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The full-length cDNA of two ferritins of Haemaphysalis flava were cloned after which recombinant Hf-FER1 and Hf-FER2 were expressed and their function was analyzed. In addition, RNA interference (RNAi) based on the injection of Hf-fer1 or Hf-fer2 dsRNA into fully engorged female ticks was performed. The cDNA encoding Hf-FER1 is 834 bp in length. It contains an iron-responsive element in the 5' untranslated region and encodes 174 amino acid residues. The full-length cDNA of Hf-FER2 contains 696 bp and encodes 199 amino acids, including a putative signal peptide sequence. Hf-FER1 and Hf-FER2 both have the ferroxidase iron center and the ferrihydrite nucleation center. The evolutionary relationship of Hf-FER1 and Hf-FER2 was established, and the predicted quaternary structures were assembled as typical spherical shells composed of 24 subunits which was demonstrated by nature PAGE. Real-time PCR showed that Hf-fer1 and Hf-fer2 were expressed in all developmental stages, with the highest expression in fully engorged females. The expression of Hf-fer1 and Hf-fer2 were relatively high in unfed larvae. Hf-fer1 was expressed in all tissues and was especially abundant in the salivary glands of fully engorged females. In contrast, the highest levels of Hf-fer2 were found in the midgut of fully engorged females, and no expression was found in the salivary glands of this life stage. Both recombinant Hf-FER1 and Hf-FER2 had iron-binding capabilities. Silencing of both Hf-fer1 and Hf-fer2 affected fecundity. Compared to the control, the percentage of ticks that laid eggs in the Hf-fer1 and Hf-fer2 RNAi groups was 73.3% and 66.7%, respectively. The silenced ticks that laid eggs had lower egg weight to body weight ratios, and the eggs had abnormal morphologies. The hatchability of eggs with normal morphology in the Hf-fer1 and Hf-fer2 silenced groups was 47.8% and 22.8%, respectively, which was significantly different from the control group (P0.005). These findings indicate that Hf-FER1 and Hf-FER2 play important roles in the iron storage of H. flava. |
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