田悅，呂天星，楊苑釗，侯鑫鑫，陳華保，張敏，龔國(guó)淑，楊春平*

苦葛皂苷對(duì)福壽螺耗氧率及排氨率的影響

田悅1，呂天星1，楊苑釗2，侯鑫鑫1，陳華保1，張敏1，龔國(guó)淑1，楊春平1*1

（1.四川農(nóng)業(yè)大學(xué)無(wú)公害農(nóng)藥研究實(shí)驗(yàn)室，成都611130；2.成都市植物園，成都610083）

為明確苦葛皂苷對(duì)福壽螺呼吸代謝的影響，測(cè)定了不同苦葛皂苷質(zhì)量濃度、不同水體培養(yǎng)溫度和不同螺質(zhì)量下苦葛皂苷處理與未處理正常福壽螺（對(duì)照）耗氧率、排氨率及氧氮比的差異。結(jié)果表明：苦葛皂苷處理組福壽螺的耗氧率及排氨率均顯著低于對(duì)照組（p＜0.05)；在水溫28℃、苦葛皂苷質(zhì)量濃度40 mg/L條件下，處理組福壽螺的耗氧率、排氨率分別為0.19、0.02 mg/(g·h)，而對(duì)照組高達(dá)0.88、0.07 mg/(g·h)；苦葛皂苷處理組福壽螺的氧氮比值顯著降低，說(shuō)明福壽螺代謝方式發(fā)生改變，由以蛋白質(zhì)及脂肪和糖類共同代謝轉(zhuǎn)向以蛋白質(zhì)代謝為主。本研究結(jié)果揭示了苦葛皂苷的作用機(jī)制可能是通過(guò)降低福壽螺的呼吸代謝水平，抑制脂肪和糖類代謝能力，迫使福壽螺通過(guò)增加蛋白質(zhì)代謝進(jìn)行免疫應(yīng)激，最終導(dǎo)致螺體能量供應(yīng)不足而中毒死亡。

福壽螺；苦葛皂苷；耗氧率；排氨率；氧氮比

福壽螺（Pomacea canaliculata）又叫蘋果螺，是兩棲淡水貝類軟體生物，廣泛分布于中國(guó)及其他亞洲國(guó)家[1-2]。福壽螺啃食水稻、甘薯等糧食作物，影響作物產(chǎn)量及品質(zhì)[3]。它還是廣州管圓線蟲（Angiostrongylus cantonensis）的中間寄主，危害人類健康[4]。因此，對(duì)福壽螺進(jìn)行綜合防治對(duì)農(nóng)業(yè)生產(chǎn)及人類健康具有重要的意義。研究發(fā)現(xiàn)，植物中富含殺螺活性物質(zhì)[5]，國(guó)內(nèi)外已對(duì)超過(guò)1 000多種植物進(jìn)行了滅螺活性篩選，其中，油茶科植物、盾葉薯蕷、黃姜、夾竹桃、商陸、血水草、麻楓樹等少數(shù)植物材料因其高活性而被人們所關(guān)注。這些植物的活性成分主要為皂苷類和生物堿類物質(zhì)，其中，皂苷類物質(zhì)是一類具有應(yīng)用前景的殺螺活性成分[6]。JOSHI等[7]和MARTINS等[8]研究表明，從藜麥（Chenopodium quinoa）中提取的皂苷類物質(zhì)能有效地防治福壽螺。現(xiàn)有的研究結(jié)果顯示，皂苷類物質(zhì)對(duì)螺體神經(jīng)系統(tǒng)、呼吸系統(tǒng)、排泄系統(tǒng)和生殖系統(tǒng)等均可造成一定程度的破壞。陳盛霞等[9]發(fā)現(xiàn)用銀杏外種皮處理釘螺，可導(dǎo)致釘螺體內(nèi)的蘋果酸脫氫酶、酸性磷酸酶等活性降低，推測(cè)其作用機(jī)制為通過(guò)干擾煙酰胺腺嘌呤二核苷酸（NADH）呼吸鏈來(lái)影響釘螺的呼吸代謝。呼吸與排泄是生物體新陳代謝的基本生理活動(dòng)，對(duì)評(píng)估水生生物在水生態(tài)系統(tǒng)中的物質(zhì)循環(huán)、生理狀態(tài)、能量流動(dòng)、代謝水平等均具有十分重要的意義[10-11]。耗氧率、排氨率及氧氮比是反映螺體呼吸代謝水平的重要指標(biāo)，其變化情況反映了殺螺藥物對(duì)福壽螺正常生命活動(dòng)的影響程度。

