蝦夷扇貝苗種培育水中可培養(yǎng)細(xì)菌群落分析

孫雪瑩,劉繼晨,李明,趙學(xué)偉,梁峻,孫丕海,馬悅欣(.大連海洋大學(xué)農(nóng)業(yè)部北方海水增養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室,遼寧大連60;.獐子島集團(tuán)股份有限公司,遼寧大連600; .大連海洋大學(xué)海珍品苗種培育基地,遼寧大連60)

蝦夷扇貝苗種培育水中可培養(yǎng)細(xì)菌群落分析

孫雪瑩1,劉繼晨1,李明2,趙學(xué)偉2,梁峻2,孫丕海3,馬悅欣1
(1.大連海洋大學(xué)農(nóng)業(yè)部北方海水增養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室,遼寧大連116023;2.獐子島集團(tuán)股份有限公司,遼寧大連116001; 3.大連海洋大學(xué)海珍品苗種培育基地,遼寧大連116023)

為研究蝦夷扇貝Patinopecten yessoensisis苗種培育過程中幼體發(fā)育5個(gè)時(shí)期苗種培育水中(受精卵期水樣W1,擔(dān)輪幼蟲期水樣W2,D型幼蟲期水樣W3,殼頂幼蟲期水樣W4,稚貝期水樣W5)可培養(yǎng)細(xì)菌的群落組成,采用傳統(tǒng)培養(yǎng)方法,從每個(gè)水樣分離細(xì)菌30株,通過16S rRNA基因測(cè)序分析鑒定細(xì)菌。結(jié)果表明:分離的150株細(xì)菌可歸為2門、3綱、6目、9科、10屬、22種;在門水平,W1~W5的優(yōu)勢(shì)門均為變形菌門,其次為厚壁菌門;在屬水平,假交替單胞菌屬為所有水樣第一優(yōu)勢(shì)屬,此外,W1的優(yōu)勢(shì)屬依次為交替單胞菌屬、弧菌屬和海桿菌屬,W2的優(yōu)勢(shì)屬依次為弧菌屬、交替單胞菌屬和Cobetia,W3的優(yōu)勢(shì)屬依次為弧菌屬、交替單胞菌屬和芽孢桿菌屬,W4的優(yōu)勢(shì)屬依次為交替單胞菌屬、芽孢桿菌屬和弧菌屬, W5的優(yōu)勢(shì)屬依次為弧菌屬、Pseudophaeobacter、芽孢桿菌屬和赤桿菌屬。研究表明,扇貝幼體不同發(fā)育時(shí)期苗種培育水中所含可培養(yǎng)細(xì)菌種類比較豐富,細(xì)菌群落結(jié)構(gòu)存在差異。

蝦夷扇貝;育苗培育水;16S rRNA基因;細(xì)菌群落

水產(chǎn)養(yǎng)殖過程中,尤其是在敏感的幼體飼養(yǎng)階段,水質(zhì)對(duì)健康動(dòng)物的生長發(fā)育至關(guān)重要。水質(zhì)指標(biāo)包括物理、化學(xué)指標(biāo),以及所含細(xì)菌的數(shù)量和類型[1]。細(xì)菌性疾病是世界范圍內(nèi)水產(chǎn)養(yǎng)殖生產(chǎn)的最大限制因素之一,其中弧菌屬細(xì)菌是最常報(bào)道的病原體[2-4]。

細(xì)菌的來源之一是苗種培育過程中使用的海水。Sandaa等[5]研究發(fā)現(xiàn),大扇貝Pecten maximus苗種培育過程中,不同養(yǎng)殖系統(tǒng)(靜態(tài)系統(tǒng)、循環(huán)水系統(tǒng)和經(jīng)過臭氧處理的循環(huán)水系統(tǒng))水中細(xì)菌群落結(jié)構(gòu)高度相似。Godoy等[6]研究表明,使用抗生素處理紫扇貝Argopecten purpuratus幼體,其養(yǎng)殖水中可培養(yǎng)細(xì)菌的數(shù)量和優(yōu)勢(shì)屬與未處理組不同,幼體存活率也不同。在蝦夷扇貝Patinopecten yessoensisis苗種培育過程中,常發(fā)生苗種大量死亡的現(xiàn)象,已證明燦爛弧菌Vibrio splendidus和塔斯馬尼亞弧菌Vibrio tasmaniensis是扇貝幼體死亡的致病菌[4],目前,尚不清楚苗種培育水中細(xì)菌是否與幼體疾病相關(guān)。本試驗(yàn)中,使用傳統(tǒng)培養(yǎng)方法和16S rRNA基因序列分析了扇貝幼體不同發(fā)育時(shí)期苗種培育水中細(xì)菌群落組成,以期為苗種培育生態(tài)系統(tǒng)的調(diào)控與管理及病害防治提供參考。

