A MicroRNA Profile Of Saliva And Their Role In Haemaphysalis Longicornis (Ixodida:Ixodidae)

Tick saliva contains many bioactive molecules that are involved in attachment to the host,blood feeding and transmission of pathogens.MicroRNAs（miRNAs）are a class of short non-coding RNAs with a length of 19-24 nucleotides.They act as regulators of gene expression by binding to their target mRNA at the post-transcriptional level and control a variety of cellular functions,including regulation of growth,metabolism,and development.The detection and characterizations of miRNAs from tick saliva may help explain the molecular mechanisms involved in the interaction between ticks,pathogens,and hosts.They may also contribute to the discovery of new targets,which can control ticks and the pathogens they transmit.An RNA library was generated from the saliva of fed adult Haemaphysalis longicornis ticks,and it contained 17.4 million clean reads of 18-30 nucleotides.Overall,319 known and 1 novel miRNA were found.The 10 most abundantly expressed miRNAs present in tick saliva were miR-100₂,miR-315,miR-184₁,miR-100-5p₂,miR-5307,miR-184-3p₃,Let-7-5p₆,miR-71₅,miR-1-3p₆,and miR-10-5p₂.The miR-375 and miR-184,are one of the abundantly expressed,was subjected to further studies in our works.Quantitative Real-Time PCR analysis（qRT-PCR）for miR-375 was taken in various tick developmental stages,as well as in different tissues isolated from adult ticks.The expression of miR-375 in different tick development stages was highest in unfed nymphs and lowest in the egg stage.In the tissues of adult ticks,miR-375 was most highly expressed in the salivary gland.To investigate the possible role of miR-375,Ant-375 was used to inhibit the miR-375.Treated group（Ant-375）had a reduced number of eggs（t（10）= 2.652,P = 0.0242）,eggs that were partially desiccated,and reduced egg hatchability（t（10）= 2.272,P = 0.044）compared to Ms-Ant and the non-injected control.Quantitative Real-Time PCR analysis（qRT-PCR）for miR-184 was taken in various tick developmental stages,as well as in different tissues isolated from adult ticks.The relative expression of miR-184 was highest in unfed nymphs and lowest in the unfed larva.The tissue distribution of miR-184 showed that it was highly expressed in the midgut.To investigate the possible role of miR-184,Ant-184 was used to inhibit the miR-184 and it was significantly down-regulated（t（4）= 12.32,P = 0.0002）.The engorged body weight was significantly reduced in treated group Ant-184 as compared to control Non-injected and Ms-Ant groups（t（22）= 2.19,P = 0.0388）.The mean of egg-laying days（33.5 ± 1.91）was significantly increased and number of eggs（t（10）= 3.147,P = 0.0137）and egg mass（t（10）= 3.4472,P = 0.0063）was reduced respectively.The eggs were also monitored during oviposition in the three groups.The eggs in half ticks of Ant-184 group became completely desiccated and have no hatching compared to control groups.To further explore the reduction in egg-laying days,number of eggs,egg mass,and hatchability we predicted the target gene Vg’s and K-10 proteins.We analyzed the expression of Vg’s（Vg1,Vg2,Vg3）and K-10 after silencing in semi-engorged ticks at 4th d,engorged tick 6th d,2nd d after engorgement and egg stage in Ant-184,Non-injected and Ms-Ant groups respectively.There was non-significant difference in the expression K-10 at different phases.The Vg1（P < 0.0001）,Vg2（P = 0.0180）,and Vg3（P = 0.0061）was up-regulated at 4th d,whereas at 6th d Vg1（P = 0.0068）was down-regulated in Ant-184 group.After engorgement at 2nd d the expression of Vg1（P = 0.004）,Vg2（P = 0.0189）and Vg3（P = 0.0149）were increased in treated group.At egg stage Vg1（P = 0.003）and Vg3（P = 0.009）level was high,while Vg2（P = 0.0034）was significantly low in Ant-184 compared to Ms-Ant and Non-injected control.In conclusion this is the first study to investigate the miRNAs profile in tick saliva.We constructed small RNA library from the saliva of fed adult Haemaphysalis longicornis ticks.This identification and characterization of miRNA in tick saliva may help to reveal the molecular mechanisms of interactions among ticks,pathogens,and hosts and suggest new target strategies to control tick-borne diseases.We first time demonstrated the role of miR-375.To explore the possible function of miR-375 in H.longicornis,Antagomir-375（Ant-375）was used to inhibit the miR-375 and inhibition of miR-375 affects the egg production and hatching.We also investigated first study to reveal the role of miR-184 in H.longicornis tick.Our data suggests the significant difference in the expression of Vg’s at different phases,after silencing of miR-184 affects the egg-laying days,number of eggs,egg mass and hatchability.Further functional characterization is required to verify other miRNAs and their exact target genes in ticks.Future experiments will include a detailed analysis of each miRNA and their target prediction,validation,and mechanism of action in the invertebrate vector.