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.

The gluconeogenic glucose-6-phosphatase gene is expressed during oxygen-limited conditions in the white shrimp Penaeus (Litopenaeus) vannamei: Molecular cloning, membrane protein modeling and transcript modulation in gills and hepatopancreas.

The white shrimp Penaeus (Litopenaeus) vannamei is the most economically important crustacean species cultivated in the Western Hemisphere. This crustacean shifts its metabolism to survive under extreme environmental conditions such as hypoxia, although for a limited time. Glucose-6-phosphatase (G6Pase) is a key enzyme contributing to maintain blood glucose homeostasis through gluconeogenesis and glycogenolysis. To our knowledge, there are no current detailed studies about cDNA or gene sequences of G6Pase from any crustacean reported. Herein we report the shrimp P. (L.) vannamei cDNA and gene sequences. The gene contains seven exons interrupted by six introns. The deduced amino acid sequence has 35% identity to other homolog proteins, with the catalytic amino acids conserved and phylogenetically close to the corresponding invertebrate homologs. Protein molecular modeling predicted eight transmembrane helices with the catalytic site oriented towards the lumen of the endoplasmic reticulum. G6Pase expression under normoxic conditions was evaluated in hepatopancreas, gills, and muscle and the highest transcript abundance was detected in hepatopancreas. In response to different times of hypoxia, G6Pase mRNA expression did not change in hepatopancreas and became undetectable in muscle; however, in gills, its expression increased after 3 h and 24 h of oxygen limitation, indicating its essential role to maintain glycemic control in these conditions.

HIF-1 is involved in the regulation of expression of metallothionein and apoptosis incidence in different oxygen conditions in the white shrimp Litopenaeus vannamei.

The white shrimp Litopenaeus vannamei is exposed to hypoxic conditions in natural habitats and in shrimp farms. Hypoxia can retard growth, development and affect survival in shrimp. The hypoxia-inducible factor 1 (HIF-1) regulates many genes involved in glucose metabolism, antioxidant proteins, including metallothionein (MT) and apoptosis. In previous studies we found that the L. vannamei MT gene expression changed during hypoxia, and MT silencing altered cell apoptosis; in this study we investigated whether the silencing of HIF-1 affected MT expression and apoptosis. Double-stranded RNA (dsRNA) was used to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia plus reoxygenation. Expression of HIF-1α, HIF-1ß and MT, and apoptosis in hemocytes or caspase-3 expression in gills, were measured at 0, 3, 24 and 48 h of hypoxia and hypoxia followed by 1 h of reoxygenation. The results showed that hemocytes HIF-1α expression was induced during hypoxia and reoxygenation at 3 h, while HIF-1ß decreased at 24 and 48 h. In normoxia, HIF-1 silencing in hemocytes increased apoptosis at 3 h and decreased at 48 h; while in gills, caspase-3 increased at 3, 24 and 48 h. In hypoxia, HIF-1 silencing decreased apoptosis in hemocytes at 3 h, but caspase-3 increased in gills. During reoxygenation, apoptosis in hemocytes and caspase-3 in gills increased. During normoxia in hemocytes, silencing of HIF-1 decreased MT expression, but in gills, MT increased. During hypoxia and reoxygenation, silencing induced MT in hemocytes and gills. These results indicate HIF-1 differential participation in MT expression regulation and apoptosis during different oxygen conditions.

Development of a Gill Assay Library for Ecological Proteomics of Threespine Sticklebacks (Gasterosteus aculeatus).

