Boosting one-step degradation of shrimp shell waste to produce chitin oligosaccharides at smart nanoscale enzyme reactor with liquid-solid system.

Chitin oligosaccharides (CTOS) possess potential applications in food, medicine, and agriculture. However, lower mass transfer and catalytic efficiency are the main kinetic limitations for the production of CTOS from shrimp shell waste (SSW) and crystalline chitin. Chemical or physical methods are usually used for pretreatment to improve chitinase hydrolysis efficiency, but this is not eco-friendly and cost-effective. To address this challenge, a chitinase nanoreactor with the liquid-solid system (BcChiA1@ZIF-8) was manufactured to boost the one-step degradation of SSW and crystalline chitin. Compared with free enzyme, the catalytic efficiency of BcChiA1@ZIF-8 on colloidal chitin was significantly improved to 142 %. SSW and crystalline chitin can be directly degraded by BcChiA1@ZIF-8 without any pretreatments. The yield of N, N'-diacetylchitobiose [(GlcNAc)2] from SSW and N-acetyl-D-glucosamine (GlcNAc) from crystalline chitin was 2 times and 3.1 times than that of free enzyme, respectively. The reason was that BcChiA1@ZIF-8 with a liquid-solid system enlarged the interface area, increased the collision frequency between enzyme and substrate, and improved the large-substrates binding activity of chitinase. Moreover, the biphasic system exhibited excellent stability, and the design showed universal applicability. This strategy provided novel guidance for other polysaccharide biosynthesis and the conversion of environmental waste into carbohydrates.

Effects of high voltage pulsed electric field on structural properties and immune reactivity of arginine kinase in Fenneropenaeus chinensis.

To evaluate the effect of high voltage pulsed electric field (PEF) treatment (10-20 kV/cm, 5-15 min) on the structural characteristics and sensitization of crude extracts of arginine kinase from Fenneropenaeus chinensis. By simulated in vitro gastric juice digestion (SGF), intestinal juice digestion (SIF) and enzyme-linked immunosorbent assay (ELISA), AK sensitization was reduced by 42.5% when treated for 10 min at an electric field intensity of 15 kV/cm. After PEF treatment, the α-helix content decreased, and the α-helix content gradually changed to ß-sheet and ß-turn. Compared to the untreated group, the surface hydrophobicity increased and the sulfhydryl content decreased. SEM and AFM analyses showed that the treated sample surface formed a dense porous structure and increased roughness. The protein content, dielectric properties, and amino acid content of sample also changed significantly with the changes in the treatment conditions. Non-thermal PEF has potential applications in the development of hypoallergenic foods.

Cloning and expression profile of the alanine aminotransferase gene from kuruma shrimp Penaeus japonicus exposed to different salinities.

Several crustaceans including shrimps change the amount of specific free amino acids to regulate the osmotic pressure in their bodies. Kuruma shrimp Penaeus japonicus also increases the concentration of alanine (Ala) in the abdominal muscle following the increase of environmental salinity. In the present study, to elucidate the mechanisms of changes in Ala accumulation of kuruma shrimp depending on salinity, we cloned the gene encoding alanine aminotransferase (ALT), an enzyme involved in Ala biosynthesis, and examined its expression profile. It was found that the full-length kuruma shrimp ALT1 cDNA consisted of 3,301 bp, encoding 514 amino acids, and that all amino acid residues important for ALT activity were conserved. Phylogenetic analysis also indicated that the ALT gene cloned in this study was classified as ALT1. Moreover, we examined the expression levels of the ALT1 gene in the abdominal muscle and the hepatopancreas of kuruma shrimp acclimated at 17‰, 34‰, and 40‰ salinities, resulting that the mRNA levels of the ALT1 genes in both tissues of the shrimp acclimated at 40‰ were significantly higher than those at 17‰ for 12 h (p < 0.05). The mRNA levels of the ALT1 gene in the abdominal muscle of the shrimp acclimated for more than 24 h tended to increase following the increase of environmental salinity. These results indicate that ALT1 is responsible for the increase of free Ala concentration in the abdominal muscle of kuruma shrimp to regulate osmotic pressure at high salinity.

