Interspecies Phylogenetic Analysis of Clonorchis sinensis in High-incidence Area

LONG Xiao Lei, WANG Shi Ping, ZHOU Shuai Feng, LI Zheng Xiang,ZHUANG Shi Feng, and HE Zhuo

1. Department of Medical Parasitology, Xiangya School of Medicine, Central South University, Changsha 410028,Hunan, China; 2. Hunan Provincial Center for Disease Control and prevention, Changsha 410005, Hunan, China

Abstract

Key words: Clonorchis sinensis; Mitochondrial genes; Cox1; Nad1; Phylogeny

INTRODUCTION

The Chinese liver fluke,Clonorchis sinensis(C. sinensis), generally parasitizes the liver and hepatic duct causes a serious foodborne parasitic disease[1,2].C. sinensiscauses mechanical stimulation and obstruction, and its metabolites and secretions[3]lead to local expansion,hyperplasia, inflammation, and even nodules in the hepatic ducts. This results in fatty degeneration,atrophy, and necrosis of adjacent liver cells and even liver cirrhosis and cancer[4-6].

C. sinensisis mainly prevalent in China, South Korea, northern Vietnam, and parts of Russia[7,8].Around 15 million humans have been estimated to be infected worldwide, with 85% in China[9]. The prevalence ofC. sinensisis subject to the combined effects of social, environmental, and control factors,and the main reason is human ingestion ofC. sinensis metacercaria[10].

Except for Xinjiang, Inner Mongolia, Gansu,Qinghai, Tibet, Ningxia, and other provinces and autonomous regions in China,C. sinensishas been spread or reported in 25 provinces and cities, with the number of patients as high as 13 million. In some areas of Hunan Province, the infection rate ofC.sinensishas always been high because of the consumption of sashimi and salted fish by the local residents and cross-pollution of raw and cooked food and tool containers[11]. In 1990,C. sinensiswas reported in 20 counties (cities and districts),including Qiyang County and Lengshuitan District, in Hunan Province. Approximately 2.1%-85.2% of the population in these areas were infected, and the age of the infected people ranged from 2 to 75 years[12].The infection rate in children (less than 15 years of age) was 19.8%, and that in adults was 2.5%. In Tongdao Coungty, Huaihua, the infection rate ofC.sinensishas always been high. In 2011, a survey ofC.sinensisin humans was conducted in Tongdao County, and the infection rate in the population was more than 70%. However, no studies have investigated the prevalence ofC. sinensisor the types of epidemic strains in this county, in fact, noC.sinensiscases have been reported in the counties and cities around Tongdao County.

Between 2016 and 2020, many surveys and studies have been conducted on human parasitic diseases in 122 counties (districts) and 14 cities (prefectures)across Hunan Province. In addition, a detailed investigation was performed on the infection status of the human population and fish metacercariae in Qiyang County, Lengshuitan District, and Tongdao County, where the infection rate was high.

The mitochondrial genome has a simple structure, maternal inheritance, lack of recombination, and rapid evolution rate; therefore,it is particularly suitable as a marker for genetic research, and it has been used by many scholars to study genetic variations between and within a variety of parasites[13,14]. Two gene fragments, partial sequences of the mitochondrial cytochrome c oxidase subunit I (Cox1) gene ofC. sinensisand nicotinamide adenine dinucleotide dehydrogenase(NADH) subunit I gene, have been used to study the genetic diversity ofC. sinensisin Qiyang County,Lengshuitan County[15,16], and Tongdao District. In this study, the genetic relationship after the sequencing was evaluated[17-20].

MATERIALS AND METHODS

Population Infection Rate

Between 2016 and 2020,C. sinensiswas monitored in 14 cities (prefectures) and 122 counties(districts) in Hunan Province. Fecal samples (> 30 g)were collected from the subjects, and a modified Kato-Katz method was used to evaluate the infection rate and infectiosity ofC. sinensis[21].C. sinensiseggs identified in the smears were counted using microscopy[22].

