氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾主要解毒酶活性與生長繁殖的影響

陳羿渠,向興,貢常委,王學(xué)貴

（四川農(nóng)業(yè)大學(xué)農(nóng)學(xué)院無公害農(nóng)藥研究實(shí)驗(yàn)室，成都 611130）

氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾主要解毒酶活性與生長繁殖的影響

陳羿渠,向興,貢常委,王學(xué)貴

（四川農(nóng)業(yè)大學(xué)農(nóng)學(xué)院無公害農(nóng)藥研究實(shí)驗(yàn)室，成都 611130）

【目的】甜菜夜蛾（Spodoptera exigua)是一種雜食性害蟲，在不同環(huán)境、藥物等選擇壓力下表現(xiàn)出不同的生長發(fā)育特點(diǎn)。氯蟲苯甲酰胺是一種新型廣譜的魚尼丁受體殺蟲劑，對(duì)鱗翅目害蟲殺蟲活性強(qiáng)。本研究旨在探究氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾幼蟲3種主要解毒酶——羧酸酯酶（CarE）、谷胱甘肽S-轉(zhuǎn)移酶(GSTs)和多功能氧化酶(MFOs)活性以及對(duì)種群繁殖的影響?！痉椒ā坎捎蔑暳匣於痉y(cè)定氯蟲苯甲酰胺對(duì)SE-Lab品系、SE-Sel品系的毒力，SE-Sel品系由SE-Lab品系經(jīng)亞致死劑量LC25連續(xù)汰選6代得到；通過浸葉法測(cè)定磷酸三苯酯（TPP）、順丁烯二酸二乙酯（DEM）、胡椒基丁醚（PBO）3種酶抑制劑與氯蟲苯甲酰胺協(xié)同對(duì)SE-Lab和SE-Sel品系的毒力增效作用，提前12 h讓試蟲取食浸漬過酶抑制劑的葉片，對(duì)照組取食用0.1% TritonX-100浸漬后的葉片，再分別測(cè)定氯蟲苯甲酰胺對(duì)使用酶抑制劑與未使用酶抑制劑試蟲的毒力；在冰上解剖試蟲的中腸和脂肪體，并通過離體酶活性測(cè)定，分析氯蟲苯甲酰胺亞致死劑量與酶抑制劑對(duì)甜菜夜蛾體內(nèi)的3種代謝解毒酶活力的影響；通過記錄試蟲各個(gè)年齡階段的生長、死亡、產(chǎn)卵量等數(shù)據(jù)，參照兩性生命表理論分析SE-Lab和SE-Sel品系的兩性生命表參數(shù)差異?！窘Y(jié)果】在3種酶抑制劑中PBO增效作用最強(qiáng)，其對(duì)甜菜夜蛾SE-Sel品系和SE-Lab品系對(duì)氯蟲苯甲酰胺的毒力增效比分別為1.58和1.69。在氯蟲苯甲酰胺亞致死劑量連續(xù)汰選下，甜菜夜蛾體內(nèi)3種解毒酶活性均被誘導(dǎo)上升，其中MFOs酶活力上升最顯著，SE-Sel品系中腸和脂肪體的MFOs活性相對(duì)于SE-Lab品系分別提高了2.07和2.10倍，而經(jīng)氯蟲苯甲酰胺亞致死劑量再次誘導(dǎo)的SE-Sel試蟲的MFOs活性亦較SE-Lab品系上升4.02和3.44倍；在使用了酶抑制劑后3種解毒酶酶活力均有下降，其中MFOs活性下降最多，其酶比活力僅為未使用酶抑制劑處理的42.3%—44.8%。與SE-Lab品系相比，SE-Sel品系成蟲的產(chǎn)卵前期和總產(chǎn)卵前期變長，而產(chǎn)卵量減少；SE-Sel品系的內(nèi)稟增長率（r）、周限增長率(λ)和凈增殖率(R0)均顯著小于SE-Lab品系，SE-Lab品系與SE-Sel品系的r分別為0.18和0.16 d-1,λ為1.20和1.17 d-1, R0為358.42和203.12 d-1。盡管SE-Sel品系的平均世代周期（T）更長，但是與SE-Lab品系無顯著差異。【結(jié)論】MFOs可能為甜菜夜蛾對(duì)氯蟲苯甲酰胺解毒代謝過程中的主要解毒酶，在其后續(xù)抗性形成中起主要作用；甜菜夜蛾在氯蟲苯甲酰胺亞致死劑量作用下，世代周期延長，繁殖力降低，種群增長減緩，氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾有持續(xù)控制作用。

甜菜夜蛾；氯蟲苯甲酰胺；亞致死劑量；增效劑；多功能氧化酶；兩性生命表

0 引言

【研究意義】甜菜夜蛾（Spodoptera exigua）是一種世界范圍分布的重要農(nóng)業(yè)害蟲，可取食多種經(jīng)濟(jì)作物[1-2]。氯蟲苯甲酰胺是美國杜邦公司研發(fā)的新一代二酰胺類超高效殺蟲劑，通過激活昆蟲魚尼丁受體，過度釋放細(xì)胞內(nèi)儲(chǔ)存的鈣離子，導(dǎo)致昆蟲癱瘓抽搐，喪失行動(dòng)能力，進(jìn)而死亡[3-4]。由于其對(duì)多種鱗翅目害蟲防控效果明顯，在田間大量使用，加速了靶標(biāo)害蟲抗藥性的產(chǎn)生[5]。目前，甜菜夜蛾對(duì)氯蟲苯甲酰胺具有抗性風(fēng)險(xiǎn)，個(gè)別種群已達(dá)到高抗性[6]。昆蟲除了直接被殺蟲劑殺死外，隨著施藥時(shí)間的增長，藥劑部分降解至劑量不足以殺死昆蟲個(gè)體的亞致死劑量，該劑量仍可引起害蟲生態(tài)學(xué)、生殖力的變化、抗藥性的發(fā)展、藥劑代謝能力變動(dòng)等[7-8]，CHI[9]創(chuàng)建了年齡-齡期兩性生命表理論，該理論在外界條件對(duì)昆蟲種群生長繁殖影響的研究中越來越多地被運(yùn)用[10-11]?！厩叭搜芯窟M(jìn)展】昆蟲產(chǎn)生抗藥性的主要機(jī)理包括表皮穿透速率降低、對(duì)殺蟲劑解毒代謝作用增強(qiáng)（代謝解毒酶主要包括多功能氧化酶（MFOs）、羧酸酯酶（CarE）及谷胱甘肽S-轉(zhuǎn)移酶（GSTs））、靶標(biāo)位點(diǎn)敏感性下降以及昆蟲行為發(fā)生改變等[12]。在甜菜夜蛾的研究中，有報(bào)道溴氰蟲酰胺亞致死劑量對(duì)甜菜夜蛾幼蟲體內(nèi)解毒酶活性有不同程度的抑制作用[13]。WANG等[14]采用多殺菌素亞致死劑量汰選所得的抗性品系，其多功能氧化酶活性是敏感品系的5.2倍；在酶抑制劑的作用下，對(duì)氰氟蟲腙高抗的甜菜夜蛾種群 CarE的增效作用明顯高于MFOs與GSTs[15]。除甜菜夜蛾外，殺蟲劑亞致死劑量對(duì)昆蟲解毒酶影響在多種鱗翅目昆蟲中均有報(bào)道，如小菜蛾（Plutella xylostella）、二化螟（Chilo suppressalis）、棉鈴蟲（Helicoverpa armigera）等[16-18]。目前關(guān)于食物、溫度等外界條件對(duì)兩性生命表基本參數(shù)影響的報(bào)道較多[19-22]，近年也有通過兩性生命表研究殺蟲劑影響昆蟲生長繁殖的相關(guān)報(bào)道，ESMAEILY等[23]分別用阿維菌素、吡蟲啉、二嗪磷和吡蚜酮，以及白花牛角瓜（Calotropis procera）乙醇提取物對(duì)煙粉虱（Bemisia tabaci）進(jìn)行處理，分析其兩性生命表數(shù)據(jù)，發(fā)現(xiàn)吡蚜酮與白花牛角瓜乙醇提取物處理的試蟲內(nèi)稟增長率（r）、周限增長率（λ）均小于其他藥劑處理；JAFARBEIGI等[24]利用兩性生命表比較了4種植物乙醇提取物與吡蚜酮亞致死劑量對(duì)煙粉虱的生長繁殖的影響，篩選出兩種對(duì)煙粉虱防治效果理想的植物提取物；RAHMANI等[25]研究了噻蟲嗪亞致死劑量對(duì)異多瓢蟲（Hippodamia variegata）兩性生命表參數(shù)的影響，結(jié)果表明噻蟲嗪的使用不利于捕食性瓢蟲的生長繁殖，其亞致死劑量對(duì)3齡幼蟲的發(fā)育歷期、成蟲壽命和產(chǎn)卵量有顯著影響；SCHNEIDER等[26]研究發(fā)現(xiàn)，將草甘膦浸泡后的草蛉（Chrysoperla externa）卵與正常卵進(jìn)行兩性生命表參數(shù)統(tǒng)計(jì)，經(jīng)過一個(gè)生長周期后，發(fā)現(xiàn)草甘膦處理組的成蟲繁殖力較對(duì)照組明顯下降?！颈狙芯壳腥朦c(diǎn)】經(jīng)氯蟲苯甲酰胺亞致死劑量連續(xù)誘導(dǎo)甜菜夜蛾，通過增效劑試驗(yàn)和對(duì)解毒代謝酶活的測(cè)定，初步確定甜菜夜蛾代謝氯蟲苯甲酰胺的解毒酶類型；采用兩性生命表研究克服了傳統(tǒng)生命表中僅突出雌蟲對(duì)種群增長的貢獻(xiàn)，而忽略雄蟲對(duì)昆蟲種群發(fā)展的貢獻(xiàn)[11,27]。試驗(yàn)對(duì)SE-Lab和SE-Sel品系采用兩性生命表進(jìn)行種群分析，研究了氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾種群參數(shù)的影響，此研究未見報(bào)道?！緮M解決的關(guān)鍵問題】明確抗性形成相關(guān)的解毒代謝酶類型后，為表達(dá)相關(guān)解毒代謝酶的基因深入分析打下基礎(chǔ)；根據(jù)生命表的研究結(jié)果，了解氯蟲苯甲酰胺對(duì)甜菜夜蛾生長發(fā)育等方面的影響，為甜菜夜蛾田間防控提供理論支持和數(shù)據(jù)支撐。

