黄海鱼类寄生异尖属线虫幼虫及内弯宫脂线虫的分子鉴定
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摘要
作者对采自2005年5月到2006年10月的黄海鱼类寄生异尖属线虫幼虫及内弯宫脂线虫进行了分子分类学鉴定,在对鱼类寄生线虫进行形态学分类的基础上,从202条隶属于30种鱼体内挑选出785条异尖属线虫幼虫用于PCR-RFLP (聚合酶链反应-限制性片段长度多态性)分析,并在具代表性的8个不同的鱼类宿主挑选出48条形态上有差别的内弯宫脂线虫进行rDNA ITS区序列测定分析。结果如下:
通过对所选异尖属线虫幼虫rDNA的ITS区进行PCR-RFLP分析,主要用内切酶Hinf1进行酶切,依据酶切片段长度的多态性和部分样品的序列测定,得出的实验结果显示黄海鱼类寄生异尖属线虫幼虫共有三个基因型:派氏异尖线虫Anisakis pegrefii,典型异尖线虫Anisakis typica及派氏异尖线虫Anisakis pegrefii与简单异尖线虫A. simplex sensu stricto的杂交体。其中Anisakis pegrefii是优势种,785个异尖属线虫幼虫样品中有770个为Anisakis pegrefii,约占总数的98%,在斑头Agrammus agrammus (Temminck & Schlegel),白姑Argyrosomus argentatus (Houttuyn),舵鲣Auxis thazard(lacépède)等29种所检鱼类体内均有发现。Anisakis typica数量较少,共3条,宿主为舵鲣Auxis thazard(lacépède),绿鳍鱼Chelidonichthys kumu。杂交体共12条,宿主为白姑Argyrosomus argentatus (Houttuyn),舵鲣Auxis thazard(lacépède),高眼鲽Cleisthenes herzenstein,大头鳕Gadus macrocephalus Tilesius,青鳞Harengula zunasi Bleeker,鲈鱼Lateolabrax japonicus (Cuvier & Valenciennes),海鳗Muraenesox cinereus (Forskal),带鱼Trichiurus haumela (Forskal),日本海魴Zeus japonicus Cuvier &Valencienn。其中Anisakis pegrefii与A. simplex sensu stricto的杂交体为国内首次发现。
选用内弯宫脂线虫的48个样品是根据其侧翼,胃盲囊和肠盲囊的比值,交合刺的长度,尾尖等形态的不同值所筛选出来的,线虫宿主为鮟鱇 Lophius litulon (Jordan),鳕鱼Gadus macrocephalus Tilesius,高眼鲽Cleisthenes herzensteini (Schmidt),六线鱼Hexagrammos otakii Jordan & Starks,黑鮶 Sebastodes fuscescens (Houttuyn),马鲅Scomberomorus niphonius (Guvie& Valenciennes),鬼鮋 Inimicus japonicus (Cuvier& Valenciennes),绵鳚Zoarces elongatus Kner8种鱼类。经过对内弯宫脂线虫的rDNA ITS-1和ITS-2进行rDNA的ITS区序列测定,结果表明来自不同宿主且形态有所差异的所有内弯宫脂线虫在分子水平上无差异,这些形态上的差异可能是由线虫不同的发育阶段及不同宿主对线虫的影响所造成的。
The larvae specimens of the genus Anisakis and adult specimens of Hysterothylacium aduncum from marine fishes in Yellow Sea were studied in molecular identification during May 2005 to October 2006. After identification based on morphological characters, a total of 785 Anisakis larvae nematodes from 203 fishes (belonging to 29 species) were analysed by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). The member of 48 adult Hysterothylacium aduncum with morphological differences belong to 8 different representative fish hosts were also analysed with rDNA ITS sequence. The main results are as follows:
The rDNA (ribosomal DNA) ITS( internal transcribed spacers) region of Anisakis larvae nematodes were examined by PCR-RFLP, and then ITS-1 and ITS-2 of the selective samples were amplified and sequenced.The RFLP patterns of 785 samples by HinfI and analysis of the ITS sequence for selective samples showed there are three species of the genus Anisakis in the Yellow Sea. They are Anisakis pegrefii, Anisakis typica and hybrid genotype between Anisakis pegrefii and A. simplex sensu stricto. Anisakis pegrefii is the dominant species in Yellow Sea, 770 out of 785 Anisakis larvae nematodes are Anisakis pegrefii, accounts for 98% of the total, and the hosts including 29 fish species such as Agrammus agrammus (Temminck & Schlegel), Argyrosomus argentatus (Houttuyn), Auxis thazard(lacépède) and