Take a part out from the fresh fecal sample to cover a 8 cm × 8 cm, 80-mesh nylon silk; scrape the fine fecal sample and place it in the round table hole of the plastic quantitation plate until it is filled up,about 38.75 mm3(4 cm × 3 cm × 1 cm); after mixing thoroughly, cover it with hydrophilic cellophane containing malachite green (mixed evenly and soaked according to the ratio of 100 mL distilled water, 100 mL pure glycerin, 1 mL malachite green(or methylene blue, 3%) and flatten it to spread the feces under the hydrophilic cellophane without overflowing the glass slide and form a uniformly thick round fecal film with a diameter of about 2 cm[22]; place the prepared modified Kato-Katz thick smear at room temperature to make it transparent before microscopic examination. IfC. sinensiseggs are discovered in the smear, it is determined to be positive, and theC. sinensiseggs are counted by microscopy.

Infection Rate of Fish Metacercariae

A preliminary investigation of the infection rate ofC. sinensiswas conducted among the human population and metacercariae of commercial freshwater fish in three counties (districts) south of Hunan, Tongdao County, Qiyang County, and Lengshuitan District, where the infection rates were relatively high. Small wild fish, especiallycrucian carp,parabramis pekinensis,cyprinuscarpio,andgrass carp(often made into sashimi), were collected from fish ponds around the epidemic area. The direct compression method was used to check for fish metacercariae: A piece of muscle (about 0.5 g)was cut from the dorsal, pectoral, and caudal fins of each fish, placed between two glass slides, and squeezed until a translucent mist was obtained, and active metacercariae were detected under a microscope and counted.

Sequencing of Cox1 and Nad1

Cats and dogs (more than 5 years of age) livers were purchased from counties and towns located to the east, west, south, north, and middle of Qiyang County, Lengshuitan District, and Tongdao County,one final host was collected from each (Figure 1)[23,24].The animal liver and bile ducts were dissected, theC. sinensisadults were washed out with distilled water, counted, and stored in 90% ethanol.

TheC. sinensisadults were puted into the ultralow temperature refrigerator for freeze-thaw,theadults was then cut into pieces with scissors, and the broken adultsC. Sinensiswere puted into ice for tissue homogenization. Nucleic acid was extracted from the homogenized tissue using the DNA Mini Kit QIAGEN Nucleic Acid Extraction Kit[25].

The partial sequences ofC. sinensis Cox1andNad1were used as the amplification sites for PCR[26,27], and the amplification conditions were as follows: 5 min pre-denaturation at 94 °C, followed by 30 s denaturation at 94 °C, 30 s annealing at 55 °C,and 30 s extension at 72 °C and finally 5 min extension at 72 °C. The PCR reaction system were Forward Primer (10 μm) 1 μL, Reverse Primer(10 μm) 1 μL, 2xTaq Master Mix 10 μL, DNA 1 μL, and finally ddH2O was added to 20 μL. The amplified products were electrophoresed with the QIAxcel Advanced automatic electrophoresis apparatus(Qiagen Germany). The selected PCR products underwent recovery and purification, and they were sent to Invertrogen Biotechnology Co., Ltd (USA). For bidirectional sequencing.

