Transcriptome analysis of immune-related genes in Sesarmops sinensis hepatopancreas in reaction to peptidoglycan challenge.

Sesarmops sinensis is a dominant omnivorous crab species, which plays an important ecological function in salt marsh ecosystems. To better understand its immune system and immune related genes under pathogen infection, the transcriptome was analyzed by comparing the data of S. sinensis hepatopancreas stimulated by PBS and PGN. A set of assembly and annotation identified 39,039 unigenes with an average length of 1105 bp, obtaining 1300 differentially expressed genes (DEGs) in all, which included 466 remarkably up-regulated unigenes and 834 remarkably down-regulated unigenes. In addition, based on mensurable real time-polymerase chain reaction and high-throughput sequencing, several immune responsive genes were found to be markedly up-regulated under PGN stimulation. In conclusion, in addition to enriching the existing transcriptome data of S. sinensis, this study also clarified the immune response of S. sinensis to PGN stimulation, which will help us to further understand the crustacean's immune system.

A complement factor I (CFI) gene mediates innate immune responses in yellow catfish Pelteobagrus fulvidraco.

This study isolated CFI gene from Pelteobagrus fulvidraco and named it PfCFI. The cDNA of PfCFI is 2374 bp long, including a 52 bp 5' untranslated sequence, a 222 bp 3' untranslated sequence, and an open reading frame (ORF) of 2100 bp encoding polypeptide consisting of 699 amino acids. Phylogenetic analysis revealed that the PfCFI was closely related to CFI of Ictalurus punctatus. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis indicate that there is the PfCFI gene which expressed in all the rest of tested tissues in varied levels, and mainly distributed in liver and least in heart. The reseachers induce the expressions level of PfCFI gene in liver, spleen, head kidney and blood at different points in time after challenged with lipopolysaccharide (LPS), and polyriboinosinic polyribocytidylic acid (poly I:C), respectively. Together these results suggested that CFI gene plays an important role in resistance to pathogens in yellow catfish immunity.

A non-mammalian Toll-like receptor 26 (TLR26)gene mediates innate immune responses in yellow catfish Pelteobagrus fulvidraco.

In this study, we identified a fish-specific Toll-like receptor (TLR) in Pelteobagrus fulvidraco, an economically important freshwater fish in China. This TLR, PfTLR26, was shown to be encoded by a 3084 bp open reading frame (ORF), producing a polypeptide 1027 amino acids in length. The PfTLR26 protein contains a signal peptide, eight leucine-rich repeat (LRR) domains, two LRR_TYP domains in the extracellular region, and a Toll/interleukin (IL)-1 receptor (TIR) domain in the cytoplasmic region, consistent with the characteristic TLR domain architecture. This predicted 117.1 kDa protein was highly homologous to those of other fish, with phylogenetic analysis revealing the closest relation to TLR26 of Ictalurus punctatus. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the PfTLR26 gene was expressed in all tissues tested, with the highest expression levels seen in the head kidney and blood, and the lowest seen in muscle. PfTLR26 exhibited significant upregulation in liver, spleen, head kidney, and blood at different time points following challenge with the common TLR agonists lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C). Taken together, these results suggest that PfTLR26 may be an important component of the P. fulvidraco innate immune system, participating in the transduction of TLR signaling under pathogen stimulation.

Transcriptomic analysis of immune-related genes in the lipopolysaccharide-stimulated hepatopancreas of the mudflat crab Helice tientsinensis.

The mudflat crab Helice tientsinensis is one of the most economically important aquaculture species in China. Nevertheless, it is susceptible to various diseases caused by viruses, bacteria and rickettsia-like organisms. A better understanding of the immune system and genes related to the responses to bacterial and viral infection is required. Herein, the hepatopancreas transcriptome of H. tientsinensis was analyzed by comparing control and lipopolysaccharide (LPS)-stimulated RNA-Seq data, yielding 91,885,038 bp and 13.78 Gb of clean reads. Following assembly and annotation, 93,207 unigenes with an average length of 883 bp were identified, of which 31,674 and 13,700 were annotated in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. Following LPS, 4845 differentially expressed genes (DEGs) were identified, of which 2491 and 2354 were up- and down-regulated, respectively. To further investigate immune-related DEGs, KEGG enrichment analysis identified immune response pathways, most notably the peroxisome and Toll-like receptor signaling pathways. Quantitative real time-PCR (qRT-PCR) confirmed the up-regulation of a random selection of DEGs. This systematic transcriptomic analysis of the innate immune pathway in H. tientsinensis expands our understanding of the immune system in crabs.

