毒死蜱對(duì)中國(guó)石龍子胚胎發(fā)育及孵出幼體表型的影響

徐純夏, 徐　衛(wèi), 陸洪良, 黨　偉

(杭州師范大學(xué)生命與環(huán)境科學(xué)學(xué)院，浙江 杭州 310036)

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毒死蜱對(duì)中國(guó)石龍子胚胎發(fā)育及孵出幼體表型的影響

徐純夏, 徐衛(wèi), 陸洪良, 黨偉

(杭州師范大學(xué)生命與環(huán)境科學(xué)學(xué)院，浙江 杭州 310036)

摘要：采用中國(guó)石龍子作為動(dòng)物模型，研究有機(jī)磷農(nóng)藥毒死蜱對(duì)其胚胎發(fā)育及幼體生長(zhǎng)的影響.結(jié)果表明，1.2 mg/g的毒死蜱完全抑制石龍子胚胎的發(fā)育，0.12 mg/g處理組卵孵化成功率顯著低于對(duì)照組.相對(duì)于對(duì)照組，0.12 mg/g處理組卵孵化期無(wú)顯著影響，孵出幼體個(gè)體顯著較小、運(yùn)動(dòng)能力較差，但飼養(yǎng)一個(gè)月的存活率和日生長(zhǎng)速率無(wú)顯著差異.

關(guān)鍵詞：卵孵化；幼體形態(tài)；運(yùn)動(dòng)表現(xiàn)；幼體發(fā)育

隨著現(xiàn)代農(nóng)業(yè)的發(fā)展，為了追求高產(chǎn)，大劑量的農(nóng)藥被用于農(nóng)業(yè)生產(chǎn)，其中膽堿酯酶抑制劑類(lèi)的化合物是最為常見(jiàn)的農(nóng)藥有效成分.毒死蜱作為一種廣泛使用的膽堿酯酶抑制劑的有機(jī)磷脂類(lèi)農(nóng)藥，自20世紀(jì)60年代被廣泛使用后，便在人類(lèi)健康和環(huán)境領(lǐng)域得到了廣泛的關(guān)注.毒死蜱在水產(chǎn)養(yǎng)殖魚(yú)類(lèi)鯉魚(yú)體內(nèi)的累積會(huì)導(dǎo)致解毒器官肝臟的衰退[1]，在家畜豬體內(nèi)的累積會(huì)導(dǎo)致肺等器官的病變，嚴(yán)重的將導(dǎo)致動(dòng)物死亡[2].這類(lèi)農(nóng)藥在動(dòng)物體內(nèi)的代謝與肝臟細(xì)胞色素P450有關(guān)[3].在以小鼠為模型的研究中發(fā)現(xiàn)，毒死蜱的累積會(huì)導(dǎo)致多個(gè)器官如肝、腎、脾中膽堿酯酶的活性顯著降低[4].同時(shí)，毒死蜱在水產(chǎn)養(yǎng)殖動(dòng)物體內(nèi)的累積刺激氧化應(yīng)激的發(fā)生，導(dǎo)致體內(nèi)大量氧自由基和其他活性氧物質(zhì)的累積，而清除這些活性氧物質(zhì)的酶類(lèi)如超氧化物歧化酶、過(guò)氧化氫酶及過(guò)氧化物酶的表達(dá)量則顯著降低[5-6].動(dòng)物體內(nèi)氧化應(yīng)激的發(fā)生，會(huì)對(duì)免疫器官造成巨大的傷害，同時(shí)抑制免疫系統(tǒng)一些重要酶類(lèi)的表達(dá)，進(jìn)而影響體液免疫或細(xì)胞免疫的進(jìn)程[7].