A data-independent acquisition (DIA) assay library for quantitative analyses of proteome dynamics has been developed for gills of threespine sticklebacks (Gasterosteus aculeatus). A raw spectral library was generated by data-dependent acquisition (DDA) and annotation of tryptic peptides to MSMS spectra and protein database identifiers. The assay library was constructed from the raw spectral library by removal of low-quality, ambiguous, and low-signal peptides. Only unique proteins represented by at least two peptides are included in the assay library, which consists of 1506 proteins, 5074 peptides, 5104 precursors, and 25,322 transitions. This assay library was used with DIA data to identify biochemical differences in gill proteomes of four populations representing different eco- and morpho-types of threespine sticklebacks. The assay library revealed unique and reproducible proteome signatures. Warm-adapted, low-plated, brackish-water fish from Laguna de la Bocana del Rosario (Mexico) show elevated HSP47, extracellular matrix, and innate immunity proteins whereas several immunoglobulins, interferon-induced proteins, ubiquitins, proteolytic enzymes, and nucleic acid remodeling proteins are reduced. Fully-plated, brackish-water fish from Westchester Lagoon (Alaska) display elevated ion regulation, GTPase signaling, and contractile cytoskeleton proteins, altered abundances of many ribosomal, calcium signaling and immunity proteins, and depleted transcriptional regulators and metabolic enzymes. Low-plated freshwater fish from Lake Solano (California) have elevated inflammasomes and proteolytic proteins whereas several iron containing and ion regulatory proteins are reduced. Gills of fully-plated, marine fish from Bodega Harbor (California) have elevated oxidative metabolism enzymes and reduced transglutaminase 2, collagens, and clathrin heavy chains. These distinct proteome signatures represent targets for testing ecological and evolutionary influences on molecular mechanisms of gill function in threespine sticklebacks. Furthermore, the gill assay library represents a model for other tissues and paves the way for accurate and reproducible network analyses of environmental context-dependent proteome dynamics in complex organisms.

Effect of salinity on the synthesis and concentration of glycine betaine in osmoregulatory tissues from juvenile shrimps Litopenaeus vannamei.

In marine animals, glycine betaine is one of the main osmolytes accumulated under osmotic stress conditions; nevertheless, in penaeids, shrimps little is known about the pathways involved in glycine betaine biosynthesis. In animal cells, glycine betaine is synthesized by the enzyme betaine aldehyde dehydrogenase (BADH). We herein investigated the salinity effect on the synthesis and concentration of glycine betaine on white shrimp Litopenaeus vannamei. Shrimps were subjected to 10, 20, 35, 40, 50, and 60 ppt salinity conditions for seven days. BADH activity increased in hepatopancreas and gills of shrimps subjected to salinities above 35 ppt salinity. In muscle, the BADH activity decreased at 35 ppt salinity. In hepatopancreas from shrimps subjected to 50 and 60 ppt salinities, BADH activity increased 1.1 and 1.7-fold. At 60 ppt salinity, BADH activity increased 1.5-fold respect to 35 ppt in gills. Glycine betaine concentration increased in hepatopancreas, gills, muscle, and hemolymph in shrimps subjected to salinities above 35 ppt. Glycine betaine concentration also increased at 20 ppt salinity, while at 10 ppt, not detected significant differences. The catch of glycine betaine from hemolymph by the cell likely is carried out to avoid protein denaturalization. Ammonia concentration in the aquarium's water only increased at salinities of 20 ppt and 10 ppt (1.1-fold relative to 35 ppt). Our data demonstrated that in L. vannamei, salinity regulates BADH activity and glycine betaine content in a tissue-specific manner.

Mitochondrial manganese superoxide dismutase from the shrimp Litopenaeus vannamei: Molecular characterization and effect of high temperature, hypoxia and reoxygenation on expression and enzyme activity.

Climate warming has been increasing ocean water temperature and decreasing oxygen concentrations, exposing aquatic organisms to environmental stress conditions. The shrimp Litopenaeus vannamei manages to survive these harsh environmental conditions by enhancing their antioxidant defenses, among other strategies. In this study, we report the mitochondrial manganese superoxide dismutase (mMnSOD) nucleotide and deduced amino acid sequences and its gene expression in L. vannamei tissues. The deduced protein has 220 amino acids with a signal peptide of 20 amino acids. Expression of mMnSOD was analyzed in hepatopancreas, gills and muscle, where gills had highest expression in normoxic conditions. In addition, shrimp were subjected to high temperature, hypoxia and reoxygenation to analyze the effect on the expression of mMnSOD and SOD activity in mitochondria. High temperature and hypoxia showed a synergistic effect in the up-regulation on expression of mMnSOD in gills and hepatopancreas. Moreover, induction in SOD activity was found in the mitochondrial fraction from gills of normoxia at high temperature, probably due to an overproduction of reactive oxygen species caused by an elevated metabolic rate due to the stress temperature. These results suggest that the combined stress conditions of hypoxia and high temperature trigger molecularly the antioxidant response in L. vannamei in a higher degree than only one stressor.

