The essential role of a single PEPCK isoform is presented as a target for disrupting tick embryogenesis.

The cattle tick Rhipicephalus microplus faces periods of nutritional stress that are inherent to its life cycle and during which starvation is a critical physiological challenge demanding specific metabolic adaptations. We used the embryonic cell line BME26 as a model to study metabolic responses during nutritional stress, to investigate the mechanisms underlying tick resilience. Our findings demonstrate that BME26 cells withstand prolonged starvation by activating gluconeogenesis, as evidenced by a marked increase in phosphoenolpyruvate carboxykinase (PEPCK) activity. Follow-up qPCR and enzymatic assays of tick tissues and BME26 cells identified the cytosolic PEPCK-C isoform as the predominantly, if not exclusively, expressed and active form, despite initial RNAseq data indicating transcripts for both PEPCK-C and PEPCK-M. Consistent with the predominant expression of the cytosolic form in R. microplus, the mitochondrial PEPCK-M isoform was present at minimal levels, exhibiting expression approximately 1,000 times lower than that of PEPCK-C. PEPCK silencing significantly reduced cell viability, particularly under starvation, demonstrating its critical role in energy homeostasis. The knockdown also downregulated the expression of fructose-1,6-bisphosphatase 1 (FBPase1) and glucose-6-phosphate dehydrogenase (G6PDH) while upregulating the autophagy-related gene ATG8. Additionally, the PEPCK inhibitor 3-mercaptopicolinic acid (3-MPA) at high concentrations caused decreased BME26 viability and reduced tick oviposition. Altogether, these results identify gluconeogenesis as a fundamental pathway for tick survival and a promising metabolic target for novel tick control strategies.