Figure 1. Sampling map for Tongdao, Qiyang, and Lengshuitan. Samples were obtained from the following towns in Tongdao: Shuangji Village of Pingtan Township (cats, sample numbers TD1 and TD2),Qiaozhai Village of Yatunpu Town (cat, sample number TD3), Shuinan Village of Jingwuzhou Town (cats,sample numbers TD4 and TD5), Xinghua Village of Wanfoshan Town (dogs, sample numbers TD6 and TD7), and Pipa Village of Shuangjiang Town (cats, sample numbers TD8 and TD9). Samples were collected from the following towns in Lengshuitan: Wujiayuan Neighborhood Committee of Yangjiaqiao Sub-district(dogs, sample numbers lST1, lST2, and lST3), Guanzitou of Yangcundian Township (cats, sample numbers lST4, lST5, and lST6), Yangshanguan Village of Shanglingqiao Town (cats, sample numbers lST7, lST8, and lST9), Hongxing Village of Renwan Street (cats, sample numbers lST10, lST11, and lST12), and Tianbatang Village of Huangyangsi Town (dog, sample number lST13). Samples were obtained from the following towns in Qiyang: Fulian Village of Maozhu Town (dogs, sample numbers QY1, QY2, and QY3), Xinlian Village of Lijiaping Town (dogs, sample numbers QY4, QY5, and QY6), Qingzhu Village of Longshan Street(dogs, sample numbers QY7, QY8, and QY9), Yanghua Village of Meixi Town (dogs, sample numbers QY10,QY11, and QY12), and Shili Village of Dazhongqiao Town (cats, sample numbers QY13, QY14, and QY15).

The complete mitochondrial genome sequence ofC. sinensisfrom Tongdao (http://www.primer3 plus.com/cgi-bin/dev/primer3plus.cgi) was used to design primers COX-F (-TTGGTTATGGGGGCTTGGTG-), COX-R(-CGCTCAGATCTCAGCAGGTT-),NAD-F(-GAGCGGCTAGATCGGGTTAG-), and NAD-R(-GTGTTGCACGCGACCAAATA-). These primers were synthesized by Invertrogen Biotechnology Co., Ltd(USA).

Data Statistics and Analysis

Excel 2019, SPSS 18.0, Instata 10.0 and other software were used for statistical analysis. In the analysis, homogeneity test of variance was conducted first. If homogeneity of variance was observed, one-way ANOVA was used for overall comparison, and then pair one-factort-test between the two groups was conducted by Dunnett method after differences were found. If the variance is not uniform, the original data should be converted into appropriate variables to meet the homogeneity test of variance, and then the converted data will be used for statistics. If the purpose of homogeneity of variance is still not achieved after the conversion of variables, the rank sum test is used for statistics, and the difference in population comparison is found,then the pair comparison is performed by Tamhane's ST2 test, which does not require homogeneity of variance.

Pair chi-squareχ2test was used to compare the difference in the detection rate ofC. sinensisinfection which detected by the three methods, and Kappa test was used for consistency analysis.P<0.05 was considered statistically significant.

Analysis of Genetic Diversity and Phylogeny

All the obtained sequences were checked using chromatograms and Contig Express and manually corrected for scoring errors. Chromas Lite version 2.1 was used to detect the sequences. DNAMAN was used to splice the sequences in both directions.

The unique haplotype was found using DNASP 5.10, and the following organisms were searched in GenBank:C. sinensisfrom Russia (FJ381664),C.sinensisfrom Guangdong (JF729303),C. sinensisfrom Gansu (JF729304),C. sinensisfrom USA(NC_012147),Opisthorchis felineus(EU921260),

Opisthorchis viverrini(JF739555),Fasciola hepatica

(NC_002546),Paragonimus westermani(NC_027673),

Schistosoma haematobium(DQ157222),Schistosoma japonicum(NC_002544),Trichobilharzia regenti

(NC_009680),Taenia solium(NC_004022)Schistosoma mekongi(NC_002529), and the outgroupGyrodactylus(NC_004022). Then, the Clustal W algorithm was used for alignment in MEGA 6.

Two independent genetic markerCox1andNad1were analyzed with the Bayesian method and BEAST 2 software to construct a phylogenetic tree, The maximum likelihood tree inferred with MEGA 6 with the best model (GTR+G+I) was used as in-put.Additionally, the maximum likelihood analysis (by J Modeltest v. 2.1.7) was performed with MEGA 6 and bootstrap resampled 1,000 times, for phylogroups.With every 1,000 generations recorded among 10,000,000, a burn-in of 25%, and final 10,000 trees summarized using the tree annotator. GTR+G+I was estimated to be the best fit substitution model. The maximum likelihood was estimated with MAGE 6 and 1,000 bootstrap values. The clades were collapsed using FigTree (http://tree.bio.ed.ac.uk/software/figtree/) with the collapse module[28].

