Abstract: Objective　To investigate the prevalence of Paragonimus infection in freshwater crabs from selected areas of Yunnan Province, and to perform molecular characterization and phylogenetic analysis of the isolated parasites based on the internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit I (COI) gene sequences. Methods　Freshwater crabs were collected from both local markets and natural streams in Zhaotong City, Pu'er City, Dehong Dai and Jingpo Autonomous Prefecture, Baoshan City, Honghe Hani and Yi Autonomous Prefecture and Lincang City of Yunnan Province from May to August 2025. Paragonimus metacercariae were isolated using a sieve and sedimentation method. After morphological identification of the metacercariae, the infection rate and infection intensity of Paragonimus in freshwater crabs were calculated. Then genomic DNA was extracted from an individual metacercaria, and ITS2 and COI gene fragments were amplified using a PCR assay and sequenced. The obtained sequences were aligned with those available in the GenBank database. Genetic distances were calculated with the software MEGA 11.0 based on the Kimura 2⁃parameter model, and phylogenetic trees were built using the neighbour⁃joining method. Results　A total of 600 freshwater crabs were collected, with 18 positive for Paragonimus metacercariae. The prevalence of Paragonimus infection was 12.00% (18/150) in freshwater crabs from Zhaotong City, while no Paragonimus infection was detected in crabs from other five prefectures (cities). Twenty metacercariae were recovered, with an overall infection intensity of 1.11 metacercariae per crab. Morphological observation revealed two types of Paragonimus metacercariae, including 19 resembling P. skrjabini, and 1 resembling P. paishuihoensis. Ten metacercariae of distinct morphotypes were randomly selected for molecular identification, and sequence alignment and phylogenetic analysis of ITS2 and COI genes identified 9 as P. skrjabini and 1 as P. paishuihoensis. The nine ITS2 gene sequences shared 98.34% to 100.00% homology with the reference sequence of P. skrjabini (GenBank accession number: KX129924.1), and the single ITS2 gene sequence showed 100.00% homology with the reference sequence of P. paishuihoensis (GenBank accession number: OM401939.1), while its COI sequence showed 98.80% homology with the reference sequence of P. paishuihoensis (GenBank accession number: AB679288.1). Phylogenetic analysis further confirmed that the 10 sequences were clustered separately into the clades of P. skrjabini and P. paishuihoensis. Conclusions　       This study reports the first detection of P. paishuihoensis in freshwater crabs from Zhaotong City, Yunnan Province, and confirms P. skrjabini as the dominant Paragonimus species in this region. Molecular identification and phylogenetic analysis based on ITS2 and COI genes are effective to distinguish Paragonimus species, which provides the scientific evidence for precise control of paragonimiasis in Yunan Province.

Key words: Paragonimus, Freshwater crab, Internal transcribed spacer 2, Cytochrome c oxidase subunit I, Molecular characterization, Yunnan Province

摘要： 目的　调查云南省部分地区淡水蟹并殖吸虫感染状况，并基于核糖体内部转录间隔区2（internal transcribed spacer 2，ITS2）和线粒体细胞色素C氧化酶亚基I（cytochrome c oxidase subunit I，COI）基因序列对分离的虫体进行分子鉴定与系统发育分析。方法　于2025年5—8月在云南省昭通市、普洱市、德宏傣族景颇族自治州、保山市、红河哈尼族彝族自治州、临沧市共6个州（市）采集市售及野外溪涧淡水蟹，通过双筛水洗沉淀法分离并殖吸虫囊蚴。对囊蚴进行形态学鉴定后，计算淡水蟹并殖吸虫感染率和感染度。提取单个囊蚴基因组DNA，经PCR扩增ITS2和COI基因片段并测序。将所得序列与GenBank数据库进行比对，使用MEGA 11.0软件基于Kimura 2⁃parameter模型计算遗传距离，并以邻接法构建系统进化树。结果　共采集淡水蟹600只，检出并殖吸虫感染阳性18只，均采集自昭通市。该市淡水蟹并殖吸虫感染率为12.00%（18/150）；其余5个州（市）均未检出阳性淡水蟹。共检获并殖吸虫囊蚴20个，总感染度为1.11个/只。形态学观察鉴定出两种囊蚴，19个囊蚴形似斯氏并殖吸虫，1个囊蚴形似白水河并殖吸虫。随机挑取两种形态不同的囊蚴，对其中10个囊蚴进行分子鉴定。ITS2和COI序列比对及系统发育分析结果均表明，在10份分子鉴定样本中，9份为斯氏并殖吸虫，1份为白水河并殖吸虫；其中，9条ITS2序列与斯氏并殖吸虫参考序列（GenBank登录号：KX129924.1）同源性为98.34%～100.00%；1条ITS2序列与白水河并殖吸虫参考序列（GenBank登录号：OM401939.1）同源性为100.00%，其COI序列与白水河并殖吸虫参考序列（GenBank登录号：AB679288.1）同源性为98.80%；系统进化树分析结果显示上述10个分子鉴定样本分别聚类于斯氏并殖吸虫和白水河并殖吸虫分支。结论　本研究首次在云南省昭通市淡水蟹中检出白水河并殖吸虫，并确认斯氏并殖吸虫为该地区的优势虫种。基于ITS2和COI基因的分子鉴定与系统发育分析可有效区分虫种，从而为当地并殖吸虫病精准防控提供科学依据。

关键词: 并殖吸虫, 淡水蟹, 核糖体内部转录间隔区2, 线粒体细胞色素C氧化酶亚基I, 分子鉴定, 云南省