胚胎發(fā)育期和幼體生長(zhǎng)期對(duì)成年后的動(dòng)物發(fā)育有著重要的影響，近年來(lái)有一些關(guān)于農(nóng)藥殘留對(duì)于動(dòng)物胚胎及幼體影響的文獻(xiàn)報(bào)道.對(duì)于胎生動(dòng)物如小鼠而言，母體暴露于毒死蜱污染的環(huán)境中對(duì)幼體T淋巴細(xì)胞的分化有長(zhǎng)期的影響[8]，同時(shí)也會(huì)影響胚胎發(fā)育過(guò)程中一些基因的甲基化，進(jìn)而影響相關(guān)基因的表達(dá)[9].對(duì)于卵生動(dòng)物而言，由于胚胎發(fā)育過(guò)程是脫離母體的，現(xiàn)在的研究多集中在農(nóng)藥污染對(duì)于胚胎發(fā)育、卵孵化以及幼體特征的影響.在一些魚(yú)類(lèi)中的研究結(jié)果表明胚胎發(fā)育過(guò)程中基因和蛋白的表達(dá)有著顯著的變化[10]，同時(shí)胚胎也會(huì)產(chǎn)生氧化應(yīng)激，但乙酰膽堿酯酶的活性并未受到影響，其他標(biāo)記分子的表達(dá)量卻發(fā)生改變[11-12].乙酰膽堿酯酶抑制劑類(lèi)農(nóng)藥如氯氰菊酯、溴氰菊酯、丙線磷和氯代芳烴類(lèi)農(nóng)藥污染也可以導(dǎo)致卵生動(dòng)物胚胎發(fā)育畸形，孵化率下降以及幼體存活率的降低[13-16].

中國(guó)石龍子(Plestiodonduinensis)廣泛分布于中國(guó)華東、華南及沿海地區(qū)，是低地田野草叢或灌木叢中常見(jiàn)的蜥蜴種類(lèi).本文以中國(guó)石龍子卵為實(shí)驗(yàn)對(duì)象，在其孵化基質(zhì)中加入不同濃度的有機(jī)磷農(nóng)藥毒死蜱，以檢測(cè)毒死蜱對(duì)石龍子卵孵化成功率、孵出幼體表型的影響，評(píng)估有機(jī)磷農(nóng)藥對(duì)卵生爬行動(dòng)物胚胎發(fā)育的毒害效應(yīng).

1材料與方法

1.1　卵收集與孵化

2013年4月、5月上旬在浙江麗水采集實(shí)驗(yàn)用的中國(guó)石龍子成體，部分捕獲個(gè)體運(yùn)至杭州師范大學(xué)兩棲爬行動(dòng)物實(shí)驗(yàn)室.石龍子經(jīng)測(cè)量、稱(chēng)重、性別鑒定后，關(guān)養(yǎng)在戶(hù)外水泥圍欄內(nèi).圍欄底部鋪有厚約15 cm的沙土，表面覆上草皮以模擬野外生境條件，提供足量黃粉蟲(chóng)幼蟲(chóng)和飲水.5月下旬將懷卵母體移入產(chǎn)卵缸中，6月初石龍子開(kāi)始產(chǎn)卵，產(chǎn)卵期間每天至少檢查產(chǎn)卵缸2次，確保新生卵能在產(chǎn)后短時(shí)間內(nèi)被收集以免因其從環(huán)境中吸收水分而導(dǎo)致重量發(fā)生變化，新生卵及時(shí)稱(chēng)重、測(cè)量長(zhǎng)徑和短徑.產(chǎn)后雌體經(jīng)稱(chēng)重和測(cè)量后放回水泥飼養(yǎng)池.至6月中旬有10條母體產(chǎn)卵，窩卵數(shù)9~20枚，每窩選取5~6枚(共計(jì)52枚)用于本實(shí)驗(yàn).剩余新生卵在其它處理下孵化，另文報(bào)道.

將購(gòu)買(mǎi)的20%毒死蜱乳油兌水稀釋至0.12和1.20 mg/g，蒸餾水為對(duì)照.以蛭石為孵化基質(zhì)，農(nóng)藥溶液或蒸餾水與干蛭石按1∶1混合成潮濕基質(zhì)，分配至3個(gè)大小一致的塑料盒中.來(lái)自同窩的新生卵隨機(jī)分配到3個(gè)不同處理.卵半埋于潮濕基質(zhì)中，胚胎面朝上.孵化盒用穿孔的塑料薄膜覆蓋，放置于溫度預(yù)先設(shè)置為(28.0±1.0) ℃的人工氣候箱(寧波萊?？萍加邢薰?內(nèi)，每3日補(bǔ)充水分以保持基質(zhì)濕度恒定.