De novo transcriptome assembly and analysis of differential gene expression following lipopolysaccharide challenge in Pelteobagrus fulvidraco.

The yellow catfish, Pelteobagrus fulvidraco, has been recognized as an important freshwater aquaculture species in Eastern and Southeast Asia. To gain a better understanding of the immune response in P. fulvidraco, we analyzed its transcriptome following stimulation with lipopolysaccharide (LPS). Phosphate buffer saline (PBS) was used as control. Following assembly and annotation, 72,152 unigenes with an average length of 1090 bp were identified. A total of 370 differentially expressed genes (DEGs) in the P. fulvidraco were observed at 12 h post LPS treatment, including 197 up-regulated genes and 173 down-regulated genes. Clusters of Orthologous Groups of proteins (KOG/COG) annotation demonstrated that a total of 18,819 unigenes classified into 26 categories. Gene ontology (GO) analysis revealed 20 biological process subcategories, 7 cellular component subcategories and 20 molecular function subcategories. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified immune responses pathways. Quantitative reverse transcription polymerase chain reaction measured the expression of 18 genes involved in the immune response. CXCL2-like chemokine (CXCL2), goose-type lysozyme (LYZ G), and cathepsin K (CTSK) were significantly up-regulated. This study enriches the P. fulvidraco transcriptome database and provides insight into the immune response of P. fulvidraco against infection.

cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae).

Hepcidin is a small, cysteine-rich antimicrobial peptide with a highly conserved ß-sheet structure that plays a vital role in innate host immunity against pathogenic organisms. In this study, a hepcidin gene was identified in Pelteobagrus fulvidraco, an economically important freshwater fish in China. The gene is named PfHep. The complete PfHep cDNA was 723 bp, including a 5'-untranslated region (UTR) of 102 bp, a 3'-UTR of 339 bp and an open reading frame of 282 bp encoding a polypeptide of 93 amino acids, which includes a predicted signal peptide and the Hepcidin domain. The predicted mature, cationic PfHep protein has a typical hepcidin RX (K/R)R motif and eight conserved cysteine residues. The deduced PfHep protein sequence has 70%, 54% and 39% percent identity with hepcidins from Ictalurus punctatus, Danio rerio, and Homo sapiens, respectively. The predicted tertiary structure of PfHep is very similar to that of hepcidin in other animals. Phylogenetic analysis revealed that PfHep is closely related to the hepcidins of I. punctatus and I. furcatus. Real-time quantitative reverse transcription-PCR showed that the PfHep gene was expressed most in liver of healthy P. fulvidraco, and expressed to some extent in all the tissues tested. After challenge with lipopolysaccharide and polyriboinosinic:polyribocytidylic acid (poly I:C), respectively, the expression levels of PfHep were markedly upregulated in liver, spleen, head kidney and blood at different time points. Together these results imply that PfHep may be an important component of the innate immune system and be involved in immune defense against invading pathogens.

Transcriptome analysis of yellow catfish (Pelteobagrus fulvidraco) liver challenged with polyriboinosinic polyribocytidylic acid (poly I:C).

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important economic freshwater species in China. However, infection by bacterial pathogenic diseases has caused high mortality and great economic loss in aquaculture. It is necessary for disease control to know more about the P. fulvidraco immune system and its related genes in response to bacterial or viral infections. In this study, the transcriptomic profiles of liver from P. fulvidraco stimulated by polyriboinosinic polyribocytidylic acid (poly I:C) was analyzed using high-throughput sequencing method. After assembly and annotation, total 67,447 unigenes were acquired, with an average length of 1091 bp. Under the infection of poly I:C, 522 differentially expressed genes (DEGs) were identified, including 307 up-regulated genes and 215 down-regulated genes. To further investigate the immune-related DEGs, Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. The result of GO enrichment indicated gene response to external stimulus, regulation of response to stimulus, cellular response to stimulus, immune response and immune system progress. Significant KEGG enrichment analysis identified major immune related pathways. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that 13 immune response genes were identified to be up-regulated after 12 h of poly I:C stimulation compared to controls. Taken together, the results of our study are beneficial for better understanding of the immune system and defense mechanisms of yellow catfish in response to poly I:C infection.

