ROS-induced moderate autophagy of haemocytes confers resistance of Mercenaria mercenaria to air exposure stress.

Air exposure (AE) is a significant environmental stressor that can lead to desiccation, hypoxia, starvation, and disruption of cellular homeostasis in marine bivalves. Autophagy is a highly conserved catabolic pathway that facilitates the degradation of damaged macromolecules and organelles, thereby supporting cellular stress responses. To date, autophagy-mediated resistance mechanisms to AE stress remain largely elusive in bivalves. In this study, we performed a multi-tool approach to investigate the autophagy-related physiological regulation in hard clams (Mercenaria mercenaria) under different duration of AE (T = 0, 1, 5, 10, 20, 30 days). We observed that autophagy of haemocytes was significantly activated on day 5. However, autophagy activity began to significantly decline from day 10 to day 30. Autophagy was significantly inhibited after antioxidant treatment, indicating that reactive oxygen species (ROS) was an endogenous inducer of autophagy. A significant decline in the survival rate of hard clams was observed after injection of ammonium chloride or carbamazepine during AE stress, suggesting that moderate autophagy was conducive for clam survival under AE stress. We also observed DNA breaks and high levels of apoptosis in haemocytes on day 10. Activation of apoptosis lagged behind autophagy, and the relationship between autophagy and apoptosis might shift from antagonism to synergy with the duration of stress. This study provides novel insights into the stress resistance mechanisms in marine bivalves.

RNA-Seq analysis and WGCNA reveal dynamic molecular responses to air exposure in the hard clam Mercenaria mercenaria.

Intertidal bivalves are constantly exposed to air due to daily and seasonal tidal cycles. The hard clam Mercenaria mercenaria is an economically important bivalve species and often subjected to air exposure for more than 10 days during long-distance transportation. Hard clam exhibits remarkable tolerance to air exposure. In this study, we performed RNA sequencing on hemocytes of M. mercenaria exposed to air for 0, 1, 5, 10, 20 and 30 days. The overall and dynamic molecular responses of hard clams to air exposure were revealed by different transcriptomic analysis strategies. As a result, most cytochrome P450 1A and 3A, and monocarboxylate transporter family members were up-regulated during air exposure. Additionally, the dominant molecular process in response to 5-d, 10-d, 20-d and 30-d air exposure was refolding of misfolded proteins in endoplasmic reticulum, lysosome-mediated degradation of phospholipids, protein metabolism and reorganization of cytoskeleton, and activation of anti-apoptotic process, respectively. Our results facilitated comprehensive understanding of the tolerance mechanisms of intertidal bivalves to air exposure.

Molecular evidence for the adaptive evolution in euryhaline bivalves.

Marine bivalves inhabiting intertidal and estuarine areas are frequently exposed to salinity stress due to persistent rainfall and drought. Through prolonged adaptive evolution, numerous bivalves have developed eurysalinity, which are capable of tolerating a wide range of salinity fluctuations through the sophisticated regulation of physiological metabolism. Current research has predominantly focused on investigating the physiological responses of bivalves to salinity stress, leaving a significant gap in our understanding of the adaptive evolutionary characteristics in euryhaline bivalves. Here, comparative genomics analyses were performed in two groups of bivalve species, including 7 euryhaline species and 5 stenohaline species. We identified 24 significantly expanded gene families and 659 positively selected genes in euryhaline bivalves. A significant co-expansion of solute carrier family 23 (SLC23) facilitates the transmembrane transport of ascorbic acids in euryhaline bivalves. Positive selection of antioxidant genes, such as GST and TXNRD, augments the capacity of active oxygen species (ROS) scavenging under salinity stress. Additionally, we found that the positively selected genes were significantly enriched in KEGG pathways associated with carbohydrates, lipids and amino acids metabolism (ALDH, ADH, and GLS), as well as GO terms related to transmembrane transport and inorganic anion transport (SLC22, CLCND, and VDCC). Positive selection of MCT might contribute to prevent excessive accumulation of intracellular lactic acids during anaerobic metabolism. Positive selection of PLA2 potentially promote the removal of damaged membranes lipids under salinity stress. Our findings suggest that adaptive evolution has occurred in osmoregulation, ROS scavenging, energy metabolism, and membrane lipids adjustments in euryhaline bivalves. This study enhances our understanding of the molecular mechanisms underlying the remarkable salinity adaption of euryhaline bivalves.

Autophagy contributes to increase the content of intracellular free amino acids in hard clam (Mercenaria mercenaria) during prolonged exposure to hypersaline environments.

