Sibship Analysis With Microsatellite In Schistosoma Japonicum Within And Among Snail Hosts

Objectives: Schistosomiasis japonica is a serious parasitic disease and a major health risk for people living in the tropical and subtropical zones of south China. China has made remarkable progress in reducing Schistosoma japonicum in humans to a relatively low level and the current bulk of tranmission occurs in the marsh and some hilly regions.Different natural populations of schistosomes are adapting to their local environments(such as definitive hosts) or selection pressures imposed by facors such as chemotherapy, then there is the potential for population subdivision to occur. Each of these relatively isolated subpopulations could then evolve a disparate set of characters for traits such as drug susceptibility and pathology. Understanding the genetic structure of S. japonicum will lead to a better understanding of the transmission dynamics of schistosomes. Schistosome population genetic structure, of which most focus on the genetic diversity within definitive populations, has played an important role in schistosome epidemiology. There are a few studies about the genetic structure of schistosomes within and among the intermediate hosts. Given this, we got infective snails from schistosomiasis endemic area through the filed survey and collected schistosomes after the procedure that cercariae infected the mice. Then microsatellite markers were used for genotyping and analyzing the genetic structure, the pedigree analysis of schistosomes within and among snails, which helps for understanding the transmission pattern of schistosomes and provides for establishing the schistosomiasis transmission dynamics model and effective control of schistosomiasis.Methods: we originally selected 17 microsatellite loci appropriate for our study from previously published articles and determined the finally selected markers after the screen test and optimization of PCR conditions.Snails were collected from the schistosomiasis field and brought back to the laboratory for infective ones. They were isolated and individually infected mice through the shedding of cercariae for collection of adults.Before the statistical analysis, we applied the GENECAP 1.2.2 to exclude the schistosomes of which multilocus genotype identical to each other. Then diversity including the number of alleles(A), allelic richness(Rs, number of alleles rarefied to the smallest sample size), observed heterozygosity(Ho), expected heterozygosity(He) and the Weir and Cockerham estimator f of Fis were calculated for each locus for the population considered as a whole using FSTAT 2.9.3. PIC for each locus were calculated by relevant formula. Hardy–Weinberg equilibrium tests at each locus were performed using GENEPOP 4.0. The observed multilocus genotype distribution among snails was tested under the assumption of random distribution(Poission goodness-of-fit test).SPAGe Di calculateed relatedness coefficients for pairs of schistosomes in and among snails. Colony 2.0 was applied here to estimate full-sib relationships between the adults.Results:1. We collected 8563 snails and 67 of which 46 successfully infected the mice were infective and 472 schistosome adults were finally collected as samples.2. After the optimization of PCR conditions and screening, 12 loci were recognized as appropriate for further study owing to their good amplification and high genetic diversity. The total number of alleles per locus ranged from 4(sjp22) to 25(sjp34), with a mean figure of 16.7. The average observed heterozygosity and expected heterozygosity were 0.633 and 0.437, with values ranging between 0.005 and 0.914, between 0.057 and 0.557, respectively. The PIC for each locus ranged from 0.1 to 0.914,with the mean figure of 0.743. Except from sjp4 and sjp22, the PIC of rest loci were larger than 0.5. Given the high genetic diversity, 12 loci were considered as suitable markers for schistosomiasis genetic study.3. Fis for each locus ranged from-0.709 to 0.779 and 3 loci(sjp32, sjp42, sjp63) conformed to Hardy-Weinberg equilibrium, while the rest showed significant departure from the equilibrium. Fis for each locus showed heterozygote excess(sjp1, sjp34, sjp28, sjp45, sjp54, jp60, sjp4, sjp18) or deficit(sjp22). The observed distribution of multilocus genotype differed significantly from expected abundance values under the assumption of random distribution(Poisson goodness-of-fit tests, p<0.005).4. Relatedness structure was studied at three hierarchical levels: pairs of all individuals(with rmean=0.164±0.159), pairs of individuals within snails(with rmean=0.670±0.24), pairs of individuals among snails(with rmean=0.150±0.131), demonstrated low relatedness for schistosomes between the snails and high relatedness within individual snails.5. Two different analyses with distinct error rates were performed by Colony, leading to the same sibship construction. Fifty-three families ranging from one to fifteen individuals were identified. Of all, fifty were found in single snails, and only three families(family 2, 8, 43) were present in two snails. On the other hand, seven snails harbored two families, one snail harbored three families and the rest snails harbored only one family. The pedigree analysis and sibship analysis demonstrated that the relatedness of schistosomes in snails is closer than among snails and schistosomes located in the same snail originated from the same parent pair, single snail tended to be infected by one miracidum. A few snails tended to be infected by several fimilies(2-3), and some families also could infect two snails.Conclusions:1. The study preliminarily determined the 12 microsatellite loci for further study on Schistosoma japonicum genetic structure and sibship analysis.2. Most snails harbour one family and some harbour more than one(snail A2P1, A3P2, A6P3, B4P3, C1P4, C3P1, C6P3, D4P3) of which snail A3P2, C3P1 were with mixed-sex infection. The former indicates less activities of definitive hosts and the latter implies more hosts and more activities, with strong ability to spread schistosomes to other definitive hosts. Moreover, A3P2 and C3P1 with mixed-sex infection are most probable to spread schistosomes.3. The closer relationship in snails is concerned with the spatial aggregation of schistosomes in standy water, concomitant immunity caused by initial schistosomes infected snails and the kin selection owing to the relatedness of schistosomes in snails.4. The pedigree analysis and sibship anlysis help for understanding the transmission pattern of schistosomes in and among snails, providng for establishing the schistosomiasis transmission dynamics model and effective control of schistosomiasis.