Studies On Distribution And Detection Methods Of Perkinsus Spp. In Mollusk Along The Coast Of China

Perkinsus spp.are protozoan parasites which are capable of infecting many marine mollusks and widely distributed throughout the world.Among them,P.marinus and P.olseni caused perkinsosis,resulting in a mass mortality,causing economic losses.In view of the serious harm to the mollusks,it is necessary to study the species diversity and host diversity of the Perkinsus distributed along the coastal of China,so that we could provide a theoretical basis for the scientific prevention and effective monitoring of the perkinsosis.In this study,we investigated the prevalence of the local mollusk in the three regions along the coastal of China(the Yellow Sea and the Bohai Sea,the East China Sea and the South China Sea),and confirmed the infection species and geographical distribution of the genus Perkinsus;through the seasonal monitoring of mollusks along the coastal areas of Guangxi province,we explored the parasitic host diversity and epidemiology of Perkinsus.At the same time,a rapid and sensitive detection technology was established.The main findings are as follows:1: Nine sampling locations were selected from three regions along the coastal of China,and 1399 samples from eight kinds of mollusks,including Crassostrea gigas,C.hongkongensis,Sinonovacula constrzcta,Ruditapes philippinarum,Scapharca subcrenata,Meretrix lyrate,Haliotis discus hannai and H.diversicolor,were detected by PCR.The results showed that three Perkinsus species were detected in Chinese waters,P.olseni,P.beihaiensis and P.chesapeaki.These Perkinsus spp.can infect a variety of mollusks,for example,P.olseni is more likely to infect the R.philippinarum while P.beihaiensis is more likely to parasitize the oysters.Geographically,P.olseni was widely distributed and had high prevalence at each sampling locations,especially distributed in the Yellow Sea and the Bohai Sea,and the East China Sea;P.beihaiensis was a dominant infection species distributed in the South China Sea.In addition,this was the first report of P.chesapeaki infecting new hosts(S.constrzcta,S.subcrenata and M.lyrata)in China.2: We investigated the distribution of the genus Perkinsus in the common mollusk species along the coastal areas of Guangxi province.Among the 14 mollusk species investigated in this study,13 mollusk species were infected with P.olseni and 8 mollusk species were infected with P.beihaiensis,7 mollusk species were infected with both Perkinsus species.Among them,the total prevalence of the P.olseni was significantly higher in Soletellina acuta,Callista erycina,R.philippinarum and S.subcrenata than in other mollusks,the total prevalence of the P.beihaiensis was significantly higher in C.hongkongensis and P.viridis than in other mollusks.In addition,the prevalence of P.beihaiensis in C.hongkongensis in winter was significantly higher than that in other seasons;the prevalence of P.olseni in C.hongkongensis was much lower than that of P.beihaiensis and had no significantly changes between different seasons.The prevalence of P.olseni in R.philippinarum was significantly higher than that of P.beihaiensis,but two Perkinsus species have no found in spring and summer.3: We developed a LF-RPA assay to detect P.beihaiensis in oyster samples.The assay was comparably sensitive to qPCR assay and complete in about 30 minutes at 37°C,and can detect down to 26 copies DNA with the result can be read by the strip;the cross amplification with other two Perkinsus species but not with other pathogen DNAs.A total of 60 oyster samples detected for P.beihaiensis infection by PCR were tested in parallel with the qPCR and LF-RPA.The LF-RPA showed 86.2% sensitivity and 77.4% specificity compared to PCR,and the overall agreement between the two methods was 81.7%.The LF-RPA showed 93.3% sensitivity and 86.7% specificity compared to qPCR,with an overall agreement between the two methods of 90.0%.The LF-RPA assay neither require a complex temperature-raising system nor require complicated product purification and agarose gel electrophoresis,shows a great potential for the detection of Perkinsus species in field application where lack of equipment or with poor condition.