Molecular pathways of osmoregulation in response to salinity stress in the gills of the scalloped spiny lobster (Panulirus homarus) within survival salinity.

Scalloped spiny lobster (Panulirus homarus) aquaculture is the preferred strategy to resolve the conflict between supply and demand for lobster. Environmental conditions, such as salinity, are key to the success of lobster aquaculture. However, physiological responses of P. homarus to salinity stress have not been well studied. This study investigated the gill histology, osmoregulation and gill transcriptome of the early juvenile P. homarus (weight 19.04 ± 3.95 g) cultured at salinity 28 (control), 18, and 38 for 6 weeks. The results showed that the gill filaments of P. homarus exposed to low salinity showed severe separation of the cuticle and epithelial cells due to water absorption and swelling, as well as the dissolution and thinning of the cuticle and the rupture of the septum that separates the afferent and efferent channels. The serum osmolarity of P. homarus varied proportionately with external medium salinity and remained consistently above ambient osmolarity. The serum Na+, Cl-, K+, and Mg2+ concentrations P. homarus exhibited a pattern similar to that of serum osmolality, while the concentration of Ca2+ remained unaffected at salinity 18 but significantly increased at salinity 38. Gill Na+/K+-ATPase activity of P. homarus increased (p < 0.05) under the both salinity stress. Salinity 18 significantly increased Glutamate dehydrogenase (GDH) and Glutamicpyruvic transaminase (GPT) activity in the hepatopancreas of P. homarus (p < 0.05). According to transcriptome analysis, versus control group (salinity 28), 929 and 1095 differentially expressed genes (DEGs) were obtained in the gills of P. homarus at salinity 18 and 38, respectively, with these DEGs were mainly involved in energy metabolism, transmembrane transport and oxidative stress and substance metabolism. In addition, the expression patterns of 8 key DEGs mainly related to amino acid metabolism, transmembrane transport and oxidative stress were verified by quantitative real-time PCR (RT-qPCR). The present study suggests that salinity 18 has a greater impact on P. homarus than salinity 38, and P. homarus demonstrates effective osmoregulation and handle with salinity fluctuations (18 to 38) through physiological and functional adaptations. This study provides an improved understanding of the physiological response strategies of P. homarus facing salinity stress, which is crucial for optimizing aquaculture practices for this species.

Developmental toxicity of the neonicotinoid pesticide clothianidin to the larvae of the crustacean Decapoda, Penaeus vannamei.

The developmental toxicity effects of neonicotinoid pesticides such as clothianidin have not been fully explored in agricultural applications. This is particularly noteworthy because such pesticides significantly impact the survival rates of invertebrates, with arthropod larvae being particularly vulnerable. This study aimed to address this research gap by specifically investigating the toxicological effects of clothianidin on the developmental stages of the larvae of the economically important aquaculture species Penaeus vannamei. In these experiments, shrimp eggs were exposed to seawater containing different concentrations of clothianidin beginning at N1, and each phase was observed and analyzed to determine its toxic impact on larval development. These results revealed that clothianidin induces an increase in deformity rates and triggers abnormal cell apoptosis. It also significantly reduced survival rates and markedly decreased body length and heart rate in the later stages of larval development (P3). Transcriptomic analysis revealed disruptions in larval DNA integrity, protein synthesis, and signal transduction caused by clothianidin. To survive prolonged exposure, larvae may attempt to maintain their viability by repairing cell structures and enhancing signal transduction mechanisms. This study offers the first empirical evidence of the toxicity of clothianidin to arthropod larvae, underscoring the impact of environmental pollution on aquatic health.

Next-generation neonicotinoid: The impact of cycloxaprid on the crustacean decapod Penaeus vannamei.

