An overview of microbial enzymatic approaches for pectin degradation.

Pectin, a complex natural macromolecule present in primary cell walls, exhibits high structural diversity. Pectin is composed of a main chain, which contains a high amount of partly methyl-esterified galacturonic acid (GalA), and numerous types of side chains that contain almost 17 different monosaccharides and over 20 different linkages. Due to this peculiar structure, pectin exhibits special physicochemical properties and a variety of bioactivities. For example, pectin exhibits strong bioactivity only in a low molecular weight range. Many different degrading enzymes, including hydrolases, lyases and esterases, are needed to depolymerize pectin due to its structural complexity. Pectin degradation involves polygalacturonases/rhamnogalacturonases and pectate/pectin lyases, which attack the linkages in the backbone via hydrolytic and ß-elimination modes, respectively. Pectin methyl/acetyl esterases involved in the de-esterification of pectin also play crucial roles. Many α-L-rhamnohydrolases, unsaturated rhamnogalacturonyl hydrolases, arabinanases and galactanases also contribute to heterogeneous pectin degradation. Although numerous microbial pectin-degrading enzymes have been described, the mechanisms involved in the coordinated degradation of pectin through these enzymes remain unclear. In recent years, the degradation of pectin by Bacteroides has received increasing attention, as Bacteroides species contain a unique genetic structure, polysaccharide utilization loci (PULs). The specific PULs of pectin degradation in Bacteroides species are a new field to study pectin metabolism in gut microbiota. This paper reviews the scientific information available on pectin structural characteristics, pectin-degrading enzymes, and PULs for the specific degradation of pectin.

Tamlana laminarinivorans sp. nov. and Tamlana sargassicola sp. nov., two novel species isolated from Sargassum, show genomic and physiological adaptations for a Sargassum-associated lifestyle.

The genus Tamlana from the Bacteroidota currently includes six validated species. Two strains designated PT2-4T and 62-3T were isolated from Sargassum abundant at the Pingtan island coast in the Fujian Province of China. 16S rRNA gene sequence analysis showed that the closest described relative of strains PT2-4T and 62-3T is Tamlana sedimentorum JCM 19808T with 98.40 and 97.98% sequence similarity, respectively. The 16S rRNA gene sequence similarity between strain PT2-4T and strain 62-3T was 98.68 %. Furthermore, the highest average nucleotide identity values were 87.34 and 88.97 % for strains PT2-4T and 62-3T, respectively. The highest DNA-DNA hybridization (DDH) value of strain PT2-4T was 35.2 % with strain 62-3T, while the DDH value of strain 62-3T was 37.7 % with T. sedimentorum JCM 19808T. Growth of strains PT2-4T and 62-3T occurs at 15-40 °C (optimum, 30 °C) with 0-4 % (w/v) NaCl (optimum 0-1 %). Strains PT2-4T and 62-3T can grow from pH 5.0 to 10.0 (optimum, pH 7.0). The major fatty acids of strains PT2-4T and 62-3T are iso-C15 : 0 and iso G-C15 : 1. MK-6 is the sole respiratory quinone. Genomic and physiological analyses of strains PT2-4T and 62-3T showed corresponding adaptive features. Significant adaptation to the growth environment of macroalgae includes the degradation of brown algae-derived diverse polysaccharides (alginate, laminarin and fucoidan). Notably, strain PT2-4T can utilize laminarin, fucoidan and alginate via specific carbohydrate-active enzymes encoded in polysaccharide utilization loci, rarely described for the genus Tamlana to date. Based on their distinct physiological characteristics and the traits of utilizing polysaccharides from Sargassum, strains PT2-4T and 62-3T are suggested to be classified into two novel species, Tamlana laminarinivorans sp. nov. and Tamlana sargassicola sp. nov. (type strain PT2-4T=MCCC 1K04427T=KCTC 92183T and type strain 62-3T=MCCC 1K04421T=KCTC 92182T).

Mutation of conserved residues in the laminarinase Lam1092 increases the antioxidant activity of the laminarin product hydrolysates.

