南海石斑鱼苗种肠道微孢子虫病病原的鉴定
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摘要
研究通过组织病理分析、超微结构观察以及分子特征分析对石斑鱼(Epinephelus spp.)苗种肠道微孢子虫病病原进行了鉴定。其为一肠孢虫属新种,命名为石斑鱼肠孢虫(Enterospora epinepheli sp.n.),专性寄生于细胞核内,发育过程与肠孢虫属模式种黄道蟹肠孢虫(Enterospora canceri)一致。早期单核裂殖体通过一层简单的电子薄膜与宿主细胞核质隔离。随后,单核裂殖体发育形成多核裂殖原质团。此时,细胞核出现明显肥大,有的甚至被裂殖子胀破。裂殖原质团进一步发育形成多核产孢体,并开始出现许多高电子密度的囊泡状结构。这些与极丝及锚状盘有关的囊泡状结构聚集在藕核周围,并组装形成微孢子虫特征性结构(挤出装置)前体。随后,产孢体原生质团通过连续分裂形成一个个孢子母细胞。孢子母细胞与细胞核直接接触,并直接发育形成成熟孢子。成熟孢子椭圆形,孢子长(1.56±0.31)μm(1.07—1.96μm),宽(1.08±0.98)μm(0.93—1.28μm)。孢壁分为3层,外壁电子密度高,厚(15.51±0.95)nm(9.87—26.18 nm),内壁为电子透明层,较外层更厚(81.13±2.71)nm(57.16—110.81 nm),最里面为孢质膜。极丝为同型极丝,共5—6圈,分2排排列。组织病理学分析发现该微孢子虫寄生于肠道上皮杯状细胞核内,肠壁脱落的内容物中也发现大量的微孢子虫。序列比对发现该种与之前报道的石斑鱼肠道微孢子虫待定种(Microsporidium sp.)序列基本一致,与其他相似性较高的种类的遗传距离在0.162—0.225。系统发育关系分析显示肠胞虫科的种类明显分为两支,石斑鱼肠孢虫和肠孢虫属其他种类及毕氏肠胞虫聚为一个独立分支,但不与该分枝中任何种类形成姊妹支。
The enteric microsporidiosis of hatchery-bred juvenile grouper, Epinephelus spp., is the most important in the mariculture area off coast of South China Sea in recent years, however, the taxonomy of the causative agent remains unknown. In this study, histopathological, ultrastructural and molecular evidences were provided to identify the aetiological agent, designated herein as Enterospora epinepheli sp. n.. The intranuclear development of the present species was consistent with Enterospora canceri, the type species of Enterospora genus. The early stages of uninucleate meronts were observed within the infected nuclei, separating from the host nucleus by a simple electron dense membrane. Later, the uninucleate meronts transformed into multinucleate plasmodia(merogonial plasmodia). At this stage,the infected nuclei were hypertrophic, or even ruptured by the multinucleate plasmodia. Sporogonial plasmodia were characterized by the appearance of multiple, small, spherical, membrane-bound vesciles. Then, multiple copies of these membrane-bound vesciles developed into the precursors of the polar filament and anchoring disk of mature spore which surrounded the diplokaryotic nuclei. With the development, sporoblasts separated from the plasmodia by successive division and direct development to mature spores. Mature spores were oval, in direct contact with host nucleus. Spores measured 1.56±0.31(1.07—1.96) μm in length and 1.08±0.98(0.93—1.28) μm in width. The spore walls were trilaminar, including an electron dense exospore coat [15.51±0.95 nm(9.87—26.18 nm) thick], surrounding a thick electron lucent endospore [(81.13±2.71) nm(57.16—110.81 nm) thick] and the plasma membrane. The polar filaments were isofilar, coiled with 5—6 turns in two rows. Histopathological analysis clearly revealed that the spores located in the nucleus of goblet cell of the intestinal epithelial and a large amount of spores appeared in the intestinal contents with the shedding necrotic infected enterocytes. Molecular analysis indicated that genetic distances of Enterospora epinepheli sp. n. form the species of Enterocytozoondiae ranged from 0.162 to 0.225 which was generally out of intraspecies variation. Phylogenetic analysis revealed that the species of Enterocytozoondiae could separate into Clade Ⅰand CladeⅡ, and Enterospora epinepheli sp. n. was an independent lineage, clustering with Enterospora hepatopenaei, E. nucleophilia, E. canceri and Enterocyozoon bieneusi within the Clade Ⅱ.