Phenylalanine hydroxylase (PAH) plays a positive role during WSSV and Vibrio parahaemolyticus infection in Litopenaeus vannamei.

Phenylalanine hydroxylase (PAH) is involved in immune defence reactions by providing the starting material, tyrosine, to synthesise catecholamines and melanin. PAH is an important metabolic enzyme of aromatic amino acids and the rate-limiting enzyme in the hydroxylation of amino acid phenylalanine to tyrosine. In the present study, a PAH gene, LvPAH, was cloned and identified from Litopenaeus vannamei. The open reading frame (ORF) of LvPAH was 1383 bp, encoding a protein of 460 amino acids comprised of an ACT domain and a Biopterin_H domain. LvPAH was constitutively expressed in healthy L. vannamei, with the highest expression levels in the eyestalk and the lowest in the hepatopancreas. Both white spot syndrome virus (WSSV) and Vibrio parahaemolyticus infection upregulated LvPAH expression in hemocytes, hepatopancreas and gills of L. vannamei. Inhibition of LvPAH resulted in a significantly lower survival rate of L. vannamei after WSSV infection than the control group, consistent with the observation that WSSV viral load was significantly higher in LvPAH-silenced L. vannamei. After a V. parahaemolyticus challenge, there was no significant difference between the survival rate of LvPAH-silenced and the control L. vannamei. However, the load of V. parahaemolyticus in LvPAH-silenced L. vannamei was significantly higher than the control population for L. vannamei. The effect of LvPAH on L. vannamei from a neuroendocrinological perspective was assessed by measuring l-DOPA, dopamine (DA) and noradrenaline (NE) levels in the hemocytes after the knockdown of LvPAH. The results showed that phenoloxidase (PO), l-DOPA and DA levels in the hemolymph of LvPAH-silenced L. vannamei were significantly decreased starting from 24hpi. In contrast, the NE levels in the hemolymph of shrimp decreased significantly at first and then increased. The results suggest that LvPAH may play an important role in antiviral and bacterial immunity in L. vannamei.

Assessment of acetylcholinesterase, catalase, and glutathione S-transferase as biomarkers for imidacloprid exposure in penaeid shrimp.

Shrimp aquaculture is a valuable source of quality seafood that can be impacted by exposure to insecticides, such as imidacloprid. Here, adult black tiger shrimp (Penaeus monodon) were used to evaluate the activity of acetylcholinesterase (AChE), catalase (CAT), and glutathione S-transferase (GST) in abdominal, head, gill, and hepatopancreas tissue as biomarkers for imidacloprid exposure. Adult P. monodon were continuously exposed to imidacloprid in water (5 µgL-1 and 30 µgL-1) or feed (12.5 µg g-1 and 75 µg g-1) for either 4 or 21 days. The imidacloprid concentration in shrimp tissues was determined using liquid chromatography-mass spectrometry after QuEChER extraction, and AChE, CAT, and GST activities were estimated by spectrophotometric assay. Imidacloprid exposure in shrimp elevated the activity of biomarkers, and the enzymatic activity was positively correlated to tissue imidacloprid accumulation, although the effects varied in a tissue-, dose- and time-dependent manner. AChE activity was correlated to imidacloprid concentration in the abdominal tissue of shrimp and was likely related to neural tissue distribution, while the activity of CAT and GST confirmed a generalised anti-oxidant stress response. AChE, CAT, and GST were valuable biomarkers for assessing shrimp response to imidacloprid exposure from dietary or water sources, and the abdominal tissue was the most reliable for exposure assessment. An elevated response in each of these biomarkers during routine monitoring could provide an early warning of shrimp stress, suggesting that investigating potential contamination by neonicotinoid pesticides would be worthwhile.

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.

Nitric Oxide Synthase Regulates Gut Microbiota Homeostasis by ERK-NF-κB Pathway in Shrimp.