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.

The glucose transporter 1 -GLUT1- from the white shrimp Litopenaeus vannamei is up-regulated during hypoxia.

During hypoxia the shrimp Litopenaeus vannamei accelerates anaerobic glycolysis to obtain energy; therefore, a correct supply of glucose to the cells is needed. Facilitated glucose transport across the cells is mediated by a group of membrane embedded integral proteins called GLUT; being GLUT1 the most ubiquitous form. In this work, we report the first cDNA nucleotide and deduced amino acid sequences of a glucose transporter 1 from L. vannamei. A 1619 bp sequence was obtained by RT-PCR and RACE approaches. The 5´ UTR is 161 bp and the poly A tail is exactly after the stop codon in the mRNA. The ORF is 1485 bp and codes for 485 amino acids. The deduced protein sequence has high identity to GLUT1 proteins from several species and contains all the main features of glucose transporter proteins, including twelve transmembrane domains, the conserved motives and amino acids involved in transport activity, ligands binding and membrane anchor. Therefore, we decided to name this sequence, glucose transporter 1 of L. vannamei (LvGLUT1). A partial gene sequence of 8.87 Kbp was also obtained; it contains the complete coding sequence divided in 10 exons. LvGlut1 expression was detected in hemocytes, hepatopancreas, intestine gills, muscle and pleopods. The higher relative expression was found in gills and the lower in hemocytes. This indicates that LvGlut1 is ubiquitously expressed but its levels are tissue-specific and upon short-term hypoxia, the GLUT1 transcripts increase 3.7-fold in hepatopancreas and gills. To our knowledge, this is the first evidence of expression of GLUT1 in crustaceans.

Detection of acute hepatopancreatic necrosis disease (AHPND) in Mexico.

Acute hepatopancreatic necrosis disease (AHPND), which has also been referred to as early mortality syndrome (EMS), initially emerged as a destructive disease of cultured shrimp species in Asia in 2009. The pathogen associated with the disease, Vibrio parahaemolyticus, subsequently spread to the Western Hemisphere and emerged in Mexico in early 2013. The spread to the Western Hemisphere is a major concern to shrimp producers in the region. To date, the only peer-reviewed published method for determining whether mortalities are due to AHPND is through histological examination. A novel PCR detection method was employed to assess samples from Mexico in order to confirm the presence of the pathogen in this country. This manuscript details the detection methods used to confirm the presence of AHPND in Mexico. Both immersion and per os challenge studies were used to expose the Penaeus vannamei to the bacteria in order to induce the disease. Histological analysis confirmed AHPND status following the challenge studies. Also provided are the details of the molecular test by PCR that was used for screening candidate V. parahaemolyticus isolates. A rapid PCR assay for detection of AHPND may help with early detection and help prevent the spread of AHPND to other countries.

Naphthalene and phenanthrene affect differentially two glutathione S-transferases (GSTs) expression, GST activity, and glutathione content in white shrimp P. vannamei.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants ubiquitous in coastal ecosystems. The white shrimp Penaeus vannamei naturally inhabits in coastal areas and is cultivated in farms located nearby the oceans. PAHs can damage shrimp health, endanger natural populations, and lower shrimp aquaculture productivity. However, crustaceans have enzymes capable of metabolizing organic xenobiotics as PAHs and to neutralize reactive oxygen species (ROS) produced during xenobiotics metabolism. An important superfamily of xenobiotic-metabolizing and antioxidant enzymes are glutathione S-transferases (GSTs). In white shrimp, some GSTs are known, but they have been scarcely studied in response to PAHs. In this study we report the molecular cloning and bioinformatic characterization of two novel nucleotide sequences corresponding to cytosolic GSTs belonging the Delta and Theta classes (GSTD and GSTT). Both proteins genes have tissue-specific patterns of expression under normal conditions, that do not necessarily relate to GST activity and glutathione content. The expression of the GSTD and GSTT, GST activity and glutathione content was analyzed in juvenile P. vannamei exposed to two PAHs, naphthalene (NAP) and phenanthrene (PHE) in sub-lethal concentrations for 96 h. GSTD expression was up-regulated by the two PAHs, while GSTT expression was only induced by NAP. In contrast, GST activity towards CDNB was only up-regulated by PHE, suggesting differential effects of PAHs at gene and protein level. On the other hand, lower reduced glutathione content (GSH) caused by PAHs indicates its utilization for detoxification or antioxidant defenses. However, the GSH/GSSG did not change by PAHs treatment, indicating that shrimp can maintain redox balance during short-term sub-lethal exposure to NAP and PHE. Despite the variations in the responses to NAP and PHE, all these results suggest that the GSTD and GSTT genes could be useful biomarkers for PAH exposure in P. vannamei.