Cycloxaprid, a new neonicotinoid pesticide, poses ecological risks, particularly in aquatic environments, due to its unique action and environmental dispersal. This study investigated the ecotoxicological effects of various concentrations of cycloxaprid on Penaeus vannamei over 28 days. High cycloxaprid levels significantly altered shrimp physiology, as shown by changes in the hepatosomatic index and fattening. Indicators of oxidative stress, such as increased serum hemocyanin, respiratory burst, and nitric oxide, as well as decreased phenol oxidase activity, were observed. Additionally, elevated activities of lactate dehydrogenase, succinate dehydrogenase, and isocitrate dehydrogenase indicated disrupted energy metabolism in the hepatopancreas. Notably, analyses of the nervous system revealed marked disturbances in neural signaling, as evidenced by elevated acetylcholine, octopamine, and acetylcholinesterase levels. Transcriptomic analysis highlighted significant effects on gene expression and metabolic processes in the hepatopancreas and nervous system. This study demonstrated that cycloxaprid disrupts neural signaling and oxidative balance in P. vannamei, potentially affecting its growth, and provides key insights into its biochemical and transcriptomic toxicity in aquatic systems.

Safety evaluation and effects of dietary phlorotannins on the growth, health, and intestinal microbiota of Litopenaeus vannamei.

Phlorotannins are phenolic compounds with diverse biological activities, yet their efficacy in aquatic animals currently remains unclear. This investigation scrutinized the influence of phlorotannins on the growth, immunity, antioxidant capacity, and intestinal microbiota in Litopenaeus vannamei, concurrently evaluating the potential adverse effects of phlorotannins on L. vannamei. A base diet without phlorotannins supplementation was used as a control, and 4 groups of diets with different concentrations (0, 0.5, 1.0, 2.0 g kg-1) of phlorotannins were formulated and fed to juvenile shrimp (0.25 ± 0.01 g) for 60 days followed by a 24-h challenge with Vibrio parahaemolyticus with triplicate in each group. Compared with the control, dietary 2.0 g kg-1 phlorotannins significantly improved the growth of the shrimp. The activities of enzymes related to cellular immunity, humoral immunity, and antioxidants, along with a notable upregulation in the expression of related genes, significantly increased. After V. parahaemolyticus challenge, the cumulative survival rates of the shrimp demonstrated a positive correlation with elevated concentrations of phlorotannins. In addition, the abundance of Bacteroidetes and functional genes associated with metabolism increased in phlorotannins supplementation groups. Phlorotannins did not elicit any detrimental effects on the biological macromolecules or histological integrity of the hepatopancreas or intestines. Simultaneously, it led to a significant reduction in malondialdehyde content. All results indicated that phlorotannins at concentrations of 2.0 g kg-1 can be used as safe feed additives to promote the growth, stimulate the immune response, improve the antioxidant capacity and intestinal health of L. vannamei, and an protect shrimp from damage caused by oxidative stress.

Dinotefuran exposure alters biochemical, metabolomic, gut microbiome, and growth responses in decapoda pacific white shrimp Penaeus vannamei.

Dinotefuran, a neonicotinoid insecticide, may impact nontarget organisms such as Decapoda P. vannamei shrimp with nervous systems similar to insects. Exposing shrimp to low dinotefuran concentrations (6, 60, and 600 µg/L) for 21 days affected growth, hepatosomatic index, and survival. Biomarkers erythromycin-N-demethylase, alanine aminotransferase, and catalase increased in all exposed groups, while glutathione S-transferase is the opposite; aminopyrin-N-demethylase, malondialdehyde, and aspartate aminotransferase increased at 60 and 600 µg/L. Concentration-dependent effects on gut microbiota altered the abundance of bacterial groups, increased potentially pathogenic and oxidative stress-resistant phenotypes, and decreased biofilm formation. Gram-positive/negative microbiota changed significantly. Metabolite differences between the exposed and control groups were identified using mass spectrometry and KEGG pathway enrichment. N-acetylcystathionine showed potential as a reliable dinotefuran metabolic marker. Weighted correlation network analysis (WGCNA) results indicated high connectivity of cruecdysone in the metabolite network and significant enrichment at 600 µg/L dinotefuran. The WGCNA results revealed a highly significant negative correlation between two key metabolites, caldine and indican, and the gut microbiota within co-expression modules. Overall, the risk of dinotefuran exposure to non-target organisms in aquatic environments still requires further attention.

Effects of myo-inositol on growth and biomarkers of environmental stress and metabolic regulation in Pacific white shrimp (Litopenaeus vannamei) reared at low salinity.

