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CO2是大气中的一种重要组成物质,也是维持各种生命体所必需的气体,据报道大气中CO2含量从1700年至今已经从280 μL/L升至397 μL/L,预计在2050年将持续上升至700 μL/L[1]。研究表明大气CO2含量不断升高会对植食性害虫生长发育及营养利用产生影响。咀嚼式昆虫取食由于CO2含量升高而造成其寄主植物体内营养物质含量发生改变受到间接影响,其蛹重、幼虫存活率以及对寄主植物的N利用率均未出现显著性影响[2];潜叶性昆虫在高CO2含量下蛹重减轻,发育时间缩短[3-4];新长沫蝉(Neophilaenus lineatus)在高CO2含量下,若虫存活率降低20%,生长发育延迟[5]。高慧璟[6]研究表明,随着CO2含量升高棉蚜(Aphis gossypii)和Q型烟粉虱(Bemisia tabaci)种群发生增加。大气CO2含量增加条件下,昆虫为了生长发育而诱导增加或抑制减少体内的解毒酶和保护酶活性。随着CO2含量增加,褐飞虱(Nilaparvata lugens)5龄幼虫体内羧酸酯酶(Carboxylesterase,CarE)和乙酰胆碱酯酶(Acetylcholinesterase,AChE)活性显著增加,碱性磷酸酶(Alkaline phosphatases,ALP)、过氧化氢酶(Catalase,CAT)和超氧化物歧化酶(Superoxide dismutase,SOD)活性显著下降[7]。高CO2含量下西花蓟马(Frankliniella occidentalis)和花蓟马(F. intonsa)成虫体内CarE、多功能氧化酶(Mixed-functional oxidase,MFO)和SOD酶活性升高,2种蓟马通过调控体内的解毒酶和保护酶活性来适应CO2含量升高的环境[8]。
舞毒蛾(Lymantria dispar)是世界性害虫,能够取食杨(Populus spp.)、柳(Salix spp.)、桦树(Betula spp.)等500多种寄主植物。使用舞毒蛾核型多角体病毒(Lymantria dispar nuclear polyhedrosis virus,LdNPV)防治舞毒蛾是无公害防治技术之一,目前大多数研究集中于各种物质对LdNPV的增效作用,如绿原酸、苏云金杆菌(Bacillus thuringiensis)、荧光增白剂,以及不同株系LdNPV对舞毒蛾的防治效果[9-12]。然而,有关CO2含量升高对LdNPV致病机制影响鲜有报道。本文测定了不同CO2含量条件下饲养的舞毒蛾3龄幼虫感染LdNPV病毒后的生长发育以及体内解毒酶和保护酶活性变化,为了解全球气候变化下昆虫病毒致病机制以及舞毒蛾暴发成灾机制提供理论依据。
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舞毒蛾卵块、人工饲料和LdNPV均来源于中国林业科学研究院森林生态与环境保护研究所。卵于(25 ± 1)℃、光照60% 14L:10D、相对湿度70% ± 5%的可控CO2人工智能气候箱中孵化(PRX-600L-CO2,赛福)。CO2处理含量为550 μL/L和750 μL/L;以397 μL/L作为对照大气CO2含量。舞毒蛾卵至3龄幼虫一直置于不同CO2含量下饲养,选取同日进入3龄且大小一致的幼虫,饥饿24 h后饲喂LdNPV溶液浸润的人工饲料,以饲喂蒸馏水浸润的人工饲料作为对照组。不同CO2含量处理组和对照组舞毒蛾3龄幼虫均在接入病毒后放入CO2含量为397 μL/L的CO2人工智能气候箱中饲养。
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LdNPV原液通过血球计数板和显微镜计数为6.4 × 106 PIB/μL,按梯度含量稀释为6.4 × 105、6.4 × 104 、6.4 × 103 、6.4 × 102 和6.4 × 10 PIB/μL,将LdNPV溶液浸润人工饲料,晾干后接入饥饿24 h、大小一致、同一天进入3龄的幼虫,对照组为蒸馏水浸润的人工饲料,每个处理设置3个重复,每个重复30头幼虫。取食24 h后开始每天统计幼虫死亡数共统计10 d。
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牛血清蛋白(BSA)、磷酸氢二钠(NaH2PO4)、磷酸二氢钠(Na2HPO4)、磷酸、乙醇均购自于天津永大化学试剂有限公司;考马斯亮蓝G-250、碘化硫代乙酰胆碱(ATCh)、二硫双对硫基苯甲酸(DTNB)、毒扁豆碱、固蓝B盐(Fast blue B salt)购自于Sigma公司;乙二胺四乙酸(EDTA)、核黄素(VB2)、对硝基苯酚磷酸二钠(PNPP)和对硝基苯酚(p-Nirophenol)均购自Amresco公司;α-乙酸萘酯(α-NA)、L-甲硫氨酸(Met)、苯酚、硝基氮蓝四唑(NBT)、过氧化氢购自于国药集团化学试剂有限公司。
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不同CO2含量下分别随机放入相同数量的舞毒蛾卵粒,一直饲养至3龄幼虫,选取大小一致、同一天进入3龄的舞毒蛾幼虫接入亚致死含量LC20 LdNPV(1.1 × 10 PIB/μL),对照组饲喂蒸馏水浸润的人工饲料。每天定时观察其生长发育状况,以蜕皮1次为标准计算发育历期(3 ~ 4龄期)、体重相对增长量((后一天幼虫质量 − 前一天幼虫质量)/幼虫个数)、每天LdNPV致死虫数。