苦葛（Pueraria peduncularis Grah.）是中國(guó)特有的一種豆科中藥植物。四川農(nóng)業(yè)大學(xué)無(wú)公害農(nóng)藥研究室研究發(fā)現(xiàn)，苦葛中的皂苷類物質(zhì)對(duì)福壽螺具有極強(qiáng)的毒殺作用[12]。在前人研究的基礎(chǔ)上，本試驗(yàn)進(jìn)一步研究了苦葛皂苷不同質(zhì)量濃度和培養(yǎng)水溫對(duì)不同大小福壽螺的耗氧率、排氨率及氧氮比的影響，以期為闡明苦葛皂苷對(duì)福壽螺的毒殺機(jī)制奠定理論基礎(chǔ)。

1 材料與方法

1.1 供試材料

供試所用福壽螺（Pomacea canaliculata）采自四川農(nóng)業(yè)大學(xué)溫江校區(qū)農(nóng)場(chǎng)。挑選符合要求的福壽螺，洗凈后在實(shí)驗(yàn)室條件下（于50 L塑料箱中培養(yǎng)，溫度、光照自然，不人工曝氣，每天換水、清理死螺及糞便，并喂食白菜）培養(yǎng)至少2周，使其適應(yīng)實(shí)驗(yàn)室環(huán)境。試驗(yàn)中用到的螺質(zhì)量為（5±0.5）g、（4±0.4）g、（3±0.3）g。

苦葛皂苷由四川農(nóng)業(yè)大學(xué)無(wú)公害農(nóng)藥研究室提供。

1.2 試驗(yàn)設(shè)計(jì)

試驗(yàn)測(cè)定不同苦葛皂苷質(zhì)量濃度、不同培養(yǎng)溫度和不同螺質(zhì)量處理下福壽螺耗氧率（RO）、排氨率（RN）及氧氮比（RO∶N）的變化。

苦葛皂苷質(zhì)量濃度處理：設(shè)20、30、40、50 mg/L 4個(gè)質(zhì)量濃度梯度，螺質(zhì)量（5±0.5）g，溫度28℃。

溫度處理：設(shè)26、27、28、29、30 ℃ 5個(gè)溫度梯度，螺質(zhì)量（5±0.5）g，苦葛皂苷質(zhì)量濃度40 mg/L。

螺質(zhì)量處理：設(shè)（5±0.5）、（4±0.4）、（3±0.3）g 3個(gè)螺質(zhì)量規(guī)格，溫度28℃，苦葛皂苷質(zhì)量濃度40 mg/L。

取相應(yīng)大小螺3頭置于500 mL碘量瓶中處理2 h，取處理后水體測(cè)定其溶解氧含量及氨氮比值。溶解氧測(cè)定采用Winkler碘量法，氨氮比值測(cè)定采用次溴酸鈉氧化法。同時(shí)將螺殼敲碎，取螺軟組織于65℃烘箱中烘干24 h至恒量，測(cè)定其軟體部干質(zhì)量。各處理均設(shè)加螺不加苦葛皂苷的空白對(duì)照，每個(gè)處理3次重復(fù)。

1.3 數(shù)據(jù)計(jì)算與分析

耗氧率、排氨率及氧氮比測(cè)定參考羅杰等[13]的方法。耗氧率（RO）、排氨率（RN）及氧氮比（RO∶N）計(jì)算方法如下：

式中：O0、Ot分別為試驗(yàn)結(jié)束時(shí)清水對(duì)照、試驗(yàn)組水體中溶解氧質(zhì)量濃度，mg/L；N0、Nt分別為試驗(yàn)結(jié)束時(shí)清水對(duì)照、試驗(yàn)組水體中氨氮質(zhì)量濃度，mg/L；t為螺處理時(shí)間，h；V為試驗(yàn)用水體積，L；n為試驗(yàn)螺個(gè)數(shù)；m為福壽螺軟組織干質(zhì)量，g。

所有數(shù)據(jù)采用DPS 9.50進(jìn)行鄧肯差異分析。

2 結(jié)果與分析

2.1 不同質(zhì)量濃度苦葛皂苷對(duì)福壽螺耗氧和排氨的影響

由圖1A可知：苦葛皂苷處理使福壽螺的呼吸耗氧量大幅降低（p＜0.05）；對(duì)照組和20 mg/L苦葛皂苷處理的福壽螺單位質(zhì)量耗氧率分別為0.88和0.24 mg/(g·h)，兩者相差3.67倍；對(duì)照組福壽螺單位質(zhì)量耗氧率與苦葛皂苷各濃度處理組間差異均有統(tǒng)計(jì)學(xué)意義（p＜0.05）。

