Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.

Interleukin 1β (IL-1β) and interleukin 8 (IL-8) are two major pro-inflammatory cytokines which play a central role in initiation of inflammatory responses against bacterial- and viral-infections. IL-1β is a member of the interleukin 1 family proteins and IL-8 is classified as a CXC-chemokine. In the current study, putative IL-1β and IL-8 counterparts were identified from a black rockfish transcriptomic database and designated as RfIL-1β and RfIL-8. The RfIL-1β cDNA sequence consists of 1140 nucleotides with a 759bp open reading frame (ORF) which encodes a 252 amino acid (aa) protein, whereas the RfIL-8 cDNA sequence (898bp) harbors a 300bp ORF encoding a 99 aa protein. Furthermore, the RfIL-1β aa sequence contains an IL-1 super family-like domain and an N-terminal IL-1 super family propeptide, while the amino acid sequence of RfIL-8 consists of a typical chemokine-CXC domain. Analysis of sequenced BAC clones containing RfIL-1β and RfIL-8 showed each gene to contain 4 exons interrupted by 3 introns. Pairwise comparison and phylogeny analysis of these cytokine sequences clearly revealed their closer relationship with other corresponding members of teleosts compared to birds and mammals. Constitutive differences in RfIL-1β and RfIL-8 mRNA expression were detected in a tissue-specific manner with the highest expression of each mRNA in spleen tissue. Two immune challenge experiments were conducted with Streptococcus iniae and polyinosinic:polycytidylic acid (poly I:C; a viral double stranded RNA mimic), and transcripts were quantified in spleen and peripheral blood cells. Significantly increased RfIL-1β and RfIL8 transcript levels were detected with almost similar profile patterns, further suggesting a putative involvement of these pro-inflammatory cytokines in the rockfish immunity.

Cell-to-cell contacts play a key role in multicellular systems and organisms. Fasciclin-1 (FAS-1) is a lipid-linked membrane associated glycoprotein that is a member of a newly recognized family of cell adhesion molecules sharing features with the immunoglobulins, cadherins, integrins, and selectins. Here, we report the identification and molecular characterization of a novel FAS-1 domain-containing cDNA from disk abalone (Haliotis discus discus), including its gene expression profile and immune response to bacterial stimuli and tissue injuries. Designated as Abfac1, the 909bp open reading frame (ORF) encodes 303 amino acid (aa) residues with a predicted molecular mass of 33kDa and isoelectric (pI) value of 4.9. The aa sequence contains two FAS-1 domains and three conserved regions, FRa motif, H-box, and FRb motif. Phylogenetic analysis showed the closest relation to Jellyfish cell adhesion protein. In healthy abalone, Abfac1 expression is highest in hepatopancreas followed by mantle and lowest in digestive gland. In immune-stimulated abalones, relative Abfac1 mRNA expression was increased in hemocytes by ~11-fold at 48h after the Vibrio parahaemolyticus infection, by 3.1-fold at 6h after the Listeria monocytogenes infection and by ~9-fold at 6h after the LPS injection. Similarly, tissue injuries caused significant increase of relative mRNA expression by 3.5-fold in hemocytes and by ~10-fold in mantle at 12h post-injury. These results suggest that the novel member of the FAS-1 domain-containing protein family, Abfac1, may be involved in immune response and cell adhesion in disk abalone.

Caspase 10 is an initiator caspase in death cascades of death receptor mediated apoptotic signaling. We identified and molecularly characterized a novel homolog of caspase 10 from black rockfish (Sebastes schlegelii) and designated as RfCasp10. The complete coding region of RfCasp10 was found to consist of 1659 bps, encoding a 553 amino acid protein with a predicted molecular mass of 61.7 kDa. The characteristic caspase family domain architecture, including death effecter domains (DEDs), was clearly identified in RfCasp10. Moreover, the RfCasp10 gene was found to contain 13 exons. Our pairwise sequence alignment confirmed the prominent sequence similarity of RfCasp10 with its fish homologs, and phylogenetic reconstruction affirmed its homology and substantial evolutionary relationship with known caspases 10 similitudes, in particular with those of teleosts. As detected by qPCR, RfCasp10 was markedly expressed in blood tissues under physiological conditions, whereas its expression was found to be upregulated under pathogenic stress, elicited by Streptococcus iniae and polyinosinic:polycytidylic acid in blood, liver, and spleen tissues. Collectively, our study suggests the plausible elicitation of RfCasp10 mediated apoptosis in immune relevant tissues of black rockfish as a host immune response to a bacterial or viral infection.