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选取20头舞毒蛾3龄幼虫,解剖中肠,用预冷的0.15 mol/L NaCl清洗食物残渣和体液,加入2 mL缓冲液,冰浴中组织研磨器(OSE-Y10,天根生化科技(北京)有限公司)匀浆,于4 ℃下高速离心,上清液即为酶液。CarE、ALP、AChE、CAT和SOD所用匀浆液和离心参数如下:保护酶系SOD和CAT匀浆液为0.05 mol/L pH7.0磷酸缓冲液(含1%PVP、0.04%苯基硫脲、10 mmol/L EDTA),4 000 rpm离心15 min。ALP匀浆缓冲液为0.1 mol/L pH7.0磷酸缓冲液,10 000 rpm离心10 min。AChE匀浆缓冲液为0.1 mol/L pH7.0磷酸缓冲液,4 000 rpm离心15 min。CarE匀浆缓冲液为0.04 mol/L pH7.0磷酸缓冲液,12 000 rpm离心15 min。
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CarE活性测定参照Van Asperen等[13]方法。反应体系为0.45 mL 0.04 mol/L磷酸缓冲液(pH7.0),1.8 mL 3 × 10− 4 mol/L α-NA,0.05 mL稀释酶液,混匀,30 ℃水浴15 min,加入0.9 mL显色剂(固蓝B盐:十二烷基硫酸钠=2:5)终止反应,加入显色剂终止反应后加入0.05 mL酶液作为空白对照,测定OD600值,每个处理重复3次,活性单位以μmol/(min·mg)表示。
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ALP活性测定参照Bessey等[14]方法。反应体系为2.3 mL 0.4 mol/L 巴比妥钠−盐酸缓冲液(pH9.6)和0.5 mL 7.5 × 10− 3 mol/L PNPP底物,0.1 mL酶液,混匀,30 ℃水浴15 min,2 mL 0.1 mol/L NaOH溶液终止反应,空白对照为终止反应后补加0.1 mL酶液,测定OD400值,每个处理重复3次,活性单位以nmol/(min·mg)表示。
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AChE活性测定参照Gorun等[15]方法。反应体系为0.1 mL 0.01 mol/L ATCh底物溶液和0.1 mL酶液,混匀,30 ℃水浴15 min,加入3.6 mL DNTB显色剂溶液,空白对照为加入显色剂后补加0.1 mL酶液,测定OD412值,每个处理重复3次。活性单位以mmol/min表示。
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CAT活性测定参照Cohen等[16]方法。反应体系为2 mL 0.1 mol/L pH7.0磷酸缓冲液,1 mL 0.08% H2O2和50 μL原酶液,空白对照以1 mL蒸馏水代替H2O2,测定OD240值,每min记数1次,记录3 min,每个处理重复3次。活性单位以ΔOD/(μg·min)表示。
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SOD活性测定参照Beauchamp等[17]方法略加改进。反应体系为3 mL,其中含有50 mmol/L、 pH7.0磷酸缓冲液,13 mmol/L甲硫氨酸,0.1 mmol/L EDTA,75 μmol/L NBT和50 μL酶液,最后加入4 μmol/L核黄素,以不加酶液管作为最大光还原管,4 000 lx光照10 min后,立即避光测定OD560值,以未光照的相同反应管作为对照管,重复测定3次,计算SOD酶活性,以达到50%抑制率所需的酶量为一个酶活性单位(U),酶活性大小以每毫克蛋白质中酶活性单位(U/mg)来表示。蛋白质含量测定参照Brandford[18]的考马斯亮蓝G-250法。
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运用SPSS22.0软件计算LdNPV对舞毒蛾3龄幼虫的致死中剂量(LC50)、亚致死剂量(LC20)和95%置信区间。不同CO2含量下生长发育指标、解毒酶和保护酶活性差异显著性采用Student-Newman-Keuls方法进行比较分析。
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LdNPV对舞毒蛾3龄幼虫的LC50和LC20分别为7.54 × 102 PIB/μL和1.1 × 10 PIB/μL(表1)。本文选用LC20剂量进行后续试验。
表 1 LdNPV对舞毒蛾3龄幼虫毒力
Table 1. Toxicity of LdNPV to 3rd instar L. dispar larvae under CO2 concentration stresses
LC50(95% 置信区间)
LC50 (95% confidence interval)/(PIB·μL− 1)LC20(95%置信区间)
