Pacific Whiteleg Shrimp Litopenaeus Research Articles

Potential role for microRNA in facilitating physiological adaptation to hypoxia in the Pacific whiteleg shrimp Litopenaeus vannamei

Hypoxia is one of the most common physiological stressors in shrimp farming. Post-transcriptional regulation by microRNAs has been recognized as a ubiquitous strategy to enable transient phenotypic plasticity and adaptation to stressful environment, but involvement of microRNAs in hypoxia stress response of penaeid shrimp remains elusive. In this study, small RNA sequencing and comparative transcriptomic analysis was conducted to construct a comprehensive microRNA dataset for the whiteleg shrimp Litopenaeus vannamei exposed to hypoxia challenge. A total of 3324 known miRNAs and 8 putative novel miRNAs were identified, providing a valuable resource for future investigation on the functional mechanism of miRNAs in shrimp. Upon hypoxia, 1213 miRNAs showed significant differential expression, and many well-known miRNAs involved in hypoxia tolerance such as miR-210, let-7, miR-143 and miR-101 were identified. Remarkably, the vast majority of these miRNAs were up-regulated, suggesting that up-regulation of miRNAs may represent an effective strategy to inhibit protein translation under stressful hypoxic condition. The differentially expressed miRNAs were potentially targeting a wide variety of genes, including those with essential roles in hypoxia tolerance such as HIF1a and p53. GO and KEGG enrichment analysis further revealed that a broad range of biological processes and metabolic pathways were over-represented. Several GO terms associated with gene transcription and translation and KEGG pathways related to cytoskeleton remodeling, immune defense and signaling transduction were enriched, highlighting the crucial roles of these cellular events in the adaptation to hypoxia. Taken together, our study revealed that the differentially expressed miRNAs may regulate host response to hypoxia by modulating the expression of stress response genes such as HIF1a and p53 and affecting key cellular events involved in hypoxia adaptation. The findings would expand our knowledge of the biochemical and molecular underpinnings of hypoxia response strategies used by penaeid shrimp, and contribute to a better understanding of the molecular mechanisms of hypoxia tolerance in decapod crustaceans.

The effects of season, aeration and light intensity on the performance of pacific whiteleg shrimp (Litopenaeus vannamei) polycultured with seaweed (Gracilaria verrucosa)

This study was aimed to determine impact of stocking season, additional oxygen supply and light intensity on performance of pacific white leg shrimp (Litopenaeus vannamei) polycultured with seaweed Gracilaria verrucosa. Three sets of experiments were used and each experiment was conducted separately in 3 different season (factor W). Three factors and the interaction, that is, stocking seasons (W1: March-June;. W2 : July-October and W3 : November-February), Oxygen supply (O, with or without supply aeration of 6.5 ppm) and light intensity (with or without addition of light 640 lux) was observed. The experiment was conducted in 16 polyethilen net place in concrete tanks (1.2 m3). Shrimp (average weight 0.1 g and lenght 1.2 cm) with density 94 shrimp/m3 and seaweed 2.188 g/m3, cultured for 94 days. Data collected included absolute growth, specific growth rate (SGR), survival rate (SR) and biomass production of shrimp and seaweed. The result showed that culture period March to June, additonal light and suply DO gave the best result with shrimp absolute growth 13.23 g, SGR 5.09 %/day, SR 99.64 % and biomass production 1.256.36 g/m3. Absolute growth of G. verrucosa was 5.223.75g, SGR 268 %/day and biomass production 12.608.55 g/m3.

Examining De Novo Transcriptome Assemblies via a Quality Assessment Pipeline

New de novo transcriptome assembly and annotation methods provide an incredible opportunity to study the transcriptome of organisms that lack an assembled and annotated genome. There are currently a number of de novo transcriptome assembly methods, but it has been difficult to evaluate the quality of these assemblies. In order to assess the quality of the transcriptome assemblies, we composed a workflow of multiple quality check measurements that in combination provide a clear evaluation of the assembly performance. We presented novel transcriptome assemblies and functional annotations for Pacific Whiteleg Shrimp (Litopenaeus vannamei ), a mariculture species with great national and international interest, and no solid transcriptome/genome reference. We examined Pacific Whiteleg transcriptome assemblies via multiple metrics, and provide an improved gene annotation. Our investigations show that assessing the quality of an assembly purely based on the assembler's statistical measurements can be misleading; we propose a hybrid approach that consists of statistical quality checks and further biological-based evaluations.

