1. Identification of the Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and their function in the degeneration of tick salivary glands
- Author
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Zhengmao Xu, Jinlin Zhou, Yanan Wang, Houshuang Zhang, Shanming Hu, Jie Cao, and Yongzhi Zhou
- Subjects
0301 basic medicine ,Rhipicephalus haemaphysaloides ,Apoptosis ,Infectious and parasitic diseases ,RC109-216 ,Tick ,Salivary Glands ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Western blot ,Proto-Oncogene Proteins ,In Situ Nick-End Labeling ,Rhipicephalus ,medicine ,Animals ,Gene silencing ,Salivary gland degeneration ,Bcl-2 ,bcl-2-Associated X Protein ,biology ,Salivary gland ,medicine.diagnostic_test ,Research ,biology.organism_classification ,Molecular biology ,030104 developmental biology ,Infectious Diseases ,medicine.anatomical_structure ,Real-time polymerase chain reaction ,Polyclonal antibodies ,Bax ,biology.protein ,Female ,Parasitology ,030217 neurology & neurosurgery - Abstract
Background The salivary glands of female ticks degenerate rapidly by apoptosis and autophagy after feeding. Bcl-2 family proteins play an important role in the apoptosis pathways, but the functions of these proteins in ticks are unclear. We studied Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and determined their functions in the degeneration of the salivary glands. Methods Two molecules containing conserved BH (Bcl-2 family homology) domains were identified and named RhBcl-2 and RhBax. After protein purification and mouse immunization, specific polyclonal antibodies (PcAb) were created in response to the recombinant proteins. Reverse transcription quantitative PCR (RT-qPCR) and western blot were used to detect the presence of RhBcl-2 and RhBax in ticks. TUNEL assays were used to determine the level of apoptosis in the salivary glands of female ticks at different feeding times after gene silencing. Co-transfection and GST pull-down assays were used to identify interactions between RhBcl-2 and RhBax. Results The RT-qPCR assay revealed that RhBax gene transcription increased significantly during feeding at all tick developmental stages (engorged larvae, nymphs, and adult females). Transcriptional levels of RhBcl-2 and RhBax increased more significantly in the female salivary glands than in other tissues post engorgement. RhBcl-2 silencing significantly inhibited tick feeding. In contrast, RhBax interference had no effect on tick feeding. TUNEL staining showed that apoptosis levels were significantly reduced after interference with RhBcl-2 expression. Co-transfection and GST pull-down assays showed that RhBcl-2 and RhBax could interact but not combine in the absence of the BH3 domain. Conclusions This study identified the roles of RhBcl-2 and RhBax in tick salivary gland degeneration and finds that the BH3 domain is a key factor in their interactions. Graphical Abstract
- Published
- 2021