梨花迁粉蝶微孢子虫对家蚕的侵染力与胚传性
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摘要
【目的】对梨花迁粉蝶Catopsilia pyranthe分离的微孢子虫进行形态与分子鉴定,探究其对非天然宿主家蚕Bombyx mori的侵染力与胚传性。【方法】从田间采集的梨花迁粉蝶中分离得到梨花迁粉蝶微孢子虫液,测定其孢子的形态、大小、体积、长短轴比,同时对该孢子虫的16S r DNA进行PCR克隆测序与分析。将梨花迁粉蝶微孢子虫Nosema sp.CP与家蚕微孢子虫N.bombycis分别对2龄起蚕、4龄起蚕进行添食感染比对,测定家蚕食下两种微孢子虫的感染率和胚种传染能力。【结果】本研究分离的梨花迁粉蝶微孢子虫形态为长椭圆形,具双核;其16S r DNA序列与已报道的梨花迁粉蝶微孢子虫的序列一致性大于99%,为梨花迁粉蝶微孢子虫。梨花迁粉蝶微孢子虫和家蚕微孢子虫对家蚕综合感染率分别是68.8%和98.3%;在继代蚁蚕中,感染梨花迁粉蝶微孢子虫和家蚕微孢子虫的雌蛾所产蚕卵次代蚁蚕检出有孢子虫的检出率分别为100%和100%,卵壳的孢子虫的检出率分别为92.9%和100%;梨花迁粉蝶微孢子虫和家蚕微孢子虫对家蚕的胚种传染力分别为9.6%和23.2%。【结论】本研究分离得到的微孢子虫为梨花迁粉蝶微孢子虫,具有微孢子虫Nosema属的典型特征。梨花迁粉蝶微孢子虫能感染危害家蚕,也具有家蚕胚种传染性,但感染率和胚传率均明显低于家蚕微孢子虫,是蚕业生产中必须防控的对象。
【Aim】To identify the morphological and molecular characteristics of microsporidium Nosema sp. CP isolated from Catopsilia pyranthe,and then to assess the infectivity and transmissibility of this Nosema sp. CP in the domestic silkworm,Bombyx mori. 【Methods】After purification of Nosema sp. CP spores isolated from wild C. pyranthe butterflies,the shape,sizes and axial ratios of the spores were measured under the microscope,respectively. The DNA fragments of 16 S rDNA from this microsporidian species were amplified by PCR and then sequenced to confirm its classification status. The Nosema sp.CP isolated and N. bombycis was fed respectively to B. mori larvae of day-1 2nd instar and day-1 4th instar to survey the infectivity and transmissibility via embryo during different developmental stages of B.mori. 【Results】Oblong shape and diplokaryotic nuclei of the isolated microsporidia in this study were visualized under the microscope. Molecular analysis of 16 S rDNA sequence showed more than 99%identity to that of the previously reported microsporidian spores isolated from C. pyranthe. The total infection rates of Nosema sp. CP and N. bombycis to B. mori were 68. 8% and 98. 3%,respectively. In the second generation of B. mori after vaccination,the detection rates of sporozoans of Nosema sp. CP and N. bombycis in newly-hatched larvae were 100% and 100%,and those of their eggshells were 92.9% and 100%,respectively. The embryo transmissibilities of Nosema sp. CP and N. bombycis to B.mori were 9. 6% and 23. 2%,respectively. 【Conclusion】The microsporidia isolated in this study are Nosema sp. CP and have typical characteristics of Nosema diplokaryotic nuclei. The Nosema sp. CP isolated can infect B. mori and has transmissibility via embryo in this insect. However,in B. mori both the infection rate and transmission rate of Nosema sp. CP isolated in this study are lower than those of N.bombycis. Thus,it is necessary to prevent and control the prevalence of Nosema sp. CP in sericultural industry.