Effect of Argemone mexicana active principles on inhibiting viral multiplication and stimulating immune system in Pacific white leg shrimp Litopenaeus vannamei against white spot syndrome virus

Argemone mexicana called as Mexican prickly poppy is a species of poppy found in Mexico and now widely naturalized in many parts of the world with broad range of bioactivities including anthelmintic, cures lepsory, skin-diseases, inflammations and bilious fevers. Plant parts of A. mexicana were serially extracted with hexane, ethyl acetate, methanol and performed antiviral and immunostimulant screening against WSSV and Vibrio harveyi respectively. The control groups succumbed to death 100% within three days, whereas the mortality was significantly (P < 0.5) reduced to 17.43 and 7.11 in the ethyl acetate extracts of stem and root treated shrimp group respectively. The same trend was reflected in the immunostimulant screening also. Different diets were prepared by the concentrations of 100 (AD-1), 200 (AD-2), 300 (AD-3) and 400 (AD-4) mg kg−1 using A. mexicana stem and root ethyl acetate extracts and fed to Pacific white leg shrimp Litopenaeus vannamei weighed about 9.0 ± 0.5 g for 30 days. The control groups fed with the normal diets devoid of A. mexicana extracts. The antiviral screening results revealed that, the ethyl acetate extract of the stem and root were effectively suppressed the WSSV and it reflected in the lowest cumulative mortality of treated shrimps. After termination of feeding trials, group of shrimps from control and each experimental group were challenged with virulent WSSV by intramuscular (IM) injection and studied cumulative mortality, molecular diagnosis by quantitative real time PCR (qRT-PCR), biochemical, haematological and immunological parameters. Control group succumbed to 100% death within four days, whereas the survival was significantly (P < 0.001) increased to 30, 45, 75 and 79% in AD1, AD-2, AD-4 and AD-5 diets fed shrimp groups respectively. qRT PCR results with positive correlation analysis revealed that, the WSSV copies were gradually decreased when increasing the A. mexicana extracts in the diets. The highest concentrations (300 and 400 mg g−1) of A. mexicana extracts in the diets helped to reduce the protein level significantly (P < 0.05) after WSSV challenge. The diets AD-3 and AD-4 also helped to decrease the coagulation time of maximum 64–67% from control groups and maintained the normal level of total haemocyte, oxyhaemocyanin level after WSSV challenge. The proPO level was significantly increased (Column: F = 35.93; P ≤ 0.001 and Row: F = 37.14; P ≤ 0.001) in the AD1-AD-4 diet fed groups from the control diet fed groups. The lowest intra-agar lysozyme activity of 1.63 mm found in control diet fed group and the activity were significantly (P < 0.05) increased to 4.86, 7.89, 9.12 and 10.45 mm of zone of inhibition respectively in AD1 to AD4 diet fed groups.

Tetraselmis chuii biomass as a potential feed additive to improve survival and oxidative stress status of Pacific white-leg shrimp Litopenaeus vannamei postlarvae