LC20 (95% confidence interval)//(PIB·μL− 1)斜率
Slopeχ2 (df) 754 (504 ~ 1 098) 11 (0.79 ~ 55.00) 2.20 ± 0.45 23 (22) -
550 μL/L和750 μL/L CO2含量下舞毒蛾3龄幼虫平均发育历期分别为5.5 d和5.7 d,但与397 μL/L含量对照组差异不显著。LdNPV侵染不同CO2含量处理下的舞毒蛾3龄幼虫,其发育历期差异均不显著,这表明CO2含量和LdNPV胁迫不影响舞毒蛾3龄幼虫发育历期(表2)。
表 2 LdNPV对CO2含量胁迫下舞毒蛾3龄幼虫发育历期影响
Table 2. Effects of LdNPV on the developmental period of 3rd instar L. dispar larvae under CO2 concentration stress
d 非LdNPV侵染处理 Non-LdNPV infection treatment LdNPV侵染处理 LdNPV infection treatment 397 CK 550 CK 750 CK 397 NPV 550 NPV 750 NPV 5.10 ± 1.41a 5.50 ± 1.32a 5.7 ± 0.83a 6.35 ± 1.43a 6.13 ± 0.80a 6.50 ± 1.20a 注:397 CK、550 CK和750 CK为蒸馏水浸湿的人工饲料饲喂397、550和750 μL/L CO2含量处理后舞毒蛾幼虫;397 NPV、550 NPV和750 NPV为LdNPV浸湿的人工饲料饲喂397、550和750 μL/L CO2含量处理后舞毒蛾幼虫。数据为平均值 ± 标准误,相同小写字母表示差异性不显著(P > 0.05),下同。Notes: 397 CK, 550 CK and 750 CK are L. dispar larvae fed on artificial diets soaked by distilled water after 397, 550 and 750 μL/L CO2 treatments, respectively. 397 NPV, 550 NPV and 750 NPV are L. dispar larvae fed on artificial diets soaked by LdNPV after 397, 550 and 750 μL/L CO2 treatments, respectively. The data in table is mean ± standard error. The same lowercase letters in a column indicate no significant difference at 0.05 level. The same below. 随着CO2含量增加,对照组舞毒蛾3龄幼虫体重累计增长率显著降低,550 μL/L和750 μL/L含量下累计7 d体重增长率比397 μL/L含量对照组分别减少37.95%和64.79%(P < 0.05)。LdNPV侵染不同CO2含量下饲养舞毒蛾3龄幼虫,397、550和750 μL/L含量处理组7 d累计体重增长率分别为81.27%、71.63%、68.41%(表3)。
表 3 感染LdNPV舞毒蛾幼虫体重累计增长率
Table 3. Cumulative growth rate of L. dispar larvae after infection by LdNPV
处理
Treatment指标
Index饲喂天数 Feeding days 1 2 3 4 5 6 7 397 CK 幼虫鲜质量 Larva fresh mass/mg 4.56 ± 0.25Be 7.34 ± 0.71Bd 8.08 ± 1.48Bd 8.57 ± 2.54Bd 9.18 ± 2.91Cc 11.64 ± 1.97Ab 13.80 ± 2.40Aa 累计增长率 Cumulative increase rate/% − 60.83 ± 9.63Be 76.62 ± 7.72Bd 86.72 ± 13.87Acd 99.55 ± 6.22Ac 152.75 ± 3.25Ab 200.24 ± 5.07Aa 397 NPV 幼虫鲜质量 Larva fresh mass/mg 4.58 ± 0.36Bd 5.70 ± 0.42Cc 6.75 ± 0.55Bb 7.33 ± 0.89BCab 7.66 ± 0.73Dab 7.72 ± 1.27Cab 8.22 ± 0.51Da 累计增长率 Cumulative increase rate/% − 25.61 ± 15.73Dd 49.15 ± 4.84Cc 62.50 ± 4.64Bb 69.70 ± 7.73Cb 71.87 ± 7.43Cb 81.27 ± 13.01Ca 550 CK 幼虫鲜质量 Larva fresh mass/mg 5.59 ± 0.29Ad 9.84 ± 0.75Ac 10.64 ± 0.85Abc 10.82 ± 1.08Ab 11.12 ± 0.79Ab 11.20 ± 1.68Ab 12.44 ± 2.36Ba 累计增长率 Cumulative increase rate/% − 75.93 ± 9.47Ac 90.02 ± 5.15Abc 93.04 ± 2.57Abc 98.68 ± 16.27Ab 99.35 ± 9.48Bb 124.25 ± 31.26Ba 550 NPV 幼虫鲜质量 Larva fresh mass/mg 5.68 ± 0.28Ab 9.61 ± 0.62Aa 9.99 ± 0.83Aa 10.15 ± 0.74Aa 10.14 ± 0.79Ba 9.83 ± 0.39Ba 9.75 ± 0.56Ca 累计增长率 Cumulative increase rate/% − 69.23 ± 8.10ABc 75.91 ± 2.39Bab 78.70 ± 1.89Ba 78.55 ± 2.78Ba 73.30 ± 3.64Cbc 71.63 ± 2.34Cbc 750 CK 幼虫鲜质量 Larva fresh mass/mg 4.51 ± 0.20Bc 6.21 ± 0.13BCb 6.60 ± 0.63Bab 6.27 ± 0.41Cb 6.75 ± 0.90Dab 7.46 ± 0.40Ca 7.70 ± 1.63Da 累计增长率 Cumulative increase rate/% − 37.82 ± 4.83Cc 46.43 ± 7. 89Cb 39.08 ± 5.04Cc 49.50 ± 8.0Db 65.83 ± 11.58CDa 70.50 ± 16.48Ca 750 NPV 幼虫鲜质量 Larva fresh mass/mg 4.96 ± 0.80ABb 7.19 ± 1.27Ba 7.32 ± 1.41Ba 7.16 ± 1.04BCa 6.95 ± 0.60Da 7.73 ± 1.05Ca 8.14 ± 0.43Da 累计增长率 Cumulative increase rate/% − 44.85 ± 5.33Cc 47.06 ± 2.31Cc 45.17 ± 5.94Cc 41.84 ± 7.86Dc 56.50 ± 5.27Db 68.41 ± 14.42Da 注:表中数据为平均值 ± 标准误,不同小写字母表示同一组不同天数差异显著性,不同大写字母表示同一天不同组差异显著性(P < 0.05)。Notes: data in table is mean ± standard error. Different lowercase letters indicate significant difference in the same group of different days. Different capital letters indicate significant differences of different groups on the same day (P < 0.05). 舞毒蛾3龄幼虫接种LdNPV 2 d,550 μL/L含量条件下幼虫死亡率显著高于397 μL/L含量对照组;750 μL/L含量条件下幼虫6 d死亡率为23.87%,显著高于397 μL/L含量对照组,比5 d增加146.67%;接种LdNPV 7 d,397、550、750 μL/L含量处理组幼虫累计死亡率分别为20.07%、23.87%和27.10%;其中,750 μL/L含量处理组幼虫累计死亡率分别比550和397 μL/L含量处理组显著增加13.53%和35.02%(P < 0.05,图1)。
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CO2含量处理下,舞毒蛾3龄幼虫体内2种解毒酶CarE和AChE活性随着CO2含量升高主要表现为诱导增加,ALP活性表现为受抑制降低。与397 μL/L CO2含量处理组相比,550 μL/L和750 μL/L CO2含量处理4 d,CarE活性分别显著增加48.12%和61.12%;AChE的活性分别显著增加25.00%和31.25%;而ALP的活性显著降低14.47%和18.72%(P < 0.05,图2)。
图 2 LdNPV对不同CO2含量胁迫下舞毒蛾3龄幼虫体内CarE、ALP和AChE活性影响
Figure 2. Effects of LdNPV on CarE, ALP and AChE activities in 3rd instar L. dispar larvae under different CO2 concentration stress
不同CO2含量条件下舞毒蛾饲养至3龄幼虫后感染LdNPV,幼虫体内的3种解毒酶活性均表现为抑制减少。LdNPV胁迫1 d,397和750 μL/L含量处理组CarE活性低于非LdNPV胁迫对照组,750 μL/L含量处理组比非LdNPV胁迫对照组显著降低28.60%(df = 1,5,F = 13.013,P = 0.023)。LdNPV感染4 d,397、550、750 μL/L CO2含量条件下幼虫体内的CarE活性分别比非LdNPV胁迫对照组显著降低61.07%、24.73%和134.74%(df = 1,5,F = 18.016,P = 0.003;df = 1,5,F = 19.172,P = 0.012;df = 1,5,F = 7.052,P = 0.001)。LdNPV胁迫1 d,397 μL/L含量处理组体内AChE活性最低,分别比550 μL/L、750 μL/L含量处理组减少16.18%和37.16%,与非LdNPV胁迫对照组差异不显著(df = 1,5,F = 1.984,P = 0.232)。LdNPV胁迫4 d,550 μL/L、750 μL/L含量处理组AChE活性均显著低于非LdNPV胁迫对照组(df = 1,5,F = 7.35,P = 0.001;df = 1,5,F = 13.225,P = 0.001)。397、550和750 μL/L含量处理组体内ALP活性分别在2 d、3 d和1 d抑制率最大,分别为81.76%、86.71%和48.91%(df = 1,5,F = 30.031,P = 0.001;df = 1,5,F = 49.196,P = 0.001;df = 1,5,F = 28.052,P = 0.006,图2)。