1 材料與方法

1.1 材料

蝦夷扇貝苗種培育在獐子島海珍品原良種場(chǎng)進(jìn)行,將經(jīng)過沙濾和沉淀處理后的海水100 L加入塑料桶中,扇貝幼體養(yǎng)殖密度為5~6只/mL,每天7:00~8:00換水1次,每次換水50 L。餌料以叉鞭金藻Dicrateria inornata為主,孵化初期,每天投喂叉鞭金藻3次(8:00、15:00、23:00),每次100 mL[(7~9)伊105cells/mL],至受精后7 d,補(bǔ)充添加10 mL[(2~4)伊106cells/mL]新月菱形藻Nitzschia closterium,隨后每天增加10 mL,至100 mL時(shí)不再增加,后續(xù)試驗(yàn)中每天投喂餌料3次,每次添加叉鞭金藻和新月菱形藻各100 mL。通過氣石充氣,水溫保持在(15依1)益,鹽度為(32依0.5)。

1.2 方法

1.2.1 樣品的采集于2014年3—4月在蝦夷扇貝幼體發(fā)育的5個(gè)時(shí)期,無菌采集苗種培育水樣1 L(3個(gè)苗種培育桶各采集500 mL混合),受精卵、擔(dān)輪幼蟲、D型幼蟲、殼頂幼蟲和稚貝時(shí)期的樣品分別標(biāo)記為W1、W2、W3、W4和W5。

1.2.2 細(xì)菌的分離、計(jì)數(shù)和純化將采集的水樣進(jìn)行梯度稀釋,取0.1 mL稀釋液涂布于2216E平板上,在15益下培養(yǎng)7 d,計(jì)數(shù)。在適當(dāng)稀釋度的平板上各隨機(jī)挑取30個(gè)菌落,于2216E平板上分離純化,獲得純培養(yǎng)菌株于冰箱中保存。

1.2.3 細(xì)菌DNA的提取、PCR擴(kuò)增和序列測(cè)定將純菌株接種到2216E液體培養(yǎng)基中培養(yǎng)過夜,使用土壤基因組DNA快速提取試劑盒[生工生物工程(上海)股份有限公司]提取細(xì)菌基因組DNA,提取方法參照試劑盒說明書。細(xì)菌16S rRNA基因片段的擴(kuò)增引物為F27/R1492,由生工生物工程(上海)股份有限公司合成,引物序列參照文獻(xiàn)[7]。PCR反應(yīng)體系及反應(yīng)程序參考文獻(xiàn)[4]。用10 g/L瓊脂糖凝膠電泳檢測(cè)PCR產(chǎn)物是否符合目標(biāo)條帶。由生工生物工程(上海)股份有限公司完成PCR產(chǎn)物的測(cè)序。

1.3 數(shù)據(jù)處理

將獲得的各菌株序列在EzBioCloud(http:// www.ezbiocloud.net/eztaxon)公共數(shù)據(jù)庫中進(jìn)行序列同源性比對(duì),從結(jié)果中取第一個(gè)相似性最高的序列。將比對(duì)后相似性大于97%的菌株作為一個(gè)種[8],反之則為兩個(gè)。為比較幼體不同發(fā)育時(shí)期苗種培育池水細(xì)菌群落差異,計(jì)算Shannon多樣性指數(shù)[9]、Pielou均勻度指數(shù)[10]和S覬rensen相似性指數(shù)[11]。

2 結(jié)果與分析

2.1 扇貝苗種培育水中異養(yǎng)細(xì)菌計(jì)數(shù)

扇貝苗種培育水中異養(yǎng)細(xì)菌計(jì)數(shù)結(jié)果如表1所示,W1的細(xì)菌數(shù)量最少,W3的細(xì)菌數(shù)量最多。

