Morphological and Molecular Characterization of Paragonimus Species Isolated from Freshwater Crabs in Southern Yunnan, China

Department of Cardiology, e Second Affiliated Hospital of Kunming Medical University, No. 374, Dianmian Road, Kunming 651010, Yunnan, China Oncology Department, People’s Hospital of Xishuangbanna Dai Autonomous Prefecture, Xishuangbanna 666100, Yunnan, China School of Basic Medical Sciences, Kunming Medical University, No. 1168, Chunrong West Road, Yuhua Street, Chenggong District, Kunming 65050, Yunnan, China


Introduction
Trematodes belonging to the genus Paragonimus are important parasites causing zoonotic infections in vertebrates, including human beings. ey are of socioeconomic importance since the route of infection is mainly food-borne [1]. e life cycle of Paragonimus is relatively complex that requires a minimum of three hosts, including a definitive host and two intermediate hosts [2]. e first and second intermediate hosts are frequently snails belonging to the families Assimineidae and Hydrobiidae and crabs belonging to the families Potamidae and Parathelphusidae [2]. Different species of Paragonimus have their own predilection for infecting specific genera of snails and crabs and hence the epidemiological prevalence of different species of Paragonimus is determined by the existence of suitable hosts. Some species of Paragonimus have been identified only from intermediate hosts in certain geographies which suggests infection and maintenance in nonhuman mammals [3,4]. e genus Paragonimus is rich in species diversity that has been reported from varied geographies, including Asia, Africa, and Central and South America spanning both tropical and temperate climates [5]. e species diversity is also reflected by the various phylogenies of Paragonimus and mainly comprises 3 species complexes. Paragonimus westermani is the most commonly reported species complex found in South and South East Asia [6]. Paragonimus heterotremus species has been predominantly isolated and reported from South East Asia while the Paragonimus skrjabini complex has been reported from China and East Asia [7,8]. Usually, the adult worms are large, plump, and resemble a coffee bean in size and shape. e most common discrimination is the patterns of lobation of the ovary and testes. For instance, the ovary of Paragonimus ohirai and Paragonimus mexicanus possesses many delicate branches, while that of Paragonimus westermani has 6 simple lobes [9,10]. e allelic diversity and ontogenetic changes have been previously studied with isozymes, which have been superseded with molecular approaches [5].
ere were previous instances of discordance in morphology and the ontogenetic changes predicted by both molecular and isozyme-based methods [11]. One of the main reasons for the observed discordance is the lack of clarity on the species boundaries and also mendelian polymorphism leading to morphological changes causing the creation of distinct species that are interfertile [12].
DNA sequence data can be used to construct phylogenetic trees that can be used to infer evolutionary trends, including insights into speciation and geographic spread [13,14]. Further, the morphological differences in adult and metacercariae that could be used to differentiate different species are also limited. is expands the potential role of DNA sequence analysis to assist in confirming species distribution and phylogenetic diversity. Currently, nuclear (internal transcribed spacer 2 (ITS2) and mitochondrial DNA (cytochrome c oxidase subunit 1 (CO1)) sequences are used for assessing the phylogeographies in various organisms [15,16].
Over 50 species of the Paragonimus have been reported worldwide, of which China has the largest distribution and number of species [17]. Due to the migration of people from rural to urban areas and food eating habits such as continuous consumption of raw or undercooked freshwater crab or meat such as wild boar or venison [2,18], China represents an ecological hotspot for the dissemination and further evolution of Paragonimus species. Moreover, the diagnosis of paragonimiasis is difficult due to nonspecific clinical symptoms [18]. Hence, frequent phylogenetic analysis of Paragonimus species from nonmammalian hosts will help understand the genetic variation and also suggest suitable lifestyle modifications for populations at risk of acquiring paragonimiasis. Yunnan province has the most endemic species of Paragonimus due to its mountainous plateau topography and unique geographical environment [17]. Hence, in this study, we report the phylogenetic diversity and evolutionary relationship of Paragonimus species isolated from crabs in southern Yunnan province, China.

