Glucose transporter GLUT1 expression is important for oriental river prawn (Macrobrachium nipponense) hemocyte adaptation to hypoxic conditions.

Crustaceans have an open vascular system in which hemocytes freely circulate in hemolymph. Hemocytes are rich in hemocyanin, a specific oxygen-transport protein in crustaceans; therefore, understanding the response of hemocytes to hypoxia is crucial. Although hemocytes take up glucose during hypoxia, the molecular mechanism of glucose uptake in crustaceans remains unclear. Herein, we identified two highly conserved glucose transporters (GLUT1 and GLUT2) in Macrobrachium nipponense (oriental river prawn) and analyzed their tissue-specific expression patterns. Our immunofluorescence assays showed that GLUT1 and GLUT2 are located on the cell membrane, with a strong GLUT1 signal in primary hemocytes under hypoxia. We found that during acute hypoxia, hypoxia-inducible factor-1α-related metabolic alterations result in decreased mitochondrial cytochrome c oxidase activity, implying a classic glycolytic mechanism. As a proof of concept, we replicated these findings in insect S2 cells. Acute hypoxia significantly induced hypoxia-inducible factor-1α, GLUT1, and pyruvate dehydrogenase kinase isozyme 1 expression in primary hemocytes, and hypoxia-induced increases in glucose uptake and lactate secretion were observed. GLUT1 knockdown induced intracellular reactive oxygen species generation and apoptosis in vitro and in vivo, resulting in increased prawn mortality and more apoptotic cells in their brains, implying a vital function of GLUT1 in hypoxia adaptation. Taken together, our results suggest a close relationship between hypoxia-mediated glycolysis and GLUT1 in hemocytes. These results demonstrated that in crustaceans, adaptation to hypoxia involves glucose metabolic plasticity.

The evolutionarily conserved hif-1/bnip3 pathway promotes mitophagy and mitochondrial fission in crustacean testes during hypoxia.

The hypoxia-inducible factor 1 (HIF-1) cascade is an ancient and strongly evolutionarily conserved signaling pathway that is involved in the hypoxic responses of most metazoans. Despite immense advances in the understanding of the HIF-1-mediated regulation of hypoxic responses in mammals, the contribution of the hif-1 cascade in the hypoxic adaptation of nonmodel invertebrates remains unclear. In this study, we used the oriental river prawn Macrobrachium nipponense for investigating the roles of hif-1-regulated mitophagy in crustacean testes under hypoxic conditions. We identified that the Bcl-2/adenovirus E1B 19-kDa interacting protein (bnip3) functions as a regulator of mitophagy in M. nipponense and demonstrated that hif-1α activates bnip3 by binding to the bnip3 promoter. Hif-1α knockdown suppressed the expression of multiple mitophagy-related genes, and prawns with hif-1α knockdown exhibited higher mortality under hypoxic conditions. We observed that the levels of BNIP3 were induced under hypoxic conditions and detected that bnip3 knockdown inhibited the mitochondrial translocation of dynamin-related protein 1 (drp1), which is associated with mitochondrial fission. Notably, bnip3 knockdown inhibited hypoxia-induced mitophagy and aggravated the deleterious effects of hypoxia-induced reactive oxygen species (ROS) production and apoptosis. The experimental studies demonstrated that hypoxia induced mitochondrial fission in M. nipponense via drp1. Altogether, the study elucidated the mechanism underlying hif-1/bnip3-mediated mitochondrial fission and mitophagy and demonstrated that this pathway protects crustaceans against ROS production and apoptosis induced by acute hypoxia.

Comparative Phosphoproteomics Reveals a Role for AMPK in Hypoxia Signaling in Testes of Oriental River Prawn (Macrobrachium nipponense).

