The Population Genetic Diversity Of Sparganum Isolates From Southwestern China Using Microsatellite Loci

The plerocercoid larvae(spargana)ofSpirometra tapeworms can parasitize in humans causing serious parasitic zoonosis: sparganosis,leading blindness,epilepsy,paralysis,and even death.Sparganosis is distributed worldwide,but most cases occur in Eastern and Southeastern Asia.China has the largest number of sparganosis cases in the world,with a total of approximately 1,000 instances of human sparganosis scattered over almost all administrative areas.In recent years,the local cases have increased and sparganosis has even been termed as emerging enzootic diseases in several districts of China.The study of genetic structure and biogeography of sparganum population is not only useful for understanding the evolution history of sparganum population but also useful for prevention and control of sparganosis in China though revealing the response relationship between sparganum population and climate changes.However,our knowledge about the genetic diversity of sparganum isolates is still fragmentary.In this study,we firstly developed 4 diversity microsatellite loci based on public database of ESTs(Expressed sequence tags)of Spirometra erinaceieuropaei.Next,we explored thegenetic diversity of sparganum isolates from 11 distinct geographical locations in Southwestern China using these microsatellites.Materials and Methods1.Collection and identification of sparganum isolates:Wild frogs were collected from 11 geographical locations(Sichuan-Nanchong,Yingshan,Guang'an,Leshan,Zigong,Luzhou,Dazhou and Liangshanzhou;Yunnan-Kunming and Baoshan;and Chongqing-Liangping.)in Southwestern Chinaduring May 2014 to September 2015.The frogs were euthanized using ethyl-ether anesthesia,weighed,and skinned.The skeletal muscles and subcutaneous tissues werecarefully observed for the presence of spargana by eyes.Then,the spargana were removed from the muscles or subcutaneous tissues and put in a Petri dish containing physiological salineto observe their movement.The number of spargana collected from infected frogs was counted.Total genomic DNA was extracted from individual sample of plerocercoid using the EasyPure Genomic DNA Kit(Transgen,China)following the manufacturer's protocol.Each isolate was identified by using barcoding method with the cox1 molecular marker.2.Microsatellites development:First,the artefactual sequences of ESTsdownloaded from public database were masked by using NCBI_BLASTand UniVec.Then,the ESTs were clustered following the appliedCD_HIT_EST online model.Next,microsatellites were selected using the MISA(MIcroSAtellite)based on the following standard: over 9 repeat units formononucleotide microsatellites,more than 4 repeat units(include 4)in case ofdi-,tri-,at least 3 repeat units for tetra-,penta-and hexa-nucleotide microsatellites,in addition,eachlength of microsatellite is no more than100 bp.In the end,theprimers were designed in Primer3 to amplify the target microsatellites.The tri-and tetra-nucleotide microsatellites with flank sequences were finally selected.Consistency and diversity of amplifiedregions were tested though temperature gradient PCR,agarose gel electrophoresis,and sequencing of small samples trials.3.Microsatellite loci amplification and sequencing:The selected microsatellite loci were amplified for each isolate with primers designed above.One portion of PCR products were used to nondenaturing polyacrylamide gel electrophoresis and silver nitrate(AgNO3)dyeing.The other PCR products were used for sequencing in the Genwiz Company(Beijing,China).Each product was sequenced inboth directions with the same primer pairs used for PCR amplification.4.Silver nitrate dyeing and data analysis: The online model of GenePop was used to calculate the observed heterozygosity(HO),the expected heterozygosity(HE)and thegenotype diversities(H).Hardy-Weinberg Equilibrium(HWE)exact test,Linkage Disequilibriumtest,theintragroup inbreeding coefficient(Fis),fixed coefficient(Fst),and total inbreeding coefficient Fitwere also calculatedin GenePop.Genetic divergence(FST coefficient)and Garza-Williamson index(G-W)were calculated by Arlequin 3.5.GenALEx 6.5 was used to evaluatethe Analysis of Molecular Variance(AMOVA)and principal coordinates analysis(PCoA).The sequences for eachlocus were aligned using the default settingsinthe program MEGA v.6.0.Haplotypes were inferredusing the PHASE algorithm implemented in DnaSP v.5.10.5.Genetic diversity analysis:Haplotype network reconstructions were performed under statistical parsimony using the software Network v.4.5Phylogeny was estimated though maximum parsimony(MP),maximum likelihood(ML)and Bayesian inference(BI)methods,respectively.MP analysis was performed in MEGA v.6.06.Confidence in each node was assessed by boot-strapping(1,000 pseudo-replicates).ML analysis was performed in PhyML v.3.0.The Ts/Tv ratio,proportion of invariable sites,and gamma distribution parameter were estimated by the software.The support of each internal branch of the phylogeny was estimated using nonparametric bootstrapping(1,000 replicates).BI analyses were performed in MrBayes v.3.2 with 10,000,000 generations,sampling trees every 100 generations.Stationarity was assessed using a convergence diagnostic.An average standard deviation of the split frequencies(ASDSF)<0.03 were used as criteria of convergence between both runs.The consensus tree was drawn after removing the first 20,000 trees(20%)as the burn-in phase.We applied Neutrality tests through the program Arlequin v3.5 as an assessment of possible population expansion.Under the assumption of neutrality,a population expansion produces a large negative value of Fu's FS test and Tajima's D.Tajima's D and Fu's FS are sensitive to bottleneck effects or population expansion,causing these values to be more significantly negative.Results1.Prevalence of spargana infections in frogs:Spargana were collected from naturally