由圖1B可知：當(dāng)苦葛皂苷質(zhì)量濃度為20 mg/L時(shí)，福壽螺單位質(zhì)量排氨率立即降低，與對(duì)照組差異有統(tǒng)計(jì)學(xué)意義（p＜0.05）；當(dāng)其質(zhì)量濃度為50 mg/L時(shí)，福壽螺單位質(zhì)量排氨率相對(duì)于其他苦葛皂苷處理組略微升高，但仍比對(duì)照低48.54%；各苦葛皂苷質(zhì)量濃度處理組間無(wú)交互關(guān)系（p＜0.05）。

從表1可以看出：在各質(zhì)量濃度苦葛皂苷處理下福壽螺氧氮比均低于對(duì)照組；隨著其質(zhì)量濃度的增加，福壽螺的氧氮比呈下降趨勢(shì)；苦葛皂苷質(zhì)量濃度為20 mg/L的處理組與對(duì)照組差異較小，但隨其質(zhì)量濃度的增加，差異逐漸增大；在50 mg/L時(shí)氧氮比達(dá)到最低，比對(duì)照組低47.35%。

圖1 不同質(zhì)量濃度苦葛皂苷對(duì)福壽螺耗氧率(RO)和排氨率(RN)的影響Fig.1 Effects of different pedunsaponin concentrations on oxygen consumption rate per unit mass(RO)and ammonia excretion rate per unit mass(RN)of P.canaliculata

表1 在不同質(zhì)量濃度苦葛皂苷處理下福壽螺的氧氮比Table 1 Oxygen-to-nitrogen ratio(RO∶N)of P.canaliculata at different pedunsaponin concentrations

2.2 不同水溫下苦葛皂苷對(duì)福壽螺耗氧和排氨的影響

各水溫下苦葛皂苷處理組福壽螺單位質(zhì)量耗氧率均顯著低于對(duì)照組（p＜0.05）（圖2A）。隨處理溫度的增加，苦葛皂苷處理組與對(duì)照組福壽螺的單位質(zhì)量耗氧量均呈先升高后下降的趨勢(shì)，且在28℃時(shí)達(dá)到最高，此時(shí)，處理組與對(duì)照組單位質(zhì)量耗氧率分別為0.19、0.88 mg/（g·h），即對(duì)照組福壽螺的單位質(zhì)量耗氧率是苦葛皂苷處理組的4.63倍。

圖2 不同溫度下福壽螺的耗氧率(RO)和排氨率(RN)Fig.2 Oxygen consumption rate of per unit mass(RO)and ammonia excretion rate of per unit mass(RN)of P.canaliculata under different temperatures

從圖2B可看出：在各水溫下，苦葛皂苷處理組福壽螺的單位質(zhì)量排氨率均低于對(duì)照組（p＜0.05）；對(duì)照組福壽螺的單位質(zhì)量排氨率隨水溫的升高呈先上升后下降再趨于平穩(wěn)，而苦葛皂苷各處理組均處于較低水平；對(duì)照組排氨率在28℃時(shí)達(dá)到最高[0.07 mg/（g·h）]，是苦葛皂苷處理組[0.02 mg/（g·h）]的3.5倍。

各溫度下福壽螺苦葛皂苷處理組氧氮比均低于對(duì)照組（表2）。在40 mg/L苦葛皂苷處理下福壽螺氧氮比呈現(xiàn)先升高后降低的趨勢(shì)。隨著溫度的升高，處理組與對(duì)照組氧氮比之間的差距呈現(xiàn)先減小后增大的趨勢(shì)，當(dāng)溫度為26、28、30℃時(shí)，對(duì)照組氧氮比分別是苦葛皂苷處理組的13.17、1.51、1.85倍。

表2 不同溫度處理下福壽螺的氧氮比Table 2 Oxygen-to-nitrogen ratio(RO∶N)of P.canaliculata under different temperatures

2.3 苦葛皂苷對(duì)不同大小福壽螺耗氧和排氨的影響

由圖3A可知：苦葛皂苷處理組中不同大小福壽螺的單位質(zhì)量耗氧率均顯著低于對(duì)照組（p＜0.05）；隨著福壽螺個(gè)體的增大，苦葛皂苷處理組的單位質(zhì)量耗氧率差異無(wú)統(tǒng)計(jì)學(xué)意義，而對(duì)照組的單位質(zhì)量耗氧率逐漸增大，且5 g福壽螺與3、4 g福壽螺之間差異有統(tǒng)計(jì)學(xué)意義（p＜0.05）。

