Objective: To compare the complete mitochondrial genome sequences of two phenotypes of Paragonimus westermani isolated from Fujian Province with different sizes of metacercariae, and perform a phylogenetic analysis of various geographical isolates of P. westermani from Asia, so as to identify the possible genetic characteristics associated with the P. westermani phenotypes.
Methods: P. westermani metacercariae with different sizes (large metacercariae, 380-420 μm in diameter; small metacercariae, 320-340 μm) isolated from freshwater crabs were used to infect dogs, and the eggs and adult worms of P. westermani were collected from the dog stool samples and lung tissues. Then, the egg size and morphology were compared. In addition, genomic DNA was extracted from the adult worms of the two phenotypes of P. westermani and used for the PCR amplification to yield the complete mitochondrial genome sequence. Sequence structure and phylogenetic analyses were performed based on the complete mitochondrial genome of P. westermani.
Results: Following infection with large and small P. westermani metacercariae, the adult worms recovered from the dog lung had a thick body, and had oral and ventral suckers. The ventral sucker was located slightly in front of the midline of the body, and testes, ovary and vitelline gland were seen in the adult worms. Following fixation, the adults appeared oval, with an approximately 1.7∶1 of the length-width ratio. The length and width of the eggs isolated from the fecal samples of dogs infected with large and small P. westermani metacercariae varied significantly, and the large metacercariae produced bigger eggs than the smaller metacercariae. Based on the morphological features of adults and eggs and the ITS2 sequences, both phenotypes were identified as P. westermani. The complete mitochondrial genome sequence analysis of adults showed almost consistent sequences in the protein-coding region of the mitochondrial genome of adult worms derived from large and small metacercariae, with a major variation seen in the former non-coding region. Sliding window analysis revealed the most polymorphic region within the ND4 gene across the mitochondrial genome from various geographical isolates of P. westermani, and phylogenetic analysis showed that both phenotypes were clustered into the Chinese branch of P. westermani, which was close to the Japanese branch and distinct from the South/Southeast Asian branch.
Conclusions: The genetic distance between the phenotypes of P. westermani isolated from Fujian Province is near at a mitochondrial genome level, with no remarkable genetic differentiation seen; however, the mutation and structural changes in the non-coding region may result in the phenotypic variations. In addition, there is a distinct variation of the evolutionary rate in the mitochondrial coding genes, suggesting the selection of appropriate molecular markers during the phylogenic researches.
［摘要］ 目的 对福建省大、小囊蚴来源的两型卫氏并殖吸虫线粒体基因组序列进行比较和系统发育分析, 探寻其可能 存在的与表型相关的遗传特征。方法 以在福建省南平市延平区太平镇溪蟹中分离出的两种大小不同的囊蚴 (直径分 别为 380～420 μm和 320～340 μm) 分别感染犬, 收集犬粪及肺部虫卵及成虫。比较两种来源虫卵大小及外形差异; 抽 提两型并殖吸虫成虫基因组DNA, 扩增并获得线粒体全基因组序列, 进行序列结构及系统发育关系分析。结果 大、小 囊蚴分别感染犬后, 所获得的成虫体肥厚, 具口、腹吸盘, 腹吸盘位于体中线稍前, 可见睾丸、卵巢和卵黄腺等结构, 固定 后呈椭圆形, 长宽比约为 1.7∶1。两组卵在长、宽上均有显著性差异, 大囊蚴感染后所获虫卵亦较大。两组成虫和卵形态 特征及ITS2 基因序列符合卫氏并殖吸虫特征。成虫线粒体基因组序列分析显示, 大、小囊蚴来源的成虫线粒体基因组 蛋白编码区序列基本一致, 主要差异区域位于前非编码区。滑动窗口分析显示, 不同地域来源的卫氏并殖吸虫线粒体基 因组间, 多态性最大区域位于ND4 基因内。系统发育分析表明, 两型并殖吸虫均聚入卫氏并殖吸虫中国地域分支, 与韩 国/日本株关系较近, 与 (东) 南亚株遗传距离较远。结论 中国福建大、小囊蚴来源的两型卫氏并殖吸虫在线粒体基因 组水平上遗传距离较近, 未表现出明显分化, 但非编码区突变和结构变化可能影响了两者在表型上的差异。此外, 线粒 体各编码基因的进化速率存在明显差异, 提示在进行系统发育等相关研究时, 应注意选择合适的分子标记。.
Keywords: Genetic differentiation; Mitochondrial genome; Paragonimus westermani; Phenotype; Phylogeny.