- Email: [email protected]
An RNA-Seq-based approach to understanding the immune system of the american lobster (Homarus americanus)
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
O-001. Tumor necrosis factor receptor-associated factor 6 (TRAF6) from mud crab participates in anti-lipopolysaccharide factors (ALFs) gene expression Shengkang Li, Wanwei Sun, Shasha Wang, Xiaobo Wen* Key Laboratory of Marine Biotechnology of Guangdong Province, Shantou University, Shantou, 515063, China Mud crab Scylla paramamosain, an importantly commercial crustacean species, is widely distributed along the coastline of southern China. However, with the development of intensive culture, frequent outbreaks of aquatic diseases have extremely constrained the sustainable development of mud crab farming. Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a cytoplasm key signal adapter protein that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp S. paramamosain TRAF6 (Sp-TRAF6) contains a 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAF6 has highest identity with PtTRAF6 (KP341006) from Portunus trituberculatus at 88%, while the similarity of other crustacean sequences was 54-55%. RT-PCR results indicated that the Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Further, Sp-TRAF6 transcripts were significantly up-regulated after immune challenge with Vibrio parahemolyticusor LPS. Our previous study had characterized two novel anti-lipopolysaccharide factor isoforms from S.paramamosain (SpALF5 and SpALF6). Both of them contain a conserved LPS-binding domain with two conservative cysteine residues, which is critical for their antimicrobial function. The in vitro binding and antimicrobial activity assays indicated that the recombinant SpALF5 and SpALF6 protein generated from prokaryotic expression system showed a varying degree of binding activity towards bacteria and fungus, and exhibited a broad spectrum of antimicrobial activities against Gram-positive, Gram-negative bacterium and fungi. Therefore, six ALF isoforms from mud crab had been reported up to now. To investigate SpTRAF6 activating SpALFs gene expression, RNA interference assay was carr ried out to examine the mRNA level of six SpALFs after silencing Sp-TRAF6 gene. The results shown that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasion via regulation of ALF gene expression in S. paramamosain. Keywords: The mud crab Scylla paramamosain; TRAF6; ALF gene expression Funding for this research was provided by Natural Science Foundation of China (41376176, 41176103) and Guangdong provincial project of Science and Technology (2012B020308007). * Corresponding author: Tel.: 0086 754 86502485; fax: 0086 754 86500614. E-mail address: [email protected] (X. Wen).
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The growth and accessibility of next-generation sequencing technologies has significantly enhanced our ability to determine the molecular mechanisms responsible for complex physiological processes in non-model organisms like crustaceans. The power and limitations of this technology are in the huge amount of data that is generated and the bioinformatic annotation of this data. Proper annotation is fundamental for understanding the molecular basis of a phenotype or condition. The American lobster (Homarus americanus) fishery is essential to the economic well-being of hundreds of small coastal Canadian and American communities. The long-term health and sustainability of this fishery is an important area of study. In addition, it is an excellent model for comparative physiological studies with other crab and shrimp species which are the basis of global aquaculture production. Understanding of decapod immune systems is critical as the most significant factor impacting sustainability and expansion of global shrimp aquaculture is disease. This study will use RNA-Seq studies of the American lobster to demonstrate an approach that significantly enhances immune-related annotation of genes involved in the crustacean immune response. In addition, it will highlight the similarities and differences between the lobster humoral immune response to Gram-positive bacterial (Aerococcus viridans var. homari) and scuticociliate (Anophryoides haemophila) challenges. Keywords: Transcriptome, American lobster, Crustacean Immunology, RNA-Seq, Gene Annotation * Corresponding author. Tel.: 902 5666028. E-mail address: [email protected] (C.K. Fraser).
