The Genetic Diversity Of Sparganum Isolates Of Spirometra Mansoni From Different Areas Of China

Spirometra mansoni(syn. S. erinaceieuropaei)(Cestoidea: Diphyllobothriidae) is one of the most important species of tapeworms. Its plerocercoid larvae(spargana) can lodge in the subcutaneous tissues and sometimes invade the abdominal cavity, eye, and central nervous system of humans causing a serious parasitic zoonosis known as sparganosis. Human infection results mainly from ingesting raw flesh of frogs and snakes infected with the plerocercoids, drinking raw water contaminated with cyclops harboring procercoids, or placing frog or snake flesh as poultice on open wound for treatment of skin ulcers or eye inflammations. Sparganosis is distributed worldwide, but most cases occur in Eastern and Southeastern Asia. China has the largest number of sparganosis cases in the world since 1999, with a total of approximately 1,000 instances of human sparganosis being reported in 27 out of 34 provinces, autonomous regions, or municipal districts. In addition, the local cases have increased in recent years and sparganosis has even been termed as emerging enzootic diseases in several districts of China. Knowledge regarding the distribution of the pathogen of this disease, the genetic characteristics of its populations in relation to local environmental conditions is valuable for the prevention and control of sparganosis in humans. Unfortunately, insufficient studies on population genetics of S. erinaceieuropaei have been carried out to date. Thus, it becomes important to analyze the population genetics and demographic history of this tapeworm, so that we can get valuable clues about the population changes and genetic variation affecting the pathogenicity.In this study, the prevalence of sparganum infection in wild frogs in 9 geographical areas in southwest China was firstly investigated. Of 276 caught frogs, 55 frogs were found to be infected with sparganum. Then, the population genetic structure of these sparganum isolates was explored based on four molecular markers(cytb, cox1, rrn S and 28 S r DNA D1). Highly genetic diversity and the genetic differentiation among sparganum isolates from different sites were revealed in the DNA polymorphism analyses. Both the phylogenetic inference and the analysis of the median-joining network supported two clades in the southwest S. erinaceieuropaei population. However, none demographic population expansion of the southwest S. erinaceieuropaei population was observed in the neutrality test, mismatch distribution analysis and Bayesian skyline plot analysis. Finally, the phylogenetic diversity of S. erinaceieuropaei from eastern, central, southern and southwest China was analyzed, the result suggested that Chinese S. erinaceieuropaei population should be divided into two groups(Group I and Group II), and they started to divergence in the middle Pliocene.Materials and methods 1. Sparganum collection: Wild frogs were collected from a field site in nine geographical locations of southwest China(Guangan, Luzhou, Mianyang, Nanchong and Nanchong Yingshan of Sichuan Province, Baoshan, Dehong, Kunming of Yunnan Province, Liangping of Chongqing Province) during May 2013 to September 2014. All spargana were collected from the wild frog, saved in the 5ml Frozen storage tube with absolute ethyl alcohol and remark it. 2. Sequencing of target genes:Total genomic DNA was extracted from individual plerocercoid using the Easy Pure Genomic DNA Kit(Transgen, China) follow the manufacturer’s protocol. Four molecular markers(cytb, cox1, rrn S and 28 S r DNA D1) were amplified by PCR. PCR products were purified using the Easy Pure PCR Purification Kit(Transgen, China) and sequenced in both directions at the Genwiz Company(Beijing, China). All sequences were deposited in the Gen Bank database. 3. Alignment and sequence saturation:The sequences for cytb and cox1 were initially aligned using the default settings in the program Clustal X v.2.0and adjusted in MEGA v.5.0 according to their amino acid sequences. For the rrn S and 28 S r DNA D1 sequences, secondary structure was inferred and used as a guide for manual sequence alignment in MEGA v.5.0. Nucleotide saturation was analyzed by plotting number of transitions(Ti) and transversions(Tv) against corrected genetic distance values in the software DAMBE v.5.2. The nucleotide composition, conserved sites, variable sites, parsimony-informative sites, and singleton sites were estimated using MEGA v.5.0. 