Mitochondrial Genomics And Phylogeny Of Three Flea Species From Dogs And Cats

Fleas are a common ectoparasites,which not only bite the host and cause dermatitis and anemia,but also cause disease as the vector of pathogenic microorganisms,causing serious public health security problems and large economic losses.Accurate identification and classification of fleas is the basis for studying flea population genetics,evolutionary biology and phylogeny.Mitochondrial genome sequence is an ideal molecular marker for ectoparasites identification,population genetics and phylogenetic studies.So far,only six mitochondrial genomes of fleas.Therefore,it is necessary to sequence,analyze and phylogenetic analysis the mitochondrial genomes of other fleas.In the first part of this study,fleas from seven provinces(autonomous regions)in China were identified.Morphological results showed that there were 59 Ctenocephalides felis,one Ctenocephalides canis and 23 Pulex irritans.The results of molecular biological identification were consistent with those of morphological identification.Analysis of ITS1 sequences of three flea species showed that the interspecific differences of ITS1 sequences were significantly greater than the intraspecific differences,suggesting that ITS1 could be used as a marker for the study of genetic variation among flea species.In the second part of this study,EF-1α,cox1 and cox2 sequences of C.felis and P.irritans were analyzed.The results showed that C.felis and P.irritans had high genetic diversity.Neutral test showed that C.felis from Guangxi zhuang Autonomous Region and Henan Province experienced equilibrium selection and bottleneck effects.There were a large number of haplotypes in C.felis and P.irritans,but the distribution of haplotypes was low,the Fst value was high,the genetic differentiation was large,and the gene flow was moderate,which may be the result of restricted gene flow between different regions.Phylogenetic analysis based on cox1+cox2 sequences showed that C.felis was divided into two different branches,and there may be two genetic lineages.In the third part of this study,the mitochondrial complete genomes of C.felis,C.canis and P.irritans and the phylogenetic relationships of the order Siphonaptera were studied.The mitochondrial genome lengths of the three flea species were 20,911 bp,20,337 bp and 15,609 bp(lacking partial non-coding region sequences),which all contained 37 genes and a long non-coding region(except C.canis).The TTT was used as the starting codon in the cox1 gene of C.felis and C.canis.The non-coding regions of the complete mitochondrial genomes of C.felis and P.irritans were more than 5 kb,with a large number of repeated sequences.The mitochondrial genomes of C.felis isolates from China and C.felis isolates from US were compared and analyzed,the results showed that both of them belonged to the C.f.felis.The amino acid sequences of 13 protein-coding genes in the mitochondrial genome of the three flea species obtained in this study and 49 representative Endopterygota insects selected from Gen Bank were used for phylogenetic analysis,and the phylogenetic position of the Siphonaptera in Endopterygota insects was reevaluated.The results based on Bayesian inference(BI)and Maximum likelihood(ML)support that the order Siphonaptera exist at the level of order and the order Siphonaptera are monophyly.The results also showed a sister relationship between Siphonaptera and orders of Diptera + Mecoptera +Megaloptera + Neuroptera.Our results refute the view that the order Siphonaptera was not monophyly and should be reduced to a secondary order,and provide a new insight into the phylogenetic relationship of the order Siphonaptera in Endopterygota insects.This study not only provided molecular markers for flea species identification and clarified the molecular phylogenetic relationship of fleas,but also elucidated the structural characteristics of mitochondrial genomes of fleas,provided new molecular data for solving the phylogenetic relationship of the order Siphonaptera.