Two hexokinases of the shrimp Penaeus (Litopenaeus) vannamei are differentially expressed during oxygen limited conditions.

The white shrimp Penaeus (Litopenaeus) vannamei is the most cultivated shrimp worldwide. Compared to other shrimp species, it has higher resistance to adverse conditions. During hypoxia, the shrimp reduces oxygen consumption and adjusts energy metabolism via anaerobic glycolysis, among other strategies. Hexokinase (HK) is the first enzyme of glycolysis and a key regulation point. In mammals and other vertebrates, there are several tissue-specific HK isoforms with differences in expression and enzyme activity. In contrast, crustacean HKs have been relatively little studied. We studied the P. vannamei HK isoforms during hypoxia and reoxygenation. We cloned two HK1 sequences named HK1-long (1455 bp) and HK1-short (1302 bp), and one HK2 (1344 bp). In normoxia, total HK1 expression is higher in hepatopancreas, while HK2 is higher in gills. Severe hypoxia (1 mg/L of DO) after 12 h exposure and 1 h of reoxygenation increased HK1 expression in both organs, but HK2 expression changed differentially. In hepatopancreas, HK2 expression increased in 6 and 12 h of hypoxia but diminished to normoxia levels after reoxygenation. In gills, HK2 expression decreased after 12 h of hypoxia. HK activity increased in hepatopancreas after 12 h hypoxia, opposite to gills. These results indicate that shrimp HK isoforms respond to hypoxia and reoxygenation in a tissue-specific manner. Intracellular glucose levels did not change in any case, showing the shrimp ability to maintain glucose homeostasis during hypoxia.

An efficient triose phosphate synthesis and distribution in wheat provides tolerance to higher field temperatures.

High temperatures in the field hinder bread wheat high-yield production, mainly because of the adverse effects of heat over photosynthesis. The Yaqui Valley, the main wheat producer region in Mexico, is a zone prone to have temperatures over 30°C. The aim of this work was to test the flag leaf photosynthetic performance in 10 bread wheat genotypes grown under high temperatures in the field. The study took place during two seasons (2019-2020 and 2020-2021). In each season, control seeds were sown in December, while heat-stressed were sown in late January to subject wheat to heat stress (HS) during the grain-filling stage. HS reduced Grain yield from 20 to 58% in the first season. HS did not reduce chlorophyll content and light-dependent reactions were unaffected in any of the tested genotypes. Rubisco, chloroplast fructose 1,6-biphosphatase (FBPase), and sucrose phosphate synthase (SPS) activities were measured spectrophotometrically. Rubisco activity did not decrease under HS in any of the genotypes. FBPase activity was reduced by HS indicating that triose phosphate flux to starch synthesis was reduced, while SPS was not affected, and thus, sucrose synthesis was maintained. HS reduced aerial biomass in the 10 chosen genotypes. Genotypes SOKWB.1, SOKWB.3, and BORLAUG100 maintained their yield under HS, pointing to a potential success in their introduction in this region for breeding heat-tolerant bread wheat.

Responses and modulation of the white shrimp Litopenaeus vannamei glutathione peroxidases 2 and 4 during hypoxia, reoxygenation and GPx4 knock-down.

