Analysis of COI and ITS2 regions of DNA obtained from Paragonimus westermani 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.

Paragonimiasis occurs in definitive hosts after ingestion of infected intermediate hosts. (1) In general, Paragonimus eggs hatch before entering snails (the first intermediate hosts). When the cercariae penetrate crustaceans such as crayfishes or crabs (the second intermediate hosts), they encyst in gills, muscles, and viscera, developing into metacercariae. After definitive hosts (e.g. humans) eat the freshwater crustaceans raw, the metacercariae excyst in the small intestine, penetrating the intestinal wall. They then traverse the diaphragm, entering the peribronchiolar tissues of the lung where they mature into adults within 8-12 weeks. (2) The genus Paragonimus constitutes a species-rich group from the tropical regions in South and Southeast Asia, Africa, and Central and South America as well as the temperate zone of North America and East Asia. (3) Almost 50 nominal species and subspecies of Paragonimus have been reported so far; (1) and approximately 16 species have been revealed to cause human diseases. Paragonimiasis mainly affects the lung. The adult worms stimulate the formation of worm capsules which then ulcerate, potentially leading to clinical symptoms such as cough, blood-tinged sputum, and pulmonary pain. Paragonimiasis could also be induced by the migration of young adult worms to the ectopic organs. (2,4,5,6,7) Among Paragonimus, the most common species is P. westermani, the oriental lung fluke mainly reported in Korea, China, Taiwan, Japan, and the Philippines. (1,8,9) Scholars have estimated that over 20 million people are currently infected with P. westermani worldwide. (1,10) Recently, researchers have attempted to reveal the genetic characteristics of P. westermani through DNA analysis. (1,8,11,12,13,14,15) Phylogenetic analyses of cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) DNA regions have revealed that P. westermani are clustered into at least two groups (East Asia and South/Southeast Asia), in association with geographically distinct distributions. (1,8,11,12,13) However, although ancient eggs have been detected in archaeological samples, very few parasitological reports regarding paleogenetics of ancient Paragonimus spp. have been published so far. The only report was our previous study on ITS2 DNA sequence of ancient P. westermani eggs obtained from a 17th century Korean mummy. (7) In that study, the ancient DNA sequence of P. westermani was very similar to that of modern P. westermani reported in East Asia, but was genetically distinct from the P. westermani sequences of Southeast Asia. (7) Nevertheless, our previous study was performed with only one sample and a single genetic marker (ITS2). Over the past several years, the genetic information of another P. westermani genetic marker (COI) has been continuously accumulated by multiple researchers. (11,12,13,16,17) Our paleoparasitological studies have also microscopically detected ancient Paragonimus eggs in many more coprolite specimens (n = 6) from the 15th to 18th century Korean mummies. (18,19) In this regard, we attempted to examine multiple genetic markers (COI and ITS2) in newly collected ancient P. westermani eggs in order to obtain more comprehensive information about the evolutionary history of P. westermani.
The specimens were ancient faeces from the intestines of mummies (Cheongdo and Hwasung) or the organic materials compiled upon the hipbones of half-mummified bodies (Dangjin, YG2-4, YG2-6 and Yongin).
To authenticate our work on ancient DNA (aDNA), we followed the Criteria of Authentication established by Hofreiter et al. (20) During aDNA analysis, we wore head caps, masks, protection gloves, and gowns. All the tools used in this study were sterilized before use. We also used specialised facilities that were exclusively dedicated to aDNA analysis. (21) We performed experiments in this facility that were developed in accordance with the Criteria. (20) The Institutional Review Board (IRB) of Seoul National University Hospital confirmed that our aDNA analyses could be exempt from board review (IRB No. 2017-001). We also followed the Vermillion Accord on Human Remains adopted by the World Archaeological Congress. (22) For DNA extraction, we followed the method described in our previous report. (23) In brief, the samples (0.3 g each) were incubated at 56ºC in 1 mL of lysis buffer (pH 8.0; including 1 % SDS; 0.1M DTT; 50 mM of EDTA; 1 mg/mL of proteinase K) for 24 h. After DNA was extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), it was then treated with chloroform/isoamyl alcohol (24:1). DNA isolation and purification were performed using a QIAmp PCR purification kit (Qiagen, Hilden, Germany). The purified DNA was eluted in 40 μl of EB elution buffer (Qiagen, Hilden, Germany). Primers for the P. westermani COI and ITS2 regions were generated by Integrated DNA Technologies, Inc. (Iowa City, IA, USA). The information of the primers used in our study is summarised in Table I. DNA quantification was performed using a Nano-Drop TM ND-1000 Spectrophotometer (Thermo Fisher Scientific, MA, USA). Extracted DNA (10 μL) was treated at 37ºC with 1 unit of uracil-DNA-glycosylase (New England Biolabs, MA, USA) for 30 min. It (40 ng) was then mixed with the reagent premix containing 10 pmol of each primer and 1X AmpliTaq Gold® 360 Master Mix (Life Technologies, CA, USA). PCR conditions were as follows: pre-denaturation at 95ºC for 10 min, 45 cycles of denaturation at 95ºC for 30 s, annealing at 54-60ºC for 30 s, extension at 72ºC for 30 s, and final extension at 72ºC for 10 min. The polymerase chain reaction (PCR) products were separated on 2.5% agarose gel (Invitrogen, CA, USA), and then stained with ethidium bromide. Negative controls (extraction controls) were also applied to the electrophoresis at the same time. Electrophoresis results were photographed using a Vilber Lourmat ETX-20.M equipped with Biocapt software (Vilber Lourmat, Collégien, France).  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 The PCR amplicons were isolated using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The bacteria were transformed using the pGEM-T Easy Vector system (Promega Corporation, Madison, USA). Transformed bacteria were grown on an agar plate containing X-GAL (40 μg/μL), ampicillin (50 μg/mL), and 0.5 mM IPTG for the 14 h. After selected colonies were grown in LB media for 12 h, the purification of cultured bacteria was performed using a QIAprep® Spin Miniprep kit (Qiagen, Hilden, Germany).