Shrimp is an important traded fishery commodity. When subjected to stress, shrimp usually suffers from oxidative stress, which leads to cell injury, senescence, and death. To maintain shrimp good health, performance and production, antioxidant and immune systems are important. Natural antioxidants found in microalgae may be used to increase the cell protection against oxidative damage, being a promising alternative to the carcinogenic synthetic antioxidants. In this study, Tetraselmis chuii was evaluated for its effect on survival, growth and oxidative stress biomarkers on Litopenaeus vannamei postlarvae (PL). The antioxidant properties of the formulated feed with T. chuii inclusion were determined using four antioxidant chemical assays. Meanwhile, the oxidative stress biomarkers on PL were analyzed by hydrogen peroxide, membrane stability and lipid peroxidation assays. Results showed that PL reared on diets supplemented with 50% T. chuii had a significantly higher (P ≤ 0.05) survival (97.6 ± 1.4%) and lower oxidative stress in terms of hydrogen peroxide content (10.08 ± 0.4 mM g−1 FW) and electrolyte leakage (10.8 ± 0.3%). The result of this study also showed that shrimp PL reared on diets supplemented with microalgal, T. chuii have high resistance to reverse salinity stress test (76.7–100%). However, no significant differences (P ≥ 0.05) were found in the growth and lipid peroxidation. Due to the positive effect on oxidative stress status, survival and resistance to salinity stress, the feeding of L. vannamei PL with diet containing at least 50% of T. chuii is recommended as a natural source of antioxidant for PL.

Antimicrobial activity of partially characterized analytes fromBacillus pumilus(B2)

This study was carried out to characterize the antimicrobial substance produced by the strain of Bacillus pumilus (B2) obtained from Novozymes Biologicals Inc. to compare its antimicrobial activity by agar well diffusion assay and bacteriocin activity assay via critical dilution method against seven different strains of Vibrio spp., specifically V. alginolyticus (A01), V. cholerae (C01), V. fluvialis (F01, F02), V herveyii (H), V. mimicus (M01), V. parahaemolyticus (P01) and V. vulnificus (V01, V02). All Vibrio spp. were isolated from the hemolymph and intestine of the white faeces disease-infected moribund pacific white-leg shrimp Litopenaeus vannamei (Boone 1931) and one strain (V. harveyi) from its diseased postlarva. The cell-free neutralized supernatant (CFNS) of B2 showed moderate thermo-stability being stable up to 70°C for 60 min with, however, reducing activity above 80°C for 20 min. B2 antimicrobials showed a stable activity within the pH ranging from 6 to 10 at room temperature and at 4°C, while residual antimicrobial activity of crude CFNS showed tolerance to salinity up to 7% of sodium chloride below 4°C. No B2 activity was obtained while exposed to proteolytic enzyme, such as proteinase k and pepsin, while its activity kept stable exposed to lipase. Initial B2 characterization for antimicrobial substance in CFNS revealed proteinaceous in nature owing to activity loss against proteolytic enzymes and no lipid moiety activity against lipase, which could be categorized as bacteriocin-like inhibitory substance having potential application against several strains of Vibrio spp. in aquaculture.

Cloning and characterization of tyrosine hydroxylase (TH) from the pacific white leg shrimp Litopenaeus vannamei, and its expression following pathogen challenge and hypothermal stress

Tyrosine hydroxylase (TH) belongs to the biopterin-dependent aromatic amino acid hydroxylase enzyme family, and it represents the first and rate-limiting step in the synthesis of catecholamines that are required for physiological and immune process in invertebrates and vertebrates. Cloned Litopenaeus vannamei TH (LvTH), containing a short alpha helix domain, a catalytic core, a regulatory domain, a phosphorylation site and two potential N-linked glycosylation sites as presented in vertebrate and insect THs without acidic region and signal peptide cleavage sites at the amino-terminal, exhibited a similarity of 60.0–61.2% and 45.0–47.0% to that of invertebrate and vertebrate THs, respectively. Further, LvTH expression was abundant in gill and haemocytes determined by quantitative real-time PCR. L. vannamei challenged with Vibrio alginolyticus at 105 cfu shrimp−1 revealed significant increase of LvTH mRNA expression in haemocytes within 30–120 min and in brain within 15–30 min followed with recuperation. In addition, shrimps exposed to hypothermal stress at 18 °C significantly increased LvTH expression in haemocytes and brain within 30–60 and 15–60 min, respectively. The TH activity and haemolymph glucose level (haemocytes-free) significantly increased in pathogen challenged shrimp at 120 min and 60 min, and in hypothermal stressed shrimp at 30–60 and 30 min, respectively. These results affirm that stress response initiates in the brain while haemocytes display later response. Further, the significant elevation of TH activity in haemolymph is likely to confer by TH that released from haemocytes. In conclusion, the cloned LvTH in our current study is a neural TH enzyme appears to be involved in the physiological and immune responses of whiteleg shrimp, L. vannamei suffering stressful stimulation.

