異色瓢蟲(chóng)脅迫對(duì)棉鈴蟲(chóng)生長(zhǎng)發(fā)育及壓力蛋白基因表達(dá)的影響

閆碩，熊曉菲，褚艷娜，李貞，巫鵬翔，楊清坡，崔維娜，徐金濤，徐麗霞，張青文，劉小俠

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異色瓢蟲(chóng)脅迫對(duì)棉鈴蟲(chóng)生長(zhǎng)發(fā)育及壓力蛋白基因表達(dá)的影響

閆碩1,2，熊曉菲3，褚艷娜1，李貞1，巫鵬翔1，楊清坡2，崔維娜4，徐金濤5，徐麗霞6，張青文1，劉小俠1

（1中國(guó)農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院，北京100193；2全國(guó)農(nóng)業(yè)技術(shù)推廣服務(wù)中心，北京100125；3北京派得偉業(yè)科技發(fā)展有限公司，北京100097；4山東省鄒城市植物保護(hù)站，山東鄒城273500；5河北省農(nóng)林科學(xué)院昌黎果樹(shù)研究所，河北昌黎066600；6昌黎縣科學(xué)技術(shù)局，河北昌黎066600）

明確不同食源異色瓢蟲(chóng)（）脅迫對(duì)棉鈴蟲(chóng)（）生長(zhǎng)發(fā)育和變態(tài)發(fā)育的影響，探討棉鈴蟲(chóng)是否能感知并分級(jí)捕食風(fēng)險(xiǎn)、能否在生長(zhǎng)發(fā)育和變態(tài)發(fā)育上體現(xiàn)出權(quán)衡效應(yīng)；明確長(zhǎng)時(shí)和短時(shí)脅迫對(duì)棉鈴蟲(chóng)壓力蛋白基因表達(dá)的影響，探討異色瓢蟲(chóng)脅迫能否引起棉鈴蟲(chóng)分子水平上的生理反應(yīng)。通過(guò)設(shè)置7種不同食源的異色瓢蟲(chóng)脅迫處理(饑餓處理、蝦卵處理、棉鈴蟲(chóng)幼蟲(chóng)處理、棉鈴蟲(chóng)卵處理、蚜蟲(chóng)處理、蚜蟲(chóng)對(duì)照處理、對(duì)照處理)，觀察記錄棉鈴蟲(chóng)在脅迫下的生長(zhǎng)發(fā)育(幼蟲(chóng)歷期、蛹?xì)v期、雌雄蛾壽命、總壽命)和變態(tài)發(fā)育(蛹重、化蛹率、羽化失敗率、卷翅率)指標(biāo)；設(shè)置長(zhǎng)時(shí)(1齡幼蟲(chóng)至3日齡成蟲(chóng))和短時(shí)(15 min至6 h)異色瓢蟲(chóng)脅迫處理，利用實(shí)時(shí)熒光定量PCR（qRT-PCR）技術(shù)檢測(cè)棉鈴蟲(chóng)壓力蛋白基因(即熱激蛋白基因)和及熱激同源蛋白基因在脅迫后的表達(dá)水平變化。在捕食性天敵異色瓢蟲(chóng)的脅迫下，棉鈴蟲(chóng)幼蟲(chóng)歷期、蛹期、雌雄蛾壽命、總壽命均顯著性縮短，蛹重和化蛹率顯著性下降，成蟲(chóng)卷翅率顯著性升高，而羽化失敗率無(wú)顯著性變化。在不同食源的天敵脅迫下，棉鈴蟲(chóng)幼蟲(chóng)歷期在異色瓢蟲(chóng)取食蚜蟲(chóng)時(shí)最短，蛹?xì)v期在瓢蟲(chóng)取食棉鈴蟲(chóng)卵時(shí)最短，總壽命在瓢蟲(chóng)取食蝦卵時(shí)最短，而雌雄蛾壽命在不同食源天敵脅迫下未表現(xiàn)出顯著性差異；棉鈴蟲(chóng)成蟲(chóng)卷翅率在瓢蟲(chóng)取食棉鈴蟲(chóng)卵時(shí)最高，而蛹重、化蛹率、羽化失敗率在不同食源天敵脅迫下未表現(xiàn)出顯著性差異。壓力蛋白基因和在短時(shí)脅迫下(：30 min至3 h；：15 min、1.5 h、2 h、6 h)表達(dá)水平顯著性上調(diào)，熱激同源蛋白基因在長(zhǎng)時(shí)脅迫下(5齡幼蟲(chóng)、預(yù)蛹、雄蛹、雌蛾階段)表達(dá)水平顯著性上調(diào)。在面對(duì)捕食性天敵異色瓢蟲(chóng)長(zhǎng)時(shí)脅迫作用下，棉鈴蟲(chóng)各生長(zhǎng)發(fā)育階段均出現(xiàn)縮短的現(xiàn)象，即棉鈴蟲(chóng)為躲避被捕食風(fēng)險(xiǎn)表現(xiàn)出了發(fā)育加速的現(xiàn)象，而快速的生長(zhǎng)發(fā)育在一定程度上干擾了變態(tài)發(fā)育，導(dǎo)致蛹重和化蛹率下降，成蟲(chóng)卷翅率提高，符合權(quán)衡效應(yīng)。棉鈴蟲(chóng)對(duì)不同食源的天敵脅迫具有不同程度的敏感性，即棉鈴蟲(chóng)對(duì)潛在的捕食風(fēng)險(xiǎn)可能存在一定的分級(jí)能力，但這種分級(jí)能力沒(méi)有得到規(guī)律性體現(xiàn)。異色瓢蟲(chóng)脅迫能夠引起棉鈴蟲(chóng)分子層面的生理反應(yīng)，導(dǎo)致壓力蛋白基因的表達(dá)上調(diào)，其中壓力蛋白基因和受到短時(shí)脅迫的反應(yīng)較為明顯，而熱激同源蛋白基因受到慢性脅迫刺激的反應(yīng)更為顯著。

