Analysis Of Host Adaptation And Genetic Differentiation Based On The Whole Genome Of Sarcoptes Scabiei And Preliminary Assessment Of The Application Of Two Antigenic Genes

Scabies is an ancient disease caused by the parasitizing of Sarcoptes scabiei in the epidermis of humans and more than 100 domestic and wild animals,causing relatively high morbidity and mortality.Scabies is also one of the top 50 pandemic disease and has officially been designated as a “neglected tropical disease” in 2017.There are several issues existed in the study of scabies mite: firstly,there’s no high-quality reference genome for Sarcoptes scabiei;secondly,knowledge about scabies mite adapting to a parasitic life is lacking,and researches concerning origin of scabies mite and its evolution is mainly based on limited markers,generally ending up with conflicting results;thirdly,microscopic examination is still the main reliable method to detect scabies,effective immunological diagnostic method is not available.The goal of this study is to present a high quality Sarcoptes scabiei var.cuniculi genome(Ssc-vcu),taking the Ssc-vcu genome as the reference genome of sarcoptes mite,and discuss the genetic basis of how scabies mite adapting to a permanent parasitic life as well the origin and evolution of scabies mite from whole genome level.Finally,coding sequence of inorganic pyrophosphatase(Ssc-PYP-1)and enolase(Ssc-eno)were selected from genome and transcriptome of Sarcoptes scabiei var.cuniculi,and the potential serodiagnostic value were evaluated by indirect ELISA based on recombinant protein.The main results were as follows:1.Chromosome-scale genome assembly of Sarcoptes scabiei var.cuniculiBy using 94.32 x of illumina data,321.09 x of Pac Bio data and 254.87 x of Hi-C data,we finally obtained a chromosome-scale genome assembly with a contig N50 size of 5.92 Mb,scaffold N50 size of 6.52 Mb,and total assembled length of 57.30 Mb.Combination usage of sequencing technologies and around 670-fold of genome sequencing guarantees high singlebase correctness of Ssc-vcu genome.CEGMA and BUSCO assessment showed that 97.98% of conserve Core Eukaryotic Genes and 89.0% of 1066 complete arthropod single-copy orthologous genes were identified,which suggested the completeness of Ssc-vcu genome assembly.With a combination of homology,Denovo and RNA-aided prediction method,9,333 protein-coding genes were found in the Ssc-vcu genome,averaging 3.78 exons and1,622.09 bp per gene.Additionally,the Ssc-vcu genome also contains 12.65% of repeat elements and 0.39% non-coding RNA sequences,including 0.36% of mi RNA、0.0084% of t RNA、0.0235% of r RNA and 0.0029% of sn RNA.2.Comparative genomics analysis revealed the genetic basis of the adaption of scabies mites to parasitic lifeSarcoptes scabiei is an ectoparasite that parasitize in the epidermis of mammalian host skin.In this study,the Ssc-vcu genome was used as reference genome of scabies mite and the genetic basis of scabies mite adapting for a parasitic life was discussed by comparative genomics.Clustering analysis suggested that the scabies genome has 104 unique gene families which include 373 genes,taking 4% of the total protein coding genes.These unique genes were mainly enriched in digestion of proteins,the process of environment information,absorption of nutrients and apoptosis.Identification four major detoxification gene families(ABC transporters,P450 s,GSTs and CCEs)showed strong contraction in CYP450 s and CCEs families.These results suggested that the enhanced ability of digesting proteins and reduced ability of detoxification might be the molecular basis of adapting to a permanent parasitic life.Characterization of 10 Hox genes in arthropod ancestor reveal that zen and abd-A were both absent in scabies mite genome,and the absence of abd-A could be the result of highly reduced segments and short opsthosoma.3.Resequencing of various mammalian hosts derived mites revealed the origin and differentiation of Sarcoptes scabieiTo investigate the conflicting issues of scabies mite classification and origin,20 sarcoptic mite samples collected from four mammalian hosts,including humans,pigs,dogs and rabbits were subjected to genome resequencing analysis.Phylogenic tree and principle component analysis showed that the scabies mites cluster together by host species,indicating that these mites primarily formed subspecies by hosts.The demographic history of scabies mites reconstructed by PSMC showed that among four hosts,human might be the primary host of sarcoptic mite and the scabies mites were then found from pig,dog and rabbit.On the other hand,selective sweep analysis among mites derived from either two hosts showed that,strong signal of selection between mites from pigs and mites from humans,358 genes which account for 3.8% of the whole genome were under selection,they were mainly enriched in “Cysteinetype peptidase activity”,“Neuroactive ligand-receptor interaction” and “Apoptosis”,and majority of the involved genes are tandem repeats;this difference might be related to the thickness of skin of humans and pigs.4.Molecular identification,prokaryotic expression of a novel PYP-1 gene in Sarcoptes scabiei and its potential as a serodiagnostic candidate by indirect-ELISAInorganic pyrophosphatases(PPases,EC 3.6.1.1)are ubiquitous enzymes that can catalyze the hydrolysis of inorganic pyrophosphate into orthophosphate,which play an important role in energy metabolism and biosynthetic reactions.In this study,prokaryotic expression and localization were performed for a novel inorganic pyrophosphatases from S.scabiei(Ssc-PYP-1),and the serodiagnostic potential was evaluated by the indirect ELISA based on recombinant Ssc-PYP-1 protein,the results showed that: native Ssc-PYP-1 was expressed in the tegument around the mouthparts and legs,in the cuticle of the mites,as well as on the fecal pellets of mites and in the integument of the mites;the sensitivity and specificity of r Ssc-PYP-1 i ELISA are 92.0% and 93.6%,and the infection of S.scabiei can be detected from 1 week post-infection,suggesting that Ssc-PYP-1 could serve as a potential serodiagnostic candidate for sarcoptic mange in rabbits and can be also used to detect early infection.5.Prokaryotic expression of alpha-enolase from Sarcoptes scabiei and the assessment of its potential as a serodiagnostic and vaccine candidateEnolase(2-phosphoglycerate hydratase,EC 4.2.1.11)is a ubiquitous enzyme which is mainly involved in glycolytic and gluconeogenesis pathways.Enolase can function in many other aspects in other organisms when its localization is changed.In this study,the alphaenolase from S.scabiei(Ssc-eno)was cloned and expressed.Localization of native Ssc-eno protein showed that the native Ssc-eno protein was intensely expressed in the tegument of the mouthparts,the legs,and the whole mites’ body,as well as in the gut and reproduction system.The indirect enzyme-linked immunosorbent assay(ELISA)based on r Ssc-eno showed 92.0%sensitivity and 95.8% specificity,and the r Ssc-eno based ELISA can also detect scabies mites infection in rabbits since one week post infection.In in vitro experiment,the anti-r Ssc-eno serum was found to have concentration-dependent acaricidal activity for larvae mites,suggesting that Ssc-eno could be a vaccine candidate.