1.2　幼體形態(tài)、功能表現(xiàn)和生長(zhǎng)

待幼體開(kāi)始出殼，每天檢查孵化盒至少兩次，收集新生幼體，隨即測(cè)定其體重、體長(zhǎng)和尾長(zhǎng).孵化期為卵產(chǎn)出時(shí)間到幼體破殼時(shí)間的間隔.1.20 mg/g處理下無(wú)幼體成功孵出，但多數(shù)胚胎已發(fā)育完整，其體重、體長(zhǎng)、尾長(zhǎng)數(shù)據(jù)以剖出卵內(nèi)死胎后測(cè)量獲得.孵出幼體運(yùn)動(dòng)表現(xiàn)在溫度為(28.0±1.0) ℃的恒溫室內(nèi)進(jìn)行.實(shí)驗(yàn)開(kāi)始前，預(yù)先將動(dòng)物置于恒溫室內(nèi)適應(yīng)2 h.測(cè)定時(shí)將石龍子放入2 m長(zhǎng)的木質(zhì)跑道一端，用毛刷驅(qū)趕使之奔跑，用SONY DCR-SR220E數(shù)碼攝像機(jī)記錄動(dòng)物的運(yùn)動(dòng)過(guò)程；每只石龍子測(cè)定一個(gè)來(lái)回.磁帶中的數(shù)據(jù)用MGI Video Wave III軟件讀出.動(dòng)物若在3 min內(nèi)拒絕跑動(dòng)，對(duì)應(yīng)數(shù)據(jù)將不用于統(tǒng)計(jì)分析.

運(yùn)動(dòng)表現(xiàn)測(cè)定后，幼體剪指編號(hào)飼養(yǎng)于鋪設(shè)約15 cm沙土的整理箱內(nèi)，并覆蓋草皮模擬野外生境.整理箱放置在(28.0±1.0) ℃的恒溫室中，一端懸掛1只60 W燈泡(光周期為早上07:00自動(dòng)開(kāi)啟，晚上19:00關(guān)閉)使動(dòng)物在箱內(nèi)能自行調(diào)節(jié)體溫，每天投喂足量小的黃粉蟲(chóng)幼蟲(chóng)和蟋蟀.飼養(yǎng)約1個(gè)月后測(cè)定動(dòng)物體重、記錄幼體死亡情況.幼體生長(zhǎng)速率以每日增重量表示.

1.3　統(tǒng)計(jì)分析

用Statistica 6.0軟件包進(jìn)行統(tǒng)計(jì)分析.對(duì)數(shù)據(jù)進(jìn)行參數(shù)統(tǒng)計(jì)分析前，分別檢驗(yàn)其正態(tài)性(Kolmogorov-Smirnov檢驗(yàn))和方差同質(zhì)性(Bartlett檢驗(yàn)).用單因子方差分析檢測(cè)毒死蜱濃度對(duì)卵孵化期、幼體表型特征(形態(tài)、運(yùn)動(dòng)表現(xiàn)、生長(zhǎng)速率)的影響；Tukey多重比較檢驗(yàn)多樣本處理間的差異.文中涉及的非參數(shù)統(tǒng)計(jì)為G檢驗(yàn).描述性統(tǒng)計(jì)值用平均值±標(biāo)準(zhǔn)誤表示，顯著性水平設(shè)置為α=0.05.

2結(jié)果

不同處理組的入孵卵重?zé)o顯著差異(F2,48=1.91,P=0.159).1.20 mg/g毒死蜱處理下，入孵卵全部無(wú)法成功孵出(表1)；0.12 mg/g處理組卵孵化成功率略低于對(duì)照組(G=54.53,df=2,P<0.001).線性回歸分析顯示孵化期與入孵卵重?zé)o顯著相關(guān)性(F1,31=3.25,P=0.081).不同濃度處理下孵出幼體的孵化時(shí)間無(wú)顯著差異(F1,31=0.09,P=0.761，表1).

