Akt is an essential player in regulating cell/organ growth at the adult stage in the hard tick Haemaphysalis longicornis

Umemiya‐Shirafuji, Rika; Tanaka, Toru; Boldbaatar, Damdinsuren; Tanaka, Tetsuya; Fujisaki, Kozo

doi:10.1016/j.ibmb.2011.12.001

Cited by 30 publications

(21 citation statements)

References 48 publications

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“…Specific mouse anti-ferritin sera [13] or anti-β-tubulin serum for control [16] were used as primary antibodies. Protein signals were detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and images were taken using the FluorChem FC2 Imaging System (Protein Simple, Santa Clara, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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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 Haemaphysalis longicornis 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, Ixodes ricinus. Here we demonstrated the role of ferritin in protecting the hard ticks from oxidative stress. Evaluation of oxidative stress in Hlfer-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 Hlfer-silenced ticks resulted in high mortality. Higher levels of MDA and protein carbonyl were detected in Hlfer-silenced ticks compared to Luciferase-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 Hlfer 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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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“…For detection of protein in the whole and different tissues of adult ticks, an antiserum previously prepared for β-actin (Liao et al, 2008) was used as the control. Alternatively, β-tubulin (Umemiya-Shirafuji et al, 2012) antiserum was used for experiments that include the egg, wherein actin band was difficult to detect, particularly in the early stage of embryogenesis. After incubation with peroxide-conjugated sheep anti-mouse IgG (1:50,000 dilution; GE Healthcare, Little Chalfont, Buckinghamshire, UK), a signal was detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare) and analyzed using FluorChem FC2 software (Cell Biosciences, Santa Clara, CA, USA).…”

Section: Protein Extraction and Western Blot Analysismentioning

confidence: 99%

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

Galay

Aung

Umemiya‐Shirafuji

et al. 2013

Journal of Experimental Biology

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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

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“…After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon Ⓡ -P; Millipore, Danvers, MA, USA). The membrane was blocked overnight with 3 % skim milk in PBS (pH 7.4) (blocking solution); it was incubated with a 1:500 dilution of anti-rHlPrx2 mouse sera in blocking solution at 37 °C for 1 h. For loading control, tubulin was detected using antiserum against recombinant H. longicornis tubulin [15]. After washing five times in PBS containing 0.05 % Tween 20 (PBS-T), the membrane was incubated with a 1:50,000 dilution of horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (Dako, Glostrup, Denmark) in blocking solution at 37 °C for 1 h. After washing five times in PBS-T, bands were detected using Amersham TM ECL TM Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) and viewed using FluorChem Ⓡ FC2 software (Alpha Innotech, San Leandro, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

2-Cys peroxiredoxin is required in successful blood-feeding, reproduction, and antioxidant response in the hard tick Haemaphysalis longicornis

et al. 2016

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BackgroundTicks are obligate hematophagous arthropods that feed on vertebrate blood that contains iron. Ticks also concentrate host blood with iron; this concentration of the blood leads to high levels of iron in ticks. The host-derived iron reacts with oxygen in the tick body and this may generate high levels of reactive oxygen species, including hydrogen peroxide (H2O2). High levels of H2O2 cause oxidative stress in organisms and therefore, antioxidant responses are necessary to regulate H2O2. Here, we focused on peroxiredoxin (Prx), an H2O2-scavenging enzyme in the hard tick Haemaphysalis longicornis.MethodsThe mRNA and protein expression profiles of 2-Cys peroxiredoxin (HlPrx2) in H. longicornis were investigated in whole ticks and internal organs, and developmental stages, using real-time PCR and Western blot analysis during blood-feeding. The localization of HlPrx2 proteins in tick tissues was also observed by immunostaining. Moreover, knockdown experiments of HlPrx2 were performed using RNA interference to evaluate its function in ticks.ResultsReal-time PCR showed that HlPrx2 gene expression in whole ticks and internal organs was significantly upregulated by blood-feeding. However, protein expression, except in the midgut, was constant throughout blood-feeding. Knockdown of the HlPrx2 gene caused significant differences in the engorged body weight, egg weight and hatching rate for larvae as compared to the control group. Finally, detection of H2O2 after knockdown of HlPrxs in ticks showed that the concentration of H2O2 significantly increased before and after blood-feeding.ConclusionTherefore, HlPrx2 can be considered important for successful blood-feeding and reproduction through the regulation of H2O2 concentrations in ticks before and after blood-feeding. This study contributes to the search for a candidate target for tick control and further understanding of the tick’s oxidative stress coping mechanism during blood-feeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1748-2) contains supplementary material, which is available to authorized users.

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Akt is an essential player in regulating cell/organ growth at the adult stage in the hard tick Haemaphysalis longicornis

Cited by 30 publications

References 48 publications

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

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

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

2-Cys peroxiredoxin is required in successful blood-feeding, reproduction, and antioxidant response in the hard tick Haemaphysalis longicornis

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