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舞毒蛾3龄幼虫体内2种保护酶的活性随着CO2含量升高主要表现为抑制减少。550和750 μL/L CO2含量下饲养4 d幼虫比397 μL/L含量对照组CAT活性分别显著减少72.24%、26.89%。LdNPV胁迫1 d,397 μL/L含量处理组SOD活性显著低于非LdNPV胁迫对照组,胁迫1 d抑制率为4.76%(df = 1,5,F = 24,P = 0.008)。LdNPV胁迫4 d,550 μL/L含量处理组SOD活性显著低于非LdNPV胁迫对照组,最大抑制率为6.06%(df = 1,5,F = 22.013,P = 0.007);750 μL/L含量处理3 d SOD活性最大,为76.88 U/mg。LdNPV胁迫4 d,750 μL/L CO2含量处理组CAT活性显著受抑制,抑制率为61.78%(df = 1,5,F = 12.138,P = 0.025);750 μL/L含量处理组CAT活性最低,分别比397、550 μL/L含量处理组显著减少40.16%和33.58%(图3)。
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19世纪以来,地球表面温度持续上升0.3 ~ 0.6 ℃,预计到22世纪将继续大幅度升高[4]。大气中CO2含量增长尤为迅速,是全球气候变暖的主要原因。CO2含量升高不仅会对土壤碳循环结构、森林物候、森林物种分布等森林生态系统方面产生影响,同时CO2含量升高也直接作用于昆虫[19]。高含量CO2(750 μL/L)使棉铃虫(Helicoverpa armigera)幼虫、蛹、成虫的历期延长,体重减轻,老熟幼虫(4 ~ 5龄)体内蛋白质含量减少,幼虫期发育变缓,取食量增加[20]。本文研究结果表明随着CO2含量升高舞毒蛾3龄幼虫的体重累计增长率减少。
昆虫核型多角体病毒是一种专性病毒,其主要作用于双翅目、膜翅目、鳞翅目森林害虫生物防治。Duan等[21]研究表明,西部云杉卷蛾(Choristoneura occidentalis)接种核型多角体病毒(CfNPV)后,6龄幼虫发育历期延长,成虫寿命缩短。席景会等[22]研究表明八字地老虎(Xestie c-nigrum)3龄幼虫感染核型多角体病毒(AnNPV)3 ~ 6 d后,体重显著减少,发育历期显著延长。本文通过对LdNPV的毒力测定,筛选出亚致死剂量LC20(1.1 × 10 PIB/μL)处理舞毒蛾3龄幼虫,结果表明随着CO2含量升高舞毒蛾3龄幼虫受到LdNPV胁迫后体重累计增长率减少、死亡率增加,可能由于高含量CO2条件下舞毒蛾幼虫降低了相对取食率[23],所以幼虫在感染病毒前期取食LdNPV的量较少导致病毒繁殖缓慢,但在高含量CO2条件下的舞毒蛾幼虫会增加食物的利用率和转化率来维持生长发育,使虫体内LdNPV大量繁殖并减缓自身的生长发育。这导致LdNPV在高含量CO2处理下舞毒蛾幼虫体内潜伏期长、致死率高。
解毒酶对昆虫分解外源化合物、维持正常生理代谢起到重要的作用。昆虫通过调节自身CarE、AChE、ALP等代谢酶活性来抵抗病原微生物等逆境环境[24]。接种家蚕核型多角体病毒(BmNPV)的非抗性家蚕(Bombyx morii)5龄幼虫后,宿主昆虫中肠CarE活性先升高再降低,AChE活性升高[25]。棉铃虫接种棉铃虫核型多角体病毒(HearNPV)后,宿主中肠ALP活性显著下降,且随着病毒含量增加,ALP活性越低[26]。本文研究表明不同CO2含量胁迫下生长的舞毒蛾3龄幼虫接种LdNPV后,CarE、AChE、ALP活性随着CO2含量增加表现为先诱导增加后抑制减少,这可能是由于高含量CO2和LdNPV共胁迫抑制舞毒蛾体内解毒酶的活性。
在受到外界环境影响的条件下,昆虫通过增加体内的负氧离子(
${\rm{O}}_2^- $ )、单线态氧(O2)、过氧化氢(H2O2)、氢氧自由基(HO)等离子对抗体内破坏自由基的保护酶[27]。受柞蚕(Antheraea pernyi)核型多角体病毒(ApNPV)侵染后,柞蚕蛹体内SOD活性呈波动性下降的趋势,CAT活性变化呈下降的趋势[28]。本文研究表明舞毒蛾3龄幼虫体内的保护酶系CAT活性随着CO2含量升高受诱导增加,SOD活性随着CO2含量升高受抑制减少。这些结果表明舞毒蛾3龄幼虫通过调节体内的CAT活性增加来适应CO2含量升高对昆虫的影响,体内的SOD活性可能由于虫体内氧自由基过剩而受抑制。高含量CO2条件下饲养的舞毒蛾3龄幼虫受LdNPV胁迫后CAT活性随着CO2含量增加先受诱导增加随后受抑制减少,SOD活性在550 μL/L含量处理组受抑制减少,在750 μL/L含量处理组受诱导增加,可能是LdNPV抑制了高含量CO2对舞毒蛾的作用机制。 -
