On the Histology of the Skin of the Lungfish Protopterus annectens after experi- mentally induced Aestivation. By G. M. Smith and C. W. Coatcs, Department of Anatomy, Yale University School of Medicine and New York Aquarium.

With 4 Text-figures.

DUBING the past year observations have been made on the histological structure of the skin of two lungfishes (Proto- pterus annectens Owen) kept for a period of almost six months under conditions of aestivation induced experimentally at the New York Aquarium. Both lungfishes had been caught in the fresh-water marshes near Beira, East Africa, and shipped directly to New York. They were adult fishes and measured approximately 14 inches in length. On July 8, 1935, each fish was placed in a separate battery jar containing a mixture of clay, loam, grass roots, and swamp plants, with enough water added to permit the fish to swim. After a few days of accommodation, the surface water was drained off slowly, so that at the end of three weeks the fish was compelled to live in soft mud at the bottom of the jar. No more water was added, with the result that the muddy contents of the jar began to dry. In the course of this drying of the mud, the lungfish made a burrow, forcing its body up to the free surface to breathe at about the rate of once an hour. Soon afterwards the surface of the mud became so hard as to prevent the extrusion of the nose for breathing. It was noticed that the fish then formed at the surface of the mud a small hole about a quarter of an inch in diameter. The fishcontinue d to make sluggish excursions to the artificial hole at the surface until finally, with the continued drying of the mud, the fish was forced into a state of immobility. Preceding this immobile state assumed for aestivation, the fish apparently exudes from its 488 G. M. SMITH AND C. W. COATES skin a very abundant mucus, so that with the hardening of the mud a mucous lined irregular chamber is formed, conforming closely to the curled body outline of the fish itself. This chamber is referred to generally as the burrow while the dried mucus adhering to the skin of the fish is called the cocoon. When an imprisoned lungfish at the end of a period of aestivation is released by chopping away the caked mud surrounding its burrow, the fish presents an external brownish translucent film, the cocoon, dry on the outside where it is in contact with dried mud and moist on the inside where it touches the skin of the fish. The cocoon, surrounding the fish from head to tail, is continued into the buccal cavity in the form of a flattened short tube which projects into the mouth for a distance of about \ inch. This opening in the cocoon permits air communication for purposes of respiration between pharynx and lungs on the one hand, and the burrow on the other, and again with the outside air by means of the small vent hole in the burrow already mentioned.1 On December 15, 1935, after a period of aestivation lasting nearly six months, both lungfishes were released from their imprisoning chamber. The block of mud containing the fish was removed from each battery jar, and pieces of mud were carefully broken away with the aid of a chisel. The lungfish was readily shelled out of its burrow and the dried filmy cocoon was picked off the surface of the fish in fragments with the aid of a thumb forceps. A piece of skin about 1 inch square was removed from each of the fishes for purposes of microscopic study. The fishes were placed in small tanks of fresh water, and it was only a few minutes later that the first spasmodic movements of the body and tail were observed. These early movements of the fish were executed while the fish was still in a curved position, and were associated with early respiratory efforts. The fishes straightened out after the lapse of twenty- four hours when they commenced to swim about and make 1 These observations on the movements of lungfishes, preliminary to and following aestivation, corroborate those by Dr. Homer W. Smith, who induced aestivation for metabolic studies. Details of this behaviour have been cited in unpublished lectures. SKIN OF PBOTOPTERUS 489 excursions to the surface of the water to breathe. The normal rate of breathing for this lungfish under aquarium conditions is about once in twenty minutes. The microscopic studies of the skin of both lungfishes was made from excised pieces of skin taken from the living fish in the right dorsal region about 3 inches caudal to the head. The pieces were removed immediately after