Uncovering Hemocyanin Subunit Heterogeneity in Penaeid Shrimp using RNA-Seq.

Aquatic crustaceans can experience low levels of O2 alone but more often in combination with high levels of CO2 both in natural estuaries and in aquaculture ponds. Hemocyanin, the respiratory pigment in many crustacean species, facilitates O2 transport and is documented to change in abundance, structure, and function in response to low O2 The impacts of high CO2 on the respiratory pigment are less clear. In this synthesis we bring together data from recently published and new RNA-Seq studies toward the aims of defining the full repertoire of hemocyanin subunits, as well as their differential expression and regulation in the Penaeoidea family in response to low O2 with or without high CO2 RNA-Seq data were collected from the hepatopancreas tissues of aquacultured Pacific whiteleg shrimp Litopenaeus vannamei, wild-caught L. vannamei, and wild-caught Atlantic brown shrimp Farfantepenaeus aztecus. De novo assembly yielded high-quality stranded transcriptomes. Manual curation of the hemocyanin subunits from all three groups of penaeid shrimp confirmed the existence of a small γ-type hemocyanin subunit (HcS), greater sequence diversity in the large γ-type hemocyanin subunit than previously identified (HcL1-3 isoforms) and expression of a β-type hemocyanin subunit (HcB) previously unidentified in Penaeid shrimp. Relative abundance of transcripts encoding these hemocyanin isoforms differed within and among the three species/strains. Exposure to low O2 induced expression of all of the subunits in aquacultured L. vannamei With concurrent exposure to high CO2, the number of γ-type hemocyanin transcripts decreased while the expression of β-type transcripts remained unchanged. Together with functional data for hemocyanins in the same shrimp species/strains, the RNA-Seq approach shows great promise to provide new insights into the connection between sequence, protein structure, and physiological function of respiratory pigments in this decapod crustacean family.

Identification and function analysis of a novel vascular endothelial growth factor, LvVEGF3, in the Pacific whiteleg shrimp Litopenaeus vannamei

VEGF signaling pathway is first discovered in mammals and proved to play important roles in the biological processes of angiogenesis, tumor migration, cell differentiation, apoptosis, host-virus interaction etc. Three members in the VEGF signaling pathway, including LvVEGFR, LvVEGF1 and LvVEGF2 in shrimp have been proved to be related with WSSV infection in our previous studies. Currently, another member of VEGF family, LvVEGF3, was isolated and its function during the WSSV infection of shrimp was studied. The deduced amino acid sequence of LvVEGF3 contained a signal peptide, a typical PDGF/VEGF domain and a cysteine-knot motif (CXCXC). Tissue distribution analysis showed that LvVEGF3 was predominantly expressed in hemocytes. The transcriptional level of LvVEGF3 in hemocytes was apparently up-regulated during WSSV infection. Silencing of LvVEGF3 with double-stranded RNA caused a reduction of the cumulative mortality rate of shrimp during WSSV infection. The expression of LvVEGFR was apparently down-regulated after LvVEGF3 silencing and up-regulated after injection of recombinant LvVEGF3 protein, suggesting an interaction between LvVEGF3 and LvVEGFR. Furthermore, the interaction between LvVEGFR and LvVEGF3 was confirmed using the yeast two-hybrid system. The results provided new insights into understanding the role of VEGF signaling pathway during virus infection.

