Gene- and Isoform-Level Responses to Extreme Acidic pH Stress in an Emerging Marine Invertebrate Model Organism Litoditis marina

Ocean acidification poses a critical threat to marine invertebrate survival and diversification, yet the post-transcriptional regulatory mechanisms underlying acid stress responses remain poorly understood. Here, we employed Oxford Nanopore long-read RNA sequencing to systematically characterize transcriptional and post-transcriptional responses to acidic pH stresses in the marine nematode Litoditis marina. Our analysis revealed 912 upregulated and 728 downregulated genes enriched in autophagy, fatty acid metabolism, and peroxisome activation, alongside 327 differential alternative splicing events and 1512 transcripts with significant usage changes under severe acidic pH stress. By integrating weighted gene co-expression network analysis with databases such as WormExp2, we found that acidic pH stress response exhibited resemblance to oxidative stress response. Specifically, we identified genes involved in the oxidative stress response, such as gpx-1, cyp-13A11, trxr-1, cyc-1, and key regulators, including hlh-30/TFEB. Genes such as gpx-1, trxr-1, and cyp-23A1 might protect L. marina from oxidative stress under acidic pH. Moreover, several ferroptosis-related genes, such as gpx-5, fat-2, and smf-1, might render L. marina vulnerable to acidic pH stress. Among the genes with splicing changes, we identified oxidative stress responding genes such as sod-4, prx-2, coq-2, coq-3, prx-10, ctl-2, gst-7, trx-1, mdt-15, and fat-2. Additionally, we discovered the preference for proximal 3′ UTR under acidic pH stress. Genes related to ferroptosis, including cyp-23A1, C07E3.9/PLA2G1B, and C53D5.5/GGT1, exhibited differential 3′ UTR usage under acidic pH stress. Our findings shift the focus from traditional gene-centric analyses to capturing the full breadth of post-transcriptional diversity, providing novel insights into post-transcriptional gene regulation in marine metazoans under environmental stress, as well as revealing that alleviating oxidative stress might increase resistance to acid pH stress.