Morphological and Molecular Characterization of Paragonimus Species Isolated from Freshwater Crabs in Southern Yunnan, China.

Miao-Miao Wang,Wen-Wei Bai,Shu-Mei-Qi He,Ming Tian,Maoshui Wang,Wen-Lin Wang,Yang Yang,Shu-De Li,Yong Meng,Qiu-Hong Shu

doi:10.1155/2021/5646291

Abstract

Paragonimus species are highly prevalent in various regions of China. The study's objective is to isolate and identify Paragonimus from natural habitats and compare the phylogenetic diversity of Paragonimus in southern Yunnan province, China. Metacercariae of Paragonimus was isolated from crabs, and morphologic identification was performed by microscopy. Metacercariae were injected into experimental Paragonimus free Sprague Dawley rats. After 114 days, adult worms and eggs were isolated from multiple organs. Morphologic identification confirmed the initial identification. DNA was extracted from 5 adult worms, and molecular characterization was performed by amplification and sequencing of CO1 and ITS2 regions, followed by phylogenetic analysis. Out of 447 crabs captured, 186 crabs were found to be infected. A total of 4 species of Paragonimus was observed from naturally infected crabs. Paragonimus microrchis (2), Paragonimus heterotremus (1), Paragonimus proliferus (1), and Paragonimus skrjabini (1) were isolated and identified. A total of 32 sequences were downloaded from the National Center for Biotechnology Information, and 5 sequences generated in the study were used for phylogenetic analysis. In the phylogenetic tree of the CO1 gene, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini were clustered with the same species, and the confidence values of their branches were >95%. A congruent phylogenetic relationship was observed with the ITS2 phylogenetic tree. In the phylogenetic tree constructed with the combined dataset of CO1 and ITS2 datasets, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini clustered with the same species, and their branch confidence values were >94%. Paragonimus microrchis clustered with Paragonimus bangkokensis in both datasets. Phylogenetic analysis revealed robustness of the double loci method as against the single-locus method with either CO1 or ITS2 alone. Paragonimus species isolated from the southern Yunnan province, China, was phylogenetically diverse, and the analysis revealed the clustering of multiple species of Paragonimus isolated from different geographic locations.

Highlights

Trematodes belonging to the genus Paragonimus are important parasites causing zoonotic infections in vertebrates, including human beings. ey are of socioeconomic importance since the route of infection is mainly food-borne [1]. e life cycle of Paragonimus is relatively complex that requires a minimum of three hosts, including a definitive host and two intermediate hosts [2]. e first and second intermediate hosts are frequently snails belonging to the families Assimineidae and Hydrobiidae and crabs belonging to the families Potamidae and Parathelphusidae [2]
E genus Paragonimus is rich in species diversity that has been reported from varied geographies, including Asia, Africa, and Central and South America spanning both tropical and temperate climates [5]. e species diversity is reflected by the various phylogenies of Paragonimus and mainly comprises 3 species complexes
In the phylogenetic tree of the CO1 gene, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini were clustered with the same species, and the confidence values of their branches were more than 95% (Figure 4)

Summary

Introduction

Trematodes belonging to the genus Paragonimus are important parasites causing zoonotic infections in vertebrates, including human beings. ey are of socioeconomic importance since the route of infection is mainly food-borne [1]. e life cycle of Paragonimus is relatively complex that requires a minimum of three hosts, including a definitive host and two intermediate hosts [2]. e first and second intermediate hosts are frequently snails belonging to the families Assimineidae and Hydrobiidae and crabs belonging to the families Potamidae and Parathelphusidae [2]. Different species of Paragonimus have their own predilection for infecting specific genera of snails and crabs and the Journal of Tropical Medicine epidemiological prevalence of different species of Paragonimus is determined by the existence of suitable hosts. Some species of Paragonimus have been identified only from intermediate hosts in certain geographies which suggests infection and maintenance in nonhuman mammals [3, 4]. E genus Paragonimus is rich in species diversity that has been reported from varied geographies, including Asia, Africa, and Central and South America spanning both tropical and temperate climates [5]. Paragonimus westermani is the most commonly reported species complex found in South and South East Asia [6]. Ere were previous instances of discordance in morphology and the ontogenetic changes predicted by both molecular and isozyme-based methods [11]. One of the main reasons for the observed discordance is the lack of clarity on the species boundaries and mendelian polymorphism leading to morphological changes causing the creation of distinct species that are interfertile [12]

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Results

Conclusion