4. Genetic structure analysis : Haplotypes of southwest S. erinaceieuropaei isolates based on the concatenated sequences of cytb, cox1, rrn S and 28 S r DNA D1 genes were inferred in Dna SP v.5.10, and the same program was used to calculate values of genetic diversity per population. Haplotype network reconstructions were performed in the software Network v.4.5. A partitioned multi-gene phylogenetic analysis of southwest S. erinaceieuropaei isolates was performed in Mr Bayes v.3.1. The approximate divergence time of the southwest sparganum isolates wase estimated based on the concatenated sequence of cytb and cox1 using an uncorrelated log-normal relaxed molecular-clock model in the software BEAST v. 1.6.1. Analyses of demographic change were estimated using mismatch distributions based on the concatenated sequences in the software Arlequin v.3.5.1.2. Population pairwise genetic distances were also estimated in the Arlequin to explore levels of genetic differentiation among the populations. To estimate the change in population size through time, and the time to the most recent common ancestor(t MRCA) of each S. erinaceieuropaei isolate, we performed a Bayesian Skyline Plot analysis(BSP) implemented in BEAST. 5. Phylogenetic analysis:The phylogenetic pattern of S. erinaceieuropaei isolates collectedfrom eastern, central, southern and southwest China was estimated using the concatenated sequences of cytb and cox1 through maximum parsimony(MP), maximumlikelihood(ML) and Bayesian inference(BI) methods, respectively. The approximate divergence time of the southwest sparganum isolates was estimated based on the concatenated sequence of cytband cox1 using an uncorrelated log-normal relaxed molecular-clockmodel in the software BEAST. Data analysis was performed using SPSS version 17.0.Results 1. Prevalence of sparganum infection in frogs: All spargana were collected from naturally infected frogs caught from a field site in nine geographical locationsof Sichuan, Yunnan, Chongqing and Guizhou Province. A total of 276 wild frogs(Rana nigromaculata) were collectedfrom nine geographical locations in southwest China. Spirometra spargana were found in 19.93%(55/276) of the examined frogs. The prevalence of sparganum infection in frogs ranged from 8.33% to 50.00%, with infection intensity of 1–31 spargana per frog in different geographical locations. 2. DNA sequence variation in southwest populations: All amplifications were successful, with fragments cytb, cox1, rrn S and 28 S r DNA D1 respectively. And the length of corresponding sequence was identical. The concatenated sequences alignmentidentified 180 polymorphic sites, of which 161 were parsimony-informative and 19 were singleton-variable. These polymorphicsites identified 32 haplotypes within 52 isolates from nine local-ities. Each sampled population and the total population has highhaplotype diversity. 3. Population structure: The results of analysis of molecular variance showedthat a little more genetic variance lay among the populations(57.31%) than within the populations(42.69%). Except FST values between SC-GA and SC-MY(FST = 0.2493), SC-GA and SC-NC(FST = 0.2198), most of FST values were above 0.25. Phylogenetic analysis revealed two main clades(Clade I and Clade II). In consistent with the phylogenetic inference, analysis of the median-joining network of the concatenated sequences generated two sub-networks with haplotypes corresponding to the two clades in the phylogenetic trees. Neutrality tests of Tajima’s D and Fu’s FS for clade I, clade II and the total population showed non-significant positive values, rejecting possible population expansion. Mismatch distribution analyses revealed multi-modal frequency distributions for clade I, clade II and the total population three phylogroups. The results of Bayesian Skyline Plot analyses also rejected sudden population expansion for all phylogroups. 4. Phylogenetic analysis and time estimation: All collected S. erinaceieuropaei isolates were grouped into two populations(Group I and Group II), and the divergence time between the two groups was estimated in the middle Pliocene. All the sparganum isolates from Henan(He N), Hunan(Hu N), Anhui(AH), Zhejiang(ZJ) and Chongqing(CQ) provinces were within the Group I, and they started to diverge in about 1.67 Myr(late Pliocene). Group II including sparganum isolates mainly from Yunnan(YN), Hainan(HN) and Guangxi(GX) provinces, and the early branching of this group was calculated in the late Pliocene too.Conclusions 1. DNA polymorphism analyses revealed that both the genetic diversity and the genetic differentiation among sparganum isolates from different sites were high. Both the phylogenetic inference and the analysis of the median-joining network based on the multi loci(cytb, cox1, rrn S and 28 S r DNA D1) supported two clades in the southwest S. erinaceieuropaei population. 2. When considered the phylogenetic diversity of S. erinaceieuropaei samples from eastern, central, southern and southwest China, the Chinese S. erinaceieuropaei population should be divided into two groups(Group I and Group II), and they are started to diverge in the middle Pliocene.