Abstract
Successful completion of the molting process requires new epidermal growth and ecdysis of the old cuticle in Haemaphysalis longicornis (H. longicornis). MicroRNAs (miRNAs) participate in the development of organisms by inhibiting the expression of their target mRNAs. In this study, a novel tick-specific miRNA was identified and denoted hlo-miR-2 that serves as a novel regulator of molting events in H. longicornis nymphs by targeting a cuticular protein. The full length of this cuticular protein was first obtained and named it CPR1. A qRT-PCR analysis showed that hlo-miR-2 and CPR1 exhibit significant tissue and temporal specificity and that their transcription levels are negatively correlated during the molting process. CPR1, as a direct target of hlo-miR-2, was identified by a luciferase reporter assay in vitro. Agomir treatment indicated that the overexpression of hlo-miR-2 significantly reduced the protein expression level of CPR1, decreased the molting rate and delayed the molting time point in H. longicornis nymphs. RNA interference (RNAi) experiments demonstrated that CPR1 was significantly associated with the molting process in H. longicornis nymphs. Phenotypic rescue experiments convincingly showed that hlo-miR-2 participated in molting events by targeting CPR1 in H. longicornis nymphs. In summary, we present evidence demonstrating that miRNAs constitute a novel important regulator of molting events in addition to hormones. The described functional evidence implicating CPR1 in molting events contributes to an improved understanding of the distinct functions of the CPR family in ticks and will aid the development of a promising application of cuticular protein RNAi in tick control.
Highlights
As a three-host tick, H. longicornis requires three hosts and undergoes two molting processes during its lifetime (Yatsenko and Shcherbata, 2014)
Because CPR1 was expressed in the epidermis and its expression changed during the normal molting process of H. longicornis hlo-miR-2 Is Controlling Molting Events nymphs, we examined the protein and mRNA expression of CPR1 and the molting timepoint and rate after CPR1 RNA interference (RNAi) to further evaluate the potential roles of this gene
The mRNA expression of CPR1 were assessed on day 2 after interference ribonucleic acid (RNA) for CPR1 (iCut) injection, and the results showed that the mRNA levels of CPR1 were significantly reduced in the iCut-treated group (0.725 ± 0.02172) compared with other group and not appreciably differ between the negative control #22 (NC) (0.996 ± 0.03407) and blank control (0.984 ± 0.02575) groups, the Western blot analysis showed that the protein expression level of CPR1 was decreased by iCut treatment compared with that found in the NC and blank control groups, which suggested that a siRNA designed to target CPR1 successfully decreased the CPR1 transcript and protein levels in H. longicornis nymphs
Summary
As a three-host tick, H. longicornis requires three hosts and undergoes two molting processes during its lifetime (Yatsenko and Shcherbata, 2014). The molting process, which can cause the generation of new epidermal growth and ecdysis of the old cuticle, is a prerequisite for the growth and development of H. longicornis ticks (Freitak et al, 2012; Zhou et al, 2013). The sharp increase in ecdysteroid (20-E) titers in the H. longicornis body during molting is consistent with the deposition of the new epicuticle and is an important factor in initiating the formation of the exuvial space (apolysis). Investigations of the effects of interference with the molting process will provide new ideas for the development of tick vaccines and the prevention and control of ticks
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