Black gill disease of Pacific white leg shrimp (Litopenaeus vannamei) by Aspergillus flavus

Objective: To study the epidemiology of black gill disease in white leg shrimp which is a major problem being faced by the commercial shrimp farmers who are culturing Litopenaeus vannamei (L. vannamei) in India. Methods: The normal and infected shrimps were collected from shrimp pond and the gill was preserved in appropriate preservative for histopathological examination and scanning electron microscope analysis. Pathogenic fungus was isolated from black gill of L. vannamei in potato dextrose agar medium. Morphological study and fungal strain identification were done by using light microscopy and scanning electron microscope. Fungal DNA was amplified by ITS4 and ITS5 primers and gene sequencing was done by Macrogen Inc., Korea. Phylogenetic tree was prepared by using MEGA 6 software. Results: Fungal spores and hyphae were observed both in internal and external gill surface of infected shrimps. Fungal spores were round in shape and mature sporangium was observed. The histopathology study showed clearly that infected gill was damaged by the fungi. Scanning electron microscopic study showed adherence of fungi in infected gill. Internal transcribed spacer gene sequencing revealed that it was caused by Aspergillus flavus. Conclusions: The outcome of the present study would help to know the cause of black gill disease and to understand the effect of pathogenic fungi in shrimp culture. This study will initiate researchers for work in field of treatment or prevention of black gill disease in commercial L. vannamei culture.

Using microsatellite markers to identify heritability of Pacific whiteleg shrimp Litopenaeus vannamei

Pacific whiteleg shrimp (Litopenaeus vannamei) is an economically relevant shrimp species in many Asian countries. The specific objective of the current research was to assess microsatellite markers in screening the fast-growth of domesticated L. vannamei stocks to establish a founder population for breeding-selection plans. The postlarvae produced by the reproduction of second generation broodstock were cultured in the same conditions throughout a five months growing period. Ninety juvenile shrimp were selected from the slow-, medium- and the fast-growth groups, and ten microsatellite markers were used to investigate their genetic diversity, and to understand the improvement of a breeding-selection scheme. Ten polymorphic loci (markers) (M1-M10) were produced at ten loci in this sample, among them Primer M8 was the highest polymorphic locus and M7 was the lowest one. A specific locus was found in the fast-growth group using Primer M5. The longest genetic distance (0.481) was determined between the fast- and medium-growth groups and the shortest (0.098) was between the slow- and medium-growth groups; therefore, the largest genetic identity (0.946) was observed between the slow- and medium-growth groups and the smallest (0.667) was observed between the medium- and fast-growth groups. The Unweighted Paired Group with Arithmetic Average (UPGMA) dendrogram based on Nei’s genetic distances provided two different groups; the first consist of the slow- and medium-growth groups and the second the fast-growth group. Selection response and realized heritability for growth were 11.55% and 31.26%, respectively. Therefore, this set of microsatellite markers would provide a useful tool in shrimp breeding schemes.

One type of VEGFR is involved in WSSV infection to the Pacific whiteleg shrimp Litopenaeus vannamei

VEGF signaling pathway plays vital roles in many physiological processes including cell proliferation, differentiation, migration, survival, cell–cell communication, vessel permeability and virus-host interaction in mammalian species. However, the VEGF signaling pathway and its biological function are still poorly understood in crustaceans. In the present study, an essential member of VEGF signaling pathway, VEGF receptor (LvVEGFR), was isolated from Penaeid shrimp Litopenaeus vannamei and its function during virus infection was analyzed. The deduced amino acid sequence of LvVEGFR possessed all common features of VEGFRs reported in other species, including a signal peptide, six IG-like domains, one immunoglobulin subtype 2 domain, a transmembrane domain, a juxtamembrane domain, a protein kinase domain separated by a kinase insert sequence, one ATP binding site and one tyrosine-protein kinase active site. LvVEGFR is mainly expressed in hemocytes and intestine. The transcriptional level of LvVEGFR could be obviously up-regulated in hemocytes and intestine after WSSV infection. Silencing of LvVEGFR gene by double-strand RNA (dsRNA) interference could not only lead to a decrease of virus copy number in WSSV infected shrimp, but also reduce the mortality of shrimp during WSSV infection. These data suggested that VEGF signaling pathway might play an important role during viral infection to shrimp.

Novel transcriptome assembly and improved annotation of the whiteleg shrimp (Litopenaeus vannamei), a dominant crustacean in global seafood mariculture.