infected frogs caught from a field site in 11 locationsof Southwestern China.A total of 347 wild frogswere collected and they were and identified asRana nigromaculata based on morphological characteristics.Spirometra spargana were found in 18.44% of the examined frogs.The prevalence of sparganum infection in frogs ranged from 6.67% to 36.36%,with infection intensity of 1–23 spargana per frog in different geographical locations.Levels of sparganum infectionsin wild frogs in Sichuan province were higher than other regions,and frogs in Nanchong of Sichuan(SC-NC)had the highest infection rate.And all sparganum isolates were identified as S.erinaceieuropaei using the cox1 sequence analysis.2.Screening of microsatellites:A total of 12,481 ESTs of spargana were adopted,in which 915 ESTs were suitable for candidate microsatellites.The probability of microsatellite site was 1 microsatellite per 1,779 bases.Most of microsatellites was the mononucleotide repeat(46.67%).In the frequency of repeats above 15,the A/T motif was principal.In contrast,the C/G motif was principal in the frequency of repeats below 15.The microsatellite with dinucleotide was about 31.26%.And themotifs of AC/GT and AG/CT were the most revealed.The microsatellite with trinucleotide was 16.72%.The AGG/CCT motifs were preponderance.However,the tetra-,penta-and hexa-nucleotide models were no more than 4.48%.Finally,5 microsatellite loci: C07,M15,I03,N11 and M20 were confirmed with diversity sits though small sample trials and sequencing.Therefore,these loci were selected for the following analyses.3.Genetic diversity analysis based on PAGE:A total of 13 alleles were identified from 64 sparganum isolates by using 5 microsatellite loci.Each locus showed 2-3 alleles(in average of 2.60).The alleles of 95% samples were less than 3(1.89 alleles/locus/isolate).The heterozygosity of the I03 was the least(only18.75%),followedby the N11(26.56%).The total alleles in SC-GA and SC-NC were the most(11 alleles),in average of 2.20.In contrast,the average allele of SC-DZ and YN-KM were the least(1.40).Isolates from SC-LS revealed diversities in each of locus.Total heterozygosity of all isolates ranged 0-0.682(in average of 0.322).The average HOof every geographical group was0.436(ranged0-1.000).HWE test indicated that each selected isolate was a random sample of the whole population(P> 0.05).Moreover,no linkage disequilibrium was appeared among all microsatellite loci in theLinkage Disequilibrium test(P> 0.05).The negative value of Fis and Fit suggested that the hybridization was existed within intragroups(HO>HE).The genetic divergence of the N11 was lower than others(Fst< 0.05).The genetic divergence of the I03 was thehighest(Fst> 0.25).C07,M20 and M15 showed moderate genetic divergence(0.05<Fst < 0.15).Also the genetic differentiation of all loci was moderate(0.05 <Fst< 0.15).The average value of theG-W index was above 0.68,indicating that there was nopopulation expansion and the population number was constant.AMOVAtest indicated that most of the observed genetic variation occurs between different populations(93%),whereas differentiation within 11 endemic populations contributed 7% to the total population.The PCoA analysis based on genetic distance and geographical distance revealed that the YN-BS + YN-KM clade was different from the remaining isolates.Therefore,there should be two groups in all isolates: the YN group and the SC+CQ group.4.Genetic diversity analysis based on sequencing data:We identified 4 haplotypes for theC07 locus,in which Hap1 contained 17isolates,Hap2 contained4 isolates,Hap3 contained5 isolates,and Hap4 only contained1 isolate.M15 contained9 haplotypes,including Hap1,Hap4,Hap6 and Hap8.M20 locus had 7haplotypes,in which Hap1contained10 isolates,and followed by Hap3(30 isolates),Hap4(13 isolates),Hap5(6 isolates),Hap2 and Hap7(2 isolates),and Hap6(only1 isolate).N11 had 10 haplotypes,Hap1contained21 isolates,Hap2contained18 isolates,Hap3contained6 isolates,Hap4contained9 isolates,Hap9contained3 isolates,Hap8 and Hap10contained2 isolates respectively,Hap5 ? Hap6 and Hap7contained1 isolates separately.The haplotype diversity(Hd)and nucleotide diversity(Pi)of C07,M15,M20 and N11 were0.175±0.061,0.00090±0.00032,0.468±0.072,0.01559±0.01007,0.553±0.057,0.00430±0.00062,0.581±0.064,and0.00494±0.00077 respectively.The results of AMOVArevealed that genetic diversity of intergroups was higher than that of intragroups.The results of phylogenetic analyses based on MP,ML and BI methods using all4 microsatellitesrevealed two main groups.Hap4 grouped into a clade when using the C07.Hap4 and Hap5 made up a single group by using the M15.Hap2 and Hap5 made up a single group by using the M20.For the N11,Hap3,Hap4,Hap6 and Hap7 revealed a monophyletic group.In agreement with the phylogenetic analyses,in the haplotype median-joining network,the haplotypes of sparganum isolates observed in the dataset also generated two subnetworks.Analysis of the median-joining network of C07 showed that Hap2 was the prominent haplotype.For M15 and M20,Hap1 and Hap3 were the prominent haplotypes respectively.Analysis of theN11 showed two prominent haplotypes: Hap1 and Hap2.Conclusions1.Spirometra spargana were found in wildRana nigromaculatafrom all 11 geographical locations in southwestern China,the average prevalence of sparganum infection was 18.44%.2.Four microsatellite loci: C07,M15,N11 and M20 were successfully screened using bioinformatic tools based on the public ESTsdatabase.3.The PCoA analysis indicated that there should be two groups in all isolates from southwestern China: the YN group and the SC+CQ group.And the genetic diversity was more affected by the geographical distance.4.The analysis of haplotypes from differentmicrosatellite loci revealed low genetic diversity level of sparganum isolates from southwestern China.5.Both of phylogenetic and haplotype network analyses revealed two genotypes in the sparganum population.