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.

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.

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.

Changes in transcriptome regulations of a marine rotifer Brachionus plicatilis under methylmercury stress.

Mercury (Hg), a heavy metal pollutant worldwide, can be transformed into methylmercury (MeHg) by various aquatic microorganisms in water, thus accumulating along the aquatic food chain and posing a particular challenge to human health. Zooplankton plays a crucial role in aquatic ecosystems and serves as a major component of the food chain. To evaluate the effects of MeHg on the rotifer Brachionus plicatilis and reveal the underlying mechanism of these effects, we exposed B. plicatilis to MeHg by either direct immersion or by feeding with MeHg-poisoned Chlorella pyrenoidesa, respectively, and conducted a transcriptomic analysis. The results showed that B. plicatilis directly exposed to MeHg by immersion showed significant enrichment of the glutathione metabolism pathway for detoxification of MeHg. In addition, the exposure to MeHg by feeding induced a significant enrichment of lysosome and notch signaling pathways of rotifers, supporting the hypothesis that MeHg can induce autophagy dysfunction in cells and disturb the nervous system of rotifers. In two different routes of MeHg exposure, the pathway of cytochrome P450 in rotifers showed significant enrichment for resisting MeHg toxicity. Our results suggest further studies on the potential mechanism and biological responses of MeHg toxicity in other links of the aquatic food chain.

The male and female genomes of golden pompano (Trachinotus ovatus) provide insights into the sex chromosome evolution and rapid growth.

INTRODUCTION: Golden pompano (Trachinotus ovatus) is economically significant important for offshore cage aquaculture in China and Southeast Asian countries. Lack of high-quality genomic data and accurate gene annotations greatly restricts its genetic breeding progress. OBJECTIVES: To decode the mechanisms of sex determination and rapid growth in golden pompano and facilitate the sex- and growth-aimed genetic breeding. METHODS: Genome assemblies of male and female golden pompano were generated using Illumina, PacBio, BioNano, genetic maps and Hi-C sequencing data. Genomic comparisons, whole genome re-sequencing of 202 F1 individuals, QTL mapping and gonadal transcriptomes were used to analyze the sex determining region, sex chromosome evolution, SNP loci, and growth candidate genes. Zebrafish model was used to investigate the functions of growth candidate gene. RESULTS: Female (644.45 Mb) and male (652.12 Mb) genomes of golden pompano were assembled and annotated at the chromosome level. Both genomes are highly conserved and no new or highly differentiated sex chromosomes occur. A 3.5 Mb sex determining region on LG15 was identified, where Hsd17b1, Micall2 and Lmx1a were putative candidates for sex determination. Three SNP loci significantly linked to growth were pinpointed, and a growth-linked gene gpsstr1 was identified by locus BSNP1369 (G â†’ C, 17489695, Chr23). Loss of sstr1a (homologue of gpsstr1) in zebrafish caused growth retardation. CONCLUSION: This study provides insights into sex chromosome evolution, sex determination and rapid growth of golden pompano.

Metabolic pathways of methylmercury in rotifer Brachionus plicatilis.

Methylmercury (MeHg) readily accumulates in aquatic organisms while transferring and amplifying in the aquatic food chains. This study firstly explores the in vivo accumulation sites and metabolic regulation of MeHg in the rotifer Brachionus plicatilis by aggregation-induced emission fluorogen (AIEgen) and metabolomics. Fluorescent image analysis by AIEgen showed that MeHg in B. plicatilis mainly occured in the ciliary corona, esophagus, mastax, stomach and intestine in the direct absorption group. In the other group, where B. plicatilis were indirectly supplied with MeHg via food intake, the accumulation of MeHg in the rotifer occurred in the ciliary corona, various digestive organs, and the pedal gland. However, the MeHg accumulated in the rotifer is difficult to metabolize outside the body. Metabolomics analysis showed that the significant enrichment of ABC transporters was induced by the direct exposure of rotifers to dissolved MeHg. In contrast, exposure of rotifers to MeHg via food intake appeared to influence carbon, galactose, alanine, aspartate and glutamate metabolisms. Besides, the disturbed biological pathways such as histidine metabolism, beta-alanine metabolism and pantothenate and CoA biosynthesis in rotifers may be associated with L-aspartic acid upregulation in the feeding group. The significant enrichment of ABC transporters and carbon metabolism in rotifers may be related to the accumulation of MeHg in the intestine of rotifers. In both pathways of MeHg exposure, the arginine biosynthesis and metabolism of rotifers were disturbed, which may support the hypothesis that rotifers produce more energy to resist MeHg toxicity. This study provides new insight into the accumulation and toxicity mechanisms of MeHg on marine invertebrates from the macro and micro perspectives.