"Candidatus Hepatobacter penaei," an intracellular pathogenic enteric bacterium in the hepatopancreas of the marine shrimp Penaeus vannamei (Crustacea: Decapoda).

The bacteria that cause necrotizing hepatopancreatitis in Penaeus vannamei adversely affect penaeid shrimp cultured in the western hemisphere. 16S rRNA and gyrase B gene analyses determined the taxonomic position of these bacteria. The name "Candidatus Hepatobacter penaei" is proposed for these pathogenic bacteria, which are members of the Rickettsiales order.

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.

Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp (Litopenaeus vannamei) infected with two different viruses: the WSSV or IHHNV.

The thioredoxin (TRX) system in crustaceans has demonstrated to act as a cell antioxidant being part of the immune response by dealing with the increased production of reactive oxygen species during bacterial or viral infection. Since the number of marine viruses has increased in the last years significantly affecting aquaculture practices of penaeids, and due to the adverse impact on wild and cultured shrimp populations, it is important to elucidate the dynamics of the shrimp response to viral infections. The role of Litopenaeus vannamei thioredoxin (LvTRX) was compared at both, mRNA and protein levels, in response to two viruses, the white spot syndrome virus (WSSV) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV). The results confirmed changes in the TRX gene expression levels of WSSV-infected shrimp, but also demonstrated a more conspicuous response of TRX to WSSV than to IHHNV. While both the dimeric and monomeric forms of LvTRX were detected by Western blot analysis during the WSSV infection, the dimer on its reduced form was only detected through the IHHNV infectious process. These findings indicate that WSSV or IHHNV infected shrimp may induce a differential response of the LvTRX protein.

Expression and silencing of Selenoprotein M (SelM) from the white shrimp Litopenaeus vannamei: effect on peroxidase activity and hydrogen peroxide concentration in gills and hepatopancreas.

Selenoprotein M (SelM), is a selenocysteine containing protein with redox activity involved in the antioxidant response. In the white shrimp Litopenaeus vannamei, SelM expression in gills is induced transiently during viral infection by the White Spot Syndrome Virus (WSSV). We report that SelM expression was detected in healthy shrimp L. vannamei in gills, muscle, hepatopancreas and pleopods, with more abundance in the hepatopancreas and gills. SelM transcripts were silenced by intramuscular injection with double-stranded RNAs (dsRNAs). In gills and hepatopancreas, all shrimp injected with long dsRNAs had lower SelM transcripts levels compared with controls. Peroxidase activity and hydrogen peroxide concentration were measured to detect effects on antioxidants. Peroxidase activity decreased upon silencing of SelM in gills, but no significant effect was detected in hepatopancreas. In contrast, total cell hydrogen peroxide concentration did not change in gills and hepatopancreas of silenced shrimp. Non-heme peroxidases are new players in the oxidative stress system that need to be addressed in detail, as well as selenium as a critical micronutrient for the antioxidant and innate immune systems in crustaceans.

Silencing of HIF-1 in WSSV-infected white shrimp: Effect on viral load and antioxidant enzymes.

Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor that induces genes involved in glucose metabolism. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive ß-subunit (HIF-1ß). The white spot syndrome virus (WSSV) induces a shift in glucose metabolism and oxidative stress. HIF-1α is associated with the induction of metabolic changes in tissues of WSSV-infected shrimp. However, the contributions of HIF-1 to viral load and antioxidant responses in WSSV-infected shrimp have been not examined. In this study, the effect of HIF-1 silencing on viral load and the expression and activity of antioxidant enzymes (superoxide dismutase-SOD, glutathione S-transferase-GST, and catalase) along with oxidative damage (lipid peroxidation and protein carbonyl) in tissues of white shrimp infected with the WSSV were studied. The viral load increased in hepatopancreas and muscle after WSSV infection, and the accumulative mortality was of 100% at 72 h post-infection. The expression and activity of SOD, catalase, and GST decreased in each tissue evaluated after WSSV infection. Protein carbonyl concentrations increased in each tissue after WSSV infection, while lipid peroxidation increased in hepatopancreas, but not in muscle. Silencing of HIF-1α decreased the WSSV viral load in hepatopancreas and muscle of infected shrimp along with shrimp mortality. Silencing of HIF-1α ameliorated the antioxidant response in a tissue-specific manner, which translated to a decrease in oxidative damage. These results suggest that HIF-1 is essential for restoring the antioxidant response, which counters the oxidative injury associated with WSSV infection.

Hypoxia drives apoptosis independently of p53 and metallothionein transcript levels in hemocytes of the whiteleg shrimp Litopenaeus vannamei.

The cellular mechanisms used by the shrimp Litopenaeus vannamei to respond to hypoxia have been studied from the energetic metabolism and antioxidant angles. We herein investigated the participation of p53 and metallothionein (MT) in the apoptotic process in response to hypoxia in shrimp hemocytes. The Lvp53 or LvMT genes were efficiently silenced by injection of double stranded RNA for p53 or MT. The effects of silencing on apoptosis were measured as caspase-3 activity and flow cytometry in hemocytes after 24 and 48 h of hypoxia (1.5 mg DO L(-1)). Hemocytes from unsilenced animals had significantly higher apoptosis levels upon both times of hypoxia. The apoptotic levels were diminished but not suppressed in dsp53-silenced but not dsMT-silenced hemocytes after 24 h of hypoxia, indicating a contribution of Lvp53 to apoptosis. Apoptosis in normoxia was significantly higher in dsp53-and dsMT-silenced animals compared to the unsilenced controls, pointing to a possible cytoprotective role of LvMT and Lvp53 during the basal apoptotic program in normoxia. Overall, these results indicate that hypoxia augments apoptosis in shrimp hemocytes and high mRNA levels of Lvp53 and LvMT are not necessary for this response.

High-Quality Draft Genomes of Two Vibrio parahaemolyticus Strains Aid in Understanding Acute Hepatopancreatic Necrosis Disease of Cultured Shrimps in Mexico.

The high-quality draft genomes of two Vibrio parahaemolyticus strains, one that causes the acute hepatopancreatic necrosis disease (AHPND) in cultured shrimps (FIM-S1708(+)), and another that does not (FIM-S1392(-)) are reported. A chromosome-scale assembly for the FIM-S1392(-) genome is reported here. The analysis of the two genomes gives some clues regarding the genomic differences between the strains.

Alternative splicing generates two lactate dehydrogenase subunits differentially expressed during hypoxia via HIF-1 in the shrimp Litopenaeus vannamei.

Metabolic adjustment to low oxygen exposure (hypoxia) in the white shrimp Litopenaeus vannamei implies a shift to anaerobic metabolism. Lactate dehydrogenase (LDH) is a key enzyme of the anaerobic metabolism described in most organisms. The structure and expression of the LDH gene, as well as the LDH isoenzymes in marine crustacean are not well defined. In the present study we characterized a gene that codes for two LDH subunits, measured their expression and detected the isoenzymes in tissues from white shrimp. We also silenced the transcriptional activator hypoxia inducible factor 1 (HIF-1) to elucidate the regulation of LDH in tissues from white shrimp in response to hypoxia. The complete LDH gene coding sequence is 7571 bp (LvanLDH) and encodes two different LDH subunits (LDHvan-1 and LDHvan-2) generated by alternative splicing and composed of 332 amino acids with conserved domains important for the function and regulation. Phylogenetic analysis shows that LvanLDH -1 and -2 are closer to the invertebrate counterparts. The LDHvan-1 transcript increased 2.5-fold after hypoxia in gills but not in hepatopancreas, while the LDHvan-2 transcript decreased 14-fold in muscle but not in gills and hepatopancreas. Three bands with LDH activity of ∼60­90 kDa were detected in hepatopancreas, while one band of ∼140 kDa was detected in gills and muscle. The silencing of HIF-1 blocked the increase of LDH mRNA and activity produced by hypoxia in gills. These results demonstrate a single gene for LDH (LvanLDH) that by alternative splicing generates two different LDH subunits (LDHvan-1 and LDHvan-2) that are expressed in a tissue-specific manner during hypoxia via the HIF-1 pathway.

