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

Abstract

Background: Ticks 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.

Methods: The 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.

Results: Real-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.

Conclusion: Therefore, 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.

Keywords: Haemaphysalis longicornis; Hydrogen peroxide; Peroxiredoxin; RNA interference.

Fig. 1

a Transcription profiles of HlPrx2 in whole ticks during blood-feeding analyzed by real-time PCR (Uf, unfed females; 1d-4d, adults partially fed for 1–4 days). b Transcription profiles of HlPrx2 in unfed and engorged tick developmental stages. c Transcription profiles of HlPrx2 in the internal organs: salivary glands, midgut, ovary, fat body, hemocytes, synganglion). L23 was used as the internal control. Data are presented as the mean ± standard deviation (SD). ^* P < 0.05; ^** P < 0.01, significant differences vs dsLuc by Welch’s t-test. Abbreviations: Uf, unfed ticks; En, engorged ticks

Fig. 2

a Protein expression profiles of HlPrx2 in whole ticks during blood-feeding as analyzed by Western blot analysis. b Protein expression profiles of HlPrx2 in developmental stages. Arrow indicates native HlPrx2 protein as distinguished from the non-specific bands below. c Protein expression profiles of HlPrx2 in the internal organs (salivary glands, midgut, ovary, fat body, hemocytes, and synganglion). For loading control, tubulin was detected. The bars show the results of band densitometry analysis for HlPrx2. The relative expression was calculated based on tubulin. Data are presented as the mean ± standard deviation (SD). ^* P < 0.05, significant differences by Welch’s t-test. Abbreviations: Uf, unfed adults; Pf, partially fed adults at day 3; En, engorged adults

Fig. 3

Localization of HlPrx2 proteins in the salivary glands, midgut, ovary, and hemocytes from engorged adult ticks using IFAT under a confocal laser scanning microscope. Anti-HlPrx2 mouse serum was used as a primary antibody. Anti-mouse IgG conjugated with Alexa Fluor 594 was used as a secondary antibody and nuclei were visualized using DAPI. Normal mouse serum was used for control. Arrows indicate the specific fluorescence. Abbreviations: SA, salivary gland acinar cells; SGG, salivary gland granular cells; SD, salivary duct. Scale-bars: 20 μm (salivary glands, midgut, and ovary) and 10 μm (hemocytes)

Fig. 4

a Knockdown confirmation of HlPrx and/or HlPrx2 genes in partially fed adult ticks. Each tick total RNA was extracted from 3 ticks pooled. The left column indicates the detection primer set; actin was used as a control. The right column indicates the size of the PCR products. b Column graph for engorged body weight in the knockdown experiment. c Column graph for egg weight after finishing oviposition by engorged adult ticks in the knockdown experiment. Horizontal lines indicate the median values. Abbreviations: dsLuc, double-stranded Luciferase-injected group; dsHlPrx2, double-stranded HlPrx2-injected group; dsHlPrx, double-stranded HlPrx-injected group; dsDouble, both double-stranded HlPrx- and HlPrx2-injected group. ^* P < 0.05; ^** P < 0.01, significant differences vs dsLuc by Welch’s t-test

Fig. 5

Concentrations of hydrogen peroxide (H₂O₂) from HlPrx and/or HlPrx2 knockdown ticks during blood-feeding. Data are presented as the ratio of H₂O₂ concentration (μM) to tick body weight or engorged body weight (mg). Horizontal bars indicate the median value. Abbreviations: dsLuc, double-stranded Luciferase-injected group; dsHlPrx2, double-stranded HlPrx2-injected group; dsHlPrx, double-stranded HlPrx-injected group; dsDouble, both double-stranded HlPrx- and HlPrx2-injected group. ^* P < 0.05; ^** P < 0.01, significant differences vs dsLuc by Welch’s t-test