The gut microbiota is a complex group of microorganisms that is not only closely related to intestinal immunity but also affects the whole immune system of the body. Antimicrobial peptides and reactive oxygen species participate in the regulation of gut microbiota homeostasis in invertebrates. However, it is unclear whether nitric oxide, as a key mediator of immunity that plays important roles in antipathogen activity and immune regulation, participates in the regulation of gut microbiota homeostasis. In this study, we identified a nitric oxide synthase responsible for NO production in the shrimp Marsupenaeus japonicus. The expression of Nos and the NO concentration in the gastrointestinal tract were increased significantly in shrimp orally infected with Vibrio anguillarum. After RNA interference of Nos or treatment with an inhibitor of NOS, L-NMMA, NO production decreased and the gut bacterial load increased significantly in shrimp. Treatment with the NO donor, sodium nitroprusside, increased the NO level and reduced the bacterial load significantly in the shrimp gastrointestinal tract. Mechanistically, V. anguillarum infection increased NO level via upregulation of NOS and induced phosphorylation of ERK. The activated ERK phosphorylated the NF-κB-like transcription factor, dorsal, and caused nuclear translocation of dorsal to increase expression of antimicrobial peptides (AMPs) responsible for bacterial clearance. In summary, as a signaling molecule, NOS-produced NO regulates intestinal microbiota homeostasis by promoting AMP expression against infected pathogens via the ERK-dorsal pathway in shrimp.

Characterization and epitope prediction of phosphopyruvate hydratase from Penaeus monodon (black tiger shrimp).

Shellfish allergies constitute an important cause of food-induced anaphylactic reactions, which pose challenges to food safety and human health worldwide. In the present study, the specific IgE (sIgE) binding characteristics of different shrimp proteins of black tiger shrimp (Penaeus monodon) to the sera of eight shrimp-allergic patients from China were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and nanoliquid chromatography time-of-flight mass spectrometry. According to the PLGS scores (>2000) and the sequence coverage (>40%), eight proteins with sIgE binding activity were identified, including myosin heavy chain type 1 (K4Q4N8), hemocyanin (G1AP69 and Q95V28), phosphopyruvate hydratase (O96656), arginine kinase (C7E3T4), tropomyosin (A1KYZ2), sarcoplasmic calcium binding protein (H7CHW2) and glyceraldehyde-3-phosphate dehydrogenase (A0A097BQP2). Among these eight proteins, phosphopyruvate hydratase was a prevalent IgE-binding protein among these Chinese patients with binding observed in 100% of sera. Moreover, 13 peptides were predicted as epitopes of phosphopyruvate hydratase. These new details help us to understand the crustacean IgE-binding proteins especially Penaeus monodon IgE-binding proteins, that would cause allergic reaction to Chinese patients. And our findings may provide essential information to improve allergy prevention and clinical treatment to shrimp allergy in China. PRACTICAL APPLICATION: This research may have diagnostic and therapeutic value for shrimp allergies in China.

Structural basis of staphylococcal Stl inhibition on a eukaryotic dUTPase.

dUTPases are key enzymes in all life kingdoms. A staphylococcal repressor protein (Stl) inhibited dUTPases from multiple species to various extents. Understanding the molecular basis underlying the inhibition differences is crucial to develop effective proteinaceous inhibitors of dUTPases. Herein, we report the complex structure of Stl N-terminal domain (StlN-ter) and Litopenaeus vannamei dUTPase domain (lvDUT65-210). Stl inhibited lvDUT65-210 through its N-terminal domain. The lvDUT65-210-StlN-ter complex structure revealed a heterohexamer encompassing three StlN-ter monomers bound to one lvDUT65-210 trimer, generating two types of Stl-dUTPase interfaces. Interface I is formed by Stl interaction with the lvDUT65-210 active-site region that is contributed by motifs I-IV from its two subunits; interface II results from Stl binding to the C-terminal motif V of the third lvDUT65-210 subunit. Structural comparison revealed both conserved features and obvious differences in Stl-dUTPase interaction patterns, giving clues about the inhibition differences of Stl on dUTPases. Noticeably, interface II is only observed in lvDUT65-210-StlN-ter. The Stl-interacting residues of lvDUT65-210 are conserved in other eukaryotic dUTPases, particularly human dUTPase. Altogether, our study presents the first structural model of Stl interaction with eukaryotic dUTPase, contributing to a more complete view of Stl inhibition and facilitating the development of proteinaceous inhibitor for eukaryotic dUTPases.