Laminarinases from the glycoside hydrolase 16 (GH16) family are hydrolases that break ß-1,3-glycosidic bonds in laminarin, which is the major storage polysaccharide present in brown algae or microalgae. We explored a laminarinase from the marine Flavobacteriaceae species Tamlana sp. PT2-4 at the structural and functional levels. Based on a homology model of Lam1092-substrate interactions, the large active groove crossing Lam1092 was deemed a reasonable pathway for the bent substrates for hydrolysis. Eight residues (Gly361, Asn364, Arg400, His466, Asp449, Glu452, Ser477 and Thr538) were selected for mutagenesis based on the interactions of Lam1092 in complex with Lam4/Lam6. Ultimately, we generated eight mutants of Lam1092, and the antioxidant activities of the hydrolysates of two mutants (G361A and H466A) showed significant improvement. These results show that the antioxidant activity of laminarin can be improved by laminarinase mutation, which will be beneficial for developing efficient approaches to engineer the substrate specificity of laminarinases and improve the application of bioactive laminarioligosaccharides.

Characterization and activity enhancement of a novel exo-type agarase Aga575 from Aquimarina agarilytica ZC1.

The novel ß-agarase gene aga575 from the agarolytic bacterium Aquimarina agarilytica ZC1 is composed of 2142 bp, and the encoded protein Aga575 has the highest amino acid sequence homology of only 65.2% with known agarases. Though carrying a domain of glycoside hydrolase family 42 in the C-terminal, Aga575 should belong to glycoside hydrolase family 50 according to the phylogenetic analysis. Gene aga575 was successfully cloned and overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein had the maximal agarase activity at pH 8.0 and 37 °C. The values Km and Vmax toward agarose were 8.4 mg/mL and 52.2 U/mg, respectively. Aga575 hydrolyzed agarose and neoagarooligosaccharides to yield neoagarobiose as the sole product. The agarose hydrolysis pattern of Aga575 indicated that it was an exo-type ß-agarase. Random mutagenesis was carried out to obtain two beneficial mutants M1 (R534G) and M2 (S4R-R424G) with higher activities. The results showed that the agarase activity of mutant M1 and M2 reached 162% and 192% of the wild-type agarase Aga575, respectively. Moreover, the activity of the mixed mutant M1/M2 (S4R-R424G-R534G) increased to 227%. KEY POINTS: • Aga575 is a novel exo-type ß-agarase degrading agarose to yield neoagarobiose as the sole product. • Though owning a domain of glycoside hydrolase family GH42, Aga575 should belong to family GH50. • The agarase activity of one mutant increased to 227% of the wild-type Aga575.

Bioconversion of bamboo shoot shells through the cultivation of the edible mushrooms Volvariella volvacea.

Bamboo shoot shell (BSS), as agricultural waste, is mostly burned or discarded, causing serious environment pollution. In this study, the degradation and utilization of BSS by the edible fungus Volvariella Volvacea was investigated. The composition of V. volvacea fruit body was determined by HPLC-MS, GC-MS and ICP-OES. The activities of CMCase and xylanase were monitored by DNS (3,5-dinitrosalicylic acid) method. Laccase activity was assayed by the oxidation reaction of ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)]. The degraded bamboo shoot shell powder was characterized by FTIR and SEM. The results showed that the mycelium of V. volvacea could degrade and utilize BSS for growth. The activities of carboxymethyl cellulase and laccase were increased during the cultivation. At the same time, the physical structure of the shell fiber becames porous and rough. Most of the products of decayed fibers contain alkanes, ethyl or methyl groups. Moreover, the biological efficiency (fruiting body yield) of V. volvacea cultivated on BSS was 1.52-fold higher than that of straw cultivation. The contents of total lipid, elaidic acid (C18:1n-9), total essential amino acids, total amino acids and iron in V. volvacea fruit bodies grown on BSS were 1.11, 1.66, 1.52, 1.60 and 1.30-fold higher than those of straw treatment, respectively. This study provides an effective method to solve the environmental pollution caused by BSS, and provides a new way for the potential utilization of BSS in edible fungi cultivation.

Winogradskyella endarachnes sp. nov., a marine bacterium isolated from the brown alga Endarachne binghamiae.