RESULTS

C. sinensis Infection Status across Hunan Province in 2016-2020

The infection rate ofC. sinensisin Hunan Province between 2016 and 2020 is shown in Table 1.The infection rate in the province, except in Yongzhou and Huaihua, was less than 1.5%. The infection rate in Yongzhou and Huaihua year-by-year showed a downward trend because of prevention,intervention, health education, and other reasons.Tongdao county, Lengshuitan District, and Qiyang County were monitored ashigh infection areas in Yongzhou and Huaihua; the systematic investigation of population infections in these three regions was of great significance in controlling the spread ofC.sinensis(Table 2).

The results showed that 1,449 people were infected in Tongdao County, with an infection rate of 26.90%; 713 people in Qiyang County, with an infection rate of 18.96%; and 699 people in Lengshuitan District, with an infection rate of 21.80%. Significant differences (χ2= 82.91,P< 0.01)were observed in the infection rates in these three regions.

Infection Status of Fish Metacercariae in Qiyang,Lengshuitan, and Tongdao

The infection of fish metacercariae byC. sinensisin Qiyang County, Lengshuitan District, and Tongdao County was investigated (Table 3). Common freshwater fish cultured in fish ponds were purchased from Tongdao County, Qiyang County,and Lengshuitan District. Differences in infectiosity

invarious freshwater fish metacercariae were investigated; specifically, both infection rate and average infection rate were the highest incrucian carp(90.41% and 73.54 piece/g, respectively),followed byParabramis pekinensis(78.26% and 46.16 piece/g, respectively),cyprinuscarpio(59.26%and 11.33 piece/g, respectively), andgrass carp(25.00% and 1.83 piece/g, respectively). The infection rates of these four fishwere significantly different (χ2= 103.56,P< 0.01); the infection rate and infectiosity were higherin Qiyang County than in the other regions.

Table 1. Infection status of C. sinensis in Hunan Province from 2016 to 2020

Table 2. Infection status of C. sinensis among populations in Tongdao, Qiyang, and Lengshuitan

Table 3. Infection of fish metacercaria in Tongdao, Qiyang, and Lengshuitan ( ± s)

Table 3. Infection of fish metacercaria in Tongdao, Qiyang, and Lengshuitan ( ± s)

(mantissa)Infection rate (%) Average infectiosity(piece/g)Tongdao County Crucian252288.0072.32 ± 38.05 Parabramispekinensis121083.3346.08 ± 22.89 cyprinuscarpio7457.147.14 ± 5.82 Grass carp4125.000.75 ± 1.00 Qiyang County Crucian181794.4477.78 ± 26.95 Parabramispekinensis10990.0049.10 ± 18.56 cyprinuscarpio8562.5010.25 ± 6.20 Grass carp5120.001.40 ± 2.09 Lengshuitan District Crucian302790.0070.52 ± 33.82 Parabramispekinensis241770.8343.28 ± 26.00 cyprinuscarpio12758.3316.58 ± 10.17 Grass carp7228.572.00 ± 2.40 Total Crucian736690.4173.54 ± 33.43 Parabramispekinensis463678.2646.16 ± 23.21 cyprinuscarpio271659.2611.33 ± 8.33 Grass carp16425.001.38 ± 1.95 RegionCategoryNumber of examinees(mantissa)Number of infected

Phylogenetic Analysis of C. sinensis

C. sinensisadults were collected from cats and dogs (age, more than 5 years) and observed under a microscope (Figure 2). The body of an adultC.sinensislong and narrow, with a flat dorsal abdomen, narrow front end, blunt and round rear end (like a sunflower seed), and no spines on the surface. The oral suckers are slightly larger than the abdominal suckers, which are located at the front 1/5 of the body. The alimentary canal is simple, with the mouth at the center of the oral sucker, the pharynx is spherical, and the esophagus is short,followed by the intestinal branches. The intestinal branches are divided into two, which reach the posterior end along both sides of the worm without confluence and with a blind end. Judging from morphology, it is the adults ofC. sinensis.