Hypoxia is one of the major stresses in aquaculture animals. Recently, we reported that hypoxia disrupts the endocrine system and inhibits testicular function of oriental river prawns (Macrobrachium nipponense), but the molecular mechanism of testes responded to hypoxia remains largely unknown. In the present study, we aimed to integrate whole phosphoproteomic profiles of hypoxia-treated testes of the oriental river prawn (Macrobrachium nipponense). We successfully isolated sperm cells and evaluated the mitochondrial morphology and function using laser confocal microscopy, flow cytometry, and biochemical analyses. Quantitative proteomics identified 117 differentially abundant phosphorylated proteins, and these proteins are mainly involved in the pathways related to cellular processes, including autophagy, apoptosis, and the FoxO signaling pathway. Protein-protein interaction analysis clustered these phosphoproteins into three groups, many of which have been suggested to impact carbohydrate metabolism, autophagy, and signal regulation in testes. Western blotting confirmed that phosphorylated proteins including AMPK, ULK1, and TP53 (of the AMPK pathway) may contribute to testicular dysfunction caused by hypoxia. Further, we investigated the potential roles of AMP-activated protein kinase (AMPK)'s in testes mitochondrial autophagy and apoptosis in M. nipponense as induced by hypoxia. Simultaneous knockdown of AMPKα in sperm cells led to a decrease in FOXO3a phosphorylation at Ser413, upregulation of caspase-3 and caspase-9 activities, and an increased apoptosis rate. These results improve our understanding of hypoxia-induced energy metabolism disorders in the testes of M. nipponense.

Transcriptome profiling and histology changes in juvenile blunt snout bream (Megalobrama amblycephala) liver tissue in response to acute thermal stress.

To understand the precise mechanism and the pathways activated by thermal stress in fish, we sampled livers from juvenile Megalobrama amblycephala exposed to control (25 °C) and test (35 °C) conditions, and performed short read (100 bp) next-generation RNA sequencing (RNA-seq). Using reads from different temperature, expression analysis identified a total of 440 differentially-expressed genes. These genes were related to oxidative stress, apoptosis, immune responses and so on. We used quantitative real-time reverse transcriptase PCR to assess the differential mRNA expression of selected genes that encode antioxidant enzymes and heat shock proteins in response to thermal stress. Fish exposed to thermal stress also showed liver damage associated with serum biochemical parameter changes. The set of genes identified showed regulatory modulation at different temperatures, and therefore could be further studied to determine how thermal stress damages M. amblycephala livers and the possible roles of reactive oxygen species in this process.

Serum biochemistry, liver histology and transcriptome profiling of bighead carp Aristichthys nobilis following different dietary protein levels.

Dietary protein plays a major role in determining the rate of fish growth and overall health. Given that the liver is an important organ for metabolism and detoxification, we hypothesized that optimal dietary protein levels may benefit liver function. Herein, we investigated the effects of dietary protein level on serum biochemistry, liver histology and transcriptome profiling of juvenile bighead carp Aristichthys nobilis fed for 8 weeks on a diet supplemented with high protein (HP, 40%), low protein (LP, 24%) or optimal protein (OP, 32%; controls). The results revealed a significant change in liver morphology in LP and HP groups compared with the OP group, coupled with increased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. RNA sequencing (RNA-Seq) analysis of the liver transcriptome yielded 47 million high-quality reads using an Illumina platform, which were de novo assembled into 80,777 unique transcript fragments (unigenes) with an average length of 1021 bp. Subsequent bioinformatics analysis identified 878 and 733 differentially expressed unigenes (DEGs) in liver in response to LP and HP diets, respectively. KEGG enrichment analysis of DEGs identified immune and metabolism-related pathways, including Toll-like receptor signaling, PI3K-Akt signaling, NF-κB signaling, complement and coagulation, peroxisome, nitrogen metabolism, PPAR signaling, and glycolysis and gluconeogenesis pathways. Transcriptome profiling results were validated by quantitative real-time PCR for 16 selected DEGs. The findings expand our understanding of the molecular mechanisms underlying the effects of dietary protein level on liver function in bighead carp.

Evaluating expression of autophagy-related genes in oriental river prawn Macrobrachium nipponense as potential biomarkers for hypoxia exposure.