This study explored the role of myo-inositol in alleviating the low salinity stress of White Shrimp (Litopenaeus vannamei). Juvenile shrimp (0.4 ± 0.02 g) in low salinity (salinity 3) water were fed diets with myo-inositol levels of 0, 272, 518, 1020 and 1950 mg/kg (crude protein is 36.82 %, crude lipid is 7.58 %), fed shrimp in seawater at a salinity of 25 were fed a 0 mg/kg myo-inositol diet as a control (Ctrl). The experiment was carried out in tanks (50 L) with satiety feeding, and the experiment lasted for 6 weeks. After sampling, the serum was used to measure immune function, the hepatopancreas homogenate was used to measure the antioxidant capacity and hepatopancreas damage state, the hepatopancreas was used for transcriptomics analysis, and the gills were used for qPCR to measure osmotic pressure regulation. The results showed that the final weight and survival of the shrimp in the 1020 mg/kg group increased significantly compared with those in the other low salinity groups, but the final weight and biomass increase were significantly lower than those in the Ctrl group. Dietary myo-inositol improved the antioxidant capacity of shrimp under low salinity. B-cell hyperplasia and hepatic duct damage were observed in the hepatopancreas in the 0 mg/kg group. The results of transcriptome analysis showed that myo-inositol could participate in the osmotic pressure regulation of shrimp by regulating carbohydrate metabolism, amino acid metabolism, lipid metabolism and other related genes. Myo-inositol significantly affected the expression of related genes in ion transporter and G protein-coupled receptor-mediated pathways. This study demonstrated that myo-inositol can not only act as an osmotic pressure effector and participate in the osmolar regulation of shrimp through the phosphatidylinositol signaling pathway mediated by G protein-coupled receptors but also relieve low salinity stress by mediating physiological pathways such as immunity, antioxidation, and metabolism in shrimp. The binomial regression analysis of biomass increases and survival showed that the appropriate amount of myo-inositol in the L. vannamei diet was 862.50-1275.00 mg/kg under low salinity.

Study on the osmotic response and function of myo-inositol oxygenase in euryhaline fish nile tilapia (Oreochromis niloticus).

To understand the role of myo-inositol oxygenase (miox) in the osmotic regulation of Nile tilapia, its expression was analyzed in various tissues. The results showed that the expression of miox gene was highest in the kidney, followed by the liver, and was significantly upregulated in the kidney and liver under 1 h hyperosmotic stress. The relative luminescence efficiency of the miox gene transcription starting site (-4,617 to +312 bp) under hyperosmotic stress was measured. Two fragments (-1,640/-1,619 and -620/-599) could induce the luminescence activity. Moreover, the -1,640/-1,619 and -620/-599 responded to hyperosmotic stress and high-glucose stimulation by base mutation, suggesting that osmotic and carbohydrate response elements may exist in this region. Finally, the salinity tolerance of Nile tilapia was significantly reduced after the knocking down of miox gene. The accumulation of myo-inositol was affected, and the expression of enzymes in glucose metabolism was significantly reduced after the miox gene was knocked down. Furthermore, hyperosmotic stress can cause oxidative stress, and MIOX may help maintain the cell redox balance under hyperosmotic stress. In summary, MIOX is essential in osmotic regulation to enhance the salinity tolerance of Nile tilapia by affecting myo-inositol accumulation, glucose metabolism, and antioxidant performance.NEW & NOTEWORTHY Myo-inositol oxygenase (MIOX) is the rate-limiting enzyme that catalyzes the first step of MI metabolism and determines MI content in aquatic animals. To understand the role of miox in the osmotic regulation of Nile tilapia, we analyzed its expression in different tissues and its function under hyperosmotic stress. This study showed that miox is essential in osmotic regulation to enhance the salinity tolerance of Nile tilapia by affecting myo-inositol accumulation, glucose metabolism, and antioxidant performance.

Quantitative proteomic and metabolomic profiling reveals different osmoregulation mechanisms of tilapia cells coping with different hyperosmotic stress.

