The molecular and functional characterization of ferritins in the hard tick Hyalomma rufipes

Abstract

Background: The protein ferritin, which plays an important role in the maintenance of iron homeostasis, is indispensable for iron detoxification, resistance to oxidative stress and innate immunity. Ticks, which are obligate blood-sucking ectoparasites, have to deal with a large amount of iron when they take a blood meal. Methods: Sequence analysis was undertaken using bioinformatics. A recombinant (r) expression vector, rferritin, was constructed for a prokaryotic expression system. A quantitative polymerase chain reaction platform was used to detect the spatial and temporal expression patterns of target genes and their responses to a low temperature environment. Knockdown of the ferritin genes through RNA interference was used to analyze their effects on physiological parameters of ticks. Results: Two ferritin genes, HrFer1 and HrFer2, were cloned from the tick Hyalomma rufipes. Their open reading frames are 519 base pairs (bp) and 573 bp in length, and number of coding amino acids 170 and 190, respectively. The phylogenetic tree showed that HrFer1 and HrFer2 have a close evolutionary relationship with the H subunit of ferritin. In vitro experiments showed that rHrFer1 and rHrFer2 had concentration-dependent iron chelating activity. The relative expression of the two ferritin genes was higher in the ovary and midgut of H. rufipes. RNA interference results demonstrated that HrFer1 and HrFer2 expression had a significant effect on engorged body weight, number of eggs laid, and mortality of H. rufipes, and that HrFer2 also had a significant effect on feeding duration. Furthermore, the relative expression of ferritin decreased significantly in a low temperature environment, suggesting that HrFer1 and HrFer2 play a regulatory role in the cold stress response of H. rufipes. Conclusions: The results of the present study improve our understanding of the involvement of ferritins in tick blood-feeding. Graphical Abstract: [Figure not available: see fulltext.].