关键词: 冠缝属吸虫, 二叶冠缝吸虫, 白琵鹭, 分离鉴定

Abstract:

Objective To identify the species of 10 trematodes isolated from a dead adult Platalea leucorodia that had been rescued in the Poyang Lake area, and to analyze the sequence characteristics and genetic and evolutionary relationships of mitochondrial DNA (mtDNA)-encoded cytochrome C oxidase subunit I (cox1) gene and ribosomal DNA (rDNA)-encoded internal transcribed spacer (ITS) sequence. Methdos A trematode worm body-shaped structure isolated from the duodenum of P. leucorodia was observed under an optical microscope, and three worms were selected for hydrochloric acid carmine staining and preliminary identification of genus and species under a microscope. In addition, genomic DNA was extracted from another three selected worm samples for PCR amplification of cox1 and ITS sequences, and the amplification products were sequenced. Sequence alignment and homology search were performed in the GenBank database, and repetitive DNA and base content were estimated. Schistosoma japonicum cox1 and ITS sequences were selected as outgroups to estimate intraspecies and interspecies genetic distances, and to build phylogenetic trees with the neighbor-joining (NJ) method. Results Isolated ten trematode worms were fleshy and thick in shape and pale red in color, with a mean length of (14.75 ± 1.89) mm. The cephalic collar was distinctly bilobed and wider than the worm body, and the head spines had blunt rod-shaped ends. Hydrochloric acid carmine staining revealed that the worm body had 58 to 59 head spines, and 4 angular spines. The cox1 gene and ITS sequences of the three trematodes have lengths of 426 and 985 bp, respectively. In the base composition of the cox1 gene sequence, the content of A + T (61.74%) was significantly higher than that of C + G (38.26%), indicating a marked AT bias. Conversely, in the base composition of the ITS gene sequence, the content of A + T (46.10%) was lower than that of C + G (53.90%), showing a GC preference. No repetitive sequences were detected in the cox1, ITS1, or 5.8S rRNA gene sequences of the trematodes, whereas a 10 bp inverted repetitive sequence was identified in the ITS2 gene sequence. The cox1 gene sequence of the trematode exhibited a 92.86% homology and the closest genetic distance (0.079) with Patagifer (GenBank accession number: OQ606511), and the ITS sequence of the trematode showed a 98.57% homology and the closest genetic distance (0.014) with P. bilobus (GenBank accession number: ON141921). The trematode shared the same small clade with trematodes of the genus Patagifer within the large clade of Echinostomatidae. Conclusion Morphological and molecular biological identification confirms the trematode isolated from P. leucorodia as P. bilobus.

Key words: Patagifer sp., Patagifer bilobus, Platalea leucorodia, Isolation and identification