This study aimed to investigate the different regulatory mechanisms of euryhaline fish under regular hyperosmotic and extreme hyperosmotic stress. The OmB (Oreochromis mossambicus brain) cells were exposed to three treatments: control, regular hyperosmotic stress and extreme hyperosmotic stress. After 12 h exposure, proteomics, metabolomics analyses and integrative analyses were explored. Both kinds of stress lead to lowering cell growth and morphology changes, while under regular hyperosmotic stress, the up-regulated processes related with compatible organic osmolytes synthesis are crucial strategy for the euryhaline fish cell line to survive; On the other hand, under extreme hyperosmotic stress, the processes related with cell apoptosis and cell cycle arrest are dominant. Furthermore, down-regulated pyrimidine metabolism and several ribosomal proteins partially participated in the lowered cell metabolism and increased cell death under both kinds of hyperosmotic stress. The PI3K-Akt and p53 signaling pathways were involved in the stagnant stage of cell cycles and induction of cell apoptosis under both kinds of hyperosmotic stress. However, HIF-1, FoxO, JAK-STAT and Hippo signaling pathways mainly contribute to disrupting the cell cycle, metabolism and induction of cell apoptosis under extreme hyperosmotic stress. SIGNIFICANCE: In the past, the research on fish osmoregulation mainly focused on the transcription factors and ion transporters of osmoregulation, the processes between osmotic sensing and signal transduction, and the associations between signaling pathways and regulation processes have been poorly understood. Investigating fish cell osmoregulation and potential signal transduction pathways is necessary. With the advancements in omics research, it is now feasible to investigate the relationship between environmental stress and molecular responses. In this study, we aimed to explore the signaling pathways and substance metabolism mode during hyper-osmoregulation in OmB cell line, to reveal the key factors that are critical to cell osmoregulation.

The interaction between lipid and vitamin D3 impacts lipid metabolism and innate immunity in Chinese mitten crabs Eriocheir sinensis.

As a fat-soluble vitamin, vitamin D3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D3: low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D3 [1α,25(OH)2D3] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH)2D3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH)2D3, mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder.

Evaluation of Dietary Essential Amino Acid Supplementation on Growth, Digestive Capacity, Antioxidant, and Intestine Health of the Juvenile Redclaw Crayfish, Cherax quadricarinatus.

The present study was an 8-week feeding trial investigating the effects of lysine and threonine supplementation in vegetable-based diets on growth, antioxidative capacity, and gut microbiota of juvenile redclaw crayfish, Cherax quadricarinatus (initial weight 11.52 ± 0.23 g). The lysine and threonine were supplemented to formulate five isonitrogenous (37%) and isolipidic (9%) diets containing 0% (control), 0.2% lysine (L0.2), 0.2% threonine (T0.2), 0.4% lysine (L0.4), and 0.4% threonine (T0.4), respectively. Compared to the control, weight gain rate (WGR) and specific growth rate (SGR) of C. quadricarinatus significantly increased with increasing dietary lysine and threonine supplementation from 0.2% to 0.4% (P < 0.05). Hepatopancreas trypsin activity significantly increased with increasing levels of lysine and threonine in diets (P < 0.05). However, the pepsin, lipase, and amylase activities were not affected by dietary levels of lysine and threonine (P > 0.05). Compared with the control, crayfish in T0.4 and L0.4 showed significantly higher glutathione peroxidase (GPx) activity (P < 0.05), lower alanine aminotransferase (ALT) activity, and lower malondialdehyde (MDA) content (P < 0.05). Supplementation with 0.4% lysine significantly changed the composition of the gut microbiota (P < 0.05), which showed a significantly increased relative abundance of Proteobacteria and decreased Firmicutes, Actinomycetes, and Pontomyces (P < 0.05). The PICRUSt analysis demonstrated that the abundance of the metabolism and cellular processes pathways in the L0.4 group were markedly decreased compared with the control (P < 0.05). Meanwhile, a tighter interaction of the microbiota community in crayfish was observed in the T0.4 experimental group. In conclusion, these results suggested that dietary supplementation with 0.4% threonine could significantly promote growth and improve microbial health in juvenile C. quadricarinatus.

Growth and Hepatopancreas Health of Juvenile Chinese Mitten Crab (Eriocheir sinensis) Fed Different Levels of Black Soldier Fly (Hermetia illucens) Larvae Meal for Fish Meal Replacement.