Biochemical characterization of the cyclooxygenase enzyme in penaeid shrimp.

Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.

Identification of a novel tailor-made chitinase from white shrimp Fenneropenaeus merguiensis.

Fenneropenaeus merguiensis (commonly named banana shrimp) is one of the most important farmed crustacean worldwide species for the fisheries and aquaculture industry. Besides its nutritional value, it is a good source of chitinase, an enzyme with excellent biological and catalytic properties for many industrial applications. In the present study, a putative chitinase-encoding cDNA was synthesized from mRNA from F. merguiensis hepatopancreas tissue. Subsequently, the corresponding cDNA was cloned, sequenced and functionally expressed in Escherichia coli, and the recombinant F. merguiensis chitinase (rFmCHI) was purified by His-tag affinity chromatography. The bioinformatics analysis of aminoacid sequence of rFmCHI displayed a cannonical multidomain architecture in chitinases which belongs to glycoside hydrolase family 18 (GH18 chitinase). Biochemical characterization revealed rFmCHI as a monomeric enzyme of molecular weight 52 kDa with maximum activity at 40 °C and pH 6.0 Moreover, the recombinant enzyme is also stable up to 60 °C, and in the pH range 5.0-8.0. Steady-state kinetic studies for colloidal chitin revealed KM, Vmax and kcat values of 78.18 µM, 0.07261 µM. min-1 and 43.37 s-1, respectively. Overall, our results aim to demonstrate the potential of rFmCHI as suitable catalyst for bioconversion of chitin waste.

Regulation of shrimp prophenoloxidase activating system by lva-miR-4850 during bacterial infection.

MicroRNAs (miRNAs) suppress gene expression and regulate biological processes. Following small RNA sequencing, shrimp hemocytes miRNAs differentially expressed in response to acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus (VPAHPND) were discovered and some were confirmed by qRT-PCR. VPAHPND-responsive miRNAs were predicted to target several genes in various immune pathways. Among them, lva-miR-4850 is of interest because its predicted target mRNAs are two important genes of the proPO system; proPO2 (PO2) and proPO activating factor 2 (PPAF2). The expression of lva-miR-4850 was significantly decreased after VPAHPND infection, whereas those of the target mRNAs, PO2 and PPAF2, and PO activity were significantly upregulated. Introducing the lva-miR-4850 mimic into VPAHPND-infected shrimps caused a reduction in the PO2 and PPAF2 transcript levels and the PO activity, but significantly increased the number of bacteria in the VPAHPND targeted tissues. This result inferred that lva-miR-4850 plays a crucial role in regulating the proPO system via suppressing expression of PPAF2 and PO2. To fight against VPAHPND infection, shrimp downregulated lva-miR-4850 expression resulted in proPO activation.

Effects of metal ions on activity and structure of phenoloxidase in Penaeus vannamei.

Phenoloxidase (PO) is a typical metal enzyme, which requires metal ions as prosthetic groups to enable the full exertion of its activity. To study how metal ions affected the activity and structure of PO enzymes, while providing reference materials for in-depth investigations, we examined the effects of different metal ions (Cu2+, Zn2+, Mg2+, Ca2+, and Ba2+) on their activities. Furthermore, Cu2+ and Mg2+ were selected for further investigation through UV spectra, intrinsic fluorescence spectroscopy, AFM, and FTIR. It was revealed that Cu2+ had a more obvious effect on PO compared to Mg2+. The PO could be activated when the concentrations of Cu2+ and Mg2+ were lower than 10-3 and 10-2 mol/L, respectively, and maximum PO activities (182.14% and 141.02%) were observed at 10-4 mol/L concentrations of Cu2+ and Mg2+. When the concentrations of Cu2+ and Mg2+ were higher than 10-2 and 10-1 mol/L, the activities PO were inhibited. The results of the UV-vis and fluorescence spectra revealed that Cu2+ shaped the tertiary structure of PO, whereas the effect of Mg2+ was slight. The AFM results demonstrated that high concentrations of Cu2+ and Mg2+ resulted in PO aggregation. FTIR analysis indicated that the total content of PO α-helices and ß-sheets decreased with higher concentrations of Cu2+ and Mg2+.

