tabteinthine APPARATUS OF LABYRINTHIO FISHES. 50i On the Construction and Purpose of the so-called Labyrinthine Apparatus of the Labyrinthic Pishes. By Doctor Nicholas Zograff, of Moscow. With Plate XLI. IN the year 1797 two Dutch sailors, Daldorf and John, sent information to the Linnean Society of London as to the remarkable capacity of Anabas scandens to crawl from one pool of water into another, to climb up bushes and trees, to spend several days without water, &c, before the beginning of a dry season. 1 Since then almost every year one or the other journal or paper has given various travellers' accounts of the interesting life and habits of these fishes. Notwithstanding the variety of these accounts they all agree in one respect, viz. in trying to show that the Labyrinthici are remarkable for their highly-developed capacity of accommodation, and for their striking viability. Cuvier and Valenciennes, for example, in their natural history of fish, tell us how the Anabus can spend hot seasons in the slime of a dry water-basin (very much like the Protopteri), or how in the markets of the East Indies the Ophiocephali will keep on moving a long time after their insides have been cleaned out by the fishmongers. Besides that, we learn from the accounts of travellers that 1 ' Transactions of the Liunean Society of London,' vol. iii, 1797.
502 NICHOLAS ZOGEAFJ?. these fishes develope more mental activity than any other species of the same class; such instances we see, for example, in their mode of nest building (Macropodus, Trichogaster), their changes of basins (Anabas, Ophiocephalus), and, lastly, in their way of getting food (Toxotes). Whilst communicating their observations on the life of Labyrinthici, travellers have not found it necessary to acquaint us with the anatomy of these fishes. I have not found in existing literature any other reports on the inner construction (anatomy) of the Labyrinthici than those published in the important works of Cuvier and Valenciennes, 1 and in the work of Wilhelm Peters, 3 describing the gill-apparatus of some of the Labyrinthici. Both these authors took interest in that striking apparatus, which, thanks to its complicated exterior, received the name of " the labyrinthine apparatus." It seems Cuvier was the first and probably the only author who examined the labyrinthine apparatus, Peters having been more intei'ested in the relation between that apparatus and the skeleton of the clavicles. Cuvier thought this apparatus to be a complex of very thin bone lamella or plates, which served, sponge-like, to retain water for the purpose of moistening the gills of the fish when in the open air. These bone lamellae, according to Cuvier, are nothing but projecting parts of the pharyngeal bones; their surface is covered with a great quantity of thin sanguiferous vessels, which apparently receive the blood from the general gill-artery. However, Cuvier says he is not quite convinced of that, having only had for his experiments samples kept in spirit, thus making it difficult to trace such thin vessels as the artery of the labyrinthine apparatus. Cuvier's views as to the construction and physiological importance of the apparatus have found credit in science, and there is not a good manual that does not describe it in the same manner as Cuvier did. As to 1 'Histoire naturelle des poissous par Cuvier et Valenciennes,' Paris, 1831, vol. vii, p. 328. 3 Wilh. Peters, " Ueber das Kiemengeriist der Labyrinthfische," ' Miiller's Aichiv fur Anatomie und Physiologie,' 1853-
LABYJUNTH1NE APPARATUS OF LABYBINTHIO PISHES. 503 the work of Peters, this author tries to prove that the labyrinthine bones correspond not to the upper pharyngeal bones, which are merely the upper fourth segments of the hindermost branchial arches, but are strongly developed third segments of those branchial arches. Peters does not say a word about the construction of the soft parts of the labyrinths. Besides these two works I have not met with any other account of this interesting apparatus. I have been able to make a detailed analysis of a labyrinthine apparatus of a Macropodus (Polyacanthus venustus, Cuv.). For my experiments I had little fishes brought up in my aquarium from the roe ejected by a female given to me by our indefatigable practical zoologist, A. S. Meschersky. Besides that (thanks to the kind permission of Professor Bogdenoff, the Director), I studied aspecimen of Anabas scandens, var. macrocephalus and Osphromenus olfax, from the collections of the Moscow Zoological Museum. All these fishes had the labyrinthine apparatus set inside the gill-operculum, and surrounded on all sides by a capsule, or walled with a thin membranous net. Its construction is the same as the lining of the cavity of the mouth; in it can be distinguished the epithelium of the surface, the connective tissue