| Schistosomiasis is a zoonotic parasitic disease caused by Genus Schistosoma infection,which seriously endangers people's health and hinders social and economic development.Schistosoma spp are dioecious trematode,in which there are lots of species infecting human and animals.Several schistosome species also overlap in their geographical and host range,which allows male and female schistosomes of different species to pair within their definitive hosts.The hybridization among different species and the production of dominant hybrid species and changes of their biological characteristics,such as host selectivity,fertility and infectivity,can lead to the evolution of Schistosoma species,regional distribution of the population,the changes of epidemic patterns,pathogenicity to human and animals.With the rapid economic globalization,trade cooperation and the exchange of different countries,increasing the opportunities of the hybridization within Genus Schistosoma,the schistosomiasis caused by hybrid strains is an emerging public health problem in our changing world.Part ? Establishment of hybrid strains of Schistosoma japonicum and Schistosoma mansoniObjective:To investigate whether Schistosoma japonicum schistosomulum and Schistosoma mansoni schistosomulum can complete the sexual reproduction in the same terminal host,the miracidium hatched from the offspring eggs infected intermediate host snail,and the larvae in the snail complete the asexual reproduction,so as to form fertility hybrid strains.Methods:Single sex cercariae had been made by single snail(Oncomelania hupensis or Biomphalaria glabrata)infected with a single miracidium,two group mice were infected with female Schistosoma japonicum cercariae and male Schistosoma japonicum cercariae,female Schistosoma japonicum cercariae and male Schistosoma mansoni cercariae,respectively.Collecting the eggs in the feces of infected mice,and the miracidium hatching from the eggs were used to infect Oncomelania hupensis and Biomphalaria glabrata.The snails are kept in the laboratory condition at 25°C by the conventional method,and the cercariae from the infected snails were used to infect mice and hatching eggs completes the lifecycle.Results:The morphological of hybrid eggs in the mice infected with female Schistosoma mansoni and male Schistosoma japonicum found in feces 52 days after infection were similar to Schistosoma mansoni eggs.The miracidium hatched from the eggs were used to infect Oncomelania hupensis and Biomphalaria glabrata snails,the hybrid cercariae from the Oncomelania hupensis and Biomphalaria glabrata had been found at the 70 and 42 days after infection,respectively.At 42 days after infection,the cercariae can develop to adults and lay eggs.The morphological of hybrid eggs in the mice infected with female japonicum and male Schistosoma mansoni found in feces 46 days after infection were similar to Schistosoma japonicum eggs.The miracidium hatched from the eggs were used to infect Oncomelania hupensis and Biomphalaria glabrata snails,the hybrid cercariae from the Oncomelania hupensis had been found at the 93 days after infection,At 36 days after infection,the cercariae can develop to adults and lay eggs.Conclusion:Two hybrid strains of female Schistosoma mansoni cercariae with male Schistosoma japonicum cercariae,or female Schistosoma japonicum and male Schistosoma mansoni cercariae in mice can develop in various stages and finish lifecycle and produce fertile offspring;The morphological characteristics of the eggs and the selectivity of the miracidium to the intermediate host snails were changed.Part ? Biological characteristics of hybrid strains of Schistosoma japonicum and Schistosoma mansoniObjective:To compare the biological characteristics of two hybrid strains of the Schistosoma mansoni cercariae and male Schistosoma japonicum cercariae,the female Schistosoma japonicum cercariae and male Schistosoma japonicum cercariae so as to evaluate the impact of changes within biological characteristics of hybrid strains on the spread of schistosomiasis.Methods:Mice were divided into four groups and infected with female and male cercariae of Schistosoma japonicum,female and male cercariae of Schistosoma mansoni,female Schistosoma japonicum cercariae and male Schistosoma japonicum cercariae,female Schistosoma japonicum cercariae and male Schistosoma mansoni cercariae,respectively.The feces from each group of mouse were collected everyday from the 20th day after infection,and the eggs was detected by direct saline smear method;when the eggs in the feces of each mouse were found,the mice were dissectted and schistosome adults were collected in the mesenteric vein,hepatic portal vein and liver of each mouse.The number of female,male,and the male-female worm pairing was counted under the anatomical microscope.The rate of the male-female worm pairing was calculated,and the lengths of the males and females were measured and