棉鈴蟲(chóng)；異色瓢蟲(chóng)；生長(zhǎng)發(fā)育；變態(tài)發(fā)育；壓力蛋白；非消耗性脅迫

0 引言

【研究意義】捕食性天敵-獵物關(guān)系是生態(tài)系統(tǒng)中最普遍的基本關(guān)系，對(duì)生態(tài)群落的發(fā)展具有重要意義[1-2]。自然界中捕食性天敵與獵物的作用關(guān)系除了直接的取食消耗（consumptive effect），還存在間接的脅迫效應(yīng)（non-consumptive effect）[3-4]。這種脅迫壓力帶來(lái)的影響在生物界中十分廣泛，對(duì)獵物具有深遠(yuǎn)的意義[5-8]。了解天敵與獵物之間的直接與間接關(guān)系，是利用天敵防治害蟲(chóng)，優(yōu)化生態(tài)系統(tǒng)結(jié)構(gòu)的必要基礎(chǔ)。【前人研究進(jìn)展】目前，關(guān)于捕食脅迫風(fēng)險(xiǎn)對(duì)獵物的影響已涉及多種昆蟲(chóng)，包括蜻蜓目[9-10]、蜉蝣目[11-12]、雙翅目[13-15]、直翅目[2,16]、同翅目[17]、鱗翅目[18-19]等昆蟲(chóng)。現(xiàn)有的研究表明，捕食脅迫會(huì)造成獵物的發(fā)育加快或者減緩[11,15,20]、致死率升高[10]、變態(tài)成功率下降[21-22]，而獵物的應(yīng)激反應(yīng)往往在捕食者取食近源物種下最強(qiáng)烈[23-24]。捕食性天敵脅迫壓力能夠引起獵物分子水平上的生理反應(yīng)，如壓力蛋白（即熱激蛋白，一般在熱激反應(yīng)方面研究較多）表達(dá)量的上調(diào)[9,25-26]，糖皮質(zhì)激素水平升高[27-28]。棉鈴蟲(chóng)（）食性雜，其寄主植物多達(dá)200多種，曾給中國(guó)棉花生產(chǎn)造成過(guò)巨大的經(jīng)濟(jì)損失[29]。Bt棉的種植雖然有效地控制了棉鈴蟲(chóng)的暴發(fā)，但其抗藥性問(wèn)題不容忽視[30-32]。生物防治作為一種有效的替代手段，符合綠色植保的發(fā)展理念，也是實(shí)現(xiàn)到2020年農(nóng)藥使用量零增長(zhǎng)行動(dòng)目標(biāo)的有力落腳點(diǎn)。目前，異色瓢蟲(chóng)（）在生物防治領(lǐng)域應(yīng)用廣泛，其適生性強(qiáng)，成蟲(chóng)和高齡幼蟲(chóng)均可大量捕食各類蚜蟲(chóng)、多種介殼蟲(chóng)、木虱、螨類、鱗翅目與鞘翅目昆蟲(chóng)的卵、低齡幼蟲(chóng)和蛹等[33-37]。【本研究切入點(diǎn)】以異色瓢蟲(chóng)和棉鈴蟲(chóng)建立的脅迫體系研究卻寥寥無(wú)幾，棉鈴蟲(chóng)受異色瓢蟲(chóng)脅迫的影響有待闡明。【擬解決的關(guān)鍵問(wèn)題】通過(guò)設(shè)置不同食源的捕食性瓢蟲(chóng)脅迫處理，探討：（1）棉鈴蟲(chóng)是否能感知外界脅迫的存在，并對(duì)異色瓢蟲(chóng)食源表現(xiàn)出敏感性；（2）脅迫下的棉鈴蟲(chóng)是否會(huì)縮短發(fā)育歷期，以躲避被捕食風(fēng)險(xiǎn)；（3）脅迫下的棉鈴蟲(chóng)是否會(huì)表現(xiàn)出一定的權(quán)衡效應(yīng)，即發(fā)育歷期的縮短或延長(zhǎng)是否會(huì)對(duì)變態(tài)發(fā)育造成一定的負(fù)面影響；（4）脅迫壓力能否引起棉鈴蟲(chóng)分子層面的生理反應(yīng)。

1 材料與方法

試驗(yàn)于2012年9月至2015年9月在中國(guó)農(nóng)業(yè)大學(xué)完成。

1.1 供試?yán)ハx(chóng)

供試棉鈴蟲(chóng)幼蟲(chóng)采自河北邯鄲棉田，在無(wú)天敵環(huán)境下，飼養(yǎng)于中國(guó)農(nóng)業(yè)大學(xué)有害生物綜合治理實(shí)驗(yàn)室。幼蟲(chóng)用人工飼料單管飼養(yǎng)[38]，成蟲(chóng)羽化后飼喂10%蜂蜜水。選取發(fā)育、繁殖情況穩(wěn)定良好的棉鈴蟲(chóng)幼蟲(chóng)進(jìn)行脅迫試驗(yàn)。捕食性天敵異色瓢蟲(chóng)采自北京市農(nóng)林科學(xué)院植物保護(hù)環(huán)境保護(hù)研究所試驗(yàn)田，室內(nèi)以豆蚜（）飼喂擴(kuò)繁，僅取異色瓢蟲(chóng)成蟲(chóng)用于脅迫試驗(yàn)。所有試蟲(chóng)均在人工氣候箱中飼養(yǎng)，飼養(yǎng)溫度（25±2）℃，相對(duì)濕度（75±5）%，光周期14L﹕10D。瓢蟲(chóng)脅迫處理在全程安靜的環(huán)境中進(jìn)行，盡量避免人為干擾。

1.2 試劑

RNAiso Plus（Trizol）、反轉(zhuǎn)錄試劑盒PrimeScript RT reagent Kit with gDNA Eraser、熒光定量染料SYBRPremix Ex TaqTM購(gòu)自Takara公司；PCR MasterMix購(gòu)自寶如億（北京）生物技術(shù)有限公司；IPTG、Amp購(gòu)自Takara公司；X-gal購(gòu)自北京拜爾迪生物有限公司；DNA膠回收試劑盒Gel Extraction Kit購(gòu)自O(shè)mega公司；克隆載體pEASY-T1 Cloning Vector、感受態(tài)細(xì)胞購(gòu)自北京全式金生物技術(shù)有限公司；PCR引物由上海生工生物工程公司合成；其他試劑均為國(guó)產(chǎn)AR級(jí)或進(jìn)口分裝AR級(jí)產(chǎn)品。

1.3 異色瓢蟲(chóng)脅迫對(duì)棉鈴蟲(chóng)生長(zhǎng)發(fā)育的影響

取同日孵化、活性良好一致的棉鈴蟲(chóng)初孵幼蟲(chóng)為試驗(yàn)材料，棉鈴蟲(chóng)以人工飼料為食物源。試驗(yàn)共設(shè)置以下7組處理用以分析異色瓢蟲(chóng)取食不同食物對(duì)棉鈴蟲(chóng)的脅迫效果，脅迫設(shè)置直至棉鈴蟲(chóng)成蟲(chóng)羽化。（1）饑餓處理：將棉鈴蟲(chóng)和人工飼料放入經(jīng)滅菌的透明試管（直徑1.8 cm×長(zhǎng)9.6 cm）中，以滅菌紗布封口，保持良好透氣性。將試管平鋪在滅菌的塑料養(yǎng)蟲(chóng)盒（22 cm×15 cm× 8 cm）底部，每盒投放30頭不供給食物的饑餓瓢蟲(chóng)，每日添加瓢蟲(chóng)以維持瓢蟲(chóng)數(shù)量；（2）蝦卵處理：同批次蝦卵購(gòu)買(mǎi)于北京市官園花鳥(niǎo)蟲(chóng)魚(yú)市場(chǎng)。將裝有棉鈴蟲(chóng)的試管平鋪在塑料養(yǎng)蟲(chóng)盒內(nèi)，每盒投放30頭異色瓢蟲(chóng)，并投放充足的蝦卵，瓢蟲(chóng)以蝦卵為食物來(lái)源；（3）棉鈴蟲(chóng)幼蟲(chóng)處理：將裝有棉鈴蟲(chóng)的試管平鋪在塑料養(yǎng)蟲(chóng)盒內(nèi)，每盒投放30頭異色瓢蟲(chóng)，并投放充足的棉鈴蟲(chóng)1齡幼蟲(chóng)，瓢蟲(chóng)以棉鈴蟲(chóng)1齡幼蟲(chóng)為食物來(lái)源；（4）棉鈴蟲(chóng)卵處理：將裝有棉鈴蟲(chóng)的試管平鋪在塑料養(yǎng)蟲(chóng)盒內(nèi)，每盒投放30頭異色瓢蟲(chóng)，并投放充足的棉鈴蟲(chóng)卵，瓢蟲(chóng)以棉鈴蟲(chóng)卵為食物來(lái)源；（5）蚜蟲(chóng)處理：將裝有棉鈴蟲(chóng)的試管平鋪在塑料養(yǎng)蟲(chóng)盒內(nèi)，每盒投放30頭異色瓢蟲(chóng)，并投放充足的蚜蟲(chóng)，瓢蟲(chóng)以蚜蟲(chóng)為食物來(lái)源；（6）蚜蟲(chóng)對(duì)照處理：將裝有棉鈴蟲(chóng)的試管平鋪在塑料養(yǎng)蟲(chóng)盒內(nèi)，不投放瓢蟲(chóng)，只投放蚜蟲(chóng)作對(duì)照，排除蚜蟲(chóng)釋放信號(hào)的干擾；（7）對(duì)照處理：將裝有棉鈴蟲(chóng)的試管平鋪在塑料養(yǎng)蟲(chóng)盒內(nèi)，不投放瓢蟲(chóng)。羽化成蟲(chóng)單獨(dú)飼養(yǎng)，繼續(xù)觀察壽命直至成蟲(chóng)死亡。7個(gè)處理彼此隔離，設(shè)置3次重復(fù)，每個(gè)重復(fù)包含50頭棉鈴蟲(chóng)，觀察統(tǒng)計(jì)棉鈴蟲(chóng)各蟲(chóng)態(tài)的發(fā)育歷期（刨除不能正常化蛹、羽化的個(gè)體），統(tǒng)計(jì)3日齡蛹重、化蛹率、羽化失敗率和成蟲(chóng)卷翅率。