由圖3B可知：對(duì)照組中5 g福壽螺的單位質(zhì)量排氨率顯著高于3 g和4 g福壽螺，且為3 g福壽螺的2.96倍；對(duì)照組福壽螺的單位質(zhì)量排氨率均顯著高于苦葛皂苷處理組（p＜0.05）。

圖3 不同質(zhì)量福壽螺的耗氧率(RO)及排氨率(RN)Fig.3 Oxygen consumption rate per unit mass(RO)and ammonia excretion rate per unit mass(RN)of P.canaliculata with different masses

對(duì)照組及苦葛皂苷處理組的氧氮比均隨福壽螺個(gè)體的增大先升高后降低；苦葛皂苷處理組的各規(guī)格福壽螺氧氮比均低于對(duì)照組，4 g螺苦葛皂苷處理組氧氮比較對(duì)照組低4.73（表3）。

表3 不同質(zhì)量福壽螺的氧氮比Table 3 Oxygen-to-nitrogen ratio(RO∶N)of P.canaliculata with different masses

3 討論和結(jié)論

物質(zhì)代謝與能量代謝密切相關(guān)，物質(zhì)代謝的異常會(huì)引發(fā)能量代謝的異常[14]。在硫化物脅迫下，日本沼蝦呼吸代謝方式發(fā)生改變，由有氧呼吸轉(zhuǎn)換為無(wú)氧呼吸，同時(shí)機(jī)體內(nèi)磷酸精氨酸在AK催化下釋放ATP為機(jī)體供能[15]。栗志民等[16]研究發(fā)現(xiàn)，皺肋文蛤?qū)}度的承受范圍存在一定的閾值，當(dāng)?shù)陀陂撝禃r(shí)，耗氧率及排氨率隨著鹽度的升高而增加，當(dāng)高于閾值時(shí)，隨著鹽度的增加而降低。在適宜的溫度范圍內(nèi)，溫度使貝類耗氧率及排氨率先增高后降低[16-17]。在本研究中，苦葛皂苷處理的福壽螺耗氧率及排氨率均低于對(duì)照組，表現(xiàn)出整體代謝水平的降低，這可能是由于苦葛皂苷對(duì)福壽螺的呼吸代謝產(chǎn)生影響，進(jìn)而影響其能量代謝。而在不同培養(yǎng)溫度及苦葛皂苷質(zhì)量濃度條件下，苦葛皂苷處理的福壽螺耗氧率及排氨率差異均不顯著，進(jìn)一步說(shuō)明苦葛皂苷在低質(zhì)量濃度下即可對(duì)福壽螺呼吸代謝表現(xiàn)出極強(qiáng)的抑制作用，且這種抑制作用不隨福壽螺生活區(qū)域水溫的變化而變化。

氧氮比（RO∶N）可表示生物體內(nèi)蛋白質(zhì)與脂肪和糖類分解代謝的比率，與新陳代謝有密切的聯(lián)系[18]。當(dāng)RO∶N＜7時(shí)，生物體代謝以蛋白質(zhì)為主；當(dāng)7＜RO∶N＜24時(shí)，以蛋白質(zhì)及脂肪和糖類共同代謝為主；當(dāng)RO∶N＞24時(shí)，生物體以脂肪和糖類代謝為主[19]。試驗(yàn)結(jié)果表明，正常福壽螺的RO∶N值范圍為7～24，其代謝方式為蛋白質(zhì)及脂肪和糖類共同代謝，而苦葛皂苷處理的福壽螺RO∶N值比對(duì)照組低，說(shuō)明福壽螺的代謝方式由以蛋白質(zhì)及脂肪和糖類共同代謝朝以蛋白質(zhì)代謝方向發(fā)展。呂繼蓉等[20]研究發(fā)現(xiàn)，免疫應(yīng)激會(huì)增強(qiáng)動(dòng)物體內(nèi)的蛋白質(zhì)分解代謝，以維持生物體內(nèi)環(huán)境穩(wěn)定。由此推測(cè)，苦葛皂苷處理可有效抑制糖類和脂肪代謝能力，促使福壽螺通過(guò)增強(qiáng)蛋白質(zhì)代謝水平進(jìn)行免疫應(yīng)激反應(yīng)，最終導(dǎo)致螺體能量供應(yīng)不足而中毒死亡。