Autophagy, a crucial process for maintaining cellular homeostasis, is under the control of several autophagy-related (ATG) proteins, and is highly conserved in most animals, but its response to adverse environmental conditions is poorly understood in crustaceans. Herein, we hypothesised that autophagy acts as a protective response to hypoxia, and Beclin 1, ATG7 and ATG8 in oriental river prawn (Macrobrachium nipponense) were chosen as potential biomarkers under hypoxia exposure; thus, their full-length cDNA sequences were cloned and characterised. Open reading frames (ORFs) of 1281, 2076 and 360 bp, encoding proteins of 427, 692 and 120 amino acid residues, respectively, were obtained. Phylogenetic analysis demonstrated the three M. nipponense proteins do not form a clade with vertebrate homologs. Protein and mRNA levels were investigated in different tissues and developmental stages, and all three were significantly upregulated in a time-dependent manner in the hepatopancreas following hypoxia stress. Biochemical and morphological analysis of hepatocytes revealed that hypoxia increased the abundance of hepatic autophagic vacuoles and stimulated anaerobic metabolism. RNA interference-mediated silencing of ATG8 significantly increased the death rate of M. nipponense juveniles under hypoxia stress conditions. Together, these results suggest that Beclin 1, ATG7 and ATG8 contribute to autophagy-based responses against hypoxia in M. nipponense. The findings also expand our understanding of the potential role of autophagy as an adaptive response against hypoxia toxicity in crustaceans. The results showed that hepatic ATG8 levels may be directly indicative of acute hypoxia in prawns, and provide insight into the time at which hypoxia exposure occurs. Autophagy-related genes expression pattern seems to be sensitive and good biomarkers of acute hypoxia exposure.

Identification and Characterization of Four Autophagy-Related Genes That Are Expressed in Response to Hypoxia in the Brain of the Oriental River Prawn (Macrobrachium nipponense).

Autophagy is a cytoprotective mechanism triggered in response to adverse environmental conditions. Herein, we investigated the autophagy process in the oriental river prawn (Macrobrachium nipponense) following hypoxia. Full-length cDNAs encoding autophagy-related genes (ATGs) ATG3, ATG4B, ATG5, and ATG9A were cloned, and transcription following hypoxia was explored in different tissues and developmental stages. The ATG3, ATG4B, ATG5, and ATG9A cDNAs include open reading frames encoding proteins of 319, 264, 268, and 828 amino acids, respectively. The four M. nipponense proteins clustered separately from vertebrate homologs in phylogenetic analysis. All four mRNAs were expressed in various tissues, with highest levels in brain and hepatopancreas. Hypoxia up-regulated all four mRNAs in a time-dependent manner. Thus, these genes may contribute to autophagy-based responses against hypoxia in M. nipponense. Biochemical analysis revealed that hypoxia stimulated anaerobic metabolism in the brain tissue. Furthermore, in situ hybridization experiments revealed that ATG4B was mainly expressed in the secretory and astrocyte cells of the brain. Silencing of ATG4B down-regulated ATG8 and decreased cell viability in juvenile prawn brains following hypoxia. Thus, autophagy is an adaptive response protecting against hypoxia in M. nipponense and possibly other crustaceans. Recombinant MnATG4B could interact with recombinant MnATG8, but the GST protein could not bind to MnATG8. These findings provide us with a better understanding of the fundamental mechanisms of autophagy in prawns.

Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense.

The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.

Molecular Cloning and Expression Analysis of Lactate Dehydrogenase from the Oriental River Prawn Macrobrachium nipponense in Response to Hypoxia.

Metabolic adaption to hypoxic stress in crustaceans implies a shift from aerobic to anaerobic metabolism. Lactate dehydrogenase (LDH) is a key enzyme in glycolysis in prawns. However, very little is known about the role of LDH in hypoxia inducible factor (HIF) pathways of prawns. In this study, full-length cDNA of LDH (MnLDH) was obtained from the oriental river prawn Macrobrachium nipponense, and was characterized. The full-length cDNA is 2267-bp with an open reading frame of 999 bp coding for a protein of 333 amino acids with conserved domains important for function and regulation. Phylogenetic analysis showed that MnLDH is close to LDHs from other invertebrates. Quantitative real-time PCR revealed that MnLDH is expressed in various tissues with the highest expression level in muscle. MnLDH mRNA transcript and protein abundance in muscle, but not in hepatopancreas, were induced by hypoxia. Silencing of hypoxia-inducible factor 1 (HIF-1) α or HIF-1ß subunits blocked the hypoxia-dependent increase of LDH expression and enzyme activity in muscle. A series of MnLDH promoter sequences, especially the full-length promoter, generated an increase in luciferase expression relative to promoterless vector; furthermore, the expression of luciferase was induced by hypoxia. These results demonstrate that MnLDH is probably involved a HIF-1-dependent pathway during hypoxia in the highly active metabolism of muscle.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense.

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), ß-act (ß-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and ß-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.