Glutathione peroxidases (GPxs) are important antioxidant enzymes that act at distinct levels of the antioxidant defense. In vertebrates, there are several glutathione peroxidase (GPx) isoforms with different cellular and tissue distribution, but little is known about their interrelationships. The shrimp Litopenaeus vannamei is the main crustacean cultivated worldwide. It is affected by environmental stressors, including hypoxia and reoxygenation that cause reactive oxygen species accumulation. Thus, the antioxidant response modulation is key for shrimp resilience. Recently, several GPx isoforms genes were identified in the L. vannamei genome sequence, but their functions are just beginning to be studied. As in vertebrates, shrimp GPx isoforms can present differences in their antioxidant responses. Also, there could be interrelationships among the isoforms that may influence their responses. We evaluated shrimp GPx2 and GPx4 expressions during hypoxia, reoxygenation, and GPx4 knock-down using RNAi for silencing, as well as the enzymatic activity of total GPx and GPx4. Also, glutathione content in hepatopancreas was evaluated. GPx2 and GPx4 presented similar expression patterns during hypoxia and reoxygenation. Their expressions decreased during hypoxia and were reestablished in reoxygenation at 6 h in non-silenced shrimp. GPx2 expression was down-regulated by GPx4 knock-down, suggesting that GPx4 affects GPx2 expression. Total GPx activity changed in hypoxia and reoxygenation at 6 h but not at 12 h, while GPx4 activity was not affected by any stressor. The GSH/GSSG ratio in hepatopancreas indicated that at early hours, the redox status remains well-modulated but at 12 h it is impaired by hypoxia and reoxygenation.

The pyruvate kinase of the whiteleg shrimp Litopenaeus vannamei: Gene structure and responses to short term hypoxia.

The shrimp Litopenaeus vannamei is the main farmed crustacean worldwide. This shrimp suffers environmental changes in oxygen availability that affect its energy metabolism. Pyruvate kinase (PK) catalyzes the last reaction of glycolysis and is key for the regulation of glycolysis and gluconeogenesis. There is ample knowledge about mammalian PK, but in crustaceans, the information is very scarce. In this study, we analyzed in silico the structures of the PK gene and protein. Also, the effects of hypoxia on gene expression, enzymatic activity, glucose, and lactate in hepatopancreas and muscle were analyzed. The PK gene is 15,103 bp and contains 11 exons and 10 introns, producing four mRNA variants by alternative splicing and named PK1, PK2, PK3 and PK4, that results in two proteins with longer C-terminus and two with a 12 bp insertion. The promoter contains putative binding sites for transcription factors (TF) that are typically involved in stress responses. The deduced amino acid sequences contain the classic domains, binding sites for allosteric effectors and potential reversible phosphorylation residues. Protein modeling indicates a homotetramer with highly conserved structure. The effect of hypoxia for 6 and 12 h showed tissue-specific patterns, with higher expression, enzyme activity and lactate in muscle, but higher glucose in hepatopancreas. Changes in response to hypoxia were detected at 12 h in expression with induction in muscle and reduction in hepatopancreas, while enzyme activity was maintained, and glucose and lactate decreased. These results show rapid changes in expression and metabolites, while enzyme activity was maintained to cope with short-term hypoxia.

Shrimp Glucose-6-phosphatase 2 (G6Pase 2): a second isoform of G6Pase in the Pacific white shrimp and regulation of G6Pase 1 and 2 isoforms via HIF-1 during hypoxia and reoxygenation in juveniles.

Animals suffer hypoxia when their oxygen consumption is larger than the oxygen available. Hypoxia affects the white shrimp Penaeus (Litopenaeus) vannamei, both in their natural habitat and in cultivation farms. Shrimp regulates some enzymes that participate in energy production pathways as a strategy to survive during hypoxia. Glucose-6-phosphatase (G6Pase) is key to maintain blood glucose homeostasis through gluconeogenesis and glycogenolysis. We previously reported a shrimp G6Pase gene (G6Pase1) and in this work, we report a second isoform that we named G6Pase2. The expression of the two isoforms was evaluated in oxygen limited conditions and during silencing of the transcription factor HIF-1. High G6Pase activity was detected in hepatopancreas followed by muscle and gills under good oxygen and feeding conditions. Gene expression of both isoforms was analyzed in normoxia, hypoxia and reoxygenation in hepatopancreas and gills, and in HIF-1-silenced shrimp. In fed shrimp with normal dissolved oxygen (DO) (5.0 mg L- 1 DO) the expression of G6Pase1 was detected in gills, but not in hepatopancreas or muscle, while G6Pase2 expression was undetectable in all three tissues. In hepatopancreas, G6Pase1 is induced at 3 and 48 h of hypoxia, while G6Pase2 is down-regulated in the same time points but in reoxygenation, both due to the knock-down of HIF-1. In gills, only G6Pase1 was detected, and was induced by the silencing of HIF-1 only after 3 h of reoxygenation. Therefore, the expression of the two isoforms appears to be regulated by HIF-1 at transcriptional level in response to oxygen deprivation and subsequent recovery of oxygen levels.

