CRAC CHANNEL PROTEINS IN Tribolium castaneum: A FUNCTIONAL PERSPECTIVE FROM RNA IN SITU HYBRIDIZATION

The calcium release-activated calcium (CRAC) channel plays a key role in maintaining and regulating intracellular Ca²⁺ homeostasis. It is formed by two essential proteins: STIM, located in the membrane of the endoplasmic reticulum, and Orai, which forms the pore in the plasma membrane. When Ca²⁺ stores in the endoplasmic reticulum are depleted, STIM undergoes conformational changes and activates Orai, promoting Ca²⁺ influx into the cytosol. Considering the critical role of Ca²⁺ in numerous cellular processes, including gene expression, cell division, growth, and apoptosis, calcium channels emerge as promising targets for pest control strategies. Although the CRAC channel is well characterized in some organisms, especially vertebrates, it remains poorly studied in others, such as Tribolium castaneum, a globally distributed stored-product pest of economic importance and a rising model organism for genetic and developmental research. This project aims to characterize the spatial expression patterns of T. castaneum STIM and Orai transcripts at various developmental stages using RNA in situ hybridization (RNA ISH). To this end, the cDNAs of both genes were amplified via PCR and cloned into the pGEM-T Easy vector. Recombinant plasmids were used to transform competent E. coli DH5-α cells, followed by plasmid extraction and sequence verification. Digoxigenin-labeled sense and antisense RNA probes were synthesized by in vitro transcription using T7 and SP6 RNA polymerases. Hybridization experiments in larvae revealed specific labeling in the abdominal segment, suggesting a role for CRAC components in the physiology of organs located in this region, such as the digestive and excretory systems. These findings represent the first evidence of CRAC channel gene expression in T. castaneum. Ongoing experiments aim to extend this analysis to embryonic stages and to investigate the functional roles of STIM and Orai through RNA interference (RNAi). This work contributes to our understanding of Ca²⁺ channel regulation in invertebrates and may offer insights into the development of novel pest control strategies targeting calcium signaling pathways.