凡纳滨对虾感染虾肝肠胞虫的群体及组织间差异性分析
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摘要
对来自河北黄骅(HH)、山东平度(PD)、江苏吴江(WJ)和山东日照(RZ)的4个凡纳滨对虾(Litopenaeus vannamei)群体进行了对虾生长参数测量,用Taq Man q PCR检测了凡纳滨对虾各群体的肝胰腺组织中和RZ群体多种组织中的虾肝肠胞虫数量(Amount of Enterocytozoon hepatopenaei,EHP)。结果显示,在主要生长相关参数中,RZ群体最优,该群体EHP载量也最低。不同群体的样本数EHP对数直方图的模式存在差异,HH和PD群体的EHP对数呈双峰分布,而WJ和RZ群体的EHP对数呈单峰分布,代表EHP在不同群体中可能存在不同的传播模式。EHP对数呈单峰分布的群体或从多峰分布的群体中分离出的高EHP对数子群体的对虾体长或体重与EHP对数呈显著的负相关。RZ群体中,各个体不同组织中EHP从高到低的顺序依次是肝胰腺>中肠>血淋巴>鳃>肌肉。肝胰腺、中肠和鳃3个组织中EHP对数相互间的相关性为99.9%的极显著水平(P<0.001);除了中肠与血淋巴和肝胰腺与血淋巴以外,其余组织间EHP对数的相关性也达到极显著(P<0.01)或显著(P<0.05)水平。用DIG标记的EHP探针对肝胰腺、肌肉、鳃、肠道组织的原位杂交显示,肝胰腺是主要的EHP感染组织,其他组织中杂交信号较弱,但各组织中有少数细胞的EHP易感。
Growth-related parameters of individuals from four populations of Litopenaeus vannameifrom Huanghua of Heibei Province(HH), Pingdu of Shandong Province(PD), Wujiang of Jiangsu Province(WJ), and Rizhao of Shandong Province(RZ) were assessed. Amount of Enterocytozoon hepatopenaei(EHP) in the hepatopancreas of individuals from all populations and EHP in multiple tissues of shrimp from the RZ population were assessed with Taq Man-based quantitative PCR. The results showed that the RZ population possessed optimal growth-related parameters and the lowest EHP among the four populations. The histograms of case-logarithmic EHPs of the four populations presented different modes. The case distribution of logarithmic EHPs from the HH and PD populations showed double peaks, while those of the WJ and RZ populations showed a single peak. The different distribution modes may indicate a different EHP spread in the four populations. The population with a single peak mode or the higher logarithmic EHP subpopulation isolated from the population with a multiple peak mode showed a significant negative correlation with shrimp body length or body weight to logarithmic EHP. The EHP detected in different tissues of the RZ population followed the order(from highest to lowest), EHP in hepatopancreas > EHP in midgut > EHP in hemolymph > EHP in gills > EHP in muscle. The logarithmic EHP in the hepatopancreas–midgut, hepatopancreas–gills, and midgut–gills had a significant correlation level above 99.9%(P<0.001), while the logarithmic EHP of the other two tissues had a significant correlation level above 99.0%(P<0.01) or above 99.5%(P<0.05), except for midgut–hemolymph and hepatopancreas–hemolymph. In-situ hybridization of a DIG-labeled EHP probe in the hepatopancreas, muscle, gills, and midgut showed that the hepatopancreas is the major target tissue of EHP infection in shrimp. Minor and weak hybridization signals were also observed in other tissues, which indicated that a few cells in those tissues were also susceptible to EHP infection in L. vannamei.
Growth-related parameters of individuals from four populations of Litopenaeus vannameifrom Huanghua of Heibei Province(HH), Pingdu of Shandong Province(PD), Wujiang of Jiangsu Province(WJ), and Rizhao of Shandong Province(RZ) were assessed. Amount of Enterocytozoon hepatopenaei(EHP) in the hepatopancreas of individuals from all populations and EHP in multiple tissues of shrimp from the RZ population were assessed with Taq Man-based quantitative PCR. The results showed that the RZ population possessed optimal growth-related parameters and the lowest EHP among the four populations. The histograms of case-logarithmic EHPs of the four populations presented different modes. The case distribution of logarithmic EHPs from the HH and PD populations showed double peaks, while those of the WJ and RZ populations showed a single peak. The different distribution modes may indicate a different EHP spread in the four populations. The population with a single peak mode or the higher logarithmic EHP subpopulation isolated from the population with a multiple peak mode showed a significant negative correlation with shrimp body length or body weight to logarithmic EHP. The EHP detected in different tissues of the RZ population followed the order(from highest to lowest), EHP in hepatopancreas > EHP in midgut > EHP in hemolymph > EHP in gills > EHP in muscle. The logarithmic EHP in the hepatopancreas–midgut, hepatopancreas–gills, and midgut–gills had a significant correlation level above 99.9%(P<0.001), while the logarithmic EHP of the other two tissues had a significant correlation level above 99.0%(P<0.01) or above 99.5%(P<0.05), except for midgut–hemolymph and hepatopancreas–hemolymph. In-situ hybridization of a DIG-labeled EHP probe in the hepatopancreas, muscle, gills, and midgut showed that the hepatopancreas is the major target tissue of EHP infection in shrimp. Minor and weak hybridization signals were also observed in other tissues, which indicated that a few cells in those tissues were also susceptible to EHP infection in L. vannamei.
