关键词: 广州管圆线虫, 福寿螺, 遗传多态性, 系统发育分析, 单倍型, 福建省

Abstract:

Objective To investigate the distribution of Pomacea and the prevalence of Angiostrongylus cantonensis infections in Pomacea in four major river systems of Fujian Province, and to analyze the population genetic polymorphisms and phylogenetic relationships of Pomacea. Methods Pomacea samples were collected from 13 sampling sites across four major river systems in Fujian Province from May to September 2025, including the Min River, Jiulong River, Jin River and Ting River, and A. cantonensis infections in Pomacea were detected using lung microscopy. One individual Pomacea was sampled from each sampling site for extraction of genomic DNA, and mitochondrial cytochrome c oxidase subunit 1 (COⅠ) gene was amplified using PCR assay, followed by sequencing. Sequencing raw reads were assembled using the SeqMan software, and the obtained target sequences were submitted to the NCBI database for sequence homology alignment using the BLAST tool. The base composition and number in DNA sequences and length of DNA sequences were analyzed with the DnaSP software, and the haplotype type and number in COⅠ gene sequences were calculated. Pomacea COⅠ gene sequences were downloaded from the GenBank. The downloaded haplotype sequences of the Pomacea COⅠ gene obtained from this study were aligned using the ClustalW program. After trimming the head and tail redundant sequences, the genetic distance was calculated among different haplotypes using the Kimura 2-parameter model with the MEGA 7.0 software, and phylogenetic trees were built using the neighbor-joining (NJ) method. Results A total of 892 Pomacea snails were collected, among which 690 individuals were randomly selected for the detection of A. cantonensis infections, with an overall prevalence rate of 4.92% (34/690). The prevalence of A. cantonensis infections was 5.62% (25/445) in Pomacea snails from Min River, 5.00% (1/20) from Ting River, 4.00% (6/150) from Jiulong River, and 4.00% (2/50) from Jin River, respectively. PCR assay showed that the Pomacea COⅠ gene was 691 bp in length. Among the obtained 13 sequences, 8 belonged to P. canaliculata and 5 belonged to P. occulta. A total of 76 polymorphic sites were detected in the 13 COⅠ gene fragments, accounting for 11.00% of total sites, and no single variable sites, insertion or deletion sites, base transitions or transversions were identified. The average contents of the four nucleotides T, A, G and C were 40.1%, 23.8%, 19.9% and 16.2%, respectively. Three haplotypes (HAP-1, HAP-2, and HAP-3) were annotated from these 13 sequences, with a haplotype diversity of 0.705 1. HAP-1 was derived from Min River, Jiulong River and Ting River, and HAP-2 was derived from Min River, Jiulong River and Jin River, while HAP-3 was derived from Min River and Jiulong River. HAP-1 showed the closest genetic distance with P. occulta (GenBank accession number: PV793390), and the farthest genetic distance of 0.232 to the outgroup Pila conica (GenBank accession number: EF514948), and HAP-2 and HAP-3 shared the closest genetic distance with P. canaliculata from the Philippines (GenBank accession number: EU528483), and the largest genetic distance with the outgroup P. conica. The genetic differentiation distance ranged from 0.051 to 0.100 among the three haplotypes, and HAP-2 and HAP-3 shared the closest phylogenetic relationship with a genetic distance of 0.051. Phylogenetic analysis showed that HAP-2 was clustered with P. canaliculata from the Philippines into the same clade, HAP-3 formed an independent clade and belonged to the same major evolutionary clade with HAP-2, while HAP-1 was clustered with P. occulta from Shanghai area into the same clade. Conclusion The infection rate of A. cantonensis in apple snails in Fujian Province has decreased significantly compared with previous years. There are two species of Pomacea, including P. canaliculata and P. occulta, distributed in different river systems, and genetic differentiation has occurred within the populations of P. canaliculata.

Key words: Angiostrongylus cantonensis, Pomacea, Genetic polymorphism, Phylogenetic analysis, Haplotype, Fujian Province