1.4 異色瓢蟲(chóng)長(zhǎng)時(shí)和短時(shí)脅迫對(duì)棉鈴蟲(chóng)壓力蛋白基因表達(dá)的影響

長(zhǎng)時(shí)脅迫取樣：取發(fā)育進(jìn)度一致、活性良好的初孵棉鈴蟲(chóng)幼蟲(chóng)600頭單頭飼養(yǎng)，棉鈴蟲(chóng)以人工飼料為食物源。將試蟲(chóng)分為2個(gè)處理：（1）長(zhǎng)時(shí)脅迫處理：置于有30頭異色瓢蟲(chóng)存在的養(yǎng)蟲(chóng)盒內(nèi)飼養(yǎng)，瓢蟲(chóng)以蚜蟲(chóng)為食物來(lái)源；（2）對(duì)照處理：置于無(wú)瓢蟲(chóng)存在的養(yǎng)蟲(chóng)盒內(nèi)飼養(yǎng)。分別在棉鈴蟲(chóng)幼蟲(chóng)5個(gè)齡期、預(yù)蛹期、第3日齡蛹期和第3日齡成蟲(chóng)期取樣，對(duì)蛹和成蟲(chóng)分雌、雄取樣。于棉鈴蟲(chóng)發(fā)育過(guò)程中的每個(gè)取樣點(diǎn)取3個(gè)生物學(xué)重復(fù)。

短時(shí)脅迫取樣：取發(fā)育進(jìn)度一致、活性良好的棉鈴蟲(chóng)3齡幼蟲(chóng)300頭單頭飼養(yǎng)，棉鈴蟲(chóng)以人工飼料為食物源。將試蟲(chóng)分為2個(gè)處理：（1）短時(shí)脅迫處理：置于有30頭異色瓢蟲(chóng)存在的養(yǎng)蟲(chóng)盒內(nèi)，瓢蟲(chóng)以蚜蟲(chóng)為食物來(lái)源；（2）對(duì)照處理：置于無(wú)瓢蟲(chóng)存在的養(yǎng)蟲(chóng)盒內(nèi)飼養(yǎng)。分別在脅迫進(jìn)行15 min、30 min、1 h、1.5 h、2 h、3 h、6 h時(shí)間點(diǎn)取樣。每個(gè)取樣點(diǎn)取3個(gè)生物學(xué)重復(fù)。

借助Trizol法對(duì)棉鈴蟲(chóng)樣本進(jìn)行RNA提取，反轉(zhuǎn)錄生成cDNA。借助實(shí)時(shí)熒光定量PCR（qRT-PCR）檢測(cè)壓力蛋白基因（熱激蛋白基因）和、熱激同源蛋白基因的表達(dá)量。根據(jù)Genbank已公布的基因序列和發(fā)表的文章[39]，設(shè)計(jì)qRT-PCR引物（表1）。在qRT-PCR之前，通過(guò)測(cè)序保證PCR產(chǎn)物是目的基因片段，PCR擴(kuò)增程序：94℃預(yù)變性5 min；94℃30 s，60℃30 s，72℃60 s，35個(gè)循環(huán)；72℃延伸10 min。PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳檢測(cè)后，將回收的目的條帶與克隆載體連接，轉(zhuǎn)化到大腸桿菌內(nèi)，37℃培養(yǎng)過(guò)夜。菌落進(jìn)行藍(lán)白斑篩選，隨機(jī)選取陽(yáng)性克隆送至北京擎科生物技術(shù)有限公司測(cè)序。基因的相對(duì)表達(dá)量檢測(cè)在Bio-RAD CFX Connect Real-Time System儀器上進(jìn)行。選取棉鈴蟲(chóng)為內(nèi)參基因[40-44]。qRT-PCR反應(yīng)程序：95℃預(yù)變性10 min；95℃，15 s，60℃，30 s，72℃，35 s，40個(gè)循環(huán)，此外再加上qRT-PCR儀器自帶的熔解步驟。檢測(cè)所取樣本中目的基因和內(nèi)參基因的Ct值，每個(gè)樣本設(shè)置3次點(diǎn)樣重復(fù)，基因相對(duì)表達(dá)量的計(jì)算采用2-ΔΔCt方法[45]進(jìn)行。

表1 實(shí)時(shí)熒光定量PCR所用引物

1.5 數(shù)據(jù)分析

數(shù)據(jù)分析采用Tukey比較和獨(dú)立樣本檢驗(yàn)方法進(jìn)行，＜0.05視為差異顯著。所有統(tǒng)計(jì)分析借助SPASS 16.0軟件包完成。

2 結(jié)果

2.1 不同食源異色瓢蟲(chóng)脅迫對(duì)棉鈴蟲(chóng)發(fā)育歷期的影響

不同食源的瓢蟲(chóng)脅迫顯著縮短了棉鈴蟲(chóng)幼蟲(chóng)歷期、蛹?xì)v期和棉鈴蟲(chóng)的總壽命，瓢蟲(chóng)以蚜蟲(chóng)為食源時(shí)，棉鈴蟲(chóng)幼蟲(chóng)歷期縮短最顯著；以棉鈴蟲(chóng)卵為食源時(shí)，棉鈴蟲(chóng)蛹?xì)v期縮短最顯著；以蝦卵為食源時(shí)，棉鈴蟲(chóng)總壽命縮短最顯著。在7個(gè)處理之間，瓢蟲(chóng)脅迫后棉鈴蟲(chóng)雌雄蛾壽命雖然有縮短的趨勢(shì)，但7個(gè)處理間無(wú)顯著性差異。在比較的5個(gè)指標(biāo)中，蚜蟲(chóng)對(duì)照處理與對(duì)照處理均不存在顯著性差異，排除了瓢蟲(chóng)取食蚜蟲(chóng)脅迫處理中可能因蚜蟲(chóng)存在而引起的干擾因素。將總脅迫處理與總對(duì)照處理進(jìn)行比較，棉鈴蟲(chóng)幼蟲(chóng)歷期、蛹?xì)v期、雌雄蛾壽命以及總壽命在脅迫因子存在下均顯著性縮短（表2）。