目前已有研究證明，抑制糖類和脂肪代謝是一個(gè)防治有害生物的有效途徑。于彩虹等[21]研究發(fā)現(xiàn)，皂角苷能顯著抑制棉鈴蟲海藻糖酶活性并使相關(guān)糖類含量降低，導(dǎo)致棉鈴蟲正常的生命活動(dòng)受到破壞。螺螨酯通過(guò)抑制螨體內(nèi)脂肪合成，破壞脂肪代謝實(shí)現(xiàn)殺死害螨的目的[22]。在殺螺機(jī)制研究方面，亦有報(bào)道表明，部分滅螺活性物質(zhì)對(duì)螺體的呼吸系統(tǒng)有較大影響，主要表現(xiàn)為線粒體破壞、生物氧化關(guān)鍵酶系活性降低[23-24]及氧化磷酸化解偶聯(lián)[25]等。因此，進(jìn)一步開展苦葛皂苷抑制糖類和脂肪代謝機(jī)制的研究，將有利于明確其滅螺機(jī)制，為苦葛皂苷作為滅螺劑的開發(fā)應(yīng)用提供理論依據(jù)。

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Effects of pedunsaponin on oxygen consumption rate and ammonia excretion rate of Pomacea canaliculata.Journal of Zhejiang University(Agric.&Life Sci.),2017,43(5):608-614

TIAN Yue1,Lü Tianxing1,YANG Yuanzhao2,HOU Xinxin1,CHEN Huabao1,ZHANG Min1,GONG Guoshu1,YANG Chunping1*
(1.Biorational Pesticide Research Lab,Sichuan Agricultural University,Chengdu 611130,China;2.Chengdu Botanical Garden,Chengdu 610083,China)

Pomacea canaliculata;pedunsaponin;oxygen consumption rate;ammonia excretion rate;ratio of oxygen to nitrogen

S 481.1

A

10.3785/j.issn.1008-9209.2016.10.241

Summary Pomacea canaliculata is an introduced aquatic animal which has transformed into a malignant aquatic animal of paddy and other aquatic plants in China.In addition,the snail also acts as an intermediate host of Angiostrongylus cantonensis,and the main causative agent of human eosinophilic encephalitis.Moreover,the snail consumes dissolved oxygen in freshwater,causing destruction of the aquatic environment.As a result,P.canaliculata was listed as an invasive species in China by the Chinese State Environmental Protection Administration in 2003.It is necessary to prevent the species from spreading further and expanding its population.Pueraria peduncularis Grah.is a traditional Chinese medicine that belongs to the Pueraria family within the Leguminosae,and distributes widely across southwestern China.In terms of agricultural activity,the herb is biologically active against a variety of agricultural pests,and has insecticidal,bactericidal,and slug-killing activities.In our previous study,we found that P.peduncularis extracts had extremely strong toxicity against the apple snail and further identified the active compound as pedunsaponin,which has LC50value of 3.89 mg/L and great molluscicidal potential.

To explore the effects of pedunsaponin on the respiratory metabolism of P.canaliculata,the oxygen consumption rate,ammonia excretion rate,and the ratio of oxygen to nitrogen(O∶N)were examined after treatment with pedunsaponin.Thecorrelations of these processes with pedunsaponin concentrations,treatment temperatures and snail sizes were also evaluated in this study.

四川省科技扶貧專項(xiàng)基金（2017NFP0062）。

楊春平（http://orcid.org/0000-0003-3433-7835），E-mail:chunping79@163.com

（First author）：田悅（http://orcid.org/0000-0002-9816-0656），E-mail:1198190076@qq.com

2016-10-24；接受日期（Accepted）：2016-12-30

The results showed that both the oxygen consumption rate and ammonia excretion rate decreased in the pedunsaponintreated groups compared with the untreated group(p＜0.05).When the temperature was 28℃ under 40 mg/L pedunsaponin,the oxygen consumption and ammonia excretion rate were 0.19 mg/(g·h)and 0.02 mg/(g·h)in the treated snails,while for the normal snails,they were 0.88 mg/(g·h)and 0.07 mg/(g·h),respectively.In addition,pedunsaponin significantly reduced the O∶N ratio of the snails,shifting away from protein,fat and carbohydrate metabolism to main protein one.

In conclusion,this study demonstrates that pedunsaponin is able to reduce the level of respiratory metabolism of P.canaliculata,which may inhibit fat and carbohydrate metabolism,thus forcing the snail to increase protein metabolism for an immune response.As a result,the snails died due to the lack of an energy supply.The study provides a theoretical basis for further research into toxic mechanism of pedunsaponin on P.Canaliculata.