Two Kinds of Ferritin Protect Ixodid Ticks from Iron Overload and Consequent Oxidative Stress

<div><p>Ticks are obligate hematophagous parasites that have successfully developed counteractive means against their hosts' immune and hemostatic mechanisms, but their ability to cope with potentially toxic molecules in the blood remains unclear. Iron is important in various physiological processes but can be toxic to living cells when in excess. We previously reported that the hard tick <i>Haemaphysalis longicornis</i> has an intracellular (HlFER1) and a secretory (HlFER2) ferritin, and both are crucial in successful blood feeding and reproduction. Ferritin gene silencing by RNA interference caused reduced feeding capacity, low body weight and high mortality after blood meal, decreased fecundity and morphological abnormalities in the midgut cells. Similar findings were also previously reported after silencing of ferritin genes in another hard tick, <i>Ixodes ricinus</i>. Here we demonstrated the role of ferritin in protecting the hard ticks from oxidative stress. Evaluation of oxidative stress in <i>Hlfer</i>-silenced ticks was performed after blood feeding or injection of ferric ammonium citrate (FAC) through detection of the lipid peroxidation product, malondialdehyde (MDA) and protein oxidation product, protein carbonyl. FAC injection in <i>Hlfer</i>-silenced ticks resulted in high mortality. Higher levels of MDA and protein carbonyl were detected in <i>Hlfer</i>-silenced ticks compared to <i>Luciferase</i>-injected (control) ticks both after blood feeding and FAC injection. Ferric iron accumulation demonstrated by increased staining on native HlFER was observed from 72 h after iron injection in both the whole tick and the midgut. Furthermore, weak iron staining was observed after <i>Hlfer</i> knockdown. Taken together, these results show that tick ferritins are crucial antioxidant molecules that protect the hard tick from iron-mediated oxidative stress during blood feeding.</p></div

Staining of HlFER for ferric iron on native PAGE gel after FAC injection.

<p>Ticks were injected with 50 or 100 µM FAC or sterilized high-purity water (0 µM) for the control group. Whole ticks were collected from 24 h to 96 h and total protein was extracted (A). Each lane of native PAGE gel was loaded with 20 µg of total protein and then the gel was stained with an equal volume of 10% K₄[Fe(CN)₆] and 10% HCl for up to 48 h. A single band stained for ferric iron. The high molecular weight marker (M), which contains ferritin from equine spleen, also stained for ferric iron, as well as the horse holoferritin (HF), used as positive control. Midguts, salivary glands, and the hemolymph (B) were also collected from ticks 72 h after injection of FAC or sterilized high-purity water. Each lane was loaded with 10 µg of protein. Western blot analysis using specific anti-HlFER sera was also performed to confirm that the band stained for ferric iron is HlFER. Arrows indicate approximately 440 kDa, which is the theoretical molecular weight of native ferritin.</p

An indirect immunofluorescent antibody test (IFAT) in the midgut after injection of FAC.

<p>Different concentrations of FAC or sterilized high-purity water (0 µM) were injected to unfed adult ticks and midguts were collected after 72 h. To visualize the extent of the stimulation of HlFER expression caused by FAC injection, frozen sections of the midgut were incubated with specific mouse anti-HlFER1 or anti-HlFER2 sera. Mouse normal serum was used as a negative control. Anti-mouse IgG conjugated with Alexa 594 was used as secondary antibody and nuclei were visualized using DAPI. Arrowheads point to areas with increased fluorescence. (Bars  = 20 µm).</p

Survival rate of <i>Hlfer</i>-silenced ticks after injection of FAC.

<p>Unfed adult female ticks were injected with <i>H. longicornis fer1</i> (<i>Hlfer1</i>), <i>H. longicornis fer2</i> (<i>Hlfer2</i>) or <i>Luciferase</i> (<i>Luc</i>) dsRNA for the control to induce RNAi. Silencing was confirmed through RT-PCR. After 4 days, 100 µM FAC was injected, and mortality was monitored. Both <i>Hlfer1</i>- and <i>Hlfer2</i>-dsRNA-injected groups had a lower survival rate compared to <i>Luc</i>. n = 30 ticks per group. The graph here represents the result of a single independent trial. Bars represent standard error. Significant difference was determined using the log-rank Mantel-Cox test (<i>P</i><0.0001, <i>Luc</i> vs. <i>Hlfer1</i> or <i>Hlfer2</i>).</p

Thiobarbituric acid reactive species (TBARS) assay for <i>Hlfer</i>-silenced ticks after blood feeding.

