The anaplerotic pyruvate carboxylase from white shrimp Litopenaeus vannamei: Gene structure, molecular characterization, protein modelling and expression during hypoxia.

Hypoxic zones are spreading worldwide in marine environments affecting many organisms. Shrimp and other marine crustaceans can withstand environmental hypoxia using several strategies, including the regulation of energy producing metabolic pathways. Pyruvate carboxylase (PC) catalyzes the first reaction of gluconeogenesis to produce oxaloacetate from pyruvate. In mammals, PC also participates in lipogenesis, insulin secretion and other processes, but this enzyme has been scarcely studied in marine invertebrates. In this work, we characterized the gene encoding PC in the white shrimp Litopenaeus vannamei, modelled the protein structure and evaluated its gene expression in hepatopancreas during hypoxia, as well as glucose and lactate concentrations. The PC gene codes for a mitochondrial protein and has 21 coding exons and 4 non-coding exons that generate three transcript variants with differences only in the 5'-UTR. Total PC expression is more abundant in hepatopancreas compared to gills or muscle, indicating tissue-specific expression. Under hypoxic conditions of 1.53 mg/L dissolved oxygen, PC expression is maintained in hepatopancreas, indicating its key role even in energy-limited conditions. Finally, both glucose and lactate concentrations were maintained under hypoxia for 24-48 h in hepatopancreas.

Longitudinal variations in the gastrointestinal microbiome of the white shrimp, Litopenaeus vannamei.

The shrimp gut is a long digestive structure that includes the Foregut (stomach), Midgut (hepatopancreas) and Hindgut (intestine). Each component has different structural, immunity and digestion roles. Given these three gut digestive tract components' significance, we examined the bacterial compositions of the Foregut, Hindgut, and Midgut digestive fractions. Those bacterial communities' structures were evaluated by sequencing the V3 hypervariable region of the 16S rRNA gene, while the functions were predicted by PICRUSt2 bioinformatics workflow. Also, to avoid contamination with environmental bacteria, shrimp were maintained under strictly controlled conditions. The pairwise differential abundance analysis revealed differences among digestive tract fractions. The families Rhodobacteraceae and Rubritalaceae registered higher abundances in the Foregut fraction, while in the Midgut, the families with a higher proportion were Aeromonadaceae, Beijerinckiaceae and Propionibacteriaceae. Finally, the Cellulomonadaceae family resulted in a higher proportion in the Hindgut. Regarding the predicted functions, amino acid and carbohydrate metabolism pathways were the primary functions registered for Foregut microbiota; conversely, pathways associated with the metabolism of lipids, terpenoids and polyketides, were detected in the Midgut fraction. In the Hindgut, pathways like the metabolism of cofactors and vitamins along with energy metabolism were enriched. Structural changes were followed by significant alterations in functional capabilities, suggesting that each fraction's bacteria communities may carry out specific metabolic functions. Results indicate that white shrimp's gut microbiota is widely related to the fraction analyzed across the digestive tract. Overall, our results suggest a role for the dominant bacteria in each digestive tract fraction, contributing with a novel insight into the bacterial community.

Granulomatous bacterial diseases in fish: An overview of the host's immune response.

Bacterial diseases represent the main impediment to the development of fish aquaculture. Granulomatous diseases caused by bacteria lead to fish culture losses by high mortality rates and slow growth. Bacteria belonging to genera Streptococcus spp., Mycobacterium sp., Nocardia sp., Francisella sp., and Staphylococcus sp. have been implicated in the development of granulomatous processes. The granuloma formation and the fish's immune response continue to be the subject of scientific research. In fish, the first defense line is constituted by non-specific humoral factors through growth-inhibiting substances such as transferrin and antiproteases, or lytic effectors as lysozyme and antimicrobial peptides, and linking with non-specific phagocyte responses. If the first line is breached, fish produce antibody constituents for a specific humoral defense inhibiting bacterial adherence, as well as the mobilization of non-phagocytic host cells and counteracting toxins from bacteria. However, bacteria causing granulomatous diseases can be persistent microorganisms, difficult to eliminate that can cause chronic diseases, even using some immune system components to survive. Understanding the infectious process leading to granulomatosis and how the host's immune system responds against granulomatous diseases is crucial to know more about fish immunology and develop strategies to overcome granulomatous diseases.

