李 鵬 習(xí)丙文, 陳 凱 謝 駿

(1. 南京農(nóng)業(yè)大學(xué)無錫漁業(yè)學(xué)院, 無錫 214081; 2. 中國水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心, 農(nóng)業(yè)部淡水漁業(yè)和種質(zhì)資源利用重點實驗室, 無錫 214081; 3. 淡水水產(chǎn)健康養(yǎng)殖湖北省協(xié)同創(chuàng)新中心, 武漢 430070)

鯉腸道寄生岳陽碘泡蟲新種的形態(tài)特征及系統(tǒng)發(fā)育分析

李 鵬1,2習(xí)丙文2,3陳 凱2謝 駿1

(1. 南京農(nóng)業(yè)大學(xué)無錫漁業(yè)學(xué)院, 無錫 214081; 2. 中國水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心, 農(nóng)業(yè)部淡水漁業(yè)和種質(zhì)資源利用重點實驗室, 無錫 214081; 3. 淡水水產(chǎn)健康養(yǎng)殖湖北省協(xié)同創(chuàng)新中心, 武漢 430070)

在洞庭湖岳陽地區(qū)開展魚類寄生蟲調(diào)查中，發(fā)現(xiàn)一種寄生于鯉Cyprinus carpioL.腸道的黏孢子蟲。該黏孢子蟲的孢囊呈白色，橢圓形，大小為(1.0±0.2) mm (0.8—1.2 mm)。成熟孢子具有殼瓣，殼面觀近似圓形，后端有4—6個“V”形褶皺；縫面觀呈紡錘形，縫脊直而粗；孢質(zhì)均勻，含有一個嗜碘泡；孢子長(9.8±0.6) μm (9.6—10.0 μm)，孢子寬(8.2±0.3) μm (8.0—8.5μm)，孢子厚(7.3±0.1) μm (7.0—7.5 μm)；2個極囊梨形，位于孢子頂端，大小相等，呈“八”字形；極囊長(4.4±0.4) μm (3.8—5.1 μm)，寬(2.7±0.2) μm (2.2—3.2 μm)，極絲4—5圈。該黏孢子蟲與腸膜碘泡蟲、丑陋圓形碘泡形態(tài)特征非常相似，但其極囊/孢子小于1/2；與文獻已報道的鯉腸道寄生北京碘泡蟲和鯉腸碘泡蟲相比較，其在孢子形態(tài)、孢子和極囊大小方面分別存在明顯差異。基于該黏孢子蟲18S rDNA基因序列(GenBank登錄號KY203795)比對分析，該黏孢子蟲與山東碘泡蟲相似率最高，僅為96%。系統(tǒng)發(fā)育分析發(fā)現(xiàn)，該黏孢子蟲與山東碘泡蟲、倪李碘泡蟲、住心碘泡蟲、Myxobolus encephalicus、Sphaerospora molnari、多涅茨尾孢蟲和Henneguya zikaweiensis聚為獨立分支，和其他已報道的黏孢子蟲親緣關(guān)系較遠。綜合形態(tài)學(xué)和18S rDNA基因序列數(shù)據(jù)，文章報道的鯉腸道寄生黏孢子蟲為碘泡蟲屬一新物種，將其命名為岳陽碘泡蟲。

鯉; 碘泡蟲; 分類; 系統(tǒng)發(fā)育

黏體動物(Myxozoa)是一種形態(tài)結(jié)構(gòu)非常簡單、個體非常微小、高度適應(yīng)寄生生活的后生動物, 在進化上與刺胞動物親緣關(guān)系較近[1—3], 可寄生在魚類、兩棲類、爬行類、鳥類及哺乳類動物各個組織器官[4,5], 是水生動物養(yǎng)殖中非常重要的寄生蟲病原。目前全世界報道的黏孢子蟲隸屬于60個屬, 約2600多種[4,6]。在黏孢子蟲傳統(tǒng)分類學(xué)研究中主要依據(jù)成熟孢子的形態(tài)特征進行種類鑒定、分類階元劃分[7]。然而, 黏孢子蟲種類繁多, 結(jié)構(gòu)簡單, 單從形態(tài)上很難區(qū)分一些相似的物種, 所以在種類鑒定中普遍存在一些同物異名或者異物同名的問題[7,10]。隨著對黏孢子蟲的研究不斷深入, 宿主和寄生部位特異性特征被逐漸發(fā)現(xiàn)并廣泛應(yīng)用到新的分類鑒定中[8—11]。近年來, 分子生物學(xué)技術(shù)在黏孢子蟲分類學(xué)研究中的得到廣泛的運用,在種類準確鑒定和闡明類群間系統(tǒng)親緣關(guān)系的研究中發(fā)揮重要作用[12]。當前, 基于黏孢子蟲形態(tài)特征和基因標記系統(tǒng)分析(如18S rRNA)相結(jié)合的方法進行新種的厘定成為主流[13], 對深入揭示我國魚類黏孢子蟲多樣性有重要意義。