Accumulation and Elimination of Enrofloxacin and Ciprofloxacin in Tissues of Shrimp Litopenaeus vannamei under Laboratory and Farm Conditions.

This study aimed to quantify the accumulation and elimination of Enrofloxacin (ENRO) and Ciprofloxacin (CIPRO) in cultivated Litopenaeus vannamei under controlled laboratory and farm conditions. Laboratory- and farm-raised shrimp were given feed supplemented with 200 mg/kg ENRO for 14 days, followed by a 16-day diet without antibiotics. The levels of ENRO and CIPRO were analyzed by High Performance Liquid Chromatography (HPLC). In the laboratory, ENRO concentrations in the muscle and hepatopancreas reached a maximum (C(max)) of 0.54 ± 0.26 µg/g and 3.52 ± 1.9 µg/g, respectively; C(max) values for CIPRO in the laboratory were 0.18 ± 0.13 µg/g (muscle) and 1.05 ± 0.20 µg/g (hepatopancreas). In farmed shrimp, C(max) values for ENRO were 0.36 ± 0.17 µg/g muscle and 1.60 ± 0.82 µg/g in the hepatopancreas; CIPRO C(max) values were 0.03 ± 0.02 µg/g (muscle) and 0.36 ± 0.08 µg/g (hepatopancreas). Two to fourteen days were necessary to eliminate both antibiotics from muscular tissue and four to more fourteen days for complete elimination of the antibiotics from the hepatopancreas. These results should be considered in terms of minimum concentrations necessary to inhibit Vibrio bacteria to determine whether the current use of this antibiotic is effective in controlling disease.

Molecular characterization of hypoxia inducible factor-1 (HIF-1) from the white shrimp Litopenaeus vannamei and tissue-specific expression under hypoxia.

Hypoxia inducible factor 1 (HIF-1) is a key transcription factor that regulates a variety of molecular responses to hypoxia. Some marine crustaceans experience changes of oxygen tension in their aquatic environment, but knowledge about the function and expression of HIF-1 is very limited. HIF-1 is a heterodimer composed by alpha and beta subunits. We report the complete cDNA sequences of HIF-1alpha and HIF-1beta from the white shrimp Litopenaeus vannamei. HIF-1alpha (LvHIF-1alpha) is 3672bp and codes for 1050 amino acids, while HIF-1beta is 2135bp (LvHIF-1beta) and 608 amino acids. Both, the alpha and beta subunits have the helix-loop-helix (bHLH) and PAS domains. HIF-1alpha also has the oxygen dependent degradation (ODD) and the C-terminal transactivation domain (C-TAD), important for regulation in normoxia. Phylogenetic analyses of the proteins indicate separation of invertebrates from vertebrates. Large differences of HIF-1alpha and HIF-1beta transcripts abundance were detected in gills, hepatopancreas and muscle under normoxia (6mg/L dissolved oxygen, DO) and hypoxia (2.5 and 1.5mg/L DO). HIF-1alpha was more abundant in gills and HIF-1beta in hepatopancreas. Large changes in response to hypoxia were detected for HIF-1alpha in gills, while HIF-1beta remained fairly constant. Glucose and lactate in hemolymph increased rapidly in hypoxia in all cases and up to 4.7 and 5.0-fold, respectively, in response to 1.5mg/L DO for 1h.