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.

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.

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.

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.

Identification of key immune and stress related genes and pathways by comparative analysis of the gene expression profile under multiple environmental stressors in pacific white shrimp (Litopenaeus vannamei).

Water salinity, pH, and nitrite concentration are considered environmental factors affecting the growth rate, survival, health, and physiological conditions of aquatic animals. The identification of key genes that are involved in the response to environmental stressors is essential for controlling stress in aquatic animals and sustainable aquaculture. In this study, RNA sequencing was performed to identify the differentially expressed genes (DEGs) and biological pathways that are involved in the response of the hepatopancreas to environmental stressors, including low salinity stress, nitrite stress, low pH stress, and high pH stress. The DEGs were enriched in biological pathways related to immune response, energy metabolism, oxidative stress response, hemostasis, and enzymatic activity of the hepatopancreas. In addition to the identification of DEGs related to each stressor, some DEGs were found to be expressed among all groups. The most important overlapping DEGs under multiple stressors were juvenile hormone esterase-like protein 2 (JHE-like), myosin light chain, C-type lectin 2, myosin-9-like, anti-lipopolysaccharide factor 1 (ALF-1), peroxisomal acyl-coenzyme An oxidase 1-like (ACX1), hepatic lectin-like, venom phosphodiesterase 2-like, hemolymph clottable protein-like (CP), cathepsin L, and Ras-like protein 2. The results of the present study provide additional information regarding the transcriptional response of the hepatopancreas to low salinity, nitrite, low pH, and high pH stress. Moreover, the discovery of several overlapping DEGs among different stressors provided a better understanding of the molecular function of the hepatopancreas.

Effects of Sulfamethoxazole and Florfenicol on Growth, Antioxidant Capacity, Immune Responses and Intestinal Microbiota in Pacific White Shrimp Litopenaeus vannamei at Low Salinity.

Antibiotic residue may pose a serious risk to aquaculture, and the culture of Litopenaeus vannamei in a low-salinity environment is a growing trend over the world. Here, we aimed to understand the combined effect of low salinity and sulfamethoxazole (SMZ) and florfenicol (FLO) antibiotics on L. vannamei. The growth performance, immune functions, antioxidant capacity and intestinal microbiota were investigated. Compared with the control group, the weight gain and survival rate significantly decreased (p < 0.05) in shrimp after they were exposed to low-salinity (salinity 3) water and the mixture of antibiotics and low-salt conditions for 28 days. The antioxidant activities of SOD and T-AOC, shown at low salinity and in the higher concentration of the SMZ treatment group (SMZH), were significantly decreased, while the GST activity was significantly increased in each treatment group in comparison with the control group. The expression of immune-related genes, including TOLL, LvIMD, PPO and HSP, in the low concentration of the SMZ treatment group (SMZL) was higher than that in the other groups. The diversity of intestine microbiota was disturbed with a lower Shannon index in the low-salinity and SMZH groups, and a higher Simpson index in the SMZH group. Proteobacteria, Actinobacteria and Bacteroidetes were the dominant phyla in the gut of L. vannamei. At the genus level, Microbacterium, Shewanella, Aeromonas, Acinetobacter, Gemmobacter, Paracoccus and Lysobacter were significantly decreased in the low-salinity group. However, the abundance of opportunistic pathogens belonging to the genus Aeromonas in the FLO group was increased. The predicted microbe-mediated functions showed that the pathway for "amino acid metabolism" and "replication and repair" was significantly inhibited in both the low-salinity and antibiotic-exposed groups. All the findings in this study indicate that the combined effect of antibiotics and low salinity on L. vannamei negatively impacted the physiological and intestinal microbiota functions.

Dietary selenium supplementation alleviates low salinity stress in the Pacific white shrimp Litopenaeus vannamei: growth, antioxidative capacity and hepatopancreas transcriptomic responses.

Se is an essential trace element associated with animal growth and antioxidant and metabolic processes. However, whether Se, especially organic Se with higher bioavailability, can alleviate the adverse effects of low salinity stress on marine economic crustacean species has not been investigated. Accordingly, juvenile Pacific white shrimp (Litopenaeus vannamei) were reared in two culture conditions (low and standard salinity) fed diets supplemented with increasing levels of l-selenomethionine (0·41, 0·84 and 1·14 mg/kg Se) for 56 d, resulting in four treatments: 0·41 mg/kg under standard seawater (salinity 31) and 0·41, 0·84 and 1·14 mg/kg Se under low salinity (salinity 3). The diet containing 0·84 mg/kg Se significantly improved the survival and weight gain of shrimp under low salinity stress and enhanced the antioxidant capacity of the hepatopancreas. The increased numbers of B and R cells may be a passive change in hepatopancreas histology in the 1·14 mg/kg Se group. Transcriptomic analysis found that l-selenomethionine was involved in the regulatory pathways of energy metabolism, retinol metabolism and steroid hormones. In conclusion, dietary supplementation with 0·84 mg/kg Se (twice the recommended level) effectively alleviated the effects of low salinity stress on L. vannamei by regulating antioxidant capacity, hormone regulation and energy metabolism.

Acute thiamethoxam exposure induces hepatotoxicity and neurotoxicity in juvenile Chinese mitten crab (Eriocheir sinensis).