表1　不同毒死蜱濃度對(duì)中國(guó)石龍子卵孵化期及孵化成功率的影響

毒死蜱顯著影響孵出幼體大小(體重：F2,45=47.11,P<0.000 1；體長(zhǎng)：F2,45=53.99,P<0.001；尾長(zhǎng)：F2,45=120.12,P<0.001).毒死蜱處理使卵內(nèi)胚胎發(fā)育至后期死亡或孵出較小幼體(圖1).孵出幼體中，0.12 mg/g處理組幼體的運(yùn)動(dòng)表現(xiàn)差于對(duì)照組幼體(平均速度：F1,26=15.57,P<0.001；疾跑速：F1,26=38.08,P<0.001).幼體飼養(yǎng)一個(gè)月后對(duì)照組和0.12 mg/g處理組存活率分別為66.7%(12/18)和56.3%(9/16)，但統(tǒng)計(jì)上無(wú)顯著差異(G=0.39,df=1,P=0.533)；存活幼體的日生長(zhǎng)速率無(wú)顯著差異(F1,17=0.21,P=0.656，圖2).

圖1　不同毒死蜱濃度對(duì)中國(guó)石龍子孵出幼體大小的影響Fig. 1　Effects of Chlorpyrifos on body size of Plestiodon chinensis hatchlings

圖2　不同毒死蜱濃度對(duì)中國(guó)石龍子孵出幼體運(yùn)動(dòng)表現(xiàn)和生長(zhǎng)的影響Fig. 2　Effects of Chlorpyrifos on locomotor performance and early growth rate of Plestiodon chinensis hatchlings

3討論

動(dòng)物胚胎通常對(duì)許多農(nóng)藥反應(yīng)敏感,包括殺蟲(chóng)劑、除草劑在內(nèi)的許多農(nóng)藥會(huì)顯著影響卵生動(dòng)物的孵化.此類(lèi)研究主要集中于魚(yú)類(lèi)及兩棲類(lèi)動(dòng)物，其胚胎暴露于受農(nóng)藥污染的水體中，會(huì)使胚胎致畸致死[13-16].農(nóng)藥對(duì)卵生爬行動(dòng)物胚胎毒害效應(yīng)的研究?jī)H見(jiàn)于少數(shù)幾個(gè)物種[17].高劑量的草甘膦導(dǎo)致紅耳龜(Trachemysscriptaelegans)孵化成功率下降，胚胎畸形率增加[18].與這些研究結(jié)果相似，高濃度的毒死蜱導(dǎo)致中國(guó)石龍子卵孵化成功率顯著降低.事實(shí)上，不同農(nóng)藥對(duì)卵生爬行動(dòng)物孵化成功率的影響并不總展現(xiàn)明顯的致死效應(yīng).在一定劑量范圍內(nèi)，二氯二苯三氯乙烷(DDE)并不影響綠海龜(Cheloniamydas)和紅耳龜卵孵化成功率[19-20]；百菌清、異丙甲草胺等農(nóng)藥暴露也不導(dǎo)致北美擬鱷龜(Chelydraserpentina)卵孵化率的顯著下降[21]；毒殺芬不影響美洲短吻鱷(Alligatormississippiensis)卵孵化率[22].除不同農(nóng)藥品種毒性、動(dòng)物胚胎大小差異導(dǎo)致卵生動(dòng)物胚胎農(nóng)藥致死效應(yīng)存在差異外，農(nóng)藥成分從環(huán)境轉(zhuǎn)移至卵內(nèi)累積的效率可能也是一個(gè)重要方面.巢址周邊基質(zhì)和農(nóng)藥的理化性質(zhì)、卵特征(如蛋殼結(jié)構(gòu)與通透性)的差異會(huì)導(dǎo)致這種遷移效率上的差別，從而顯示出胚胎致死效應(yīng)的種間差異[23-24].例如，存在于巢址基質(zhì)中的有機(jī)氯農(nóng)藥及重金屬容易通過(guò)北美牛蛇(Pituophismelanoleucus)、東方強(qiáng)棱蜥(Sceloporusundulatus)等柔性卵而在卵內(nèi)累積[25-26]，但一些污染物僅少量會(huì)通過(guò)紅耳龜?shù)群駳せ蛴矚ぢ?中國(guó)石龍子的卵屬于殼薄的柔性卵，有機(jī)磷農(nóng)藥成分容易透過(guò)卵殼而在卵內(nèi)累積；新生卵內(nèi)胚胎相對(duì)較小而脆弱，對(duì)有機(jī)磷農(nóng)藥的反應(yīng)也極為敏感.事實(shí)上，本研究設(shè)置的毒死蜱濃度遠(yuǎn)高于土壤殘留濃度(7×106~3.5×105mg/g)[27]而接近于0.4~1.3 mg/g的田間實(shí)際噴灑濃度，較高濃度的毒死蜱對(duì)敏感的石龍子胚胎產(chǎn)生明顯的致死效應(yīng)是可預(yù)見(jiàn)的.