Growth, biochemical, antioxidants, metabolic enzymes and hemocytes population of the shrimp Litopenaeus vannamei exposed to acidified seawater.

Acidification in the marine environment has become a global issue that creates serious threats to marine organisms. In the present study, we evaluated the effect of CO2 driven acidification on the shrimp Litopenaeus vannamei post-larvae (PL). L. vannamei PL were exposed to six different CO2 driven acidified seawater, such as 8.2 (control), pH 7.8 (IPCC-predicted ocean pH by 2100), 7.6, 7.4, 7.2 and 7.0 with corresponding pCO2 level of 380.66, 557.53, 878.55, 1355.48, 2129.46, and 3312.12 µatm for seven weeks. At the end of the acidification experiment, results revealed that the survival, growth, feed index, biochemical constituents, chitin, minerals (Na, K, and Ca), and hemocyte populations of shrimps were found to be significantly decreased in CO2 driven acidified seawater which indicates the negative impacts of acidified seawater on these parameters in L. vannamei. Further, the level of antioxidants, lipid peroxidation, and metabolic enzymes were significantly higher in the muscle of shrimps exposed to acidified seawater suggests that the L. vannamei under oxidative stress and metabolic stress. Among the various acidified seawater tested, pH 7.6 to 7.0 produced a significantly adverse effect on shrimps. Hence, the present study concluded that the elevated level of seawater acidification can produce harmful effects on the biology and physiology of the commercially important shrimp L. vannamei PL.

GSK3ß Plays a Negative Role During White Spot Syndrome Virus (WSSV) Infection by Regulating NF-κB Activity in Shrimp Litopenaeus vannamei.

Glycogen synthase kinase-3 (GSK3), a cytoplasmic serine/threonine-protein kinase involved in a large number of key cellular processes, is a little-known signaling molecule in virus study. In this study, a GSK3 protein which was highly similar to GSK3ß homologs from other species in Litopenaeus vannamei (designated as LvGSK3ß) was obtained. LvGSK3ß was expressed constitutively in the healthy L. vannamei, at the highest level in the intestine and the lowest level in the eyestalk. White spot syndrome virus (WSSV) reduced LvGSK3ß expression was in immune tissues including the hemocyte, intestine, gill and hepatopancreas. The inhibition of LvGSK3ß resulted in significantly higher survival rates of L. vannamei during WSSV infection than the control group, and significantly lower WSSV viral loads in LvGSK3ß-inhibited L. vannamei were observed. Knockdown of LvGSK3ß by RNAi resulted in increases in the expression of LvDorsal and several NF-κB driven antimicrobial peptide (AMP) genes (including ALF, PEN and crustin), but a decrease in LvCactus expression. Accordingly, overexpression of LvGSK3ß could reduce the promoter activity of LvDorsal and several AMPs, while the promoter activity of LvCactus was increased. Electrophoretic mobility shift assays (EMSA) showed that LvDorsal could bind to the promoter of LvGSK3ß. The interaction between LvGSK3ß and LvDorsal or LvCactus was confirmed using co-immunoprecipitation (Co-IP) assays. In addition, the expression of LvGSK3ß was dramatically reduced by knockdown of LvDorsal. In summary, the results presented in this study indicated that LvGSK3ß had a negative effect on L. vannamei by mediating a feedback regulation of the NF-κB pathway when it is infected by WSSV.