Morphological differences were detected among theC. sinensisadults from the three regions. The some adults in Qiyang County are similar to those in Lengshuitan District. The some adults of Tongdao county was more slender and brighter in color.

The mitochondrial genesCox1andNad1ofC.sinensiswere used as genetic markers for amplification. The results showed that the length ofCox1andNad1are approximately 1,000 bp and 800 bp, respectively, which isconsistent with the predicted values; the electrophoresis results showed clear bands with no tailing phenomenon or unspecific bands (Figure 3).

Analysis of Genetic Diversity and Phylogeny

To verify the amplification accuracy of the two genes, the PCR products were sequenced and compared with homologous sequences from GenBank, and the amplified products were theCox1andNad1sequences ofC. sinensis.

A phylogenetic tree was constructed usingCox1as the genetic marker andGyrodactylusas the outgroup forC. sinensisand other trematodes in Tongdao County, Qiyang County, and Lengshuitan District as well as Guangdong Province, Gansu Province, China, the United States, and Russia(Figure 4A).C. sinensisspecimens from Qiyang and Lengshuitan were clustered together without any regional differences; both exhibited high homology with those from Guangdong Province and Gansu Province, China, with a high bootstrap value. The regional clustering in Tongdao County was high and demonstrated low homology with that in Qiyang County and Lengshuitan District but high homology with that in the United States and Russia. The relationships of the other liver flukes, schistosomes,andGyrodactyluswithC. sinensisgradually decreased, which is consistent with the taxonomic records. No significant specificity was observed in the genetic relationships of the parasiticC. sinensisin different definitive hosts (dog and cat).

Figure 2. Adult Clonorchis sinensisobtained from hosts. (A) Naked eye of C. sinensis. (B) Clonorchis sinensisfrom Tongdaounder a microscope (magnification, 10×). (C) Clonorchis sinensisfrom Qiyang under a microscope (magnification, 10×). (D) Clonorchis sinensisfrom Lengshuitan under a microscope(magnification, 10×).

WithNad1as the genetic marker (Figure 4B),C.sinensisfrom Qiyang County and Lengshuitan District showed few regional differences and high homology with those from Guangdong Province and Gansu Province, China. the regional clustering in Tongdao County was high and demonstrated high homology with that in the United States and Russia. The relationships of the other liver flukes, schistosomes,andGyrodactyluswithC. sinensisgradually decreased, which is consistent with the taxonomic records. The parasiticC. sinensisin different definitive hosts (dog and cat) showed no significant specificity in the genetic relationship. This is consistent with the phylogenetic relationship results withCox1as the genetic marker.

Figure 3. Electrophoresis map of Cox1 gene and Nad1 gene. Description: Lame: 3kb ladder, LameA9, A10,A11, A12, B1, B2, B3 and B4 are Cox1 gene amplification results, and Lame B5, B6, B7, B8, B9, B10, B11,B12 are Nad1 gene amplification results.

Figure 4. Phylogenetic relationships of C. sinensis with Cox1 and Nad1 as the genetic marker. (A)Phylogenetic relationships of C. sinensis with Cox1 as the genetic marker. (B) Phylogenetic relationships of C. sinensis with Nad1 as the genetic marker.

DISCUSSION

According to the investigation results ofC.sinensisinfection in the population of 122 counties(districts), 14 cities (prefectures) in the Hunan Province from 2016 to 2020, it can be seen that the infection ofC. sinensishas been decreasing year by year since 2016, The high infection rates ofC.sinensisamong the populations in Qiyang County and Lengshuitan District were related to the high consumption of sashimi and salted fish in these areas[29]. Recently, the infection rates ofC. sinensishave decreased in these areas because of improvement indietetic hygiene and scientific prevention[30], but theyare still significantly higher than the infection rates in other areas of Hunan Province. Sashimi is part of the daily diet of people in Tongdao County, Huaihua, so it is important to investigateC. sinensisinfection in the population and intermediate hosts. The results of this study showed that the infection rate ofC. sinensisin Tongdao County is high. Of the four fish commonly used for making sashimi, the infection rate incruciancarp was the highest, which could be attributed to its small size and phototropism. This study filled the gap of systematic report on the infection ofC. sinensisbetween the population and the intermediate host in Tongdao County.

