Hindgut Bacterial Flora Analysis in Oriental Honey Buzzard (Pernis ptilorhynchus).

The intestinal microbiome is known to affect host health through various effects on nutrition and immunity. The oriental honey buzzard (OHB) is a raptor that feeds on bees and wasps. Due to its restricted diet, we reasoned that the OHB may have a unique microbiome. The aim of this study was to characterize the structure of the intestinal flora of oriental honey buzzards and to investigate the difference of intestinal bacterial flora between individuals in the wild and those reared in captivity. We investigated the intestinal microbiome of seven wild buzzards (Wild), one zoo-reared (Zoo), and one individual reared in captivity for one month (Rearing). Average operational taxonomic units in Wild and Rearing were 69.4 and 113, respectively. Diversity indices such as ACE, Chao 1, Shannon, and Alpha were significantly lower in the Wild than in the Rearing samples. These results suggest that the variety of Wild microbiome is remarkably low. At the phylum level, the composition of the microbiome was similar in all three groups, with firmicutes and bacteroidetes predominating. The third most abundant bacterium in Wild was Proteobacteria, whereas it was Actinobacteria in Rearing and unclassified bacteria in Zoo. Thus, microbiome composition is affected even with just one month of human rearing.

Establishment of a kit-negative cell line of melanocyte precursors from mouse neural crest cells.

We previously established a mouse neural crest cell line named NCCmelb4, which is positive for Kit and negative for tyrosinase. NCCmelb4 cells were useful to study the effects of extrinsic factors such as retinoic acids and vitamin D(3) on melanocyte differentiation, but in order to study the development of melanocytes from multipotent neural crest cells, cell lines of melanocyte progenitors in earlier developmental stages are needed. In the present study, we established an immortal cell line named NCCmelb4M5 that was derived from NCCmelb4 cells. NCCmelb4M5 cells do not express Kit and are immortal and stable in the absence of Kit ligand. They are positive for melanocyte markers such as tyrosinase-related protein 1 and DOPAchrome tautomerase and they contain stage I melanosomes. Interestingly, glial fibrillary acidic protein, which is a marker for glial cells, is also positive in NCCmelb4M5 cells, while NCCmelb4 cells are negative for this protein. Immunostaining and a cell ELISA assay revealed that 12-O-tetradecanoylphorbol 13-acetate (TPA) and cholera toxin (CT) induce Kit expression in NCCmelb4M5 cells. Real-time polymerase chain reaction analysis also demonstrated the induction of Kit mRNA by TPA and CT. Microphthalmia-associated transcription factor mRNA is simultaneously enhanced by the same treatment. Kit induced by TPA/CT in NCCmelb4M5 cells disappeared after the cells were subcultured and incubated without TPA/CT. These findings show that NCCmelb4M5 cells have the potential to differentiate into Kit-positive melanocyte precursors and may be useful to study mechanisms of development and differentiation of melanocytes in mouse neural crest cells.

Establishment by an original single-cell cloning method and characterization of an immortal mouse melanoblast cell line (NCCmelb4).

We devised a unique new single-cell cloning method which uses microscope cover glasses and established a melanoblast cell line derived from mouse neural crest cells. A microscope cover glass was nicked and broken into small pieces and put on a dish. Culture medium and a suspension of 20-30 cells/ml were dropped in the dish. After 1-3 d, a piece of glass to which only one cell was adhered was picked up and transferred to another dish containing culture medium. The greatest advantage of this method is that the derivation of a colony from a single cell can be directly confirmed by microscopy and there is no risk of migratory cells being contaminated by other colonies. Using this single-cell cloning method, in this study we established a cell line derived from a neural crest cell line (NCC-S4.1) and designated it as NCCmelb4. When the culture medium was supplemented with stem cell factor (SCF) alone, NCCmelb4 cells were KIT-positive and tyrosinase-negative melanocyte precursors; they remained at an immature and undifferentiated stage. When the medium was supplemented with phorbol 12-o-tetradecanoyl-13-acetate (TPA) + cholera toxin (CT), the cell morphology changed and became L-3,4-dihydroxyphenylalanine (DOPA)-positive. This observation indicates that the NCCmelb4 cells are capable of further differentiation with suitable stimulation. NCCmelb4 cells derived from the mouse neural crest has characteristics of melanocyte precursors (melanoblasts), and is a cell line which can be utilized to study differentiation-inducing factors and growth factors without the effects of feeder cells.