【Aim】To identify the morphological and molecular characteristics of microsporidium Nosema sp. CP isolated from Catopsilia pyranthe,and then to assess the infectivity and transmissibility of this Nosema sp. CP in the domestic silkworm,Bombyx mori. 【Methods】After purification of Nosema sp. CP spores isolated from wild C. pyranthe butterflies,the shape,sizes and axial ratios of the spores were measured under the microscope,respectively. The DNA fragments of 16 S rDNA from this microsporidian species were amplified by PCR and then sequenced to confirm its classification status. The Nosema sp.CP isolated and N. bombycis was fed respectively to B. mori larvae of day-1 2nd instar and day-1 4th instar to survey the infectivity and transmissibility via embryo during different developmental stages of B.mori. 【Results】Oblong shape and diplokaryotic nuclei of the isolated microsporidia in this study were visualized under the microscope. Molecular analysis of 16 S rDNA sequence showed more than 99%identity to that of the previously reported microsporidian spores isolated from C. pyranthe. The total infection rates of Nosema sp. CP and N. bombycis to B. mori were 68. 8% and 98. 3%,respectively. In the second generation of B. mori after vaccination,the detection rates of sporozoans of Nosema sp. CP and N. bombycis in newly-hatched larvae were 100% and 100%,and those of their eggshells were 92.9% and 100%,respectively. The embryo transmissibilities of Nosema sp. CP and N. bombycis to B.mori were 9. 6% and 23. 2%,respectively. 【Conclusion】The microsporidia isolated in this study are Nosema sp. CP and have typical characteristics of Nosema diplokaryotic nuclei. The Nosema sp. CP isolated can infect B. mori and has transmissibility via embryo in this insect. However,in B. mori both the infection rate and transmission rate of Nosema sp. CP isolated in this study are lower than those of N.bombycis. Thus,it is necessary to prevent and control the prevalence of Nosema sp. CP in sericultural industry.
引文
Chen SL,Gao JH,Yang RG,Gao X,Zhang JX,Zhu F,2015a.Influence of microsporidium on female silkworm moth after mating of the female and male silkworm moth.Guizhou Agricultural Science,43(2):115-117.[陈世良,高建华,杨荣贵,高翔,张金祥,朱峰,2015a.家蚕微孢子虫感染雄蛾交配对雌蛾的影响.贵州农业科学,43(2):115-117]
Chen SL,Yang RG,Gao JH,Gao X,Zhu F,Luo SG,Zhang JX,2015b.Effects of microsporidium isolated from Pieris rapae L.on embryo transmissibility and quality of sericulture.Jiangsu Agricultural Sciences,43(8):241-243.[陈世良,杨荣贵,高建华,高翔,朱峰,罗顺高,张金祥,2015b.菜粉蝶微孢子虫对家蚕胚种传染性与蚕桑质量影响研究.江苏农业科学,43(8):241-243]
Chen YT,Wu ZQ,Chen W,2013.Flight activity and egg-laying behavior in the Catopsilia pyranthe L.(Lepidoptera:Pieridae).Journal of Biosafety,22(3):212-214.[陈燕婷,吴珍泉,陈伟,2013.梨花迁粉蝶成虫的产卵行为.生物安全学报,22(3):212-214]
