| Ticks are obligate hematophagous ectoparasitic that widely parasitized mammals,poultry,and amphibians.It is one of the most important arthropod vectors of human and animals.Not only they can cause skin and vascular damage of hosts during long-term blood sucking,but also transmitting tick-borne diseases.It is also during this process that the tick can transmit serious public pathogens such as Rickettsia,Ehrlichia,Borrelia,and protozoans,which can cause severe losses in animal production and health problems for humans.To date,the eradication of ticks mainly depends on chemical insecticides in the world,but the long-term use of chemical drugs has led to the increase of drug resistance,and the problem of drug residues seriously endangers the ecological environment.Ticks function genes as protective antigen have been found in abundance for developing of tick vaccine.However,the diversity of pathogens in ticks and the complex physiological characteristics of the host animal immune defense system lead to the protective antigen not effective in the control of ticks.In addition,these anti-tick vaccines have different resistance to different species ticks.Therefore,understanding the physiological development characteristics of ticks and the interrelationship between ticks and pathogens can provide scientific theoretical basis for the prevention and control of ticks and prevention of tick borne diseases.In the present study,tick samples were collected from Guangxi province of China.Identification and classification of ticks were performed by morphological details.DNA was extracted from these ticks to evaluate the presence of piroplasma by reverse line blot hybridization assay.Furthermore,the different development stage of Haemaphysalis flava were collected from host animals in our lab.Their morphological ultrastructure were described in detail.The life cycle and physiological characteristics were understood by rearing in the laboratory.Phylogenetic analysis were performed by homogenous conservative genes multiple-sequence alignments.To better understand the molecular basis of H.flava,high-throughput transcriptome sequencing technique was carried out to analyze differentially expressed genes(DEGS)between larvae and nymph development stage.Based on RNA-seq results,a novel cysteine protease(HfCL)were identified and cloned from H.flava nymph.The transcriptional levels of HfCL at different developmental stages were detected by qRT-PCR.Recombinant protein rHfCL enzyme activity assay were peformed by hydrolysing specific fluorescence substrate.The localization of HfCL in tick tissue were conducted by specific antibody.Recombinant protein rHfCL inhibited the Babesia parasites in vitro culture.The study results showed:(1)The identification of 671 ticks was conducted by morphological details.The results showed Rhipicephalus sanguineus 430/671(64.08%),R.haemaphysaloides 11/671(1.64%)and H.longicornis 219/671(32.63%),R.microplus 9/671(1.34%)and H.cornigera 2/671(0.29%).DNA was extracted from these ticks to evaluate the presence of piroplasma by a RLB hybridization assay,which used the hyper-variable region 4 of the piroplasmic 18S rRNA genes as probes to report Theileria and Babesia species.The RLB results indicated that 144/671(21.46%)of the ticks were infected with piroplasma species belonging to the genera Theileria or Babesia.Theileria buffeli,Babesia vogeli,T.annulata,and T.luwenshini were present in ticks at frequencies of 60/671(8.94%),21/671(3.13%),6/671(0.89%),and 6/671(0.89%),respectively.Mixed-infections with two or more piroplasma species were present in 8/671(1.19%)of the tick samples.(2)H.flava belong to the three host ticks.It raised in laboratory conditions with the optimum growth temperature for 28±1℃,relative humidity 88%.Optical microscopy and scanning electron microscopy were employed to observe the external ultrastructure of larvae,nymph,and adult tick.Its morphological characteristics mainly included the capitulum of square ratio,no eyes,short and blunt of coraua,the long pedipalp enveloped hypostome,heart-shaped of gentital orifice,anus groove arounded the anus,the anal valves is divided into two half-moon,spiracular plate formed a subcircular,female scutum covered with a subcircular covering half the back.And then,16s rRNA and cox 1gene sequence alignment with homologous species sequence and constructed phylogenetic tree,respectively.The results showed there was no difference in the evolution of the different region H.flava species.H.flava is closely related to the evolution of H.qinghaiensis and H.formosensis,forming an independent branch,and is far away from the species of the Rhipicephalus genus.(3)RNA-seq analysis between unfed larvae and nymph two cDNA library showed that the number of Raw Reads is 62538796,71175770,respectively.Clean Reads are59871570 and 69519916,respectively.Using Nr,GO,KEGG databases to annotate the transcriptome after assmbled,unigene was obtained by FPKM algorithm for differential expression analysis.Total of 56 genes were up-regulated at nymph stage.And 24 genes were up-regulated at larvae stage.Those differentially expressed genes mainly focuse on cuticular protein,endochitinase,cysteine,endonuclease,exonuclease,hypothetical protein,deoxyribonuclease,cytochrome enzyme,protease activating factor,cystatin.(4)According to RNA-seq database,we isolated and characterized a novel gene encoding cathepsin from H.flava nymph by RACE method.Cloning and sequencing results showed that the full cDNA of HfCL was 1216 bp,containing a 1041 bp open reading frame that encodes 346 amino acids,a 38 bp 5’-UTR,and a 137 bp 3’-UTR.To clarify the genetic identity of HfCL,the amino acid sequences of 27 orthologs in other invertebrates and vertebrates were obtained from the Genbank database for phylogenetic analyses that showed HfCL located on papain-like superfamily belong to cathepsin L-like family.Real-time PCR results showed HfCL transcripts profiling were detected in different development stages of H.flava including egg,larvae,nymph,adult female,and adult male.Nymph and adult compared with larvae with a significant difference(P<0.001),while eggs compared with larvae with a non-significant difference.In addition,the recombinant plasmid of pET28a-HfCL was successfully expressed in Transetta competent cell.36 kDa purification recombinant protein(rHfCL)was immuned mice and obtained specificity of polyclonal antibodies.Fluorescence immunohistochemistry assay results showed the expression of the HfCL was localized in both midgut and salivary glands.Enzyme activity results demonstrated that the specific fluorescence substrates were effectively hydrolyzed by the purified rHfCL under the optimal pH 6 and the recombinant protease concentration is 16μM.In vitro growth inhibition assays of Babesia parasites indicated that a certain dose of rHfCL protease could inhibit the Babesia merozoite growth and survival of B.microti and B.gibsoni.In this study,the potential risk of piroplasma infection ticks was evaluated by reverse line blot hybridization from Guangxi province of China.High-throughput transcriptome sequencing technology was used to analyze the differentially expressed genes between unfed larvae and nymph.We isolated and characterized a novel gene encoding cathepsin L from H.flava nymph and study gene functions to provide molecular basis for the prevention and control of ticks and tick born transmitted piroplasmosis. |
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