The combination of hypoxia and high temperature affects heat shock, anaerobic metabolism, and pentose phosphate pathway key components responses in the white shrimp (Litopenaeus vannamei).

Due to global warming, world water bodies have higher temperatures and lower oxygen concentrations that affect aquatic species including the white shrimp Litopenaeus vannamei. This species withstands these conditions, but the information of the physiological responses that allow them to survive are scarce. We analyzed the effects of high temperature, hypoxia, reoxygenation, and the combination of these factors on the relative expression of selected genes: HSF1, Hsp70, p53, TIGAR, HIF-1α, and VEGF1-3 in gills of L. vannamei. Additionally, glucose, lactate, NADP, and NADPH were determined. HSF1 was up-regulated in the high temperature and oxygen stress conditions, but Hsp70 was up-regulated only in reoxygenation at both temperatures. HIF-1α was also up-regulated by reoxygenation in both temperatures. Meanwhile, the VEGF genes were not altered by the stress conditions, since none of them changed expression drastically. p53 relative expression remained stable at the tested stress conditions, which prompts to the maintenance of antioxidant defenses. TIGAR expression was induced in normoxia and hypoxia at high temperature, which induced NADPH content helping to scavenge reactive oxygen species (ROS). Additionally, high temperature caused higher glucose and lactate content in normoxia and hypoxia, indicating carbohydrate mobilization and a switch to anaerobic metabolism. The results showed that HSF1, the anaerobic metabolism and the pentose phosphate pathway (PPP) are crucial for the shrimp response to these abiotic stress conditions and contribute to their survival.

The bifunctional 6-phosphofructokinase-2/fructose-2,6-bisphosphatase from the shrimp Litopenaeus vannamei: Molecular characterization and down-regulation of expression in response to severe hypoxia.

Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.

Combined hypoxia and high temperature affect differentially the response of antioxidant enzymes, glutathione and hydrogen peroxide in the white shrimp Litopenaeus vannamei.

Low oxygen concentration in water (hypoxia) and high temperature are becoming more frequent due to climate change, forcing animals to endure stress or decease. Hypoxia and high temperature stress can lead to reactive oxygen species (ROS) accumulation and oxidative damage to the organisms. The shrimp Litopenaeus vannamei is the most cultivated crustacean worldwide. The aim of this study was to evaluate the expression and enzymatic activity of glutathione peroxidase (GPx), catalase (CAT) and cytosolic manganese superoxide dismutase (cMnSOD) in gills and hepatopancreas from L. vannamei in response to two combined stressors: hypoxia and reoxygenation at control and high temperature (28 vs 35 °C, respectively). In addition, glutathione and hydrogen peroxide content were analyzed. The changes were mainly tissue-specific. In gills, cMnSOD expression and enzymatic activity increased in response to the interactions between oxygen variation and thermal stress, while GPx and CAT were maintained. More changes occurred in GPx, CAT and MnSOD in hepatopancreas than in gills, mainly due to the effect of the individual stress factors of thermal stress or oxygen variations. On the other hand, the redox state of glutathione indicated that during high temperature, changes in the GSH/GSSG ratio occurred due to the fluctuations of GSSG. Hydrogen peroxide concentration was not affected by thermal stress or oxygen variations in hepatopancreas, whereas in gills, it was not detected. Altogether, these results indicate a complex pattern of antioxidant response to hypoxia, reoxygenation, high temperature and their combinations.

Cloning, expression, purification and biochemical characterization of recombinant metallothionein from the white shrimp Litopenaeus vannamei.