We present a new transcriptome assembly of the Pacific whiteleg shrimp (Litopenaeus vannamei), the species most farmed for human consumption. Its functional annotation, a substantial improvement over previous ones, is provided freely. RNA-Seq with Illumina HiSeq technology was used to analyze samples extracted from shrimp abdominal muscle, hepatopancreas, gills and pleopods. We used the Trinity and Trinotate software suites for transcriptome assembly and annotation, respectively. The quality of this assembly and the affiliated targeted homology searches greatly enrich the curated transcripts currently available in public databases for this species. Comparison with the model arthropod Daphnia allows some insights into defining characteristics of decapod crustaceans. This large-scale gene discovery gives the broadest depth yet to the annotated transcriptome of this important species and should be of value to ongoing genomics and immunogenetic resistance studies in this shrimp of paramount global economic importance.

White spot syndrome virus (WSSV) genome stability maintained over six passages through three different penaeid shrimp species.

White spot syndrome virus (WSSV) replicates rapidly, can be extremely pathogenic and is a common cause of mass mortality in cultured shrimp. Variable number tandem repeat (VNTR) sequences present in the open reading frame (ORF)94, ORF125 and ORF75 regions of the WSSV genome have been used widely as genetic markers in epidemiological studies. However, reports that VNTRs might evolve rapidly following even a single transmission through penaeid shrimp or other crustacean hosts have created confusion as to how VNTR data is interpreted. To examine VNTR stability again, 2 WSSV strains (PmTN4RU and LvAP11RU) with differing ORF94 tandem repeat numbers and slight differences in apparent virulence were passaged sequentially 6 times through black tiger shrimp Penaeus monodon, Indian white shrimp Feneropenaeus indicus or Pacific white leg shrimp Litopenaeus vannamei. PCR analyses to genotype the ORF94, ORF125 and ORF75 VNTRs did not identify any differences from either of the 2 parental WSSV strains after multiple passages through any of the shrimp species. These data were confirmed by sequence analysis and indicate that the stability of the genome regions containing these VNTRs is quite high at least for the WSSV strains, hosts and number of passages examined and that the VNTR sequences thus represent useful genetic markers for studying WSSV epidemiology.

Crystal structure of the shrimp proliferating cell nuclear antigen: structural complementarity with WSSV DNA polymerase PIP-box.

DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV) that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA). This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein). The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems.

The cytochrome c oxidase and its mitochondrial function in the whiteleg shrimp Litopenaeus vannamei during hypoxia

Cytochrome c oxidase (COX), which is located in the inner membrane of mitochondria, is a key constituent of the electron transport chain that catalyzes the reduction of oxygen. The Pacific whiteleg shrimp Litopenaeus vannamei is constantly exposed to hypoxic conditions, which affects both the central metabolism and the mitochondrial function. The purpose of this study was to isolate shrimp mitochondria, identify the COX complex and to evaluate the effect of hypoxia on the shrimp mitochondrial function and in the COX activity. A 190 kDa protein was identified as COX by immunodetection techniques. The effect of hypoxia was confirmed by an increase in the shrimp plasma L-lactate concentration. COX activity, mitochondrial oxygen uptake and protein content were reduced under hypoxic conditions, and gradually restored as hypoxia continued, this suggests an adaptive mitochondrial response and a highly effective COX enzyme. Both mitochondrial oxygen uptake and COX activity were completely inhibited by KCN and sodium azide, suggesting that COX is the unique oxidase in L. vannamei mitochondria.

Crystal structure of shrimp arginine kinase in binary complex with arginine—a molecular view of the phosphagen precursor binding to the enzyme

Arginine kinase (AK) is a key enzyme for energetic balance in invertebrates. Although AK is a well-studied system that provides fast energy to invertebrates using the phosphagen phospho-arginine, the structural details on the AK-arginine binary complex interaction remain unclear. Herein, we determined two crystal structures of the Pacific whiteleg shrimp (Litopenaeus vannamei) arginine kinase, one in binary complex with arginine (LvAK-Arg) and a ternary transition state analog complex (TSAC). We found that the arginine guanidinium group makes ionic contacts with Glu225, Cys271 and a network of ordered water molecules. On the zwitterionic side of the amino acid, the backbone amide nitrogens of Gly64 and Val65 coordinate the arginine carboxylate. Glu314, one of proposed acid-base catalytic residues, did not interact with arginine in the binary complex. This residue is located in the flexible loop 310-320 that covers the active site and only stabilizes in the LvAK-TSAC. This is the first binary complex crystal structure of a guanidine kinase in complex with the guanidine substrate and could give insights into the nature of the early steps of phosphagen biosynthesis.

