Identification Of Bloodfeeding-related MiRNAs In Haemaphysalis Longicornis

Haemaphysalis longicornis,as one of the most prevalent species of ticks widely distributed in north and south of China,can spread various pathogens,such as Babesia sp.,spirochetes,nebulia virus and so on,with great significance in veterinary medicine and public hygiene.Micro RNA(miRN A)is a small non-coding RNA found in eukaryotes,which can degrades the target mRNA or inhibit its translation for regulating gene functions.The objective of this study is to identify the bloodfeeding-related miRNAs in Haemaphysalis longicornis in order to provide a scientific basis for the development of new drugs or vaccines against ticks.Ten differently expressed miRNAs in Haemaphysalis longicornis before and after bloodfeeding were identified by real-time PCR.The injection of miRN A interferons disturbed the regulating effect of relative miRN As.And three bloodfeeding-related miRN As including miR-278,Let-7 and Bantam were successfully screened from these ten miRNAs through the animal experiment.We amplified the precursor sequences of these three miRN As by PCR method;detected the expression quantity of these three miRN As in four different development stages(egg,Larva,nymph and adult)of the tick and four different organs(ovary,fat bady,midgut and salivary gland)of adult tick by using fluorescence quantitative PC R;and detected the differential expression of miR-278 between the engorged nymph infected with Babesia microti and noninfected nymph;and detected the biological activities of miR-278 in H.longicornis by injecting antagomiR-278 and agomiR-278.The results showed that the precursor sequences of miR-278,Let-7 and Bantam in the tick were obtained;there were significant differences in the expression quantity of three miRN As with the highest expression quantity in the fat bodies of nymphal and adult ticks,but the expression quantity of Bantam was lower than that of other two miRN As;and the expression of miR-278 decreased significantly in the engorged nymph infected with Babesia microti;The inhibition of miR-278 expression made the 24-hour uptake rate of adult H.longicornis to reduce to 5 %,the mortality rate up to 72.5 %,and the bloodbleeding rate to 27.5 %.The results indicated that the expressions of three miRN As have stage-and organ-specificity.The miR-278 has a particularly significant effect on the uptake rate and lethality of adult H.longicornis,and has a certain influence on the tick infected with Babesia microti.Under the current unitary method of tick and tick-borne disease prevention and control,we explored the bloodfeeding mechanism of H.longicornis at miRN A level,which may provide new ideas and approaches for the development of new tick-proof agents or vaccines.