Ocean acidification and acid rain, caused by modern industries’ fossil fuel burning, lead to a decrease in the living environmental pH, which results in a series of negative effects on many organisms. However, the underlying mechanisms of animals’ response to acidic pH stress are largely unknown. In this study, we used the nematode Caenorhabditis elegans as an animal model to explore the regulatory mechanisms of organisms’ response to pH decline. Two major stress-responsive pathways were found through transcriptome analysis in acidic stress environments. First, when the pH dropped from 6.33 to 4.33, the worms responded to the pH stress by upregulation of the col, nas, and dpy genes, which are required for cuticle synthesis and structure integrity. Second, when the pH continued to decrease from 4.33, the metabolism of xenobiotics by cytochrome P450 pathway genes (cyp, gst, ugt, and ABC transporters) played a major role in protecting the nematodes from the toxic substances probably produced by the more acidic environment. At the same time, the slowing down of cuticle synthesis might be due to its insufficient protective ability. Moreover, the systematic regulation pattern we found in nematodes might also be applied to other invertebrate and vertebrate animals to survive in the changing pH environments. Thus, our data might lay the foundation to identify the master gene(s) responding and adapting to acidic pH stress in further studies, and might also provide new solutions to improve assessment and monitoring of ecological restoration outcomes, or generate novel genotypes via genome editing for restoring in challenging environments especially in the context of acidic stress through global climate change.

Nematodes play key roles in marine ecosystem. Although oceans cover 71% of the Earth’s surface, none of marine model nematode has been reported. Here, we constructed the first inbred line of free-living marine nematode Litoditis marina , sequenced and assembled its genome. Furthermore, we successfully applied CRISPR/Cas9 genome editing in L. marina . Comparative genomics revealed that immunity and oxygen regulation genes are expanded, which is probably central to its sediment adaptation. While L. marina exhibits massive gene contractions in NHRs, chemoreceptors, xenobiotics detoxification and core histones, which could explain the more defined marine environment. Our experiments showed that dozens of H4 genes in Caenorhabditis elegans might contribute to its adaptation to the complex terrestrial environments, while two H4 genes in L. marina are involved in salinity stress adaptation. Additionally, ninety-two conserved genes appear to be positively selected in L. marina , which may underpin its osmotic, neuronal and epigenetic changes in the sea. With short generation time, highly inbred lines, and genomic resources, our report brings L. marina a promising marine animal model, and a unique satellite marine model to the well-known biomedical model nematode C. elegans . This study will underpin ongoing work on animal functional genomics, environmental adaptation and developmental evolution.

Maintenance of osmotic homeostasis is essential for all organisms, especially for marine animals in the ocean with 3% salinity or higher. However, the underlying molecular mechanisms that how marine animals adapt to high salinity environment compared to their terrestrial relatives, remain elusive. Here, we investigated marine animal’s genome-wide transcriptional responses to salinity stresses using an emerging marine nematode model Litoditis marina. We found that the transthyretin-like family genes were significantly increased in both hyposaline and hypersaline conditions, while multiple neurotransmitter receptor and ion transporter genes were down-regulated in both conditions, suggesting the existence of conserved strategies for response to stressful salinity environments in L. marina. Unsaturated fatty acids biosynthesis related genes, neuronal related tubulins and intraflagellar transport genes were specifically up-regulated in hyposaline treated worms. By contrast, cuticle related collagen genes were enriched and up-regulated for hypersaline response. Given a wide range of salinity tolerance of the marine nematodes, this study and further genetic analysis of key gene(s) of osmoregulation in L. marina will likely provide important insights into biological evolution and environmental adaptation mechanisms in nematodes and other invertebrate animals in general.

The marine nematode Litoditis marina is widely distributed in intertidal zones around the globe, yet the mechanisms underlying its broad adaptation to salinity remain elusive. In this study, we applied ONT long-read sequencing technology to unravel the transcriptome responses to different salinity conditions in L. marina. Through ONT sequencing under 3‰, 30‰ and 60‰ salinity environments, we obtained 131.78 G clean data and 26,647 non-redundant long-read transcripts, including 6464 novel transcripts. The DEGs obtained from the current ONT lrRNA-seq were highly correlated with those identified in our previously reported Illumina short-read RNA sequencing data. When we compared the 30‰ to the 3‰ salinity condition, we found that GO terms such as oxidoreductase activity, cation transmembrane transport and ion transmembrane transport were shared between the ONT lrRNA-seq and Illumina data. Similarly, GO terms including extracellular space, structural constituents of cuticle, substrate-specific channel activity, ion transport and substrate-specific transmembrane transporter activity were shared between the ONT and Illumina data under 60‰ compared to 30‰ salinity. In addition, we found that 79 genes significantly increased, while 119 genes significantly decreased, as the salinity increased. Furthermore, through the GO enrichment analysis of 214 genes containing DAS, in 30‰ compared to 3‰ salinity, we found that GO terms such as cellular component assembly and coenzyme biosynthetic process were enriched. Additionally, we observed that GO terms such as cellular component assembly and coenzyme biosynthetic process were also enriched in 60‰ compared to 30‰ salinity. Moreover, we found that 86, 125, and 81 genes that contained DAS were also DEGs, in comparisons between 30‰ and 3‰, 60‰ and 30‰, and 60‰ and 3‰ salinity, respectively. In addition, we demonstrated the landscape of alternative polyadenylation in marine nematode under different salinity conditions This report provides several novel insights for the further study of the mechanisms by which euryhalinity formed and evolved, and it might also contribute to the investigation of salinity dynamics induced by global climate change.

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.

Ocean acidification and acid rain, caused by modern industrial fossil fuels burning, lead to decrease of living environmental pH, which results in a series of negative effects on many organisms. However, the underlying mechanisms of animals’response to acidic pH stress are largely unknown. In this study, we used the nematode Caenorhabditis elegans as an animal model to explore the regulatory mechanisms of organisms’response to pH decline. Two major stress-responsive pathways were found through transcriptome analysis in acidic stress environments. Firstly, when the pH dropped from 6.33 to 4.33, the worms responded to the pH stress by up-regulation of the col , nas and dpy genes, which are required for cuticle synthesis and structure integrity. Secondly, when the pH continued to decrease from 4.33, the metabolism of xenobiotics by cytochrome P450 pathway genes ( cyp, gst, ugt, and ABC transporters) played a major role in protecting the nematodes from the toxic substances probably produced by the more acidic environment. At the same time, cuticle synthesis slowed down might due to its insufficient protective ability. Moreover, the systematic regulation pattern we found in nematodes, might also be applied to other invertebrate and vertebrate animals to survive in the changing pH environments. Thus, our data might lay the foundation to identify the master gene(s) responding and adaptation to acidic pH stress in further studies, and might also provide new solutions to improve assessment and monitoring of ecological restoration outcomes, or generate novel genotypes via genome editing for restoring in challenging environments especially in the context of acidic stress through global climate change.