二种吸虫尾蚴生物学特征及分子生物学鉴定

Biological characterization and molecular identification of cercaria of two fluke species

目的研究江西省纹沼螺吸虫类尾蚴及逸出特征,为吸虫类尾蚴防制提供科学依据。方法于2023年9—11月,从江西省信丰县某一鱼塘内分三次采集纹沼螺,采用尾蚴逸出法鉴定尾蚴感染情况;每天6:00—18:00,连续2 d,每间隔2 h在恒温状态下记录纹沼螺中尾蚴溢出数量,显微镜下观察溢出尾蚴形态特征。分别提取不同尾蚴DNA,采用PCR法扩增吸虫尾蚴内转录间隔区2(ITS2),对扩增产物进行测序,序列经Blast比对,以华支睾吸虫为外群,使用MEGA 7.0软件,采用邻接法构建基于ITS2基因序列的系统进化树,确定逸出尾蚴种类。结果共采集纹沼螺1982只,逸出2种尾蚴,总阳性率为1.21%(24/1982)。尾蚴a呈纺锤形,前端钝圆,后端略尖,该尾蚴在8∶00—12∶00无尾蚴逸出,12∶00后少量逸出,逸出率在16∶00后逐渐增高,18∶00达到峰值,且18∶00—次日6∶00大量逸出;尾蚴b分体、尾两部分,尾部长于体部,末端分叉,活动时呈“人”或“Y”字形。8∶00—12∶00尾蚴逸出率逐渐增高,12∶00达到峰值,12∶00之后逐渐回落,16∶00后几乎无尾蚴逸出,夜间不逸出,该尾蚴逸出率可能与光照有关。基因测序及序列比对结果显示,尾蚴a ITS2基因与Lecithodendrium linstowi ITS2基因同源性最高,为92.43%。尾蚴b ITS2基因与Holostepha⁃nus dubinini ITS2基因同源性最高,为99.35%。结论江西省纹沼螺可感染不同种类尾蚴,不同尾蚴的逸出规律及生物学特征存在较为明显差异,分子生物学结果分析尾蚴a可能为枝腺属吸虫,尾蚴b可能为日本全冠吸虫。通过了解尾蚴的生物学特征,对不同种类尾蚴采取相应防治措施,对吸虫防控具有潜在价值。

Objective To understand the characteristics of cercariae and their shedding from Parafossarulus striatulus in Jiangxi Province,providing a scientific basis for the control of trematode cercariae.Methods From September to November 2023,P.striatulus snails were collected three times from a fish pond in Xinfeng County,Jiangxi Province.Cercaria infection was identified by the cercarial shedding method.From 06:00 to 18:00 each day over two consecutive days,the number of released cercariae was recorded under constant temperature at an interval of 2 hours.Morphological characteristics of the cercariae were observed under the microscope.DNA from different cercariae was extracted,and internal transcribed spacer region 2(ITS2)was amplified by PCR.The amplified products were sequenced,and the sequences were then compared by Blast.With Clonorchis sinensis as the outgroup,a phylogenetic tree based on the ITS2 gene sequence was constructed by the neighbor-joining method using MEGA 7.0 software to determine the species of escaped cercariae.Results A total of 1982 P.striatulus snails were collected,with two species of cercariae emerging,showing an overall positive rate of 1.21%(24/1982).Cercaria a was oval-shaped,with a blunt front end and slightly pointed rear end.No cercaria shedded from 8:00 to 12:00,and a small amount was released after noon.The shedding rate gradually increased after 16:00,reached a peak at 18:00,and gradually declined after 20:00.Cercaria b consisted of body and tail parts,with the tail longer than the body and bifurcated at the end,displaying movement in“man”or“Y”shapes.The shedding rate of cercaria b gradually increased between 08:00 and 12:00,peaked at 12:00,decreased gradually thereafter,and almost no cercariae emerged after 16:00,with no escape at night,possibly related to light exposure.Gene sequencing and sequence comparison showed that the ITS2 gene of cercaria a had the highest homology with Lecithodendrium linstowi at 92.43%,whereas cercaria b showed the highest homology with Holostephanus dubinini at 99.35%.Conclusions P.striatulus snails in Jiangxi Province can be infected with different species of cercariae,which display significant differences in the biological characteristics and shedding pattern.Molecular analysis shows that cercaria a may belong to the genus of Lecithodendrium,and cercaria b may be H.dubinini.Understanding the biological characteristics of cercariae could be valuable for implementing corresponding control measures for different species of trematodes.