利用封闭CO2人工气候箱探讨了LdNPV对不同CO2含量条件下生长的舞毒蛾个体生长发育直接影响机制,以及在高含量CO2和LdNPV的胁迫下舞毒蛾体内解毒酶和保护酶的变化。研究结果表明,不同含量CO2条件下饲养到舞毒蛾3龄幼虫接种LdNPV后,随着CO2含量升高,幼虫体重累计增长率减少、死亡率增加,LdNPV在高含量CO2处理的舞毒蛾幼虫体内潜伏期长、致死率高。幼虫受LdNPV胁迫后解毒酶系CarE、AChE活性在550 μL/L CO2含量处理组最高,ALP活性随着CO2含量升高呈先上升后下降再上升的趋势;保护酶系SOD活性在397 μL/L含量最高,CAT活性随着CO2含量升高而升高。这些初步研究结果为进一步了解全球气候变化下害虫响应机制、病原微生物病毒致病机理以及害虫治理策略制定提供理论依据。
Effects of LdNPV on growth, development and biochemical enzymatic activities of Lymantria dispar under CO2 concentration stress
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摘要:
目的以森林害虫舞毒蛾为对象,研究CO2介导下LdNPV病毒对舞毒蛾生长发育、解毒酶和保护酶的影响。 方法利用密闭式CO2人工气候箱研究了不同CO2含量(397、550和750 μL/L)条件下饲养的舞毒蛾幼虫,LdNPV对其生长发育指标的影响,并采用分光光度计法测定体内保护酶和解毒酶活性。 结果高CO2含量胁迫下舞毒蛾3龄幼虫体重累计增长率降低。LdNPV胁迫导致不同CO2含量(397、550和750 μL/L)条件下饲养的舞毒蛾3龄幼虫体重累计增长率分别比对照组增加81.27%、71.63%和68.41%;随着CO2含量升高幼虫感染LdNPV死亡率增加,750 μL/L高含量处理组死亡率为27.09%。高CO2含量胁迫下舞毒蛾3龄幼虫体内解毒酶CarE和AChE活性随着CO2含量升高而诱导增加,ALP活性随CO2含量增加抑制下降;保护酶CAT活性随着CO2含量升高诱导增加,而SOD活性随着CO2含量升高抑制减少。高CO2含量条件下生长的舞毒蛾3龄幼虫接种LdNPV后,随着CO2含量升高体内CarE、ALP、AChE和CAT活性被抑制,而SOD活性表现为诱导增加。 结论不同CO2含量下舞毒蛾饲养至3龄幼虫接种LdNPV,随着CO2含量增加,幼虫体重累计增长率减少、死亡率增加。舞毒蛾幼虫CarE、AChE、ALP和CAT活性随着LdNPV处理时间和CO2含量增加而主要表现为抑制减少;SOD活性则主要表现为诱导增加。CO2含量升高可能通过影响生长发育和生理生化增加LdNPV对舞毒蛾幼虫的致病力。 Abstract:ObjectiveIn this study, the forest pest Lymantria dispar was used to study the effects of LdNPV on the development, detoxifying enzymes and protective enzymes of Lymantria dispar larvae under CO2 concentration stress. MethodThe effects of LdNPV on the growth and development of L. dispar larvae under different CO2 concentrations (397, 550 and 750 μL/L) reared into the closed CO2 artificial climate chamber, and in vivo detoxifying enzymes and protective enzyme activities of L. dispar larvae were also determined by spectrophotometry. Result The cumulative growth rate of fresh mass of the 3rd instar larvae of L. dispar decreased under high CO2 concentration stress. The cumulative increase rates of fresh mass of the 3rd instar L. dispar larvae infected by LdNPV were higher than the control by 81.27%, 71.63% and 68.41%, respectively. The mortality rate increased with increasing CO2 concentration, and the cumulative mortality rate of 750 μL/L CO2 treatment was 27.09%. Under the high CO2 concentration stress, the activities of detoxifying enzymes CarE and AChE in the 3rd instar larvae of L. dispar