Tong Hao

Molting is a critical physiological process for the growth and development of Eriocheir sinensis. Any disruption in this process can significantly affect both survival rates and crab quality. The regulatory mechanisms of molting vary across different stages of the molting cycle and remain poorly understood. In this study, ATAC-seq and RNA-seq were combined to identify the integrated differentially expressed genes (IDEGs) in muscle across adjacent stages of the molting cycle. A total of 17, 491, 84, and 491 IDEGs were identified in the comparisons of inter-molt_vs_pre-molt, pre-molt_vs_molt, molt_vs_post-molt, and post-molt_vs_inter-molt stages, respectively. GO enrichment analysis of these IDEGs revealed several key signaling pathways involved in each adjacent molting stage. The GPCR signaling, steroid hormone-mediated signaling, and smoothened signaling pathways were all active across three molting transitions (pre-molt_vs_molt, molt_vs_post-molt, and post-molt_vs_inter-molt). Among them, the GPCR pathway played a dominant role throughout the process. Further structural analysis and RT-qPCR validation identified eight GPCRs involved in molting regulation: GRM7 and moody were specific to the post-molt_vs_inter-molt stage; Kpna6, ADRB2, and SSTR2 were unique to the pre-molt_vs_molt stage; FMRFaR and gpr161 functioned in both post-molt_vs_inter-molt and pre-molt_vs_molt stages; and mth2 was active in both post-molt_vs_inter-molt and molt_vs_post-molt stages. These findings improve the understanding of molting regulation and provide potential targets for further genetic improvement in E. sinensis.

Temperature is one of the critical factors influencing the survival, growth, and reproduction of organisms. The molting and developmental mechanisms of crustaceans are highly sensitive to temperature, yet the regulatory mechanisms underlying their thermal adaptation remain unclear. In this work, transcriptome sequencing was performed to analyze the gene expression profiles of Eriocheir sinensis under normal temperature (22 °C) and high-temperature (27 °C and 32 °C) conditions. A total of 377 differentially expressed genes (DEGs) were identified, including 149 up-regulated and 227 down-regulated genes. Through Gene Ontology (GO) enrichment analysis of these DEGs, 11 significantly temperature-regulated signaling pathways were identified, including the estrogen and androgen receptor signaling pathways, and two neurotransmission signaling pathways. These findings suggest that temperature may influence sex regulation in E. sinensis, while the dopamine receptor and neuropeptide signaling pathways may play a role in its thermal adaptation. Further validation via RT-qPCR of DEGs involved in neurotransmission signaling pathways revealed that crustacean hyperglycemic hormone (CHH) and excitatory amino acid transporter 3 (EAA3) genes are likely involved in the thermal adaptation of E. sinensis. In addition, the hemolymph glucose levels associated with the elevated temperatures were detected and consistent variations between glucose levels and CHH expressions were found. This indicates that the eyestalk CHH is strongly correlated with the hemolymph glucose levels and likely mediates the response to temperature changes by regulating blood glucose in E. sinensis. The results of this study not only provide key molecular targets for elucidating the mechanisms by which temperature affects molting and development in E. sinensis, but also establish a theoretical foundation for further research into thermal adaptation strategies in crustaceans.

Vibrio parahaemolyticus is a pathogenic bacterium widely distributed in marine environments, posing significant threats to aquatic organisms and human health. The overuse and misuse of antibiotics has led to the development of multidrug- and pan-resistant V. parahaemolyticus strains. There is an urgent need for novel antibacterial therapies with innovative mechanisms of action. In this work, a genome-scale metabolic network model (GMSN) of V. parahaemolyticus, named VPA2061, was reconstructed to predict the metabolites that can be explored as potential drug targets for eliminating V. parahaemolyticus infections. The model comprises 2061 reactions and 1812 metabolites. Through essential metabolite analysis and pathogen–host association screening with VPA2061, 10 essential metabolites critical for the survival of V. parahaemolyticus were identified, which may serve as key candidates for developing new antimicrobial strategies. Additionally, 39 structural analogs were found for these essential metabolites. The molecular docking analysis of the essential metabolites and structural analogs further investigated the potential value of these metabolites for drug design. The GSMN reconstructed in this work provides a new tool for understanding the pathogenic mechanisms of V. parahaemolyticus. Furthermore, the analysis results regarding the essential metabolites hold profound implications for the development of novel antibacterial therapies for V. parahaemolyticus-related disease.

