Rapid Molecular Diagnoses Of Species And Genetic Introgression Of Apple Snails(Gastropoda:Pomacea) In China

Apple snails(Ampullariidae: Pomacea)are freshwater snails,which are native to South America.In the last decades,they have been introduced to a number of countries of Asia,North America,Oceania,and Europe.They pose seriously threat to aquatic agricultural products,ecological balance and human health in invaded areas.Since their initial introduction to Taiwan in 1980 s,apple snails have become major pests in rice paddies in southern China.Three Pomacea species have been reported distribution in China,i.e.P.canaliculata,P.maculata,and Pomacea sp.Due to the similar shell morphology among different species,as well as the geographical diversity within each species,it is difficult to diagnose the Pomacea spp.accurately and rapidly based on external morphology only.In addition,studies have shown that P.canaliculata and P.maculata are admixed during the course of pervasion.Genetic introgression was detected in invasive populations of P.canaliculata and P.maculata.However,genetic introgression of Chinese populations of apple snails has not been studies previously.This study collected samples from the main distribution ranges of apples snails in southern China,including 31 geographical populations in 12provinces/autonomous regions(Jiangsu,Zhejiang,Jiangxi,Hunan,Sichuan,Chongqing,Guizhou,Yunnan,Fujian,Guangxi,Guangdong,Hainan).We established a technique for accurate and rapid diagnoses among P.canaliculata,P.maculata,and Pomacea sp.Furthermore,the genetic introgression patterns of P.canaliculata and P.maculata and their distribution in China were explored.The effects of genetic introgression on the fitness of apple snails were revealed through the comparison of morphological and biological characteristics of eggs.This study is of great significance for understanding the invasive mechanism of apple snails and developing effective pest management measures.The main results and conclusions are as follows:1.Rapid molecular species diagnoses of Pomacea spp.In this study,the complete mitochondrial genome sequences of P.canaliculata,P.maculata,and Pomacea sp.were downloaded from NCBI.Based on multiple alignment of the mitochondrial genome sequences,we designed primers fromsequence variation regions for the three Pomacea spp.Specificity and sensitivity of the primers was tested,and then a multiplex PCR method was established.The results showed that the multiplex PCR assay amplified 1238 bp,901 bp and 571 bp fragments from different geographical populations of P.canaliculata,P.maculata and Pomacea sp.,respectively.This multiplex PCR assay with species-specific primers can be applied in detection tissues and samples of different developmental stages.Also,this method does not need sequencing,which shows advantage in fast,accurate,and efficient in detection of multiple targets of Pomacea spp.2.Genetic introgression of apple snails in ChinaWe cloned and sequenced nuclear EF1?(elongation factor 1-alpha)fragments of 128 apple snails collected from 30 geographical populations in China.The maximum likelihood phylogenetic tree showed that EF1? sequences obtained in this study clustered into two clusters.Each cluster was grouped with the published P.canaliculata(C type)and P.maculata(M type)EF1? sequences downloaded from GenBank,respectively.Samples with EF1? clones containing both C and M type sequences were hybrids(H type).Based on multiple alignment and analysis of the obtained EF1? sequences,specific primers were designed for both the C and M type sequences.We established a multiplex PCR method to detect hybrids between P.canaliculata and P.maculata based on the evaluation of specificity and sensitivity of the primers.Comparing with the published sequence cloning and PCR-RFLP methods,which cost about 5 days or 4-5 h,the detection time of the multiplex PCR method developed this study was reduced to 2-3 h in detecting genetic introgression patterns of P.canaliculata and P.maculata.Detection of 276 snails collected from 30 geographical populations in 12provinces/autonomous regions of China using the combined EF1? sequencing and multiplex PCR detection technology showed that genetic introgression of P.canaliculata and P.maculata populations were widespread in China.There were different introgression patterns,including C,M and H types of P.canaliculata and C and H types of P.maculata.We did not detect M type individuals in P.maculata.The H type of P.canaliculata is the most widely distributed detected in 28 geographical populations,which accounted for 61.6% of the total sampling number.It is speculated that genetic introgression may enhance the invasive fitness of P.canaliculata and take advantage of heterosis.3.The effects of genetic introgression on biological characteristics of apple snails eggsWe collected egg clutches from the West Lake of Hangzhou,Jianggan of Hangzhou,and Wenling of Taizhou.We also obtained the Japanese population of "pure" P.canaliculata and the American population of "pure" P.maculata.Species and introgression patterns were identified based on the methods developed above.Egg numbers,hatching rates,and shell heights of hatchlings of clutches were compared among the three Chinese populations and both the “pure” apple snail species populations.It was shown that genetic introgression increased the fecundity and fitness of P.canaliculata population,which was one of the reasons for the widespread distribution of P.canaliculata in China.In addition,results of the laboratory crossbreeding experiment between "pure" P.canaliculata population in Japan and the "purebred" P.maculata population in Amercia,showed that the P.canaliculata and P.maculata did not mate,or their hybridized eggs could not hatch.The reproductive isolation results between P.canaliculata and P.maculata indicated that their interspecific genetic introgression may have occurred prior to introduction.