Investigation On The Metacercariae Of Prosthogonimus Spp.in Heilongjiang Dragonflies And Analysis Of The Complete Mitochondrial And Ribosomal Sequences Of Adult Flukes

Prosthogonimus spp.is a common parasite of poultry and wild birds,which seriously affects the health of them.Currently,most of the studies on Prosthogonimus spp.in poultry belongs to case analysis as well as disease prevention and treatment,while the studies on the intermediate host and the complete mitochondrial and ribosomal sequences of Prosthogonimus spp.are relatively few.Therefore,this study investigates the infection of metacercaria of Prosthogonimus spp.in dragonflies in Heilongjiang,obtain adults through animal reduction experiments,amplifies and analysis the complete mitochondrial genome and ribosome sequence and discuss the genetic relationship between Prosthogonimus spp.and other trematodes.From June 2019 to September 2020,dragonfly samples were collected at different locations in 12 prefectures and cities in Heilongjiang.A total of 12 species,6809 dragonflies were collected.The abdominal muscle compression method was used to check the infection of the progenitor metacercaria in different places and different species of dragonflies,and the infection rate was calculated.The collected metacercaria were identified by morphology,animal infection experiment and molecular biology methods.Adults were obtained from animal infection experiments and then subjected to morphological and molecular biological identification to determine the accuracy of metacercaria identification.The complete mitochondrial genome and ribosomal sequence were amplified by PCR,and the phylogenetic tree of 12 tandem protein coding genes in mitochondria and 18 S in ribosomal was constructed by Maximum parsimony(MP),Maximum likelihood(ML)and Bayesian(BI)methods respectively,so as to explore its genetic relationship with other trematodes.The results showed that it was found that dragonflies in Heilongjiang Province contained three kinds of metacercaria of Prosthogonimus cuneatus,Prosthogonimus pellucidus and Prosthogonimus sp.;The total infection rate of dragonflies metacercariae was 22.0%(1498/6809),the highest in Heihe,46.6%(48/103);Of the 12 species of dragonflies collected,6 species were detected with fluke metacercaria.The three dragonflies with the highest infection rate of metacercariae were the Sympetrum depressiusculum 30.6%(190/621),the Pantala flavescens 22.8%(541/2371),and the Sympetrum frequens 22.3%(376/1689).Among the three metacercariae of Prosthogonimus spp.,the number of infections of Prosthogonimus sp.and P.pellucidus were more widely distributed.Pantala flavescens was the dragonfly species with the largest number of infections of the three Prosthogonimus spp.metacercaria.After the animals were infected,two adults of P.cuneatus and P.pellucidus were obtained.The the length of complete mitochondrial amplification of P.cuneatus and P.pellucidus were 14 829 bp and 15 013 bp,respectively.They contained12 protein coding genes(PCGs),22 t RNAs,2 r RNA and 1 non-coding region,and the AT content of them were 64.47% and 65.34%,respectively.The most frequently used start codon and stop codon of them were ATG and TAG,respectively.Phylogenetic analysis of the concatenated,aligned amino acid sequences of the 12 PCGs showed Prosthogoniminae to be more closely related to Dicrocoeliidae,than it was to Plagiorchiidae and Brachycladiidae in the same suborder.The the length of complete ribosomal sequence of P.cuneatus and P.pellucidus were 10 713 bp and 9501 bp,respectively,including18 S,ITS1,5.8S,ITS2 and IGS,and the AT content were 49.87% and 49.52%,respectively.A total of 15 pairs of forward repeat sequences and 34 pairs of inverted repeat sequences were found in the complete ribosomal sequence of P.cuneatus,and 27 pairs of forward repeat sequences and 37 pairs of inverted repeat sequences were found in the complete ribosomal sequence of P.pellucidus.Phylogenetic analysis using 18 S as a gene marker showed that P.cuneatus and P.pellucidus clustered together with the same genus of Prosthogonimus ovatus and Schistogonimus rarus of the same family.The genetic relationship with Brachycladiidae and Auridistomidae two families of the same suborder was closer than that of other families.This study has ascertained the infection of Prosthogonimus spp.metacercariae in dragonflies in Heilongjiang,and provided a reliable basis for the prevention and control of Prosthogonimosis.In this study,the complete mitochondrial genomes and ribosomal sequences of P.cuneatus and P.pellucidus were determined for the first time and their sequence characteristics and evolutionary relationship were analyzed,which laid the foundation for further research on the molecular classification,population genetics and evolution of Prosthogonimus spp..