TEXT-FIGS. 1 and 2. Fig. 1.—Skin of normal non-aestivating lungfish Protopterus annectens showing large and greatly distended mucous cells. X325. Fig. 2.—Skin of Protopterus annectens immediately after a period of aestivation lasting nearly six months. Small mucous cells (MO.) covered by flattened epithelium (E.); matrix cells (A.); basement membrane (B.) ; Corium (c.); Melanophores (D.). X 325. freeing the fishes from their burrows of dried mud and before immersion in water. The skin of the normal non-aestivating Protopterus annectens is composed of a stratified epithelium resting on a delicate basemenAmembrane, below which is a loosely ar- ranged fibrous corimt where are embedded the scales. Very conspicuous in the epidermis are the mucous cells, which under normal physiological conditions supply abundant mucus for the lubrication of the surface of the body, and which produce large amounts of mucus during the phases of burrow and cocoon formation. These large mucous cells, greatly distended with droplets of mucus, are found to occupy nearly the full depth of the epithelium (Text-fig. 1). At times they are arranged in closest approximation with each other, with only an occasional elongated supporting epithelial cell separating two adjacent 490 G. M. SMITH AND C. W. COATES mucous cells. The cell membrane enclosing the fine droplets of mucus is a very delicate one. The nucleus of the mucous cell is pushed toward the base of the cell, and often appears trans- versely elongated, flattened, or crescentic. Near the surface of the epithelium, the cell membrane of mucous cells is extremely thin, permitting readily a rupture of the cell with a discharge

TEXT -FIGS. 3 and 4. Fig. 3.—Skin of Protopterus annectens (lungfish 2) immedi- ately after a period of aestivation lasting nearly six months. X 325. Fig. 4.—Skin excised from same lungfish 2 after seven months' exist- ence under aquatic conditions subsequent to aestivation, showing a return to normal non-aestivating structural arrangement of epithelium, x 325. of mucus. In the case of greatly distended cells, the cell mem- brane may be pointed or conical shaped at the free surface, a shape assumed possibly just prior to rupture. The histological preparations indicate that incompletely distended mucous cells which have not as yet reached the surface, are covered by a single layer of flattened or oval shaped cells which mark the outer limit of the epidermis. Near the basement membrane of the epidermis are a varying number of smaller polyhedral cells which may be designated as matrix cells, and which very likely represent the mother cell from which the mucous cell is derived. Melanophores are seen only occasionally in the epidermis of lungfishes under normal conditions, but under pathologic condi- tions of cutaneous melanosis, the melanophores may increase greatly in number (Smith and Coates, 1936). The corium of Protopterus annectens, as in many teleosts, shows a collection of melanophores lying directly under the epidermis. SKIN OP PROTOPTERUS 491 The skin of the two lungfishes subjected experimentally to a six-month period of aestivation, contrasts sharply with the appearance of the normal epidermis as seen under aquarium conditions (Text-figs. 2 and 3). The entire epidermis is narrowed and atrophic looking, due largely to disuse and the inactivity of mucus production. The mucous cells are small and spherical in form, and cell membranes at times appear thicker than during normal activity. Epithelial cells cover the mucous cells at the surface of the epidermis where it lies in contact with the cocoon. Matrix cells lying in the region of the basement membrane are numerous and form a very definite zone. Melanophores show no appreciable changes during aestivation either in the corium or in the epidermis, where there are occasional cells with their irregularly spread dendrites lying among epithelial cells as under normal active aquatic conditions. Text-fig. 3 shows the skin of lungfish 2, in a state of inactivity after six months of aestivation. Text-fig. 4 shows the condition of the skin excised from the same living fish after a seven- months' existence under aquatic conditions following this period of aestivation, with a resulting return to a normal structural arrangement. SUMMARY. Structural changes are described in the skin of two lungfishes (Protopterus annectens Owen) following experimental induction of aestivation. These changes relate chiefly to in- activity of mucous cells during aestivation.

EBFERENCE Smith, G. M., and Coates, C. W.—"Cutaneous Melanosia in Lungfishes (Lepidosirenidae)", 'Biol. Bulletin', vol. 71, p. 282, 1936.