A 56-day feeding trial assessed the effects of black soldier fly larvae meal (BSFLM) on the growth performance and hepatopancreas health of juvenile Eriocheir sinensis. Six isoproteic and isolipidic diets with 0% (FM), 10% (BSFLM10), 20% (BSFLM20), 30% (BSFLM30), 40% (BSFLM40), or 50% (BSFLM50) replacement of fish meal by BSFLM were formulated. Compared to FM, replacing 10%-40% of fish meal with BSFLM did not significantly affect the weight gain rate (WGR) or specific growth rate (SGR), while BSFLM50 significantly decreased the WGR and SGR. Crabs fed BSFLM50 had significantly lower T-AOC activity than those fed other diets, and crabs fed BSFLM30, BSFLM40, or BSFLM50 had significantly lower activities of antioxidant enzymes (SOD and GSH-Px) in the hepatopancreas than those fed FM or BSFLM10. Compared to FM, BSFLM10, BSFLM20, and BSFLM30 did not affect the relative expression of genes related to the nonspecific immunity, while BSFLM40 and BSFLM50 upregulated the relative expression of these genes. Furthermore, histological analysis showed that the hepatopancreas was deformed in the BSFLM50 group, with widened lumens and loss of basal membrane integrity. In summary, BSFLM replacing 50% of fish meal reduced growth and structural damage to the hepatopancreas. An immune response was activated when the replacement level was over 30%. Therefore, the replacement level of dietary fish meal by BSFLM is recommended to be not more than 30% of the juvenile E. sinensis feed.

Identifying genes involved in the secretory physiological response to feeding in Pacific White Shrimp (Litopenaeus vannamei) using transcriptomics.

The physiological response to feeding is important for production aspects that include feed utilization and growth, and the responses require the action of numerous secretory factors. However, as an important aquaculture animal, the secretory response of Pacific White Shrimp (Litopenaeus vannamei) after feeding has not been comprehensively characterized. In this study, transcriptome analysis showed that 3172 differentially expressed genes were involved in the post-feeding response, including 289 new genes not annotated in the L. vannamei reference genome. Subsequently, 715 differentially expressed secretory reference genes and 18 new differentially expressed secretory genes were obtained through the identification of signal peptides in secreted proteins. Functional classification revealed that differentially expressed secretory genes were enriched in pathways pertaining to lipid metabolism (20 genes), carbohydrate metabolism (21 genes), glycan biosynthesis and metabolism (27 genes), digestive system (40 genes), and transport and metabolism (43 genes). The 14 pathways most enriched by differentially expressed secretory genes involved 83 genes, 71 of which encoded enzymes involved in food digestion and metabolism. Specific enzymes such as lipase 3-like and NPC intracellular cholesterol transporter 1-like in lipid metabolism, alpha-amylase-like and glucosylceramidase-like in carbohydrate metabolism, and cysteine proteinase 4-like and trypsin-1-like in the digestive system were found to be differentially expressed. Furthermore, we discovered a new gene, MSTRG.2504, that participates in the digestive system and carbohydrate metabolism. The study provides valuable insights into the secretory response (especially metabolism-related enzymes) to feeding in L. vannamei, uncovering the significant roles of both known and new genes. Furthermore, this study will improve our understanding of the feeding physiology of L. vannamei and provide a reference basis for further feeding endocrine research in the future.

Identification of key genes and molecular pathways associated with claw regeneration in mud crab (Scylla paramamosain).

The mud crab (Scylla paramamosain) possesses extensive regenerative abilities, enabling it to replace missing body parts, including claws, legs, and even eyes. Studying the genetic and molecular mechanisms underlying regenerative ability in diverse animal phyla has the potential to provide new insights into regenerative medicine in humans. In the present study, we performed mRNA sequencing to reveal the genetic mechanisms underlying the claw regeneration in mud crab. Several differentially expressed genes (DEGs) were expressed in biological pathways associated with cuticle synthase, collagen synthase, tissue regeneration, blastema formation, wound healing, cell cycle, cell division, and cell migration. The top GO enrichment terms were microtubule-based process, collagen trimer, cell cycle process, and extracellular matrix structural constituent. The most enriched KEGG pathways were ECM-receptor interaction and focal adhesion. The genes encoding key functional proteins, such as collagen alpha, cuticle protein, early cuticle protein, arthrodial cuticle protein, dentin sialophosphoprotein (DSPP), epidermal growth factor receptor (EGFR), kinesin family member C1 (KIFC1), and DNA replication licensing factor mcm2-like (MCM2) were the most significant and important DEGs suspected to participate in claw regeneration. The findings of this research offer a comprehensive and insightful understanding of the genetic and molecular mechanisms underlying claw regeneration in S. paramamosain. By elucidating the specific genes and molecular pathways implicated in this process, our study contributes significantly to the broader field of regenerative biology and offers potential avenues for further exploration in crustacean limb regeneration.