2.2 扇貝苗種培育水中細(xì)菌群落組成分析

經(jīng)16S rRNA基因序列比對(duì),150株細(xì)菌歸屬于2門,即變形菌門Proteobacteria和厚壁菌門Fir-micutes;3綱,即酌變形菌綱酌-Proteobacteria、琢變形菌綱琢-Proteobacteria和芽孢桿菌綱Bacillus (圖1);6目,即交替單胞菌目Alteromonadales、弧菌目Vibrionales、海洋螺菌目Oceanospirillales、芽孢桿菌目Bacillales、紅桿菌目Rhodobacterales和鞘脂單胞菌目Sphingomonadales;9科,即假交替單胞菌科Pseudoalteromonadaceae、交替單胞菌科Alteromonadaceae、弧菌科Vibrionaceae、Marino-bacter_f、鹽單胞菌科Halomonadaceae、芽孢桿菌科Bacillaceae、希瓦氏菌科Shewanellaceae、紅桿菌科Rhodobacteraceae和赤桿菌科Erythrobacteraceae (圖2);10屬,即假交替單胞菌屬Pseudoaltero-monas、交替單胞菌屬Alteromonas、弧菌屬Vibrio、海桿菌屬M(fèi)arinobacter、Cobetia、芽孢桿菌屬Bacil-lus、希瓦氏菌屬Shewanella、亞硫酸桿菌屬Sulfito-bacter、Pseudophaeobacter和赤桿菌屬Erythrobacter (圖3)。

表1 扇貝苗種培育水中異養(yǎng)細(xì)菌數(shù)量Tab.1Heterotrophic bacterial counts in the water of scal-lop larva rearing tanks

圖1 扇貝苗種培育水中可培養(yǎng)細(xì)菌的群落組成(綱水平)Fig.1Culturable bacterial community composition in the water of scallop larva rearing tanks(in class)

從圖1可知,扇貝苗種培育水中酌-變形菌綱為優(yōu)勢(shì)綱,除W1外,其余4個(gè)時(shí)期水樣還含有芽孢桿菌綱,只有W4和W5中含有琢-變形菌綱。

交替單胞菌目為W1~W5中第一優(yōu)勢(shì)目,占總數(shù)的比例依次為83.33%、73.33%、73.33%、80.00%和56.67%。其中,弧菌目為W1(13.33%)、W2(16.67%)、W3(20.00%)和W5(16.67%) 4個(gè)時(shí)期水樣的第二優(yōu)勢(shì)目,芽孢桿菌目(10.00%)為W4的第二優(yōu)勢(shì)目,W5的優(yōu)勢(shì)菌目還有紅桿菌目(13.33%)。

從圖2可知:假交替單胞菌科是W1~W5中第一優(yōu)勢(shì)科;W1和W2的其余優(yōu)勢(shì)科依次為交替單胞菌科(23.33%和16.67%)和弧菌科(13.33%和16.67%),W3的其余優(yōu)勢(shì)科依次為弧菌科(20.00%)和交替單胞菌科(13.33%), W4的其余優(yōu)勢(shì)菌科依次為交替單胞菌科(13.33%)和芽孢桿菌科(10.00%),W5的其余優(yōu)勢(shì)菌科依次為弧菌科(16.67%)和紅桿菌科(13.33%)。

圖2 扇貝苗種培育水中可培養(yǎng)細(xì)菌的群落組成(科水平)Fig.2Culturable bacterial community composition in the water of scallop larva rearing tanks(in fam ily)

從圖3可知:假交替單胞菌屬為W1~W5中第一優(yōu)勢(shì)屬,所占比例依次為53.33%、56.67%、53.33%、66.67%和56.67%;W1的其余優(yōu)勢(shì)屬依次為交替單胞菌屬(23.33%)、弧菌屬(13.33%)和海桿菌屬(6.67%);W2的其余優(yōu)勢(shì)屬依次為交替單胞菌屬(16.67%)、弧菌屬(16.67%)和Cobetia(6.67%);W3的其余優(yōu)勢(shì)屬依次為弧菌屬(20.00%)、交替單胞菌屬(13.33%)和芽孢桿菌屬(6.67%);W4的其余優(yōu)勢(shì)屬依次為交替單胞菌屬(13.33%)、芽孢桿菌屬(10.00%)和弧菌屬(6.67%);W5的其余優(yōu)勢(shì)屬依次為弧菌屬(16.67%)、Pseudophae-obacter(10.00%)、芽孢桿菌屬(6.67%)和赤桿菌屬(6.67%)。

