MicroRNA-275 and its target Vitellogenin-2 are crucial in ovary development and blood digestion of Haemaphysalis longicornis

Abstract

Background: The hard tick Haemaphysalis longicornis is widely distributed in eastern Asia, New Zealand and Australia and is considered the major vector of Theileria and Babesia, harmful parasites to humans and animals. Female ticks need successful blood meals to complete the life-cycle. Therefore, elucidation of the underlying molecular mechanisms of H. longicornis development and reproduction is considered important for developing control strategies against the tick and tick-borne pathogens. Methods: Luciferase assays were used to identify the targets of micro RNA miR-275 in vitro. RNAi of Vitellogenin (Vg) was used in phenotype rescue experiments of ticks with miR-275 inhibition, and these analyses were used to identify the authentic target of miR-275 in vivo. The expression of miR-275 in different tissues and developmental stages of ticks was assessed by real-time PCR. To elucidate the functions of miR-275 in female ticks, we injected a miR-275 antagomir into female ticks and observed the phenotypic changes. Statistical analyses were performed with GraphPad5 using Student’s t-test. Results: In this study, we identified Vg-2 as an authentic target of miR-275 both in vitro and in vivo by luciferase assays and phenotype rescue experiments. miR-275 plays the regulatory role in a tissue-specific manner and differentially in developmental stages. Silencing of miR-275 resulted in blood digestion problems, substantially impaired ovary development and significantly reduced egg mass (P < 0.0001). Furthermore, RNAi silencing of Vg-2 not only impacted the blood meal uptake (P < 0.05) but also the egg mass (P < 0.05). Significant rescue was observed in miR-275 knockout ticks when RNAi was applied to Vg-2. Conclusion: To our knowledge, this study is the first demonstration that miR-275 targets Vg-2 in H. longicornis and regulates the functions of blood digestion and ovary development. These findings improve the molecular understanding of tick development and reproduction.