A Gram-negative, aerobic, rod-shaped, non-flagellated and motile by gliding bacterium HL2-2T, was isolated from the surface of the brown alga Endarachne binghamiae in China. The 16S rRNA gene sequence analysis showed that this strain was affiliated with the genus Winogradskyella in the family Flavobacteriaceae and presented great similarity with the type strain Winogradskyella litoriviva KMM 6491T (97.9 % sequence similarity). The whole genome of strain HL2-2T comprised 3.6 Mbp with a G+C content of 31.9 mol%. The average nucleotide identity between strain HL2-2T and Winogradskyella litoriviva KMM 6491T was 83.7 %. Growth of the isolated strain was observed from 20-40 °C (optimum, 30 °C), at pH ranged from 5.5 to 8.0 (optimum, pH 6.0) and in the presence of 0-5 % (w/v) NaCl (optimum, 0-2 %). The major fatty acids (>10 % of the total) were C16 : 0, iso-C15 : 0 and the predominant menaquinone was MK-6. The combined phylogenetic, physiological and chemotaxonomic analysis show that the strain HL2-2T represents a novel species belonging to the genus Winogradskyella, for which the name Winogradskyella endarachnes sp. nov. is proposed, and which the type strain is HL2-2T (=CICC 24857T=KCTC 72882T).

Enteromorpha prolifera Diet Drives Intestinal Microbiome Composition in Siganus oramin.

Enteromorpha prolifera (E. prolifera) contains complex sulfated polysaccharides that are resistant to biological degradation. Most organisms cannot digest biomass of E. prolifera, except Siganus oramin (S. oramin). This study was conducted to identify the bacteria in the intestine of S. oramin facilitating the digestion of E. prolifera polysaccharides (EPP). Metagenomic sequencing analysis of the S. oramin intestinal microbiota revealed that E. prolifera diet increased the number of Firmicutes, replacing Proteobacteria to be the dominant bacteria. The proportion of Firmicutes increased from 38.8 to 58.6%, with Bacteroidetes increasing nearly fivefold from 5 to 23.7%. 16S rDNA high-throughput sequencing showed that EPP-induced Bacteroidetes increased significantly in the intestinal flora of S. oramin cultivated in vitro. Metatranscriptome analysis showed that EPP induced more transferase, polysaccharide hydrolase, glycoside hydrolase, and esterases expressed in vitro, and most of them were taxonomically annotated to Bacteroidetes. Compared with the aggregation of GH family genes in metagenomic sequencing analysis in vivo, EPP induced more CBM32, GH2, GT2, GT30, and GH30 families gene expression in vitro. In general, We found that the bacteria in intestinal tract of S. oramin responsible for digestion of E. prolifera were Firmicutes and Bacteroidetes, while Bacteroidetes was the dominant bacteria involved in EPP degradation in vitro cultures. Compared with in vivo experiments, only GH family genes were mostly involved, we detected a more complete and complex EPP degradation pathway in vitro. The results may benefit the further study of biodegradation of E. prolifera and has potential implications for the utilization of E. prolifera for biotechnology.

Tamlana fucoidanivorans sp. nov., isolated from algae collected in China.

A Gram-stain-negative, aerobic, non-motile and rod-shaped marine bacterium, CW2-9T, was isolated from algae collected from Fujian Province in PR China. 16S rRNA gene sequence analysis showed that this strain was affiliated with the genus Tamlana in the family Flavobacteriaceae of the class Flavobacteriia and was very similar to the type strain Tamlana sedimentorum MCCC 1A10799T (96.3 % sequence similarity). The whole genome of strain CW2-9T comprised 3 997 513 bp with a G+C content of 34.3 mol%. The average nucleotide identity value between strain CW2-9T and T. sedimentorum MCCC 1A10799T was 73.8 %. Growth was observed from 15 to 40 °C (optimum, 30 °C), at pH from pH 5.0 to 10.0 (pH 8.0) and in the presence of 0-4 % (w/v) NaCl (0-1 %). The major fatty acids (>10 % of the total) were iso-C15 : 0, iso G-C15 : 1, iso-C17 : 0 3-OH and anteiso-C15 : 0. The predominant menaquinone was MK-6. The combined phylogenetic, physiological and chemotaxonomic data indicate that strain CW2-9T represents a novel species in the genus Tamlana, for which the name Tamlana fucoidanivorans sp. nov. is proposed. The type strain is CW2-9T (=CICC 24749T=KCTC 72389T).