1 材料與方法

試驗(yàn)于 2016年在四川農(nóng)業(yè)大學(xué)農(nóng)學(xué)院無公害農(nóng)藥研究實(shí)驗(yàn)室完成。

1.1 供試蟲源及飼養(yǎng)

甜菜夜蛾SE-Lab品系（SE-Lab）：2011年從中國農(nóng)業(yè)大學(xué)昆蟲生理生化與分子毒理學(xué)實(shí)驗(yàn)室引種連續(xù)飼養(yǎng)至今，實(shí)驗(yàn)室飼養(yǎng)條件為溫度（25±1）℃，相對(duì)濕度（65±5）%，光周期14L﹕10D。初孵幼蟲在含有人工飼料的指形管內(nèi)（直徑2.0 cm，高8.0 cm）飼養(yǎng)至化蛹，將蛹置于 0.5%次氯酸鈉溶液中浸泡消毒后，放入養(yǎng)蟲籠內(nèi)保濕培養(yǎng)待其羽化，成蟲羽化后飼喂10%蜂蜜水為其產(chǎn)卵提供營養(yǎng)，養(yǎng)蟲籠內(nèi)放入有褶皺的硫酸紙供成蟲產(chǎn)卵，定期收集卵塊，并用0.5%次氯酸鈉溶液對(duì)卵塊浸泡消毒后晾干，放入封口袋中，待幼蟲孵化。

甜菜夜蛾SE-Sel品系（SE-Sel）：以SE-Lab品系為基礎(chǔ)品系，4齡幼蟲時(shí)以飼料混毒法飼喂含LC25（SE-Lab品系亞致死劑量）氯蟲苯甲酰胺人工飼料，48 h后繼續(xù)飼喂新鮮飼料，連續(xù)汰選6代后繼續(xù)飼養(yǎng)，飼養(yǎng)方法同SE-Lab品系。

1.2 供試藥劑與儀器

95%氯蟲苯甲酰胺原藥（美國杜邦公司）；磷酸三苯酯（TPP）、順丁烯二酸二乙酯（DEM）、胡椒基丁醚（PBO）（上海aladdin試劑公司）；TritonX-100、α-乙酸萘酯（α-NA）、α-萘酚、固藍(lán)B鹽、對(duì)硝基苯甲醚、1-氯-2, 4-二硝基苯（CDNB）（成都艾科達(dá)化學(xué)試劑有限公司）；二硫蘇糖醇（DTT）、苯甲基磺酰氟（PMSF）、還原型谷胱甘肽、NADPH、牛血清蛋白（北京索萊寶科技有限公司）；毒扁豆堿（eserine）（Sigma-Aldrich公司）；乙二胺四乙酸二鈉鹽（EDTANa2）、十二烷基硫酸鈉（SDS）、考馬斯亮藍(lán)G250、丙三醇、85%磷酸、NaH2PO4、Na2HPO4、NaOH、37%鹽酸（成都市科龍化工試劑廠）。

紫外可見分光光度計(jì)UV-3000型（上海美譜達(dá)儀器有限公司）、全自動(dòng)酶標(biāo)儀680型（Bio-Rad公司）、臺(tái)式高速大容量冷凍離心機(jī) 5810型（Eppendorf 公司）。

1.3 生物測(cè)定

飼料混毒法：參照余慧靈等[13]方法，用丙酮將配置氯蟲苯甲酰胺1 g·L-1母液，用0.1% TritonX-100將母液稀釋成梯度濃度，取1 mL稀釋后的藥液加入至50 g人工飼料中混勻，試驗(yàn)工作濃度為0.005、0.01、0.02、0.04、0.08、0.16 μg·g-1，并設(shè)0.1% TritonX-100水溶液為空白對(duì)照，在細(xì)胞培養(yǎng)板中加入2.5 g已配好的混毒飼料，接入長勢(shì)均勻的甜菜夜蛾4齡初期幼蟲（試蟲體重范圍 3.78—4.65 mg/頭），每個(gè)濃度處理15頭，每個(gè)處理3次重復(fù)。

浸葉法：參照LAI等[6]方法，將已經(jīng)配置好的氯蟲苯甲酰胺1 g·L-1母液用0.1% TritonX-100稀釋成梯度濃度，試驗(yàn)工作濃度為0.2、0.4、0.6、0.8、1.0、1.2 mg·L-1，后將甘藍(lán)葉片（Φ=5.0 cm）在藥液中浸泡20 s，待其晾干后放入塑料培養(yǎng)皿（Φ=9.0 cm）中，每個(gè)培養(yǎng)皿接入4齡初期甜菜夜蛾幼蟲（試蟲體重范圍3.78—4.65 mg/頭）5頭，每個(gè)濃度處理15頭，每個(gè)處理重復(fù)3次，用0.1% TritonX-100處理的葉片做對(duì)照。解毒酶抑制劑測(cè)定時(shí)，用丙酮提前將酶抑制劑配制為10 g·L-1的母液，再用0.1% TritonX-100將母液稀釋為100 mg·L-1的工作液，測(cè)定毒力前12 h讓甜菜夜蛾幼蟲取食經(jīng)過酶抑制劑浸泡后的甘藍(lán)葉片，對(duì)照組取食用0.1% TritonX-100浸泡后的甘藍(lán)葉片，再采用浸葉法進(jìn)行毒力測(cè)定。生物測(cè)定48 h后檢查試蟲死亡率，用軟毛筆觸碰蟲體，不能協(xié)調(diào)運(yùn)動(dòng)的試蟲視為死亡。

