Identification And Genetic Diversity Of The Thelazia Callipaeda Isolates From China

Thelazia callipaeda can parasitize in the conjunctival sac of domestic animals,wild predators and humans,leading to conjunctivitis,conjunctival hyperemia,lacrimation and itchiness,severe corneal ulcers,and even blindness.In 1910,Thelazia callipaeda was first discovered in conjunctival sac of an India dog.human thelaziasis were found in Beijing and Fuzhou of China,in 1917.At present,there are many reports of the thelaziasis in many parts of the world,but most cases have occurred in the eastern and southeastern parts of Asia.Therefore,Thelazia callipaeda are also known as the oriental eyeworms.China is the country with the most cases of Thelazia callipaeda,and there has been known more than 600 cases.In recent years,an increase in T.callipaeda infections has been reported in animals and humans living in China,and it has become a newly emerging animal-borne parasitic disease.Despite the threat of Thelaziacallipaedain many places of China,the systematic identification and genetic polymorphism studies of Thelaziacallipaeda are still weak.In this study,we first conducted morphological identification of clinical isolates of 3 cases of Thelazia callipaeda in central China,meanwhile conducted a de novo sequencing of their mitochondrial genomes.and then the genetic diversity analysis was performed on the isolates from 11 geographical locations of China,which laid a foundation for the prevention and control strategies of Thelazia callipaeda.Materials and Methods1 Three clinical isolates of T.callipaeda were harvested in different geographical locations of central China and examined under light microscopy at different magnifications and under the scanning electron microscope.The key morphological sites were photographed,described,and recorded,According to the classification search table,its species identification.2 Mitogenome sequence and assembly : The mitochondrial genome of Thelazia callipaeda was amplified by long-PCR and was sequenced on an Illumina Hi Seq 2000.Using the Fast QC v.0.11.5 to assess the quality of original sequencing.And then using the CLC Genomic Workbench v.7.0.4 to map the modified sequences against two reference mt genomes of T.callipaeda?NC₀18363 and AP017700?.Finally,MITOS was used to annotate mitochondrial genes,Submit to Gen Bank database.3 Genetic diversity analysis of Thelazia callipaeda:Using several molecular markers?cytb,cox1,12 S r DNA,ITS1 and 18 S r DNA?to analyze genetic diversity of T.callipaeda.Analysing the number of haplotypes,haplotype diversity?Hd?,and nucleotide diversity?Pi?of each molecular marker with the help of Dna SP v5.10.A median-joining network between haplotypes are drawn throughing Network v5.0 software.The levels of genetic differentiation among the populations was explored by Pairwise FST values.Using mismatch distributions in the software Arlequin to evaluate demographic changes.At the same time,we use Tajima's D and Fu's FS assesses to test the possible of population expansion.In view of molecular markers cox1,we analyse genetic variability comparative analysis of T.callipaeda isolates in the world.4 Phylogenetic analysis: We have used the maximum parsimony?MP?and Bayesian?BI?methods to estimate phylogenetic analysis of cox1 haplotypes of the T.callipaeda.MP analysis was performed in PAUP* 4b10 and BI analysis was performed in Mr Bayes v3.2.Results1 T.callipaeda morphology: for the females: T.callipaeda was characterized by the presence of a buccal capsule;In the posterior half of the body,immature eggs or germ cells filled the uterine tubules.The phasmids and an anus were present on the tip of the tail;The transversally striated cuticle was more corrugated on the anterior of the body than on the caudal end;The vulva with a short vulvar flap was located in the anterior region of the body.For the males: In contrast with the femal,the buccal capsule was inconspicuous for the male,and their mouth opening showed a cycle or ellipse profile;Two dissimilar spicules revealed in the males,one was the anterior extremity of the longer left spicule,the other was the typical crescent shape of the shorter right spicule;The caudal end was ventrally curved;There were 5pairs of postcloacal papillae in the male.2.Characteristics and comparison of mt genomes of T.callipaeda: The mitochondrial genome of the T.callipaeda has 2 r RNA genes,22 t RNA genes and 12 PCGs.All genes have the same arrangement and coding direction,and the length of protein-coding genes are the same.The mt genomes,some genes have overlapping regions.22 t RNA genes range in length from 53 to 66 bases.There is a long?316 bp?noncoding region between the cox3 and alanine?trn A?genes.The similarity of mitochondrial genomic sequences from the T.callipaeda from different regions was 98.22%.The highest similarity between protein-coding genes sequences is nad4 L,and the lowest is atp6.The most similar amino acid sequence is cox1,the lowest is nad6.The sequence similarity of mitochondrial ribosome size subunit r DNA?rrn L,rrn S?was89.95% and 96.71%,respectively.3 Genetic diversity and genetic variability of T.callipaeda isolates: Through studying 32 isolates from 11 areas in China we find the higher genetic diversity.A total 21 haplotypes were identified for the cox1 gene in different regions of the world.Hap1 for the T.callipaeda in European countries.,and Haps2 – 8 and haps10 – 12 for the T.callipaeda were mostly found in domestic dogs.Hap9 and haps13 – 21 were mainly found in clinical isolates of T.callipaeda.37 nucleotide variations were found in the alignment of 21 haplotypes.The resultsof the analysis of molecular variance show that the differences between European and Asian countries' populations are greater than those between China,South Korea,and Japan.Analysis of the distribution of defects based on all markers shows that population expansion effects are not supported.4 Phylogeny reconstruction: In the family of spirurid nematodes,the branches formed by the Gnathostomatidae and Dracunculidae,Philometridae,Physalopteridae,Thelaziidae,Gongylonematidae,Setariidae,and Onchocercidae are sister groups.Among the larger branches,the earliest differentiated species were Dracunculidae.The Onchocercidae as a monophyletic group was the last diverged.There are two sub-populations of FST divided from European and Asian countries,which began to differentiate in the mid Pleistocene.Conclusion1 According to morphological characteristics by light microscopy and scanning electron microscopy,all clinical isolates were identified as T.callipaeda.2 The nad6 gene showed high sequence variability among all isolates,which is worth considering for future population genetic studies of T.callipaeda.3 Five nucleotide positions in the cox1 sequences can distinguish individuals from Europe and Asia;Differences between populations in Europe and Asia are greater than those between China,Korea,and Japan.4 There are two sub-populations of FST divided from European and Asian countries,which began to differentiate in the mid Pleistocene.5 Phylogenetic analysis showed that the phylogenetic relationships of Thelaziidae,Onchocercidae,Setariidae,Gongylonematidae,Physalopteridae,Dracunculidae and Philometridae were relatively close.