so on.The number of Anisakis typica were very few, only 3 of 785 Anisakis larvae nematodes were found, and its hosts are Auxis thazard(lacépède), Chelidonichthys kumu. Twelve hybrid genotype were also identified,and its hosts are Argyrosomus argentatus (Houttuyn),Auxis thazard (lacépède), Cleisthenes herzensteini,Gadus mac-rocephalus Tilesius,Harengula zunasi Bleeker, Lateola- brax japonicus (Cuvier&Valenciennes),Muraenesox cinereus (Forskal), Trichiurus haumela (Forskal), Zeus japonicus Cuvier &Valencienn. This hybrid genotype is first reported in China.
Forty-eight adult Hysterothylacium aduncum were selected according to different morphological index value such as lateral alae width, ratio of the intestinal caecum to ventricular appendix, spicule length and morphology of tail tip.The adult Hysterothylacium aduncum were collected from 8 fish species, they are Lophius litulon (Jordan), Gadus macrocephalus Tilesius, Cleisthenes herzensteini (Schmidt), Hexagrammos otakii Jordan & Starks,Sebastodes fuscescens (Houttuyn),Scomberomorus niphonius (Guvie& Valenciennes), Inimicus japonicus (Cuvier & Valenciennes), Zoarces elongatus Kner. The result of analysis of rDNA ITS-1 and ITS-2 sequences indicated all sequences from 48 samples are identical although the samples from different host showed morphological differences. Then we can conclude that these certain metrical and morphological differences are probably associated with the state of development of these parasites and are also influenced by different numbers of the specimens examined from individual fish host species.
通过对所选异尖属线虫幼虫rDNA的ITS区进行PCR-RFLP分析,主要用内切酶Hinf1进行酶切,依据酶切片段长度的多态性和部分样品的序列测定,得出的实验结果显示黄海鱼类寄生异尖属线虫幼虫共有三个基因型:派氏异尖线虫Anisakis pegrefii,典型异尖线虫Anisakis typica及派氏异尖线虫Anisakis pegrefii与简单异尖线虫A. simplex sensu stricto的杂交体。其中Anisakis pegrefii是优势种,785个异尖属线虫幼虫样品中有770个为Anisakis pegrefii,约占总数的98%,在斑头Agrammus agrammus (Temminck & Schlegel),白姑Argyrosomus argentatus (Houttuyn),舵鲣Auxis thazard(lacépède)等29种所检鱼类体内均有发现。Anisakis typica数量较少,共3条,宿主为舵鲣Auxis thazard(lacépède),绿鳍鱼Chelidonichthys kumu。杂交体共12条,宿主为白姑Argyrosomus argentatus (Houttuyn),舵鲣Auxis thazard(lacépède),高眼鲽Cleisthenes herzenstein,大头鳕Gadus macrocephalus Tilesius,青鳞Harengula zunasi Bleeker,鲈鱼Lateolabrax japonicus (Cuvier & Valenciennes),海鳗Muraenesox cinereus (Forskal),带鱼Trichiurus haumela (Forskal),日本海魴Zeus japonicus Cuvier &Valencienn。其中Anisakis pegrefii与A. simplex sensu stricto的杂交体为国内首次发现。
选用内弯宫脂线虫的48个样品是根据其侧翼,胃盲囊和肠盲囊的比值,交合刺的长度,尾尖等形态的不同值所筛选出来的,线虫宿主为鮟鱇 Lophius litulon (Jordan),鳕鱼Gadus macrocephalus Tilesius,高眼鲽Cleisthenes herzensteini (Schmidt),六线鱼Hexagrammos otakii Jordan & Starks,黑鮶 Sebastodes fuscescens (Houttuyn),马鲅Scomberomorus niphonius (Guvie& Valenciennes),鬼鮋 Inimicus japonicus (Cuvier& Valenciennes),绵鳚Zoarces elongatus Kner8种鱼类。经过对内弯宫脂线虫的rDNA ITS-1和ITS-2进行rDNA的ITS区序列测定,结果表明来自不同宿主且形态有所差异的所有内弯宫脂线虫在分子水平上无差异,这些形态上的差异可能是由线虫不同的发育阶段及不同宿主对线虫的影响所造成的。