Metallothioneins (MTs) are cysteine rich proteins with antioxidant capacity that participate in the homeostasis and detoxification of metals and other cellular processes, and help to counteract the oxidative stress produced by Reactive Oxygen Species (ROS). The production of ROS increases during several stress conditions, including metal intoxication and hypoxia (oxygen deficiency). During hypoxia the expression of the MT gene is induced in the shrimp Litopenaeus vannamei; however, the MT protein coded by this gene has not been purified nor characterized. In this work, the coding sequence of L. vannamei MT was cloned and overexpressed in Escherichia coli as a fusion protein, containing an intein and a chitin binding domain (CBD). The MT was purified by chitin affinity chromatography and its antioxidant capacity and ability to bind cadmium (Cd) and copper (Cu) were evaluated. This MT has an antioxidant capacity of 27.23 µM equivalent to Trolox in a 100 µg/mL solution. Addition of CdCl2 to the culture media augments 273-fold the Cd content, while addition of CuCl2 increases Cu content 569-fold in the purified MT. Thus, the shrimp MT gene codes for a functional protein that has antioxidant capacity and binds Cu and Cd.

Purification and partial biochemical characterization of recombinant lactate dehydrogenase 1 (LDH-1) of the white shrimp Litopenaeus vannamei.

Lactate dehydrogenase (LDH) is a key enzyme to produce energy during hypoxia by anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, two protein subunits (LDH-1 and LDH-2) were previously identified, deduced from two different transcripts that come from the same LDH gene by processing via mutually exclusive alternative splicing. LDH-1 contains exon five and LDH-2 contains exon six and the two proteins differ only in 15 amino acid residues. Both subunits were independently cloned and overexpressed in E. coli as a fusion protein containing a chitin binding domain. Previously, recombinant LDH-2 was successfully purified and characterized, but LDH-1 was insoluble and aggregated forming inclusion bodies. We report the production of soluble LDH-1 by testing different pHs in the buffers used to lyse the bacterial cells before the purification step and the characterization of the purified protein to show that the cDNA indeed codes for a functional and active protein. The recombinant native protein is a homotetramer of approximately 140 kDa composed by 36 kDa subunits and has higher affinity for pyruvate than for lactate. LDH-1 has an optimum pH of 7.5 and is stable between pH 8.0 and 9.0; pH data analysis showed two pKa values of 6.1 ±â€¯0.15 and 8.8 ±â€¯0.15 suggesting a histidine and asparagine, respectively, involved in the active site. The enzyme optimal temperature was 44 °C and it was stable between 20 and 60 °C. LDH-1 was slightly activated by NaCl, KCl and MgCl2 and fully inhibited by ZnCl2.

Regulation of glyceraldehyde-3-phosphate dehydrogenase by hypoxia inducible factor 1 in the white shrimp Litopenaeus vannamei during hypoxia and reoxygenation.

Hypoxia is a frequent source of stress in the estuarine habitat of the white shrimp Litopenaeus vannamei. During hypoxia, L. vannamei gill cells rely more heavily on anaerobic glycolysis to obtain ATP. This is mediated by transcriptional up-regulation of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The hypoxia inducible factor 1 (HIF-1) is an important transcriptional activator of several glycolytic enzymes during hypoxia in diverse animals, including crustaceans. In this work, we cloned and sequenced a fragment corresponding to the 5' flank of the GAPDH gene and identified a putative HIF-1 binding site, as well as sites for other transcription factors involved in the hypoxia signaling pathway. To investigate the role of HIF-1 in GAPDH regulation, we simultaneously injected double-stranded RNA (dsRNA) into shrimp to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia followed by reoxygenation, and then measured gill HIF-1α, HIF-1ß expression, GAPDH expression and activity, and glucose and lactate concentrations at 0, 3, 24 and 48 h. During normoxia, HIF-1 silencing induced up-regulation of GAPDH transcripts and activity, suggesting that expression is down-regulated via HIF-1 under these conditions. In contrast, HIF-1 silencing during hypoxia abolished the increases in GAPDH expression and activity, glucose and lactate concentrations. Finally, HIF-1 silencing during hypoxia-reoxygenation prevented the increase in GAPDH expression, however, those changes were not reflected in GAPDH activity and lactate accumulation. Altogether, these results indicate that GAPDH and glycolysis are transcriptionally regulated by HIF-1 in gills of white shrimp.

