Potential mechanisms underlying embryonic developmental toxicity caused by benzo[a]pyrene in Japanese medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widely distributed in air, water, and sediments; however, limited data are available regarding their potential adverse effects on the early life stages of fish. In this study, we evaluated the embryonic teratogenicity and developmental toxicity of BaP in Japanese medaka (Oryzias latipes) using a nanosecond pulsed electric field (nsPEF) technique and predicted their molecular mechanisms via transcriptome analysis. The gas chromatography/mass spectrometry analyses revealed that the BaP was efficiently incorporated into the embryos by nsPEF treatment. The embryos incorporating BaP presented typical teratogenic and developmental effects, such as cardiovascular abnormalities, developmental abnormalities, and curvature of backbone. DNA microarray analysis revealed several unique upregulated genes, such as those involved in cardiovascular diseases, various cellular processes, and neural development. Furthermore, the gene set enrichment and network analyses found several genes and hub proteins involved in the developmental effects of BaP on the embryos. These findings suggest a potential mechanism of teratogenicity and developmental toxicity caused by exposure to BaP. The nsPEF and transcriptome analyses in combination can be effective for evaluating the potential effects of chemical substances on medaka embryos.

Influence of cognitive impairment on cardiac mortality after percutaneous coronary intervention in very elderly patients: a retrospective observational study.

BACKGROUND: Cognitive impairment (CI) increases cardiac mortality among very elderly patients. Percutaneous coronary intervention (PCI) for ischemic heart disease (IHD) patients is considered a favorable strategy for decreasing cardiac mortality. Here, we investigated the influence of CI on cardiac mortality after PCI in very elderly patients. METHODS: We performed a retrospective observational analysis of patients who received PCI between 2012 and 2014 at the South Miyagi Medical Center, Japan. IHD patients over 80 years old who underwent the Mini-Mental State Examination for CI screening during hospitalization and/or who had been diagnosed with CI were included. Participants were divided into CI and non-CI groups, and cardiac mortality and incidence of adverse cardiac events in a 3-year follow-up period were compared between groups. Statistical analyses were performed using the t-test, χ2 test, and multivariable Cox regression analysis, with major comorbid illness and conventional cardiac risk factors as confounders. RESULTS: Of 565 patients, 95 were included (41 CI, 54 non-CI). Cardiac mortality during the follow-up period was significantly higher in the CI group (36%) compared with the non-CI group (13%) (OR = 4.3, 95% CI: 1.56-11.82, P < 0.05). CI was an independent cardiac prognostic factor after PCI and, for CI patients, living only with a CI partner was an independent predictor of cardiac death within three years. CONCLUSIONS: CI significantly affected cardiac prognosis after PCI in very elderly patients, particularly those living with a CI partner. To improve patients' prognoses, social background should be considered alongside conventional medical measures.

A SNP in a Steroidogenic Enzyme Is Associated with Phenotypic Sex in Seriola Fishes.

Vertebrate sex development consists largely of two processes: "sex determination," the initial bifurcation of sexual identity, and "sex differentiation," which subsequently facilitates maleness or femaleness according to the sex determination signal. Steroid hormones promote multiple types of sexual dimorphism in eutherian mammals and avians [1-3], in which they are indispensable for proper sex differentiation. By contrast, in many poikilothermic vertebrates, steroid hormones have been proposed to be key players in sex determination as well as sex differentiation [4-8]. This hypothesis was introduced more than 50 years ago but has never been rigorously tested due to difficulties in discriminating the roles of steroids in sex determination and differentiation. We found that a missense SNP in the gene encoding the steroidogenic enzyme 17ß-hydroxysteroid dehydrogenase 1 (Hsd17b1) was perfectly associated with ZZ/ZW sex determination in Seriola fishes. Biochemical analyses revealed that a glutamate residue present specifically in Z-type HSD17B1 attenuated interconversion between 17-keto and 17ß-hydroxy steroids relative to the allelic product from the W chromosome, which harbors glycine at that position, by disrupting the hydrogen bond network between the steroid and the enzyme's catalytic residues. Hsd17b1 mRNA is constitutively expressed in undifferentiated and differentiating gonads of both genotypic sexes, whereas W-type mRNA is expressed only in genotypic females. Meanwhile, Cyp19a1 is predominantly expressed in differentiating ovary. We conclude that the combination of Hsd17b1 alleles determines sex by modulating endogenous estrogen levels in Seriola species. These findings strongly support the long-standing hypothesis on steroids in sex determination.

