云南省异盘并殖吸虫的形态和分子标记研究
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摘要
[目的]在形态学分类的基础上,结合DNA序列分析的方法深入研究异盘并殖吸虫的虫种独立性,及其在并殖科吸虫中的分类学地位,建立基因进化树,确定云南省异盘并殖吸虫与其他地区相同或不同虫种间的亲缘关系,逐步澄清并殖科吸虫的虫种争议,探讨更加客观和易于鉴定的分类学方法,并为进一步研究各虫种的生物学特性、地理分布、对人体的致病性,以及开展相关的防治工作奠定重要基础,为云南省并殖吸虫的研究和并殖吸虫病的防治提供有价值的科学依据。
[方法]从异盘并殖吸虫流行区采集溪蟹,经分类鉴定后分离囊蚴,感染实验动物大白鼠获得成虫和虫卵。观察囊蚴、成虫及虫卵形态,并测量其大小；扫描电镜观察成虫的皮棘形态特征。用试剂盒分别提取囊蚴和成虫的DNA，利用ITS2和CO1引物体外扩增获得ITS2和CO1基因序列,通过GenBank的Blast程序和DNASTAR的MagAlign程序进行基因的同源性分析,使用MAGE 4.0软件构建基因进化树。
[结果]本次共采集到两类溪蟹,分类地位隶属于溪蟹总科,溪蟹科,溪蟹属,分别为景洪溪蟹和毛足溪蟹；景洪溪蟹头胸甲表面较隆,暗红色,分区明显,前侧缘具有细齿10余个；两螯不对称,以右螯为大；雄性第1腹肢粗壮,倒数第2节长度约为末节的3.6倍,末缘似山峰样起伏,尖锐指向外方；雄性尾节三角形,雌性尾节半圆形。毛足溪蟹头胸甲表面扁平,暗褐色,具有短刚毛,前侧缘具有细齿13-15个；两螯稍不对称；第3颚足外肢有短鞭；雄性第1腹肢扁平,倒数第2节长度约为末节的3.0倍；雄性腹部呈三角形,尾节长三角形；雌性腹部卵圆形,尾节半圆形。
异盘并殖吸虫活体成虫肉红色,作蛭样运动。轻压固定后呈椭圆形,虫体宽长比为1：1.56-2.12。口腹吸盘比为1.62-2.09：1,口吸盘明显大于腹吸盘。卵巢多位于腹吸盘左侧，前缘位于腹吸盘水平前后,分支细而多；子宫管状蟠曲成团,与卵巢并列、大于卵巢,其前缘多超过腹吸盘水平,并与腹吸盘部分或完全重叠。睾丸分别位于卵巢和子宫团的下方,中心部较小,分支末端常有显著膨大。异盘并殖吸虫囊蚴多呈椭圆形,具有二层囊壁,外壁薄而均匀,内壁远较外壁厚且两极增厚明显,内外壁之间的间隙明显。异盘并殖吸虫虫卵多呈卵圆形,金黄色,绝大多数两侧对称；卵壳厚薄不均匀,末端增厚明显；卵盖位于较宽一端,肩峰多明显；扫描电镜观察成虫体表被有单生型皮棘,虫体前中部皮棘密集,排列规则；虫体后部皮棘稀疏,排列不规则,形状似尖刀状或三角形；口吸盘内壁为纵向皱襞,腹吸盘内壁为环形皱襞。
通过Blast同源性比较,来自中国云南与印度Arunachal和Manipur地区的异盘并殖吸虫ITS2基因同源性为98%,与泰国Saraburi地区的异盘并殖吸虫ITS2同源性99%,与中国云南的丰宫和印度的卫氏并殖吸虫ITS2同源性分别为95%和93%；来自中国云南与泰国、越南、中国广西地区的异盘并殖吸虫CO1基因同源性为98%,与来自印度的异盘并殖吸虫CO1基因同源性为91%。ITS2和CO1基因进化树显示已报道的所有异盘并殖吸虫聚为一支,与其它虫种间差异明显。
[结论]云南的景洪溪蟹和毛足溪蟹是异盘并殖吸虫的第二中间宿主。家猫对异盘并殖吸虫的易感性明显优于SD大鼠,家猫是异盘并殖吸虫的理想实验动物模型；而大鼠则不理想。云南省存在的异盘并殖吸虫与其它地区报道的异盘并殖吸虫无明显形态学差异。DNA序列分析显示来自不同地区的异盘并殖吸虫同源性极高,进化树上聚类为一支,独立于其它虫种之外。
[Objective] Based on morphological classification and combined with DNA sequence analysis, This paper aimed to further study the independence of Paragonimus heterotremus, taxonomic status in paragonimus and reconstruction neighbor-joining trees of P. heterotremus in order to gradually clarify the controversy of classification in Paragonimus, investigate more objective methods for taxonomy and identification, and make a foundation for studying the biologic characteristics of Paragonimus, geographical distribution, pathogenicity to human, prevention and treatment of paragonimiasis, which can offer an important scientific information to research the paragonimus and prevent paragonimiasis in Yunnan province.