were induced with the increase of CO2 concentration, and the ALP activity was inhibited with the increase of CO2 concentration; the activity of protective enzyme CAT was induced with the increase of CO2 concentration while SOD activity was inhibited with increasing CO2 concentration. However, the activities of CarE, ALP, AChE and CAT in 3rd instar larvae of L. dispar infected by LdNPV were inhibited with the increase of CO2 concentration, and the activity of SOD was induced with the increase of CO2 concentration. ConclusionThe duration and cumulative mortality rate of L. dispar larvae infected by LdNPV increased while cumulative growth rate decreased after different CO2 concentraion treatments. The activities of CarE, AChE, ALP and CAT in 3rd instar larvae of L. dispar decreased with the increase of both the treatment time and CO2 concentration, and the activities of SOD were increased. Therefore, elevated concentration of CO2 may increase the virulence of LdNPV to the L. dispar larvae by affecting the growth, development and biochemical enzymes. -
Key words:
- Lymantria dispar /
- CO2 concentration /
- LdNPV /
- growth and development /
- detoxifying enzyme /
- protective enzyme
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表 1 LdNPV对舞毒蛾3龄幼虫毒力
Table 1. Toxicity of LdNPV to 3rd instar L. dispar larvae under CO2 concentration stresses
LC50(95% 置信区间)
LC50 (95% confidence interval)/(PIB·μL− 1)LC20(95%置信区间)
LC20 (95% confidence interval)//(PIB·μL− 1)斜率
Slopeχ2 (df) 754 (504 ~ 1 098) 11 (0.79 ~ 55.00) 2.20 ± 0.45 23 (22) 表 2 LdNPV对CO2含量胁迫下舞毒蛾3龄幼虫发育历期影响
Table 2. Effects of LdNPV on the developmental period of 3rd instar L. dispar larvae under CO2 concentration stress
d 非LdNPV侵染处理 Non-LdNPV infection treatment LdNPV侵染处理 LdNPV infection treatment 397 CK 550 CK 750 CK 397 NPV 550 NPV 750 NPV 5.10 ± 1.41a 5.50 ± 1.32a 5.7 ± 0.83a 6.35 ± 1.43a 6.13 ± 0.80a 6.50 ± 1.20a 注:397 CK、550 CK和750 CK为蒸馏水浸湿的人工饲料饲喂397、550和750 μL/L CO2含量处理后舞毒蛾幼虫;397 NPV、550 NPV和750 NPV为LdNPV浸湿的人工饲料饲喂397、550和750 μL/L CO2含量处理后舞毒蛾幼虫。数据为平均值 ± 标准误,相同小写字母表示差异性不显著(P > 0.05),下同。Notes: 397 CK, 550 CK and 750 CK are L. dispar larvae fed on artificial diets soaked by distilled water after 397, 550 and 750 μL/L CO2 treatments, respectively. 397 NPV, 550 NPV and 750 NPV are L. dispar larvae fed on artificial diets soaked by LdNPV after 397, 550 and 750 μL/L CO2 treatments, respectively. The data in table is mean ± standard error. The same lowercase letters in a column indicate no significant difference at 0.05 level. The same below. 表 3 感染LdNPV舞毒蛾幼虫体重累计增长率