A ferritin gene from Procambarus clarkii, molecular characterization and in response to heavy metal stress and lipopolysaccharide challenge.

Ferritin plays important roles in iron storage, detoxification, and immune response. Here, a ferritin gene (PcFer) was identified in Procambarus clarkii, an economically important freshwater crayfish. Full-length PcFer cDNA was 1022-bp, including a 135-bp 5'-untranslated region (UTR) with a typical iron responsive element, a 374-bp 3'-UTR, and a 513-bp open reading frame encoding a polypeptide of 170 amino acids which contained the Ferritin domain. PcFer has ion binding sites, a ferrihydrite nucleation center, and an iron ion channel. PcFer is phylogenetically closely-related to Pacifastacus leniusculus and Eriocheir sinensis ferritins. Real-time quantitative reverse-transcription PCR analysis showed that PcFer was expressed in all tested P. clarkii tissues, and expressed most in hepatopancreas. After challenge with various heavy metals and lipopolysaccharide, respectively, the hepatopancreatic expression levels of PcFer were markedly upregulated. These results suggest that expression of PcFer might be involved in immune defense and protection of P. clarkii against heavy metal stress.

Molecular identification and expression analysis of a goose-type lysozyme (LysG) gene in yellow catfish Pelteobagrus fulvidraco.

Lysozymes, innate immunity molecules, play a vital role in immune response to pathogens. The yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae) is an economically important fish in China. The aim of this study was to quantify expression of the P. fulvidraco LysG gene (a g-type lysozyme) in response to pathogen-associated molecular patterns (PAMP) challenge. First, the P. fulvidraco LysG gene (PfLysG) was cloned and characterized. The full-length cDNA of PfLysG is 1323 bp, including a 5'-untranslated region (UTR) of 131 bp, a 3'-UTR of 634 bp, and an open reading frame of 558 bp encoding a polypeptide of 185 amino acids, which contains a transglycosylase SLT domain (Pfam01464). The predicted molecular weight of the protein is 20.52 kDa with a pI of 9.08. Two catalytic residues and seven N-acetyl-D-glucosamine binding sites are conserved in the sequence and there is no predicted signal peptide. The deduced PfLysG protein sequence has 84%, 76% and 69% percent identity with the LysGs from Ictalurus furcatus, Danio rerio, and Salmo salar, respectively. The predicted tertiary structure of PfLysG is very similar to that from other animals. Phylogenetic analysis showed that PfLysG is closely related to those from Teleostei. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that PfLysG was expressed in all examined tissues and most highly expressed in head kidney, spleen, and intestine. After simulated pathogen challenge with lipopolysaccharide and polyriboinosinic polyribocytidylic acid, respectively, the mRNA expression of PfLysG was upregulated significantly at different time points. The results suggest that the identified g-type lysozyme of P. fulvidraco is involved in innate immune responses.

An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924 bp, including a 5'-untranslated region (UTR) of 38 bp, 3'-UTR of 980 bp and an open reading frame (ORF) of 906 bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A. japonicus against thermal stress.

Identification of differentially expressed genes in the spleens of polyriboinosinic polyribocytidylic acid (poly I:C)-stimulated yellow catfish Pelteobagrus fulvidraco.

The yellow catfish, Pelteobagrus fulvidraco (Siluriformes: Bagridae) is an economically important fish in China. However, genomic research and resources on this species are largely unavailable and still in infancy. In the present study, we constructed a cDNA library following poly I:C injection to screen for immune response genes in the spleens of P. fulvidraco using suppression subtractive hybridization (SSH). A total of 420 putative expressed sequence tag (EST) clones were identified at 24 h post-injection, which contain 103 genes consisting of 25 immune response genes, 12 cytoskeleton genes, 7 cell cycle and apoptosis genes, 7 respiration and energy metabolism genes, 7 transport genes, 26 metabolism genes, 10 stress response genes, 9 translational regulation genes, and 71 unknown genes. Real-time quantitative reverse transcription-PCR (qRT-PCR) results revealed that a set of randomly selected immune response genes were identified to be up-regulated after 24 h of poly I:C stimulation compared to controls. Our study provides an annotation of immune genes in detail and insight into fish immunity.

Characterization of immune-related genes in the yellow catfish Pelteobagrus fulvidraco in response to LPS challenge.