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.

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.

Dietary Methionine Level Impacts the Growth, Nutrient Metabolism, Antioxidant Capacity and Immunity of the Chinese Mitten Crab (Eriocheir sinensis) under Chronic Heat Stress.

This study examined whether diets with high dietary methionine levels could alleviate chronic heat stress in Chinese mitten crab Eriocheir sinensis. Crabs were fed three dietary methionine levels of 0.49%, 1.29% and 2.09% for six weeks. The analyzed methionine concentration of diets was 0.48%, 1.05% and 1.72%, respectively. Crabs were fed three different supplemental concentrations of dietary methionine at 24 °C and 30 °C, respectively. The trial was divided into six groups with five replicates in each group, and 40 juvenile crabs (initial average weight 0.71 ± 0.01 g) in each replicate. During the trial, crabs were fed twice daily (the diet of 4% of the body weight was delivered daily). The effects of dietary methionine level on nutrient metabolism, antioxidant capacity, apoptosis factors and immunity were evaluated at a normal water temperature of 24 °C and high temperature of 30 °C. Feed conversion ratio decreased under chronic heat stress. Chronic heat stress increased weight gain, specific growth rate, molting frequency, and protein efficiency ratio. The survival of crabs decreased under chronic heat stress, whereas a high level of dietary methionine significantly improved survival. Chronic heat stress induced lipid accumulation and protein content reduction. The high-methionine diet decreased lipid in the body and hepatopancreas, but increased protein in the body, muscle and hepatopancreas under chronic heat stress. Simultaneously, the high dietary methionine levels mitigated oxidative stress by reducing lipid peroxidation, restoring the antioxidant enzyme system, decreasing apoptosis and activating immune function under chronic heat stress. This study suggests that supplementing 1.72% dietary methionine could alleviate the adverse effects of a high water temperature in E. sinensis farming.

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.

Methylmercury bioaccumulation in water flea Daphnia carinata by AIEgen.

Mercury ion (Hg2+) is a toxic heavy metal ion and Hg2+ is convertible to methylmercury (MeHg) by many aquatic microorganisms, leading to bioaccumulation and biomagnification in aquatic organisms, which can interfere with brain development and function in humans. This study employs a newly developed AIEgen (Aggregation-induced emission fluorogen) to quantify and visualise the process of MeHg bioaccumulation in vivo on the species of water flea Daphnia carinata. Two approaches to MeHg absorption were taken, either by direct incubation in a MeHg solution or by indirect consumption of algae contaminated with MeHg. We analysed the relationship between the ratio of photoluminescence (PL) intensities (I585/I480) and MeHg concentration (CMeHg) and generated a master curve for determining MeHg concentration based on the measurement of PL intensities. Fluorescent image analysis showed the occurrence of MeHg in D. carinata to be mainly in the compound eyes, optic nerve and carapace. This study indicates that MeHg absorption can be quantified and visualised in the body of zooplankton, and the MeHg transfer to zooplankton is more likely through direct exposure than via indirect food intake. The accumulation of MeHg in the eye and the nervous system could be the cause of the high mortality of D. carinata exposed to MeHg in water.

Gamma-aminobutyric acid enhances hypoxia tolerance of juvenile Chinese mitten crab (Eriocheir sinensis) by regulating respiratory metabolism and alleviating neural excitotoxicity.