Marine bivalves in intertidal zones and land-based seawater ponds are constantly subjected to a wide range of salinity fluctuations due to heavy rainfall, intense drought, and human activities. As osmoconformers, bivalves rely primarily on rapid release or accumulation of free amino acids (FAAs) for osmoregulation. Euryhaline bivalves are capable of withstanding hyposaline and hypersaline environments through regulation of physiology, metabolism, and gene expression. However, current understanding of the molecular mechanisms underlying osmoregulation and salinity adaptation in euryhaline bivalves remains largely limited. In this study, RNA-seq, WGCNA and flow cytometric analysis were performed to investigate the physiological responses of hard clams (Mercenaria mercenaria) to acute short-term hyposalinity (AL) and hypersalinity (AH), and chronic long-term hyposalinity (CL) and hypersalinity (CH) stress. We found that amino acids biosynthesis was significantly inhibited and aminoacyl-tRNA biosynthesis was augmented to decrease intracellular osmolarity during hyposaline exposure. Under CH, numerous autophagy-related genes (ATGs) were highly expressed, and the autophagy activity of gill cells were significantly up-regulated. A significant decrease in total FAAs content was observed in gills after NH4Cl treatment, indicating that autophagy was crucial for osmoregulation in hard clams during prolonged exposure to hypersaline environments. To prevent premature or unnecessary apoptosis, the expression of cathepsin L was inhibited under AL and AH, and inhibitors of apoptosis was augmented under CL and CH. Additionally, neuroendocrine regulation was involved in salinity adaption in hard clams. This study provides novel insights into the physiological responses of euryhaline marine bivalves to hyposaline and hypersaline environments.

Effect of heat and hypoxia stress on mitochondrion and energy metabolism in the gill of hard clam.

Aquatic animals suffer from heat and hypoxia stress more frequently due to global climate change and other anthropogenic activities. Heat and hypoxia stress can significantly affect mitochondrial function and energy metabolism. Here, the response and adaptation characteristics of mitochondria and energy metabolism in the gill of the hard clam Mercenaria mercenaria under heat (35 °C), hypoxia (0.2 mg/L), and heat plus hypoxia stress (35 °C, 0.2 mg/L) after 48 h exposure were investigated. Mitochondrial membrane potentials were depolarized under environmental stress. Mitochondrial fusion, fission and mitophagy played a key role in maintain mitochondrion function. The AMPK subunits showed different expression under environmental stress. Acceleration of enzyme activities (phosphofructokinase, pyruvate kinase and lactic dehydrogenase) and accumulation of anaerobic metabolites in glycolysis and TCA cycle implied that the anaerobic metabolism might play a key role in providing energy. Accumulation of amino acids might help to increase tolerance under heat and heat combined hypoxia stress. In addition, urea cycle played a key role in amino acid metabolism to prevent ammonia/nitrogen toxicity. This study improved our understanding of the mitochondrial and energy metabolism responses of marine bivalves exposed to environmental stress.

Genome-wide analysis of the hard clam mitogen-activated protein kinase kinase gene family and their transcriptional profiles under abiotic stress.

Mitogen-activated protein kinase kinase (MAPKK) was the hub component of the Mitogen-activated protein kinase (MAPK) signaling pathway and played an important role in the cellular response to environmental stress. In this study, we identified five MmMAPKK genes in hard clam Mercenaria mercenaria and found that all MmMAPKK genes contain a conserved protein kinase domain. The MmMAPKK genes derived from dispersed duplication were unevenly distributed in three chromosomes. Although the genome size was highly variable among different bivalve mollusks, the number of MAPKK genes was relatively stable. Phylogenetic analysis showed that bivalve MAPKK was divided into five clades, and amino acid sequences of MAPKK from the same clade consisted of similar conserved motifs. The syntenic analysis demonstrated that MmMAPKKs had the highest number of homologous gene pairs with Cyclina sinensis. MmMAPKKs were ubiquitously expressed in all examined tissues, and all MmMAPKK genes were highly expressed in the ovary. MmMAPKK genes showed stress-specific expression under envirionmental stress. MmMAPKK7 showed an upregulated in heat and heat plus hypoxia stress while MmMAPKK1 showed an upregulated in hypoxic stress groups. Dynamic changes of MmMAPKK7, MmMAPKK6 and MmMAPKK1 in hemocytes were observed in response to air exposure. MmMAPKK4 significantly downregulated after air exposure for five days. MmMAPKK7 and MmMAPKK6 might participate in adaptation to low salinity stress. Our results provided useful information about MAPKK and laid a foundation for further studies on MAPKK evolution in the bivalve.