鯉Cyprinus carpioL.是東亞地區(qū)重要的養(yǎng)殖品種, 在中國已經(jīng)有4000多年的養(yǎng)殖歷史[14]。截至目前已經(jīng)報道的寄生于鯉不同組織器官的黏孢子蟲種類約75種[6]。其中一些種類可造成養(yǎng)殖魚苗和成魚的大量死亡[15,16], 如引起鯉魚養(yǎng)殖中腸道“巨囊病”的吉陶單極蟲(Thelohanellus kitauei), 大量寄生于鯉鰓絲造成宿主死亡的野鯉碘泡蟲(Myxobolus koi)。我國魚類物種多樣性高、分布廣泛, 隨著魚類寄生蟲調(diào)查的深入開展, 一些新的寄生黏孢子蟲種類不斷被發(fā)現(xiàn)和報道。作者在進行黏孢子蟲調(diào)查和分類研究過程中, 從洞庭湖鯉腸道中發(fā)現(xiàn)一種黏孢子蟲新種, 本文詳細描述和分析了該黏孢子蟲的形態(tài)特征和系統(tǒng)發(fā)育關(guān)系。

1 材料與方法

1.1 樣品采集

2015年9月在湖南岳陽洞庭湖區(qū)魚碼頭購買野生鯉進行寄生蟲調(diào)查, 按照《魚病診斷與防治手冊》[17]檢查每一條魚的體表、鰓絲、口腔、腸道,將取得的孢囊保存于裝有100%酒精的1.5 mL離心管中, 標注時間、地點、寄生部位, 帶回實驗室。

1.2 形態(tài)學(xué)檢查

用滅菌的解剖針刺破孢囊, 滴少量蒸餾水, 并且用移液槍吸取5 μL黏孢子蟲白色漿液均勻涂抹于載玻片上, 用Olympus CX-31顯微鏡100倍油鏡檢查。黏孢子蟲形態(tài)采用圖森(TAC-9.0C)圖像軟件進行拍攝。參考《中國動物志(黏體門: 黏孢子綱)》[6]形態(tài)數(shù)據(jù)測量標準, 隨機選取25個黏孢子蟲孢子個體, 采用軟件自帶測量工具進行形態(tài)特征數(shù)據(jù)測量。

1.3 18S rDNA基因序列擴增

從100%酒精中取出保存的孢囊, 置于2 mL離心管中加入200 μL蒸餾水復(fù)水, 共換水3次。基因組DNA提取采用Mag-MK磁珠法(磁珠法深加工產(chǎn)品基因組DNA抽提試劑盒, 上海生工)。18S rDNA基因序列片段擴增采用引物ERI-B1 (ACCTGGT TGATCCTGCCAG)和ERI-B10 (CCTCCGCAG GTTCACCTACGG)[9]。PCR反應(yīng)體系(50 μL)組成為10×ExTaqBuffer 5 μL, dNTP Mix 4 μL, 正反向引物(10 μmol/L) 2 μL, 模板3 μL, TaKaRa ExTaq(5 U/μL) 0.3 μL, 用RNase-free Water將體系補加到50 μL。反應(yīng)程序設(shè)置為94℃預(yù)變性5min; 94℃變性30s, 56℃退火45s, 72℃延伸60s, 30個循環(huán); 72℃末端延伸7min。PCR陽性產(chǎn)物送到生工(上海)生物工程股份有限公司測序。