With climate change and intensive aquaculture development, environmental hypoxia in aquaculture water has become a common challenge for many aquatic species. Therefore, it is crucial to improve the hypoxic tolerance of animals through nutritional strategies. This study explored the positive role of dietary gamma-aminobutyric acid (GABA) supplementation in enhancing hypoxia tolerance of juvenile Eriocheir sinensis through respiratory regulation and alleviation of hypoxia-induced neural excitotoxicity. Acute hypoxia stress significantly up-regulated the mRNA expression level of hypoxia-inducible factor 1α, oxygen consumption rate and anaerobic respiratory metabolism-related enzyme activities. On the other hand, aerobic respiratory metabolism-related enzyme activities were significantly decreased. However, dietary GABA supplementation remodeled the respiratory metabolism pattern of juvenile crabs exposed to hypoxia stress. In addition, acute hypoxic stress significantly increased the contents of free glutamate and GABA in the nervous tissue. The expression levels of N-Methyl-d-aspartate-related receptor genes and calcium-dependent degradation enzyme-related genes were significantly up-regulated. Similarly, neuronal apoptosis rates, expression levels of apoptosis-related genes, and vesicular glutamate transporter genes were also significantly increased. The high-affinity neuronal glutamate transporter decreased significantly in the crabs exposed to hypoxia stress. However, dietary GABA supplementation could effectively prevent acute hypoxia stress-induced neural excitotoxicity. Furthermore, dietary GABA could significantly improve the redox status in vivo exposed to hypoxia stress. In conclusion, acute hypoxia stress can affect respiratory metabolism and redox state and induce neural excitotoxicity in juvenile E. sinensis. GABA supplementation could improve hypoxia tolerance through multiple physiological regulation pathways.

New insights into the influence of myo-inositol on carbohydrate metabolism during osmoregulation in Nile tilapia (Oreochromis niloticus).

A two-factor (2 × 3) orthogonal test was conducted to investigate the effects of dietary myo-inositol (MI) on the osmoregulation and carbohydrate metabolism of euryhaline fish tilapia (Oreochromis niloticus) under sustained hypertonic stress (20 practical salinity units [psu]). 6 diets containing either normal carbohydrate (NC, 30%) or high carbohydrate (HC, 45%) levels, with 3 levels (0, 400 and 1,200 mg/kg diet) of MI, respectively, were fed to 540 fish under 20 psu for 8 weeks. Dietary MI supplementation significantly improved growth performance and crude protein content of whole fish, and decreased the content of crude lipid of whole fish (P < 0.05). Curled, disordered gill lamella and cracked gill filament cartilage were observed in the gill of fish fed diets without MI supplementation. The ion transport capacity in gill was significantly improved in the 1,200 mg/kg MI supplementation groups compared with the 0 mg/kg MI groups (P < 0.05). Moreover, the contents of Na+, K+, Cl- in serum were markedly reduced with the dietary MI supplementation (P < 0.05). The fish fed 1,200 mg/kg MI supplementation had the highest MI content in the gills and the lowest MI content in the serum (P < 0.05). Additionally, the fish fed with 1,200 mg/kg MI supplementation had the highest MI synthesis capacity in gills and brain (P < 0.05). Dietary MI markedly promoted the ability of carbohydrate metabolism in liver (P < 0.05). Moreover, fish in the 1,200 mg/kg MI groups had the highest antioxidant capacity (P < 0.05). This study indicated that high dietary carbohydrate would intensify stress, and impair the ability of osmoregulation in tilapia under a long-term hypersaline exposure. The supplementation of MI at 1,200 mg/kg in the high carbohydrate diet could promote carbohydrate utilization and improve the osmoregulation capacity of tilapia under long-term hypertonic stress.

Dietary gamma-aminobutyric acid (GABA) improves non-specific immunity and alleviates lipopolysaccharide (LPS)-induced immune overresponse in juvenile Chinese mitten crab (Eriocheir sinensis).

Inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is an immunomodulator to inhibit immune-mediated pro-inflammatory response and has been used to treat various immune-related diseases in mammals. However, the immunoregulatory effect of GABA in crustaceans has not been reported. This study evaluates the regulatory effect of dietary GABA supplementation on the innate immune status and immunoregulatory potential in lipopolysaccharide (LPS)-induced immune response in juvenile Eriocheir sinensis. Juvenile crabs were fed with six diets supplemented with graded GABA levels (0, 40, 80, 160, 320 and 640 mg/kg dry matter) for 8 weeks and then 24 h LPS challenge test was carried out. The results showed that dietary GABA supplementation significantly decreased mortality at 4 and 8 weeks. Moreover, the hemocyanin content, acid phosphatase, and alkaline phosphatase activities significantly increased in the crabs fed GABA supplementation compared with the control. On the contrary, the alanine aminotransferase and alanine aminotransferase activities in serum decreased significantly in the GABA supplementation groups compared with the control. Similarly, superoxide dismutase activity, glutathione content, and the transcriptional expression of the antioxidant-related genes and immune-related genes were significantly higher in the GABA supplementation groups than in the control. In addition, the mRNA expressions of anti-lipopolysaccharide factors (ALF 1, ALF 2, ALF 3) and inflammatory signaling pathways related genes (TLR, Myd88, Relish, LITAF, P38-MAPK, ADAM17) were significantly up-regulated in LPS stimulation groups compared with PBS treatment. Meanwhile, pro-apoptosis-related genes' mRNA expressions were significantly up-regulated, and anti-apoptosis-related genes were significantly down-regulated under LPS stimulation compared with PBS treatment. However, GABA pretreatment effectively alleviated LPS-induced immune overresponse and apoptosis. Therefore, this study demonstrates that dietary GABA supplementation could be used as an immunomodulator to improve the non-specific immunity and antioxidant capacity and alleviate the immune-mediated immune overresponse of juvenile E. sinensis.

Visualising the Emerging Platform of Using Microalgae as a Sustainable Bio-Factory for Healthy Lipid Production through Biocompatible AIE Probes.

Nowadays, a particular focus is using microalgae to get high-valued health beneficiary lipids. The precise localisation of the lipid droplets (LDs) and biochemical changes are crucial to portray the lipid production strategy in algae, but it requires an in vivo tool to rapidly visualise LD distribution. As a novel strategy, this study focuses on detecting lipid bioaccumulation in a green microalga, Chlamydomonas reinhardtii using the aggregation-induced emission (AIE) based probe, 2-DPAN (C24H18N2O). As the messenger molecule and stress biomarker, hydrogen peroxide (H2O2) activity was detected in lipid synthesis with the AIE probe, TPE-BO (C38H42B2O4). Distinctive LDs labelled with 2-DPAN have elucidated the lipid inducing conditions, where more health beneficiary α-linolenic acid has been produced. TPE-BO labelled H2O2 have clarified the involvement of H2O2 during lipid biogenesis. The co-staining procedure with traditional green BODIPY dye and red chlorophyll indicates that 2-DPAN is suitable for multicolour LD imaging. Compared with BODIPY, 2-DPAN was an efficient sample preparation technique without the washing procedure. Thus, 2-DPAN could improve traditional fluorescent probes currently used for lipid imaging. In addition, the rapid, wash-free, multicolour AIE-based in vivo probe in the study of LDs with 2-DPAN could advance the research of lipid production in microalgae.

Neural excitotoxicity and the toxic mechanism induced by acute hypoxia in Chinese mitten crab (Eriocheir sinensis).

Hypoxia can induce neural excitotoxicity in mammals, but this adverse effect has not been investigated in aquatic animals to date, especially in crustaceans. This study explored the induction effect and toxic mechanism of acute hypoxia stress (1.0 ± 0.1 mg dissolved oxygen /L) for 24 h on neural excitotoxicity in juvenile Chinese mitten crab, Eriocheir sinensis. The results showed that hemolymph glucose and serum lactic acid content were significantly increased, and the mRNA expression of crustacean hyperglycemic hormone and hypoxia-inducible factor 1α were significantly up-regulated in the hypoxia group compared with control. RNA-Seq results confirmed that acute hypoxia stress had a more significant impact on carbohydrate metabolism than lipid and protein metabolism. In addition, the TUNEL assay showed that the apoptosis rate of nerve cells was significantly higher in the hypoxia group than in the control, and similar trends were observed in the expression of apoptosis-related genes. RNA-Seq results also showed that acute hypoxia stress-induced neuronal apoptosis by regulating multiple apoptosis-related pathways. Moreover, free glutamate and GABA contents in the nerve tissue of thoracic ganglia were significantly higher in the hypoxia group than in the control group. Furthermore, the mRNA expression of NMDA related receptors was significantly up-regulated in the hypoxia group compared with the control. Similar trends were observed in the expression of calcium-dependent degrading enzymes and endogenous antioxidant-related proteins or enzymes. Meanwhile, the mRNA expression level of high-affinity neuronal glutamate transporter in the hypoxia group was significantly up-regulated compared with the control, whereas the vesicular glutamate transporter was significantly down-regulated. Furthermore, NMDA-R antagonists (MK-801 and Ro25-6981) injection showed that NMDA-R served as the bridge and core position of glutamate-induced neural neurotoxicity. This study provides a new perspective and theoretical guidance for exploring the regulation of hypoxic tolerance in E. sinensis.