1.4 解毒酶比活力測(cè)定

1.4.1 羧酸酯酶活性測(cè)定 參照余慧靈等[13,28]方法，將甜菜夜蛾的冷凍組織與 1 mL磷酸鹽緩沖液（0.04 mol·L-1，pH 7.0）冰浴勻漿，并在4℃ 10 000×g條件下離心10 min，取上清液為待測(cè)酶液低溫儲(chǔ)存?zhèn)溆?。體系反應(yīng)體積為3.2 mL，含有50 μL稀釋后的酶液，0.45 mL磷酸鹽緩沖液與1.8 mL的0.3 mmol·L-1的α-NA溶液（含有0.3 mmol·L-1毒扁豆堿）。30℃溫育15 min后加入0.9 mL顯色液，對(duì)照組在反應(yīng)結(jié)束后再添加酶液，在600 nm比色測(cè)定OD值。制作α-萘酚標(biāo)準(zhǔn)曲線與蛋白質(zhì)含量標(biāo)準(zhǔn)曲線，計(jì)算比活力（mmol·min-1·mg-1pro）。

1.4.2 谷胱甘肽 S-轉(zhuǎn)移酶活性測(cè)定 參照余慧靈等[13,29]方法，將甜菜夜蛾的冷凍組織與 1 mL含 1.0 mmol·L-1EDTA的磷酸鹽緩沖液（0.04 mol·L-1，pH 7.0）冰浴勻漿，并在4℃ 10 000×g條件下離心10 min，取上清液為待測(cè)酶液低溫儲(chǔ)存?zhèn)溆谩ｓw系中含有30 μL CDNB（15 mmol·L-1），790 μL磷酸鹽緩沖液，30 μL還原型谷胱甘肽（30 mmol·L-1）和50 μL酶液，在340 nm比色記錄OD值5 min的變化情況，根據(jù)酶液蛋白質(zhì)含量標(biāo)準(zhǔn)曲線，計(jì)算比活力（mmol·min-1·mg-1pro）。

1.4.3 多功能氧化酶 O-脫甲基活力測(cè)定 參照余慧靈等[13,30]方法有改動(dòng)，在將甜菜夜蛾的冷凍組織中加3 mL磷酸鹽緩沖液（0.1 mol·L-1，pH 7.8，含0.1 mmol·L-1DTT，0.1 mmol·L-1EDTA，0.1 mmol·L-1PMSF，20%甘油）冰上勻漿，勻漿液于4℃ 10 000×g離心10 min，取上清液為酶液低溫儲(chǔ)存?zhèn)溆?。在酶?biāo)板中加入100 μL對(duì)硝基苯甲醚（2 mmol·L-1），90 μL酶液再加入10 μL NADPH（0.5 mmol·L-1），30℃溫育10 min。在波長405 nm處測(cè)定OD值。對(duì)照組在溫育后加入 NADPH，制作對(duì)硝基苯酚制作標(biāo)準(zhǔn)曲線與酶液蛋白質(zhì)含量標(biāo)準(zhǔn)曲線，計(jì)算比活力（nmol·min-1·mg-1pro）。

1.4.4 蛋白質(zhì)含量的測(cè)定 參照 BRADFORD[31]考馬斯亮藍(lán)G250法測(cè)定，用各處理低溫儲(chǔ)存的酶液進(jìn)行蛋白測(cè)定。

1.5 兩性生命表

采用SE-Lab與SE-Sel品系的F1代構(gòu)建生命表。參照郝強(qiáng)等[19]方法，在雌蟲產(chǎn)卵期的第2天隨機(jī)收集5個(gè)卵塊，用0.5%次氯酸鈉溶液浸泡30 s后晾干，放入封口袋中，待卵塊孵化后挑取 30頭初孵幼蟲；將150頭初孵幼蟲用人工飼料飼養(yǎng)，放入指形管（每管放入一頭蟲），待其化蛹；用0.5%次氯酸鈉溶液對(duì)蛹行進(jìn)浸泡消毒，放入六孔細(xì)胞培養(yǎng)板中保濕培養(yǎng)待蛹羽化，并將羽化后的成蟲編號(hào)，根據(jù)雌雄個(gè)體進(jìn)行一比一配對(duì)，放入圓柱形一次性帶蓋塑料杯（上口直徑×下口直徑×高=9.0 cm×5.0 cm×17.0 cm）飼養(yǎng)，提供10%蜂蜜水供成蟲補(bǔ)充營養(yǎng)繁殖產(chǎn)卵，如有未配對(duì)的成蟲，則從初期5個(gè)隨機(jī)收集卵塊中成長的成蟲中挑選健康的異性成蟲進(jìn)行配對(duì)；每天準(zhǔn)確記錄甜菜夜蛾各個(gè)階段的生長狀況、產(chǎn)卵情況，直至成蟲死亡。試蟲飼養(yǎng)環(huán)境條件為溫度（25±1）℃、相對(duì)濕度（65±10）%和光周期14L﹕10D。

1.6 數(shù)據(jù)處理

1.6.1 生命表參數(shù) 試蟲各階段年齡時(shí)間、成蟲存活時(shí)間及產(chǎn)卵量等原始數(shù)據(jù)參照年齡-階段兩性生命表理論[9]進(jìn)行統(tǒng)計(jì)，并用程序TWOSEX-MSChart[32]計(jì)算相關(guān)生命表參數(shù)，生命表參數(shù)中x表示年齡，j表示試蟲發(fā)育階段；年齡-階段存活率（sxj）；種群特定年齡存活率（lx）；計(jì)算公式：

種群特定年齡繁殖力（mx）指整個(gè)種群在年齡 x的平均產(chǎn)卵數(shù)量，雌蟲年齡-階段繁殖力（fxj），計(jì)算公式：

特定年齡-階段壽命期望值（exj）是指在x年齡j階段的個(gè)體可以繼續(xù)存活的時(shí)間，年齡-階段生殖值（vxj）就是在x年齡j階段的個(gè)體為種群的增長的貢獻(xiàn)值；種群特定年齡存活率（lx）與種群特年齡繁殖力（mx）的乘積為種群特異性年齡繁殖值（lxmx）。內(nèi)稟增長率（r）指種群在理想狀態(tài)下的最大種群增長率；周限增長率（λ）指種群在理想狀態(tài)下，種群內(nèi)平均每個(gè)個(gè)體能產(chǎn)生的后代數(shù)；凈增殖率（R0）指?jìng)€(gè)體的總后代數(shù)；平均世代周期（T）指當(dāng)一個(gè)種群達(dá)到穩(wěn)定增長速率時(shí)，增加到 R0所需要的時(shí)間。計(jì)算公式：

生命表參數(shù)數(shù)據(jù)使用Sigma plot 12.5作圖，生命表參數(shù)的平均值和標(biāo)準(zhǔn)誤用bootstrap技術(shù)[33]來估算，用Paired bootstrap test（TWOSEX-MSChart）程序[32]計(jì)算甜菜夜蛾發(fā)育歷期、繁殖值和種群參數(shù)間的差異顯著性。

1.6.2 生物測(cè)定數(shù)據(jù)分析 采用POLO-Plus10.0軟件計(jì)算毒力參數(shù)。采用SPSS Statistics 19.0軟件分析酶活性等的差異顯著性。

