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

Abstract

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

The moulting arthropod: a complete genetic toolkit review

“…For Myriapoda too the picture remains far from complete, where EcR-RXR have been detected in a few centipede and millipede genomes (Chipman et al, 2014;Qu et al, 2015Qu et al, , 2020, but without follow-up experimental validations. Furthermore, our understanding of EcR and RXR in Chelicerata remains limited: while we have genomic evidence for the presence of both the heterodimer components for Acari (T. urticae, Dermatophagoides farinae, I. scapularis, P. citri, Neoseiulus cucumeris), Araneae (Stegodyphus mimosarum), Scorpiones (Mesobuthus martensii), Xiphosura (Limulus polyphemus, Carcinoscorpius rotundicauda, Tachypleus tridentatus) (Qu et al, 2015;Schumann et al, 2018;Zhang et al, 2019b), recent studies that experimentally characterised them are limited to few spider, mite, and tick species (Honda et al, 2017;Li et al, 2017;Nicewicz et al, 2021;Lu et al, 2021;Li et al, 2022a;Yang et al, 2023). Further analysis of EcR and RXR in these groups is required to assess if in chelicerates the predominant bioactive hormone is 20E or ponA (Figure 2).…”

“…For instance, programmed cell death is involved in metamorphic remodelling of head projections in horned beetles (Tettamanti & Casartelli, 2019). Remarkably, salivary gland degeneration depends on EcR-induced caspase-dependent apoptosis in the tick Rhipicephalus haemaphysaloides (Lu et al, 2021) and histolytic processes have been described in maturation of copulatory organs in male P. tepidariorum spiders (Quade et al, 2019).…”

The use of RNA interference for the management of arthropod pests in livestock farms

“…Wang et al (2019) injected dsRNA into Rhipicephalus haemaphysaloides Supino (Acari: Ixodidae), demonstrating how the silencing of the RH‐Hsc70 gene, expressing an heat shock protein in adult ticks, resulted in a reduction in blood meal fulfilment, and an increase in mortality rates. Lu et al (2021) silenced the functional ecdysone receptor ( RhECR / RhUSP ) of 20‐hydroxyecdysone from the salivary gland of the R. haemaphysaloides obtaining the inhibition of blood supply and curbed salivary gland degeneration by suppressing caspase‐dependent apoptosis.…”

A Novel Rhipicephalus microplus Estrogen Related Receptor (RmERR), a Molecular and In Silico Characterization of a Potential Protein Binding Estrogen

“…In contrast, some hormones and their cellular receptors have been identified in ticks, including molting and neuro hormones (ecdysteroids), involved in molting, development, and reproduction (vitellogenesis) [ 10 , 11 , 12 , 13 , 14 ]. For example, during the life cycle and after engorgement, ecdysteroid hormones trigger the salivary glands degeneration in Rhipicephalus haemaphysaloides [ 12 ].…”

“…In contrast, some hormones and their cellular receptors have been identified in ticks, including molting and neuro hormones (ecdysteroids), involved in molting, development, and reproduction (vitellogenesis) [ 10 , 11 , 12 , 13 , 14 ]. For example, during the life cycle and after engorgement, ecdysteroid hormones trigger the salivary glands degeneration in Rhipicephalus haemaphysaloides [ 12 ]. There even exist reports proposing that the tick microbiome is responsive to the hormone 20-hydroxyecdysone, which regulates vitellogenesis and the egg development in Dermacentor variabilis [ 9 ].…”