To study the genetic relationships ofC. sinensisin Tongdao County, Qiyang County, and Lengshuitan District, the mitochondrial genesCox1andNad1were selected as markers[31]and sequenced from adult specimens collected from different areas of Tongdao County, Qiyang County, and Lengshuitan District. The sequence alignment and phylogenetic analysis showed that the parasitic species in Qiyang County and Lengshuitan Distric had a high degree of homology, which could be attributable to the similar latitudes and regional closeness. However, the parasitic species in Tongdao were different from those in Qiyang and Lengshuitan, which have no high homology with the domestic parasitic species, but similar to those in the United States and Russia. This may be related to species invasion and needs to be further studied in the future. This is the first phylogenetic analysis ofC. sinensisin the highincidence area of Hunan Province, China.

The results obtained by designing primers forCox1andNad1showed a high degree of consistency,indicating that bothCox1andNad1are stable genetic markers for the phylogenetic analysis ofC.sinensis. The homology analysis ofC. sinensisshowed that latitude and longitude and geographical clustering are important. It is suggested thatC.sinensisin the Tongdao County may have the biological characteristics of high latitude species, and have similar antigenicity. It is also suggested thatC.sinensisin the Tongdao County may originate from the high latitude region of Eurasia, Russia. This will lay a foundation for further exploration of its control direction, gene traceability and detection methods.

The phylogenetic analysis ofC. sinensisadults detected in different definitive hosts (dogs and cats)showed thatC. sinensishas no specificity in the parasitism of mammalian hosts, and it does not have different gene homology because of changes in the hosts.

A combination of molecular biology and morphology is more reliable than morphological identification. Morphological description is greatly affected by subjective factors and measurement errors. Differences in the genomes ofC. sinensisexplain the differences in the morphology ofC.sinensisin Tongdao County, suggesting that the antigenicity ofC. sinensisin Tongdao County may be closer to that of the species in high-latitude regions[32]. The results of this study have laid the foundation for further natural selection and evolutionary analyses ofC. sinensisin Tongdao.C.sinensisin Tongdao may have originated from highlatitude areas, providing clues for studying the evolution and mutation adaptation ofC. sinensisin high- and low-latitude areas and research on group evolution, individual differences, detection methods,control drugs, and vaccines forC. sinensisin this area.

CONCLUSION

The infection rate ofC. sinensiswas higher in Qiyang County, Lengshuitan District and Tongdao County of Hunan province, China. Mitochondrial genesCox1andNad1were used as genetic markers to study the phylogeny ofC. sinensis. The results showed that theC. sinensisin Tongdao county was far from the other area of Hunan Province, which might be related to the migration and invasion of the species. Based on this method, mitochondrial genesCox1andNad1were selected as good genetic markers for the phylogenetic analysis ofC. sinensis.The results of molecular genetic marker analysis were basically the same, which again confirmed the reliability of the experimental study. Taking into account the complexity of the species ofC. sinensisin Hunan Province, it is recommended to reduce the infection rate of parasites in terms of controlling local transmission and preventing external sources.

DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

ACKNOWLEDGMENTS

We are grateful to the Hunan Provincial Center for Disease Control and Prevention for providingC.sinensisepidemiological survey data. We thank the Xiangya School of Medicine, Central South University for their technical assistance and participation. We also thank the Centers for Disease Control and Prevention of Tongdao County, Qiyang County and Lengshuitan District for providing metacercaria and adults ofC. sinensis.

Received: May 1, 2021;

Accepted: August 17, 2021