The similar nervous system structure between crustaceans and insects and the high-water solubility of thiamethoxam can lead to the more severe toxicity of thiamethoxam to crustaceans. However, the effects of thiamethoxam on crustaceans are unclear. Therefore, a 96-h acute toxicity test was performed to explore the hepatotoxicity and neurotoxicity effects of thiamethoxam on Chinese mitten crab (Eriocheir sinensis) at concentrations 0 µg/L, 150 µg/L and 300 µg/L. The antioxidant and detoxification systems (including phases I and II) were significantly activated after exposure of juvenile crabs to thiamethoxam for 24 h in 300 µg/L group, whereas the toxic activation effect in 150 µg/L group was delayed. Moreover, a similar pattern was observed for the transcription levels of immune-related genes. Further analysis of inflammatory signaling pathway-related genes showed that thiamethoxam exposure with 300 µg/L for 24 h may induce a pro-inflammatory response through the NF-κB pathway. In contrast, the gene expression levels in 150 µg/L group were significantly upregulated compared with 0 µg/L group after 96 h. In addition, although the acute exposure of 150 µg/L thiamethoxam did not seem to induce significant neurotoxicity, the acetylcholinesterase activity was significantly decreased in 300 µg/L group after thiamethoxam exposure for 96 h. Correspondingly, thiamethoxam exposure with 300 µg/L for 24 h resulted in significantly downregulated transcriptional levels of synaptic transmission-related genes (e.g. dopamine-, gamma-aminobutyric acid- and serotonin-related receptors). Therefore, thiamethoxam may be harmful and cause potential toxic threats such as neurotoxicity and metabolic damage to crustaceans.

Growth, Metabolite, Antioxidative Capacity, Transcriptome, and the Metabolome Response to Dietary Choline Chloride in Pacific White Shrimp Litopenaeus vannamei.

To determine the response of Pacific white shrimp Litopenaeus vannamei to different levels of dietary choline, juvenile white shrimp (1.75 ± 0.09 g) were fed six semi-purified diets supplemented with 0 (control), 2000, 4000, 6000, 8000, and 12,000 mg/kg choline chloride for eight weeks. Growth performance, whole-body composition, serum characteristics and hepatopancreatic antioxidant indexes were evaluated. Meanwhile, serum metabolome and hepatopancreas transcriptome were performed to examine the overall difference in metabolite and gene expression. The weight gain, survival, specific growth rate, condition factor and hepatosomatic index were not affected by dietary choline levels. The shrimp fed 6000 mg/kg dietary choline chloride gained the maximal whole-body crude protein, which was significantly higher than that of shrimp fed with 12,000 mg/kg dietary choline. Serum total cholesterol of shrimp fed 6000 mg/kg dietary choline was higher than that in shrimp fed 4000 mg/kg choline. Dietary choline significantly decreased malondialdehyde content, superoxide dismutase, and glutathione peroxidase activities in shrimp hepatopancreas. Compared with the shrimp fed 6000 mg/kg dietary choline chloride, the glycerophospholipid metabolism pathway was significantly enriched in the shrimp fed 0 mg/kg dietary choline chloride, and the choline content and bile salt-activated lipase-like expression were upregulated. The expression of trypsin-1-like in protein digestion and absorption pathway was significantly downregulated in the shrimp fed 12,000 mg/kg dietary choline chloride. Apolipoprotein D might be a potential biomarker in shrimp, and dietary choline played an important role in lipid metabolism, especially in the reduction of oxidative damage in L. vannamei. Based on the results of weight gain and degree of oxidative damage, 1082 mg/kg dietary choline could meet the growth requirement of L. vannamei, but 2822 mg/kg dietary choline was needed to reduce peroxidation damage.

Growth and health status of Pacific white shrimp, Litopenaeus vannamei, exposed to chronic water born cobalt.

Cobalt (Co) is an important component of vitamin B12, but is toxic to aquatic animals at a high level. In this study, the Pacific white shrimp, Litopenaeus vannamei were exposed to three Co concentrations (0, 100, and 1000 µg/L) for 4 weeks. The survival and condition factor in shrimp exposed to the Co treatments were not different from the control, but the shrimp exposed to 100 µg Co/L gained more weight than in other two groups, and the shrimp exposed to 1000 µg Co/L gained less weight than in other groups. The SOD and GSH-PX activities were higher in shrimp exposed to 100 µg Co/L, but lower in the shrimp exposed to 100 µg Co/L compared with the control, respectively. The MDA contents in the hepatopancreas decreased in the 100 µg Co/L, but increased in the 1000 µg Co/L. The serum lysozyme decreased with ambient cobalt, was lower in the shrimp exposed to 1000 µg Co/L than in other two groups. The expression of C-type lectin 3 was down-regulated by Co concentrations. The Toll and immune deficiency in shrimp exposed to 100 µg Co/L was higher than in other two groups. The mucin-1 was lower in the 1000 µg Co/L group than in other two groups, but mucin-2 and mucin-5AC were higher in the 1000 µg Co/L group than in the control. With increasing Co concentration, Shannon and Simpson indexes of the intestinal microbial communities were decreased. The abundance of pathogenic bacteria (Ruegeria and Vibrio) increased in both Co groups. This study indicates that chronic exposure to waterborne cobalt could affect growth, cause oxidative stress, stimulate the immune response, damage intestinal histology, and reshape intestinal microbiota community L. vannamei.