The ecdysteroid receptor regulates salivary gland degeneration through apoptosis in Rhipicephalus haemaphysaloides

Background It is well established that ecdysteroid hormones play an important role in arthropod development and reproduction, mediated by ecdysteroid receptors. Ticks are obligate hematophagous arthropods and vectors of pathogens. The salivary gland plays an essential role in tick growth and reproduction and in the transmission of pathogens to vertebrate hosts. During tick development, the salivary gland undergoes degeneration triggered by ecdysteroid hormones and activated by apoptosis. However, it is unknown how the ecdysteroid receptor and apoptosis regulate salivary gland degeneration. Here, we report the functional ecdysteroid receptor (a heterodimer of the ecdysone receptor [EcR] and ultraspiracle [USP]) isolated from the salivary gland of the tick Rhipicephalus haemaphysaloides and explore the molecular mechanism of ecdysteroid receptor regulation of salivary gland degeneration. Methods The full length of RhEcR and RhUSP open reading frames (ORFs) was obtained from the transcriptome. The RhEcR and RhUSP proteins were expressed in a bacterial heterologous system, Escherichia coli. Polyclonal antibodies were produced against synthetic peptides and were able to recognize recombinant and native proteins. Quantitative real-time PCR and western blot were used to detect the distribution of RhEcR, RhUSP, and RhCaspases in the R. haemaphysaloides organs. A proteomics approach was used to analyze the expression profiles of the ecdysteroid receptors, RhCaspases, and other proteins. To analyze the function of the ecdysteroid receptor, RNA interference (RNAi) was used to silence the genes in adult female ticks. Finally, the interaction of RhEcR and RhUSP was identified by heterologous co-expression assays in HEK293T cells. Results We identified the functional ecdysone receptor (RhEcR/RhUSP) of 20-hydroxyecdysone from the salivary gland of the tick R. haemaphysaloides. The RhEcR and RhUSP genes have three and two isoforms, respectively, and belong to a nuclear receptor family but with variable N-terminal A/B domains. The RhEcR gene silencing inhibited blood-feeding, blocked engorgement, and restrained salivary gland degeneration, showing the biological role of the RhEcR gene in ticks. In the ecdysteroid signaling pathway, RhEcR silencing inhibited salivary gland degeneration by suppressing caspase-dependent apoptosis. The heterologous expression in mammalian HEK293T cells showed that RhEcR1 interacts with RhUSP1 and induces caspase-dependent apoptosis. Conclusions These data show that RhEcR has an essential role in tick physiology and represents a putative target for the control of ticks and tick-borne diseases. Graphical Abstract

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

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

Identification of the Bcl-2 and Bax Homologs From the Tick Rhipicephalus Haemaphysaloides and Their Function in the Degeneration of Tick Salivary Glands

BackgroundThe salivary gland of female ticks degenerates 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 the tick Rhipicephalus haemaphysaloides and determined their functions in the degeneration of the salivary glands.MethodsThe full-length cDNA of the RhBcl-2 and RhBax genes were obtained by transcriptome analysis and RhBcl-2 and RhBax were expressed in E. coli. Two molecules containing conserved BH (Bcl-2 family homology) domains were identified and named RhBcl-2 and RhBax. After protein purification and mouse immunizations, specific polyclonal antibodies (PcAb) were created in response to the recombinant protein. Reverse transcription quantitative PCR (RT-qPCR) and western blot were used to detect the existence of RhBcl-2 and RhBax in ticks. TUNEL assays were used to determine the apoptosis level in the salivary glands at different feeding times after gene silencing. The interactions between RhBcl-2 and RhBax were identified by co-transfection and GST pull-down assays. The RT-qPCR assay demonstrated that the transcription of RhBax genes increased significantly during the engorged periods of the tick developmental stages (engorged larval, nymph, and adult female ticks). ResultsTranscriptional levels of RhBcl-2 and RhBax in the salivary glands increased more significantly than other tissues post engorgement. RhBcl-2 treatment significantly restrained tick feeding. In contrast, RhBax interference had no effect on tick feeding. TUNEL staining showed that apoptosis levels were significantly reduced after interfering with RhBcl-2 expression. Co-transfection and GST pull-down assays showed that RhBcl-2 and RhBax could combine with each other, but failed to combine without the BH3 domain. ConclusionsThis study identified the roles of RhBcl-2 and RhBax in tick salivary gland degeneration and clarifies their interactions.