2 結(jié)果

2.1 氯蟲苯甲酰胺對(duì)甜菜夜蛾幼蟲的亞致死劑量

氯蟲苯甲酰胺對(duì)SE-Lab品系48 h的亞致死劑量LC25為0.017 μg·g-1，對(duì)SE-Sel品系48 h亞致死劑量LC25為0.041 μg·g-1。因此，甜菜夜蛾SE-Lab品系經(jīng)過LC25亞致死劑量連續(xù)汰選6代后，其對(duì)氯蟲苯甲酰胺的敏感度降至汰選前的48.5%（表1）。

2.2 酶抑制劑對(duì)甜菜夜蛾幼蟲毒力的影響

對(duì)SE-Lab與SE-Sel品系分別用酶抑制劑進(jìn)行處理，SE-Lab品系經(jīng)過TPP和DEM處理后抗性倍數(shù)分別增加1.06、1.47倍，經(jīng)過PBO處理抗性倍數(shù)增加1.58倍。SE-Sel品系在TPP和DEM處理后抗性倍數(shù)分別增加1.05、1.51倍，經(jīng)過PBO處理后增加1.69倍。其中PBO和DEM在SE-Sel品系的增效效果高于SE-Lab品系（表2）。

2.3 氯蟲苯甲酰胺亞致死劑量與增效劑對(duì)甜菜夜蛾幼蟲體內(nèi)主要代謝解毒酶活性的影響

各處理中腸3種解毒酶活性普遍高于脂肪體中酶

表1 氯蟲苯甲酰胺對(duì)甜菜夜蛾4齡幼蟲的毒力Table 1 Toxicity of chlorantraniliprole to the 4th instar larvae of S. exigua

表2 氯蟲苯甲酰胺與氯蟲苯甲酰胺+酶抑制劑對(duì)甜菜夜蛾4齡幼蟲毒力

增效比SR (synergism ratio)：試蟲品系對(duì)氯蟲苯甲酰胺LC50與在使用增效劑后的氯蟲苯甲酰胺LC50的比值LC50of a strain treated with chlorantraniliprole alone divided by LC50of the same strain treated with chlorantraniliprole plus enzyme inhibitors

Table 2 Responses of the 4th instar larvae to chlorantraniliprole or chlorantraniliprole plus enzyme inhibitors in the SE-Lab and SE-Sel strains of S. exigua活性，且SE-Sel品系3個(gè)解毒酶活性為SE-lab品系的1.07—2.10倍，其中多功能氧化酶增長倍數(shù)最高，中腸和脂肪體分別增長2.07與2.10倍；氯蟲苯甲酰胺LD50劑量對(duì)SE-Sel品系再次誘導(dǎo)處理48 h后酶活性表明，再次誘導(dǎo)處理的試蟲3種解毒酶活性較SE-Sel品系均有有不同程度的增加，尤其以多功能氧化酶增長最明顯，在中腸和脂肪體中分別比SE-Lab品系增加了3.44與4.02倍，羧酸酯酶與谷胱甘肽S-轉(zhuǎn)移酶為SE-Lab品系的1.30—2.61倍；而以增效劑+氯蟲苯甲酰胺LD50劑量處理SE-Sel品系試蟲，解毒酶活結(jié)果表明，試蟲酶活性較只用藥劑的酶活性有顯著降低，尤其以PBO處理的多功能氧化酶效果最為明顯。在使用酶抑制劑后，3種解毒酶活性均有降低，其中多功能氧化酶活性下降最多，其酶活力值僅為未使用酶抑制劑處理的 42.3%—44.8%（表3）。

表3 各處理的甜菜夜蛾中腸與脂肪體解毒酶比活力

Table 3 The specific activities of detoxification enzymes in midgut and fatbody of S. exigua

表中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤，同列數(shù)據(jù)后含有相同字母表示差異不顯著，括號(hào)中為每個(gè)處理數(shù)值與第一行處理數(shù)據(jù)的比值 Data in the table were represented as mean±SE. The same letter after the data in the same column indicated no significant difference. Data in brackets indicated induced fold compared with data of first row；*酶抑制劑Enzyme inhibitors：使用需要測(cè)定的3種解毒酶的專性抑制劑，羧酸酯酶使用TTP，谷胱甘肽S-轉(zhuǎn)移酶使用DEM，多功能氧化酶使用PBO Specific inhibitors of three detoxifying enzymes needed to be measured. Carboxylesterases used TTP, glutathione S-transferases used DEM, multifunctional oxidases used PBO

2.4 氯蟲苯甲酰胺亞致死劑量與甜菜夜蛾兩性生命表

2.4.1 氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾生長發(fā)育與繁殖情況的影響 在3齡、4齡與預(yù)蛹以外的時(shí)期，SE-Lab與SE-Sel品系生長發(fā)育歷期均有顯著差異；SE-Sel品系的成蟲前期較SE-Lab品系顯著增加，SE-Sel品系的產(chǎn)卵前期（APOP）及總產(chǎn)卵前期（TPOP）較 SE-Lab品系也有所延長；兩個(gè)品系雄蟲的壽命并無顯著差異，但雌蟲壽命SE-Lab品系顯著低于SE-Sel品系，在SE-Sel品系中雄蟲比例較SE-Lab品系有所增加；兩個(gè)品系的平均單雌產(chǎn)卵量有顯著差異，SE-Lab品系為995.61粒，SE-Sel品系為743.12粒（表4）。

2.4.2 氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾存活率與繁殖力的影響 年齡-階段特征存活率（sxj）曲線（圖1）表示甜菜夜蛾從初產(chǎn)卵活到年齡x和階段j的可能性，在各個(gè)體之間復(fù)雜的生長發(fā)育階段形成了大量的時(shí)間重疊，自5齡幼蟲開始，后期的生長中，SE-Sel品系較 SE-Lab品系存活率有一個(gè)明顯的下降；計(jì)算各品系中由卵完全發(fā)育為成蟲的概率，SE-Sel品系為51.3%，SE-Lab品系為76.7%。

圖2中SE-Sel與SE-Lab品系的種群特定年齡存活率（lx）在生長階段的前期與后期各有一個(gè)較陡的斜線，這兩個(gè)時(shí)期為種群死亡高發(fā)期，對(duì)應(yīng)年齡階段為卵過度到2齡幼蟲的時(shí)期與成蟲后期，其中SE-Sel品系死亡率更高；兩個(gè)品系的種群特定年齡繁殖力（mx）呈現(xiàn)先增后降的趨勢(shì)，并在30—38 d達(dá)到繁殖高峰，SE-Sel品系的繁殖高峰產(chǎn)卵為70.25粒，SE-Lab品系為88.40粒，雌蟲年齡-階段繁殖力（fxj）在SE-Lab品系呈現(xiàn)先增后降的曲線，但在SE-Sel中在雌蟲產(chǎn)卵初期即出現(xiàn)產(chǎn)卵峰，之后產(chǎn)生一定程度波動(dòng)。

特定年齡-階段壽命期望值（exy）曲線（圖3）表示年齡x階段j的個(gè)體預(yù)期能存活的總時(shí)間。隨著年齡的增長，壽命期望值會(huì)隨之降低，SE-Lab品系的壽命期望值高于SE-Sel品系。

年齡-階段生殖值（vxj）曲線（圖4） 表示年齡x階段j的個(gè)體對(duì)未來種群的貢獻(xiàn)，兩個(gè)品系的雌成蟲隨著齡期的增加而達(dá)到生殖高峰，SE-Lab品系在29 d達(dá)到高峰，生殖力為634.6粒，SE-Sel品系在產(chǎn)卵早期即達(dá)到最高峰，在25 d時(shí)達(dá)到700.1粒。