Development of specific enzyme-linked immunosorbent assays for multiple vitellogenins in marbled sole, Pleuronectes yokohamae.

Non-competitive, enzyme-linked immunosorbent assays (ELISAs) for three distinct sole vitellogenins (VtgAa, VtgAb and VtgC) were designed using their purified lipovitellin (Lv) products and corresponding digoxigenin-labeled, anti-Lv polyclonal antibodies, primarily for employment in monitoring estrogenic pollution of the environment. The working range of the ELISAs was from 0.97 to 1,000 ng/mL for all Vtg subtypes. Each ELISA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed ELISAs were lower than 10%. Three Vtg subtypes were induced in serum of immature fish by estradiol-17ß (E2) injection (0.5 mg/kg body weight). All Vtg subtypes were induced one day after the injection, reaching peak levels (Lv equivalents) within three days, as follows: 39.1 ±â€¯28.9 µg/mL (VtgAa), 57.9 ±â€¯30.7 µg/mL (VtgAb) and 12.6 ±â€¯4.8 µg/mL (VtgC). In wild-caught males, VtgAa, VtgAb and VtgC were detected in ranges from 0.26 to 1.21, 0.19 to 8.69, and 0.17 to 53.50 µg/mL, respectively, over various sampling periods. In vitellogenic females sampled in January, the average level of VtgAb (8,744.43 ±â€¯733.93 µg/mL) was significantly higher than for VtgAa (150.33 ±â€¯22.35 µg/mL) or VtgC (57.08 ±â€¯6.00 µg/mL); thus VtgAb appeared to be the most dominant Vtg subtype. The present study entails the first report on development of subtype-specific Vtg ELISAs in marbled sole, which empowers us to detect and monitor estrogenic contamination in aquatic environments inhabited by this species.

Influence of atrial fibrillation on cardiac prognosis in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD), a known risk factor for the development of congestive heart failure (CHF), was recently shown to predict the prevalence of atrial fibrillation (AF). Here, we explore the influence of AF on cardiac prognosis in COPD patients. METHODS: A total of 339 consecutive patients who underwent spirometry from 2010 to 2013 for various reasons were retrospectively examined. Based on the diagnostic criteria, patients were stratified into COPD and non-COPD groups, which were both further divided into those with AF (chronic AF or paroxysmal AF) or sinus rhythm (SR) based on previous electrocardiography results. Significances of differences in cardiac events were assessed by the chi-square test. Multivariate logistic regression analyses and Cox proportional hazard models were applied to evaluate the influence of AF on cardiac events. RESULTS: Of the 339 patients, 190 were diagnosed with COPD, with 42 of these were having AF. During the mean follow-up period of 7.4 ± 0.8 years, CHF developed more frequently in COPD patients with AF than in COPD patients without AF [50% vs 7%; odds ratio (OR) 12.4, 95% confidence interval (CI): 5.25-29.49, p < 0.05]. AF was an independent predictor of CHF development (OR 20.4, 95% CI: 6.55-79.80, p < 0.05) and cardiac mortality (OR 2.8, 95% CI: 1.79-4.72, p < 0.05). Moreover, positive correlations were found between the severity of pulmonary obstruction with AF and CHF development (R = 0.69, p < 0.05), as well as cardiac mortality (R = 0.78.p < 0.05). CONCLUSIONS: These results suggest that AF may be strongly associated with cardiac mortality and CHF in COPD patients.

Oil spill off the coast of Guimaras Island, Philippines: Distributions and changes of polycyclic aromatic hydrocarbons in shellfish.