1.4 序列分析

樣品測序數(shù)據(jù)采用SeqMan (Lasergen package,DNAStar Inc.)生物軟件檢查和拼接。采用Blastn(http://blast.ncbi.nlm.nih.gov/)在GenBank數(shù)據(jù)庫中檢索同源序列。序列多重比對采用CLUSTAL 1.8(默認設(shè)置)。序列一致性和DNA堿基替換最佳模型分析采用MEGA7.0軟件包Modeltest。序列系統(tǒng)進化分析分別采用最大似然法(Maximum likelihood, ML)和貝葉斯法(Bayesian Inferences, BI)。最大似然法模型參數(shù)設(shè)置為(T93+G+I), 分支節(jié)點的支持率采用Bootstrop 1000次檢驗; 核苷酸頻率為(A=0.2651、C=0.2349、G=0.2350、T=0.2650); 6種核苷酸替換率分別為[AC]=0.081, [AG]=0.338,[AT]=0.086, [CG]=0.076, [CT]=0.338, [GT]=0.081;不變位點的比例為0.3872, 伽馬分布值為0.3825。貝葉斯法分析在MrBayes (Ronquist and Huelaenbeck 2003)軟件進行(參數(shù)設(shè)置為nruns=4, nst=6,rates=invagamma, ngen=5000000), 每100代對系統(tǒng)樹進行抽樣, 以后驗概率來表示分支的可信性。

2 結(jié)果

2.1 岳陽碘泡蟲, 新種Myxobolus yueyangensis sp. nov. (圖1)

孢囊特征: 孢囊呈白色, 橢圓形, 寄生鯉魚腸道肌層組織(圖2), 直徑約為1.0 ± 0.2 (0.8—1.2) mm(n=10)。

圖1 岳陽碘泡蟲(殼面、縫面, 比例尺=5 μm)Fig. 1 The schematic drawing of Myxobolus yueyangensis sp.nov., scale bar=5 μm

圖2 岳陽碘泡蟲寄生于腸道組織切片(比例尺=500 μm)Fig. 2 Histopathological section of intestine of common carp infected by Myxobolus yueyangensis, scale bar=500 μm

孢子特征: 成熟孢子具有碘泡蟲屬典型特征,殼面觀近似圓形, 前端稍尖(圖1), 殼片表面光滑,后端有4—6個“V”形褶皺(圖3a), 囊間突小而明顯,孢質(zhì)均勻, 含有一個嗜碘泡; 縫面觀呈紡錘形(圖1、 圖3b), 縫脊直而粗(圖3c); 孢子長9.8±0.6 (9.6—10.0) μm, 孢子寬8.2±0.3 (8.0—8.5) μm; 孢子厚7.3±0.1 (7.0—7.5) μm; 2個極囊梨形, 大小相同, 稍微呈“八”字形叉開并列于孢子前段, 在其頂端存在一個半圓形凸起(圖3d); 極囊長4.4±0.4 (3.8—5.1) μm,極囊寬2.7±0.2 (2.2—3.2) μm, 極絲圈數(shù)4—5圈。

圖3 岳陽碘泡蟲在顯微鏡下的孢子形態(tài), 比例尺=5 μmFig. 3 Spores of Myxobolus yueyangensis under light microscope,scale bar=5 μm

宿主: 鯉Cyprinus carpioL. (Cyprinidae)

寄生部位: 腸道

采樣地點: 湖南岳陽洞庭湖區(qū) (29°37′N,113°09′E)

感染率: 20% (2/10)

模式標本分子序列: GenBank序列收錄號KY203795。

樣本保存: 100%酒精固定標本保存于中國水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心魚病研究室, 標本號MYY201509111, MYY201509112。

種名詞源: 該黏孢子蟲發(fā)現(xiàn)于岳陽(Yueyang),故命名為岳陽碘泡蟲(Myxobolus yueyangensis)。

2.2 18S rDNA基因序列分析

采用黏孢子蟲通用引物成功擴增出18S rDNA基因序列片段2051 bp。該序列在NCBI中BLAST分析, 與以下幾個種類相似性最高: 山東碘泡蟲(Myxobolus shantungensis, KJ725079) 96% 、倪李碘泡蟲(Myxobolus nielii, JQ690358) 95%、住心碘泡蟲(Myxobolus hearti, GU574808) 95%、Myxobolus encephalicus(JQ801549) 95%。