Glutamine Synthetase (GS): A Key Enzyme for Nitrogen Assimilation in The Macroalga Gracilariopsis lemaneiformis (Rhodophyta).

This study aimed to address the importance of glutamine synthetase II (GSII) during nitrogen assimilation in macroalga Gracilariopsis lemaneiformis. The cDNA full-length sequence of the three glGSII genes was revealed to have the 5' m7 G cap, 5'-untranslated region, open reading frame (ORF), 3'-untranslated region, and a 3' poly (A) tail. The three glGSIIs were classified into plastid glGS2 and cytosolic glGS1-1 and glGS1-2, having conserved GSII domains but different cDNA sequences. The complicated 5' end flanking region indicates complex function of glGS genes. glGS1 genes were significantly up-regulated under the different NH4+ : NO3- ratio (i.e., 40:10, 25:25, 10:40, and 0:50) except glGS2 which dramatically up-regulated under the low NH4+ : NO3- ratio (i.e., 10:40 and 0:50) during different cultivation times. These different expression patterns perhaps are due to the different biological roles of GS1 and GS2 in the gene family. Furthermore, hypothetical working model of nitrogen assimilation pathway exhibiting the role of glGS1 and glGS2 is proposed. Finally, glGS2 was expressed in Escherichia coli BL21 (DE3), and the optimal conditions for culture (15°C, overnight), purification (500 mM imidazole washing), and activity (pH 7.4, 37°C) were established. This study lays a very important foundation for exploring the role of GS in nitrogen assimilation in algae and plants.

Analysis of Litopenaeus vannamei hemocyanin interacting proteins reveals its role in hemolymph clotting.

Hemocyanin is the main component of hemolymph plasma proteins and possesses diverse immunological properties and immunomodulatory functions. However, the interacting networks of hemocyanin in shrimp immune response remain poorly understood. In this study, 39 potential hemocyanin interacting partners were identified from Litopenaeus vannamei plasma by co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Gene Ontology (GO) analysis showed that most of the identified interactors were cell proteins involved in metabolic process and binding. Among these identified proteins, transglutaminase (TGase), a crucial regulator in hemolymph clotting cascade, was chosen for further studies. Far-Western blot and His-pull down assays revealed that hemocyanin directly interacted with TGase. Further analysis demonstrated that hemocyanin and TGase followed similar expression patterns upon pathogen infection. Moreover, in vivo knockdown of hemocyanin led to a significant decrease in TGase expression, as well as inhibited hemolymph clotting. Taken together, these data suggest that hemocyanin might positively regulate hemolymph clotting by modulating TGase in shrimp. SIGNIFICANCE: The interaction networks among immune-related factors is critical for the innate immune response in invertebrates. We report for the first time, proteins that potentially interact with hemocyanin, which led to the identification of 39 possible hemocyanin-proteins including the clotting-related factor TGase. Further studies demonstrated that hemocyanin directly interacted with TGase and modulated its expression, therefore affecting the formation of hemolymph clotting. These findings not only extend our knowledge of the immune interaction networks but also contribute to shrimp disease control and prevention.

Identification and characterization of glycosylation sites on Litopenaeus vannamei hemocyanin.

The respiratory glycoprotein hemocyanin has been implicated in immune-related functions. Using lectin blotting, we show that the binding of shrimp (Litopenaeus vannamei) hemocyanin to concanavalin A decreases markedly with O-glycosidase treatment but not with PNGase F. Twelve O-glycosylation sites, three on the large hemocyanin subunit and nine on the small hemocyanin subunit (HMCs), were identified by LC-MS/MS. Importantly, when the glycosylation sites at Thr-537, Ser-539, and Thr-542 on the C terminus of HMCs were replaced with alanine, the resultant mutant hemocyanin had reduced carbohydrate content, coupled with a fourfold reduction in bacterial agglutination and 0.2-fold reduction in antibacterial activities toward Vibrio parahaemolyticus and Staphylococcus aureus. These results suggest that the glycosylation sites on shrimp hemocyanin are closely related to its immunological functions.

Identification and immune-related analysis of SNPs in Litopenaeus vannamei Toll3 receptor.