The effect of dietary supplementation of Astragalus membranaceus and Bupleurum chinense on the growth performance, immune-related enzyme activities and genes expression in white shrimp, Litopenaeus vannamei.

A 56-day feeding trial was conducted to investigate the effects of dietary supplementation of Astragalus membranaceus or/and Bupleurum chinense on the growth performance, immune enzymes, and related gene expression of Pacific white shrimp (Litopenaeus vanammei). Six experimental diets were formulated and supplemented with two levels (0.25% and 0.5%) of each herb and their combination. At the end of the trial, the specific growth rate and feed conversion ratio of shrimp were significantly (P < 0.05) improved by herbal diets. Besides, the activities of immune-related enzymes such as superoxide dismutase (SOD), alkaline phosphatase (AKP), and lysozyme in serum and hepatopancreas were significantly (P < 0.05) elevated in shrimp fed A. membranaceus or/and B. chinense. The high expression levels of immune deficiency (IMD), lysozyme, and Toll-like receptor mRNA directly or indirectly reflected the activation effect of innate immune in shrimp by dietary A. membranaceus or/and B. chinense. However, no significant difference (P > 0.05) among the herbal incorporated treatments was detected on the growth performance and immune response. In conclusion, the results suggest that A. membranaceus and B. chinense could be used as a beneficial feed additives and alternatives to antibiotics for white shrimp aquaculture.

Comparative Transcriptome Analysis of Litopenaeus vannamei Reveals That Triosephosphate Isomerase-Like Genes Play an Important Role During Decapod Iridescent Virus 1 Infection.

Decapod iridescent virus 1 (DIV1) results in severe economic losses in shrimp aquaculture. However, little is known about the physiological effect of DIV1 infection on the host. In this study, we found that the lethal dose 50 of DIV1-infected Litopenaeus vannamei after 48, 72, 96, and 156 h were 4.86 × 106, 5.07 × 105, 2.13 × 105, and 2.38 × 104 copies/µg DNA, respectively. In order to investigate the mechanisms of DIV1 infection, a comparative transcriptome analysis of hemocytes from L. vannamei, infected or not with DIV1, was conducted. The BUSCO analysis showed that the transcriptome was with high completeness (complete single-copy BUSCOs: 57.3%, complete duplicated BUSCOs: 41.1%, fragmentation: 0.8%, missing: 0.8%). A total of 168,854 unigenes were assembled, with an average length of 601 bp. Based on homology searches, Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and cluster of orthologous groups of proteins (KOG) analysis, 62,270 (36.88%) unigenes were annotated. Among them, 1,112 differentially expressed genes (DEGs) were identified, of which 889 genes were up-regulated and 223 genes were down-regulated after DIV1 infection. These genes were mainly annotated to the major metabolic processes such as fructose and mannose metabolism, carbon metabolism, and inositol phosphate metabolism. Among these metabolic pathways, the triosephosphate isomerase (TPI) family was the most eye-catching DEG as it participates in several metabolic processes. Three types of TPI, LvTPI-like, LvTPI-Blike, and LvTPI-Blike1, were obtained for gene silencing by RNA interference. The results showed that LvTPI-like and LvTPI-Blike1 silencing caused a high mortality rate among L. vannamei. However, LvTPI-like and LvTPI-Blike silencing reduced DIV1 replication in DIV1-infected L. vannamei. All the results indicated that TPI-like genes play an important role during DIV1 infection, which provides valuable insight into the infection mechanism of DIV1 in shrimp and may aid in preventing viral diseases in shrimp culture.

Genomic Characterization and Expression of Juvenile Hormone Esterase-Like Carboxylesterase Genes in Pacific White Shrimp, Litopenaeus vannamei.