系統(tǒng)發(fā)育分析表明該黏孢子蟲與山東碘泡蟲、倪李碘泡蟲、住心碘泡蟲、Myxobolus encephalicus、Sphaerospora molnari(AF378345)、多涅茨尾孢蟲(Henneguya doneci, LC011456)和Henneguya zikaweiensis(KR020026)親緣關(guān)系較近, 聚為穩(wěn)定的獨立分支。該分支類群中的種類分別寄生在鯽、鯉不同器官, 沒有呈現(xiàn)出一致的寄生器官特異性。

3 討論

碘泡蟲屬物種非常豐富, 目前已報道的種類多達856[18,19]種, 隨著研究范圍的擴大和調(diào)查強度的提高, 一些新的種類不斷被報道。近年來國內(nèi)學(xué)者在鯽(Carassius auratus)中陸續(xù)發(fā)現(xiàn)和命名多個碘泡蟲新種類, 如: 丑陋圓形碘泡蟲(Myxobolus turpisrotundus)、洪湖碘泡蟲(Myxobolus honghuensis)、射陽碘泡蟲(Myxobolus sheyangensis)、Myxobolus pronini等[20—23]。本文描述了1種寄生在鯉魚腸道的碘泡蟲屬新種, 岳陽碘泡蟲。該碘泡蟲與文獻記錄的鯉科魚類腸道寄生碘泡蟲或者其他碘泡蟲種類都有明顯的形態(tài)特征差異(表1)。岳陽碘泡蟲與寄生于雅羅魚腸黏膜層的Myxobolus gayeae、草魚腸道的餅形碘泡蟲(Myxobolus artus)、鯉魚腸道的北京碘泡蟲(Myxobolus pekingensis)和鯉腸碘泡蟲(Myxobolus cyprinicola)的孢子形態(tài)較為近似。然而,M. gayeae、北京碘泡蟲在孢子和極囊大小方面明顯大于岳陽碘泡蟲, 餅形碘泡蟲的則明顯小于岳陽碘泡蟲。此外, 岳陽碘泡蟲的極絲盤繞圈數(shù)明顯少于M. gayeae、北京碘泡蟲和鯉腸碘泡蟲。18S rDNA基因序列分析進一步表明岳陽碘泡蟲與Gen-Bank已登錄黏孢子蟲都不一致, 其中與山東碘泡蟲的序列相似率較高(96%), 但遠低于種內(nèi)相似水平。因此綜合形態(tài)學(xué)和DNA18S rDNA基因序列分析, 本文所報道的鯉腸道寄生黏孢子蟲應(yīng)為碘泡蟲屬一新物種。

基于18S rDNA基因序列的系統(tǒng)發(fā)育分析表明本文報道的岳陽碘泡蟲與山東碘泡蟲、M. encephalicus、倪李碘泡蟲、住心碘泡蟲親緣關(guān)系較近(圖4), 然而, 它們在孢子、極囊大小和形態(tài)及極絲圈數(shù)方面明顯不同[25,27—29]。岳陽碘泡蟲所處的分支譜系中(圖4), 倪李碘泡蟲(M. nielii)、多涅茨尾孢蟲(H. doneci)、住心碘泡蟲(M. hearti)、莫氏球孢蟲(S. molnari)和Henneguya zikaweiensis分別寄生于鯽鰓和心臟組織,M. encephalicus和M. shantungensis分別寄生于鯉腦和鰓組織。在該分支譜系中來自鯽、鯉的黏孢子蟲分別具有較近的親緣關(guān)系,并且呈現(xiàn)一定的宿主特異性。

表1 岳陽碘泡蟲與相似種類形態(tài)特征比較Tab. 1 The measurement comparison of spore of Myxobolus yueyangensis sp. nov. with other morphologically similar species from fish intestine

圖4 基于18S rDNA基因序列構(gòu)建的鯉科魚類寄生黏孢子蟲貝葉斯法(BI)系統(tǒng)發(fā)育樹Fig. 4 Phylogenetic tree of Myxozoans based on 18S rDNA sequences based on the Bayesian Inferences (BI) method