Tolls and Toll-like receptors (TLRs), as innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens, play a critical role in antimicrobial immune responses. Here, we report on single nucleotide polymorphisms (SNPs) of Litopenaeus vannamei Toll3 (LvToll3). Multiple sequence alignment of the L. vannamei Toll3 Leucine rich repeat C-terminal domain (LvToll3-LRR-CT) with other L. vannamei Tolls LRR-CT domains showed 39.23% - 43.96% homology at the nucleic acid level and 20.31% - 30.00% identity at the amino acid level. Analysis of different shrimp tissues by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) revealed that LvToll3-LRR-CT had genetic polymorphisms at both the genomic deoxyribonucleic acid (gDNA) and complementary deoxyribonucleic acid (cDNA) levels. Further, high-throughput sequencing analysis confirmed the presence of 8 non-synonymous SNP (nsSNP) and 1 nsSNPs with frequency greater than 1% at the gDNA level, while 13 nsSNPs and 2 nsSNPs with frequency greater than 1% at the cDNA level. In silico analysis revealed that the α-helix secondary structure and tertiary structure of LvToll3 changed when 3 SNPs (C2039T, T2041C, T2228C) were mutated. Interestingly, 2 novel bands on PCR-DGGE, which were identified as 2 nsSNPs (C2140A, T2186A) were observed following challenge with Streptococcus iniae but not with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). Moreover, the secondary and tertiary structures of LvToll3 changed when the nsSNP T2186A was mutated. The present findings therefore provide novel insight into the molecular basis of shrimp innate immune response to pathogens through the generation of specific SNPs.

Litopenaeus vannamei attenuates white spot syndrome virus replication by specific antiviral peptides generated from hemocyanin.

Recent studies have shown that hemocyanin plays immune-related functions apart from its canonical respiratory function. While shrimp hemocyanin is found to generate antimicrobial peptides, antiviral related peptides have not been reported. In the present study, the serum of white spot syndrome virus (WSSV) infected Litopenaeus vannamei analyzed by two-dimensional gel electrophoresis, revealed 45 consistently down-regulated protein spots and 10 up-regulated protein spots. Five of the significantly up-regulated spots were identified as hemocyanin derived peptides. One of the five peptides, designated LvHcL48, was further characterized by analyzing its primary sequence via Edman N-terminal sequencing, C-terminal sequencing and amino acid sequence alignment. LvHcL48 was found to be a 79 amino acid fragment (aa584-662) from the C-terminal domain of L. vannamei hemocyanin protein (ADZ15149). Both in vivo and in vitro functional studies revealed that LvHcL48 has immunological activities, as recombinant LvHcL48 protein (rLvHcL48) significantly inhibited the transcription of the WSSV genes wsv069 and wsv421 coupled with a significant reduction in WSSV copy numbers. Further analysis showed that LvHcL48 could interact with the WSSV envelope protein 28 (VP28). Our present data therefore reveals the generation of an antiviral hemocyanin derived peptide LvHcL48 from WSSV infected shrimp, which binds to the envelope protein VP28 of WSSV.

Enhanced Fermentable Sugar Production from Enteromorpha Polysaccharides by the Crude Enzymes of Vibrio sp. H11.

In recent years, large-scale outbreaks of the green alga Enteromorpha prolifera in China's offshore waters have posed a serious threat. This study aimed to improve Enteromorpha polysaccharide (EP) enzymatic sugar production using the hydrolase system of Vibrio sp. H11, an EP-utilizing microbial strain. Strain H11 was found to contain 711 carbohydrate-related genes, and 259 genes belong to glycoside hydrolases that have the potential to hydrolyze EP. To maximize the capability of strain H11 to hydrolyze EP, both the culture medium and the composition were optimized. Response surface methodology analysis showed that maximal enzymatic production from strain H11 was 8.43 U/mL after 26-h incubation. When 50 g/L of EP were treated with crude H11 enzyme, the concentration of fermentation sugars increased by 36.12%. Under these conditions, the hydrolysates were capable of generating 3,217 mL/L of biogas and 6.74 g/L of biosolvents, with increases of 28.17 and 7.29%, respectively, compared to controls. The combined application of the H11 enzymatic system and anaerobic fermentation has the potential to improve the comprehensive application of EP.