Effects of Dietary ß-Glucan Feeding Strategy on the Growth, Physiological Response, and Gut Microbiota of Pacific White Shrimp, Litopenaeus vannamei, under Low Salinity.

An eight-week feeding trial was conducted to investigate the effects of a dietary ß-glucan application strategy on the growth performance, physiological response, and gut microbiota of Pacific white shrimp (Litopenaeus vannamei) (0.49 ± 0.17 g) under low salinity. Six feeding strategies were established, including a continuous ß-glucan-free diet group (control), a continuously fed group with a 0.1% ß-glucan diet (T1), and groups with the following intermittent feeding patterns: 1 day of ß-glucan diet and 6 days of ß-glucan-free diet (T2), 2 days of ß-glucan diet and 5 days of ß-glucan-free diet (T3), 3 days of ß-glucan diet and 4 days of ß-glucan-free diet (T4), and 4 days of ß-glucan diet and 3 days of ß-glucan-free diet (T5) each week. No significant differences in growth performance among all the groups were found, although the condition factor was significantly higher in the T3 group than in the T1 and T5 groups (p < 0.05). The T-AOC and GPX activities were significantly lower in the T3 group than in the control group (p < 0.05). The MDA content was also significantly lower in the T2 group than in the T3 and T4 groups (p < 0.05). Additionally, the mRNA expression of the Pen3a gene was significantly upregulated in the hepatopancreas of the T4 group compared to the control and T5 groups (p < 0.05), and the Toll gene was also significantly upregulated in the T3 group compared to the T1 and T2 groups (p < 0.05). Dietary ß-glucan induced changes in the alpha diversity and composition of the gut microbiota in different feeding strategies. The beta diversity of the gut microbiota in the T2 group was significantly different from that in the control group. The results of a KEGG analysis showed that gut function in the carbohydrate metabolism, immune system, and environmental adaptation pathways was significantly enhanced in the T3 group. These findings provide evidence that the intermittent feeding strategy of ß-glucan could alleviate immune fatigue, impact antioxidant ability, and change gut microbiota composition of L. vannamei under low salinity.

Identification of key overlapping DEGs and molecular pathways under multiple stressors in the liver of Nile tilapia (Oreochromis niloticus).

The identification of key genes and molecular pathways that are involved in the response to stressors is crucial for controlling stress in fish and sustainable aquaculture. Environmental stressors can induce stress responses in aquatic animals, resulting in compromised immune function, inhibited growth, and increased mortality rates. mRNA-seq analysis provides a powerful tool to identify key genes and pathways associated with stress response. In the present study, mRNA-seq analysis was employed to identify key overlapping differentially expressed genes (DEGs) and molecular pathways under salinity, nitrite, copper, and pH stress in the liver of Nile tilapia (Oreochromis niloticus). The pathways associated with the immune response, oxygen transport, homeostasis, and oxidative stress were enriched across all stressors. The top KEGG pathways were complement and coagulation cascades, PPAR signaling pathway, and cardiac muscle contraction. The top GO enrichment terms were oxidoreductase activity, aerobic respiration, endopeptidase inhibitor activity, endopeptidase regulator activity, heme binding, and iron ion binding. The complement genes (C3, C4, C5, factor B, and factor H), alpha-2-macroglobulin (A2M), hemoglobin subunit epsilon (HBE), hemoglobin subunit alpha (HBA), coagulation factor genes (XI and X) and the cytochrome c oxidase (COX) gene family (cox1, cox2, cox3, cytochrome P450) were identified as key shared genes across multiple stressors. The discovery of these genes and molecular pathways provided a better understanding of the molecular mechanism underlying the stress response in Nile tilapia. The results of the present study can facilitate the development of stress management strategies in Nile tilapia.

Reducing Dietary Protein Content by Increasing Carbohydrates Is More Beneficial to the Growth, Antioxidative Capacity, Ion Transport, and Ammonia Excretion of Nile Tilapia (Oreochromis niloticus) under Long-Term Alkalinity Stress.

