Characterization and functional analysis of a tandem-repeat galectin-9 in large yellow croaker Larimichthys crocea.

Galectins are a family of endogenous lectins with ß-galactosides affinity, playing significant roles in the innate immunity of vertebrates and invertebrates. In this report, a new galectin-9 cDNA was identified and characterized in large yellow croaker Larimichthys crocea (designated as LcGal-9). The complete cDNA sequence of LcGal-9 was 1795 bp, with an open reading frame (ORF) of 1032 bp encoding 343 amino acids. The putative LcGal-9 protein contained two carbohydrate recognition domains (CRDs) connected by a linker peptide, with each carrying two conserved ß-galactoside binding motifs H-NPR and WG-EE-, and it possessed neither a signal peptide nor a transmembrane domain. LcGal-9 protein shared 43-74% identity with galectin-9 sequences from other species. The qRT-PCR analysis revealed that LcGal-9 mRNA was constitutively expressed in all tissues examined, predominately expressed in liver, spleen, gill, kidney, head-kidney and intestine. Western blot analysis showed that LcGal-9 protein was highly expressed in liver, spleen, intestine, kidney, head-kidney, skin, gill, and heart, but not detected in muscle and plasma. LcGal-9 mRNA transcripts were induced by poly I:C in the liver (from 6 h to 48 h), spleen (at 12 h) and head-kidney (at 12 h and 24 h). In contrast, Vibrio parahaemolyticus caused a significant down-regulation in these three tissues, except for in spleen of 48 h and head-kidney of 3 h. Post-infection with Cryptocaryon irritans, the transcripts were dramatically up-regulated in gill, skin, spleen and head-kidney during initial infection period, while significant down-regulation afterward was also observed both in spleen and head-kidney. The recombinant LcGal-9 (named as rLcGal-9) purified from Escherichia coli BL21 (DE3) demonstrated hemagglutination against human, rabbit and L. crocea in a Ca(2+)-independent manner, which was inhibited by α-Lactose and LPS. The results of bacterial agglutination assays showed that rLcGal-9 was able to agglutinate Gram-negative bacteria V. alginolyticus and Aeromonas hydrophila in a Ca(2+)-independent manner. By immunohistochemistry assay, significant increases of LcGal-9 protein appeared in the spleen stimulated with poly I:C (for 12 h) and V. parahaemolyticus (for 48 h) compared with the control. Based on the collective data, LcGal-9 might play an important role in innate immune responses, especially defense against Gram-negative bacteria in L. crocea.

Expression profiles and interaction suggest TBK1 can be regulated by Nrdp1 in response to immune stimulation in large yellow croaker Larimichthys crocea.

TBK1 has been extensively studied in mammals because of its important roles as a molecular bridge, linking the TLRs (TLR3 and TLR4) and RLRs signals to activate transcriptional factors IRF3 and IRF7 for IFN-I production. However, the information on molecular and functional characteristics of TBK1 in teleosts is limited. In this study, the molecular characterization and immune response of TBK1 in Larimichthys crocea (named as LcTBK1) as well as its interaction with Nrdp1 were investigated. Sequence analysis demonstrated that LcTBK1 included four functional motifs, the N-terminal protein kinase domain and ATP-binding site, middle ULD and C-terminal coiled-coil domain. The tissue expression profiles indicated that LcTBK1 gene was constitutively expressed in the twelve tissues examined, with high expression in brain. Temporal expression analysis showed that LcTBK1 mRNA was obviously increased in the liver after injection of LPS, Poly I:C and inactive Vibrio parahaemolyticus, however, declined at some time points in spleen and head-kidney. Furthermore, we found that LcTBK1 can interact with LcNrdp1, an E3 ubiquitin ligase that involved in immune response to Cryptocaryon irritans infection in L. crocea. The qPCR showed that LcNrdp1 was also significantly up-regulated in liver, down-regualted at some time points in spleen and head-kidney after LPS, Poly I:C and inactive V. parahaemolyticus injection, although the expression patterns of the two genes after the three treatments were different in change magnitude and up-regulation timespan. These results suggested that LcTBK1 was involved in L. crocea defense against the pathogen infection and can be regulated by Nrdp1 in PPRs signaling pathway of fishes.

Characterization and expression analysis of BCA2 gene in large yellow croaker, Larimichthys crocea.

BCA2, as an E3 ubiquitin ligase, is an important anti-virus immune factor in mammals. Up to date, there are not any related reports on BCA2 protein in fishes yet. In the present investigation, BCA2 in large yellow croaker Larimichthys crocea (named as LcBCA2) was identified and characterized. The full-length cDNA of LcBCA2 was 1571 bp, including an ORF of 888 bp encoding a polypeptide of 295 amino acids. The putative LcBCA2 protein contained a RING-H2 motif at C terminal. The LcBCA2 transcripts were broadly distributed in all detected tissues, with high expression in muscle, moderate in blood, skin, heart, liver and spleen, weak in other tissue as indicated by qPCR analysis. Significant increases were observed in skin, gill and spleen after infection of Cryptocaryon irritans, and in spleen and head-kidney after inactivated Vibrio. parahaemolyticus, LPS and Poly I:C stimulations. Tissue localization by in-situ hybridization showed that LcBCA2 mainly expressed in the spleen of the fish in the test group. Our findings showed that LcBCA2 inclined to sharply increase in immune organs, especially in head-kidney after bacterial and viral stimulations, while in locations (skin and gill) of parasites infections, suggesting that BCA2 may play an important role in fish defense against bacteria, virus and parasites infections, but the immune mechanisms is are different.