The larvae specimens of the genus Anisakis and adult specimens of Hysterothylacium aduncum from marine fishes in Yellow Sea were studied in molecular identification during May 2005 to October 2006. After identification based on morphological characters, a total of 785 Anisakis larvae nematodes from 203 fishes (belonging to 29 species) were analysed by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). The member of 48 adult Hysterothylacium aduncum with morphological differences belong to 8 different representative fish hosts were also analysed with rDNA ITS sequence. The main results are as follows:
The rDNA (ribosomal DNA) ITS( internal transcribed spacers) region of Anisakis larvae nematodes were examined by PCR-RFLP, and then ITS-1 and ITS-2 of the selective samples were amplified and sequenced.The RFLP patterns of 785 samples by HinfI and analysis of the ITS sequence for selective samples showed there are three species of the genus Anisakis in the Yellow Sea. They are Anisakis pegrefii, Anisakis typica and hybrid genotype between Anisakis pegrefii and A. simplex sensu stricto. Anisakis pegrefii is the dominant species in Yellow Sea, 770 out of 785 Anisakis larvae nematodes are Anisakis pegrefii, accounts for 98% of the total, and the hosts including 29 fish species such as Agrammus agrammus (Temminck & Schlegel), Argyrosomus argentatus (Houttuyn), Auxis thazard(lacépède) and so on.The number of Anisakis typica were very few, only 3 of 785 Anisakis larvae nematodes were found, and its hosts are Auxis thazard(lacépède), Chelidonichthys kumu. Twelve hybrid genotype were also identified,and its hosts are Argyrosomus argentatus (Houttuyn),Auxis thazard (lacépède), Cleisthenes herzensteini,Gadus mac-rocephalus Tilesius,Harengula zunasi Bleeker, Lateola- brax japonicus (Cuvier&Valenciennes),Muraenesox cinereus (Forskal), Trichiurus haumela (Forskal), Zeus japonicus Cuvier &Valencienn. This hybrid genotype is first reported in China.
Forty-eight adult Hysterothylacium aduncum were selected according to different morphological index value such as lateral alae width, ratio of the intestinal caecum to ventricular appendix, spicule length and morphology of tail tip.The adult Hysterothylacium aduncum were collected from 8 fish species, they are Lophius litulon (Jordan), Gadus macrocephalus Tilesius, Cleisthenes herzensteini (Schmidt), Hexagrammos otakii Jordan & Starks,Sebastodes fuscescens (Houttuyn),Scomberomorus niphonius (Guvie& Valenciennes), Inimicus japonicus (Cuvier & Valenciennes), Zoarces elongatus Kner. The result of analysis of rDNA ITS-1 and ITS-2 sequences indicated all sequences from 48 samples are identical although the samples from different host showed morphological differences. Then we can conclude that these certain metrical and morphological differences are probably associated with the state of development of these parasites and are also influenced by different numbers of the specimens examined from individual fish host species.
引文
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