Sequence analysis of Paragonimus internal transcribed spacer 2 and cyclooxygenase 1 genes in freshwater crabs in Henan Province / 中国血吸虫病防治杂志

OBJECTIVE@#To investigate the sequences of internal transcribed spacer 2 (ITS2) and cyclooxygenase 1 (COX1) genes of Paragonimus metacercariae in freshwater crabs in Henan Province, identify the species of Paragonimus and evaluate its genetic relationships with Paragonimus isolates from other provinces in China.@*METHODS@#Freshwater crabs were collected from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province from 2016 to 2021, and Paragonimus metacercariae were detected in freshwater crabs. Genomic DNA was extracted from Paragonimus metacercariae, and the ITS2 and COX1 genes were amplified using PCR assay, followed by sequencing of PCR amplification products. The gene sequences were spliced and aligned using the software DNASTAR, and aligned with the sequences of Paragonimus genes in the GenBank. Phylogenetic trees were created using the MEGA6 software with the Neighbor-Joining method based on ITS2 and COX1 gene sequences, with Fasciola hepatica as the outgroup.@*RESULTS@#The detection rates of Paragonimus metacercariae were 6.83% (11/161), 50.82% (31/61), 18.52% (5/26), 8.76% (12/137), 14.29% (9/63), 17.76% (19/105), 18.50% (32/173) and 42.71% (41/96) in freshwater crabs from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province, with a mean detection rate of 19.46% (160/822), and a mean infection intensity of 0.57 metacercariae/g. The amplified ITS2 and COX1 gene fragments of Paragonimus were approximately 500 bp and 450 bp in lengths, respectively. The ITS2 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (99.8% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: MW960209.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with P. skrjabini from Sichuan Province (GenBank accession number: AY618747.1), Guangxi Zhuang Autonomous Region (GenBank accession number: AY618729.1) and Hubei Province (GenBank accession number: AY618751.1), and P. miyazaki from Fujian Province (GenBank accession number: AY618741.1) and Japan (GenBank accession number: AB713405.1). The COX1 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (90.0% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: AY618798.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with all P. skrjabini and clustered into the same sub-clade with P. skrjabini from Hubei Province (GenBank accession numbers: AY618782.1 and AY618764.1).@*CONCLUSIONS@#Paragonimus species from freshwater crabs in Henan Province were all characterized as P. skrjabini, and the ITS2 and COX1 gene sequences had the highest homology to those of P. skrjabini from Hubei Province. The results provide insights into study of Paragonimus in Henan Province and China.

Divergent long-terminal-repeat retrotransposon families in the genome of Paragonimus westermani

To gain information on retrotransposons in the genome of Paragonimus westermani, PCR was carried out with degenerate primers, specific to protease and reverse transcriptase (rt) genes of long-terminal-repeat (LTR) retrotransposons. The PCR products were cloned and sequenced, after which 12 different retrotransposon-related sequences were isolated from the trematode genome. These showed various degrees of identity to the polyprotein of divergent retrotransposon families. A phylogenetic analysis demonstrated that these sequences could be classified into three different families of LTR retrotransposons, namely, Xena, Bel, and Gypsy families. Of these, two mRNA transcripts were detected by reverse transcriptase-PCR, showing that these two elements preserved their mobile activities. The genomic distributions of these two sequences were found to be highly repetitive. These results suggest that there are diverse retrotransposons including the ancient Xena family in the genome of P. westermani, which may have been involved in the evolution of the host genome.

Phylogenetic relationship of ribosomal ITS2 and mitochondrial COI among diploid and triploid Paragonimus westermani isolates

We compared patterns of intraspecific polymorphism of two markers with contrasting modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), in the lung fluke, diploid and triploid Paragonimus westermani from three geographical regions of Korea. The genetic distances between three populations of Korean diploid and triploid P. westermani showed no significant difference in the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (mtCOI) and ribosomaal second internal transcribed spacer (ITS2) genes. A highly resolved strict-consensus tree was obtained that illustrated phylogenetically useful information of the ITS2 and mtCOI sequences from diploid and triploid P. westermani.

Paragonimiasis in Yongjia County, Zhejiang Province, China: clinical, parasitological and karyotypic studies on Paragonimus westermani.

Paragonimiasis in Yongjia County, Zhejiang Province, China, has been of such public health concern that a specialized Institute for Paragonimiasis Control has been established there. The study reported here involved both parasitological research on Paragonimus westermani in the endemic areas and a clinical analysis of 94 cases of paragonimiasis. Eggs were found in sputum, feces and brain tissue in 45 cases. Twenty patients, 19 of whom where children under 10 years of age, showed hepatic injury. Treatment with praziquantel or bithionol had satisfactory results in all cases. Dimensions of the parasite eggs, metacercariae from freshwater crabs and adult flukes suggested that the form of P. westermani in Yongjia is the 'small type': a suggestion supported by karyotypic studies on gonad cells from adult flukes collected from experimentally infected animals as all were found to be diploid. In conclusion, the 'small type' or diploid form of P. westermani is the cause of paragonimiasis pulmonary symptoms in Yongjia.

The karyotype of the lung fluke, Paragonimus siamensis.

The karyotype of chromosomes obtained from the germ cells of Paragonimus siamensis was analyzed by air-drying technique. The result revealed the diploid number of chromosomes to be 2n=22. The entire chromosomes consisted of one large-sized metacentric, four medium-sized subtelocentrics, three small-sized metacentrics or submetacentrics and three small-sized submeta-centrics or subtelecentrics. The relative arm lengths of the chromosomes were 20.13+/-1.21, 13.16+/-0.42, 11.07+/-0.48, 10.37+/-0.84, 9.29+/-0.52, 6.50+/-0.39, 6.42+/-0.34, 6.27+/-0.28, 6.04+/-0.58, 5.65+/-0.39 and 5.03+/-0.40% respectively. These data were comparable with those of other related species.