[Methods] Crabs were collected from endemic areas of Paragonimus heterotremus. Metacercaria were separated, identified and fed to SD rats, from which adult worms and eggs were obtained. Metacercaria, adult worms and eggs were observed and measured. Morphological characteristic spines of adult worms were observed with scanning electron microscope (SEM). With the help of reagent kits, DNA was extracted from metacercaria and adult worms, then ITS2 and CO1 gene were amplified with gene primer. Analysis of gene identity was finished with Blast program of GenBank and DNASTAR software MagAlign program and neighbor-joining trees were constructed with MEGA4.0 software.
[Results] Two kinds of crabs, both of which belong to potamodiea, potamidea and potamon, were collected in this research. These kind of crabs were identified with Potamon chinghungense and Potamon hispidum. Morphological characteristic of Potamon chinghungense shows swelling carapace, dark red color, clear region, about 10 pieces of serration in antero-lateral border, and asymmetric cheliped leg which indicates that right one was bigger than the left. The first pleopod of male shows sturdy and the length of Penultimate section is 3.6 times bigger than the final section. The end edge of the first pleopod which seems like protruding peaks points to outside. The last section of male's abdomen shows triangle, but female shows oval. Morphological characteristic of Potamon hispidum shows flat carapace, dark brown color, enjoying short bristle hair, about 13-15 pieces of serration in antero-lateral border, and asymmetric cheliped leg. Exopod of the third maxilliped has short whips. The first pleopod of male shows flat and the length of penultimate section is 3.0 times bigger than the final section. The male's abdomen shows triangle and the last section indicates long triangle. The female's abdomen shows oval and the last section indicates semicircular in shape.
Living adult worms of P. heterotremus shows pink in color, leech-like movement. After low-pressing and fixing, it shows elliptic in shape. To the whole adult worms, the average rate of width to length is 1:1.56-2.12. The average rate of oral sucker to ventral sucker is 1.62-2.09:1, so oral suckers are apparently bigger than ventral suckers. In this species, ovary is mostly located at the left of ventral suckers, and the front edge line is located at the level of ventral suckers, and also shows more thin branches, Uterus convolute into a group, parallels arrangement with ovary, but the size of which indicates bigger than the size of ovary. Front edge line of uterus mostly exceed to the level of ventral sucker, even partly or completely overlap. Testis are located below uterus and ovary and the central part shows small. The end of the branches swell apparently. The living metacercaria of P. heterotremus show elliptic in shape and have a thin outer cyst wall and thick inner cyst wall. The inner cyst wall thicks apparently in the bipolar and the gap between inner and outer cyst wall indicates clear. Eggs of P. heterotremus are always oval in shape, golden yellow in color, most of which is symmetry. The thickness of egg shows uneven with apparently thicker end edge. The eggs' cover always locates to the relative width end with a clear acromion. With scanning electron microscope observation, the surface of adult worms covers single spines which show rule and intensive at the central part, but irregular and thin at the end part. The shape of spines is dagger-like or triangle. The oral sucker has longitudinal folds and the ventral sucker has circular folds.
The result of Blast homology analysis indicates that P. heterotremus homology of ITS2 gene between Yunnan, China and Arunacha、Manipur, India is 98%, but identity between Yunnan, China and Saraburi, Thailand is 99%. Homology of ITS2 gene between P. heterotremus and P. proliferus, all of which come from Yunnan, China is 95%, but comparison between P. heterotremus from Yunnan and P. westermani from India shows 93%. P. heterotremus homology of CO1 gene between Yunnan, China、Thailand、Vietnam and Guangxi, China is 98%. But to India, this data changes to 91%. The neighbor-joining trees of ITS2 and CO1 gene indicates that all species of P. heterotremus cluster to one branch and separate apart from other species.