The sesquiterpenoid methyl farnesoate (MF), a juvenile hormone (JH) analog, plays important roles in many physiological processes of crustaceans, such as morphogenesis, molting and reproduction. Juvenile hormone esterase-like (JHE-like) carboxylesterase (CXE) is a key enzyme in MF degradation, playing a significant role in regulating MF titer. However, its function is barely known in shrimp. In this study, a total of 21 JHE-like CXEs (LvCXEs) were characterized in Pacific white shrimp Litopenaeus vannamei, based on the full genome and multi-transcriptomic data. LvCXE has a conserved triplet catalytic site (Ser-Glu-His) and a characteristic GxSxG motif. Most LvCXEs were highly expressed in the hepatopancreas, which was the main site for MF degradation. LvCXEs containing a GESAG motif showed a specific expansion in the L. vannamei genome. Those GESAG-containing LvCXEs presented differential expressions at different larvae stages and different molting stages of L. vannamei, which suggested their potential functions in development and molting. Additionally, when the transcription level of CXEs was inhibited, it could lead to failed molt and death of L. vannamei. When we further detected the expression levels of the key ecdysone responsive transcription factors including LvE75, LvBr-C, LvHr3 and LvFtz-f1 after the CXE inhibitor was injected into L. vannamei, they all showed apparent down-regulation. These results suggested that the expansion of LvCXEs in the L. vannamei genome should contribute to the regulation of metamorphosis at larvae stages and frequent molting during the growth of L. vannamei.

Effects of chlorpyrifos on the crustacean Litopenaeus vannamei.

Shrimps can be used as indicators of the quality of aquatic systems exposed to a variety of pollutants. Chlorpyrifos is one of the most common pesticides found in environmental samples. In order to evaluate the effects of chlorpyrifos, adult organisms of Litopenaeus vannamei were exposed to two sublethal concentrations of the pesticide (0.7 and 1.3 µg/L) for four days. The LC50 (96-hours) value was determined and Lipid oxidation levels (LPO) and the activities of catalase (CAT), glutathion peroxidase (GPx), glutathion-S-transferase (GST) were assessed on the muscle, hepatopancreas and gills from the exposed organisms. In addition, inhibition of acetylcholinesterase (AChE) was determined in the brain. LC50 (96-hours) was 2.10 µg/L of chlorpyrifos. Catalase activity and LPO were elevated in the three tissues, whereas a decrease of AChE activities in the brain and an increase of GST activity in the hepatopancreas were observed.

Cloning and characterization of DOPA decarboxylase in Litopenaeus vannamei and its roles in catecholamine biosynthesis, immunocompetence, and antibacterial defense by dsRNA-mediated gene silencing.

Catecholamines (CAs) play critical roles in regulating physiological and immunological homeostasis in invertebrates and vertebrates under stressful environments. DOPA decarboxylase (DDC), an enzyme responsible for the decarboxylation step of dopamine synthesis, participates in neurotransmitter metabolism and innate immunity. In shrimp, two genes encoding CA-related enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, were further identified and characterized as neuroendocrine-immune regulators. In this study, full-length complementary DNA of DDC cloned from the thoracic ganglia of shrimp, Litopenaeus vannamei, (LvDDC) was predicted to encode a 452-amino acid protein with a pyridoxal-dependent decarboxylase-conserved domain, and this deduced protein of LvDDC was phylogenetically closely related to insect DDC. LvDDC messenger RNA expression was analyzed by a semiquantitative RT-PCR and a real-time quantitative RT-PCR and found to be abundant in the hepatopancreas and nervous system but at low levels in haemocytes, heart, stomach, and gills. To determine the role of LvDDC, double-stranded (ds)RNA was used for in vivo assessments. LvDDC-depleted shrimp revealed significant increases in the total haemocyte count, hyaline cells, granular cells, phenoloxidase activity, and respiratory bursts of haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency toward Vibrio alginolyticus. Further, decreased LvDDC mRNA expression was accompanied by decreases in dopamine, glucose, and lactate levels in haemolymph. In shrimp that received LvDDC-dsRNA for 3 days and were then challenged with V. alginolyticus, the survival rate of LvDDC-depleted shrimp was significantly higher than that of shrimp that received diethyl pyrocarbonate-water or non-targeted dsRNA. In conclusion, the cloned LvDDC was responsible for controlling dopamine synthesis, which then regulated physiological and immune responses in L. vannamei.