Arabinoxylans and gelled arabinoxylans used as anti-obesogenic agents could protect the stability of intestinal microbiota of rats consuming high-fat diets.

This study evaluated the effect of using arabinoxylans (AX) and gelled arabinoxylans (AxGel) as anti-obesogenic agents on the faecal microbiota of rats fed with a high-fat (HF) diet. Results revealed that the HF content in diet caused obesity in rats and alterations in the taxonomic and functional profiles of faecal microbiota. However, these effects were lessened when AX and AxGel were used as ingredients of the HF diet. Metabolisms of amino acids and energy, as well as genetic information processing, were negatively affected when the rats consumed the HF diet; however, this effect was not observed if AX and AxGel were included as part of the diet formulation. Results suggest that AX may act as a prebiotic agent. Therefore, AX and AxGel could be considered as hypothetical protectors of the intestinal microbiota against HF consumption.

Proteomic profiling of integral membrane proteins associated to pathogenicity in Vibrio parahaemolyticus strains.

Vibrio parahaemolyticus has been recognized as the causal agent of early mortality syndrome and is currently considered an emerging shrimp disease causing losses of millions in the aquaculture industry. Integral membrane proteins are widely recognized as pathogenicity factors involved in essential mechanisms for V. parahaemolyticus infection, which makes them attractive as therapeutic targets. However, their physico-chemical properties and weak expression has resulted in under-representation of these proteins in conventional bottom-up proteomics, making integral membrane proteomics a challenging task. Integral membrane proteins from a bacterial strain isolated from the hepatopancreases of white shrimp with early mortality syndrome and identified by 16S rRNA sequencing as V. parahaemolyticus and an ATCC strain that is pathogenic for humans were obtained by a sequential extraction method and subjected to relative quantification and identification by isobaric Tags for Relative and Absolute Quantitation. A homology database search resulted in identification of more than two hundred proteins, 35 of which are recognized as pathogenic factors showed statistically significant differential accumulation between the strains. These proteins are mainly associated with adherence, secretion systems, cell division, transport, lysogenization, movement and virulence. Identification of pathogenicity-related proteins in V. parahaemolyticus provides valuable information for developing strategies based on molecular mechanisms that inhibit these proteins, which may be useful therapeutic targets for assisting the shrimp and aquaculture industry.

From microbes to fish the next revolution in food production.

Increasing global population and the consequent increase in demand for food are not a new story. Agroindustrial activities such as livestock help meet this demand. Aquaculture arose decades ago and revolutionized the agroindustrial activity as a significant food generator. However, like livestock, aquaculture is based on finite resources and has been accused of being unsustainable. Abandoning aquaculture is not an option considering the food, foreign exchange, and employment it generates, and therefore must be reinvented. Among the many alternatives suggested to make aquaculture more sustainable, microorganisms have been highlighted as a direct food source for cultured fish and crustaceans, a strategy that promises to revolutionize aquaculture by eliminating waste. Considering waste, as part of a cycle, it can increase stock densities and reduce emissions of contaminants and operational costs.

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.

Effect of ante-mortem hypoxia on the physicochemical and functional properties of white shrimp (Litopenaeus vannamei) muscle stored on ice.