雖然低劑量的農(nóng)藥并不一定直接造成卵生動(dòng)物的胚胎死亡，但可能會(huì)對(duì)其孵出幼體具有深遠(yuǎn)影響.0.12 mg/g處理組的石龍子卵孵化成功率并不明顯低于對(duì)照組，但其孵出幼體明顯較小、運(yùn)動(dòng)表現(xiàn)和早期生長(zhǎng)情況較差.不同農(nóng)藥顯著影響卵生動(dòng)物幼體表型的結(jié)果已被頻繁地報(bào)道[28-30].卵生爬行動(dòng)物胚胎農(nóng)藥毒性試驗(yàn)的結(jié)果通常是一致的.例如，毒殺芬和硫丹暴露分別使美洲短吻鱷和寬吻凱門(mén)鱷(Caimanlatirostris)孵出幼體減小[22,31]；高濃度的甲萘威會(huì)顯著降低游蛇的游泳能力[32].幼體大小、運(yùn)動(dòng)表現(xiàn)和生長(zhǎng)速率與其適合度相關(guān)聯(lián)，因此可以推測(cè)環(huán)境中的農(nóng)藥殘留可能會(huì)通過(guò)影響孵出幼體表型而影響其生存.

大多數(shù)有機(jī)農(nóng)藥屬于內(nèi)分泌干擾物，在動(dòng)物胚胎或體內(nèi)積累引起機(jī)體內(nèi)分泌失衡而造成活動(dòng)異常.值得注意的是內(nèi)分泌干擾物以模擬類(lèi)固醇化合物來(lái)干擾類(lèi)固醇的合成與活性而阻斷正常的性別決定途徑，使胚胎發(fā)育過(guò)程中性器官形成發(fā)生逆轉(zhuǎn)[22,32].一些內(nèi)分泌干擾物會(huì)導(dǎo)致紅耳龜、佛羅里達(dá)紅腹龜(Chrysemysnelsoni)、美洲短吻鱷等雄性胚胎雌性化[33-36].毒死蜱在一些動(dòng)物中表現(xiàn)出類(lèi)似雌激素作用已有報(bào)道[37]，但是否會(huì)影響爬行動(dòng)物的性別決定有待于進(jìn)一步確定.

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Effects of Chlorpyrifos on the Embryonic Development and Hatchling

Phenotype of Chinese skink,Plestiodonchinensis

XU Chunxia, XU Wei, LU Hongliang, DANG Wei

(College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China)

Abstract:Taking Chinese skink (Plestiodonchinensis)as the animal model, the effects of organophosphorus pesticide chlorpyrifos on embryonic development and hatchling phenotypes are studied. The results indicate that 1.2 mg/g high concentration of chlorpyrifos seriously inhibits embryonic development, and egg hatching success of 0.12 mg/g treated group is significantly lower than that of the control group. The incubation period of 0.12 mg/g treated group is not different from the control group. However, the hatchlings from 0.12 mg/g treated group are significantly smaller and performed worse than those from the control group. In the first month, hatchling survival rate and growth rate have no significant difference.

Key words:egg incubation; larval morphology; locomotor performance; larval development

文章編號(hào):1674-232X(2016)01-0040-06

中圖分類(lèi)號(hào):Q955

文獻(xiàn)標(biāo)志碼:A

doi:10.3969/j.issn.1674-232X.2016.01.008

通信作者：黨偉(1984—)，女，助理研究員，博士，主要從事爬行動(dòng)物生態(tài)學(xué)研究.E-mail:dang.wei@hotmail.com

基金項(xiàng)目：浙江省自然科學(xué)基金項(xiàng)目(LQ12C03003).

收稿日期：2015-05-13