Species identification and ITS sequence analysis of Prosthogonimus in Grus japonensis

doi:10.12140/j.issn.1000-7423.2025.01.015

Abstract

Abstract:

Objective To identify the species of Prosthogonimus in Grus japonensis and analyze the ribosomal DNA internal transcribed spacer (ITS) gene sequence. Methods Prosthogonimus samples were collected from the G. japonensis cloaca in Zhalong National Nature Reserve, Heilongjiang Province. The ITS gene of Prosthogonimus was amplified using PCR assay, and the amplified product was sequenced. The sequencing results were aligned with sequences recorded in the NCBI database using the BLAST tool. Repetitive sequences were identified using the Repeat Masker tool, and sequence similarity analysis was performed using the MegAlign software. Sequence alignments were conducted using the MEGA 7.0, Clustal X, and Paup software, and a phylogenetic tree was created using the maximum parsimony method. Results A total of 5 Prosthogonimus specimens were collected from G. japonensis, and five ITS gene sequences were obtained. Three sequences (GenBank accession numbers: PQ634967, PQ634968 and PQ634969) were 1 240 bp in size and showed 98.2% sequence similarity with P. cuneatus (GenBank accession number: OQ344776), which were identified as P. cuneatus. The other two sequences (GenBank accession numbers: PP956934 and PP956935) were 1 183 bp in size and showed 97.7% sequence similarity with P. pellucidus (GenBank accession number: KP192732), which were identified as P. pellucidus. There were no repetitive sequences in 5.8S or ITS2 genes of P. cuneatus and P. pellucidus, and there were 16 and 17 repetitive sequences the ITS1 gene of P. cuneatus and P. pellucidus, respectively. Phylogenetic analysis revealed that the P. cuneatus from this study was firstly clustered with P. cuneatus from Turdusmerula and Anasplatyrhynchos (Czech Republic isolate) into a subclade, and then clustered with P. pellucidus from this study into a clade. Conclusion The Prosthogonimus species infecting G. japonensis in the Zhalong National Nature Reserve are P. cuneatus and P. pellucidus, with 16 and 17 repetitive sequences in the P. cuneatus and P. pellucidus ITS1, respectively. P. cuneatus and P. pellucidus share a closer phylogenetic relationship with the Czech Republic isolate of P. cuneatus.

Key words: Prosthogonimus cuneatus, Prosthogonimus pellucidus, ITS sequences, Grus japonensis

CLC Number:

R383.2

CAO Yu, LI Ye, JIN Zhenhua, WANG Likun, GAO Zhongyan, ZHANG Xianguang, JIANG Botao. Species identification and ITS sequence analysis of Prosthogonimus in Grus japonensis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(1): 97-102.

Figures/Tables 3

Table 1

Table 2

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重复序列（3'→5'） Repetitive sequence (3'→5')	长度/bp Length/bp	位置/bp Position/bp	重复次数Repetition
GGTAGAGCGCCTCAGTTCTATCAAACACTGCAGCCTCGTCTGCCTACGG-TAGAGCGCCTCAGTTCTATCAAAC	73	1，93	2
GGTAGAGCGCCTCAGTTCTATCAAACACTGCAGCCTCGTCTGCCTACGG-TAGAGCGCCTCAGTTCTATC	69	1，93，140	3
AGTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATCGGAAACTCTGT	49	220，318	2
AGTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATCGGAAACTCTG	48	220，269，318	3
GTCTGCCTACGGTAGAGCGCCTCAGTTCTATCAAACACTGCAGCCTC	47	83，130	2
GTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATCGGAAACTCTG	47	220，270，319	3
TCTGCAGTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATC	43	215，362	2
AGTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATC	38	269，318，367	3
GTCTGCCTACGGTAGAGCGCCTCAGTTCTATCAAAC	36	38，83，130	3
CTGCCTACGGTAGAGCGCCTCAGTTCTATCGGAAA	35	179，228，277，326	4
GTCTGCCTACGGTAGAGCGCCTCAGTTCTATC	32	38，83，130，178	4
TCTGCCTACGGTAGAGCGCCTCAGTTCTATC	31	39，84，131，178	4
CTGCCTACGGTAGAGCGCCTCAGTTCTATC	30	40，85，132，179，228，277，326，375	8
GGTAGAGCGCCTCAGTTCTATCAAAC	26	1，48，93，140,	4
GGTAGAGCGCCTCAGTTCTATC	22	1，48，93，140，187，236，285，334，383	9
GTTGTCAGTC	10	525，590	2

重复序列（3'→5'）Repetitive sequence (3'→5')	长度/bp Length/bp	位置/bp Position/bp	重复次数 Repetition
CTGCCTACGGTAGAGCGCCTCAGTTCTATC	30	224，371	2
CGGTAGAGCGCCTCAGTTCTATCGGAAA	28	231，280	2
AGTCTTGCCTGCCTACGGTAGAGCGC	26	314，363	2
CGGTAGAGCGCCTCAGTTCTATC	23	231，280，378	3
TCAGTTCTATCGGAAACTCTG	21	292，341	2
TCTGCAGTCTTGCCTGCCT	19	260，358	2
CTGCCTACGGTAGAGCGC	18	224，322，371	3
GAAAATCTGCAGTCTTG	17	206，255	2
TCAGTTCTATCGGAAA	16	243，341	2
TGCCTACGGTAGAG	14	83，225，323，372	44
AGTCTTGCCTGCCT	14	265，314，363	3
TCTGCAGTCTTG	12	211，260，358	3
CGGTAGAGCGC	11	231，280，329，378	4
AGAGCGCGTCA	11	186，333	2
TCAGTTCTATC	11	243，292，341，390	4
GTTGTCAGTC	10	521，586	2
GGTAGAGAGC	10	1，90	2