The enteric microsporidiosis of hatchery-bred juvenile grouper, Epinephelus spp., is the most important in the mariculture area off coast of South China Sea in recent years, however, the taxonomy of the causative agent remains unknown. In this study, histopathological, ultrastructural and molecular evidences were provided to identify the aetiological agent, designated herein as Enterospora epinepheli sp. n.. The intranuclear development of the present species was consistent with Enterospora canceri, the type species of Enterospora genus. The early stages of uninucleate meronts were observed within the infected nuclei, separating from the host nucleus by a simple electron dense membrane. Later, the uninucleate meronts transformed into multinucleate plasmodia(merogonial plasmodia). At this stage,the infected nuclei were hypertrophic, or even ruptured by the multinucleate plasmodia. Sporogonial plasmodia were characterized by the appearance of multiple, small, spherical, membrane-bound vesciles. Then, multiple copies of these membrane-bound vesciles developed into the precursors of the polar filament and anchoring disk of mature spore which surrounded the diplokaryotic nuclei. With the development, sporoblasts separated from the plasmodia by successive division and direct development to mature spores. Mature spores were oval, in direct contact with host nucleus. Spores measured 1.56±0.31(1.07—1.96) μm in length and 1.08±0.98(0.93—1.28) μm in width. The spore walls were trilaminar, including an electron dense exospore coat [15.51±0.95 nm(9.87—26.18 nm) thick], surrounding a thick electron lucent endospore [(81.13±2.71) nm(57.16—110.81 nm) thick] and the plasma membrane. The polar filaments were isofilar, coiled with 5—6 turns in two rows. Histopathological analysis clearly revealed that the spores located in the nucleus of goblet cell of the intestinal epithelial and a large amount of spores appeared in the intestinal contents with the shedding necrotic infected enterocytes. Molecular analysis indicated that genetic distances of Enterospora epinepheli sp. n. form the species of Enterocytozoondiae ranged from 0.162 to 0.225 which was generally out of intraspecies variation. Phylogenetic analysis revealed that the species of Enterocytozoondiae could separate into Clade Ⅰand CladeⅡ, and Enterospora epinepheli sp. n. was an independent lineage, clustering with Enterospora hepatopenaei, E. nucleophilia, E. canceri and Enterocyozoon bieneusi within the Clade Ⅱ.
引文
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[18]Stentiford G D,Bateman K S,Longshaw M,et al.Enterospora canceri n.gen.,n.sp.,intranuclear within the hepatopancreatocytes of European edible crab Cancer pagurus[J].Diseases of Aquatic Organism,2007,75:61-72
[19]Bojko J,Clark F,Bass D,et al.Parahepatospora carcini n.gen.,n.sp.,a parasite of invasive Carcinus maenas with intermediate features of sporogony between the Enterocytozoon clade and other microsporidia[J].Journal of Invertebrate Pathology,2017,143:124-134
[20]Stentiford G D,Bateman K S,Dubuffet A,et al.Hepatospora eriocheir(Wang and Chen 2007)gen.et comb.Nov.infecting invasive Chinese mitten crabs(Eriocheri sinensis)in Europe[J].Journal of Invertebrate Pathology,2011,108:156-166
[21]Tourtip S,Wongtripop S,Stentiford G D,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporidia:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationship[J].Journal of Invertebrate Pathology,2009,102:21-29
[22]Boakye D W,Jarpenlak P,Prachumwat A,et al.Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia[J].Environmental Microbiology,2017,19(5):2077-2089
[2]Diamant A,Goren M,Yokes M B,et al.Dasyatispora levantinae gen.et sp.nov.,a new microsporidian parasite from the common stingray Dasyatis pastinaca in the eastern Mediterranean[J].Diseases of Aquatic Organism,2010,91(2):137-150