The sinking of the Solar 1 tanker caused serious heavy oil pollution around Guimaras Island, Philippines. In the present study, variations of parent polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs (alkPAHs) in some shellfish were investigated around Guimaras Island and other small islands from 3months to 5years after the spill. The total PAHs and alkPAHs in shellfish were detected in high concentrations at 448 and 33,666ng/g dry weight, respectively, in November 2006. The concentrations of alkPAHs gradually decreased, while the parent PAHs in shellfish degraded more slowly than the alkPAHs, which was likely due to the persistent characteristics of PAHs. The risks based on European Union regulations were insignificant in 2008, but total PAHs in shellfish were still over 8 times higher at the investigated sites in November 2011 than that before the oil spill.

Distributions of nitrated polycyclic aromatic hydrocarbons in the sediment of Osaka Bay, Japan.

The distributions of 15 nitrated polycyclic aromatic hydrocarbons (NPAHs) in sediments collected at 44 sites throughout Osaka Bay, Japan were examined. The highest total NPAHs, with a concentration of 1949ng/kg dry weight, were detected near the city of Amagasaki. Some sites near the cities of Osaka, Kishiwada, sand Sakai registered ng/kg levels of NPAHs, but individual NPAH concentrations were relatively lower than those in previous studies. The sources were estimated using principal component analysis, and NPAHs were derived from exhaust gases of automobiles and industries at some sites. However, our results suggest that it is difficult to estimate the source, especially in coastal areas near big cities and large industrial areas, because the generation pathways between parent polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs are slightly different, and the ratio of PAHs and NPAHs could not be accurately reflect the characters of sources in sediments especially at coastal areas with large-scale industrial zones.

Oxidative stress and genotoxicity biomarker responses in tilapia (Oreochromis niloticus) exposed to environmental concentration of 1-nitropyrene.

The present study aimed to assess whether environmental 1-nitropyrene (1-NP) concentration will induce genotoxicity and oxidative damages in tilapia, lives in estuarine and brackish water. Tilapia were exposed to waterborne 1-NP. Cellular antioxidant enzyme activity of glutathione peroxidase and oxidative damage, i.e., lipid peroxidation, protein and DNA oxidation were used as biomarkers of oxidative stress, while the micronucleus test was used for evaluation of chromosomal damage and was used as an indication of genotoxicity. Results showed that all biomarkers for oxidative stress positively responded, and micronucleus and other nuclear abnormalities frequencies significantly increased (p<0.001). This study showed that environmentally relevant 1-NP concentration in test water (0.15ng/L) and in fish (3ng/kg) induced genotoxicity and oxidative stress. Micronuclei and other nuclear abnormalities were probably formed as a result of oxidative stress. In conclusion, exposure to lower waterborne 1-NP concentration can pose a risk to freshwater and estuarine organisms through accumulation.

Evaluation of 2,4-dichlorophenol exposure of Japanese medaka, Oryzias latipes, using a metabolomics approach.

In this study, the metabolic effects of waterborne exposure of medaka (Oryzias latipes) to nominal concentrations of 20 (L group) and 2000 µg/L (H group) 2,4-dichlorophenol (DCP) were examined using a gas chromatography/mass spectroscopy (GC/MS) metabolomics approach. A principal component analysis (PCA) separated the L, H, and control groups along PC1 to explain the toxic effects of DCP at 24 h of exposure. Furthermore, the L and H groups were separated along PC1 at 96 h on the PCA score plots. These results suggest that the effects of DCP depended on exposure concentration and time. Changes in tricarboxylic cycle metabolites suggested that fish exposed to 2,4-DCP require more energy to metabolize and eliminate DCP, particularly at 96 h of exposure. A time-dependent response in the fish exposed to DCP was observed in the GC/MS data, suggesting that the higher DCP concentration had greater effects at 24 h than those observed in response to the lower concentration. In addition, several essential amino acids (arginine, histidine, lysine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, and valine) decreased after DCP exposure in the H group, and starvation condition and high concentration exposure of DCP could consume excess energy from amino acids.