Each amplified strand was sequenced using an ABI Prism BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Waltham, USA) and the 3730xl Automatic Sequencer (Applied Biosystems, Waltham, USA). The obtained DNA sequences were aligned by MEGA7 program. (24) The consensus sequences were compared to those available in GenBank using NCBI/BLAST tools. (25) The web browser module and Alignment Explorer of MEGA7 were used to retrieve sequences homologous to those of interest from NCBI GenBank database.
The evolutionary relationship of P. westermani and other taxa from NCBI GenBank was inferred from the Phylogeny Reconstruction analysis implemented in MEGA7. (24) We used the Maximum Likelihood (ML) method. Selected parameters of our method followed the Tamura-Nei model (26) for COI or the Kimura 2-parameter model (27) for ITS2; we selected Complete Deletion for Gaps/Missing data treatment, Uniform Rates for Rates among Sites, and Nearest-Neighbor-Interchange (NNI) for the ML Heuristic Method. To estimate the reliability of the tree, we performed a bootstrap test. (28) The number of bootstrap replicates was 1000.
To select the specimens used for this experiment, we screened all the mummy coprolite samples using PCR with P. westermani-specific primers (COI1 and ITS2-1 in Table I). In agarose gel electrophoresis, the amplified products specific for P. westermani primer sets COI1 (106 bp) and ITS2-1 (133 bp) were detected in only two samples (Cheongdo and YG2-4) while negative controls (extraction controls) did not exhibit any amplified bands (Fig. 1). Thus, we used Cheongdo and YG2-4 mummy specimens for subsequent experiments.
To obtain the consensus sequences of COI and ITS2 regions, we repeated cloning and sequencing for each specific amplicon. From these trials, 9-12 clone sequences were successfully acquired for the COI (390 bp) and ITS2 (417 bp) regions. Next, multiple sequence alignment was performed using Clustal W implemented in MEGA7. We then obtained consensus sequences after alignment of the cloned sequences [Supplementary data ( Fig. 1)]. The consensus sequences of the P. westermani COI and ITS2 regions from the Cheongdo and YG2-4 mummies were identical [Supplementary data (Fig. 2)].
Next, we performed phylogenetic analysis of P. westermani COI and ITS2 regions (Fig. 2). Every P. westermani taxon in the COI and ITS2 regions was clearly distinct from other Paragonimus species (P. siamensis, P. skrjabini, P. paishuihoensis and P. mexicanus). P. westermani sequences were clustered into several groups. In the case of the COI region, Group I included P. westermani sequences from East Asia (Korea, Japan, and China) whereas Group II included those from South and Southeast Asia (India, Philippines, and Thailand) and formed another cluster. We note that some sequences reported from India and Sri Lanka formed a separate cluster of the third separate group (Group III) (Fig. 2A). P. westermani COI sequences obtained from Korean mummies in this study evidently belong to the East Asia group. Our result was similar to those of the phylogenetic analyses conducted by Iwagami et al. (13,17) and Devi et al., (8,16) which yielded that the P. westermani COI region was clustered into East, Southeast, and South Asia groups ( Fig. 2A). Like the COI region, P. westermani ITS2 sequences reported from East Asia (Korea, Japan, and China; Group I), Southeast Asia (Thailand; Group II), and South Asia (India; Group III) were also separately clustered in the phylogenetic analysis (Fig. 2B). The P. westermani ITS2 sequences from Cheongdo and YG2-4 Korean mum- mies in our study belonged to the East Asia group as well. Taken together, our phylogenetic tree of the ITS2 region was generally similar to those reported in previous studies conducted by Iwagami et al. (13,17) and Devi et al. (8,16) However, different from the COI region, there was also a unique finding of our examination of the ITS2 region of P. westermani. In brief, some P. westermani ITS2 sequences from South Asia (India: AB938198.1, JN656208.1, JN656205.1) and Southeast Asia (Vietnam: LC144902.1, FJ434982.1) were clustered with those of the East Asia group in this study (Fig. 2B). Similar findings have rarely been reported except for the research conducted by Doanh et al. (12) According to the paleoparasitological estimation, the prevalence of P. westermani infection in the Joseon society might reach as high as 33.3%. (18,19) Since Joseon people commonly enjoyed raw crayfish or raw crab dishes, (7,29,30) it is understandable that they were heavily infected by P. westermani. In this study, using coprolite samples obtained from Joseon dynasty mummies, we successfully analysed the COI and ITS2 regions of aDNA from P. westermani. As very few studies have examined ancient P. westermani DNA so far, (7) the current study significantly expands the existing gene pool of P. westermani paleogenetics. Nevertheless, we admit that aDNA reports of much wider geo-historical scope are still required to improve our knowledge about the exact evolutionary history of P. westermani.