Expression and presentation of immune-related membrane proteins of fish by a cell surface display platform using insect cells.

Cell surface display is a useful platform to examine the interactions between two proteins of interest, such as immune receptors and ligands. This technique is also useful for studies on the immune receptors of lower vertebrates and invertebrates. However, in many cases, the commonly used cell culture temperature is relatively high for proteins from such organisms. Since insect cells can be cultured at lower temperatures than many other cells, and since they are equipped with "quality control" system, which is advantageous for the presentation of properly folded proteins, we anticipated that the insect cell surface display system could be more suitable for that type of research. In the present study, multiple cloning site of the commercially available expression vector pIB/V5-His was modified, and whether this vector could be useful to present fish immune-related membrane proteins was investigated. Using this plasmid, fugu's CD8α and CC chemokine receptor 7 could be presented on the cell surface. The clones of the lamprey variable lymphocyte receptors obtained previously by the yeast surface display (YSD) system as hen's egg lysozyme (HEL) binders also could be presented on the cell surface and bound to HEL. These results suggest that functional immune-related membrane proteins can be presented on the insect cell surface, indicating that this system is useful for immunological studies on exothermal animals.

Variations in bile tolerance among Lactococcus lactis strains derived from different sources.

Lactococcus lactis subsp. lactis has been isolated from the intestines of marine fish and is a candidate probiotic for aquaculture. In order to use the bacterium as a probiotic, properties such as bile tolerance need to be assessed. Here, we compared bile tolerance in L. lactis strains derived from different sources. Three L. lactis subsp. lactis strains from marine fish (MFL), freshwater fish (FFL), and cheese starter (CSL) were used along with an Lactococcus lactis subsp. cremoris strain from cheese starter (CSC). The four strains were grown under various culture conditions: deMan-Rogosa-Sharpe (MRS) broth containing bile salts/acids, MRS agar containing oxgall, and phosphate-buffered saline (PBS) containing fish bile. Survival/growth of the strains in the presence of sodium cholate and sodium deoxycholate varied in the order MFL, CSL > CSC > FFL; in the presence of sodium taurocholate, the order was MFL > CSL > CSC > FFL. In liquid media containing various concentrations of oxgall, survival of the strains was observed in the order MFL > CSL > FFL and CSC. The survival of MFL was not affected by bile collected from the goldfish (Carassius auratus subsp. auratus) or the puffer fish (Takifugu niphobles), although the other strains showed significant inhibition of growth. It is a novel and beneficial finding that MFL has the highest resistance to bile acid.

Larval pufferfish protected by maternal tetrodotoxin.

Marine pufferfish contain tetrodotoxin (TTX), an extremely potent neurotoxin. All species of the genus Takifugu accumulate TTX in the liver and ovaries, although the tissue(s) in which it is localized can differ among species. TTX is the major defense strategy the pufferfish appears to use against predators. TTX is also used as a male-attracting pheromone during spawning. Here we demonstrate an additional (and unexpected) use of maternal TTX in the early larval stages of the Takifugu pufferfish. Predation experiments demonstrated that juveniles of all the species of fish used as predators ingested pufferfish larvae, but spat them out promptly. Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) analysis revealed that the pufferfish larvae contain a small quantity of TTX, which is not enough to be lethal to the predators. Immunohistochemical analysis with anti-TTX monoclonal antibody revealed that the TTX is primarily localized in the body surface of the larvae as a layer of protection. Our study showed the female parent of the Takifugu pufferfish vertically transfers TTX to the larvae through its accumulation in the ovaries, and subsequent localization on the body surface of the larvae.

Difference in the localization of tetrodotoxin between the female and male pufferfish Takifugu niphobles, during spawning.

In order to understand the sexual differences in TTX-usage in the pufferfish, Takifugu niphobles, localization of TTX and toxin amount in tissues of mature male and female specimens were investigated by immunohistochemical methods using anti-TTX antibody and LC/MS analysis. Subsequently, differences in the immunohistochemical signals were compared with the amount of TTX. The paraffin-embedded sections of the skin, muscle, liver, gonad and intestinal tract were subjected to anti-TTX monoclonal antibody based on the fluorescent immunohistochemical techniques. Immuno-positive reaction was observed in the skin and liver in males, and the skin and ovary in females. In the skin, TTX was localized at the epidermis, the basal cell layer, the mucous cells and the sacciform cells, and with intense immunoreaction at the flat epithelial cell layer and the sacciform cells. The signal from the liver cells was stronger in males than in females. The intensity of the signal from the tissues correlated with the toxin amounts therein. These results suggest that tissue distributions of TTX and toxin amount in the pufferfish were sex-dependent.