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

ABSTRACT: 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.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.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.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.

SUBMITTER: Kusakisako K

PROVIDER: S-EPMC4992251 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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

Kusakisako Kodai K Galay Remil Linggatong RL Umemiya-Shirafuji Rika R Hernandez Emmanuel Pacia EP Maeda Hiroki H Talactac Melbourne Rio MR Tsuji Naotoshi N Mochizuki Masami M Fujisaki Kozo K Tanaka Tetsuya T

Parasites & vectors 20160819

<h4>Background</h4>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 regul ...[more]

PMID: 27542835

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Project description:BackgroundTicks are obligate hematophagous parasites important economically and to health. Ticks consume large amounts of blood for their survival and reproduction; however, large amounts of iron in blood could lead to oxidative stress. Ticks use several molecules such as glutathione S-transferases (GSTs), ferritins, and peroxiredoxins to cope with oxidative stress. This study aimed to identify and characterize the GSTs of the hard tick Haemaphysalis longicornis in order to determine if they have a role in coping with oxidative stress.MethodsGenes encoding GSTs of H. longicornis were isolated from the midgut CDNA library. Genes have been cloned and recombinant GSTs have been expressed. The enzymatic activities, enzyme kinetic constants, and optimal pH of the recombinant GSTs toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined. The gene transcription and protein expression profiles were determined in the whole ticks and internal organs, and developmental stages using real time RT-PCR and Western blotting during blood feeding. The localization of GST proteins in organs was also observed using immunofluorescent antibody test (IFAT).ResultsWe have isolated two genes encoding GSTs (HlGST and HlGST2). The enzymatic activity toward CDNB is 9.75 ± 3.04 units/mg protein for recombinant HlGST and 11.63 ± 4.08 units/mg protein for recombinant HlGST2. Kinetic analysis of recombinant HlGST showed K m values of 0.82 ± 0.14 mM and 0.64 ± 0.32 mM for the function of CDNB and GSH, respectively. Meanwhile, recombinant HlGST2 has K m values of 0.61 ± 0.20 mM and 0.53 ± 0.02 mM for the function of CDNB and GSH, respectively. The optimum pH of recombinant HlGST and recombinant HlGST2 activity was 7.5-8.0. Transcription of both GSTs increases in different developmental stages and organs during blood-feeding. GST proteins are upregulated during blood-feeding but decreased upon engorgement in whole ticks and in some organs, such as the midgut and hemocytes. Interestingly, salivary glands, ovaries, and fat bodies showed decreasing protein expression during blood-feeding to engorgement. Varying localization of GSTs in the midgut, salivary glands, fat bodies, ovaries, and hemocytes was observed depending on the feeding state, especially in the midgut and salivary glands.ConclusionsIn summary, a novel GST of H. longicornis has been identified. Characterization of the GSTs showed that GSTs have positive correlation with the degree and localization of oxidative stress during blood-feeding. This could indicate their protective role during oxidative stress.

Project description:BackgroundTicks can transmit numerous tick-borne pathogens and cause a huge economic loss to the livestock industry. Tick vaccines can contribute to the prevention of tick-borne diseases by inhibiting tick infestation or reproduction. Subolesin is an antigenic molecule proven to be a potential tick vaccine against different tick species and even some tick-borne pathogens. However, its effectivity has not been verified in Haemaphysalis longicornis, which is a widely distributed tick species, especially in East Asian countries. Therefore, the purpose of this study was to evaluate the effectivity of subolesin vaccination against H. longicornis in a rabbit model.MethodsHaemaphysalis longicornis (Okayama strain, female, adult, parthenogenetic strain) and Japanese white rabbits were used as the model tick and animal, respectively. The whole open reading frame of H. longicornis subolesin (HlSu) was identified and expressed as a recombinant protein using E. coli. The expression was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the immunogenicity of rHlSu against anti-H. longicornis rabbit serum was confirmed using Western blotting. After vaccination of rHlSu in rabbits, experimental infestation of H. longicornis was performed. Variables related to blood-feeding periods, pre-oviposition periods, body weight at engorgement, egg mass, egg mass to body weight ratio, and egg-hatching periods were measured to evaluate the effectiveness of subolesin vaccination.ResultsThe whole open reading frame of HlSu was 540 bp, and it was expressed as a recombinant protein. Vaccination with rHlSu stimulated an immune response in rabbits. In the rHlSu-vaccinated group, body weight at engorgement, egg mass, and egg mass to body weight ratio were statistically significantly lower than those in the control group. Besides, egg-hatching periods were extended significantly. Blood-feeding periods and pre-oviposition periods were not different between the two groups. In total, the calculated vaccine efficacy was 37.4%.ConclusionsVaccination of rabbits with rHlSu significantly affected the blood-feeding and reproduction in H. longicornis. Combined with findings from previous studies, our findings suggest subolesin has the potential to be used as a universal tick vaccine.

Project description:BACKGROUND:Close relationships between ticks and microbial communities are important for tick fitness and pathogen colonization and transmission. Haemaphysalis longicornis, distributed widely in China, can carry and transmit various pathogens and pose serious damages to public health and economics. However, little is known about the broader array of microbial communities and symbionts in H. longicornis under natural conditions. In the present study, we investigated the composition of bacterial communities associated with H. longicornis and evaluated the putative symbionts. METHODS:The eubacterial 16S rRNA gene clone libraries of H. longicornis were constructed and analyzed by restriction fragment length polymorphism (RFLP) and DNA sequencing. In addition, diagnostic PCR was performed to assess the prevalence, vertical transmission and infection sites of the symbionts in H. longicornis. RESULTS:Vertically-transmitted symbionts, potential pathogens and allochthonous nonpathogenic bacteria were identified from the field-collected H. longicornis. Three types of symbionts (Coxiella-like, Arsenophonus-like and Rickettsia-like symbionts) were identified in a single host simultaneously. A series of analyses revealed the vertical transmission, prevalence, and infection sites of these symbionts. However, only Coxiella-like bacteria were transmitted stably in the laboratory-reared ticks. In addition, we identified a novel Coxiella-like agent with 95.31% sequence similarity to the taxon described previously. CONCLUSIONS:The present study demonstrated that natural H. longicornis harboured a diverse array of microbial communities. Three types of symbionts were identified in a single host simultaneously. Moreover, high prevalence, vertical transmission and the infection sites supported an obligate symbiotic association between Coxiella symbiont and its host. The role of Coxiella symbiont in the host fitness and the interaction among microbial communities remained to be elucidated. Our investigation of microbial communities in the ticks revealed the complexity of ecological interactions between host and microbe and provided insight for the biological control of ticks.

Dataset Information

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

Publications

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

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