compared.Eggs in the liver tissues were collected by nylon bag collection method.The size of the hybrid eggs was measured,the hatching rate of the eggs was counted by miracidium hatching method,and the infection rate of the Oncomelania hupensis and Biomphalaria glabrata snails was calculated by quantitative infection of miracidium.The prepatent period of cercariae in the intermadiate host snails were observed.Results:In the terminal host mice,the prepatent period of Schistosoma japonicum,Schistosoma mansoni eggs in feces was 36.09±1.98 days and 44.74±1.98days.The prepatent period of female Schistosoma mansoni and male Schistosoma japonicum infection group eggs in feces was 54.30±2.23 days.The prepatent period of female Schistosoma japonicum and male Schistosoma mansoni infection group eggs in feces was 48.74±2.09 days.There was a statistically significant difference in the prepatent period between female Schistosoma mansoni and male Schistosoma japonicum infection group and Schistosoma mansoni group(t=16.87 P(27)0.001)),There was a statistically significant difference in the prepatent period between female Schistosoma japonicum and male Schistosoma mansoni infection group and Schistosoma japonicum group(t=21.08 P(27)0.001).The length and width of the eggs of female Schistosoma mansoni and male Schistosoma japonicum infection group and Schistosoma mansoni eggs were122.79±8.92,59.77±5.14mm and 115.21±8.75,56.24±5.49mm,respectively.And the hatching rate of eggs was2.40%,5.50%,the size of hybrid eggs was significantly larger than that of Schistosoma mansoni(P(27)0.01),the hatching rate was lower than that of Schistosoma mansoni(P(27)0.05);The length and width of the eggs of the female Schistosoma japonicum and male Schistosoma mansoni infection group and Schistosoma japonicum were 76.64±3.97,57.56±2.97mm,and 72.90±3.50,52.99±1.88mm,respectively.The hatching rate of eggs was4.85%,5.55%and,respectively.The size of hybrid eggs was significantly larger than that of Schistosoma japonicum.(P(27)0.01),the hatching rate was lower than that of Schistosoma japonicum(P(27)0.05).The rates of male-female worm pairing in Schistosoma japonicum group,Schistosoma mansoni group,female Schistosoma japonicum and male Schistosoma japonicum infection group,female Schistosoma mansoni and male Schistosoma japonicum infection group were 84%,74.64%,60.85%,40.00%(P(27)0.05).The length of pairing female Schistosoma japonicum adults was 18.87±1.66mm,the length of the unpairing female adults was 11.13±1.69mm.The length of the pairing female Schistosoma mansoni adults was 13.36±0.92mm,the length of the unpaired females was 8.26±0.94mm.The difference between the length of female adults after male-female worm pairing and unpairing in the same Schistosome has statistically significant(P(27)0.01).The length of pairing female and male adults of the 4th group of female Schistosoma japonicum and male Schistosoma mansoni was 6.50±0.77,7.07±0.63mm,respectively,The length of the unpairing female and male aduls was6.75±0.79,7.07±0.75mm,respectively.The length of pairing female and male adults of the 2nd group of female Schistosoma mansoni and male Schistosoma japonicum was 5.40±0.91,8.17±0.89mm,and the length of unpairing female and male aduls was5.36±0.80,8.07±0.75mm.The length of female and male adults of male-female worm pairing between different Schistosome species was significantly shorter than that of the same Schistosome species(P(27)0.05).The hybrid miracidium from the group of female Schistosoma mansoni and male Schistosoma japonicum can infect Oncomelania hupensis and Biomphalaria glabrata,the infection rates were 4.61%and 5.11%,respectively.The hybrid miracidium from the group of female Schistosoma japonicum and male Schistosoma mansoni can only infect Oncomelania hupensis,the infection rate was 1.44%;the infection rates of snails infected with Schistosoma japonicum and Schistosoma mansoni miracidium were 8.94%and 11.33%,respectively,There was a statistically significant difference between the four groups(c~2=131.25,P(27)0.01).The prepatent period of hybrid cercariae of the group of female Schistosoma mansoni and male Schistosoma japonicum in Biomphalaria glabrata and Oncomelania hupensis,and the prepatent period of Schistosoma mansoni in Biomphalaria glabrata was 43.38±1.19,70.14±1.79,37.36±2.16 days,respectively.There was a statistically significant difference between the hybrid cercariae and Schistosoma mansoni cercariae(P(27)0.01);The prepatent period of the hybrid cercariae of the group of female Schistosoma japonicum and the male Schistosoma mansoni in Oncomelania hupensis,and the prepatent period of the Schistosoma japonicum cercariae in Oncomelania hupensis were 96.54±3.15,64.45±2.84,respectively,There was a statistically significant difference between the hybrid cercariae and Schistosom japonicum cercariae(P(27)0.01).Conclusion:The rate of male-female worm pairing in different Schistosome species