Metabolic response of Mercenaria mercenaria under heat and hypoxia stress by widely targeted metabolomic approach.

In the context of global climatic changes, marine organisms have been exposed to environmental stressors including heat and hypoxia. This calls for the design of multi-stressors to uncover the impact of oceanic factors on aquatic organisms. So far, little is known about the metabolic response of marine organisms, especially bivalves, to the combined effects of heat and hypoxia. In this study, we employed widely targeted metabolomic analysis to study the metabolic response of gills in hard clam, a heat- and hypoxia-tolerant bivalve. A total of 810 metabolites were identified. Results showed that the heat group (HT) and heat plus hypoxia group (HL) had a higher number of differential metabolites than the hypoxia group (LO). Glycolysis was affected by the heat and heat plus hypoxia stress. Moreover, anaerobic metabolic biomarkers were accumulated marking the onset of anaerobic metabolism. Environmental stresses may affect Tricarboxylic acid (TCA) cycle. Accumulation of carnitine and glycerophospholipid may promote fatty acid ß oxidation and maintain cell membrane stability, respectively. The high content of oxidized lipids (i.e., Leukotriene) in HL and HT groups implied that the organisms were under ROS stress. The significantly differential metabolites of organic osmolytes and vitamins might relieve ROS stress. Moreover, accumulation of thermoprotective osmolytes (monosaccharide, Trimethylamine N-oxide (TMAO)) accumulation was helpful to maintain protein homeostasis. This investigation provided new insights into the adaptation mechanisms of hard clam to heat, hypoxia and combined stress at the metabolite level and highlighted the roles of molecules and protectants.

Metabolomics and biochemical assays reveal the metabolic responses to hypo-salinity stress and osmoregulatory role of cAMP-PKA pathway in Mercenaria mercenaria.

Hypo-salinity events frequently occur in marine ecosystem due to persistent rainfall and freshwater inflow, reducing the cytosol osmolarity and triggering cellular stress responses in aquatic organisms. Euryhaline bivalves have developed sophisticated regulatory mechanisms to adapt to salinity fluctuations over a long period of evolution. In this study, we performed multiple biochemical assays, widely targeted metabolomics, and gene expression analysis to investigate the comprehensive metabolic responses to hypo-salinity stress and osmoregulation mechanisms in hard clam Mercenaria mercenaria, which is a euryhaline bivalve species widely cultured in China. During hypo-salinity stress, increased vacuoles appeared in gill filaments. The Na+ and Cl- concentrations in gills significantly decreased because of the up-regulation of Na+/K+-ATPase (NKA) activity. The cAMP content dramatically decreased at 5 d post hypo-salinity stress. Meanwhile, the gene expression levels of adenylate cyclase, proteinkinase A, and sodium and calcium channel proteins were evidently down-regulated, suggesting that cAMP-PKA pathway was inhibited to prevent ambient inorganic ions from entering the gill cells. Antioxidant metabolites, such as serine and Tyr-containing dipeptides, were significantly up-regulated to resist oxidative stress. Glycerolipid metabolism was strengthened to stabilize membrane structure when hypo-salinity stress was prolonged to 5 days. At 1 d post hypo-salinity stress, an increase in alanine and lactate contents marked the initiation of anaerobic metabolism. Acylcarnitines accumulation indicated that fatty acids ß-oxidation was promoted to provide energy for osmoregulation. The potential biomarkers of hypo-salinity stress were identified in hard clams. This study provides novel insights into the metabolic regulatory mechanisms to hypo-salinity stress in euryhaline bivalves.

Depression promotes prostate cancer invasion and metastasis via a sympathetic-cAMP-FAK signaling pathway.

Depression drives cancer progression and induces poor clinical outcome. However, the mechanisms underlying depression and cancer outcomes are unclear. In this work, we investigated 98 prostate cancer patients and found that patients with high score of psychological depression were correlated with tumor invasion and metastasis. We found focal adhesion kinase (FAK) was increased in cancer patients with metastatic features and high score of depression. FAK knockdown completely blocked depression-promoted tumor invasion in orthotopic transplantation tumors. In Hi-myc mice and a murine model of depression, sympathetic activation was detected in the prostate tissue. Further we showed that FAK activation was dependent on a cAMP-PKA signaling pathway. Our results demonstrated that the activation of a sympathetic-FAK signaling pathway in prostate cancer patients with high degrees of depression facilitates tumor invasion. We suggest that blocking ß2AR with propranolol or inhibiting FAK activation with PF562 271 may be novel strategies for depressed patients with invasive prostate cancer.