Phosphoenolpyruvate carboxykinase cytosolic and mitochondrial isoforms are expressed and active during hypoxia in the white shrimp Litopenaeus vannamei.

Hypoxic zones in marine environments are spreading around the world affecting the survival of many organisms. Marine animals have several strategies to respond to hypoxia, including the regulation of gluconeogenesis. Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of gluconeogenesis. The objective of this work was to study two isoforms of PEPCK, one mitochondrial (PEPKC-M) and one cytosolic (PEPCK-C), from the white shrimp Litopenaeus vannamei and the response to hypoxia. Both PEPCK isoforms are 72 kDa proteins and have 92% identity at the amino acid level. The mitochondrial isoform has a N-terminal signal peptide for mitochondrial import. Gene expression and enzymatic activity in subcellular fractions were detected in gills, hepatopancreas and muscle in normoxic and hypoxic conditions. Expression of PEPCK-C was higher than PEPCK-M in all the tissues and induced in response to hypoxia at 48 h in hepatopancreas, while the enzymatic activity of PEPCK-M was higher than PEPCK-C in gills and hepatopancreas, but not in muscle and also increased in response to hypoxia in hepatopancreas but decreased in gills and muscle. During limiting oxygen conditions, shrimp tissues obtain energy by inducing anaerobic glycolysis, and although gluconeogenesis implies energy investment, due to the need to maintain glucose homeostasis, these gluconeogenic enzymes are active with contrasting behaviors in the cytosol and mitochondrial cell compartments and appear to be up-regulated in hepatopancreas indicating this tissue pivotal role in gluconeogenesis during the response to hypoxia.

White shrimp Litopenaeus vannamei recombinant lactate dehydrogenase: Biochemical and kinetic characterization.

Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pKa of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants Km for NADH was 23.4 ± 1.8 µM, and for pyruvate was 203 ± 25 µM, while Vmax was 7.45 µmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia.

Role of HIF-1 on phosphofructokinase and fructose 1, 6-bisphosphatase expression during hypoxia in the white shrimp Litopenaeus vannamei.

HIF-1 is a transcription factor that controls a widespread range of genes in metazoan organisms in response to hypoxia and is composed of α and ß subunits. In shrimp, phosphofructokinase (PFK) and fructose bisphosphatase (FBP) are up-regulated in hypoxia. We hypothesized that HIF-1 is involved in the regulation of PFK and FBP genes in shrimp hepatopancreas under hypoxia. Long double stranded RNA (dsRNA) intramuscular injection was utilized to silence simultaneously both HIF-1 subunits, and then, we measured the relative expression of PFK and FBP, as well as their corresponding enzymatic activities in hypoxic shrimp hepatopancreas. The results indicated that HIF-1 participates in the up-regulation of PFK transcripts under short-term hypoxia since the induction caused by hypoxia (~1.6 and ~4.2-fold after 3 and 48h, respectively) is significantly reduced in the dsRNA animals treated. Moreover, PFK activity was significantly ~2.8-fold augmented after 3h in hypoxia alongside to an ~1.9-fold increment in lactate. However, when animals were dsRNA treated, both were significantly reduced. On the other hand, FBP transcripts were ~5.3-fold up-regulated in long-term hypoxic conditions (48h). HIF-1 is involved in this process since FBP transcripts were not induced by hypoxia when HIF-1 was silenced. Conversely, the FBP activity was not affected by hypoxia, which suggests its possible regulation at post-translational level. Taken together, these results position HIF-1 as a prime transcription factor in coordinating glucose metabolism through the PFK and FBP genes among others, in shrimp under low oxygen environments.