Molecular cloning and expression analysis of scd1 gene from large yellow croaker Larimichthys crocea under cold stress.

Desaturation of fatty acids is an important adaptation mechanism to maintain membrane fluidity under cold stress. To comprehend the mechanism of adaptation to low temperatures in fish, we investigated stearoyl-CoA desaturase 1 (SCD1) endocrine expression in the process of cold acclimation from 15°C to 7°C in Larimichthys crocea. The cDNA and genomic sequences of scd1 were cloned and characterized and named as Lcscd1. The cDNA encoded an iron-containing protein of 337 amino acids with functional motifs. The full-length genome sequence of Lcscd1 was composed of 2556 nucleotides, including five exons and four introns. Tissue expression profiles by qPCR and western blot analysis revealed that Lcscd1 was highly expressed in the liver, followed by the brain. The expression of Lcscd1 mRNA in the liver was firstly down-regulated from 15°C to 11°C, and then up-regulated until the first day of 7°C, followed by a decline until the last day. In the brain, the expression showed no significant change from 15°C to 9°C, but then significantly increased until the last day of 7°C. SCD1 protein expression in the liver decreased from 15°C to the first day of 7°C, and then gradually recovered to the starting level. In the brain, SCD1 protein expression maintained rising trends in the whole process. Immunoelectron microscopic analysis showed that SCD1 was localized in fat granules, mitochondria and granular endoplasmic reticulum of hepatic cells, but only in mitochondria of encephalic cells. The results above suggested that SCD1 expression was responsive to both cold and starvation stresses in the liver, but only to cold stress in the brain. In conclusion, these findings suggested that SCD1 may be involved in fish adaptation to cold stress.

Characterization of E3 ubiquitin ligase neuregulin receptor degradation protein-1 (Nrdp1) in the large yellow croaker (Larimichthys crocea) and its immune responses to Cryptocaryon irritans.

Neuregulin receptor degradation protein-1 (Nrdp1) was recently identified in humans as an important immune factor responding to the challenge of virus, LPS or cytokine. Its role in fish immune defense and whether it is involved in anti-parasite immunity have not been proven yet. In this report, the full-length cDNA sequence and genomic structure of Nrdp1 in the large yellow croaker Larimichthys crocea (LcNrdp1) were identified and characterized. The full-length cDNA of LcNrdp1 was 1248bp, including a 5' untranslated region (UTR) of 32bp, a 3' UTR of 259bp and an open reading frame (ORF) of 937bp, encoding a polypeptide of 318 amino acid residues. The full-length genomic DNA sequence of LcNrdp1 was composed of 2635 nucleotides, including four exons and three introns. The putative LcNrdp1 protein had no signal peptide sequence and contained a characteristic Nrdp1 consensus motif C3HC3D ring finger and a Coiled-coil domain. Phylogenetic analysis showed that Nrdp1 in fish was closer with that in other vertebrates (79%-90% amino acid identity) than in invertebrates and bacteria (27%-65%). In fishes, Nrdp1 in large yellow croaker was closer with that in Takifugu rubripes. The expression profile showed that LcNrdp1 was constitutively expressed in all tested tissues, especially highly expressed in brain, muscle and kidney. Post-infection (PI) with Cryptocaryon irritans, an increased expression of LcNrdp1 was induced in infection sites (skin and gill), whereas in immune organs, the expression of LcNrdp1 was up-regulated in spleen (except the 1st d and 10th d PI) but suppressed in head kidney. These results suggested that LcNrdp1 might play an important immune role in the finfish L. crocea in the defense against the parasite C. irritans.

A microstructural study of individual nacre tablet of Pinctada maxima.

Nacre tablets from the shell of Pinctada maxima were studied with SEM, TEM and STEM. The systematic nanolath morphology on the (001) surface of nacre tablets was observed after acidic etching and mechanical polishing. The nanolaths were along the [100] crystallographic orientation of aragonite crystal. The (010) and (100) cross section surfaces of the nacre tablets showed nanolath and nanograin morphologies, respectively, which was consistent with [100] crystallographic orientation of nanolath on the (001) surface. Sheet-like defects with low mass density were observed on the (001) plane inside nacre tablets and were considered to be the cause of nanolath morphology revealed on the surfaces by acidic etching and mechanical polishing. On the other hand, large block [110] twins that divide the nacre tablets into two sectors were identified. The implication of these twins on the understanding to the crystallization mechanism of nacre tablets was discussed.