[Conclusion] Potamon chinghungense and Potamon hispidum collected from Yunnan are the second intermediate host of Paragonimus heterotremus. Cat's susceptibility to P. heterotremus is better than SD rat's. Cat is the appropriate experimental definitive host, but SD rat is not. Paragonimus heterotremus collected from Yunnan indicates no apparently different from the same species from other regions. DNA sequence analysis shows highly identity of P. heterotremus from different areas, clusters to one branch in neighbor-joining trees and separates apart from other species.
[方法]从异盘并殖吸虫流行区采集溪蟹,经分类鉴定后分离囊蚴,感染实验动物大白鼠获得成虫和虫卵。观察囊蚴、成虫及虫卵形态,并测量其大小；扫描电镜观察成虫的皮棘形态特征。用试剂盒分别提取囊蚴和成虫的DNA，利用ITS2和CO1引物体外扩增获得ITS2和CO1基因序列,通过GenBank的Blast程序和DNASTAR的MagAlign程序进行基因的同源性分析,使用MAGE 4.0软件构建基因进化树。
[结果]本次共采集到两类溪蟹,分类地位隶属于溪蟹总科,溪蟹科,溪蟹属,分别为景洪溪蟹和毛足溪蟹；景洪溪蟹头胸甲表面较隆,暗红色,分区明显,前侧缘具有细齿10余个；两螯不对称,以右螯为大；雄性第1腹肢粗壮,倒数第2节长度约为末节的3.6倍,末缘似山峰样起伏,尖锐指向外方；雄性尾节三角形,雌性尾节半圆形。毛足溪蟹头胸甲表面扁平,暗褐色,具有短刚毛,前侧缘具有细齿13-15个；两螯稍不对称；第3颚足外肢有短鞭；雄性第1腹肢扁平,倒数第2节长度约为末节的3.0倍；雄性腹部呈三角形,尾节长三角形；雌性腹部卵圆形,尾节半圆形。
异盘并殖吸虫活体成虫肉红色,作蛭样运动。轻压固定后呈椭圆形,虫体宽长比为1：1.56-2.12。口腹吸盘比为1.62-2.09：1,口吸盘明显大于腹吸盘。卵巢多位于腹吸盘左侧，前缘位于腹吸盘水平前后,分支细而多；子宫管状蟠曲成团,与卵巢并列、大于卵巢,其前缘多超过腹吸盘水平,并与腹吸盘部分或完全重叠。睾丸分别位于卵巢和子宫团的下方,中心部较小,分支末端常有显著膨大。异盘并殖吸虫囊蚴多呈椭圆形,具有二层囊壁,外壁薄而均匀,内壁远较外壁厚且两极增厚明显,内外壁之间的间隙明显。异盘并殖吸虫虫卵多呈卵圆形,金黄色,绝大多数两侧对称；卵壳厚薄不均匀,末端增厚明显；卵盖位于较宽一端,肩峰多明显；扫描电镜观察成虫体表被有单生型皮棘,虫体前中部皮棘密集,排列规则；虫体后部皮棘稀疏,排列不规则,形状似尖刀状或三角形；口吸盘内壁为纵向皱襞,腹吸盘内壁为环形皱襞。
通过Blast同源性比较,来自中国云南与印度Arunachal和Manipur地区的异盘并殖吸虫ITS2基因同源性为98%,与泰国Saraburi地区的异盘并殖吸虫ITS2同源性99%,与中国云南的丰宫和印度的卫氏并殖吸虫ITS2同源性分别为95%和93%；来自中国云南与泰国、越南、中国广西地区的异盘并殖吸虫CO1基因同源性为98%,与来自印度的异盘并殖吸虫CO1基因同源性为91%。ITS2和CO1基因进化树显示已报道的所有异盘并殖吸虫聚为一支,与其它虫种间差异明显。
[结论]云南的景洪溪蟹和毛足溪蟹是异盘并殖吸虫的第二中间宿主。家猫对异盘并殖吸虫的易感性明显优于SD大鼠,家猫是异盘并殖吸虫的理想实验动物模型；而大鼠则不理想。云南省存在的异盘并殖吸虫与其它地区报道的异盘并殖吸虫无明显形态学差异。DNA序列分析显示来自不同地区的异盘并殖吸虫同源性极高,进化树上聚类为一支,独立于其它虫种之外。
[Objective] Based on morphological classification and combined with DNA sequence analysis, This paper aimed to further study the independence of Paragonimus heterotremus, taxonomic status in paragonimus and reconstruction neighbor-joining trees of P. heterotremus in order to gradually clarify the controversy of classification in Paragonimus, investigate more objective methods for taxonomy and identification, and make a foundation for studying the biologic characteristics of Paragonimus, geographical distribution, pathogenicity to human, prevention and treatment of paragonimiasis, which can offer an important scientific information to research the paragonimus and prevent paragonimiasis in Yunnan province.
