Analysis of COI and ITS2 regions of DNA obtained from <i>Paragonimus westermani</i> eggs in ancient coprolites on Joseon dynasty mummies

The genetic information of ancient Paragonimus westermani, the oriental lung fluke infecting over 20 million people worldwide, has not been thoroughly investigated thus far. We analysed genetic markers (COI and ITS2) of P. westermani from coprolite specimens (n = 6) obtained from 15th to 18th century Korean mummies. Our results indicated that all P. westermani sequences were generally distinct from the other species of the genus Paragonimus. The sequences were clustered into three groups: Group I for East Asia; Group II for South and Southeast Asia; and Group III for India and Sri Lanka. In this study, we found that ancient P. westermani sequences in Korea belong to Group I, adding invaluable information to the existing knowledge of Paragonimus paleogenetics.

Key words:
ancient parasite; Paragonimus westermani; ancient DNA

Authorship

Jong Ha Hong

Seoul National University College of Medicine, Laboratory of Bioanthropology, Paleopathology and History of Diseases, Seoul, South KoreaSeoul National University College of MedicineSouth KoreaSeoul, South KoreaSeoul National University College of Medicine, Laboratory of Bioanthropology, Paleopathology and History of Diseases, Seoul, South Korea

Chang Seok Oh

Seoul National University College of Medicine, Institute of Forensic Science, Seoul, South KoreaSeoul National University College of MedicineSouth KoreaSeoul, South KoreaSeoul National University College of Medicine, Institute of Forensic Science, Seoul, South Korea

Min Seo

Dankook University College of Medicine, Department of Parasitology, Cheonan, South KoreaDankook University College of MedicineSouth KoreaCheonan, South KoreaDankook University College of Medicine, Department of Parasitology, Cheonan, South Korea

Dong Hoon Shin ⁺+ Corresponding author: cuteminjae@gmail.com

http://orcid.org/0000-0001-8032-1266

+ Corresponding author: cuteminjae@gmail.com

DHS and MS designed this study as leading authors; MS performed microscopic examination; JHH and OCS performed the molecular experiments; JHH, MS, and DHS analysed the data and wrote the manuscript.

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Figures | Tables

Figures (2)
Tables (2)

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Fig. 1:
agarose gel electrophoresis for the polymerase chain reaction (PCR) amplicons of Paragonimus westermani aDNA from Cheongdo and YG2-4 mummies. Note specific amplicons 1 (106 bp), 2 (150 bp), 3 (136 bp), 4 (114 bp), and 5 (183 bp) of the COI region and 1 (133 bp), 2 (169 bp), 3 (129 bp), 4 (165 bp), and 5 (170 bp) of ITS2 region.

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Fig. 2:
maximum likelihood tree of Paragonimus (A) COI and (B) ITS2 DNA region. The percentage of replicate trees in which the associated taxa clustered in the bootstrap test are marked next to the branches. The ancient P. westermani sequences revealed in this study are represented by red dots.

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TABLE I
List of primers used for amplification of Paragonimus westermani DNA in this study

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TABLE II
BLAST searching results for the coverage and percent identity of each taxon comparing to the consensus sequence of Paragonimus westermani COI and ITS2 from Korean mummies. GenBank accession numbers and geographical information are also indicated

Fig. 1: agarose gel electrophoresis for the polymerase chain reaction (PCR) amplicons of Paragonimus westermani aDNA from Cheongdo and YG2-4 mummies. Note specific amplicons 1 (106 bp), 2 (150 bp), 3 (136 bp), 4 (114 bp), and 5 (183 bp) of the COI region and 1 (133 bp), 2 (169 bp), 3 (129 bp), 4 (165 bp), and 5 (170 bp) of ITS2 region.

Fig. 2: maximum likelihood tree of Paragonimus (A) COI and (B) ITS2 DNA region. The percentage of replicate trees in which the associated taxa clustered in the bootstrap test are marked next to the branches. The ancient P. westermani sequences revealed in this study are represented by red dots.

