关键词: 淡水螺类, 吸虫, 形态学, 分子生物学, 感染情况, 拉萨

Abstract:

Objective To investigate the species of freshwater snails and the prevalence of trematode infections in freshwater snails in Lahsa City, and to analyze the phylogeny of freshwater snails and trematodes. Methods Freshwater snail samples were collected from Chengguan District, Duilongdeqing District, Linzhou County, Dazi District and Qushui County of Lhasa City from June to July 2024, and characterized for species, and the morphology and infection of trematodes were observed using microscopy. Genomic DNA was extracted from freshwater snails and trematodes for amplification of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene of freshwater snails and the internal transcribed spacer 2 (ITS2) of trematodes using PCR assay, and the PCR amplification products were sequenced. Raw reads were assembled using the DNASTAR software, and the obtained target sequences were submitted to the NCBI database for sequence alignment using the BLAST tool. Phylogenetic trees were constructed using the neighbor-joining and maximum likelihood methods, and genetic distances were calculated using the software MEGA 11.0. Results A total of 1 022 freshwater snails were collected and 8 genera were morphologically identified. Multiple developmental stages of trematode larvae were observed using microscopy, including miracidia, sporocysts, rediae, cercariae, and metacercariae. PCR amplification displayed the CO1 fragment from freshwater snails with approximately 740 bp in length and the ITS2 fragment from trematodes with approximately 630 bp in length, which were consistent with the expected size of target fragments. Gene sequencing and sequence alignment revealed that the CO1 sequence of freshwater snails exhibited the highest sequence identity with those of Physella acuta (98.66%), Ampullaceana lagoti (99.23%), Bellamya aeruginosa (99.29%), Galba pervia (100%), Gyraulus sp. (98.78%), Orientogalba viridis (98.91%), Radix Auricularia (99.38%), R. makhrovi (100%) and Tibetoradix khamensis (99.65%), and phylogenetic analysis revealed 8 clusters, with species of each genus clustering independently into a single clade. The ITS2 sequence of trematodes exhibited 100% sequence identity with Diplostomum mergi, Cotylurus syrius, Apatemon sp., Plagiorchis maculosus, Fasciola hepatica, F. gigantica, and Paramphistomum cervi, and showed the highest sequence identity with Schistosoma turkestanicum (99.80%), Echinostoma revolutum (99.84%), and Notocotylidae sp. (98.78%), respectively. Phylogenetic analysis revealed 9 clusters, with species of each genus clustering independently into a single clade, and all clades were supported by high bootstrap values, generating stable monophyletic clades. The overall prevalence of trematode infects was 59.59% (609/1 022) in freshwater snails across five counties (districts), with the highest prevalence in Qushui County (73.80%, 138/187), and the lowest in Chengguan District (39.24%, 31/79). Dazi District exhibited the greatest species diversity of freshwater snails (7 species), whereas Duilongdeqing District and Linzhou County harbored the highest trematode species (9 species each). In addition, Qushui County had the fewest species of both freshwater snails (1 species) and trematodes (2 species). Conclusion Freshwater snail species are abundant in the high-altitude waters of Lhasa City, with diverse trematode species and high prevalence of trematode infections.

Key words: Freshwater snail, Trematode, Morphology, Molecular biology, Infection prevalence, Lhasa