2.4.3 氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾生命表種群參數(shù)的影響 SE-Sel品系的內(nèi)稟增長率（r）、周限增長率（λ）、凈增殖率（R0）分別為0.18、1.20與358.42 d-1，SE-Lab品系分別為0.16、1.17與203.12 d-1，且SE-Sel品系顯著低于SE-Lab品系；SE-Sel品系平均世代周期（T）與Se-Lab品系差異不顯著（表5）。

表4 甜菜夜蛾在氯蟲苯甲酰胺亞致死劑量作用下的基本生活史參數(shù)Table 4 Parameters of life history of sublethal of chlorantraniliprole on developmental duration of S. exigua

圖1 氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾年齡-階段特征存活率(sxj)的影響Fig. 1 The effects of chlorantraniliprole sublethal dosage on age-stage specific survival rate (sxj) of S. exigua

圖 2 氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾種群特定年齡存活率(lx)、雌蟲年特定年齡繁殖力(fx9)、種群特定年齡繁殖力(mx)和種群特定年齡繁殖值(lxmx)的影響Fig. 2 The effects of chlorantraniliprole sublethal dosage on age-specific survival rate (lx), female age-specific fecundity (fx9), age-specific fecundity of total population (mx), and age-specific maternity (lxmx) of S. exigua

圖3 氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾年齡-階段特征壽命期望值(exj)的影響Fig. 3 The effects of chlorantraniliprole sublethal dosage on age-stage specific life expectancies (exj) of S. exigua

表5 氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾生命表參數(shù)的影響Table 5 The effects of sublethal dosage of chlorantraniliprole on population parameters of S. exigua

3 討論

殺蟲劑亞致死劑量處理對(duì)昆蟲生長發(fā)育、繁殖力及抗性等有相應(yīng)程度的影響[34]。刑靜等[35]報(bào)道，氯蟲苯甲酰胺亞致死劑量處理小菜蛾3齡幼蟲24、48和72 h，試蟲體內(nèi)的細(xì)胞色素P450 O-脫乙基酶（ECOD）和谷胱甘肽S-轉(zhuǎn)移酶（GSTs）酶活性均被抑制；在使用PBO、DEM、TPP酶抑制劑后，SE-Lab品系和抗性品系的小菜蛾的抗性均有相應(yīng)的增加，其中 DEM增效效果最佳，對(duì)抗性品系的小菜蛾使用氯蟲苯甲酰胺短期誘導(dǎo)后，其GSTs酶活性也顯著增加[36]；據(jù)余慧靈等[13]報(bào)道，以LC10和LC25劑量處理甜菜夜蛾3齡幼蟲24 h，3種解毒酶活性均有相應(yīng)的增加。本試驗(yàn)中，SE-Sel品系的GSTs與MFOs酶活性較SE-Lab有明顯的增加，而在用亞致死劑量氯蟲苯甲酰胺誘導(dǎo)SE-Sel品系后3種解毒酶活性均有一定程度的上升，其中MFOs活性增長倍數(shù)最高，根據(jù)昆蟲抗藥性機(jī)理，多功能氧化酶可能與甜菜夜蛾對(duì)氯蟲苯甲酰胺產(chǎn)生抗性有關(guān)，并與表達(dá)相關(guān)解毒酶的基因相關(guān)[37-38]。

兩性生命表中研究雄蟲的參數(shù)，在傳統(tǒng)生命表中是相對(duì)缺乏的，ZHANG等[39]用氯蟲苯甲酰胺亞致死劑量處理棉鈴蟲后，F(xiàn)1代雄蟲比例增加；DELPUECH等[40]研究表明，在亞致死劑量藥劑壓力下，昆蟲間的信息素傳遞受到影響，使雌成蟲生殖力降低使后代雄蟲數(shù)量的增加，從而影響種群的性別分布。由于卵子受精是由中樞神經(jīng)系統(tǒng)控制的，而殺蟲劑通過影響神經(jīng)傳導(dǎo)干擾卵子受精致使受精卵的減少，來控制昆蟲性別比例變化[41]。本研究中，SE-Sel品系雄成蟲與雌成蟲的性別比例較 SE-Lab品系有所增加，與前人研究結(jié)果變化趨勢(shì)一致。宋月芹等[42]研究表明，用氯蟲苯甲酰胺LC10劑量處理試蟲后，亞洲玉米螟（Ostrinia furnacalis）成蟲壽命變長且雌成蟲壽命明顯延長；楊洪等[43]用氯蟲苯甲酰胺 LC25劑量處理白背飛虱（Sogatella furcifera）后，雌成蟲壽命都有一定的縮短；而LAI等[44]采用氯蟲苯甲酰胺亞致死劑量處理甜菜夜蛾幼蟲后，則發(fā)現(xiàn)成蟲壽命沒有顯著變化。本研究表明，SE-Sel品系雌成蟲壽命相對(duì)于SE-Lab品系雌成蟲有一定程度的增加。因此，不同昆蟲在不同環(huán)境下，經(jīng)不同藥劑處理可能對(duì)昆蟲雌成蟲壽命有不同影響。

圖4 氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾的年齡-階段特征生殖能力(vxj)的影響Fig. 4 The effects of chlorantraniliprole sublethal dosage on age-stage specific reproductive values (vxj) of S. exigua

殺蟲劑亞致死劑量對(duì)昆蟲所產(chǎn)生的效應(yīng)與施用殺蟲劑種類和濃度水平、受藥昆蟲的種類和生理狀況有關(guān)系。GUO等[45]報(bào)道，小菜蛾取食含LC25劑量氯蟲苯甲酰胺的甘藍(lán)葉片后，世代周期增長，凈增殖率明顯降低，此現(xiàn)象有跨代效應(yīng)，在F1代中仍然出現(xiàn)，統(tǒng)計(jì)試蟲死亡率發(fā)現(xiàn)經(jīng)藥劑處理試蟲的 F1代較對(duì)照組顯著增加；HAN等[46]用氯蟲苯甲酰胺LC10、LC25劑量處理小菜蛾后，發(fā)現(xiàn)試蟲發(fā)育歷期明顯增長，雌成蟲產(chǎn)卵量也有相應(yīng)程度的降低；陳瓊等[47]用氯蟲苯甲酰胺LC25處理甜菜夜蛾后也得到相似結(jié)果。但是，也有殺蟲劑在誘導(dǎo)昆蟲親代后出現(xiàn)猖獗現(xiàn)象并促進(jìn)昆蟲繁殖率的報(bào)道，如綠盲蝽（Apolygus lucorum）[48]、大螟（Sesamia inferens）[49]和小菜蛾[50]等。本研究中，SE-Sel品系經(jīng)過連續(xù)6代的藥劑亞致死劑量處理，較傳統(tǒng)通過藥劑處理1代的亞致死效應(yīng)研究，更類似于田間環(huán)境下殺蟲劑對(duì)連續(xù)多代害蟲的影響，SE-Sel品系在各年齡階段的存活率與雌成蟲繁殖力較 SE-Lab品系有明顯的下降的現(xiàn)象，該現(xiàn)象可能與氯蟲苯甲酰胺處理的跨代效應(yīng)有關(guān)。由于連續(xù)的藥劑誘導(dǎo)，試蟲對(duì)殺蟲劑耐受能力增強(qiáng)，但甜菜夜蛾的幼蟲存活率、繁殖力、內(nèi)稟增長率、周限增長率、凈增殖率等種群參數(shù)均有下降。YIN等[51]使用多抗菌素亞致死劑量連續(xù)5代誘導(dǎo)小菜蛾，小菜蛾的種群增長受到抑制，其繁殖力、內(nèi)稟增長率、周限增長率、凈增殖率等參數(shù)均有一定程度下降；劉澤文等[52]研究發(fā)現(xiàn)，田間采集的抗吡蟲啉褐飛虱（Nilaparvata lugens）種群與敏感品系相比，相對(duì)適合度有明顯下降，田間種群表現(xiàn)出繁殖不利現(xiàn)象；陳朗杰等[53]通過對(duì)抗性品系與敏感品系的橘小實(shí)蠅（Bactrocera dorsalis）的種群生物學(xué)參數(shù)進(jìn)行比較，發(fā)現(xiàn)高抗性品系的繁殖力和種群世代增長量受到抑制，中抗品系尤為明顯?？顾幮栽黾恿撕οx抗性個(gè)體在藥劑選擇壓力下的存活率，但由于藥劑的持續(xù)壓力使害蟲的生存繁殖力下降，以至于抗性種群與敏感種群相比并沒有明顯生存競(jìng)爭優(yōu)勢(shì)。氯蟲苯甲酰胺亞致死劑量的連續(xù)處理雖然對(duì)甜菜夜蛾的種群增長有抑制或減緩的作用，但也會(huì)使其逐漸的形成抗藥性。