Fish are considered an excellent model for studies in comparative immunology as they are a representative population of lower vertebrates linked to invertebrate evolution. To gain a better understanding of the immune response in fish, we constructed a subtractive cDNA library from the head kidney of lipopolysaccharide-stimulated yellow catfish (Pelteobagrus fulvidraco) using suppression subtractive hybridization (SSH). A total of 300 putative EST clones were identified which contained 95 genes, including 27 immune-related genes, 7 cytoskeleton-related genes, 3 genes involved in the cell cycle and apoptosis, 9 respiration and energy metabolism-related genes, 7 genes related to transport, 24 metabolism-related genes, 10 genes involved in stress responses, seven genes involved in regulation of transcription and translation and 59 unknown genes. Using real-time quantitative reverse transcription PCR, a subset of randomly selected genes involved in the immune response to lipopolysaccharide challenge were investigated to verify the reliability of the SSH data which identified 16 up-regulated genes. The genes identified in this study provide novel insight into the immune response in fish.

Differentially expressed genes in head kidney of Pelteobagrus fulvidraco following Vibrio cholerae challenge.

The yellow catfish (Pelteobagrus fulvidraco) is a freshwater fish with high economic value in eastern China. Nevertheless, pathogens causing bacterial diseases in P. fulvidraco have brought about huge economic loss and high mortality in artificial aquaculture. For disease control, it is critical to further understand the immune system of yellow catfish and immune-related genes with which they respond to pathogenic infections. In this study, high-throughput sequencing methods were used to analyze the transcriptomic spectrum of the head kidney from P. fulvidraco challenged by Vibrio cholera. A total of 45,544 unique transcript fragments (unigenes) were acquired after assembly and annotation, with an average length of 1,373 bp. Additionally, 674 differentially expressed genes (DEGs) were identified after stimulation with V. cholerae, 353 and 321 genes were identified as remarkably up- or downregulated, respectively. To further study the immune-related DEGs, we performed KEGG enrichment and GO enrichment. The results showed gene regulation of response to stimulus, immune response, immune system progress, response to external stimuli and cellular response to stimuli. Analysis of KEGG enrichment is important to identify chief immune related pathways. Real-time quantitative reverse transcription-PCR (qRT-PCR) results indicated 10 immune response genes that were found to be upregulated compared to a control group after 6 h of V. cholerae challenging. In summary, the results of our study are helpful to determine the defense mechanisms and immune system responses of yellow catfish in reaction to bacterial challenges.

Molecular identification and expression analysis of natural resistance-associated macrophage protein (Nramp) gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae).

Natural resistance associated macrophage protein genes (Nramp) is one of the important candidate genes responsible for regulating immune response against pathogen infection. The aim of the present was to quantify expression of Nramp gene in response to pathogen infection. Here, a Nramp was identified and molecularly characterized from Pelteobagrus fulvidraco (PfNramp). The obtained 3134 bp cDNA fragment of PfNramp comprised a 5'-untranslated region (UTR) of 81 bp, a 3'-UTR of 1403 bp and an open reading frame (ORF) of 1650 bp, encoding a polypeptide of 549 amino acids that contained a typical structural features of Nramp domain (Pfam01566). BLAST analysis exhibited that PfNramp shared sequence similarity to other organisms, in particular to Ictalurus furcatus (92%), Danio rerio (82%), and Homo sapiens (77%). Phylogenetic analysis revealed that PfNramp is close to Teleostei. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that PfNramp was expressed in all examined tissues, with the highest abundance in liver. The mRNA expression of PfNramp was remarkably increased at different time points after lipopolysaccharide (LPS), and polyriboinosinic polyribocytidylic acid (poly I:C) challenge. These results suggest that PfNramp is an inducible protein in the innate immune reactions of P. fulvidraco and probably in other fish species.

A myeloid differentiation factor 88 gene from yellow catfish Pelteobagrus fulvidraco and its molecular characterization in response to polyriboinosinic polyribocytidylic acid and lipopolysaccharide challenge.

Myeloid differentiation factor 88 (MyD88) is an adaptor protein of Toll-like receptor (TLR) signalling pathways that activates the innate immune system. Herein, MyD88 was identified in the economically important freshwater fish Pelteobagrus fulvidraco. The complete 2156 bp PfMyD88 cDNA includes a 147 bp 5'-untranslated region (UTR), a 1133 bp 3'-UTR, and an open reading frame (ORF) of 876 bp encoding a 291 residue protein containing Death and Toll/interleukin-1 receptor (TIR) domains. The deduced protein sequence shares 88.8%, 73.8% and 59.3% identity with orthologs in Ictalurus punctatus, Danio rerio and Homo sapiens, respectively. qRT-PCR revealed expression in all tested tissues, highest in trunk kidney, followed by spleen, and lowest in muscle. After challenge with lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid (Poly I:C), PfMyD88 expression was up-regulated in blood, liver, head kidney and spleen. Thus, PfMyD88 acts in innate immunity in P. fulvidraco.