TABLE I List of primers used for amplification of Paragonimus westermani DNA in this study

Region	Set	Primer	5’ to 3’	Annealing temp. (ºC)	Length (bp)
COI	COI1	COI-1F	GGG CAT CCG GAG GTG TAT GT	54	106
		COI-1R	TTC GGG TAC TAC GGG CTG G
	COI2	COI-2F	CTG ACC AAC AAC GAT TCC T	54	150
		COI-2R	TCC CGT GAC AGA ACT AAA GA
	COI3	COI-3F	GTC TGG GTA GTG TTG TGT GG	54	136
		COI-3R	AGC ATG AAC AAC CAA GAG AA
	COI4	COI-4F	TTA GTT CTG TCA CGG GAG TG	57	114
		COI-4R	GAA TTC ACC ACA AAA CAG GA
	COI5	COI-5F	TTC TCT TGG TTG TTC ATG CT	54	183
		COI-5R	GAC GTA ATG AAA ATG AGC C
ITS2	ITS2-1	ITS2-1F	GCG CAG CCA ACT GTG TGA A	57	133
		ITS2-1R	GGC GTC GCG ATA GTT TAT
	ITS2-2	ITS2-2F	TTA ATG CGA ACT GCA TAC TG	54	169
		ITS2-2R	AAG ACC AGA TTG GGG AGA T
	ITS2-3	ITS2-3F	GGT CGG CTT ATA AAC TAT CG	54	129
		ITS2-3R	CCC GAG TAT GTT AGG GAA A
	ITS2-4	ITS2-4F	AAT CTG GTC TTG TGC CTG T	60	165
		ITS2-4R	AAA CCA CAG ATG AAG ACA GG
	ITS2-5	ITS2-5F	GTG GCT CAG TGA ATG ATT TAT	54	170
		ITS2-5R	CCG CTT AGT GAT ATG CTT A

TABLE II BLAST searching results for the coverage and percent identity of each taxon comparing to the consensus sequence of Paragonimus westermani COI and ITS2 from Korean mummies. GenBank accession numbers and geographical information are also indicated

DNA region	Species	Coverage	Percent identity	Accession number	Geographical region
COI	P. westermani	100%	100%	U97205.1	Japan
		100%	99%	AF333280.1	South Korea
		100%	99%	AF540958.1	South Korea
		100%	99%	U97208.1	Japan
		98%	99%	AY140680.1	China
		100%	98%	U97209.1	China
		99%	96%	FJ434988.1	Vietnam
		100%	93%	JN656169.1	India
		99%	86%	KM280646.1	India
		99%	90%	U97213.1	The Philippines
		99%	89%	AB354224.1	Thailand
		92%	84%	AY240940.1	Sri Lanka
	P. siamensis	99%	95%	AB354231.1	Thailand
	P. paishuihoensis	99%	84%	AB679289.1	Laos
ITS2	P. westermani	100%	100%	KC417492.1	China
		100%	100%	AB713404.1	China
		100%	100%	AB938198.1	India
		100%	99%	AF333277.1	South Korea
		100%	99%	JN656205.1	India
		100%	99%	JN656208.1	India
		100%	99%	U96908.1	Taiwan
		100%	99%	LC144902.1	Vietnam
		100%	99%	AB354217.1	Thailand
		100%	98%	DQ836243.1	India
		100%	98%	JN656199.1	India
		100%	97%	DQ351845.1	India
		100%	97%	AB354214.1	Thailand
		97%	99%	FJ434982.1	Vietnam
		87%	100%	JF500452.1	Ancient South Korea
		87%	100%	U96907.1	Japan
		87%	98%	U96909.1	Malaysia
		87%	97%	AF159604.1	Thailand
		68%	95%	AY240942.1	Sri Lanka
	P. siamensis	100%	96%	AB354222.1	Thailand
	Euparagonimus cenocopiosus	87%	93%	AF159601.1	China

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Analysis of COI and ITS2 regions of DNA obtained from <i>Paragonimus westermani</i> eggs in ancient coprolites on Joseon dynasty mummies

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[1] + Corresponding author: cuteminjae@gmail.com