本研究從解毒酶及生命表參數(shù)兩方面分析氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾的影響，可為進(jìn)一步從分子角度研究與抗藥性相關(guān)的解毒代謝酶基因提供依據(jù)；兩性生命表研究可更直觀地了解連續(xù)亞致死劑量的氯蟲苯甲酰胺對(duì)甜菜夜蛾各齡期的生命參數(shù)影響，是對(duì)傳統(tǒng)生命表研究的重要補(bǔ)充，為田間害蟲的藥劑防控提供理論依據(jù)。

4 結(jié)論

多功能氧化酶可能為甜菜夜蛾對(duì)氯蟲苯甲酰胺解毒代謝過程中的主要解毒酶，在其后續(xù)抗性形成中起主要作用；甜菜夜蛾在氯蟲苯甲酰胺亞致死劑量作用下，世代周期延長，繁殖力降低，種群增長減緩，氯蟲苯甲酰胺亞致死劑量對(duì)甜菜夜蛾有持續(xù)控制作用。

[1] JAKUBOWSKA A K, LYNN D E, HERRERO S, VLAK J M, VAN OERS M M. Host-range expansion of Spodoptera exigua multiple nucleopoly hedrovirus to Agrotis segetum larvae when the midgut is bypassed. Journal of General Virology, 2010, 91(4): 898-906.

[2] WU G, GUO J Y, WAN F H, XIAO N W. Responses of three successive generations of beet armyworm, Spodoptera exigua, fed exclusively on different levels of gossypol in cotton leaves. Journal of Insect Science, 2010, 10: Article 165.

[3] LI Y X, MAO M Z, LI Y M, XIONG L X, LI Z M, XU J Y. Modulations of high-voltage activated Ca2+channels in the central neurones of Spodoptera exigua by chlorantraniliprole. Physiological Entomology, 2011, 36(3): 230-234.

[4] TEIXEIRA L A, ANDALORO J T. Diamide insecticides: Global efforts to address insect resistance stewardship challenges. Pesticide Biochemistry and Physiology, 2013, 106(3): 76-78.

[5] SAIL A A, BRUNNER J F. Assessment of resistance risk in obliquebanded leafroller (Lepidoptera: Tortricidae) to the reduced-risk insecticides chlorantraniliprole and spinetoram. Journal of Economic Entomology, 2010, 103(4): 1378-1385.

[6] LAI T C, LI J, SU J Y. Monitoring of beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) resistance to chlorantraniliprole in China. Pesticide Biochemistry and Physiology, 2011, 101(3): 198-205.

[7] RAKOTONDRAVELO M L, ANDERSON T D, CHARLTON R E, ZHU K Y. Sublethal effects of three pesticides on activities of selected target and detoxification enzymes in the aquatic midge, Chironomus tentans (Diptera: Chironomidae). Archives of Environmental Contamination and Toxicology, 2006, 51(3): 360-366.

[8] VOJOUDI S, SABER M, HEJAZI M J,TALAEI-HASSANLOUI R. Toxicity of chlorpyrifos, spinosad and abamectin on cotton bollworm, Helicoverpa armigera and their sublethal effects on fecundity and longevity. Bulletin of Insectology, 2011, 64(2): 189-193.

[9] CHI H. Life-table analysis incorporating both sexes and variable development rates among individuals. Environmental Entomology, 1988, 17(1): 26-34.

[10] CHI H, SU H Y. Age-stage, two-sex life tables of Aphidius gifuensis (Ashmead) (Hymenoptera: Braconidae) and its host Myzus persicae (Sulzer) (Homoptera: Aphididae) with mathematical proof of the relationship between female fecundity and the net reproductive rate. Environmental Entomology, 2006, 35(1): 10-21.

[11] HUANG Y B, CHI H. The age-stage, two-sex life table with an offspring sex ratio dependent on female age. Journal of Agricultural and Forest, 2011, 60(4): 337-345.

[12] 劉喃喃, 朱芳, 徐強(qiáng), PRIDGEON J W, 高希武. 昆蟲抗藥性機(jī)理:行為和生理改變及解毒代謝增強(qiáng). 昆蟲學(xué)報(bào), 2006, 49(4): 671-679.

LIU N N, ZHU F, XU Q, PRIDGEON J W, GAO X W. Behavioral change, physiological modification, and metabolic detoxification: mechanisms of insecticide resistance. Acta Entomologica Sinica, 2006, 49(4): 671-679. (in Chinese)

[13] 余慧靈, 向興, 袁貴鑫, 陳羿渠, 王學(xué)貴. 溴氰蟲酰胺亞致死劑量對(duì)甜菜夜蛾生長發(fā)育及體內(nèi)解毒酶活性的影響. 昆蟲學(xué)報(bào), 2015, 58(6): 634-641.

YU H L, XIANG X, YUAN G X, CHEN Y Q, WANG X G. Effects of sublethal doses of cyantraniliprole on the growth and development and the activities of detoxifying enzymes in Spodoptera exigua (Lepidoptera: Noctuidae). Acta Entomologica Sinica, 2015, 58(6): 634-641. (in Chinese)

[14] WANG W, MO J C, CHENG J A, ZHUANG P J, TANG Z H. Selection and characterization of spinosad resistance in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). Pesticide Biochemistry and Physiology, 2006, 84(3): 180-187.

[15] TIAN X R, SUN X X, SU J Y. Biochemical mechanisms for metaflumizone resistance in beet armyworm, Spodoptera exigua. Pesticide Biochemistry and Physiology, 2014, 113(1): 8-14.

[16] 賈變桃, 焦鵬, 楊素梅. 虱螨脲亞致死濃度對(duì)小菜蛾保護(hù)酶系和解毒酶系活力的影響. 植物保護(hù)學(xué)報(bào), 2016, 43(2): 293-299.

JIA B T, JIAO P, YANG S M. Effects of sublethal concentrations of lufenuron on endogenous protective and detoxifying enzymes in the diamondback moth, Plutella xylostella (L.). Journal of Plant Protection, 2016, 43(2): 293-299. (in Chinese)

[17] 歐善生, 梁沛, 宋敦倫, 史雪巖, 高希武. 氯蟲苯甲酰胺亞致死劑量對(duì)棉鈴蟲生長發(fā)育和解毒酶活性的影響. 植物保護(hù), 2012, 38(4): 1-8.

OU S S, LIANG P, SONG D L, SHI X Y, GAO X W. Effects of sublethal dosage of chlorantraniliprole on development and detoxifying enzymes activity of Helicoverpa armigera. Plant Protection, 2012, 38(4): 1-8. (in Chinese)

[18] 黃誠華, 姚洪渭, 葉恭銀, 程家安. 氟蟲腈亞致死劑量處理對(duì)二化螟和大螟幼蟲體內(nèi)解毒酶系活力的影響. 中國水稻科學(xué), 2006, 20(4): 447-450.