Jiang MG,Luo ML,Tang QK,Huang XH,Tang MR,An CM,Deng ZB,2009.Investigation and analysis of microsporidian pollution in environments of large scale silkworm eggs production.Guangxi Agricultural Sciences,40(10):1346-1349.[蒋满贵,罗梅兰,汤庆坤,黄旭华,唐茂荣,安春梅,邓忠榜,2009.蚕种生产大环境中微孢子虫污染情况调查分析.广西农业科学,40(10):1346-1349]
Liao S,Fang D,Zheng X,Nong C,Lu K,1992.Studies on the character of a microsporidian parasite from Phyllobrotica armte Baly and pathogenicity to the silkworm,Bombyx mori.J.South China Agric.Univ.,13(4):53.[廖森泰,方定坚,郑祥明,农朝志,卢铿明,1992.蓝叶甲微孢子虫的特性及其对家蚕病原性研究.华南农业大学学报,13(4):53]
Liu T,Xu JS,Li T,Pan GQ,He Q,Li XY,Zhou ZY,2014.Identification and phylogenetic analysis of MITE transposons in a microsporidia isolate from Papilio machaon Linnaeus.Science of Sericulture,40(1):52-58.[刘铁,许金山,李田,潘国庆,何强,李雪燕,周泽演,2014.从金凤蝶分离的一株微孢子虫的全基因组MITEs转座子鉴定与系统进化分析.蚕业科学,40(1):52-58]
Ma QZ,Wang YL,2006.Discussion on the relationship between Pebrine disease and environment toxic source.Sericulture in China,27(1):11-14.[马群忠,王永亮,2006.试论家蚕微粒子病与环境毒源的关系.中国蚕业,27(1):11-14]
Pan HM,Cui HJ,Wan YJ,Yang B,Pan J,Zeng HM,1999.Primary research on the diversity of microsporidia isolated from Pieris rapae L.and their infectivity to the silkworm.Sichuan Canye,(3):10-11.[潘慧敏,崔红娟,万永继,杨彪,潘杰,曾华明,1999.菜粉蝶微孢子的多态性及对家蚕感染性的初步研究.四川蚕业,(3):10-11]
Sun S,Zong H,Yang X,2001.Study on the infection and pathogenesis of Nosema bombycis in Pieris rapae.Journal of Sichuan Normal University(Natural Science),24(1):69-71.[孙胜,宗浩,杨小蓉,2001.家蚕微孢子(Nosema bombycis)对菜青虫(Pieris rapae)的感染与致病性研究.四川师范大学学报(自然科学版),24(1):69-71]
Yang Q,Xu X,Lu K,Zheng X,Fang D,2001.Research on the infection of two species of microsporidia isolated from Pieris rapae L.(I)Pathogenic morphology and pathogenicity to the silkworm.Guangdong Sericulture,35(2):16-19.[杨琼,徐兴耀,卢铿明,郑祥明,方定坚,2001.感染菜粉蝶的两种微孢子虫的研究(I)病原形态及对家蚕的病原性.广东蚕业,35(2):16-19]
Zheng L,Chen S,Ma Z,Xu J,Zhou Z,2014.Phylogenetic characterization of a microsporidia Nosema sp.CP from mottled emigrant(Catopsilia pyranthe).Journal of Chongqing Normal University(Natural Science),31(5):42-47.[郑丽金,陈世良,马振刚,许金山,周泽扬,2014.从梨花迁粉蝶中所得微孢子虫的系统进化分析.重庆师范大学学报(自然科学版),31(5):42-47]
Zheng X,Yang Q,Fang D,Liao S,Zou Y,Huang B,2003.Studies on insect microsporidia resources in Guangdong Province and cross infection among some insects.Science of Sericulture,29(40):382-383.[郑祥明,杨琼,方定坚,廖森泰,邹宇晓,黄炳辉,2003.广东省昆虫微孢子虫资源调查及交叉感染的研究.蚕业科学,29(40):382-383]
Chen SL,Yang RG,Gao JH,Gao X,Zhu F,Luo SG,Zhang JX,2015b.Effects of microsporidium isolated from Pieris rapae L.on embryo transmissibility and quality of sericulture.Jiangsu Agricultural Sciences,43(8):241-243.[陈世良,杨荣贵,高建华,高翔,朱峰,罗顺高,张金祥,2015b.菜粉蝶微孢子虫对家蚕胚种传染性与蚕桑质量影响研究.江苏农业科学,43(8):241-243]