The thioredoxin (Trx) system plays a significant role in cellular antioxidative defense by dismutating the surpluses of reactive oxygen species. Thus, the role of thioredoxin reductase (TrxR) cannot be ignored, owing to its participation in initiating the Trx enzyme cascade. Here, we report the identification and molecular characterization of a teleostean TrxR (RbTrxR-3) ortholog that showed high similarity with the TrxR-3 isoforms of other vertebrates. The complete RbTrxR-3 coding sequence comprised 1800 nucleotides, encoding a 600-amino acid protein with a predicted molecular mass of ~66 kDa. RbTrxR-3 consisted of 16 exons separated by 15 introns and had a total length of 12,658 bp. In silico analysis of the RbTrxR-3 protein sequence revealed that it possesses typical TrxR domain architecture. Moreover, using multiple sequence alignment and pairwise sequence alignment strategies, we showed that RbTrxR-3 has high overall sequence similarity to other teleostean TrxR-3 proteins, including highly conserved active site residues. Phylogenetic reconstruction of RbTrxR-3 affirmed its close evolutionary relationship with fish TrxR-3 orthologs, as indicated by its clustering pattern. RbTrxR-3 transcriptional analysis, performed using quantitative polymerase chain reaction (qPCR), showed that RbTrxR-3 was ubiquitously distributed, with the highest level of mRNA expression in the blood, followed by the gill, and liver. Live bacterial and viral stimuli triggered the modulation of RbTrxR-3 basal transcription in liver tissues that correlated temporally with that of its putative substrate, rock bream thioredoxin1 under the same conditions of pathogenic stress. Finally, resembling the typical function of TrxR protein, purified recombinant RbTrxR-3 showed detectable dose-dependent thiol reductase activity against 5,5'-dithiobis (2-nitrobenzoic) acid. Taken together, these results suggest that RbTrxR-3 plays a role in the host Trx system under conditions of oxidative and pathogenic stress.

Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a newly described negative immune regulator, whose enigmatic biological functions are not clearly understood. In the present study, the TNFAIP8L2 homolog of rock bream (Oplegnathus fasciatus) was identified and characterized. The genomic composition of rock bream TNFAIP8L2 (~6.7 kb) represented a tripartite arrangement in which three exons are interrupted by two introns. The rock bream TNFAIP8L2 transcript (1974 bp) possessed a coding sequence of 561 bp encoding a peptide of 186 amino acids. The predicted rock bream TNFAIP8L2 protein was 21.1kDa and revealed the typical features of known TNFAIP8L2 members including the DED-like domain. Rock bream TNFAIP8L2 was composed of six α-helices and demonstrated a distinct folding pattern of the TNFAIP8L2 family. It showed a certain degree of homology and phylogenetic relationship with the corresponding tilapia counterpart. Based on an interspecies genomic organizational comparison of TNFAIP8L2 orthologs, they could be classified into two classes, with teleost and non-teleost origin respectively. While teleost TNFAIP8L2s manifest a tripartite arrangement, non-teleost counterparts demonstrate a dipartite structure suggesting the loss of an intron during the post-piscine speciation. Promoter proximal region of rock bream TNFAIP8L2 consisted of multiple immune responsive cis-regulatory elements. Analysis of basal transcription in eleven tissues revealed its constitutive expression in examined tissues with highest magnitude in the head kidney. The modulated temporal mRNA expression of rock bream TNFAIP8L2 in head kidney post-challenges with stimulants (LPS and poly I:C) and pathogens (Streptococcus iniae and irido virus) was stimulant-specific. Additionally, a drastic down-regulation of rock bream TNFAIP8L2 mRNA level occurred in blood cells collected from experimentally injured animals, and it was accompanied by a contemporaneous down-regulation of cytokines, TNF-α and TGFβ3. All these findings imply that rock bream TNFAIP8L2 is potentially responsible for immune and inflammatory modulation in rock bream.