HUANG C H, YAO H W, YE G Y, CHENG J A. Effects of sublethal dose of fipronil on detoxifying enzymes in the larvae of Chilo suppressalis and Sesamia inferens. Chinese Journal of Rice Science, 2006, 20(4): 447-450. (in Chinese)

[19] 郝強(qiáng), 黃倩, 梁煒博, 貢常委, 王學(xué)貴. 不同溫度下斜紋夜蛾的兩性生命表. 昆蟲學(xué)報(bào), 2016, 59(6): 654-662.

HAO Q, HUANG Q, LIANG W B, GONG C W, WANG X G. Age-stage two-sex life tables of Spodoptera litura (Lepidoptera: Noctuidae) at different temperatures. Acta Entomologica Sinica, 2016, 59(6): 654-662. (in Chinese)

[20] TUAN S J, LI N J, YEH C C, TANG L C, CHI H. Effects of green manure cover crops on Spodoptera litura (Lepidoptera: Noctuidae) populations. Journal of Economic Entomology, 2014, 107(3): 897-905.

[21] JHA R K, TUAN S J, CHI H, TANG L C. Life table and consumption capacity of corn earworm, Helicoverpa armigera, fed asparagus, Asparagus officinalis. Journal of Insect Science, 2014, 14: Article 34.

[22] 王海鴻, 薛瑤, 雷仲仁. 恒溫和波動(dòng)溫度下西花薊馬的實(shí)驗(yàn)種群生命表. 中國農(nóng)業(yè)科學(xué), 2014, 47(1): 61-68.

WANG H H, XUE Y, LEI Z R. Life tables for experimental populations of Frankliniella occidentalis (Thysanoptera: Thripidae) under constant and fluctuating temperature. Scientia Agricultura Sinica, 2014, 47(1): 61-68. (in Chinese)

[23] ESMAEILY S, SAMIH M A, ZARABI M, JAFARBEIGI F. Sublethal effects of some synthetic and botanical insecticides on Bemisia tabaci (Hemiptera: Aleyrodidae). Journal of Plant Protection Research, 2014, 54(2): 171-178.

[24] JAFARBEIGI F, SAMIH M A, ZARABI M, ESMAEILY S. Age stage two-sex life table reveals sublethal effects of some herbal and chemical insecticides on adults of Bemisia tabaci (Hem.: Aleyrodidae). Psyche A Journal of Entomology, 2014, 2014(3): 1-9.

[25] RAHMANI S, BANDANI A R. Sublethal concentrations of thiamethoxam adversely affect life table parameters of the aphid predator, Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae). Crop Protection, 2013, 54(12): 168-175.

[26] SCHNEIDER M I, SANCHEZ N, PINEDA S, CHI H, RONCO A. Impact of glyphosate on the development, fertility and demography of Chrysoperla externa (Neuroptera: Chrysopidae): Ecological approach. Chemosphere, 2009, 76(10): 1451-1455.

[27] HUANG Y B, CHI H. Age-stage, two-sex life tables of Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) with a discussion on the problem of applying female age-specific life tables to insect populations. Insect Science, 2012, 19(2): 263-273.

[28] ASPEREN K V. A study of housefly esterases by means of a sensitive colorimetric method. Journal of Insect Physiology, 1962, 8(4): 401-416.

[29] HABIG W H, JAKOBY W B. Assays for differentiation of glutathione S-transferases. Methods in Enzymology, 1981, 77: 398-405.

[30] ROSE R L, BARBHAIYA L, ROE R M, ROCK G C, HODGSON E. Cytochrome P450-associated insecticide resistance and the development of biochemical diagnostic assays in Heliothis virescens. Pesticide Biochemistry and Physiology, 1995, 51(3): 178-191.

[31] BRADFORD M M A. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 25(1): 248-256.

[32] CHI H. TWOSEX-MSChart: A computer program for the age stage, two-sex life table analysis. National Chung Hsing University, Taichung, Taiwan. 2016. http://140.120.197.173/Ecology/.

[33] HUANG Y B, CHI H. Life tables of Bactrocera cucurbitae (Diptera: Tephritidae): with an invalidation of the jackknife technique. Journal of Applied Entomology, 2013, 137(5): 327-339.

[34] DESNEUX N, DECOURTYE A, DELPUECH J M. The sublethal effects of pesticides on beneficial arthropods. Annual Review of Entomology, 2007, 52(1): 81-106.

[35] 邢靜, 梁沛, 高希武. 亞致死濃度氯蟲苯甲酰胺對(duì)小菜蛾藥劑敏感度和解毒酶活性的影響. 農(nóng)藥學(xué)學(xué)報(bào), 2011, 13(5): 464-470.

XING J, LIANG P, GAO X W. Effects of sublethal concentrations ofchlorantraniliprole on insecticide susceptibility and detoxifying enzyme activity in Plutella xylostella. Chinese Journal of Pesticide Science, 2011, 13(5): 464-470. (in Chinese)

[36] HU Z D, FENG X, LIN Q S, CHEN H Y, LI Z Y, YIN F, LIANG P, GAO X W. Biochemical mechanism of chlorantraniliprole resistance in the diamondback moth, Plutella xylostella, Linnaeus. Journal of Integrative Agriculture, 2014, 13(11): 2452-2459.

[37] ZHONG D, CHANG X, ZHOU G, HE Z, FU F, YAN Z, ZHU G, XU T, BONIZZONI M, WANG MH, CUI L, ZHENG B, CHEN B, YAN G. Relationship between knockdown resistance, metabolic detoxification and organismal resistance to pyrethroids in Anopheles sinensis. PLoS ONE, 2013, 8(2): e55475.

[38] LI X C, SCHULER M A, BERENBAUM M R. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annual Review of Entomology, 2007, 52(1): 231-253.

[39] ZHANG R M, DONG J F, CHEN J H, QING E J, CUI J J. The sublethal effects of chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae). Journal of Integrative Agriculture, 2013, 12(3): 457-466.

[40] DELPUECH J M, GAREAU E, TERRIER O, FOUILLET P. Sublethal effects of the insecticide chlorpyrifos on the sex pheromonal communication of Trichogramma brassicae. Chemosphere, 1998, 36(8): 1775-1785.

[41] DELPUECH J M, MEYET J. Reduction in the sex ratio of the progeny of a parasitoid wasp (Trichogramma brassicae) surviving the insecticide chlorpyrifos. Archives of Environmental Contamination and Toxicology, 2003, 45(2): 203-208.

[42] 宋月芹, 董鈞鋒, 孫會(huì)忠. 亞致死濃度氯蟲苯甲酰胺可降低亞洲玉米螟的種群增長. 昆蟲學(xué)報(bào), 2013, 56(4): 446-451.

SONG Y Q, DONG J F, SUN H Z. Chlorantraniliprole at sublethal concentrations may reduce the population growth of the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Pyralidae). Acta Entomologica Sinica, 2013, 56(4): 446-451. (in Chinese)

[43] 楊洪, 王召, 金道超. 氯蟲苯甲酰胺對(duì)白背飛虱實(shí)驗(yàn)種群的亞致死效應(yīng). 昆蟲學(xué)報(bào), 2012, 55(10): 1161-1167.

YANG H, WANG Z, JIN D C. Sublethal effects of chlorantraniliprole on the experimental populations of the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae). Acta Entomologica Sinica, 2012, 55(10): 1161-1167. (in Chinese)

[44] LAI T C, SU J A. Effects of chlorantraniliprole on development and reproduction of beet armyworm, spodoptera exigua, (Hübner). Journal of Pest Science, 2011, 84(3): 381-386.

[45] GUO L, DESNEUX N, SONODA S, LIANG P, HAN P, GAO X W. Sublethal and transgenerational effects of chlorantraniliprole on biological traits of the diamondback moth, Plutella xylostella L. Crop Protection, 2013, 48(2): 29-34.

