 mg·L^-1和0.988 mg·L^-1。LC₂₀组中PPO、Apidaecins、Defensin-1和Hymenoptaecin基因表达量与对照组相比有显著(P<0.05)上调趋势,LC₅₀组中AChE-2、PPO、GST、Cyp450、Apidaecins和Defensin-1表达量有显著上调趋势,Abaecin和Hymenoptaecin基因表达量有显著下调趋势,且LC₅₀组中的abaecin和Hymenoptaecin表达量均显著低于LC₂₀组,AChE-2、PPO、GST、Cyp450和Apidaecins基因表达量显著高于LC₂₀组。LC₂₀组与对照组相比,AChE、CAT和SOD活性均显著下降;LC₅₀组与对照组相比,AChE、PPO、CAT和SOD活性均显著下降。呋虫胺抑制Abaecin和Hymenoptaecin基因表达,诱导Apidaecins、Defensin-1、AChE-2、PPO、GST和Cyp450基因的表达,影响正常的免疫系统。AChE、PPO、CAT和SOD活性的下降预示着呋虫胺对蜜蜂解毒系统的损害,本试验为探究呋虫胺对意大利蜜蜂生理和代谢的影响提供理论依据。

关键词: 意大利蜜蜂, 呋虫胺, 亚致死效应, 免疫解毒, 酶活性

Abstract:

To determine sublethal doses of dinotefuran to Apis mellifera ligustica and explore the effects of sublethal doses of dinotefuran on the expression of immune detoxification genes and enzyme activities on Apis mellifera ligustica, in this study, the feeding trials was used to determine the virulence curve of dinotefuran to Apis mellifera ligustica. The effects of LC₂₀ and LC₅₀ dinotefuran on the relative expressions of Abaecin, Apidaecins, Defensin-1, Hymenoptaecin, AChE-2, PPO, GST and Cyp450 mRNA were investigated by qRT-PCR. Determination of changes in the activities of PPO, AChE, SOD, GST, CAT in Apis mellifera ligustica. The LC₂₀ and LC₅₀ of dinotefuran on Apis mellifera ligustica determined by feeding trials were 0.458 mg·L^-1 and 0.988 mg·L^-1. Compared with the control group, the expression levels of PPO, Apidaecins, Defensin-1, Hymenoptaecin genes were significantly (P<0.05) up-regulated in the LC₂₀ group. Apidaecins, In LC₅₀ group, the expression of AChE-2, PPO, GST, Cyp450, Apidaecins, Defensin-1 showed a significant up-regulation trend, and the expression of Hymenoptaecin and Abaecin showed a significant down-regulation trend. In additon, the expression levels of Abaecin and Hymenoptaecin in the LC₅₀ group were significantly lower than those in the LC₂₀ group. The expression levels of AChE-2, PPO, GST, Cyp450, Apidaecins in the LC₅₀ group were significantly higher than those in the LC₂₀ group. Compared with the control group, the activities of AChE, CAT and SOD of LC₂₀ group decreased significantly. Compared with the control group, the activities of AChE, PPO, CAT and SOD of LC₅₀ group decreased significantly. In conclusion, dinotefuran inhibits gene expression of Abaecin and Hymenoptaecin, and has an inducing effect on Apidaecins, Defensin-1, AChE-2, PPO, GST, Cyp450 affects the normal immune system of Apis mellifera ligustica. The decline of AChE, PPO, CAT and SOD enzyme activities indicates the damage of dinotefuran to the detoxification system of Apis mellifera ligustica. Our stduy provides a further basis for further research on the effects of sublethal dose dinotefuran on the metabolism and physiology of Apis mellifera ligustica.

Key words: Apis mellifera ligustica, dinotefuran, sublethal effect, immune detoxification, enzyme activity

中图分类号:

S893.3

张宇昊, 马卫华, 刘晋佳, 马秀梅, 姜玉锁. 亚致死剂量呋虫胺对意大利蜜蜂采集蜂免疫解毒相关基因表达和酶活性的影响[J]. 浙江农业学报, 2023, 35(3): 575-581.

ZHANG Yuhao, MA Weihua, LIU Jinjia, MA Xiumei, JIANG Yusuo. Effects of sublethal doses of dinotefuran on genes expression and enzymes activities related to immune and detoxification in foragers of Apis mellifera ligustica[J]. Acta Agriculturae Zhejiangensis, 2023, 35(3): 575-581.

图/表 4

表1 本实验所用引物对序列信息

Table 1 Sequence information of primer pairs used in this experiment

基因名称 Gene name	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')
Abaecin	ATCTTCGCACTACTCGCCAC	CTGACCAGGAAACGTTGGAA
Apidaecin	AGATTGTCGATCGGTCCACG	TCTGGTTCGAGCATGCTACC
Defensin-1	GTTGAGGATGAATTCGAGCC	TTAACCGAAACGTTTGTCCC
Hymenoptaecin	ATATCCCGACTCGTTTCCGA	TCCCAAACTCGAATCCTGCA
AChE-2	GACGCGAAGACCATATCCGT	TCTGTGTCCTTGAAGTCCGC
PPO	ATGTGGATGGCCGCAACATA	CGCCATATTTCCGGTGAGGA
GST	TGCATATGCTGGCATTGATT	TCCTCGCCAAGTATCTTGCT
Cyp450	CAAAATGGTGTTCTCCTTACCG	ATGGCAACCCATCACTGC
β-actin	ATGCCAACACTGTCCTTTCTGG	GACCCACCAATCCATACGGA

图1 呋虫胺对意大利蜜蜂急性毒性的毒力回归曲线 y为呋虫胺浓度的常用对数值;x为死亡概率。

Fig.1 Toxicity regression curve of dinotefuran acute toxicity to Apis mellifera ligustica y, Common logarithm value of dinotefuran concentration; x, Probit value to mortality.

图2 意大利蜜蜂Hymenoptaecin、abaecin、Apidaecin、Defensin-1、AChE-2、PPO、GST和Cyp450和在不同浓度呋虫胺处理下的mRNA表达量 CK,50%蔗糖水;LC20,0.458 mg·L-1呋虫胺蔗糖溶液;LC50,0.988 mg·L-1呋虫胺蔗糖溶液。下同。同一基因下,柱上无相同字母的表示处理间差异显著(P<0.05)。

Fig.2 Expression patterns of Hymenoptaecin, Abaecin, Apidaecin, Defensin-1, AChE-2,PPO,GST and Cyp450 mRNA in different concentrations of dinotefuran on Apis mellifera ligustica CK, 50% sucrose solution; LC20, 0.458 mg·L-1 dinotefuran sucrose solution; LC50, 0.988 mg·L-1 dinotefuran sucrose solution. The same as below. Bars marked without the same letters indicate significant (P<0.05) difference within treatments under the same gene.

图3 LC20和LC50呋虫胺胁迫后意大利蜜蜂采集蜂免疫解毒酶系活性柱上无相同字母的表示处理间差异显著(P<0.05)。

Fig.3 Immune and detoxification enzyme activity in foragers of Apis mellifera ligustica after LC20 and LC50 dinotefuran stress Bars marked without the same letters indicate significant (P<0.05) difference within treatments.

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亚致死剂量呋虫胺对意大利蜜蜂采集蜂免疫解毒相关基因表达和酶活性的影响

Effects of sublethal doses of dinotefuran on genes expression and enzymes activities related to immune and detoxification in foragers of Apis mellifera ligustica

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