Table 3. Cumulative growth rate of L. dispar larvae after infection by LdNPV
处理
Treatment指标
Index饲喂天数 Feeding days 1 2 3 4 5 6 7 397 CK 幼虫鲜质量 Larva fresh mass/mg 4.56 ± 0.25Be 7.34 ± 0.71Bd 8.08 ± 1.48Bd 8.57 ± 2.54Bd 9.18 ± 2.91Cc 11.64 ± 1.97Ab 13.80 ± 2.40Aa 累计增长率 Cumulative increase rate/% − 60.83 ± 9.63Be 76.62 ± 7.72Bd 86.72 ± 13.87Acd 99.55 ± 6.22Ac 152.75 ± 3.25Ab 200.24 ± 5.07Aa 397 NPV 幼虫鲜质量 Larva fresh mass/mg 4.58 ± 0.36Bd 5.70 ± 0.42Cc 6.75 ± 0.55Bb 7.33 ± 0.89BCab 7.66 ± 0.73Dab 7.72 ± 1.27Cab 8.22 ± 0.51Da 累计增长率 Cumulative increase rate/% − 25.61 ± 15.73Dd 49.15 ± 4.84Cc 62.50 ± 4.64Bb 69.70 ± 7.73Cb 71.87 ± 7.43Cb 81.27 ± 13.01Ca 550 CK 幼虫鲜质量 Larva fresh mass/mg 5.59 ± 0.29Ad 9.84 ± 0.75Ac 10.64 ± 0.85Abc 10.82 ± 1.08Ab 11.12 ± 0.79Ab 11.20 ± 1.68Ab 12.44 ± 2.36Ba 累计增长率 Cumulative increase rate/% − 75.93 ± 9.47Ac 90.02 ± 5.15Abc 93.04 ± 2.57Abc 98.68 ± 16.27Ab 99.35 ± 9.48Bb 124.25 ± 31.26Ba 550 NPV 幼虫鲜质量 Larva fresh mass/mg 5.68 ± 0.28Ab 9.61 ± 0.62Aa 9.99 ± 0.83Aa 10.15 ± 0.74Aa 10.14 ± 0.79Ba 9.83 ± 0.39Ba 9.75 ± 0.56Ca 累计增长率 Cumulative increase rate/% − 69.23 ± 8.10ABc 75.91 ± 2.39Bab 78.70 ± 1.89Ba 78.55 ± 2.78Ba 73.30 ± 3.64Cbc 71.63 ± 2.34Cbc 750 CK 幼虫鲜质量 Larva fresh mass/mg 4.51 ± 0.20Bc 6.21 ± 0.13BCb 6.60 ± 0.63Bab 6.27 ± 0.41Cb 6.75 ± 0.90Dab 7.46 ± 0.40Ca 7.70 ± 1.63Da 累计增长率 Cumulative increase rate/% − 37.82 ± 4.83Cc 46.43 ± 7. 89Cb 39.08 ± 5.04Cc 49.50 ± 8.0Db 65.83 ± 11.58CDa 70.50 ± 16.48Ca 750 NPV 幼虫鲜质量 Larva fresh mass/mg 4.96 ± 0.80ABb 7.19 ± 1.27Ba 7.32 ± 1.41Ba 7.16 ± 1.04BCa 6.95 ± 0.60Da 7.73 ± 1.05Ca 8.14 ± 0.43Da 累计增长率 Cumulative increase rate/% − 44.85 ± 5.33Cc 47.06 ± 2.31Cc 45.17 ± 5.94Cc 41.84 ± 7.86Dc 56.50 ± 5.27Db 68.41 ± 14.42Da 注:表中数据为平均值 ± 标准误,不同小写字母表示同一组不同天数差异显著性,不同大写字母表示同一天不同组差异显著性(P < 0.05)。Notes: data in table is mean ± standard error. Different lowercase letters indicate significant difference in the same group of different days. Different capital letters indicate significant differences of different groups on the same day (P < 0.05). -
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