Effects of oxygenated polycyclic aromatic hydrocarbons on the early life stages of Japanese medaka.

Oxygenated polycyclic aromatic hydrocarbons (oxyPAHs) are directly discharged into the atmosphere with exhaust gas from diesel engine automobiles and industry and are also generated by photo-oxidation and/or microbial metabolism of parent polycyclic aromatic hydrocarbons (PAHs). They are widely distributed in environment, especially around urban areas. We evaluated the effects of exposure to oxyPAHs as acenaphthenequinone (ANQ), 7,12-benz(a)anthracenequinone (BAQ), 1,4-naphthoquinone (NAQ), and 9,10-phenanethrenequinone (PHQ) in Japanese medaka (Oryzias latipes) embryos. ANQ (>2720 µg/L) caused caving in the yolk sac, and BAQ (>22.4 µg/L) caused tanning of the oil droplet. Additionally, NAQ (>314 µg/L) and PHQ (>734 µg/L) stopped development of medaka embryos beginning 2 days after the start of exposure. The hatched larvae from embryos exposed to ANQ, BAQ, NAQ, or PHQ at 518, 9.86, 36.5, or 80.7 µg/L, respectively, exhibited incomplete development of the cephalic region, incomplete palate, unabsorbed and hypertrophied yolk sac, tubular heart, altered axial curvature, and poor swimming ability. These symptoms were similar to those observed in blue sac disease, which is caused by exposure to PAHs.

Photo-induced toxicity and oxidative stress responses in Tigriopus japonicus exposed to nitro-polycyclic aromatic hydrocarbons and artificial light.

Photo-induced toxicity is an important phenomenon in ecotoxicology because sunlight reaches many organisms in their natural habitats. To elucidate whether sunlight enhances the toxicity of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs), the acute toxicities of 10 nitro-PAHs and the related compound 1-nitropyrene (1-NP) to Tigriopus japonicus were assessed in darkness or under light conditions. In addition, the relationships among the toxicity of 1-NP to T. japonicus, lighting condition, and the concentration of reactive oxygen species (ROS) formed were investigated in the presence or absence of the ROS scavenger ascorbic acid in the test solutions. Light irradiation increased the toxicity of all tested nitro-PAHs except 1,5-dinitronaphthalene. Among the compounds tested, 1-NP was the most phototoxic: it was more than 1000 times more toxic under the light conditions than in darkness. In contrast, at the same light levels, pyrene was not phototoxic. Light irradiation induced the generation of ROS in the 1-NP exposure groups, and the immobilization rate of T. japonicus increased with the amount of ROS produced. The addition of ascorbic acid to the test solutions suppressed both the generation of ROS and the light-induced immobilization of T. japonicus. To accurately assess the ecotoxicologic risk of nitro-PAHs, their overall photo-induced toxicity must be considered.

Toxic evaluations of sediments in Tokyo Bay, Japan, using Japanese medaka embryos.

Toxic risks of sediments collected from seven sites in Tokyo Bay were evaluated using Japanese medaka embryos. Those sediments with slight pore water were placed in grass petri dishes without overlying water. The most remarkable effect in the field sediment was to cause hatching delay in embryos, and the longest time until hatching took was 12.5 ± 1.6 days post-fertilization (dpf), although that in control group was 10.1 ± 0.7 dpf. A significant delay in hatching was observed at four sites. Because total carbon concentrations were relatively high in sediments at three of these four sites, several chemicals were expected to be residues in these sites and could cause their delay. Although extreme mortality was not observed at all sites, sediments collected from the site close to Kawasaki city induced 10 % mortality. Polycyclic aromatic hydrocarbon (PAH) concentrations were remarkably high at this site compared with other sites, and thus PAH toxicities could be causing the mortality. Concentration of heavy metals such as cadmium, copper, lead, and zinc in sediments were also determined, but no clear relationship was found between toxicities to embryos and the distribution of their concentrations.

Recurrent Acute Myocardial Infarction in a Patient with Severe Coronary Artery Ectasia: Implication of Antithrombotic Therapy.