Alkalinity stress is the main stress experienced by aquatic animals in saline-alkali water, which hinders the aquaculture development and the utilization of water resources. The two-factor (2 × 3) test was adopted to study the influence of dietary protein to carbohydrate ratios on the energy metabolism of Nile tilapia (Oreochromis niloticus) under different alkalinity stress levels. Three diets with different protein-carbohydrate ratios (P27/C35, P35/C25, and P42/C15) were fed to fish cultured in freshwater (FW, 1.3 mmol/L carbonate alkalinity) or alkaline water (AW, 35.7 mmol/L carbonate alkalinity) for 50 days. Ambient alkalinity decreased tilapia growth performance. Although ambient alkalinity caused oxidative stress and enhanced ion transport and ammonia metabolism in tilapia, tilapia fed the P27/C35 diet showed better adaptability than fish fed the other two diets in alkaline water. Further metabolomic analysis showed that tilapia upregulated all the pathways enriched in this study to cope with alkalinity stress. Under alkalinity stress, tilapia fed the P27/C35 diet exhibited enhanced pyruvate metabolism and purine metabolism compared with tilapia fed the P42/C15 diet. This study indicated that ambient alkalinity could significantly decrease growth performance and cause oxidative stress and osmotic regulation. However, reducing dietary protein content by increasing carbohydrates could weaken stress and improve growth performance, ion transport, and ammonia metabolism in tilapia under long-term hyperalkaline exposure.

Adverse effects of thiamethoxam on the behavior, biochemical responses, hepatopancreas health, transcriptome and intestinal flora of juvenile Chinese mitten crab (Eriocheir sinensis).

Frequent detection of thiamethoxam in global surface waters has provoked great concern in environmental safety, as thiamethoxam exhibits high toxicity to aquatic arthropods. However, little systematic investigation has been conducted on the chronic toxicity of thiamethoxam to crustaceans. This study exposed Eriocheir sinensis to thiamethoxam (0, 0.5, 5 and 50 µg/L) in water for 28 days. No significant difference in mortality was observed among all groups. A high concentration of thiamethoxam (50 µg/L) impaired the righting ability of E. sinensis. Thiamethoxam significantly increased antioxidant enzyme activities (superoxide dismutase, total antioxidant capacity and glutathione peroxidase) and malondialdehyde levels. Simultaneously, detoxification enzyme activities (aminopyrine N-demethylase, erythromycin N-demethylase and glutathione-S-transferase) increased under chronic thiamethoxam stress. In addition, thiamethoxam caused immune and hepatopancreas damage. Moreover, thiamethoxam induced intestinal flora dysbiosis by altering the microbiome structure. The reduced complexity of the gut microbiota further illustrated that thiamethoxam could disrupt the stability of the microbiota ecological network. The transcriptomic results revealed that the number of downregulated DEGs increased in a dose-dependent manner, and most downregulated DEGs were enriched in energy metabolism-related pathways. These results indicate that thiamethoxam can adversely affect the crab behavior, biochemistry, intestinal microflora and transcriptomic responses.

Growth, Biochemical Characteristics, Flesh Quality, and Gut Microbiota of the Pacific White Shrimp (Penaeus vannamei) Fed a Defatted Superworm (Zophobas atratus) Larvae Meal.

