MiR-275 And Its Target Vg-2 Affects Blood Digestion And Ovary Development In Haemaphysalis Longicornis

The Haemaphysalis longicornis is a hematophagous ectoparasite and widely distributed in eastern Asian,New Zealand,Australia and many provinces of China.It is considered the major vector of Theileria and Babesia,harmful parasites to humans and animals.microRNA is a class of non-coding small RNA in length 18~25.In recent years,studies suggested that miRNA play an important role in hematophagous arthropod,especially in blood meal and reproduction.Therefore,elucidation of the underlying molecular mechanisms of H.longicornis development and reproduction is needed.Expression of analysis the mi R-275 from developmental stages and various tissues in H.longicornis by real-time PCR,the results showed that the miR-275 expressed in all stages and tissues,and there are significantly different in stages and tissues,which suggested that miR-275 exhibit an apparent specific tissue.In the unfed stage,the expression of miR-275 is highest in midgut,the expression of midgut is 1.91-fold of salivary glands and 1.28-fold of ovary respectively;in the fed stage,mi R-275 significantly increased in ovary and midgut,increased 2.72-fold and 1.67-fold respectively,decreased 0.806-fold in salivary gland.miR-275 significantly increased in fed ticks which suggested that miR-275 may involved in ovary development and blood digestion.Analysis of miR-275 in different developmental stages showed that the expression level of fed females ticks and eggs were significantly higher than those of other stages which suggested that mi R-275 is crucial for egg and fed female ticks.Using RNAhybrid programs to predict the target gene of the miR-275,the result showed that there a two canonical binding sites with Vg-2 in open reading frame.The luciferase reporter assay result showed that the treatment luciferase activity was 66.08%,significantly different with control group,thus the result imply that the Vg-2 is an authentic target of miR-275.Injection of miR-275 antagomir and missense into unfed female ticks,the result showed that the antagomir could efficiently inhibit endogenous miR-275(p<0.05);analysis of physiological index from each group of ticks,the results showed that silencing of miR-275 resulted in blood digestion problems,substantially impaired ovary development and significantly reduced egg mass(p<0.0001)compare with negative and blank control.This results suggested that mi R-275 play an important role in ovarian development and blood digestion.To further identify the authentic target gene of miR-275 in vivo,phenotype rescue experiment was conducted through Vg RNAi with Ant-275 background in female ticks.The results showed that co-injected Ant-275/dsRNA could partially alleviate phenotype.The Ant-275/dsRNA females ticks body weight significantly increased after blood meal compared with dsVg ticks and the deposition of eggs were more than dsVg ticks but still lower than non-injection group,this experiment results suggested that Vg-2 is an authentic target of mi R-275 in vivo.RNAi of Vg seriously caused female ticks inability to blood feeding and lay eggs,which suggest that Vg is important to bloodsucking and lay eggs in ticks.