Parasitological Methods.
e Paragonimus metacercariae were isolated from naturally infected primary freshwater crabs belonging to the genus Indochinamon, the second intermediate hosts, from Tongchang Town, Jinping County, Yunnan Province, China. e habitat included fast-moving streams with no adjacent vegetation. e identification of the secondary hosts was done according to the classification method of "Chinese Medical Crustaceans" [19]. e crabs were smashed in a mortar followed by sieving and washed with distilled water into a sedimentation cup. e debris was passed through the first filter mesh of pore size 200 microns, and later the filtered liquid and sediment were passed through a filter pore size of 1000 microns to collect the sediment. e supernatant obtained during this process was discarded after every 20 minutes, and the same step was repeated four to five times until the supernatant was clear.
e sediment obtained at the bottom of the cup was then placed in a glass dish for microscopic biological observation. e metacercariae of Paragonimus were counted under the microscope and a part of the sediment with the metacercariae were fixed with 70% absolute ethanol and stored in a refrigerator at 4°C with 96% ethanol for molecular biological experiments.

Experimental Infection.
Freshly isolated, live metacercariae were then injected intraperitoneally (15 metacercariae per rat) into paragonimiasis-negative Sprague Dawley (SD) rats (purchased from the Laboratory Animal Department of Kunming Medical University). All the animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1985). All experimental protocols were approved by the Animal Care and Use Committee of Kunming Medical University (reference: KMMU2015002). Experimental animal inoculation was performed based on the sampling point. After subcutaneous injection, five SD rats were sacrificed on the second, fourth, and sixth week to confirm infection of rats with cercariae of Paragonimus. After 114 days of injection, the SD rats were dissected to isolate the cysts, eggs, and adult worms from the muscles, abdomen, liver, thoracic cavity, and lungs of SD rats. e isolated adult worms were used for genomic DNA extraction and for preparing permanent slides for microscopic confirmation by fixing them onto glass slides with alcohol, formalin, and acetic acid.

Microscopic Identification.
e different characteristic features such as the shape, body size, measurement, and position of the suckers were used for microscopic and morphologic characterization.

Molecular Analysis.
Genomic DNA was extracted from both the adult worms in SD rats and the metacercariae extracted from crab using QIAamp DNA Mini kit (QIA-GEN, Hilden, Germany). e whole process was carried out in strict accordance with the manufacturer's instructions. e final elution of DNA was done with 100 µL of distilled water. e extracted total genomic DNA was quantified and stored in the refrigerator at−20°C until further use.

Polymerase Chain Reaction
Amplification of CO1 and ITS2. Polymerase chain reaction (PCR) was performed with primers targeting a fragment of the CO1 gene and the ITS2 region synthesized by Shanghai Bioengineering Co., Ltd. e primers used for amplifying CO1 gene fragments were CO1F-5′GAGGTGTATGTCCTGATTTTGCC-3′ and CO1R-5′GACCTCACCCAATGACCCTGCAACA3′, and the primers for amplifying ITS2 gene fragments were ITS2F-5′GGGTACCGGATCACTCGCTCGGTG3′ and ITS2R-5′GGGGATCCTGGTTGCCTTAGTCTCCGC3′ [20]. PCR was performed in 25 µL volume with 2 µL template DNA corresponding to 0.1 ng and 1 µL of primers (10 µmol/µL), 2.5 µL of 10X PCR buffer, 1 µL of 10 mM deoxynucleotide triphosphates (dNTP), 0.1 µL (0.5 units) of Taq enzyme (5 U/ ul), and 17.4 µL of PCR grade water. e setup of PCR was done in an ice bath. e PCR amplification was conducted in TaKaRa PCR instrument (Baolingbao Biology Co., Ltd., China), and the amplification conditions were as follows: initial denaturation of 95°C for 3 minutes followed by 35 cycles of denaturation at 93°C for 1 minute, annealing at 48°C (for CO1)/60°C (for ITS2) for 1 minute, and extension at 72°C for 1 minute followed by a final extension at 72°C for 5 minutes. e expected length of the PCR fragments was 500-750 base pairs. Detection of PCR amplified products was done by agarose gel electrophoresis with 1.5% agarose gel immersed in 1.0% Tris-Acetate-EDTA buffer stained with ethidium bromide. e purity and quantity were estimated by imaging the gel in a gel documentation system (Bio-Rad company). e PCR products were then subjected to bidirectional sequencing using the same PCR primers by Shanghai Biotechnology Co., Ltd. (Hitachi fluorescent DNA sequencer SQ-3000). e forward and reverse sequences were then manually curated and aligned with the Dnastar v7.1 software, and the consensus sequence was used for bioinformatic analysis. e initial quality check of the sequences was done by checking the coverage and alignment with previously submitted sequences in National Center for Biotechnology Information (NCBI) using the BLAST tool. Previously submitted sequences of CO1 and ITS2 were retrieved from NCBI and compared with the sequences obtained in this study with ClustalX software with default parameters. Phylogenetic analysis was done as per the Kimura 2-parameter model. e neighbor-joining method (NJ) and maximum parsimony (MP) method were used to construct the phylogenetic tree. e genetic distances of the Kimura-2parameter model were calculated, considering all substitutions and missing/gaps as unambiguous changes. Test of phylogenetic accuracy was done with bootstrap replicates of 1000. e analysis was performed with MEGA5.0 software. e cut-off value for the consensus tree was set to 75%. . e size of the metacercariae was found to be 0.419 mm * 0.398 mm. e average thicknesses of the outer wall and inner wall were found to be 0.004 mm and 0.012 mm, respectively, using the microscopic technique. Further, the larvae were surrounded by two larger excretory cysts and intestinal branches.