綜上所述,不同幼體發(fā)育時(shí)期苗種培育水中(W1~W5)異養(yǎng)細(xì)菌的群落組成存在差異,除各時(shí)期第一優(yōu)勢(shì)門(變形菌門)、綱(酌-變形菌綱)、目(交替單胞菌目)、科(假交替單胞菌科)和屬(假交替單胞菌屬)一致外,W4中第二優(yōu)勢(shì)目發(fā)生改變,W3和W5中第二優(yōu)勢(shì)科屬與其他3個(gè)水樣不同,W4和W5中第三優(yōu)勢(shì)科發(fā)生演替,W3、W4和W5中第三優(yōu)勢(shì)屬發(fā)生改變且各不相同。

所有菌株與數(shù)據(jù)庫中相關(guān)菌株的16S rRNA基因序列相似性為99%。按16S rRNA基因序列相似性大于97%的菌株歸于同一種計(jì)[9],150株細(xì)菌可歸為22個(gè)種。扇貝苗種培育水中種水平細(xì)菌群落組成見表2。

從Shannon指數(shù)和Pielou指數(shù)可以看出,幼體不同發(fā)育時(shí)期苗種培育水中細(xì)菌多樣性稍有變化,細(xì)菌種類分布較為均勻(表3)。S覬rensen指數(shù)表明,幼體不同發(fā)育時(shí)期苗種培育水中細(xì)菌群落存在差異,其中W5與W1、W2、W3和W4,W4與W1細(xì)菌群落差異較為明顯(表4)。

圖3 扇貝苗種培育水中可培養(yǎng)細(xì)菌的群落組成(屬水平)Fig.3Culturable bacterial community composition in the water of scallop larva rearing tanks(in ge-nus)

3 討論

3.1 扇貝苗種培育水中異養(yǎng)細(xì)菌數(shù)量及群落變化

隨著蝦夷扇貝幼體的生長發(fā)育,其苗種培育水中異養(yǎng)細(xì)菌數(shù)量不斷變化,受精卵期,水樣中細(xì)菌數(shù)量為102CFU/mL,從擔(dān)輪幼蟲期到稚貝期水中細(xì)菌數(shù)量穩(wěn)定在104CFU/mL。本試驗(yàn)中所得細(xì)菌數(shù)量與紫扇貝幼體養(yǎng)殖水中細(xì)菌數(shù)量[6]接近。大扇貝幼體發(fā)育早期階段(4~11 d),靜態(tài)養(yǎng)殖系統(tǒng)水中細(xì)菌群落略有波動(dòng),DGGE圖譜中優(yōu)勢(shì)條帶數(shù)目只有輕微變化[5]。tRFLP(限制性片段長度多態(tài)性)數(shù)據(jù)表明,太平洋牡蠣Crassostrea gigas苗種培育階段,不同天清潔海水(經(jīng)泡沫分離和過濾處理,加幼體或餌料之前)細(xì)菌群落顯著不同[1]。本研究中,蝦夷扇貝幼體不同發(fā)育時(shí)期苗種培育水中細(xì)菌群落組成存在差異,細(xì)菌群落演替的驅(qū)動(dòng)力之一可能是進(jìn)水細(xì)菌群落的變化[1,5],餌料(叉鞭金藻和新月菱形藻相關(guān)優(yōu)勢(shì)菌包括芽孢桿菌屬、希瓦氏菌屬、弧菌屬和假交替單胞菌屬等,未發(fā)表資料)和不同生長階段幼體攜帶細(xì)菌的群落變化也可能影響水體細(xì)菌群落的演替[12]。

表2 扇貝苗種培育水中可培養(yǎng)細(xì)菌的群落組成(種水平)Tab.2Culturable bacterial community com position in the water of scallop larva rearing tanks(in species)

表3 扇貝苗種培育水中細(xì)菌群落的Shannon指數(shù)和Pielou指數(shù)Tab.3Shannon and Pielou indices of bacterial community in the water of scallop larva rearing tanks

表4 扇貝苗種培育水中細(xì)菌群落的S覬rensen指數(shù)Tab.4S覬rensen index of bacterial community in the wa-ter of scallop larva rearing tanks