O-003. Lipid metabolism during WSSV infection in shrimp Cheng-Shun Cheng, Han-Ching Wang* Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan Abstract Global metabolic changes in white spot syndrome virus (WSSV)-infected shrimp were recently clarified with proteomics and metabolomics. Interestingly, rerouting of host metabolism (known as Warburg effect in cancer cells) increased availability of energy and biosynthetic building blocks in host cells in shrimp after WSSV infection at the genome replication stage (12 hpi). Moreover, WSSV switched lipid metabolism of host from lipolysis at 12 hpi to lipogenesis at 24 hpi and used it to complete viral replication as well as morphogenesis. Lipolysis was induced by WSSV in hepatopancreas at replication stage (12 hpi), which released free fatty acids that were soon taken up by WSSV target tissues (e.g. hemocytes and stomach). Lipolysis was switched to hemocytes and stomach until lipid in hepatopancreas was exhausted at a late stage of viral infection (24 hpi). Furthermore, betaoxidation in shrimp may be triggered during WSSV infection. We determined that inhibition of beta-oxidation, or inhibition of fatty acid synthesis (FAS, a key enzyme of lipogenesis), prevented WSSV from completing its replication cycle. Therefore, we inferred that alteration of lipid metabolism might be essential for WSSV virion formation. Our study provided new insights into important changes in host lipid metabolism triggered by an invertebrate virus. Keywords: Litopenaeus vannamei, White spot syndrome virus, Warburg effect, Lipolysis, Lipogenesis * Corresponding author. Tel.: þ886 6 2757575 ext 65603 810; fax: þ886 6 276 6505. E-mail address: [email protected] (H.-C. Wang).
O-002. An RNA-Seq-based approach to understanding the immune system of the american lobster (Homarus americanus) Clark K. Fraser*, Spencer J. Greenwood Department of Biomedical Sciences and AVC Lobster Science Centre, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada
O-004. Antimicrobial peptides in crustaceans: Molecular and functional characterization Rosamma Philip 1, *,§, Afsal V. V 1, *, Swapna P. Antony 2, *, Sruthy K. S 1, *, Wilsy Wilson 1, *, Divya T. Babu 1, *, Jayesh P 1,*, I S Bright Singh 2, *
O-001. Tumor necrosis factor receptor-associated factor 6 (TRAF6) from mud crab participates in anti-lipopolysaccharide factors (ALFs) gene expression Shengkang Li, Wanwei Sun, Shasha Wang, Xiaobo Wen* Key Laboratory of Marine Biotechnology of Guangdong Province, Shantou University, Shantou, 515063, China Mud crab Scylla paramamosain, an importantly commercial crustacean species, is widely distributed along the coastline of southern China. However, with the development of intensive culture, frequent outbreaks of aquatic diseases have extremely constrained the sustainable development of mud crab farming. Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a cytoplasm key signal adapter protein that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp S. paramamosain TRAF6 (Sp-TRAF6) contains a 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAF6 has highest identity with PtTRAF6 (KP341006) from Portunus trituberculatus at 88%, while the similarity of other crustacean sequences was 54-55%. RT-PCR results indicated that the Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Further, Sp-TRAF6 transcripts were significantly up-regulated after immune challenge with Vibrio parahemolyticusor LPS. Our previous study had characterized two novel anti-lipopolysaccharide factor isoforms from S.paramamosain (SpALF5 and SpALF6). Both of them contain a conserved LPS-binding domain with two conservative cysteine residues, which is critical for their antimicrobial function. The in vitro binding and antimicrobial activity assays indicated that the recombinant SpALF5 and SpALF6 protein generated from prokaryotic expression system showed a varying degree of binding activity towards bacteria and fungus, and exhibited a broad spectrum of antimicrobial activities against Gram-positive, Gram-negative bacterium and fungi. Therefore, six ALF isoforms from mud crab had been reported up to now. To investigate SpTRAF6 activating SpALFs gene expression, RNA interference assay was carr ried out to examine the mRNA level of six SpALFs after silencing Sp-TRAF6 gene. The results shown that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasion via regulation of ALF gene expression in S. paramamosain. Keywords: The mud crab Scylla paramamosain; TRAF6; ALF gene expression Funding for this research was provided by Natural Science Foundation of China (41376176, 41176103) and Guangdong provincial project of Science and Technology (2012B020308007). * Corresponding author: Tel.: 0086 754 86502485; fax: 0086 754 86500614. E-mail address: [email protected] (X. Wen).