2.2 不同食源異色瓢蟲(chóng)脅迫對(duì)棉鈴蟲(chóng)變態(tài)發(fā)育的影響

不同食源的瓢蟲(chóng)脅迫處理顯著提高了棉鈴蟲(chóng)成蟲(chóng)卷翅率，瓢蟲(chóng)以棉鈴蟲(chóng)卵為食源時(shí)，棉鈴蟲(chóng)成蟲(chóng)卷翅率提高最顯著；以棉鈴蟲(chóng)幼蟲(chóng)為食源時(shí)，成蟲(chóng)卷翅率提高程度最小。在7個(gè)處理之間，瓢蟲(chóng)脅迫后棉鈴蟲(chóng)蛹重和化蛹率有下降趨勢(shì)、羽化失敗率有提高趨勢(shì)，但7個(gè)處理間無(wú)顯著性差異。在比較的5個(gè)指標(biāo)中，蚜蟲(chóng)對(duì)照處理與對(duì)照處理均不存在顯著性差異，排除了瓢蟲(chóng)取食蚜蟲(chóng)脅迫處理中可能因蚜蟲(chóng)存在而引起的干擾因素。將總脅迫處理與總對(duì)照處理進(jìn)行比較，在脅迫因子存在下，棉鈴蟲(chóng)蛹重和化蛹率均顯著下降，成蟲(chóng)卷翅率顯著性上升（表3）。

表2 不同食源異色瓢蟲(chóng)脅迫對(duì)棉鈴蟲(chóng)發(fā)育歷期的影響

數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤，不同字母表示處理間差異顯著（Tukey比較，＜0.05）。下同

Each value was the mean±SE of three collections. Different letters indicated significant differences among treatments according to Tukey comparison test (＜0.05). The same as below

表3 不同食源異色瓢蟲(chóng)脅迫對(duì)棉鈴蟲(chóng)變態(tài)發(fā)育的影響

2.3 異色瓢蟲(chóng)長(zhǎng)時(shí)脅迫對(duì)壓力蛋白基因表達(dá)的影響

對(duì)比脅迫與對(duì)照處理的棉鈴蟲(chóng)各個(gè)蟲(chóng)態(tài)壓力蛋白基因的表達(dá)量（圖1），結(jié)果表明脅迫與對(duì)照處理的相對(duì)表達(dá)量隨蟲(chóng)齡變化趨勢(shì)較為一致，在1—2齡階段低水平表達(dá)，3—4齡階段出現(xiàn)表達(dá)峰值（脅迫處理：9,20=17.277，＜0.001；對(duì)照處理：9,20=15.212，＜0.001）。脅迫處理中3齡幼蟲(chóng)的表達(dá)量顯著升高（=7.980，df=4，=0.001），而其他發(fā)育階段，脅迫與對(duì)照處理間不存在顯著性差異（1齡：=0.406，df=4，=0.705；2齡：=0.251，df=4，=0.814；4齡：=2.136，df=4，=0.100；5齡：=1.893， df=4，=0.131；預(yù)蛹：=0.008，df=4，=0.994；雌蛹：=1.951，df=4，=0.123；雄蛹：=1.393，df=4，=0.236；雌蛾：=1.258，df=4，=0.277；雄蛾：=2.356，df=4，=0.078）。對(duì)表達(dá)量檢測(cè)結(jié)果表明（圖1），在對(duì)照處理中，在蛹期和成蟲(chóng)期表達(dá)水平較高（9,20=81.327，＜0.001），在棉鈴蟲(chóng)各個(gè)生長(zhǎng)發(fā)育階段，脅迫與對(duì)照處理之間均無(wú)顯著性差異（1齡：=1.782，df=4，=0.149；2齡：=4.098，df=2.014，=0.054；3齡：=2.737，df=4，=0.052；4齡：=1.185，df=4，=0.302；5齡：=0.650，df=2.182，=0.577；預(yù)蛹：=0.374，df=4，=0.728；雌蛹：=1.017，df=4，=0.367；雄蛹：=0.657，df=4，=0.547；雌蛾：=0.741，df=2.022，=0.535；雄蛾：=2.348，df=2.044，=0.141）。

在對(duì)照處理中，熱激同源蛋白基因在蛹期和成蟲(chóng)期表達(dá)水平高（9,20=299.092，＜0.001）。棉鈴蟲(chóng)在進(jìn)入5齡后，脅迫處理中表達(dá)有上調(diào)趨勢(shì)，其中在棉鈴蟲(chóng)5齡幼蟲(chóng)期、預(yù)蛹期、雄蛹期、雌蛾期，脅迫處理中表達(dá)水平顯著高于對(duì)照處理（5齡：=11.48，df=4，＜0.001；預(yù)蛹期：=39.998，df=4，＜0.001；雄蛹期：=3.046，df=4，=0.038；雌蛾期：=3.976，df=4，=0.016）。而在棉鈴蟲(chóng)其他發(fā)育階段，脅迫與對(duì)照處理間無(wú)顯著性差異（1齡：=0.538，df=4，=0.619；2齡：=2.392，df=4，=0.075；3齡：=1.583，df=2.261，=0.240；4齡：=0.048，df=4，=0.964；雌蛹：=2.638，df=2.239，=0.106；雄蛾：=3.184，df=2.013，=0.085）（圖1）。

1st：1齡幼蟲(chóng)1st instar larvae；2nd：2齡幼蟲(chóng)2nd instar larvae；3rd：3齡幼蟲(chóng)3rd instar larvae；4th：4齡幼蟲(chóng)4th instar larvae；5th：5齡幼蟲(chóng)5th instar larvae；PP：預(yù)蛹prepupa；FP：雌蛹female pupae；MP：雄蛹male pupae；FM：雌蛾female moth；MM：雄蛾male moth