引文
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Chayaburakul K,Nash G,Pratanpipat P,et al.Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand.Diseases of Aquatic Organisms,2004,60(2):89-96
Han JE,Tang KFJ,Pantoja CR,et al.Detection of a new microsporidium Perezia sp.in shrimps Penaeus monodon and P.indicus by histopathology,in situ hybridization and PCR.Diseases of Aquatic Organisms,2016,120(2):165-171
Liu BB,Yang B,Lv XW,et al.Detection and quantification of infectious hypodermal and hematopoietic necrosis virus(IHHNV)and Enterocytozoon hepatopenaei(EHP)of Litopenaeus vannamei by real-time PCR.Progress in Fishery Sciences,2017,38(2):158-166[刘宝彬,杨冰,吕秀旺,等.凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量PCR检测.渔业科学进展,2017,38(2):158-166]
Liu YM,Qiu L,Sheng AZ,et al.Quantitative detection method of Enterocytozoon hepatopenaei using Taqman probe real-time PCR.Journal of Invertebrate Pathology,2018,151:191-196
Liu YM,Qiu L,Chen DY,et al.The relationship between body length and weight in Enterocytozoon hepatopenaei detected Litopenaeus vannamei populations.Progress of Fishery Sciences,2017,38(4):96-103[刘雅梅,邱亮,程东远,等.检出虾肝肠孢虫(Enterocytozoon hepatopenaei)的凡纳滨对虾(Litopenaeus vannamei)群体的体长和体重关系.渔业科学进展,2017,38(4):96-103]
Liu Z,Zhang QL,Wan XY,et al.Development of real-time PCRassay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates.Progress in Fishery Sciences,2016,37(2):119-126[刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测.渔业科学进展,2016,37(2):119-126]
Lom J,DykováI.Ultrastructure of Nucleospora secunda n.sp.(Microsporidia),parasite of enterocytes of Nothobranchius rubripinnis.European Journal of Protistology,2002,38(1):19-27
Lu XM,Jin W.Recent advances in taxonomy of phylum Microspora.Bulletin of Science and Technology,1999,15(2):119-125[鲁兴萌,金伟.微孢子虫分类学研究进展.科技通报,1999,15(2):119-125]
Luo YH,Shi J,Fang L,et all,Qian D.Development and application of a Taq Man real-time PCR assay for the detection of Enterocytozoon hepatopenaei.Veterinary Science in China,2016,46(7):847-852[骆云慧,石坚,方磊,等.虾肝肠胞虫Taq Man实时荧光定量PCR检测方法的建立及应用.中国兽医科学,2016,46(7):847-852]
Phelps NB,Goodwin AE.Validation of a quantitative PCRdiagnostic method for detection of the microsporidian Ovipleistophora ovariae in the cyprinid fish Notemigonus crysoleucas.Diseases of Aquatic Organisms,2007,76(3):215-221
Rajendran KV,Shivam S,Praveena PE,et al.Emergence of Enterocytozoon hepatopenaei(EHP)in farmed Penaeus(Litopenaeus)vannamei in India.Aquaculture,2016,454:272-280
Salachan PV,Jaroenlak P,Thitamadee S,et al.Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis(HPM)caused by Enterocytozoon hepatopenaei(EHP).BMC Veterinary Research,2017,13(1):1-7
Santin M.Enterocytozoon bieneusi.Springer Berlin Heidelberg,2001,218-220
Santhoshkumar S,Sivakumar S,Vimal S,et al.Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei(EHP)in naturally and experimentally EHP-infected whiteleg shrimp,Litopenaeus vannamei(Boone,1931),in India.Journal of Fish Diseases,2016,40(4):529-539
Tang KFJ,Han JE,Aranguren LF,et al.Dense populations of the microsporidian Enterocytozoon hepatopenaei(EHP)in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp.Journal of Invertebrate Pathology,2016,140:1-7
Tang KFJ,Pantojaa CR,Redmana RM,et al.Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei(EHP),a microsporidian parasite infecting penaeid shrimp.Journal Invertebrate Pathology,2015,130:37-41
Tourtip S,Wongtripop S,Stentiford GD,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationships.Journal of Invertebrate Pathology,2009,102(1):21-29