is lower than that of the same species.The male-female worm pairing in the same Schistosome species can promote the development of female schistosomulum,but have no effect on the development of male schistosomulum.The lengths of female and male adults are significantly shorter than those of the same species.The morphology of the hybrid eggs of female Schistosoma japonicum and male Schistosoma mansoni infection group was similar to the eggs of Schistosoma japonicum,the hatching rate of eggs decreased,the preptent period of eggs in the feces was longer than Schistosoma japonicum.The hybrid miracidium can infect the Oncomelania hupensis,and the infection rate decreased;The morphology of the hybrid eggs of female Schistosoma mansoni and male Schistosoma japonicum infection group was similar to the eggs of Schistosoma mansoni,the hatching rate of eggs decreased,the prepatent period of eggs in the feces was longer than Schistosoma mansoni.The hybrids miracidium can infect Oncomelania hupensis and Biomphalaria glabrata,and the infection rate decreased,and the prepatent period of hybrid cercariae in Oncomelania hupensis and Biomphalaria glabrata were delayedPart ? The molecular markers of the hybrid strain of Schistosoma japonicum and Schistosoma mansoniObjective:To explore the molecular basis of the relationship between the hybrid strains and the parental strains.Methods:The schistosome STRs were obtained by literature search and laboratory establish,the schistosome STRs were used to establish an STR amplification system for Schistosoma japonicum and Schistosoma mansoni.The multiple reaction system of Schistosoma japonicum and Schistosoma japonicum was used to identify the species of adults of male-female worm pairing and hybrid miracidium,cercariae and adult.The homology between the hybrids progeny and the parents was analyzed by genomic resequencing.Results:The female adult DNA of the male-female worm pairing in the mixed infection group of female Schistosoma japonicum and male Schistosoma mansoni could be amplified in the multiplex PCR reaction system of Schistosoma japonicum,and the male adult DNA could be amplified in the multiplex PCR reaction system of Schistosoma mansoni;The female adult DNA of the male-female worm pairing in the mixed infection group of female Schistosoma mansoni and male Schistosoma japonicum can be amplified in the multiplex PCR reaction system of Schistosoma mansoni,and the male adult DNA can be amplified in the multiplex PCR reaction system of Schistosoma japonicum.Hybrid miracidium,cercariae and adults of the mixed infection group of female Schistosoma mansoni and male Schistosoma japonicum can be amplified in the multiplex PCR reaction system of Schistosoma mansoni,but can not be amplified in the multiple PCR reaction system of Schistosoma japonicum;Hybrid miracidium,cercariae and adults of the mixed infection group of female Schistosoma japonicum and male Schistosoma mansoni can be amplified in the multiplex PCR reaction system of Schistosoma japonicum,but can not be amplified in the multiple PCR reaction system of Schistosoma mansoni.Homology analysis:The percentage of hybrid adults of female schistosoma mansoni and male Schistosoma japonicum(derived from Oncomelania hupensis)with at least 1 and 4 bases covered by Schistosoma japonicum and schistosoma mansoni genomes was 94.49%,91.91%and 2.44%,1.51%,respectively;The percentage of hybrid adults of female schistosoma mansoni and male Schistosoma japonicum(derived from Biomphalaria glabrata)with at least 1 and 4 bases covered by Schistosoma japonicum and schistosoma mansoni genomes was 2.36%,1.58%,and97.20%,94.53%,respectively;The percentage of hybrid adults of female schistosoma japonicum and male Schistosoma mansoni(derived from Oncomelania hupensis)with at least 1 and 4 bases covered by Schistosoma japonicum and schistosoma mansoni genomes was 95.45%,91.62%,and 2.33%,1.47%,respectively.Conclusion:The male-female worm pairing adults of the mixed group of female Schistosoma mansoni and male Schistosoma japonicum,female Schistosoma japonicum and male Schistosoma mansoni were different species,one was from Schistosoma japonicum,the other was from Schistosoma mansoni.Hybrid miracidium,cercariae and adults DNA can only be amplified in their maternal multiplex PCR reaction system.Hybrid offsprings of female Schistosoma mansoni and male Schistosoma japonicum from the Oncomelania hupensis have high homology with Schistosoma japonicum,and hybrid offsprings from the Biomphalaria glabrata have high homology with Schistosoma mansoni;Hybrid offsprings of female Schistosoma japonicum and males Schistosoma mansoni has high homology with the female Schistosoma japonicum.It is suggested that the hybrid reproduction between Schistosoma japonicum and Schistosoma mansoni lacks evidence of genetic communication,which seems to be related to parthenogenesis. |
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