Molecular responses of amphipod (Parhyale darvishi), to PH stress in Persian Gulf

Abstract Climate change is driving more frequent and extreme pH fluctuations in intertidal habitats, yet the molecular mechanisms by which small crustaceans cope with acid–base stress remain poorly understood. In this study, we evaluated the transcriptional responses of the intertidal amphipod Parhyale darvishi to acute low-pH (6.0) and high-pH (9.0) challenges, simulating the extremes observed in tide pools. Following a 7-day acclimatization in aerated seawater (salinity 40–42 ppt, 24–25 °C, 12:12 h light:dark), individuals (4–7 mm length) were randomly assigned to one of three treatments: control (ambient pH 7.50–7.60), low pH (adjusted to 6.0 with 20 mL 37% HCl), or high pH (adjusted to 9.0 with 3 mL NaOH), each with two 1-L replicates containing 50 animals. After 0h, 12h and 24 h of exposure, total RNA was extracted and reverse-transcribed to cDNA. Real-time PCR assays quantified expression of five target genes: catalase (CAT), glutathione S-transferase (GST), Na⁺/K⁺-ATPase, apoptosis signal-regulating kinase 1 (ASK1), and caspase-3, with tubulin serving as the reference gene. Both pH stressors elicited significant transcriptional changes relative to controls. Under low pH, antioxidant genes CAT and GST were upregulated by approximately 2.5- and 2.1-fold, respectively, indicating activation of oxidative defense pathways. In contrast, high pH induced a more moderate antioxidant response (1.8- and 1.5-fold for CAT and GST) but triggered a pronounced apoptotic signal, with caspase-3 expression increasing nearly 3-fold. Na⁺/K⁺-ATPase transcripts rose under both treatments, reflecting osmoregulatory adjustments, while ASK1 exhibited a stronger induction in acid-stressed amphipods, suggesting stress-activated kinase signaling. These findings demonstrate that P. darvishi mounts distinct molecular responses to acid versus alkaline challenges, engaging antioxidant defenses under low pH and apoptosis-related pathways under high pH. Such differential gene expression profiles provide mechanistic insight into how intertidal amphipods cope with rapid pH swings, and underscore the utility of molecular biomarkers for assessing the resilience of coastal invertebrates under future acidification and alkalinization scenarios.

Abstract Climate change is driving more frequent and extreme pH fluctuations in intertidal habitats, yet the molecular mechanisms by which small crustaceans cope with acid–base stress remain poorly understood. In this study, we evaluated the transcriptional responses of the intertidal amphipod Parhyale darvishi to acute low-pH (6.0) and high-pH (9.0) challenges, simulating the extremes observed in tide pools. Following a 7-day acclimatization in aerated seawater (salinity 40–42 ppt, 24–25 °C, 12:12 h light:dark), individuals (4–7 mm length) were randomly assigned to one of three treatments: control (ambient pH 7.50–7.60), low pH (adjusted to 6.0 with 20 mL 37% HCl), or high pH (adjusted to 9.0 with 3 mL NaOH), each with two 1-L replicates containing 50 animals. After 0h, 12h and 24 h of exposure, total RNA was extracted and reverse-transcribed to cDNA. Real-time PCR assays quantified expression of seven target genes: catalase (CAT), glutathione S-transferase (GST), Na⁺/K⁺-ATPase, apoptosis signal-regulating kinase 1 (ASK1), and caspase-3, with tubulin serving as the reference gene. Both pH stressors elicited significant transcriptional changes relative to controls. Under low pH, antioxidant genes CAT and GST were upregulated by approximately 2.5- and 2.1-fold, respectively, indicating activation of oxidative defense pathways. In contrast, high pH induced a more moderate antioxidant response (1.8- and 1.5-fold for CAT and GST) but triggered a pronounced apoptotic signal, with caspase-3 expression increasing nearly 3-fold. Na⁺/K⁺-ATPase transcripts rose under both treatments, reflecting osmoregulatory adjustments, while ASK1 exhibited a stronger induction in acid-stressed amphipods, suggesting stress-activated kinase signaling. These findings demonstrate that P. darvishi mounts distinct molecular responses to acid versus alkaline challenges, engaging antioxidant defenses under low pH and apoptosis-related pathways under high pH. Such differential gene expression profiles provide mechanistic insight into how intertidal amphipods cope with rapid pH swings, and underscore the utility of molecular biomarkers for assessing the resilience of coastal invertebrates under future acidification and alkalinization scenarios.