[Methods] Crabs were collected from endemic areas of Paragonimus heterotremus. Metacercaria were separated, identified and fed to SD rats, from which adult worms and eggs were obtained. Metacercaria, adult worms and eggs were observed and measured. Morphological characteristic spines of adult worms were observed with scanning electron microscope (SEM). With the help of reagent kits, DNA was extracted from metacercaria and adult worms, then ITS2 and CO1 gene were amplified with gene primer. Analysis of gene identity was finished with Blast program of GenBank and DNASTAR software MagAlign program and neighbor-joining trees were constructed with MEGA4.0 software.
[Results] Two kinds of crabs, both of which belong to potamodiea, potamidea and potamon, were collected in this research. These kind of crabs were identified with Potamon chinghungense and Potamon hispidum. Morphological characteristic of Potamon chinghungense shows swelling carapace, dark red color, clear region, about 10 pieces of serration in antero-lateral border, and asymmetric cheliped leg which indicates that right one was bigger than the left. The first pleopod of male shows sturdy and the length of Penultimate section is 3.6 times bigger than the final section. The end edge of the first pleopod which seems like protruding peaks points to outside. The last section of male's abdomen shows triangle, but female shows oval. Morphological characteristic of Potamon hispidum shows flat carapace, dark brown color, enjoying short bristle hair, about 13-15 pieces of serration in antero-lateral border, and asymmetric cheliped leg. Exopod of the third maxilliped has short whips. The first pleopod of male shows flat and the length of penultimate section is 3.0 times bigger than the final section. The male's abdomen shows triangle and the last section indicates long triangle. The female's abdomen shows oval and the last section indicates semicircular in shape.
Living adult worms of P. heterotremus shows pink in color, leech-like movement. After low-pressing and fixing, it shows elliptic in shape. To the whole adult worms, the average rate of width to length is 1:1.56-2.12. The average rate of oral sucker to ventral sucker is 1.62-2.09:1, so oral suckers are apparently bigger than ventral suckers. In this species, ovary is mostly located at the left of ventral suckers, and the front edge line is located at the level of ventral suckers, and also shows more thin branches, Uterus convolute into a group, parallels arrangement with ovary, but the size of which indicates bigger than the size of ovary. Front edge line of uterus mostly exceed to the level of ventral sucker, even partly or completely overlap. Testis are located below uterus and ovary and the central part shows small. The end of the branches swell apparently. The living metacercaria of P. heterotremus show elliptic in shape and have a thin outer cyst wall and thick inner cyst wall. The inner cyst wall thicks apparently in the bipolar and the gap between inner and outer cyst wall indicates clear. Eggs of P. heterotremus are always oval in shape, golden yellow in color, most of which is symmetry. The thickness of egg shows uneven with apparently thicker end edge. The eggs' cover always locates to the relative width end with a clear acromion. With scanning electron microscope observation, the surface of adult worms covers single spines which show rule and intensive at the central part, but irregular and thin at the end part. The shape of spines is dagger-like or triangle. The oral sucker has longitudinal folds and the ventral sucker has circular folds.
The result of Blast homology analysis indicates that P. heterotremus homology of ITS2 gene between Yunnan, China and Arunacha、Manipur, India is 98%, but identity between Yunnan, China and Saraburi, Thailand is 99%. Homology of ITS2 gene between P. heterotremus and P. proliferus, all of which come from Yunnan, China is 95%, but comparison between P. heterotremus from Yunnan and P. westermani from India shows 93%. P. heterotremus homology of CO1 gene between Yunnan, China、Thailand、Vietnam and Guangxi, China is 98%. But to India, this data changes to 91%. The neighbor-joining trees of ITS2 and CO1 gene indicates that all species of P. heterotremus cluster to one branch and separate apart from other species.
[Conclusion] Potamon chinghungense and Potamon hispidum collected from Yunnan are the second intermediate host of Paragonimus heterotremus. Cat's susceptibility to P. heterotremus is better than SD rat's. Cat is the appropriate experimental definitive host, but SD rat is not. Paragonimus heterotremus collected from Yunnan indicates no apparently different from the same species from other regions. DNA sequence analysis shows highly identity of P. heterotremus from different areas, clusters to one branch in neighbor-joining trees and separates apart from other species.
引文
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