Identification of the Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and their function in the degeneration of tick salivary glands

• Published in: Parasites & vectors 2021-08, Vol.14 (1), p.386-12, Article 386
• Main Authors: Hu, Shanming, Wang, Yanan, Xu, Zhengmao, Zhou, Yongzhi, Cao, Jie, Zhang, Houshuang, Zhou, Jinlin
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Apoptosis; Apoptotic proteins; Arachnids; Assaying; Autophagy; Bax; BAX protein; Bcl-2; Bcl-2 protein; bcl-2-Associated X Protein - isolation & purification; bcl-2-Associated X Protein - physiology; Cell division; Chromatography; Cloning; Degeneration; Degeneration (Pathology); Developmental stages; DNA; Domains; Engorgement; Exocrine glands; Feeding; Female; Females; Gene expression; Gene silencing; Genetic aspects; Glands; Homology; Identification; Identification and classification; Immunization; In Situ Nick-End Labeling; Interference; Larvae; Lymphoma; Maximum likelihood method; Mice; Nucleotide sequence; Nymphs; Ovaries; Parasitological research; Pathogens; PCR; Phagocytosis; Physiological aspects; Polyclonal antibodies; Protein expression; Protein purification; Proteins; Proto-Oncogene Proteins - isolation & purification; Proto-Oncogene Proteins - physiology; Recombinants; Reverse transcription; Rhipicephalus - metabolism; Rhipicephalus haemaphysaloides; Salivary gland; Salivary gland degeneration; Salivary glands; Salivary Glands - metabolism; Salivary Glands - pathology; Ticks; Transcription; Transfection; Water purification
• ISSN: 1756-3305; 1756-3305
• DOI: 10.1186/s13071-021-04879-z

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. 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. 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. 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.