2.4 異色瓢蟲(chóng)短時(shí)脅迫對(duì)壓力蛋白基因表達(dá)的影響

無(wú)脅迫因子存在下，棉鈴蟲(chóng)3齡幼蟲(chóng)3個(gè)壓力蛋白基因的表達(dá)水平比較穩(wěn)定（：6,14=1.650，=0.206；：6,14=5.590，=0.004；：6,14=2.412，=0.082）。短時(shí)脅迫后，棉鈴蟲(chóng)3齡幼蟲(chóng)和的表達(dá)有上調(diào)趨勢(shì)，在脅迫開(kāi)始30 min至3 h內(nèi)表達(dá)量顯著提升（30 min：=5.269，df=4，=0.006；1 h：=3.538，df=4，=0.024；1.5 h：=4.559，df=2.02，=0.044；2 h：=4.478，df=4，=0.011；3 h：=3.183，df=4，=0.033）；在脅迫開(kāi)始15 min、1.5 h、2 h和6 h時(shí)間點(diǎn)表達(dá)量顯著提升（15 min：=3.057，df=4，=0.038；1.5 h：=4.391，df=4，=0.012；2 h：=13.06，df=4，＜0.001；6 h：=10.895，df=4，＜0.001）。而短時(shí)脅迫處理后，棉鈴蟲(chóng)的表達(dá)水平?jīng)]有出現(xiàn)顯著性變化（15 min：=0.777，df=2.008，=0.518；30 min：=0.660，df=2.180，=0.572；1 h：=1.462，df=4，=0.217；1.5 h：=0.310，df=4，=0.772；2 h：=1.042，df=2.029，=0.406；3 h：=2.094，df=4，=0.104；6 h：=3.035，df=2.132，=0.086）。綜合短時(shí)脅迫各時(shí)間點(diǎn)數(shù)據(jù)，結(jié)果表明棉鈴蟲(chóng)3齡幼蟲(chóng)受到短時(shí)脅迫后，和表達(dá)增強(qiáng)，表達(dá)無(wú)顯著性變化（：=6.520，df=40，＜0.001；：=3.884，df=40，＜0.001；：=0.383，df=32.867，=0.704）（圖2）。

圖2 異色瓢蟲(chóng)短時(shí)脅迫對(duì)棉鈴蟲(chóng)Hsp70、Hsp90、Hsc70基因表達(dá)的影響

3 討論

捕食脅迫作用可能引起獵物發(fā)育加快或者減慢，獵物選擇哪種策略與其生態(tài)特點(diǎn)相關(guān)，同時(shí)取決于哪種策略有利于其增加存活率[14-15,20]。本試驗(yàn)表明捕食性天敵異色瓢蟲(chóng)的脅迫導(dǎo)致棉鈴蟲(chóng)各蟲(chóng)態(tài)發(fā)育歷期及成蟲(chóng)壽命的縮短，此現(xiàn)象在筆者實(shí)驗(yàn)室之前的研究中也有類似發(fā)現(xiàn)[19]。異色瓢蟲(chóng)傾向于取食棉鈴蟲(chóng)低齡幼蟲(chóng)，對(duì)高齡幼蟲(chóng)的捕食現(xiàn)象偶然才會(huì)發(fā)生，棉鈴蟲(chóng)蛹多位于土中被隔離，且蛹?xì)べ|(zhì)地硬，不利于瓢蟲(chóng)捕食，成蟲(chóng)由于生活棲境的改變而免于被瓢蟲(chóng)捕食的風(fēng)險(xiǎn)[35-36]。因此，加快幼蟲(chóng)期的生長(zhǎng)發(fā)育對(duì)于棉鈴蟲(chóng)來(lái)說(shuō)，有助于快速脫離被捕食的高風(fēng)險(xiǎn)蟲(chóng)態(tài)，逃避捕食風(fēng)險(xiǎn)，增加存活率，這一變化對(duì)種群發(fā)展具有積極意義。而棉鈴蟲(chóng)蛹和成蟲(chóng)發(fā)育歷期縮短的現(xiàn)象，有可能與加快發(fā)育進(jìn)度的策略有關(guān)，也有可能是其他表型變化的副產(chǎn)物。獵物在脅迫下發(fā)育變緩或加快，紅眼樹(shù)蛙（）幼蟲(chóng)階段受到捕食性蝽（spp.）脅迫時(shí)會(huì)加快發(fā)育，而受到覓食水生蜘蛛（spp.）脅迫時(shí)，則會(huì)減緩發(fā)育[20]；黑腹果蠅（）在面對(duì)龜紋瓢蟲(chóng)（）捕食性脅迫的體系中，也表現(xiàn)了不同程度的發(fā)育加快現(xiàn)象[15]。

獵物對(duì)捕食脅迫的適應(yīng)性反應(yīng)建立在獵物對(duì)脅迫風(fēng)險(xiǎn)的感知和識(shí)別的基礎(chǔ)之上，獵物是否能夠識(shí)別環(huán)境中的捕食風(fēng)險(xiǎn)，是否有對(duì)風(fēng)險(xiǎn)進(jìn)行分級(jí)的能力，將決定獵物采取何種策略[46-49]。天敵取食食源的不同往往為天敵貼上了某種化學(xué)信號(hào)標(biāo)簽，不同食源的天敵對(duì)某一種特定獵物來(lái)說(shuō)所暗示的捕食風(fēng)險(xiǎn)不同。本試驗(yàn)設(shè)置7種食源的捕食性瓢蟲(chóng)，棉鈴蟲(chóng)幼蟲(chóng)歷期、蛹?xì)v期和壽命在不同食源的捕食性瓢蟲(chóng)脅迫下具有顯著性差異，暗示棉鈴蟲(chóng)對(duì)不同食源的瓢蟲(chóng)脅迫具有一定的敏感性，但在發(fā)育歷期的指標(biāo)上，這種敏感性沒(méi)有得到規(guī)律性體現(xiàn)，這可能與棉鈴蟲(chóng)對(duì)風(fēng)險(xiǎn)評(píng)估的敏感性、選取指標(biāo)的靈敏性等因素有關(guān)。Brodin等[24]設(shè)置不同食源的捕食者泉蜓（）脅迫一種豆娘（）幼蟲(chóng)，食物充足條件下，的行為防御反應(yīng)在捕食者取食同源物種的處理下最強(qiáng)烈，這種影響在蜻蜓幼蟲(chóng)發(fā)育早期顯著，而后期不顯著；Chivers等[23]通過(guò)對(duì)豆娘混合種群（spp.）幼蟲(chóng)設(shè)置不同食源捕食者梭子魚(yú)（）證實(shí)，豆娘對(duì)豆娘食源環(huán)境刺激反應(yīng)顯著比對(duì)照強(qiáng)烈，而對(duì)黃粉蟲(chóng)（）食源環(huán)境反應(yīng)不明顯。這反應(yīng)了蜻蜓和豆娘幼蟲(chóng)對(duì)脅迫程度具有一定的識(shí)別能力，將天敵取食食源的不同定為不同的風(fēng)險(xiǎn)等級(jí)，進(jìn)而采取不同強(qiáng)烈程度的適應(yīng)性反應(yīng)。因?yàn)楂C物與其近源獵物擁有共同天敵的概率較大，當(dāng)近源獵物遭遇捕食時(shí)，釋放出的危險(xiǎn)信號(hào)對(duì)獵物的警示作用更強(qiáng)，進(jìn)而更容易采取防御措施抵御或逃避被捕食的風(fēng)險(xiǎn)。