BACKGROUND Acute myocardial infarction (AMI) can be caused not only by plaque rupture/erosion, but also by many other mechanisms. Thromboembolism due to atrial fibrillation and coronary thrombosis due to coronary artery ectasia are among the causes. Here we report on a case of recurrent myocardial infarction with coronary artery ectasia. CASE REPORT Our case was a 78-year-old woman with hypertension. Within a one-month interval, she developed AMI twice at the distal portion of her right coronary artery along with coronary artery ectasia. On both events, emergent coronary angiography showed no obvious organic stenosis or trace of plaque rupture at the culprit segment after thrombus aspiration. After the second acute event, we started anticoagulation therapy with warfarin to prevent thrombus formation. In the chronic phase, we confirmed, by using coronary angiography, optimal coherence tomography and intravascular ultrasound, that there was no plaque rupture and no obvious thrombus formation along the coronary artery ectasia segment of the distal right coronary artery, which suggested effectiveness of anticoagulant. Furthermore, by Doppler velocimetry we found sluggish blood flow only in the coronary artery ectasia lesion but not in the left atrium which is generally the main site of systemic thromboembolism revealed by transesophageal echocardiography. CONCLUSIONS These results suggest that the two AMI events at the same coronary artery ectasia segment were caused by local thrombus formation due to local stagnant blood flow. Although it has not yet been generally established, anticoagulation therapy may be effective to prevent thrombus formation in patients with coronary artery ectasia regardless of the prevalence of atrial fibrillation.

Biomagnification of endocrine disrupting chemicals (EDCs) by Pleuronectes yokohamae: Does P. yokohamae accumulate dietary EDCs?

We evaluated the potential for biomagnification of endocrine disrupting chemicals (EDCs) such as nonylphenol (NP), octylphenol (OP), bisphenol A (BP), and natural estrogens such as estrone (E1) and 17ß-estradiol (E2) in a benthic fish, Pleuronectes yokohamae. The assimilation efficiencies (AE) of most EDCs ranged from 88 to 96% suggesting that they were efficiently incorporated and assimilated into P. yokohamae, except for NP (50%). However, the biomagnification factor (BMF) values were <1.0 suggesting that the compounds were not biomagnifying. Additionally, three of the target EDCs were not detected (BP, E1 and E2). Glucuronidation activity towards BP (11.44 ± 2.5 nmol/mg protein/min) and E2 (12.41 ± 3.2 nmol/mg protein/min) was high in the intestine suggesting that EDCs were glucuronidated prior to excretion into bile. Thus, we conclude that biomagnification of dietary EDCs is reduced in P. yokohamae because of effective glucuronidation.

Acute Toxicity of Water-Accommodated Fraction and Chemically Enhanced WAF of Bunker C Oil and Dispersant to a Microalga Tetraselmis tetrathele.

This study assessed the toxicity of water-accommodated fraction (WAF) and chemically enhanced WAF (CEWAF) of bunker C oil and dispersant (DISP) to a microalga, Tetraselmis tetrathele. The 72-h median effective concentration (72-h EC50) of CEWAF and DISP were determined at 3.30% and 2.40%, respectively. The no observed effect concentration (NOEC) of CEWAF to T. tetrathele was at 2.0% and lowest observed effect concentration (LOEC) was at 3.0% while NOEC and LOEC of DISP to T. tetrathele were determined at 1.0% and 2.0%, respectively. The addition of dispersant to oil increased the amount of total PAH present in the CEWAF test solutions. DISP alone was highly toxic, and the toxicity of CEWAF was primarily caused by the presence of dispersant.

Reproductive toxicity of 1-nitronaphthalene and 1-nitropyrene exposure in the mummichog, Fundulus heteroclitus.