59
The growth and accessibility of next-generation sequencing technologies has significantly enhanced our ability to determine the molecular mechanisms responsible for complex physiological processes in non-model organisms like crustaceans. The power and limitations of this technology are in the huge amount of data that is generated and the bioinformatic annotation of this data. Proper annotation is fundamental for understanding the molecular basis of a phenotype or condition. The American lobster (Homarus americanus) fishery is essential to the economic well-being of hundreds of small coastal Canadian and American communities. The long-term health and sustainability of this fishery is an important area of study. In addition, it is an excellent model for comparative physiological studies with other crab and shrimp species which are the basis of global aquaculture production. Understanding of decapod immune systems is critical as the most significant factor impacting sustainability and expansion of global shrimp aquaculture is disease. This study will use RNA-Seq studies of the American lobster to demonstrate an approach that significantly enhances immune-related annotation of genes involved in the crustacean immune response. In addition, it will highlight the similarities and differences between the lobster humoral immune response to Gram-positive bacterial (Aerococcus viridans var. homari) and scuticociliate (Anophryoides haemophila) challenges. Keywords: Transcriptome, American lobster, Crustacean Immunology, RNA-Seq, Gene Annotation * Corresponding author. Tel.: 902 5666028. E-mail address: [email protected] (C.K. Fraser).
O-003. Lipid metabolism during WSSV infection in shrimp Cheng-Shun Cheng, Han-Ching Wang* Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan Abstract Global metabolic changes in white spot syndrome virus (WSSV)-infected shrimp were recently clarified with proteomics and metabolomics. Interestingly, rerouting of host metabolism (known as Warburg effect in cancer cells) increased availability of energy and biosynthetic building blocks in host cells in shrimp after WSSV infection at the genome replication stage (12 hpi). Moreover, WSSV switched lipid metabolism of host from lipolysis at 12 hpi to lipogenesis at 24 hpi and used it to complete viral replication as well as morphogenesis. Lipolysis was induced by WSSV in hepatopancreas at replication stage (12 hpi), which released free fatty acids that were soon taken up by WSSV target tissues (e.g. hemocytes and stomach). Lipolysis was switched to hemocytes and stomach until lipid in hepatopancreas was exhausted at a late stage of viral infection (24 hpi). Furthermore, betaoxidation in shrimp may be triggered during WSSV infection. We determined that inhibition of beta-oxidation, or inhibition of fatty acid synthesis (FAS, a key enzyme of lipogenesis), prevented WSSV from completing its replication cycle. Therefore, we inferred that alteration of lipid metabolism might be essential for WSSV virion formation. Our study provided new insights into important changes in host lipid metabolism triggered by an invertebrate virus. Keywords: Litopenaeus vannamei, White spot syndrome virus, Warburg effect, Lipolysis, Lipogenesis * Corresponding author. Tel.: þ886 6 2757575 ext 65603 810; fax: þ886 6 276 6505. E-mail address: [email protected] (H.-C. Wang).
O-002. An RNA-Seq-based approach to understanding the immune system of the american lobster (Homarus americanus) Clark K. Fraser*, Spencer J. Greenwood Department of Biomedical Sciences and AVC Lobster Science Centre, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada
O-004. Antimicrobial peptides in crustaceans: Molecular and functional characterization Rosamma Philip 1, *,§, Afsal V. V 1, *, Swapna P. Antony 2, *, Sruthy K. S 1, *, Wilsy Wilson 1, *, Divya T. Babu 1, *, Jayesh P 1,*, I S Bright Singh 2, *