3.2 扇貝苗種培育水中細(xì)菌群落組成

在綱水平,蝦夷扇貝苗種培育水中優(yōu)勢(shì)綱為酌-變形菌綱,該結(jié)果與對(duì)大扇貝苗種培育系統(tǒng)水中相關(guān)細(xì)菌群落的研究結(jié)果一致[5],酌-變形菌綱也是紫扇貝幼體養(yǎng)殖水中的主要優(yōu)勢(shì)菌[6,13]。從蝦夷扇貝苗種培育水中還可分離出屬于芽孢桿菌綱和琢-變形菌綱的細(xì)菌,芽孢桿菌綱也存在于紫扇貝幼體養(yǎng)殖水中[6],琢-變形菌綱為太平洋牡蠣苗種培育用清潔海水中第一優(yōu)勢(shì)綱[1],也可在大扇貝苗種培育系統(tǒng)進(jìn)水口水樣中檢測(cè)到[5]。在屬水平,共分離到10個(gè)屬。其中假交替單胞菌屬、交替單胞菌屬和弧菌屬曾從包括蝦夷扇貝在內(nèi)的魚和貝類養(yǎng)殖場(chǎng)周圍海水中分離出[14]。大扇貝苗種培育系統(tǒng)水中可檢測(cè)到假交替單胞菌屬和弧菌屬[5]。從紫扇貝幼體養(yǎng)殖水中可分離出交替單胞菌屬、芽孢桿菌屬和海桿菌屬[6]。地中海牡蠣養(yǎng)殖海水中存在交替單胞菌屬/假交替單胞菌屬/希瓦氏菌屬群[4]。在近岸海水中均可發(fā)現(xiàn)Cobetia、亞硫酸桿菌屬、Pseudophaeobacter和Erythrobacter[15-18]。

已有研究表明,弧菌是扇貝幼體主要的致病菌[4,19-21]。在苗種培育過程中,蝦夷扇貝幼體死亡現(xiàn)象發(fā)生在D形幼蟲到殼頂幼蟲過渡期和整個(gè)殼頂幼蟲期。Liu等[4]通過人工感染試驗(yàn)證實(shí),燦爛弧菌是幼體大量死亡的致病菌。本試驗(yàn)中,從D形幼蟲期苗種培育水中分離出的燦爛弧菌可能是幼體病原菌的來源,因?yàn)橥瑫r(shí)進(jìn)行的高通量測(cè)序數(shù)據(jù)分析表明,該菌的豐度在擔(dān)輪幼蟲和D型幼蟲階段明顯高于受精卵、殼頂幼蟲和稚貝3個(gè)階段,而且幼體攜帶該菌的豐度是D型幼蟲和殼頂幼蟲階段,高于其他3個(gè)階段[22]。燦爛弧菌也是紫扇貝幼體大量死亡的病原菌[3]。Vibrio neocaledonicus在擔(dān)輪幼蟲、D型幼蟲和殼頂幼蟲期出現(xiàn),可能存在潛在致病性,該菌曾從弧菌病暴發(fā)的菲律賓蛤仔Ruditapes philippinarum幼體中分離出[23]。除弧菌外,假交替單胞菌屬的某些菌株也可能是潛在致病菌,如假交替單胞菌LT13菌株在攻毒試驗(yàn)中會(huì)導(dǎo)致10~16 d大扇貝幼體大量死亡[20]。另外,尤其要注意的是從水中分離出與人類病原菌炭疽桿菌Bacillus anthracis相似性為99%的菌株。因此,在扇貝苗種培育過程中應(yīng)十分重視對(duì)包括細(xì)菌在內(nèi)的水質(zhì)監(jiān)測(cè)。

