Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis

Abstract

SUMMARYTicks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis. Supplementary material available online at

Evaluation and comparison of the potential of two ferritins as anti-tick vaccines against Haemaphysalis longicornis

“…SDS-PAGE showed that the purified rHlFER1 and rHlFER2 have a similar molecular mass of around 26 kDa (Figure 1) as described previously [17]. An antigenspecific ELISA was conducted to monitor the antibody titer of individual rabbits, and then the average antibody titer for each group from the two trials was calculated ( Figure 2).…”

“…RNAi is a good technique for screening candidate antigens for vaccine development [12]. Our previous gene silencing studies showed that HlFERs are crucial to the blood feeding and reproduction of H. longicornis [17], providing protection against ironmediated oxidative stress [18] and making them good candidate vaccine antigens. Our vaccination experiments showed that both rHlFER1 and rHlFER2 are highly immunogenic, inducing host antibody production.…”

“…Our previous studies on two ferritins of the hard tick H. longicornis using RNAi showed that both are physiologically important in blood feeding and reproduction [17] by preventing iron overload and the occurrence of oxidative stress [18]. Furthermore, a previous study showed that FER2 vaccination had considerable effects on infestation of I. ricinus and R. microplus [19].…”

“…These prompted us to evaluate and compare the potential of two HlFERs as targets for the control of H. longicornis. HlFERs are abundant in different tissues of the tick, with HlFER1 being intracellular in nature, while HlFER2 is secretory [17]. Being concealed antigens, these proteins are not normally encountered by the host immune system during blood feeding, and thus a high level of host antibodies against HlFERs is probably necessary for the blockade of the HlFER function [23].…”

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control

“…The significance of proteins involved in iron metabolism to tick feeding and reproduction has been also demonstrated using RNAi in two hard tick species, I. ricinus [111] and H. longicornis [112]. Silencing two types of the iron storage protein ferritin greatly reduced the ticks' capacity to engorge and produce eggs, also affecting post-blood meal survival due to occurrence of iron-mediated oxidative stress [113].…”