[3]Lin H R.The establishment of grouper cultural technology and the ideas for sustained development of grouper aquaculture industry[J].Journal of Fujian Fisheries,2012,34(1):1-10[林浩然.石斑鱼鱼类养殖技术体系的创建和石斑鱼养殖产业持续发展的思考.福建水产,2012,34(1):1-10]
[4]Abdel-Ghaffar F,Abdel-Rahman B,Mehlhorn H,et al.Microsprodian parasites:a danger facing marine fishes ofthe Red sea[J].Parasitology Research,2011,108219-225
[5]Azevedo C,Abdel-Baki A A S,Rocha S,et al.Ultrastructure and phylogeny of Glugea Arabica n.sp.(Microsporidia)infecting the marine fish Epinephelus polyphekadion from the Red sea[J].European Journal of Protistology,2016,52:11-21
[6]Wu H B,Wu Y S,Wu Z H.Occurrence of a new Microsporidium in the abdominal cavity of Epinephelus akaara[J].Acta Hydrobiologica Sinica,2005,29(2):150-154[吴后波,吴英松,吴灶和.微孢子虫一新种的描述及分子鉴定.水生生物学报,2005,29(2):150-154]
[7]Xu L W,Liu X H,Zhang J Y,et al.Outbreak of enteric microsporidiosis of hatchery-bred juvenile groupers,Epinephelus spp.,associated with a new intranuclear microsporidian in China[J].Journal of Fish Diseases,2016,40(2):183-189
[8]Hall T A.Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows95/98/NT[J].Nucleic Acids Symposium Seriese,1999,41:95-98
[9]Thompson J D,Gibson T J,Plewniak F,et al.The CLUSTAL-X window interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25:4876-4882
[10]Posada D.j Model Test:phylogenetic model averaging[J].Molecular Biology and Evolution,2008,25:1253-1256
[11]Guindon S,Dufayard J F,Lefort V,et al.New algorithms and methods to estimate maximum likelihood phylogenies:assessing the performance of Phy ML 3.0[J].Systematic Biology,2010,59:307-321
[12]Ronquist F,Huelsenbeck J P.Mr Bayes 3:Bayesian phylogenetic inference under mixed models[J].Bioinformatics,2003,19:1572-1574
[13]Page R D M.TREEVIEW:an application to display phylogenetic trees on personal computers[J].Computer Application in Biosciences:CABIOS,1996,12:357-358
[14]Tamura K,Stecher G,Peterson D,et al.MEGA 6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30(12):2725-2729
[15]Weiss L M,Becnel J J.Microsporidia pathogenes of opportunity[A].In:Elizabeth S D(Eds.),Mammlian Animal Model of Human Microsporidiosis[C].Wiley online.2014,327-340
[16]Weiss L M,Becnel J J.Microsporidia pathogenes of opportunity[A].In:Stentiford G D,Dunn A M(Eds.),Microsporidia in Aquatic Invertebrates[C].Wiley online.2014,579-604
[17]Palenzuela O,Redondo M J,Cali A,et al.A new intranuclear microsporidium,Enterospora nucleophile n.sp.,causing an emaciative syndrome in a piscine host(Sparus aurata),prompts the redescription of family Enterocytozoonidae[J].International Journal of Parasitology,2014,44:189-203
[18]Stentiford G D,Bateman K S,Longshaw M,et al.Enterospora canceri n.gen.,n.sp.,intranuclear within the hepatopancreatocytes of European edible crab Cancer pagurus[J].Diseases of Aquatic Organism,2007,75:61-72
[19]Bojko J,Clark F,Bass D,et al.Parahepatospora carcini n.gen.,n.sp.,a parasite of invasive Carcinus maenas with intermediate features of sporogony between the Enterocytozoon clade and other microsporidia[J].Journal of Invertebrate Pathology,2017,143:124-134
[20]Stentiford G D,Bateman K S,Dubuffet A,et al.Hepatospora eriocheir(Wang and Chen 2007)gen.et comb.Nov.infecting invasive Chinese mitten crabs(Eriocheri sinensis)in Europe[J].Journal of Invertebrate Pathology,2011,108:156-166
[21]Tourtip S,Wongtripop S,Stentiford G D,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporidia:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationship[J].Journal of Invertebrate Pathology,2009,102:21-29
[22]Boakye D W,Jarpenlak P,Prachumwat A,et al.Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia[J].Environmental Microbiology,2017,19(5):2077-2089