Bruce LD,Redman RM,Lightner DV,et al.Application of gene probes to detect a penaeid shrimp baculovirus in fixed tissue using in situ hybridization.Diseases of Aquatic Organisms,1993,17(3):215-221
Chayaburakul K,Nash G,Pratanpipat P,et al.Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand.Diseases of Aquatic Organisms,2004,60(2):89-96
Han JE,Tang KFJ,Pantoja CR,et al.Detection of a new microsporidium Perezia sp.in shrimps Penaeus monodon and P.indicus by histopathology,in situ hybridization and PCR.Diseases of Aquatic Organisms,2016,120(2):165-171
Liu BB,Yang B,Lv XW,et al.Detection and quantification of infectious hypodermal and hematopoietic necrosis virus(IHHNV)and Enterocytozoon hepatopenaei(EHP)of Litopenaeus vannamei by real-time PCR.Progress in Fishery Sciences,2017,38(2):158-166[刘宝彬,杨冰,吕秀旺,等.凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量PCR检测.渔业科学进展,2017,38(2):158-166]
Liu YM,Qiu L,Sheng AZ,et al.Quantitative detection method of Enterocytozoon hepatopenaei using Taqman probe real-time PCR.Journal of Invertebrate Pathology,2018,151:191-196
Liu YM,Qiu L,Chen DY,et al.The relationship between body length and weight in Enterocytozoon hepatopenaei detected Litopenaeus vannamei populations.Progress of Fishery Sciences,2017,38(4):96-103[刘雅梅,邱亮,程东远,等.检出虾肝肠孢虫(Enterocytozoon hepatopenaei)的凡纳滨对虾(Litopenaeus vannamei)群体的体长和体重关系.渔业科学进展,2017,38(4):96-103]
Liu Z,Zhang QL,Wan XY,et al.Development of real-time PCRassay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates.Progress in Fishery Sciences,2016,37(2):119-126[刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测.渔业科学进展,2016,37(2):119-126]
Lom J,DykováI.Ultrastructure of Nucleospora secunda n.sp.(Microsporidia),parasite of enterocytes of Nothobranchius rubripinnis.European Journal of Protistology,2002,38(1):19-27
Lu XM,Jin W.Recent advances in taxonomy of phylum Microspora.Bulletin of Science and Technology,1999,15(2):119-125[鲁兴萌,金伟.微孢子虫分类学研究进展.科技通报,1999,15(2):119-125]
Luo YH,Shi J,Fang L,et all,Qian D.Development and application of a Taq Man real-time PCR assay for the detection of Enterocytozoon hepatopenaei.Veterinary Science in China,2016,46(7):847-852[骆云慧,石坚,方磊,等.虾肝肠胞虫Taq Man实时荧光定量PCR检测方法的建立及应用.中国兽医科学,2016,46(7):847-852]
Phelps NB,Goodwin AE.Validation of a quantitative PCRdiagnostic method for detection of the microsporidian Ovipleistophora ovariae in the cyprinid fish Notemigonus crysoleucas.Diseases of Aquatic Organisms,2007,76(3):215-221
Rajendran KV,Shivam S,Praveena PE,et al.Emergence of Enterocytozoon hepatopenaei(EHP)in farmed Penaeus(Litopenaeus)vannamei in India.Aquaculture,2016,454:272-280
Salachan PV,Jaroenlak P,Thitamadee S,et al.Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis(HPM)caused by Enterocytozoon hepatopenaei(EHP).BMC Veterinary Research,2017,13(1):1-7
Santin M.Enterocytozoon bieneusi.Springer Berlin Heidelberg,2001,218-220
Santhoshkumar S,Sivakumar S,Vimal S,et al.Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei(EHP)in naturally and experimentally EHP-infected whiteleg shrimp,Litopenaeus vannamei(Boone,1931),in India.Journal of Fish Diseases,2016,40(4):529-539
Tang KFJ,Han JE,Aranguren LF,et al.Dense populations of the microsporidian Enterocytozoon hepatopenaei(EHP)in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp.Journal of Invertebrate Pathology,2016,140:1-7
Tang KFJ,Pantojaa CR,Redmana RM,et al.Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei(EHP),a microsporidian parasite infecting penaeid shrimp.Journal Invertebrate Pathology,2015,130:37-41
Tourtip S,Wongtripop S,Stentiford GD,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationships.Journal of Invertebrate Pathology,2009,102(1):21-29