Adult Worm.
After experimental infection, from each SD rat, 2 to 4 adult worms were retrieved ( Figure 1). Adult worms were distributed in both lungs and muscles but seldom found in the liver and brain of affected SD rats. A total of 5 fully developed adult flukes recovered from SD rats were used for morphological and molecular analysis. e average size of the gravid adult worms was 6.7 mm * 3.8 mm.
e tegmental spines were mainly clustered into clusters of 4-6 around the ventral sucker. e spines around the oral sucker were short, small, and solitary. e size of the oral and ventral suckers was 0.43 mm * 0.53 mm and 0.75 mm * 0.79 mm, respectively. e reproductive organs were observed as large uterine masses, with a size of 2.695 mm * 2.107 mm. e testicle is in the inferior half of the worm with dimensions of 1.72 mm * 1.38 mm.
Morphological identification of the metacercariae isolated from the infected crabs and adult worms from the SD rats led to the identification of 4 species of Paragonimus: Paragonimus proliferus, Paragonimus microrchis, Paragonimus heterotremus, and Paragonimus skrjabini.

Molecular Identification.
A total of 5 samples (all adult worms) of Paragonimus from southern Yunnan province were used for molecular phylogenetic analyses. e sequences were aligned with those of several related species within genus Paragonimus obtained from GenBank. PCR amplification of the CO1 and ITS2 regions, followed by agarose gel electrophoresis, revealed amplicons of about 500 and 750 bp in length, respectively. BLAST searches using our sequences as queries found matches with 100% coverage in GenBank. Further, only the CO1 sequence of Paragonimus microrchis differed slightly from previously published sequences. All the other sequences obtained in this study revealed 100% identity to previously published sequences.

Base Composition of DNA Sequence in Paragonimus
Species.
e base composition ratio of the same gene in different species was identical. However, the ratios of DNA Journal of Tropical Medicine base composition between CO1 and ITS2 among different isolates were different. In the CO1 gene, the content of base T was very high, with an average of 43.2%, far exceeding the base composition of ITS2, which is consistent with the abundant base A and base T content of the mitochondrial genome (Table 1).

Genetic Distance between ITS2 and CO1 of Paragonimus Species.
e average genetic distance of Paragonimus species for the ITS2 sequence was 0.056, while the average genetic distance for the CO1 sequence was 2.3 times ITS2 with 0.128. e genetic distances of adult worms had extremely high sequence similarity though the distances between DNA and the corresponding species were the smallest (Figures 2 and 3).