The effect of ante-mortem hypoxia on the physicochemical and functional properties of raw and cooked white shrimp was studied. Hue angle was greater (p ≤ 0.05) for stressed raw shrimp compared to control (greener color); whereas a lower angle was detected for cooked stressed shrimp (redder/orange coloration). In addition, hue angle increased (p ≤ 0.05) over the ice storage period for control and stressed shrimp (raw and/or cooked). Muscle hardness and shear force showed no differences when comparing control and stressed shrimp (raw and/or cooked). However, during ice storage, shear force increased (p ≤ 0.05) by 22% and 9% for control and stressed raw shrimp, respectively; in contrast, shear force and muscle hardness decreased for cooked shrimp (p ≤ 0.05). Control showed more (p ≤ 0.05) elasticity than stressed cooked shrimp. Stressed raw shrimp showed a water holding capacity 10.8% lower (p ≤ 0.05) than control. However, during the storage, water holding capacity increased (p ≤ 0.05) reaching similar values to control after day 4. Muscle protein solubility of stressed shrimp was 31% lower than control; however, no differences (p > 0.05) were observed after the second day. The thermal stability of myosin (T max) showed differences (p ≤ 0.05) among control and stressed shrimp, whereas no differences for ΔH were observed. Results showed the influence of ante-mortem hypoxia on the physicochemical and functional properties of white shrimp muscle.

Experimental infection and detection of necrotizing hepatopancreatitis bacterium in the American lobster Homarus americanus.

Necrotizing hepatopancreatitis bacterium (NHPB) is an obligated intracellular bacteria causing severe hepatopancreatic damages and mass mortalities in penaeid shrimp. The worldwide distribution of penaeid shrimp as alien species threatens the life cycle of other crustacean species. The aim of the experiment was to evaluate the possibility of experimentally infecting the American lobster (Homarus americanus) with NHPB extracted from shrimp hepatopancreas. Homogenates from infected shrimp were fed by force to lobsters. Other group of lobsters was fed with homogenates of NHPB-free hepatopancreas. After the 15th day from initial inoculation, the presence of NHPB was detected by polymerase chain reaction in feces and hepatopancreas from lobsters inoculated with infected homogenates. Necrotized spots were observed in the surface of lobster hepatopancreas. In contrast, lobsters fed on NHPB-free homogenates resulted negative for NHPB. Evidence suggests the plasticity of NHPB which can infect crustacean from different species and inhabiting diverse latitudes. Considering the results, the American lobster could be a good candidate to maintain available NHPB in vivo.

Shrimp invertebrate lysozyme i-lyz: gene structure, molecular model and response of c and i lysozymes to lipopolysaccharide (LPS).

The invertebrate lysozyme (i-lyz or destabilase) is present in shrimp. This protein may have a function as a peptidoglycan-breaking enzyme and as a peptidase. Shrimp is commonly infected with Vibrio sp., a Gram-negative bacteria, and it is known that the c-lyz (similar to chicken lysozyme) is active against these bacteria. To further understand the regulation of lysozymes, we determined the gene sequence and modeled the protein structure of i-lyz. In addition, the expression of i-lyz and c-lyz in response to lipopolysaccharide (LPS) was studied. The shrimp i-lyz gene is interrupted by two introns with canonical splice junctions. The expression of the shrimp i-lyz was transiently down-regulated after LPS injection followed by induction after 6 h in hepatopancreas. In contrast, c-lyz was up-regulated in hepatopancreas 4 h post-injection and slightly down-regulated in gills. The L. vannamei i-lyz does not contain the catalytic residues for muramidase (glycohydrolase) neither isopeptidase activities; however, it is known that the antibacterial activity does not solely rely on the enzymatic activity of the protein. The study of invertebrate lysozyme will increase our understanding of the regulatory process of the defense mechanisms.

An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: cloning, characterization and expression analysis.

Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.

Transcriptome analysis of gills from the white shrimp Litopenaeus vannamei infected with White Spot Syndrome Virus.