A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells' Proliferation.

Antimicrobial peptides play important roles in the immune response to pathogens and tumor cells; for this reason, they are being exploited for therapeutic use. In this study, we describe a Litopenaeus vannamei hemocyanin-derived peptide, denoted B11, which shares similar features with other anticancer peptides and attenuates the proliferation of cancer cells. Cell viability assay revealed that B11 significantly inhibited the proliferation of human cervical (HeLa), human hepatocellular carcinoma (HepG2), and human esophageal cancer (EC109) cancer cell lines, but not normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE) cells or THLE-3), by inducing morphological changes, nuclear condensation, and margination, features which are indicative of apoptosis. Besides, peptide B11-induced apoptosis was confirmed by isothiocyanate-labeled Annexin V/propidium iodide (Annexin V-FITC/PI) double staining of HeLa cells. Moreover, cell uptake studies, confocal microscopy, and Western blot analysis revealed that rhodamine-labeled B11 permeated HeLa cells and localized to the mitochondria, causing mitochondria dysfunction through lost mitochondrial membrane potential, which consequently triggered the induction of apoptosis. Increased expression levels of caspase-9, caspase-3, and Bax (Bcl-2-associated X) proteins, coupled with a decrease in Bcl-2 (B-cell lymphoma 2) protein, confirmed that peptide B11 induced apoptosis via the mitochondrial pathway. Thus, the hemocyanin-derived peptide, B11, inhibits the proliferation of cancer cells by causing mitochondrial dysfunction and inducing apoptotic cell death, for which reason it could be explored as an anticancer peptide.

Functional domains of Litopenaeus vannamei transglutaminase and their involvement in immunoregulation in shrimp.

Shrimps, which mainly rely on their innate immune system to response to infectious pathogens, have clottable proteins as an important component of this system. While transglutaminases (TGase) are found in Litopenaeus vannamei and constitute part of the coagulation system, the specific immune-related roles played by its functional domains in the immunoregulation of shrimp has not been well understood. In the present study, we report that the Ig-like domain of L. vannamei transglutaminase (TGase-C) is the main immune-related domain among the three functional domains, as it had higher bacterial agglutinative activity against Vibrio parahaemolyticus and Streptococcus iniae. Using Co-immunoprecipitation and LC-MS/MS analysis, TGase-C was shown to interact with 474 proteins, of which 52 proteins were annotated to L. vannamei. More than half of the L. vannamei annotated proteins have immune-related functions, including apoptosis. Further analysis using pull-down assay revealed that TGase-C interacted with CAP-3 (a homologue of caspase 3). In addition, siRNA-mediated knockdown of LvTGase significantly (p < 0.01) increased the expression level of LvCAP-3 coupled with a significant (p < 0.01) increase in caspase 3/7 activity, suggesting that probably LvTGase participates in shrimp immune response by modulating the activity of LvCAP-3. These findings thus suggest the Ig-like functional domain of L. vannamei's transglutaminase is the domain that is involved in immunoregulation in shrimp.

SNPs in the Toll1 receptor of Litopenaeus vannamei are associated with immune response.

Tolls and Toll-like receptors (TLRs) are important regulators in the innate immune system and their genetic variations usually affect the host's susceptibility/resistance to pathogen infections. In this study, we report on the single nucleotide polymorphisms (SNPs) of Toll1 in Litopenaeus vannamei (LvToll1) and how this is associated with immune response. PCR-DGGE analysis revealed genetic polymorphisms in LvToll1 at both the genomic DNA (gDNA) and cDNA levels. Using high-throughput sequencing, 223 SNPs were identified at the gDNA level, of which 145 were non-synonymous SNP (nsSNP), with 3 nsSNPs having frequency over 1%. On the other hand, 60 SNPs were identified at the cDNA level including 38 nsSNPs and 4 nsSNPs with frequency over 1%. Upon challenging shrimps with Streptococcus iniae, Vibrio parahaemolyticus and white spot syndrome virus (WSSV), LvToll1 was shown to generate 6, 4 and 4 novel bands, respectively when analyzed with PCR-DGGE. Sequencing analysis of these bands showed that they contained 6, 4 and 2 nsSNPs, respectively. Moreover, the nsSNP C1526T was detected in S. iniae-resistant but not in susceptible shrimps. Most significantly, the C1526T mutation could shorten the α-helix of the LRR domain and was predicted to affect the function of LvToll1, indicating that SNP C1526T might be associated with shrimp's resistance to pathogen infections. In sum, our findings here reveal that the genetic polymorphisms of Toll receptor are linked with the immune response to pathogen infections in L. vannamei.