Mitochondrial genome of Argopecten irradians reveals higher-level phylogenetic relationships in Anisomyaria.

The complete mitochondrial genome of Argopecten irradians strain Zhongkehong was sequenced and annotated: it is 16,212 bp in length and contains twelve protein-coding genes (atp8 is absent, as in most species in Anisomyaria), two ribosomal RNA genes, and 21 transfer RNA genes (trnS is absent and there are two copies of trnF). The heavy strand has an overall A + T content of 57.3%; GC and AT skews are 0.249 and -0.262, respectively, indicating more Gs and more Ts than Cs and As. Phylogenetic analysis based on Bayesian Inference and Maximum Likelihood of the twelve protein-coding genes shows that A. irradians has close relationships with A. purpuratus and A. ventricosus; this indicated that A. irradians belongs to the Pectinidae family. The Pectinidae was sister to (Ostreidae + Mytilidae). This work provides general information on the evolution of cultured scallops.

Characterization of the complete mitochondrial genome of the red crayfish, Procambarus clarkii (Decapoda: Cambaridae).

Here we present the complete mitochondrial (mt) genome of Procambarus clarkii (Decapoda: Cambaridae) and provide its annotation. The complete mt genome was determined to be 15 929 bp and contains 22 tRNA genes, 13 protein-coding genes (PCGs), two rRNA genes, and a D-loop region. The nucleotide composition was biased toward A + T nucleotides (72.91%) and the AT skew of this mt genome was slightly negative. All the 22 tRNA genes displayed a typical clover-leaf structure, with the exception of trnS1 (AGN). About 13 PCGs were initiated by ATN codons, except for cox1 and nad2 genes which were initiated by ACG and GTG, respectively. Six of the 13 PCGs harbor the incomplete termination codon by T or TA. The D-loop region of the mt genome was 1188 bp in length and the A + T content was 81.08%. Phylogenetic analysis showed that the placement of P. clarkii was within the Cambaridae. This mt genome sequence will provide a better understanding for crayfish evolution in the future.

Identification of the complete mitochondrial genome of the pacific white shrimp Litopenaeus vannamei (Decapoda: Penaeidae).

The complete mitochondrial genome (mitogenome) of Litopenaeus vannamei was determined to be 15 989 bp in size, it contains 22 tRNA genes, 13 protein-coding genes (PCGs), two rRNA genes and a D-loop region. The nucleotide composition was biased toward A + T nucleotides (67.8%) and the AT skew of this mitogenome was slightly negative (-0.025). All tRNA genes displayed a typical clover-leaf structure, except for trnS1 (AGN). Thirteen PCGs were initiated by ATN codons, except for cox1 gene which was initiated by ACG. Four of the 13 PCGs had the incomplete termination codon by T. The rrnL was 1371 bp and the rrnS was 853 bp, and the AT content of the combined rRNA genes was 70.9%. The D-loop region of the mitogenome was 985 bp in length and the A + T content was 82.7%, and no tandem repeat was found in this region. Phylogenetic analysis showed that the placement of L. vannamei was within the Penaeidae.

The complete mitochondrial genome of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae).

The complete mitochondrial genome (mitogenome) of Tribolium castaneum (Coleoptera: Tenebrionidae) was determined to be 15,883 bp (GenBank accession No. KM009121), which contains 22 tRNA genes, 13 protein-coding genes (PCGs), 2 rRNA genes and a major non-coding A + T-rich region. It has the typical gene organization and order of mitogenomes from ancestral insects. The nucleotide composition was also biased toward A + T nucleotides (71.72%) and the AT skew of this mitogenome was slightly positive. All of the 22 tRNA genes displayed a typical clover-leaf structure, with the exception of trnS1 (AGN). Thirteen PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which was initiated by AAT. Eight of the 13 PCGs harbor the incomplete termination codon by T or TA. The A + T-rich region of the mitogenome was 1237 bp in length and the A + T content was 82.30%.