Abstract
RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks.
Highlights
Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans
Results Double-stranded RNA (dsRNA)-mediated gene silencing of H. longicornis SRB (HlSRB), HlVg-1, and HlVgR
DsRNA of HlSRB was injected into female ticks individually or in combination with different exogenous dsRNAs of ticks, namely HlVg-1, a yolk protein precursor expressed only at the midgut of H. longicornis [7], and HlVgR, a receptor localized only in the oocyte surface of H. longicornis [6], as well as firefly luciferase as a control [16]
Summary
Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans. They are considered to be second to mosquitoes as vectors of human diseases and are the most important arthropods transmitting pathogens to domestic animals [1]. Four different methods have been used to deliver dsRNA for RNAi in ticks to date: injection, soaking, feeding, and virus production of dsRNA [2]. We have confirmed that RNAi can be a powerful tool for gene silencing of the hard tick, H. longicornis, by the injection [3,5,6,7,8] and soaking methods [9]
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