Artemia is a typical animal used for the study of the diapause mechanism. The research on the regulation mechanism of diapause mainly focuses on the occurrence and maintenance of diapause. There are few studies on the mechanism of embryonic pause termination (EDT), especially for its transcriptional regulation mechanism. This study integrated transcriptional regulatory data from ATAC-seq and gene expression data from RNA-seq to explore the transcriptional regulatory mechanisms involved in the EDT process. Through integrated analysis, four important transcription factors (TFs), SVP, MYC, RXR, and SMAD6, were found to play a role in the EDT process, in which SVP, MYC, and RXR were upregulated, while SMAD6 was downregulated in the EDT stage. Through co-expression analysis, a transcription regulatory network for these four TFs was constructed and the functions of the TFs were analyzed. The expression of the TFs was further verified by RT-qPCR. Through functional analysis, SVP was found to be predominantly involved in cell adhesion and signal transduction. MYC probably played a role in protein binding. RXR may function in the process of RNA binding and the transfer of phosphorus-containing groups. Smad6 regulated the signal transduction, cell adhesion, and oxidation–reduction processes. The expression of the key TFs was verified by RT-qPCR. The results of this work provide important clues for the mechanism of transcriptional regulation in the EDT process of Artemia.

Embryonic diapause is a common evolutionary adaptation observed across a wide range of organisms. Artemia is one of the classic animal models for diapause research. The current studies of Artemia diapause mainly focus on the induction and maintenance of the embryonic diapause, with little research on the molecular regulatory mechanism of Artemia embryonic reactivation. The first 5 h after embryonic diapause breaking has been proved to be most important for embryonic reactivation in Artemia. In this work, two high-throughput sequencing methods, ATAC-seq and RNA-seq, were integrated to study the signal regulation process in embryonic reactivation of Artemia at 5 h after diapause breaking. Through the GO and KEGG enrichment analysis of the high-throughput datasets, it was showed that after 5 h of diapause breaking, the metabolism and regulation of Artemia cyst were quite active. Several signal transduction pathways were identified in the embryonic reactivation process, such as G-protein-coupled receptor (GPCR) signaling pathway, cell surface receptor signaling pathway, hormone-mediated signaling pathway, Wnt, Notch, mTOR signaling pathways, etc. It indicates that embryonic reactivation is a complex process regulated by multiple signaling pathways. With the further protein structure analysis and RT-qPCR verification, 11 GPCR genes were identified, in which 5 genes function in the embryonic reactivation stage and the other 6 genes contribute to the diapause stage. The results of this work reveal the signal transduction pathways and GPCRs involved in the embryonic reactivation process of Artemia cysts. These findings offer significant clues for in-depth research on the signal regulatory mechanisms of the embryonic reactivation process and valuable insights into the mechanism of animal embryonic diapause.

Eriocheir sinensis is an economically important aquatic animal. Its regulatory mechanisms underlying many biological processes are still vague due to the lack of systematic analysis tools. The protein–protein interaction network (PIN) is an important tool for the systematic analysis of regulatory mechanisms. In this work, a novel machine learning method, DGO-SVM, was applied to predict the protein–protein interaction (PPI) in E. sinensis, and its PIN was reconstructed. With the domain, biological process, molecular functions and subcellular locations of proteins as the features, DGO-SVM showed excellent performance in Bombyx mori, humans and five aquatic crustaceans, with 92–96% accuracy. With DGO-SVM, the PIN of E. sinensis was reconstructed, containing 14,703 proteins and 7,243,597 interactions, in which 35,604 interactions were associated with 566 novel proteins mainly involved in the response to exogenous stimuli, cellular macromolecular metabolism and regulation. The DGO-SVM demonstrated that the biological process, molecular functions and subcellular locations of proteins are significant factors for the precise prediction of PPIs. We reconstructed the largest PIN for E. sinensis, which provides a systematic tool for the regulatory mechanism analysis. Furthermore, the novel-protein-related PPIs in the PIN may provide important clues for the mechanism analysis of the underlying specific physiological processes in E. sinensis.

Artemia is a widely distributed small aquatic crustacean, renowned for its ability to enter a state of embryonic diapause. The embryonic diapause termination (EDT) is closely linked to environmental cues, but the precise underlying mechanisms remain elusive. In this study, ATAC-seq and RNA-seq sequencing techniques were employed to explore the gene expression profiles in Artemia cysts 30 min after EDT. These profiles were compared with those during diapause and 5 h after EDT. The regulatory mechanisms governing the EDT process were analyzed through Gene Ontology (GO) enrichment analysis of differentially expressed genes. Furthermore, the active G-protein-coupled receptors (GPCRs) were identified through structural analysis. The results unveiled that the signaling transduction during EDT primarily hinges on GPCRs and the cell surface receptor signaling pathway, but distinct genes are involved across different stages. Hormone-mediated signaling pathways and the tachykinin receptor signaling pathway exhibited heightened activity in the ‘0–30 min’ group, whereas the Wnt signaling pathway manifested its function solely in the ‘30 min–5 h’ group. These results imply a complete divergence in the mechanisms of signal regulation during these two stages. Moreover, through structural analysis, five GPCRs operating at different stages of EDT were identified. These findings provide valuable insights into the signal regulation mechanisms governing Artemia diapause.