[46] HAN W S, ZHANG S F, SHEN F Y, LIU M, REN C C, GAO X W. Residual toxicity and sublethal effects of chlorantraniliprole on Plutella xylostella (Lepidoptera: Plutellidae). Pest Management Science, 2012, 68(8): 1184-1190.

[47] 陳瓊, 黃水金, 秦文婧. 氯蟲苯甲酰胺對(duì)甜菜夜蛾的亞致死效應(yīng)研究. 江西農(nóng)業(yè)大學(xué)學(xué)報(bào), 2011, 33(4): 690-695.

CHEN Q, HUANG S J, QIN W J. Sublethal effects of chlorantraniliprole in Spodoptera exigua. Acta Agriculturae Universitis Jiangxiensis, 2011, 33(4): 690-695. (in Chinese)

[48] TAN Y, BIONDI A, DESNEUX N, GAO X W. Assessment of physiological sublethal effects of imidacloprid on the mirid bug Apolygus lucorum (Meyer-Dür). Ecotoxicology, 2012, 21(7): 1989-1997.

[49] 楊國慶, 李麗, 戈林泉, 吳進(jìn)才. 三唑磷和毒死蜱亞致死劑量對(duì)大螟種群增長和卵黃蛋白含量的影響. 應(yīng)用昆蟲學(xué)報(bào), 2014, 51(6): 1582-1588.

YANG G Q, LI L, GE L Q, WU J C. Effects of sublethal doses of triazophos and chlorpyrifos on the population growth and yolk protein content of Sesamia inferens (Walker). Chinese Journal of Applied Entomology, 2014, 51(6): 1582-1588. (in Chinese)

[50] MAHMOUDVAND M, ABBASIPOUR H, GARJAN A S, BANDANI A R. Sublethal effects of hexaflumuron on development and reproduction of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Insect Science, 2011, 18(6): 689-696.

[51] YIN X H, WU Q J, LI X F, ZHANG Y J, XU B Y. Demographic changes in multigeneration Plutella xylostella (Lepidoptera: Plutellidae) after exposure to sublethal concentrations of spinosad. Journal of Economic Entomology, 2009, 102(1): 357-365.

[52] 劉澤文, 劉成君, 張洪偉, 韓召軍. 褐飛虱抗吡蟲啉品系生物適合度研究. 應(yīng)用昆蟲學(xué)報(bào), 2003, 40(5): 419-422.

LIU Z W, LIU C J, ZHANG H W, HAN Z J. Relative biological fitness of imidacloprid resistant strains of Nilaparvata lugens. Chinese Journal of Applied Entomology, 2003, 40(5): 419-422. (in Chinese)

[53] 陳朗杰, 劉昕, 吳善俊, 朱弋凡, 曾玲, 陸永躍. 桔小實(shí)蠅抗敵百蟲品系的實(shí)驗(yàn)種群生物學(xué)比較研究. 昆蟲學(xué)報(bào), 2015, 58(8): 864-871.

CHEN L J, LIU X, WU S J, ZHU Y F, ZENG L, LU Y Y. A comparative study of the population biology of trichlorfon- resistant strains of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritdae). Acta Entomologica Sinica, 2015, 58(8): 864-871. (in Chinese)

（責(zé)任編輯 岳梅）

Effects of Sublethal Doses of Chlorantraniliprole on the Detoxification Enzymes Activities and the Growth and Reproduction of Spodoptera exigua

CHEN YiQu, XIANG Xin, GONG ChangWei, WANG XueGui
(Biorational Pesticide Research Laboratory, College of Agronomy, Sichuan Agricultural University, Chengdu 611130)

Spodoptera exigua; chlorantraniliprole; sublethal doses; synergists; MFOs; age-stage two-sex life tables

2016-12-12；接受日期：2017-02-27

國家公益性行業(yè)（農(nóng)業(yè)）科研專項(xiàng)（201203038）

聯(lián)系方式：陳羿渠，Tel：028-86290977；E-mail：chenyiqu123@163.com。通信作者王學(xué)貴，Tel：028-86290977；E-mail：wangxuegui@sicau.edu.cn

Abstract:【Objective】 Spodoptera exigua is a polyphagous pest and shows different characteristics of growth and development under the selective pressure of different environments, drugs and so on. Chlorantraniliprole, a novel insecticide that acts on ryanodine receptors, has highly activity to those insects in Lepidopteran. The objective of this study is to explore the effects of S. exigua larvae treated by the sublethal doses of chlorantraniliprole on the toxicities, the activities of three main detoxifying enzymes, including carboxylesterase (CarE), glutathione S-transferase (GSTs), and mixed-function oxidase (MFOs), and population breeding.【Method】 The toxicities of chlorantraniliprole on SE-Lab and SE-Sel strains were detected by incorporation bioassay and the SE-Sel strain was achieved with continuous selecting with LC25sublethal doses for six generations from SE-Lab strain, then the synergistic effects of enzyme inhibitors (TPP, DEM, PBO) with chlorantraniliprole on the SE-Lab and SE-Sel strains were assayed using the dip-leaf method. The toxicities of chlorantraniliprole on the insects which were fed the leaves soaked with the solution of enzyme inhibitor or 0.1% TritonX-100 as blank control before 12 h were assayed by the dip-leaf method. The midgut and fatbody of tested insects were dissected on the ice and the effects of the sublethal doses of chlorantraniliprole and enzyme inhibitors on three metabolic detoxification enzyme activities were analyzed by the determination of detoxification enzyme activities. According to the age-stage two-sex life tables theory, the growth, mortality, fecundity data of the tested insects were also recorded to analyze the differences of the age-stage two-sex life table parameters between the strains of SE-Lab and SE-Sel. 【Result】 The synergistic effect of the PBO was the strongest among the three enzyme inhibitors and the synergic ratios on the strains of SE-Sel and SE-Lab reached 1.58- and 1.69-fold, respectively. The activities of the three detoxification enzymes, which were induced by the continuous selection of the sublethal doses of chlorantraniliprole, were promoted and the MFOs activities were the most significant, which of the SE-Sel strain in the midgut and fatbody were enhanced by 2.07- and 2.10-fold, meanwhile, the MFOs activities of the insects of SE-Sel induced by the sublethal dose of chlorantraniliprole again were also promoted by 4.02- and 3.44-fold in the midgut and fatbody compared to those in SE-Lab strain, respectively. The enzyme activities of three detoxifying enzymes were decreased when the tested insects were treated with enzyme inhibitors and the descend range of MFOs activity among the three enzymes was maximum, which was only 42.3%-44.8% compared to the treatment not treated with the enzyme inhibitor. The adult preoviposition period and total preoviposition period of F1generation of SE-Sel strains became shorter and spawning quantity of became higher compared to those in F1generation of SE-Lab. SE-Sel strains had the longest mean generation time and least amount of eggs. The intrinsic rate of increase (r), finite rate of increase (λ) and net reproductive rate (R0) of SE-Sel strain were significantly lower than those in SE-Lab strain. The r, λ and R0values of SE-Lab and SE-Sel strains were 0.18 and 0.16 d-1, 1.20 and 1.17 d-1, 358.42 and 203.12 d-1, respectively. Even though the mean generation times of SE-Sel strain was longer than SE-Lab strain, there was no significant difference between the two strains.【Conclusion】MFOs may be the major detoxication enzyme on the metabolic detoxification of chlorantraniliprole in the S. exigua and involved in the formation of resistance. A longer generation period, lower fecundity and slower population growth of S. exigua were demonstrated when it was treated by a continuous selection of lethal dose of chlorantraniliprole. The sublethal doses of chlorantraniliprole have a continuous control effect on S. exigua.