This study evaluated the effects of defatted superworm (Zophobas atratus) larvae meal (DBWLM) as an alternative protein ingredient for juvenile Pacific white shrimp (Penaeus vannamei). Six isonitrogenous and isolipidic experimental diets were characterized by replacing 0%, 15%, 30%, 45%, 60%, and 75% fish meal (DBWLM0, DBWLM15, DBWLM30, DBWLM45, DBWLM60, and DBWLM75, respectively) with DBWLM on a w/w basis and feeding them to juvenile shrimp (0.34 ± 0.04 g) for 56 days. The results showed that the replacement of up to 75% fish meal by DBWLM had no negative effect on the growth performance of P. vannamei. The survival of shrimp in the DBWLM30 group was the highest, and the weight gain, specific growth rate, feed conversion ratio, condition factor, and apparent digestibility coefficients of dry matter in the DBWLM15 group were the highest. The substitution of DBWLM for fish meal significantly increased the elasticity of flesh, improved the total content of umami amino acids in flesh (aspartic acid, glutamic acid, glycine, and alanine), promoted lipid metabolism in shrimp, and reduced serum lipid levels. With the increase in DBWLM level, serum acid phosphatase, alkaline phosphatase activity, and intestinal inflammatory gene expression (IGF-1 and IL-6) were inhibited, malondialdehyde content decreased, and total antioxidant capacity level and superoxide dismutase activity increased significantly. Histological sections of the hepatopancreas showed that when 60% or more fish meal was replaced, the hepatopancreas atrophied and had irregular lumen distortion, but the cell membrane was not damaged. Microbiome analysis showed that the abundance of Bacteroidetes and Firmicutes increased and the abundance of Proteobacteria decreased in the DBWLM replacement group, and it was rich in "metabolism"-related functional pathways. It is worth mentioning that the expression of amino-acid-related enzymes was upregulated in the DBWLM15 and DBWLM30 groups, and the DBWLM75 group inhibited the biosynthesis of steroids and hormones. To conclude, the replacement of 15%-45% fish meal with DBWLM can result in better growth and immune status, improved meat elasticity, and reduced inflammation in P. vannamei. However, it is recommended that the replacement level should not exceed 60%, otherwise it will cause atrophy of hepatopancreas cells.

Caspar negatively regulates anti-bacterial immunity by controlling the nuclear translocation of Relish in Chinese mitten crab (Eriocheir sinensis).

Caspar, a homolog of the Fas-associated factor 1 (FAF1) family, contains an N-terminal ubiquitin interaction domain, a ubiquitin-like self-association domain, and a C-terminal ubiquitin regulatory domain. Caspar has been reported to be involved in the antibacterial immunity of Drosophila, which is unclear whether it is involved in the antibacterial immune process of crustaceans. In this article, we identified a Caspar gene in Eriocheir sinensis and named it EsCaspar. EsCaspar positively respond to bacterial stimulation and downregulate the expression of certain associated antimicrobial peptides by inhibiting the nuclear translocation of EsRelish. Thus, EsCaspar might be a suppressor of the immune deficiency (IMD) pathway that prevents over-activation of the immune system. Indeed, excess EsCaspar protein in crabs reduced resistance to bacterial infection. In conclusion, EsCaspar is a suppressor of the IMD pathway in crabs that plays a negative regulatory role in antimicrobial immunity.

Comprehensive transcriptional and metabolomic analysis reveals the neuroprotective mechanism of dietary gamma-aminobutyric acid response to hypoxic stress in the Chinese mitten crab (Eriocheir sinensis).

Hypoxia is one of the serious stress challenges that aquatic animals face throughout their life. Our previous study found that hypoxia stress could induce neural excitotoxicity and neuronal apoptosis in Eriocheir sinensis, and observed that gamma-aminobutyric acid (GABA) has a positive neuroprotective effect on juvenile crabs under hypoxia. To reveal the neuroprotective pathway and metabolic regulatory mechanism of GABA in E. sinensis exposed to hypoxia stress, an 8-week feeding trial and acute hypoxia challenge were performed. Subsequently, we performed a comprehensive transcriptomic and metabolomic analysis of the thoracic ganglia of juvenile crabs. Differential genes and differential metabolites were co-annotated to 11 KEGG pathways, and further significant analysis showed that only the sphingolipid signaling pathway and the arachidonic acid metabolism pathway were significantly enriched. In the sphingolipid signaling pathway, GABA treatment significantly increased long-chain ceramide content in thoracic ganglia, which exerted neuroprotective effects by activating downstream signals to inhibit hypoxia-induced apoptosis. Moreover, in the arachidonic acid metabolism pathway, GABA could increase the content of neuroprotective active substances and reduce the content of harmful metabolites by regulating the metabolism of arachidonic acid for inflammatory regulation and neuroprotection. Furthermore, the decrease of glucose and lactate levels in the hemolymph suggests the positive role of GABA in metabolic regulation. This study reveals the neuroprotective pathways and possible mechanisms of GABA in juvenile E. sinensis exposed to hypoxia stress and inspires the discovery of new targets for improving hypoxia tolerance in aquatic animals.