Phylogenetic Analysis.
A total of 30 gene sequences were downloaded from NCBI and were selected based on coverage for this analysis ( Table 2). In the phylogenetic tree of the CO1 gene, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini were clustered with the same species, and the confidence values of their branches were more than 95% (except for the support degree of the branches where sample 30 was located at 88%) ( Figure 4). Paragonimus microrchis clustered with Paragonimus bangkokensis ( Figure 5). A congruent phylogenetic relationship was observed with the ITS2 phylogenetic tree. In the phylogenetic tree constructed with the combined dataset of CO1 and ITS2 datasets, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini still clustered with the same species, and their branch confidence values were more than 94%. Paragonimus microrchis remained clustered with Paragonimus bangkokensi (Figure 6).

Discussion
China is known for the endemic diversity of Paragonimus species, wherein 45 genera and 311 species have been identified till 2018 [21]. A previous paper reported that Yunnan alone has the highest number of species (48) and genera [14] and the second-highest Shannon index (2.21%) [22]. is number has recently increased to 58 species [23]. Hence, we determined the occurrence and phylogenetic diversity of Paragonimus species in southern Yunnan province, China. In the current study, out of the sequenced samples, 2 were Paragonimus microrchis and 1 each of Paragonimus heterotremus, Paragonimus skrjabini, and Paragonimus proliferus.
Since the differential identification features of cercariae are not well marked, most often, the species identification is done at the metacercariae stage from the secondary intermediate hosts [24]. In this study, morphological identification made at the metacercariae stage and adult stage were concordant with each other, further substantiating the role of morphological identification. is was also confirmed with PCR amplification and sequencing of CO1 and ITS2 region.
Phylogenetic analysis with previously published sequences and the sequences generated in this study revealed concordance between the single locus (CO1 or ITS2) and double loci (CO1 and ITS2) phylogenetic analysis. e phylogenetic analysis revealed the presence of 3 distinct clusters of Paragonimus consisting of Paragonimus siemensis and Paragonimus westermani in cluster 1; Paragonimus

Journal of Tropical Medicine
By comparing the results of constructing adjacent NJ and MP phylogenetic trees from CO1, ITS2, CO1, and ITS2 datasets, it is obvious that different datasets and treebuilding methods have a great influence on the robustness of phylogenetic trees. In phylogenetic trees constructed from single-gene datasets, the number of branches with more than 75% support is less than that constructed from joint datasets, especially self-exhibition, which deserves significant improvement. e phylogenetic tree constructed by the MP method using CO1 and ITS2 datasets has high robustness, and Paragonimus macrorchis and Paragonimus heterotremus were clustered together, which is supported by 91%.
Among  reported clustering of isolates based on geographic location, which was in accordance with the current study [25]. Paragonimus skrjabini was previously reported from Gansu, Shanxi, Yunnan, Guangxi, Guizhou, Sichuan, and Jiangxi provinces of China. In contrast, P. heterotremus has been reported only from Yunnan and Guangxi provinces [2]. Phylogenetic analysis has varied values in different organisms. In Paragonimus, where there is no consensus on identification with both morphological and molecular characteristics, phylogenetic analysis helps in determining the phylogeographies [26,27]. e results of our study further substantiated the conservative phylogeographies revealed through analysis of Cox1 and ITS2 regions. Previous literature on the identification of Paragonimus species among individuals consuming freshwater crabs is scarce. A recent study reported that 8 adult humans were infected with P. heterotremus, which were recovered from their lung masses on examination in Xishuangbanna, Yunnan [28]. In such a case, phylogenetic analysis is helpful in the identification of various species of organisms. e study has certain limitations. e study sites were localized to southern Yunnan province in China; hence, the results may not be extended to other parts of China. Furthermore, we used the maximum parsimony method, which is infrequently used despite the robustness mainly because of the nature of assumptions deployed in the method. Nevertheless, the maximum likelihood method used in other studies also has drawbacks.
In conclusion, we obtained 4 morphologically different species of Paragonimus from southern Yunnan province, China. Phylogenetic analysis using the polymorphisms in two different loci revealed considerable variations in the species of Paragonimus identified in different geographic locations with clustering of Paragonimus based on geographic location.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors have no conflicts of interest to declare.