Jude Juventus Aweya

Litopenaeus vannamei notch affects lipopolysaccharides induced reactive oxygen species

&NA; Notch signaling pathway was originally discovered in the development stage of drosophila but has recently been found to play essential roles in innate immunity. Most previous studies on Notch have focused on mammals, whereas, in this study, we employed the shrimp Litopenaeus vannamei as a model to study the functions of Notch in invertebrate innate immune system. Our results showed that LvNotch was highly expressed in hemocytes and could be strongly induced by lipopolysaccharides (LPS) injection. Small interfering RNA (siRNA)‐mediated knockdown of LvNotch could significantly increase LPS induced L. vannamei mortality, which might be due to the fact that LPS induced ROS was greatly enhanced in LvNotch knockdown shrimps. Further, quantitative polymerase chain reaction (qPCR) analysis revealed that LvNotch could affect the expression of multiple genes, including dorsal, relish, anti‐lipopolysaccharide factor 1 (ALF1), ALF3 and NADH dehydrogenases which were upregulated, and Hypoxia‐inducible factor (HIF, &agr;/&bgr;) which were downregulated in LPS treated shrimps. In summary, LvNotch is important in the control of inflammation‐induced ROS production in shrimp. HighlightsWe applied a featured invertebrate animal model‐shrimp to study Notch function for the first time.Notch can affect inflammation related genes and generate ROS with LPS injection in shrimp.Notch from L. vannamei (LvNotch) was cloned and characterized.

Scavenger receptor B promotes bacteria clearance by enhancing phagocytosis and attenuates white spot syndrome virus proliferation in Scylla paramamosian

ABSTRACT Phagocytosis and apoptosis are key cellular innate immune responses against bacteria and virus in invertebrates. Class B scavenger receptors (SRBs), which contain a CD36 domain, are critical pattern recognition receptors (PRRs) of phagocytosis for bacteria and apoptotic cells. In the present study, we identified a member of SRB subfamily in mud crab Scylla paramamosain, named Sp‐SRB. The full‐length cDNA of Sp‐SRB is 2593 bp with a 1629 bp open reading frame (ORF) encoding a putative protein of 542 amino acids, and predicted to contain a CD36 domain with two transmembrane regions at the C‐ and N‐terminals. Real‐time qPCR analysis revealed that Sp‐SRB was widely expressed in all tissues tested, and the expression of Sp‐SRB was up‐regulated upon challenge with Vibrio parahaemolyticus, white spot syndrome virus (WSSV), lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (PolyI:C). Moreover, in vitro experiments indicated that recombinant Sp‐SRB protein (rSp‐SRB) could bind to fungi, Gram‐positive and Gram‐negative bacteria. RNA interference of Sp‐SRB resulted in significant reduction in the expression level of phagocytosis related genes, antimicrobial peptides (AMPs) and Toll‐like receptors (TLRs), which consequently led to impairment in both bacterial clearance and the phagocytotic activity of hemocytes. In addition, we found that Sp‐SRB had the ability to attenuate the replication of WSSV proliferation in mud crab S. paramamosain. Collectively, this study has shown that Sp‐SRB contributed to bacteria clearance by enhancing phagocytosis and up‐regulating the expression of AMPs possibly in a TRLs (SpToll 1 and SpToll 2)‐dependent manner. Besides, Sp‐SRB inhibited the replication of WSSV in S. paramamosian probably through enhancement of hemocytes phagocytosis of apoptotic cells. HIGHLIGHTSFull‐length of Sp‐SRB with 2593 bp was isolated from mud crab.Sp‐SRB promoted bacteria clearance by enhancing phagocytosis and up‐regulating the expression of AMPs.Sp‐SRB inhibited the replication of WSSV in S. paramamosian.

Trypsin of Litopenaeus vannamei is required for the generation of hemocyanin-derived peptides

ABSTRACT Hemocyanin is a copper containing respiratory glycoprotein in arthropods and mollusks, which also have multiple functions in vivo. Recent studies have shown that hemocyanin could generate several peptides, which play important roles in shrimp innate immunity. However, how these hemocyanin‐derived peptides are generated is still largely unknown. In this study, we report for the first time that the generation of hemocyanin‐derived peptides was closely correlated with trypsin expression in shrimp hepatopancreas following infection with different bacteria. RNA interference (RNAi) mediated knockdown of trypsin or treatment with the serine protease inhibitor, aprotinin, resulted in significant decrease in the levels of hemocyanin‐derived peptides. Moreover, recombinant trypsin (rTrypsin) was able to hydrolyse hemocynin in vitro with the hydrolysate having a high bacterial agglutination activity while the denatured hemocynin untreated with rTrypsin lost its agglutination activity. Taken together, our current results showed that the generation of hemocyanin‐derived peptides correlates with an increase trypsin expression. HighlightsFirst to show that Litopenaeus vannamei hemocyanin‐derived peptides generation requires trypsin or a trypsin‐like protease.Recombinant trypsin can digest hemocyanin in vitro, yielding a hydrolysate which possess bacterial agglutination activity.

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.