天敵脅迫作用引入的表型變化，有可能在防御反應(yīng)與發(fā)育表現(xiàn)上反應(yīng)出權(quán)衡效應(yīng)。本試驗(yàn)結(jié)果表明加快的發(fā)育進(jìn)度引起了變態(tài)成功率不同程度的降低，蛹重和羽化率顯著下降，卷翅率顯著上升，表明了過(guò)快的生長(zhǎng)發(fā)育對(duì)變態(tài)過(guò)程產(chǎn)生了負(fù)面的影響，與權(quán)衡理論相符。但5種食源的捕食性瓢蟲(chóng)處理之間，只有卷翅率存在顯著性差異，而其他變態(tài)指標(biāo)沒(méi)有顯著性變化，沒(méi)有體現(xiàn)出不同食源天敵捕食脅迫的敏感性。權(quán)衡效應(yīng)也在其他昆蟲(chóng)中被證實(shí)，一種蜉蝣（）在有捕食性魚(yú)類存在的溪流中表現(xiàn)出變態(tài)后個(gè)體變小，繁殖力下降的現(xiàn)象[11]；一種蚊子（）暴露于捕食性魚(yú)信號(hào)的環(huán)境下，存活率提高但發(fā)育減緩，變態(tài)時(shí)體型變小，并且成蟲(chóng)對(duì)饑餓的耐受力減弱[50]。翅的對(duì)稱性是壓力反應(yīng)的可靠指示指標(biāo)，在捕食性天敵脅迫下，艷麗絲蟌（）會(huì)降低發(fā)育的穩(wěn)定性，提高后翅的不對(duì)稱性[22]。Mangel等[51]認(rèn)為生物體最大化的生長(zhǎng)率能夠在之后的生活史中表現(xiàn)出一定代價(jià)，因?yàn)檫^(guò)快的生長(zhǎng)速率降低了細(xì)胞與免疫功能的功效，使其對(duì)生理壓力的抵抗力降低。綜上所述，在面對(duì)捕食性瓢蟲(chóng)長(zhǎng)時(shí)脅迫作用下，棉鈴蟲(chóng)為了躲避捕食風(fēng)險(xiǎn)和適應(yīng)環(huán)境，表現(xiàn)出了發(fā)育加速的現(xiàn)象，而快速的生長(zhǎng)發(fā)育在一定程度上干擾了變態(tài)發(fā)育。

自然界中多變的脅迫因子能給生物的生長(zhǎng)發(fā)育、種群發(fā)展與進(jìn)化造成重要影響，天敵脅迫作用對(duì)獵物生理生化方面的影響涉及諸多方面[52-56]。本試驗(yàn)設(shè)計(jì)長(zhǎng)時(shí)脅迫和短時(shí)脅迫兩種水平的脅迫處理，檢測(cè)2種重要的壓力蛋白基因和，以及熱激同源蛋白基因的表達(dá)量水平，以探討天敵脅迫是否能夠誘導(dǎo)棉鈴蟲(chóng)體內(nèi)壓力蛋白基因的表達(dá)變化。在長(zhǎng)時(shí)脅迫下，棉鈴蟲(chóng)自5齡幼蟲(chóng)起，的表達(dá)有上調(diào)趨勢(shì)；在短時(shí)脅迫下，和的表達(dá)有上調(diào)趨勢(shì)。可見(jiàn)捕食脅迫，無(wú)論是慢性作用還是急性作用，都不同程度地影響了棉鈴蟲(chóng)體內(nèi)壓力蛋白基因的表達(dá)水平，使其產(chǎn)生了一定的應(yīng)激反應(yīng)。棉鈴蟲(chóng)壓力蛋白基因與受到短時(shí)脅迫的反應(yīng)較為明顯，而熱激同源蛋白基因受到慢性脅迫刺激的反應(yīng)更為顯著，這可能與三者執(zhí)行的功能不同有關(guān)。盡管捕食壓力對(duì)生物來(lái)說(shuō)是非常普遍的選擇壓力，但是目前有關(guān)脅迫壓力響應(yīng)的分子機(jī)制尚不清楚。在蛋白水平上，心斑綠蟌（）在捕食性魚(yú)5 d的脅迫處理下，表達(dá)水平未受影響，而表達(dá)水平顯著上調(diào)[9]；以魚(yú)激素作為捕食者存在的化學(xué)信號(hào)，大型水蚤（）在捕食性天敵的脅迫處理下，在脅迫6 h表達(dá)顯著上調(diào)，24 h后回落[26]；鯽魚(yú)（）在藍(lán)鰓太陽(yáng)魚(yú)（）脅迫下，視葉中在脅迫6 h高水平表達(dá)，在12 h表達(dá)水平回落，暗示視覺(jué)在脅迫中起到重要的作用[25]。綜上所述，面對(duì)捕食性天敵脅迫，不同種類壓力蛋白的表達(dá)水平與脅迫處理時(shí)間、獵物物種有關(guān)，但總體來(lái)說(shuō)，脅迫下的壓力蛋白含量呈現(xiàn)出上調(diào)或不變的趨勢(shì)。本試驗(yàn)結(jié)果與這一趨勢(shì)相符，進(jìn)一步證明捕食性瓢蟲(chóng)脅迫壓力能夠引起棉鈴蟲(chóng)分子層面的生理反應(yīng)，這種生理反應(yīng)與應(yīng)對(duì)脅迫壓力可能有著密切關(guān)系。而且，棉鈴蟲(chóng)在脅迫壓力作用下的分子生理反應(yīng)機(jī)制，很可能與其在脅迫壓力下的生長(zhǎng)發(fā)育、變態(tài)發(fā)育等表型變化相關(guān)。

4 結(jié)論

在面對(duì)捕食性瓢蟲(chóng)長(zhǎng)時(shí)脅迫作用下，棉鈴蟲(chóng)為了躲避被捕食風(fēng)險(xiǎn)表現(xiàn)出了發(fā)育加速的現(xiàn)象，而快速的生長(zhǎng)發(fā)育在一定程度上干擾了變態(tài)發(fā)育，導(dǎo)致蛹重和羽化率顯著下降，卷翅率顯著上升。棉鈴蟲(chóng)對(duì)不同食源的天敵捕食脅迫具有不同程度的敏感性，即棉鈴蟲(chóng)對(duì)潛在的捕食風(fēng)險(xiǎn)可能存在一定的分級(jí)能力，但這種分級(jí)能力沒(méi)有得到規(guī)律性體現(xiàn)。捕食性瓢蟲(chóng)脅迫壓力能夠引起棉鈴蟲(chóng)分子層面的生理反應(yīng)，其壓力蛋白基因與受到短時(shí)脅迫的反應(yīng)較為明顯，表現(xiàn)為基因表達(dá)的上調(diào)，而熱激同源蛋白基因受到慢性脅迫刺激的反應(yīng)更為顯著，同樣表現(xiàn)出表達(dá)上調(diào)的現(xiàn)象，暗示三者在棉鈴蟲(chóng)體內(nèi)執(zhí)行的功能存在差異，這種生理反應(yīng)（基因表達(dá)水平的變化）可能與應(yīng)對(duì)脅迫壓力有著密切的關(guān)系。

[1] Frank D A. Evidence for top predator control of a grazing ecosystem., 2008, 117(11): 1718-1724.

[2] Hawlena D, Schmitz O J. Herbivore physiological response to predation risk and implications for ecosystem nutrient dynamics., 2010, 107(35): 15503-15507.

[3] Abrams P A. Implications of dynamically variable traits for identifying, classifying and measuring direct and indirect effects in ecological communities., 1995, 146(1): 112-134.

[4] Schmitz O J. Direct and indirect effects of predation and predation risk in old field interaction webs., 1998, 151(4): 327-342.

[5] Werner E E, Peacor S D. A review of trait-mediated indirect interactions in ecological communities., 2003, 84(5): 1083-1100.

[6] Nelson E H, Matthews C E, Rosenheim J A. Predators reduce prey population growth by inducing changes in prey behavior., 2004, 85(7): 1853-1858.