在黏孢子蟲傳統(tǒng)形態(tài)分類系統(tǒng)中, 孢子后端的尾突是碘泡科尾孢蟲屬和碘泡蟲屬的重要區(qū)分特征[25]。近年來, 分子系統(tǒng)發(fā)育分析發(fā)現(xiàn)尾孢蟲屬和碘泡蟲屬的種類經(jīng)常交織在不同的分支類群中, 傳統(tǒng)形態(tài)分類類群沒有準確的反映出它們真實進化親緣關(guān)系[30—32]。在本文構(gòu)建的系統(tǒng)進化樹中, 多涅茨尾孢蟲、H. zikaweiensis與山東碘泡蟲、M. encephalicus、倪李碘泡蟲和住心碘泡蟲穩(wěn)定的聚為獨立分支(圖4)。在Bahri[33]和El-Mansy[34]發(fā)現(xiàn)M.bizerti與M. heteromorphs孢子基部存在與尾孢蟲相似的尾突, 從而對尾突作為區(qū)分碘泡蟲屬和尾孢蟲屬分類特征的有效性提出質(zhì)疑。Liu等[20]在種丑陋圓形碘泡蟲(Myxobolus turpisrotundus)同一個孢囊中發(fā)現(xiàn)存在少數(shù)的碘泡蟲孢子基部具有細小的尾突, 并對兩種形態(tài)的碘泡蟲分別測序, 序列相似性達到99.9%, 證明了這2種不同形態(tài)的孢子屬于同一物種的不同發(fā)育階段。截止目前已經(jīng)報道的發(fā)現(xiàn)具有尾突的碘泡蟲數(shù)量較少, 而引入物種的數(shù)量對系統(tǒng)發(fā)育樹的進化樹分析結(jié)果有著較大的影響, 尾突能否作為區(qū)分碘泡蟲屬和尾孢蟲屬的重要依據(jù)還需發(fā)現(xiàn)和引入更多的物種加以驗證。

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MORPHOLOGICAL AND MOLECULAR CHARACTERISTIC OF MYXOBOLUS YUEYANGENSIS SP. NOV. (MYXOZOA: MYXOBOLIDAE) FROM THE INTESTINE OF COMMON CARP (CYPRINUS CARPIO) IN CHINA

LI Peng1,2, XI Bing-Wen2,3, CHEN Kai2and XIE Jun1
(1. Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China; 2. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Science, Wuxi 214081, China; 3. Hubei Collaborative Innovation Center for Freshwater Aquaculture, Wuhan 430070, China)

Myxobolus yueyangensissp. nov. parasitic in the intestine muscularis of common carpCyprinus carpiowith the form of round or ellipsoidal plasmodia, was found during a survey on fish myxozan diversity in Yueyang(29°37′N, 113°09′E). Mature spores were suborbicular in frontal view, and fusiform in back view, measuring 9.8±0.6(9.6—10) μm long, 8.2±0.3 (8.0—8.5) μm wide, and 7.3±0.1 (7.0—7.5) μm thick. The surface of spores was smooth,and a round iodinophilous vacuole was found in its uniform protoplasm. Two Polar capsules were pyriform with an apophysis at its top and end closed to apex of spore, measuring 4.4±0.4 (3.8—5.1) μm in length, 2.7±0.2 (2.2—3.2) μm in width. Polar filaments coiled 4 to 5 turns.M. yueyangensisis morphologically distinct from all otherMyxobolusspecies. The BLAST search indicated that the newly obtained small subunit ribosomal RNA (ssrRNA) gene sequence did not match any available sequences in GenBank, and showed the high similarity withMyxobolus yueyangensisof 96%.In the phylogenetical tree,M. yueyangensiswas closely clustered withMyxobolus yueyangensis. Based on the comparison and analysis of spore morphology, host specificity, tissue or organ parasitized specificity, and 18S rDNA sequence, the myxosporean from the intestine ofC. carpiodescribed in the study should be a new species of genusMyxobolus.

Common carp;Myxobolus; Intestine; 18S rDNA

S941.5

A

1000-3207(2017)06-1251-06

2016-12-13;

2017-05-10

國家自然科學(xué)基金(31302222); 現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項資金(CARS-45)資助 [Supported by the National Natural Sciences Foundation of China (31302222); the Earmarked Fund for China Agriculture Research System (CARS-45)]

李鵬(1992—), 男, 江蘇南通人; 碩士研究生; 研究方向為魚類寄生蟲。E-mail: 1574944091@qq.com

習(xí)丙文, E-mail: xibw@ffrc.cn

10.7541/2017.155