Integrated networks have become a new interest in genome-scale network research due to their ability to comprehensively reflect and analyze the molecular processes in cells. Currently, none of the integrated networks have been reported for higher organisms. Eriocheir sinensis is a typical aquatic animal that grows through ecdysis. Ecdysone has been identified to be a crucial regulator of ecdysis, but the influence factors and regulatory mechanisms of ecdysone synthesis in E. sinensis are still unclear. In this work, the genome-scale metabolic network and protein–protein interaction network of E. sinensis were integrated to reconstruct a metabolic–protein interaction integrated network (MPIN). The MPIN was used to analyze the influence factors of ecdysone synthesis through flux variation analysis. In total, 236 integrated reactions (IRs) were found to influence the ecdysone synthesis of which 16 IRs had a significant impact. These IRs constitute three ecdysone synthesis routes. It is found that there might be alternative pathways to obtain cholesterol for ecdysone synthesis in E. sinensis instead of absorbing it directly from the feeds. The MPIN reconstructed in this work is the first integrated network for higher organisms. The analysis based on the MPIN supplies important information for the mechanism analysis of ecdysone synthesis in E. sinensis.

Introduction: As global temperatures continue to rise, extreme weather phenomena such as El Niño and the Southern Oscillation (ENSO) near the equatorial Pacific Ocean are occurring more frequently and leading to tropical cyclones, droughts, and a series of extreme weather disasters. Accurately predicting ENSO in advance can greatly reduce the serious damage to human society, economy, and ecological environment. However, existing methods often neglect the data relation between geographical regions and meteorological factors, hindering the accuracy of ENSO prediction.Methods: To overcome this problem, we propose a residual network with geographical and meteorological attention to capture important geographical information and explore the spatio-temporal correlation of different meteorological factors. Specifically, we propose two main attention modules: (1) the Geographical Semantic Information Enhancement Module (GSIEM), which selectively attends to important geographical regions and filters out irrelevant noise through a spatial-axis attention map, and (2) the Meteorological Factors Discriminating Enhancement Module (MFDEM), which aims to learn the spatio-temporal dependency of different meteorological factors using a learnable channel-axis weight map. We then integrate our proposed two attention modules into the backbone using residual connection, enhancing the model's prediction ability.Results: We conducted extensive experimental comparisons and ablation studies to evaluate the performance of our proposed method. The results show that our method outperforms existing state-of-the-art methods in ENSO prediction, with a significant improvement in prediction accuracy.Discussion: Our proposed method effectively captures geographical and meteorological information, facilitating accurate ENSO prediction. The attention modules we proposed can effectively filter out irrelevant noise and learn the spatio-temporal dependency of different meteorological factors, contributing to the superior performance of our model. Overall, our study provides a novel approach for ENSO prediction and has great potential for practical applications.

Discontinuous muscle growth during molting is an important feature of Eriocheir sinensis. Molting is a physiological process completed by the cooperation of multiple organs. Signal transmission is critical for the accurate regulation of each step in molting. However, the knowledge of the signal transduction mechanism in the molting process of E. sinensis is presently very limited. In this work, the chromatin accessibility and gene expression of the muscle in E. sinensis in pre-molt (D) and post-molt (A) stages were sequenced by assay of transposase accessible chromatin sequencing (ATAC-seq) and RNA-seq, respectively. The differentially expressed genes (DEGs) in the muscle before and after molting were analyzed by combining ATAC-seq and RNA-seq, especially the G-protein coupled receptor (GPCR) genes in the process of signal transduction. The results showed that there were 616 common DEGs in ATAC-seq and RNA-seq in A vs. D stages, of which 538 were upregulated and 78 were downregulated. In the 19 DEGs included in the signaling transduction process, 13 were located in the GPCR signaling pathway and all were upregulated in A stages, which indicated that GPCRs play a leading role in muscle signal transmission during post-molt stage in molting. In these genes, the structure of the proteins encoded by 10 membrane-located genes with transmembrane activity was further analyzed. Six candidate GPCR genes were finally identified and further verified by real-time quantitative PCR (qRT-PCR). The GPCRs include metabotropic glutamate receptor 7, Mth-like 4, and Mth2 proteins. These results show the existence of GPCRs in the muscle of E. sinensis and, for the first time, found their dominant role in the signal transduction process during molting. It provides important clues for the study of muscle discontinuous growth and molting mechanism of E. sinensis.