Comparative transcriptomic analysis of shrimp hemocytes in response to acute hepatopancreas necrosis disease (AHPND) causing Vibrio parahemolyticus infection.

The recent emergence of acute hepatopancreas necrosis disease (AHPND) in shrimps has posed a major challenge in the shrimp aquaculture industry. The Pir toxin proteins carried by some strains of Vibrio parahaemolyticus are believed to play essential roles in the pathogenesis of AHPND. However, few studies have so far explored how the host immune system responds to these bacteria. In this study, AHPND V. parahaemolyticus (with Pir) and non-AHPND V. parahaemolyticus (without Pir) were injected into two groups of shrimps, and the hemocytes collected for comparative transcriptomic analyses. A total of 1064 differentially expressed genes (DEGs) were identified, of which 910 were up-regulated and 154 were down-regulated. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that many DEGs were involved in a number of biological processes such as cellular process, metabolic process and single-organism process in the AHPND V. parahaemolyticus injected group than the non-AHPND V. parahaemolyticus injected group. Among these, major metabolic processes such as carbohydrate metabolism, lipid metabolism and amino acid metabolism were further identified as the major responsive gene groups. We observed that genes involved in cell growth and anti-apoptosis including src, iap2, cas2, cytochrome P450, gst and cytochromecoxidase were strongly activated in the AHPND V. parahaemolyticus group than in the non-AHPND V. parahaemolyticus group. Collectively, our results unveiled that shrimp hemocytes respond to AHPND related strain of Vibrio parahaemolyticus infection at the transcriptional level, which is useful in furthering our understanding of AHPND.

Litopenaeus vannamei hemocyanin exhibits antitumor activity in S180 mouse model in vivo.

Hemocyanin is a multifunctional glycoprotein, which also plays multiple roles in immune defense. While it has been demonstrated that hemocyanin from some mollusks can induce potent immune response and is therefore undergoing clinical trials to be used in anti-tumor immunotherapy, little is currently known about how hemocyanin from arthropods affect tumors. In this study we investigated the anti-tumor activity of hemocyanin from Litopenaeus vannamei on Sarcoma-180 (S180) tumor-bearing mice model. Eight days treatment with 4mg/kg bodyweight of hemocyanin significantly inhibited the growth of S180 up to 49% as compared to untreated. Similarly, histopathology analysis showed a significant decrease in tumor cell number and density in the tissues of treated mice. Moreover, there was a significant increase in immune organs index, lymphocyte proliferation, NK cell cytotoxic activity and serum TNF-α level, suggesting that hemocyanin could improve the immunity of the S180 tumor-bearing mice. Additionally, there was a significant increase in superoxide dismutase (SOD) activity and a decrease in the level of malondialdehyde (MDA) in serum and liver, which further suggest that hemocyanin improved the anti-oxidant ability of the S180 tumor-bearing mice. Collectively, our data demonstrated that L. vannamei hemocyanin had a significant antitumor activity in mice.

Synergistic enzymatic saccharification and fermentation of agar for biohydrogen production.

Nowadays, marine biomass is gradually considered as another utilizable material for the sustainable bioenergy development. In the present study, galactose, the main component of agar polysaccharide, was utilized for the biohydrogen production by Enterobacter sp. CN1. The highest hydrogen yield of 303.2mL/g was obtained in the cultivation media containing 5.87g/L of galactose, together with initial pH of 7.3 and incubation temperature of 36°C, after the response surface methodology (RSM) analysis. After the saccharification process by the agarase (AgaXa) and neoagarobiose hydrolase (NH852), the agar hydrolysate obtained was further applied to generate biohydrogen by strain CN1. Under the synergistic enzymatic saccharification and fermentation process, the production of biohydrogen was obtained to be 5047±228mL/L from 50g/L of agar, resulting in 3.86-fold higher than the control without enzymatic pretreatment.