A Histological Study of the Development of the Scales of the Largemouth Black Bass (Micropterus salmoides) By MARY ALICE DIETRICH (From Simmons College, Boston, Massachusetts) With one plate (fig. i) SUMMARY 1. The scale method of age determination in largemouth black bass, Micropterus salmoides, is valid at least up to and probably including the third year of the fish's life. A more careful study of older fish might reveal that it was valid beyond that point. 2. The annulus in a largemouth black bass is formed by a brief but gradual slowing down of growth and then its quick resumption. The annulus appears as a wide band on the anterior edge of the scale, and as a dark line (circulus) cutting across some inner dark lines (also circuli) on the lateral edges. 3. The scale is mesodermal in origin, and lies in a pocket surrounded by dermal tissue, which in turn is covered externally by a thin layer of epidermis. 4. The bony layer is formed by osteoblastic cells on its outer surface in a manner similar to the formation of dermal bone. Excess bony material is accumulated in ridges or circuli. 5. The fibrillary plate is formed by the fibroblastic cells on its inner surface. INTRODUCTION ALTHOUGH the method of determining age in teleost fish by counting JTX. rings is generally accepted as valid, work is still being done in the field in an attempt to accumulate further evidence bearing on the subject. It has been the purpose of this study to gain more information of a histological nature regarding the early development of scales. Largemouth black bass, Micropterus salmoides salmoides Lac^pede (Bailey and Hubbs, 1949), was used in this work because of its particular importance as one of the leading game fish. The scale of a largemouth black bass is of typical ctenoid type. It varies somewhat in shape in different parts of the body, but generally resembles that shown in fig. 1. The scale is considered to be mesodermal in origin, as it seems to be formed in a manner similar to that of dermal bone. It has a lobed anterior end, which is buried in the dermis of the skin, and a toothed posterior end, which is exposed, and overlaps the anterior parts of the next, more posterior scales. A thin layer of epidermis originally covers the dermal layer, but as the scale grows, the posterior edge moves backwards, and the epidermis will remain either as a thin coat over the exposed toothed part, or will be rubbed off (Hyman, 1947). [Quarterly Journal of Microscopical Science, Vol. 94, part 1, pp. 71-82, March 1953.] 72 Dietrich—The Scales of the Largemouth Black Bass A general surface view of a scale will show the following parts: a. focus, which is a small clear area in the centre and presumably represents the original scale (Van Oosten, 1928); circuit, which are concentric (or nearly concentric) ridges around the focus; radii, which run from the region of the focus to the anterior margin; and annuli, which are zones of retarded growth (not shown in fig. 1). The annuli are so called because they are generally regarded as being formed during the winter months when the growth of the fish is retarded because of scarcity of food and low temperature of the water. This general assumption, however, varies not only among the authorities who study fish growth, but also from one species of fish to another. Thus, a careful study of annulus- formation should be made for each species before using annuli as a certain indication of age. In largemouth black bass, annulus-formation seems to vary from April (eleventh month) into July (fourteenth month), the majority being formed in April and May (Roach and Evans, 1948). Everhart (1948) found that in his work with the smallmouth black bass (Micropterus dolomieu Lacepede), experimental annuli were formed in February and March. This unexplained variation within a genus is one indication of the need of more specific studies, both in the laboratory and in the field. HISTORICAL REVIEW Concentric rings or zones in various parts have been used for a long time as indicators of variation in growth caused by the changes in season (Thomp- son, 1948). In fish these rings have been observed in scales, otoliths, and bones, and are used not only as an indication of age, but also as a key to the growth of the animal itself, and thus to the environment in which it lived. Some general observations of scales were made in the eighteenth and early nineteenth centuries, but most of the specific studies correlating scales with growth have been done in the last 50 years. The older work has been reviewed by Van Oosten (1928), and will not be discussed here. Professor D'Arcy Thompson, in his book on Growth andform (1948), pre- sents interesting information on the deposition of