[7] Preisser E L, Bolnick D I, Benard M F. Scared to death? The effects of intimidation and consumption in predator-prey interactions., 2005, 86(2): 501-509.

[8] Pangle K L, Peacor S D, Johannsson O E. Large nonlethal effects of an invasive invertebrate predator on zooplankton population growth rate., 2007, 88(2): 402-412.

[9] Slos S, Stoks R. Predation risk induces stress proteins and reduces antioxidant defense., 2008, 22(4): 637-642.

[10] McCauley S J, Rowe L, Fortin M J. The deadly effects of “nonlethal” predators., 2011, 92(11): 2043-2048.

[11] Peckarsky B L, Taylor B, McIntosh A R, McPeek M A, Lytle D A. Variation in mayfly size at metamorphosis as a developmental response to risk of predation., 2001, 82(3): 740-757.

[12] Dahl J, Peckarsky B L. Developmental responses to predation risk in morphologically defended mayflies., 2003, 137(2): 188-194.

[13] Ball S L, Baker R L. Predator-induced life history changes: antipredator behavior costs or facultative life history shifts?, 1996, 77(4): 1116-1124.

[14] Hechtel L J, Juliano S A. Effects of a predator on prey metamorphosis: plastic responses by prey or selective mortality?, 1997, 78(3): 838-851.

[15] 李燕平, 戈峰. 龜紋瓢蟲(chóng)的捕食脅迫作用對(duì)連續(xù)三代果蠅發(fā)育與繁殖的影響. 昆蟲(chóng)知識(shí), 2010, 47(1): 139-145.

Li Y P, Ge F. Effect of prey stress fromon development and fecundity ofin successive three generations., 2010, 47(1): 139-145. (in Chinese)

[16] Hawlena D, Kress H, Dufresne E R, Schmitz O J. Grasshoppers alter jumping biomechanics to enhance escape performance under chronic risk of spider predation., 2011, 25(1): 279-288.

[17] Kunert G, Otto S, Rose U, Gershezon J, Weiser W. Alarm pheromone mediates production of winged dispersal morphs in aphids., 2005, 8(6): 596-603.

[18] Thaler J S, McArt S H, Kaplan L. Compensatory mechanisms for ameliorating the fundamental trade-off between predator avoidance and foraging., 2012, 109(30): 12075-12080.

[19] Xiong X F, Michaud J P, Li Z, Wu P X, Chu Y N, Zhang Q W, Liu X X. Chronic, predator-induced stress alters development and reproductive performance of the cotton bollworm,., 2015, 60(6): 827-837.

[20] Vonesh J R, Warkentin K M. Opposite shifts in size at metamorphosis in response to larval and metamorph predators., 2006, 87(3): 556-562.

[21] Stoks R, De Block M, McPeek M A. Alternative growth and energy storage responses to mortality threats in damselflies., 2005, 8(12): 1307-1316.

[22] Stoks R. Food stress and predator-induced stress shape developmental performance in a damsel?y., 2001, 127(2): 222-229.

[23] Chivers D P, Wisenden B D, Smith R J F. Damselfly larvae learn to recognize predators from chemical cues in the predator’s diet., 1996, 52(2): 315-320.

[24] Brodin T, Mikolajewski D J, Johansson F. Behavioural and life history effects of predator diet cues during ontogeny in damsel?y larvae., 2006, 148(1): 162-169.

[25] Kagawa N, Ryo K, Mugiya Y. Enhanced expression of stress protein 70 in the brains of goldfish,, reared with bluegills,., 1999, 21(2): 103-110.

[26] Pauwels K, Stoks R, De Meester L. Coping with predator stress: interclonal differences in induction of heat-shock proteins in the water flea., 2005, 18(4): 867-872.

[27] Cockrem J F, Silverin B. Sight of a predator can stimulate a corticosterone response in the great tit ()., 2002, 125(2): 248-255.

[28] Barcellos L J G, Ritter F, Kreutz L C, Quevedo R M, da Silva L B, Bedin A C, Finco J, Cericato L. Whole-body cortisol increases after direct and visual contact with a predator in zebrafish,., 2007, 272(1/4): 774-778.

[29] 張青文. 有害生物綜合治理學(xué). 北京: 中國(guó)農(nóng)業(yè)大學(xué)出版社, 2007.

Zhang Q W.. Beijing: China Agricultural University Press, 2007. (in Chinese)

[30] Tabashnik B E, Carrière Y, Dennehy T J, Morin S, Sisterson M S, Roush R T, Shelton A M, Zhao J Z. Insect resistance to transgenic Bt crops: lessons from the laboratory and field., 2003, 96(4): 1031-1038.

[31] 陳海燕, 楊亦樺, 武淑文, 楊亞軍, 吳益東. 棉鈴蟲(chóng)田間種群Bt毒素Cry1Ac抗性基因頻率的估算. 昆蟲(chóng)學(xué)報(bào), 2007, 50(1): 25-30.

Chen H Y, Yang Y H, Wu S W, Yang Y J, Wu Y D. Estimated frequency of resistance alleles to Bt toxin Cry1Ac in the field populations of(Hübner) from Northern China., 2007, 50(1): 25-30. (in Chinese)

[32] Tay W T, Soria M F, Walsh T, Thomazoni D, Silvie P, Behere G T, Anderson C, Downes S. A brave new world for an old world pest:(Lepidoptera: Noctuidae) in Brazil., 2013, 8(11): e80134.

[33] 王小藝, 沈佐銳. 異色瓢蟲(chóng)的應(yīng)用研究概況. 昆蟲(chóng)知識(shí), 2002, 39(4): 255-261.

Wang X Y, Shen Z R. Progress of applied research on multicolored Asian ladybird beetle., 2002, 39(4): 255-261. (in Chinese)

[34] Koch R L. The multicolored Asian lady beetle,: a review of its biology, uses in biological control, and non-target impacts., 2003, 3(1): 32.

[35] Michaud J P, Olsen L E. Suitability of Asian citrus psyllid,, as prey for ladybeetles., 2004, 49(4): 417-431.

[36] Evans E W. Lady beetles as predators of insects other than Hemiptera., 2009, 51(2): 255-267.

[37] Weber D C, Lundgren J G. Assessing the trophic ecology of the Coccinellidae: their roles as predators and as prey., 2009, 51(2): 199-214.

[38] Wu K J, Gong P Y. A new and practical artificial diet for the cotton bollworm., 1997, 4(3): 277-282.

[39] 楊靜, 韓召軍. 棉鈴蟲(chóng)3種蛻皮相關(guān)基因的RNA干擾效應(yīng)比較. 南京農(nóng)業(yè)大學(xué)學(xué)報(bào), 2014, 37(1): 81-86.

Yang J, Han Z J. Comparison of silencing effect of three ecdysis involved genes in cotton bollworm,., 2014, 37(1): 81-86. (in Chinese)

[40] 閆碩, 朱家林, 朱威龍, 潘李隆, 張青文, 劉小俠. 棉鈴蟲(chóng)-微管蛋白基因的克隆、序列分析及表達(dá)模式檢測(cè). 中國(guó)農(nóng)業(yè)科學(xué), 2013, 46(9): 1808-1817.