本研究中,從扇貝苗種培育水中分離出的若干交替單胞菌、芽孢桿菌、弧菌、假交替單胞菌和Pseudophaeobacter arcticus,可能對(duì)扇貝幼體產(chǎn)生有益的影響。Douillet等[24]研究表明,交替單胞菌CA2菌株可提高太平洋牡蠣Crassostrea gigas幼體的存活率和生長率。麥?zhǔn)辖惶鎲伟?444菌株可顯著提高新西蘭綠唇貽貝Perna canaliculus幼體的存活率,并增強(qiáng)其對(duì)病原菌弧菌DO1菌株的抵抗力[25],該菌株和Phaeobacter gallaeciensis可保護(hù)大扇貝幼體免受病原菌燦爛弧菌和Vibrio coralliilyticus的感染,保護(hù)歐洲牡蠣Ostrea edulis幼體免受V.coralliilyticus和Vibrio pectenicida的感染[26]。Phaeobacter sp.S4或短小芽孢桿菌Bacillus pumilus RI06-95可以保護(hù)美洲牡蠣Crassostrea virginica幼體免受病原菌Vibrio tubiashii RE22和Roseovarius crassostreae CV919-312T攻毒導(dǎo)致的死亡[27]。含弧菌C33菌株、假單胞菌11菌株和芽孢桿菌B2菌株的混合菌能夠提高紫扇貝幼體存活率,使其在不使用抗生素的情況下順利度過浮游幼體階段[28]。弧菌OY15菌株可使美洲牡蠣幼體經(jīng)病原菌弧菌B183菌株攻毒后48 h存活率提高[29],并且無論是否用B183菌株攻毒,該菌株也能顯著提高美洲牡蠣在幼體變態(tài)過程中的存活率[30]。假交替單胞菌D41菌株可增強(qiáng)大扇貝幼體對(duì)燦爛弧菌感染的抵抗力,該菌株和P.gallaeciensis能保護(hù)太平洋牡蠣幼體免受V.coralliilyticus的感染[25]。本試驗(yàn)中分離的屬于交替單胞菌屬、芽孢桿菌屬、弧菌屬和假交替單胞菌屬和P.arcticus的菌株是否可作為益生菌應(yīng)用在蝦夷扇貝苗種培育中有待進(jìn)一步研究。

本研究表明,扇貝幼體不同發(fā)育時(shí)期苗種培育水中可培養(yǎng)細(xì)菌種類比較豐富,細(xì)菌群落結(jié)構(gòu)存在差異。

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Characterization of culturable bacterial diversity in the water of Yesso scallop Patinopecten yessoensisis larva rearing tanks

SUN Xue-ying1,LIU Ji-chen1,LIMing2,ZHAO Xue-wei2,LIANG Jun2,SUN Pi-hai3,MA Yue-xin1
(1.Key Laboratory ofMariculture and Stock Enhancement in North China蒺s Sea,Ministry of Agriculture,Dalian Ocean University,Dalian 116023,Chi-na;2.Zhangzidao Island Group Company Limited,Dalian 116001,China;3.Seafood Seedling Breeding Base,Dalian Ocean University,Dalian 116023,China)

This study was aimed to examine the culturable bacterial community in the water of larva rearing tanks during Yesso scallop Patinopecten yessoensisis larval development stages(fertilized egg W1,trochophora W2,D-shaped larvaeW3,umbo larvaeW4,and juvenile scallop W5).Thirty strainswere isolated from each water sample by Zobell2216E,and 150 bacterial strainswere identified by polymerase chain reaction and partial16S rRNA gene sequencing.Results showed thatall isolateswere found to be in 2 phyla,3 classes,6 orders,9 families,10 genera and 22 species.At phylum level,Proteobacteria was themost dominant phylum in 5 water samples.At genus lev-el,the first dominantgenus in all sampleswas Pseudoalteromonas,followed by Alteromonas,Vibrio and Marinobact-er atW1 stage,Vibrio,Alteromonas and Cobetia atW2 stage,Vibrio,Alteromonas and Bacillus atW3 stage,Altero-monas,Bacillus and Vibrio atW4 stage,and Vibrio,Bacillus,Pseudophaeobacter and Erythrobacter at W5 stage. The findings show that there are abundant bacterial species and different community structure in the water of larva rearing tanks across different larval development stages.

Patinopecten yessoensisis;water in a larva rearing tank;16S rRNA gene;bacterial community

S917.1

A

10.16535/j.cnki.dlhyxb.2017.03.011

2095-1388(2017)03-0323-06

2016-06-02

國家科技支撐計(jì)劃項(xiàng)目(2013BAD23B01);獐子島集團(tuán)資助項(xiàng)目(99801214)

孫雪瑩(1989—),女,碩士研究生。E-mail:sunxueying1109@163.com

馬悅欣(1963—),女,博士,教授。E-mail:mayuexin@dlou.edu.cn