Chen YT,Wu ZQ,Chen W,2013.Flight activity and egg-laying behavior in the Catopsilia pyranthe L.(Lepidoptera:Pieridae).Journal of Biosafety,22(3):212-214.[陈燕婷,吴珍泉,陈伟,2013.梨花迁粉蝶成虫的产卵行为.生物安全学报,22(3):212-214]
Jiang MG,Luo ML,Tang QK,Huang XH,Tang MR,An CM,Deng ZB,2009.Investigation and analysis of microsporidian pollution in environments of large scale silkworm eggs production.Guangxi Agricultural Sciences,40(10):1346-1349.[蒋满贵,罗梅兰,汤庆坤,黄旭华,唐茂荣,安春梅,邓忠榜,2009.蚕种生产大环境中微孢子虫污染情况调查分析.广西农业科学,40(10):1346-1349]
Liao S,Fang D,Zheng X,Nong C,Lu K,1992.Studies on the character of a microsporidian parasite from Phyllobrotica armte Baly and pathogenicity to the silkworm,Bombyx mori.J.South China Agric.Univ.,13(4):53.[廖森泰,方定坚,郑祥明,农朝志,卢铿明,1992.蓝叶甲微孢子虫的特性及其对家蚕病原性研究.华南农业大学学报,13(4):53]
Liu T,Xu JS,Li T,Pan GQ,He Q,Li XY,Zhou ZY,2014.Identification and phylogenetic analysis of MITE transposons in a microsporidia isolate from Papilio machaon Linnaeus.Science of Sericulture,40(1):52-58.[刘铁,许金山,李田,潘国庆,何强,李雪燕,周泽演,2014.从金凤蝶分离的一株微孢子虫的全基因组MITEs转座子鉴定与系统进化分析.蚕业科学,40(1):52-58]
Ma QZ,Wang YL,2006.Discussion on the relationship between Pebrine disease and environment toxic source.Sericulture in China,27(1):11-14.[马群忠,王永亮,2006.试论家蚕微粒子病与环境毒源的关系.中国蚕业,27(1):11-14]
Pan HM,Cui HJ,Wan YJ,Yang B,Pan J,Zeng HM,1999.Primary research on the diversity of microsporidia isolated from Pieris rapae L.and their infectivity to the silkworm.Sichuan Canye,(3):10-11.[潘慧敏,崔红娟,万永继,杨彪,潘杰,曾华明,1999.菜粉蝶微孢子的多态性及对家蚕感染性的初步研究.四川蚕业,(3):10-11]
Sun S,Zong H,Yang X,2001.Study on the infection and pathogenesis of Nosema bombycis in Pieris rapae.Journal of Sichuan Normal University(Natural Science),24(1):69-71.[孙胜,宗浩,杨小蓉,2001.家蚕微孢子(Nosema bombycis)对菜青虫(Pieris rapae)的感染与致病性研究.四川师范大学学报(自然科学版),24(1):69-71]
Yang Q,Xu X,Lu K,Zheng X,Fang D,2001.Research on the infection of two species of microsporidia isolated from Pieris rapae L.(I)Pathogenic morphology and pathogenicity to the silkworm.Guangdong Sericulture,35(2):16-19.[杨琼,徐兴耀,卢铿明,郑祥明,方定坚,2001.感染菜粉蝶的两种微孢子虫的研究(I)病原形态及对家蚕的病原性.广东蚕业,35(2):16-19]
Zheng L,Chen S,Ma Z,Xu J,Zhou Z,2014.Phylogenetic characterization of a microsporidia Nosema sp.CP from mottled emigrant(Catopsilia pyranthe).Journal of Chongqing Normal University(Natural Science),31(5):42-47.[郑丽金,陈世良,马振刚,许金山,周泽扬,2014.从梨花迁粉蝶中所得微孢子虫的系统进化分析.重庆师范大学学报(自然科学版),31(5):42-47]
Zheng X,Yang Q,Fang D,Liao S,Zou Y,Huang B,2003.Studies on insect microsporidia resources in Guangdong Province and cross infection among some insects.Science of Sericulture,29(40):382-383.[郑祥明,杨琼,方定坚,廖森泰,邹宇晓,黄炳辉,2003.广东省昆虫微孢子虫资源调查及交叉感染的研究.蚕业科学,29(40):382-383]