<p>Whole bodies or midguts of ticks injected with <i>Hlfer1</i>, <i>Hlfer2</i>, or <i>Luc</i> dsRNA were collected after dropping from the host. Individual whole ticks or pooled midguts were weighed before being homogenized. After ultrasonication, the supernatants were obtained and boiled with TBARS reagent. Upon cooling and centrifugation, the absorbance of the supernatants was measured at OD₅₃₂. The relative amount of MDA was calculated based on the sample weight and expressed as nmol/g. Both <i>Hlfer1</i>- and <i>Hlfer2</i>-silenced ticks had higher MDA levels in both whole ticks or midguts than the control (<i>Luc</i>) ticks. Values are means of 30 samples for each group ± SE. *<i>P</i><0.05, significantly different vs. control, Student's <i>t-</i>test.</p

Detection of malondialdehyde (MDA) from <i>Hlfer</i>-silenced ticks after blood feeding or injection of FAC.

<p>Total protein was extracted from whole ticks (A) and midguts (B) after blood feeding and whole ticks 72 h after injection of 100 µM FAC (C). Western blot analysis was performed and the membrane was incubated with a specific anti-MDA antibody. Tubulin was used as internal control. The relative content of MDA (clearest band) to tubulin was calculated after band densitometry analysis. Both <i>Hlfer1</i>- and <i>Hlfer2</i>-silenced ticks had significantly higher MDA compared to the control (<i>Luc</i>) group. Data represent the means of three independent trials ± SE. *<i>P</i><0.05, significantly different vs. <i>Luc</i>, Student's <i>t</i>-test.</p

Detection of protein carbonyl groups (PC) from <i>Hlfer</i>-silenced ticks after blood feeding or injection of FAC.

<p>Total protein was extracted from whole ticks (A) and midguts (B) after blood feeding and whole ticks 72 h after injection of 100 µM FAC (C). After the transfer of proteins to a membrane through Western blot, the membrane was incubated first with dinitrophenylhydrazine (DNPH) for pre-derivitazation of the carbonyl group, before incubation with a specific anti-DNP antibody. Tubulin was used as internal control. The relative content of PC (strongest band) to tubulin was calculated after band densitometry analysis. Both <i>Hlfer1</i>- and <i>Hlfer2</i>-silenced ticks had significantly higher PC levels compared to the control (<i>Luc</i>) group. Data represent the means of three independent trials ± SE. *<i>P</i><0.05, significantly different vs. <i>Luc</i>, Student's <i>t</i>-test.</p

Protein expression of <i>H. longicornis</i> ferritins in midguts (A) and hemolymph (B) of unfed ticks injected with different concentrations of FAC.

<p>Midguts and hemolymph were collected at 24 µM or 100 µM FAC or sterilized high purity water for the control group (0 µM). Hemocyte was separated from the hemolymph by centrifugation. Western blot analysis was performed using specific anti-sera against <i>H. longicornis</i> FER1 (HlFER1) or <i>H. longicornis</i> FER2 (HlFER2). Tubulin was used as an internal control. The relative expression of HlFER1 and HlFER2 was calculated based on tubulin after band densitometry analysis. Significant increase in expression was particularly found in HlFER1. Data represent the means of three independent trials ± SE. Statistical significance (*<i>P</i><0.05) was determined using the Mann-Whitney test.</p

Evaluation of iron accumulation in <i>Hlfer</i>-silenced ticks.

<p>Ferric iron accumulation was evaluated by staining HlFER on native PAGE (A). Total protein was extracted from whole bodies and midguts after blood feeding and whole bodies 72 h after FAC injection. The amount of protein was adjusted based on the tubulin profile after Western blotting. Weak staining was observed in <i>Hlfer1</i>-silenced ticks. Ferrozine assay for ferrous iron 72 h after injection of 100 µM FAC to unfed <i>Hlfer</i>-silenced ticks. (B). Ten ticks from each group were homogenized and total protein concentration was measured. Ferrous iron was extracted with concentrated HCl and then detected using ferrozine. Absorbance was measured at OD₅₅₀. Relative ferrous iron content was calculated based on protein concentration. <i>Hlfer2</i>-silenced ticks had significantly higher ferrous iron content than the control (<i>Luc</i>) group. *<i>P</i><0.05, significantly different vs. <i>Luc</i>, Student's <i>t</i>-test.</p