Yan S, Zhu J L, Zhu W L, Pan L L, Zhang Q W, Liu X X. Molecular cloning, sequence analysis and expression pattern detection of-tubulin gene from(Hübner)., 2013, 46(9): 1808-1817. (in Chinese)

[41] Yan S, Ni H, Li H T, Zhang J, Liu X X, Zhang Q W. Molecular cloning, characterization, and mRNA expression of twogenes in(Lepidoptera: Noctuidae)., 2013, 106(1): 450-462.

[42] Yan S, Zhu J L, Zhu W L, Zhang X F, Li Z, Liu X X, Zhang Q W. The expression of three opsin genes from the compound eye of(Lepidoptera: Noctuidae) is regulated by a circadian clock, light conditions and nutritional status., 2014, 9(10): e111683.

[43] 閆碩, 劉彥君, 張馨方, 秦萌, 劉慧, 朱家林, 李貞, 張青文, 劉小俠. 棉鈴蟲(chóng)復(fù)眼中生物鐘基因的晝夜表達(dá)模式. 中國(guó)農(nóng)業(yè)科學(xué), 2017, 50(19): 3733-3744.

YAN S, LIU Y J, ZHANG X F, QIN M, LIU H, ZHU J L, LI Z, ZHANG Q W, LIU X X. Daily expression ofgene in compound eye of., 2017, 50(19): 3733-3744. (in Chinese)

[44] YAN S, LIU Y J, ZHU J L, CUI W N, ZHANG X F, YANG Y H, LIU X M, ZHANG Q W, LIU X X. Daily expression of two circadian clock genes in compound eye of: evidence for peripheral tissue circadian timing., 2017, DOI: 10.1111/1744-7917.12541.

[45] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtmethod., 2001,25(4): 402-408.

[46] Harvell C D. The ecology and evolution of inducible defenses., 1990, 65(3): 323-340.

[47] Lively C M, Hazel W N, Schellenberger M J, Michelson K S. Predator-induced defense: variation for inducibility in an intertidal barnacle., 2000, 81(5): 1240-1247.

[48] Schoeppner N M, Relyea R A. Detecting small environmental differences: risk-response curves for predator-induced behavior and morphology., 2008, 154(4): 743-754.

[49] Schoeppner N M, Relyea R A. Interpreting the smells of predation: how alarm cues and kairomones induce different prey defences., 2009, 23(6): 1114-1121.

[50] van Uitregt V O, Hurst T P, Wilson R S. Reduced size and starvation resistance in adult mosquitoes,, exposed to predation cues as larvae., 2012, 81(1): 108-115.

[51] Mangel M, Stamps J. Trade-offs between growth and mortality and the maintenance of individual variation in growth., 2001, 3(5): 583-593.

[52] Clinchy M, Zanette L, Boonstra R, Wingfield J C, Smith J N M. Balancing food and predator pressure induces chronic stress in songbirds., 2004, 271(1556): 2473-2479.

[53] Beckerman A P, Wieski K, Baird D J. Behavioural versus physiological mediation of life history under predation risk., 2007, 152(2): 335-343.

[54] Poledník L, ?ehulka J, Kranz A, Poledníková K, Hlavá? V, Kazihnitková H. Physiological responses of over-wintering common carp () to disturbance by Eurasian otter ()., 2008, 34(3): 223-234.

[55] Steiner U K, Van Buskirk J. Predator-induced changes in metabolism cannot explain the growth/predation risk tradeoff., 2009, 4(7): e6160.

[56] Thaker M, Lima S L, Hews D K. Alternative antipredator tactics in tree lizard morphs: hormonal and behavioural responses to a predator encounter., 2009, 77(2): 395-401.

（責(zé)任編輯 岳梅）

Effects of Predator-Induced Stress fromon the Development and Stress Protein Gene Expression of

YAN Shuo1,2, XIONG XiaoFei3, CHU YanNa1, LI Zhen1, WU PengXiang1, YANG QingPo2, CUI WeiNa4, XU JinTao5, XU LiXia6, ZHANG QingWen1, LIU XiaoXia1

(1College of Plant Protection, China Agricultural University, Beijing 100193;2National Agricultural Technology Extension and Service Center, Beijing 100125;3Beijing PAIDE Science and Technology Development Co., Ltd., Beijing 100097;4Zoucheng Plant Protection Station, Zoucheng 273500, Shandong;5Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Changli 066600, Hebei;6Changli County Bureau of Science and Technology, Changli 066600, Hebei)

The objective of this study is to determine the effects of predator-induced stress fromwith various feeding resources on the development and metamorphose of, illustrating that whethercan perceive and classify the predation risk, and show the trade-off characteristic between the development and metamorphose, and to determine the effects of long-term and short-term stress on the stress protein gene expression of, illustrating that whether the predator-induced stress fromcan induce the physiological reactions ofat molecular level.The development (larval and pupal duration, female and male longevity, and total longevity) and metamorphose (pupae weight, pupation rate, fail eclosion rate, and wrinkled-wing rate) indicators ofwere observed and recorded under the predator-induced stress fromwith 7 kinds of feeding resources (hungry treatment, shrimp egg treatment, cotton bollworm larva treatment, cotton bollworm egg treatment, aphid treatment, CK treatment with aphid, and CK treatment). Long-term (first instar larvae to 3-day-old moth) and short-term (15 min to 6 h) stress treatments were set up, and the changes of stress protein genes (heat shock protein genes)and, heat shock cognate protein geneexpression were determined under the predator-induced stress by quantitative real-time PCR (qRT-PCR).Under the predator-induced stress from, larval and pupal duration, female and male longevity, and total longevity ofshortened, pupal weight and pupation rate decreased, and wrinkled-wing rate increased significantly, whereas the fail eclosion rate was not influenced by the predator-induced stress. Under the predator-induced stress fromwith various food sources, the larval duration ofwas the shortest when predators consumed aphids, the pupal duration was the shortest when predators consumed cotton bollworm eggs, and the total longevity was the shortest when predators consumed shrimp eggs, whereas the female and male longevity were not influenced by the diets of. The wrinkled-wing rate ofwas the highest when predators consumed cotton bollworm eggs, whereas the pupal weight, pupation rate, and fail eclosion rate were not influenced by the diets of. Stress protein genesandwere up-regulated after short-term stress (: 30 min to 3 h;: 15 min, 1.5 h, 2 h and 6 h), whereas heat shock cognate protein genewas up-regulated after long-term stress (the stages of 5th instar larvae, prepupa, male pupae and male moth).Under the long-term stress from, all developmental stages ofshortened, and the development ofbecame faster to avoid the predation risk. The developmental acceleration might disturb the metamorphose ofto some degree, leading the smaller pupal weight, lower pupation rate, and higher wrinkled-wing rate, which was according with the trade-off hypothesis. The sensitiveness ofto predator-induced stress were different among various diets of, andmight be able to classify the potential predation risk, however this ability showed a degree of uncertainty. The predator-induced stress fromcould induce the physiological reactions ofat molecular level, leading the higher expression levels of stress protein genes.andexpressions were more affected by the short-term stress, whereasexpression was more affected by long-term stress.

;; development; metamorphose; stress protein; non-consumptive effect

2017-05-25；接受日期：2017-07-14

國(guó)家自然科學(xué)基金（31572018）

閆碩，E-mail：yanshuo2011@foxmail.com。通信作者劉小俠，E-mail：liuxiaoxia611@cau.edu.cn