Glycosylation of hemocyanin in Litopenaeus vannamei is an antibacterial response feature

Hemocyanin is an important multifunctional non-specific immune molecule. In this study, we purified lectin binding and non-lectin binding hemocyanin from Litopenaeus vannamei using Concanavalin A (ConA) lectin affinity chromatography (designated HMC-C and HMC-NC, respectively). Analysis of the carbohydrate content showed that HMC-C had about 20 times as much carbohydrate as HMC-NC. 54 and 42 peaks were observed in HMC-C and HMC-NC by HPLC, which reduced to 49 and 6 peaks, respectively, when digested with trypsin and repurified with ConA lectin column. Further, the agglutinative activity of HMC-C against two pathogenic bacteria, Vibrio alginolyticus and Vibrio fluvialis, was about 8-fold and 4-fold, respectively, to that of HMC-NC. While the antibacterial activity of HMC-NC was about 30% lower compared with HMC-C. Similarly, when HMC was deglycosylated using O-glycosidase, its agglutinative activity reduced about 4-8 fold. Most importantly, when shrimps were challenged with V. alginolyticus or V. fluvialis, the glycan content of hemocyanin increased dramatically and remained high at the earlier time points (24-72h) post infection, only decreasing after 96 hpi. Taken together, these results suggest that hemocyanin glycosylation plays an important role in its antibacterial properties.

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

ABSTRACT Tolls and Toll‐like receptors (TLRs) are important regulators in the innate immune system and their genetic variations usually affect the hosts 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 &agr;‐helix of the LRR domain and was predicted to affect the function of LvToll1, indicating that SNP C1526T might be associated with shrimps 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. HighlightsWe found for the first time that Litopenaeus vannamei Toll1 (LvToll1) possessed extraordinary SNPs.It could be modulated by various pathogen infections.The SNP C1526T might be associated with the shrimps resistance to infections.

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

ABSTRACT 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 cytochrome c oxidase 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. HIGHLIGHTSWe found 1064 genes differentially expressed between the AHPND and Non‐AHPND V. parahemolyticus infected hemocytes in Litopenaeus vannamei.Among this, the genes involved in the major metabolism processes, cell growth and anti‐apoptosis were further identified as the major responsive genes in response to the AHPND V. parahemolyticus.Apart from the hepatopancreas, shrimp hemocytes also respond to AHPND V. parahemolyticus infection.

Identification and molecular characterization of the Pim1 serine/threonine kinase homolog in Litopenaeus vannamei

ABSTRACT The Pim1 serine/threonine kinase is associated with multiple cellular functions including proliferation, survival, differentiation, apoptosis, tumorigenesis, immune regulation and inflammation in vertebrates. However, little is known about the role of Pim1 in invertebrate immunity. In this study, we identified and characterized for the first time, a Pim1 (LvPim1) gene in Litopenaeus vannamei, with a full‐length cDNA of 2352 bp and a 1119 bp open reading frame (ORF) encoding a putative protein of 372 amino acids, which contains a typical serine/threonine kinase domain. Sequence and phylogenetic analysis revealed that LvPim1 shared a close evolutionary relationship with Pim1 from vertebrates. Real‐time qPCR analysis showed that LvPim1 was widely expressed in all tissues tested; with its transcript level induced in hepatopancreas and hemocytes upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), thus, suggesting its probable involvement in shrimp immune response. Moreover, knockdown of LvPim1 resulted in increased hemocytes apoptosis; shown by high caspase3/7 activity, coupled with increase in pro‐apoptotic LvCaspase3 and LvCytochrome C, and decrease in pro‐survival LvBcl2, LvIAP1, and LvIAP2 mRNA expression in hemocytes. Finally, LvPim1 knockdown renders shrimps more susceptible to V. parahaemolyticus infection. Taken together, our present data strongly suggest that LvPim1 is involved in modulating shrimp resistance to pathogen infection, promote hemocytes survival, and therefore plays a role in shrimp immune response. HIGHLIGHTLitopenaeus vannamei pim1 was identified as a LPS responsive gene.Litopenaeus vannamei pim1 played important roles for hemocytes anti‐apoptosis during inflammation.Litopenaeus vannamei Pim1 is involved in shrimp immune response by modulating apoptosis.

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

ABSTRACT 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. HighlightsWe found five hemocyanin‐derived peptides ˜10 kDa in size from WSSV‐challenged shrimp.a novel peptide LvHcL48 with 8.9 kDa could inhibit WSSV transcription and proliferation.LvHcL48 might bind to the viral envelope protein VP28 of WSSV.

Protective effects of Sargassum horneri against ammonia stress in juvenile black sea bream, Acanthopagrus schlegelii

Ammonia is an important environmental toxic pollutant that represents a biochemical and physiological hazard to living organisms, especially in intensive culture systems with high stocking density. This study aimed at evaluating the protective effects of Sargassum horneri (SH) on ammonia stress in black sea bream. Four hundred and eighty fingerlings with approximate mean body weight 12.0u2009±u20090.1xa0g were randomly distributed into four experimental groups in triplicate. Each group was stocked with 40 fish and fed with isonitrogenous (42% crude protein) and isolipid (12% crude lipid) experimental diets containing either 0% SH (control), 3% SH, 6% SH, or 9% SH in the feed. At the end of 8xa0weeks of experimental feeding, fish were exposed to ammonia for 24xa0h. The results indicated that ammonia stress led to an increase in oxidative stress as shown by elevation in the levels of cortisol and liver lipid peroxidative damage markers as well as decrease in antioxidant biomarkers. Furthermore, humoral immunity was suppressed during the stress period. However, fish supplied with S. horneri had significant improvement in immune response, antioxidant capacity, and resistance against ammonia stress, particularly in the 6% SH group. Therefore, dietary S. horneri supplementation at an optimum level of 6% could increase the resistance of juvenile black sea bream to ammonia-induced stress.