Isotocin receptor-adenylyl cyclase signaling mediates pH compensation in marine fish exposed to CO₂-induced acidification

Marine fish survival is threatened by ocean acidification, but the hormonal mechanisms for pH compensation are not well understood, limiting mechanistic understanding of stress responses in marine fish. We examined isotocin signaling in marine medaka ( Oryzias melastigma) exposed to year-2100 ocean acidification conditions (pCO₂ ~0.14 kPa, pH 7.6). Our analysis revealed that isotocin receptor b (ITRb) was selectively upregulated at 6 hours post-exposure in adult gills, though it showed only a non-significant trend at 5 dpf embryos, while adenylyl cyclase 5 (ADCY5) showed hypercapnia responsiveness primarily at hatching. Using immunofluorescence and confocal microscopy, we found that both ITRb and ADCY5 proteins localize to the basolateral membrane of NKA-positive ionocytes, partially separated from apical H⁺-secretion machinery. Knockdown experiments showed that ITRb-ADCY5 coupling is crucial for pH compensation, with individual knockdown moderately reducing H⁺ secretion and combined knockdown causing severe impairment (>70% reduction) and decreasing transcription of acid-secretion genes ( nhe3, ca2, rhcgb) by 44-60%. Paradoxically, double knockdown triggered a 2-fold cAMP increase that failed to restore function, whereas wild-type embryos maintained stable cAMP levels across pH conditions, consistent with the hypothesis that ITRb-ADCY5 coupling may organize cAMP production within specific basolateral microdomains, though direct subcellular imaging would be required to validate this compartmentalization model. The developmental asynchrony between ITRb (5 dpf) and ADCY5 (hatching) responses indicates life-stage-specific vulnerabilities. Our findings reveal that basolateral ITRb-ADCY5 coupling represents a critical control point for pH compensation capacity.