Cloning and expression analysis of grouper (Epinephelus coioides) M-CSFR gene post Cryptocaryon irritans infection and distribution of M-CSFR(+) cells.

The M-CSF/M-CSFR system plays a central role in the cell survival, proliferation, differentiation and maturation of the monocyte/macrophage lineage. In present study, we cloned the sequence of the M-CSFR cDNA from the orange-spotted grouper (Epinephelus coioides). Sequence analysis reveals that ten cysteines in the extracellular immunoglobulin-like (Ig-like) domains of EcM-CSFR are conserved in fish and mammals, its nine possible N-glycosylation sites are conserved in fish but not mammals, 7 of 8 identified mammal M-CSFR intracellular autophosphorylation tyrosine sites was found in EcM-CSFR. Real-time PCR showed that the constitutive expression level of EcM-CSFR was the highest in the spleen, less in the gill, kidney, head kidney and liver, least in the blood, skin, gut and thymus. A rabbit anti-EcM-CSFR polyclonal antibody against the recombinant EcM-CSFR extracellular domain was developed and it was efficient in labeling the monocytes and macrophages isolated from the head kidney. Immunochemistry analysis showed that M-CSFR(+) cells located in all tested paraffin-embedded tissues and M-CSFR(+) cell centres with the characteristic of melano-macrophage centres(MMCs) was found in the spleen, head kidney, kidney, gut and liver. All these results indicate the widespread distribution of macrophages in grouper tissues and its importance in fish immune system. In Crytocaryon irritans infected grouper, EcM-CSFR was transient up-regulated and rapidly down-regulated in skin, gill, head kidney and spleen. The possible activation mechanism of macrophage via EcM-CSFR signal transduction in the fish anti-C. irritans infection was discussed.

Orange-spotted grouper (Epinephelus coioides) TLR2, MyD88 and IL-1ß involved in anti-Cryptocaryon irritans response.

Cryptocaryon irritans is one of the most important ectoparasites of marine fish, and can have a devastating effect on aquacultured fish populations. The role of TLR signaling pathways in anti-parasitic immune responses is poorly understood in fish. In this paper, we first cloned Epinephelus coioides MyD88 full-length cDNA (EcMyD88) and its respective gene. The open reading frame (ORF) of cDNA is 873bp encoding 291 amino acid residues. Similar to other species, the EcMyD88 gene contains of five conserved exons and four diverse introns. The constitutive expression of EcMyD88 was detected in the gill, trunk kidney, head kidney, spleen, and heart in high concentrations, while the skin, brain, liver, and muscles contained much lower titers, indicating that EcMyD88 may play a crucial role in host innate immunologic surveillance. To identify the potential role of TLR pathways in fish anti-C. irritans immune responses, we chose three important molecules involved in anti-parasite responses, TLR2, MyD88 and IL-1ß to indicate TLR pathway's signal-in, signal transduction, and signal-out functions, respectively. The expression profile of these three genes was detected in grouper infected by C. irritans. Results showed these molecules each experience significant changes within the skin, gill (two infected mucosal sites), head kidney and spleen (two systematic immune organs) after C. irritans infection. These findings indicate the TLR signaling pathway may play an important role in host defense against C. irritans.