Expression, purification, crystallization and X-ray crystallographic studies of different redox states of the active site of thioredoxin 1 from the whiteleg shrimp Litopenaeus vannamei.

Thioredoxin (Trx) is a 12 kDa cellular redox protein that belongs to a family of small redox proteins which undergo reversible oxidation to produce a cystine disulfide bond through the transfer of reducing equivalents from the catalytic site cysteine residues (Cys32 and Cys35) to a disulfide substrate. In this study, crystals of thioredoxin 1 from the Pacific whiteleg shrimp Litopenaeus vannamei (LvTrx) were successfully obtained. One data set was collected from each of four crystals at 100 K and the three-dimensional structures of the catalytic cysteines in different redox states were determined: reduced and oxidized forms at 2.00 Å resolution using data collected at a synchrotron-radiation source and two partially reduced structures at 1.54 and 1.88 Å resolution using data collected using an in-house source. All of the crystals belonged to space group P3212, with unit-cell parameters a = 57.5 (4), b = 57.5 (4), c = 118.1 (8) Å. The asymmetric unit contains two subunits of LvTrx, with a Matthews coefficient (VM) of 2.31 Å(3) Da(-1) and a solvent content of 46%. Initial phases were determined by molecular replacement using the crystallographic model of Trx from Drosophila melanogaster as a template. In the present work, LvTrx was overexpressed in Escherichia coli, purified and crystallized. Structural analysis of the different redox states at the Trx active site highlights its reactivity and corroborates the existence of a dimer in the crystal. In the crystallographic structures the dimer is stabilized by several interactions, including a disulfide bridge between Cys73 of each LvTrx monomer, a hydrogen bond between the side chain of Asp60 of each monomer and several hydrophobic interactions, with a noncrystallographic twofold axis.

Antioxidant enzyme activity in pacific whiteleg shrimp (Litopenaeus vannamei) in response to infection with white spot syndrome virus

White spot syndrome virus is a virulent pathogen that affects most crustaceans. Reactive oxygen species are produced by the immune system that defends the host against foreign microorganisms. This study examined the antioxidant enzymes and oxidative damage in tissues (hemolymph, digestive gland, gills, and muscle) of Pacific whiteleg shrimp (Litopenaeus vannamei) infected with the disease by intramuscular injection of the virus. Viral loads were confirmed by real-time PCR. The levels of oxidative damage (lipid peroxidation and protein carbonyl), the activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase) were quantified in uninfected and infected tissues. Lipid peroxidation and protein carbonyl levels were higher in infected specimens compared to uninfected controls. A significant reduction in antioxidant enzyme activities was found at 48h post-infection in all tissues analyzed. These results suggest that oxidative stress and tissue damage via inactivation of antioxidant enzymes in infected shrimps result in system failure and sudden death.

Dynamics of free amino acids in the hemolymph of Pacific whiteleg shrimp Litopenaeus vannamei exposed to different types of stress

We analyzed the dynamics of amino acids, ammonia-N, and carbohydrates in hemolymph when Pacific whiteleg shrimp Litopenaeus vannamei was subjected to air-exposure and low-salinity stresses. Glycine, l-arginine, and d- and l-alanine levels in hemolymph were increased under both stress conditions. Ammonia-N, a product of amino acid catabolism, also increased in level. These results suggest that the above-mentioned amino acids are used as energy sources. Levels of total carbohydrates, the sum of glucose and other carbohydrates, in the hemolymph showed different dynamics between the two types of stress and were not always high. This suggests that other energy sources, such as amino acids, are important when animals are subjected to stress. It is possible that particular amino acids act as energy sources under various stress conditions.