After pregnant mummichog were implanted with 1-nitronaphthalene or 1-nitropyrene via cholesterol pellet, we investigated the effects of the chemicals on embryo normality, hatchability and days to hatch of normal embryos, and growth and survival of hatched larvae from normal embryos of the implanted mummichog. Hatchability was the parameter most sensitive to the effects of both 1-nitronaphthalene and 1-nitropyrene. The 4-week lowest-observed-effect concentration (LOEC) of 1-nitronaphthalene, based on the actual concentrations in the eggs in the test, was 447 ng g(-1) wet wt.; and the LOEC and no-observed-effect concentration (NOEC) of 1-nitropyrene were 958 and 344 ng g(-1) wet wt., respectively. The 4-week LOEC of 1-nitronaphthalene, based on the concentration in the water, was estimated at 4.8 µg L(-1) by using the reported bioconcentration factor; and the LOEC and NOEC of 1-nitropyrene, based on the concentration in the water, were estimated at 3.1 and 8.6 µg L(-1), respectively. The reported environmental concentrations of 1-nitropyrene and 1-nitronaphthalene are over three magnitudes lower than the toxicity values we obtained. Therefore, the effects of environmental levels of 1-nitropyrene and 1-nitronaphthalene on fish reproduction, not including genomic effects on embryos, appear to be almost negligible. However, DNA damage has been detected in marine organisms exposed to 1-nitropyrene. Further studies of the genotoxicity of nitrated polycyclic aromatic hydrocarbons at environmental levels are therefore needed to evaluate their ecotoxicological risks.

Effects of chlorpyrifos on the metabolome of the freshwater carp, Cyprinus carpio.

This study investigated the effects of waterborne chlorpyrifos with concentrations of 1 and 100 µg/L for L and H-groups, respectively, on metabolome profiles of carp plasma using (1)H-NMR. Principal component analysis suggests that chlorpyrifos exposure firstly affected in L and H-groups on day 2 or 4, and followed a second effect in both exposure groups on day 14. Levels of metabolites related to the energy production in the body, such as glucose, glycerol, valine, leucine, isoleucine, lactate, alanine, 3-D-hydroxybutyrates and acetoacetate, significantly changed by exposures of chlorpyrifos. Those results suggest that energy production was severely affected in carp. The exposure could also be highly elevated ammonia levels especially in H-group due to severe convulsion in muscle caused by the inhibition of acetylcholinesterase activity.

Simulated distribution and ecotoxicity-based assessment of chemically-dispersed oil in Tokyo Bay.

To assess risks of chemically-dispersed oil to marine organisms, oil concentrations in the water were simulated using a hypothetical spill accident in Tokyo Bay. Simulated oil concentrations were then compared with the short-term no-observed effect concentration (NOEC), 0.01 mg/L, obtained through toxicity tests using marine diatoms, amphipod and fish. Area of oil concentrations higher than the NOEC were compared with respect to use and non-use of dispersant. Results of the simulation show relatively faster dispersion near the mouth of the bay compared to its inner sections which is basically related to its stronger water currents. Interestingly, in the inner bay, a large area of chemically-dispersed oil has concentrations higher than the NOEC. It seems emulsifying oil by dispersant increases oil concentrations, which could lead to higher toxicity to aquatic organisms. When stronger winds occur, however, the difference in toxic areas between use and non-use of dispersant is quite small.

Distributions of polycyclic aromatic hydrocarbons and alkylated polycyclic aromatic hydrocarbons in Osaka Bay, Japan.

Contaminations in sediments by polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs were investigated at 44 sites in Osaka Bay, Japan. Concentrations of total PAHs and alkylated PAHs were in the range 6.40-7800 ng/g dry weights and 13.7-1700 ng/g dry weights, respectively. The PAH concentrations tended to be higher along the shoreline in the vicinities of big ports, industrialized areas, and densely populated regions such as the cities of Osaka and Kobe. The major sources appeared to be pyrogenic or both pyrogenic and petrogenic at most of the sites. PAH concentrations were remarkably high at a site near Kobe, where the concentrations of dibenzo(a,h)anthracene and benzo(g,h,i)perylene exceeded the effects-range-medium concentration and eight PAHs were above the corresponding effects-range-low concentrations. Those PAHs may have been derived from the great fire associated with the large earthquake in 1995.

Two kinds of ferritin protect ixodid ticks from iron overload and consequent oxidative stress.

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.