Abstract:

Objective To characterize the trematode species isolated from Grus japonensis in the Heilongjiang Zhalong Nature Reserve, and to analyze the phylogenetic relationships of the trematode using ribosomal internal transcribed spacer (ITS) and 28S DNA sequences as molecular markers. Methods Trematodes were isolated from intestines of naturally deceased G. japonensis in the Heilongjiang Zhalong Nature Reserve and stained with hydrochloric acid-carmine to observe the morphological characteristics of trematodes. DNA was extracted from trematodes, and ITS and 28S sequences were amplified using PCR assay. PCR amplification products were sequenced, followed by sequence analysis, similarity analysis and repetitive sequence analysis. In addition, phylogenetic trees were built using the maximum parsimony method. Results The isolated trematodes had enlarged bodies, and well-developed, distinctly bilobed head collars. The length of the ITS sequence was 1 007 bp, and the lengths of ITS1, 5.8S and ITS2 sequences were 389, 161 and 457 bp, respectively. The G + C content of the ITS sequence was 52.14%. The length of 28S sequence was 983 bp and the G + C content was 56.79%. The ITS2 sequence had 2 forward repeat sequences with 11 bp in length and the 28S sequence had no repeat sequences. The ITS and 28S sequences of isolated trematodes shared 94.39% and 98.27% sequence similarities with those of P. bilobus (GenBank accession numbers: ON141931 and KT956945), and clustered into the same sub-clade with Patagifer species. The isolated trematodes were therefore identified as P. bilobus. Conclusion The trematodes isolated from G. japonensis in the Heilongjiang Zhalong Nature Reserve are P. bilobus.

Key words: Patagifer spp., Patagifer bilobus, Grus japonensis, Morphology, Molecular identification