We report the analysis of 872 cDNA clones from a WSSV-infected white shrimp Litopenaeus vannamei gill cDNA library. Comparison against the GenBank protein and nucleotide sequences identified 87% (E < or = 10(-2)) as previously known genes, while 13% are novel sequences. The 601 ESTs (87%) represent transcripts of 276 genes. These genes were categorized into 12 groups according to their functions. The more abundant categories were (1) ribosomal proteins (21%), (2) WSSV transcripts and sequences without homology to proteins deposited in the non-redundant database (15%), (3) hypothetical proteins (12%) which include genes never described in shrimp and (4) metabolism related proteins (9%). We also found genes involved in stress and immune response; and only one involved in ion transport. Full-length sequences of keratinocyte associated protein 2 (KCP2), selenoprotein M (SelM), chicadae, prohibitin and oncoprotein nm23 are reported. Their mRNAs steady state levels in addition to ferritin, changed at different times post-WSSV infection as estimated by RT-PCR. These results suggest that WSSV alters gene expression in gills and has led to the identification of novel white shrimp specific genes.

The cytosolic manganese superoxide dismutase from the shrimp Litopenaeus vannamei: molecular cloning and expression.

Manganese containing superoxide dismutase (SOD) is normally a nuclear-encoded mitochondrial enzyme in eukaryotic organisms; however, a cytoplasmic manganese SOD (cMnSOD) was found in crustaceans that use hemocyanin as oxygen carrier. The complete cDNA and deduced amino acid sequence of a cMnSOD from Litopenaeus vannamei were determined. The coding sequence predicts a 287 residues protein with a unique 61 amino acids extension at the N-terminus and lacking a mitochondrial-targeting sequence. Phylogenetic analysis clusters cMnSODs and mitochondrial MnSODs in two separate groups. cMnSOD transcripts were detected in hemocytes, heart, hepatopancreas, intestine, nervous system, muscle, pleopods and gills. Since hemocytes are key defense cells and their reactions produce superoxide radicals, the infection by white spot syndrome virus on the cMnSOD transcript levels were investigated and found to increase transiently 1h post-infection and then decrease as the viral infection progressed to levels significantly lower than uninfected controls by 12h post-infection.

Purification and characterization of alpha 2-macroglobulin from the white shrimp (Penaeus vannamei).

alpha(2)-Macroglobulin (alpha(2)M) is a broad-spectrum protease-binding protein abundant in plasma from vertebrates and several invertebrate phyla. This protein was purified from cell-free hemolymph of the white shrimp, Penaeus vannamei, using Blue-Sepharose and Phenyl-Sepharose chromatography. The shrimp alpha(2)M is a 380 kDa protein, a homodimer of two apparently identical subunits of approximately 180 kDa linked by disulphide bridges. The amino acid sequence of the N-terminus is similar to the Limulus alpha(2)M counterpart. The shrimp alpha(2)M has a wide inhibition spectrum against different proteinase types including trypsin, leucine amino peptidase, chymotrypsin, elastase and papain. The secondary structure of shrimp alpha(2)M is mainly beta-sheet (36%), with a characteristic minimum elipticity at 217 nm. Evidence for a thiolester-mediated inhibition mechanism of proteases by alpha(2)M was provided by inactivation with methylamine.

In the spiny lobster (Panulirus interruptus) the prophenoloxidase is located in plasma not in haemocytes.

In the spiny lobster (Panulirus interruptus), unlike other crustaceans most of the prophenoloxidase (proPO) was detected in cell-free plasma (86.3%). In spite of its location, lobster proPO activating system has a similar activation mechanism to other crustacean proPO systems. Haemocyte lysate was able to activate the plasma proPO indicating location of the prophenoloxidase activating enzyme (PPAE) in haemocytes. Lobster haemocyte PPAE was isolated by affinity chromatography and its participation as activating enzyme was demonstrated. This enzyme is a serine-proteinase that transforms the inactive form (proPO) to an active one (phenoloxidase). The PPAE was also present in the cell-free supernatant of haemocytes previously incubated with Vibrio alginolyticus.