inorganic material in living organisms. He discusses the physico-chemical phenomenon of Liesegang's rings, which are periodic depositions from solutions. He offers this pheno- menon as a possible explanation of the formation of 'age-rings', particularly in otoliths. He says further that although there is much supporting evidence for the scale method of age determination, that in light of the Liesegang phenomenon, we should not assume without doubt that these periodic rings or zones are accurate indicators of external conditions. Before considering the histology of teleost scales, perhaps one further item should be noted in a general consideration of the validity of the scale method. The rate of growth in fish decreases with age (Lea, 1910), and consequently the older annuli are closer together, and often difficult to distinguish. Added to this is the possibility of various discrepancies, as suggested by Lee (1920), due to contraction of the previous year's layer, partial absorption of the scale growing edge radius Focus / circulus FIG. I. Photograph of a whole mount of a scale at 9 months. Alizarin red S M.A. DIETRICH Dietrich—The Scales of the Largemouth Black Bass 73 during spawning, and the formation of 'accessory' rings. All these possible variations serve further to emphasize the necessity for careful study of the growth of each species of fish and its scale before the scale method can be applied to it with any degree of certainty. A detailed historical review of histological studies is given by Neave (1940), who indicates that the structure of a fish scale has been interpreted differently by different investigators. In general, the scale is considered to consist of a thin layer of bone, supported by one to many layers of material forming a fibrillary plate, and covered on both sides by one or more layers of cells. However, there has been much disagreement as to scale origin, the cause of the formation of the ridges and of the annuli on the scale, the order in which the various layers in the scale were deposited, and the function and consistency of the various parts. Because Neave's paper has been the major source of ideas for this work, I shall restate his conclusions briefly : (1) The teleost scale is covered by two layers of cells on the external surface, and one layer internally; the latter is continuous with the deeper external layer. (2) The intimate layers are osteoblasts and osteocytes, and are concerned with the formation of the bony layer. (3) The fibrillary plate is laid down as collagen, but soon becomes infil- trated with an albumoid, ichthylepidin. (4) The scale is mesodermal in tissue relationships. (5) The ridges, formed between cell rows of the deeper external cell layer, are not products of specific cells, but depositions of excess bone-forming materials in the intercellular fluid not used by the growing margin. (6) The radii represent lines of flexibility, as the bony layer is absent, and the underlying fibrillary plate lacks ichthylepidin. MATERIAL AND METHODS All the specimens for this investigation were furnished by the Ohio Division of Conservation, Section of Fish Management, under the supervision of Mr. Lee S. Roach. The material was collected at designated intervals, begin- ning with the day of hatching on 17 May 1949, and preserved in 10 per cent, formalin. Collections were made daily for the first 10 days, weekly for the next 6 samples, and then monthly. In November, the school was divided, and half was kept in an outdoor tank and half indoors. Thereafter, a monthly sample was taken from each half of the school. Ideally, all the specimens used should have been from one school to reduce the individual variation factor. However, this was not possible, because after the January sample was taken, the tank in which the outdoor half of the school was located was flooded during some heavy rains, and lost. Subsequent out- door representatives were taken from another school of the same age. A few checks were made also on scales collected from older fish in previous years. 74 Dietrich—The Scales of the Largemouth Black Bass bony layer fibhllary plate anterior trough anterior \^ FIG. a. A, detail of scale near anterior end. Composite drawing of sections of 9-month scale. Alizarin red S and Heidenhain's iron haematoxylin. B, portion of outer surface. View of 1 \- month scale from outer side. Heidenhain's iron haematoxylin. c, same as B, but nearer anterior edge in growing margin. The material was differentiated with alizarin red S in a method after Williams (1946), and stored in xylene. Cross-sections were made by the celloidin-parafRn double imbedding method (Bensley and Bensley, 1938), and, except for a few trial sections, were cut at IOJU.