The reactions of the mature erythrocytes of seventeen species of fishes to the vital dyes neutral red, Janus green B, and brilliant cresyl blue, have been studied. In most teleosts the primary vital granules are readily demonstrated by neutral red and consist of one or two small granules eccentrically placed near one pole of the nucleus, but in the menhaden, alewife, and mackerel the primary granules are most numerous and are either arranged in a single definite line about the nucleus or scattered irregularly throughout the cytoplasm. In the elasmobranchs the granules are large, numerous, and scattered. In a majority of the teleosts the primary granules may be demonstrated as basophilic bodies in dry films stained by Wright's method and are also frequently seen in fresh unstained preparations.Secondary or induced granules may also appear in the cytoplasm of cells exposed for long periods to the dye. The degree of induction of new bodies does not appear to depend entirely on external factors but is determined to a large extent by factors inherent in the cytoplasm of the given species. In general the mitochondria are filamentous, reduced in number, and lie in chose contact with the surface of the nucleus.The reticular substance in all mature erythrocytes of the fishes is greatly reduced and appears either as short irregular filaments or as minute granular remnants. It is best demonstrated with brilliant cresyl blue.An attempt is made to compare the relative degree of differentiation attained by the mature erythrocytes of the several classes of vertebrates. The following criteria have been considered: changes in nuclear-cytoplasmic ratio; chromatin distribution in the nucleus; involution of the nucleoli; loss of basophilia; changes in the form, distribution, and volume of mitochondrial substance; reduction of reticular substance; amount of primary granules; and degree of induction of secondary granules. Of these criteria the degree of persistence of reticulation has been found to be the most consistent, and on this basis the several classes of vertebrates are arranged in the following ascending order of relative differentiation attained by their erythrocytes at maturity: amphibians, reptiles, fishes, birds, and mammals. This arrangement is also supported by the behavior of the nucleoli, which persist in the erythrocytes f amphibians and reptiles but are not usually demonstrated in the mature cells of fishes and birds.The history of the primary and secondary granules is less regular and consequently less useful for measuring the relative differentiation attained by the cells of different classes of vertebrates. However, within a given class of vertebrates it is concluded that the presence of a large number of primary granules or the rapid induction of new granules in mature cells may be regarded as supplementary evidence of a lesser degree of differentiation. The presence of primary granules or the degree of induction of new granules, however, cannot always be correlated with the degree of persistence of reticulation.It is also concluded on the basis of this survey of the vertebrate erythrocyte that primary granules, secondary granules, and patterns of reticulation as revealed by vital dyes, must be regarded as three separate entities which are not genetically related.
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