Combined effects of polystyrene microplastics and copper on antioxidant capacity, immune response and intestinal microbiota of Nile tilapia (Oreochromis niloticus).

Microplastics (MPs) coexist with other pollutants (such as heavy metals) in water, adversely impacting aquatic organisms, which might cause unpredictable ecological risks. This study aims to evaluate the effect of copper (Cu2+) and polystyrene microplastics (PS-MPs) on antioxidant capacity, immune response and intestinal microbiota of Nile tilapia. Cu2+ and PS-MPs co-exposure enhanced Cu2+ bioaccumulation in the liver of fish compared with Cu2+-alone exposure. Fish exposed to PS-MPs and Cu2+ displayed histopathologic alterations in the liver, intestine and gill. Exposure at low concentrations of Cu2+ in the C0 and CP0 groups can improve antioxidant capacity and immune response, while oxidative damage and inflammation existed in the high concentration of Cu2+ groups. Intestinal microbiota results showed that the diversity and structure were changed by Cu2+ and PS-MPs exposure, and harmful bacterium even increased at high concentration of Cu2+ and PS-MPs exposure groups. All in all, PS-MPs aggravate the accumulation of Cu2+ and lead to perturbations in biological systems of Nile tilapia.

Aggregation-induced emission luminogens for lipid droplet imaging.

Lipid droplets (LDs) are evolutionarily conserved organelles involved in energy homeostasis and versatile intracellular processes in different cell types. Their importance is ubiquitous, ranges from utilization as the biofunctional components to third-generation biofuel production from microalgae, while morphology and functional perturbations could also relate to the multiple diseases in higher mammals. Biosynthesis of lipids can be triggered by multiple factors related to organismal physiology and the surrounding environment. An early prediction of this might help take necessary actions toward desired outcomes. In vivo visualization of LDs can give molecular insight into regulatory mechanisms and the underlying connections with other cellular structures. Traditional bioprobes for LDs detection often suffer from different dye-specific limitations such as aggregation-caused quenching and self-decomposition phenomena that hinder the research advancement. The emergence of lipid-specific nanoprobes with aggregation-induced emission (AIE) attributes in recent years is promising in remunerative characteristics with defined bioimaging properties. By utilizing the easy synthetic techniques and exploiting the unique physical features of these molecules, highly selective, stable, biocompatible and facile fluorescent probes could be fabricated for lipid detection. This chapter will provide up-to-date insight into the recent advances in lipid-specific AIE-based probes to enhance the opportunities for basic research related to the distinct roles of LDs in living organisms.

Microalgae-Derived Health Supplements to Therapeutic Shifts: Redox-Based Study Opportunities with AIE-Based Technologies.

Reactive oxygen species (ROS) are highly reactive molecules, serve the normal signaling in different cell types. Targeting ROS as the chemical signals, different stress based strategies have been developed to synthesis different anti-inflammatory molecules in microalgae. These molecules could be utilized as health supplements in human. To provoke the ROS-mediated defence systems, their connotation with the associated conditions must be well understood, therefore, proper tools for studying ROS in natural state are essential. The in vivo detection of ROS with phosphorescent probes offers promising opportunities to study these molecules in a non-invasive manner. Most of the common problems in the traditional fluorescent probes are lower photostability, excitation intensity, slow responsiveness, and the microenvironment that challenge their performance. Some ROS-specific aggregationinduced emission luminogens (AIEgens) with pronounced spatial and temporal resolution have recently demonstrated high selectivity, rapid responsiveness, and efficacies to resolve the aggregation-caused quenching issues. The nanocomposites of some AIE-photosensitizers can also improve the ROS-mediated photodynamic therapy. These AIEgens could be used to induce bioactive components in microalgae through altering the ROS signaling, therefore are more auspicious for biomedical research. This study reviews the prospects of AIEgen-based technologies to understand the ROS mediated bio-physiological processes in microalgae for better healthcare benefits.