cut is, and scattered here and there pigmented cells. From the above we have the right to draw the conclusion that the sides or partitions of this capsule are only projecting cutaneous coverings of the inner surface of the gill-operculum. Indeed, if we examine under a moderate power the inner surface of a well-prepared gill-operculum of a Macropodus, we shall see on it a little narrow slit leading to the capsule of the labyrinthine apparatus, and connecting its cavities with the mouth and gill-cavities of the fish. The same relation betwixt the gill-cover and the labyrinthine capsule exists in the other fishes which I have examined, viz. Osphromenus olfax and Anabas scandens. The labyrinthine apparatus rises up into the cavity of the capsule from its inner side, which touches the exterior sides of the branchial arches (fig. 2). Notwithstanding Cuvier's
504 NICHOLAS ZOGEAFF. very accurate description of the exterior view of the labyrinthine apparatus in several species of the Labyrinthici, his description seems to be deficient in thorough acquaintance with the other sides of the apparatus, i. e. relative to dorsal, pectoral, front and back surfaces of the fish, the study of which is very important, as we shall see later, for the purpose of forming a just idea of the designation or purpose of that organ. I shall begin with the apparatus of Anabas. Cuvier describes it as a complex of numerous very thin bone lamellae lying over one another, and joined in the middle by a piece by which they are fastened to the branchial arches. The space between these lamellae, according to Cuvier, is so small as to enable them easily to detain water. If we take an exterior view of the labyrinthine apparatus we can readily agree with what Cuvier has said about its construction and purpose; but, taking a profile view of the same (fig. 3), we are prone to suspect the truth of Cuvier's explanation. Indeed, the labyrinthine apparatus of Anabas does not consist of numerous laminae, but only of three 1, thin wavy lamellae, one over the other, which become the larger the nearer they are to the point of fastening to the branchial arches; besides that the distance between each lamellae is much wider than it seems to be when seen from above. Measured with a pair of compasses these distances or intervals were found to vary from 1*5 mm. to 2'75 mm. in breadth. We leave the reader to judge if an apparatus consisting of three lamellae, of which the uppermost is 5 mm. long and 4 mm. broad, the middle one from 7 to 8 mm. broad, and the undermost 11 mm. long and 9 mm. wide, can detain much water. There are three spaces to detain water: one between the first and second lamellae with a surface of about 15 square mm. and about 1*5 deep; another between the second and third lamellae with a surface of about 60 square mm. and 2'5 mm. deep; a third one between the hard lamella and the sides of the pouch of the labyrinthical apparatus with a surface of about 80 square mm., and varying in depth according to wrinkles or 1 After ' Giinther's Manual of Ichthyology,' 3 5.
LABYRINTHINE APPARATUS OF LABYRINTHIO PISHES. 505 furrows formed here by the sides of the pouch. Experiments show that water is not detained in sufficient quantities between small plates with such a distance between them. I have tried to fasten together covering glasses in such a manner as to keep their sides parallel, with a distance from 1 to 3 mm. between each, and have found that with a surface of the glasses equal to 324 square mm., and with a distance of 1 mm. between each, the surface of water detained therein was equal to about 80 square mm.; with a distance of 2 mm. the surface of detained water was still less, and with a distance equal to 3 mm. the water was accumulated on a surface exceeding a little 0*5 mm. just round the cork that kept the glasses together. The quantity of water between the lamellae was again still smaller if, instead of glasses, cartoon or British paper was used. Consequently the apparatus of Anabas, if applied for the purpose attributed to it by Cuvier, would in any case function imperfectly. The apparatus of a Macropodus is still less fit to function in that way. It also consists of three parallel lamellae or plates (see figure 4) which are placed in such a manner as to be quite unable to obtain water. The (first) top plate is so bent that its back part only remains parallel to the middle one; the third one forms a rectangle with it; the lower part is elongated more towards the pectoral surface of the fish, whereas two top lamellae have grown in the opposite direction (see drawings 4 and 1). The labyrinthine apparatus of Osphromenus olfax is so much like the apparatus of Anabas, that the aforesaid can be wholly applied to it. But what are, then, properly the functions of the labyrinthine apparatus of the Labyrinthici? A study of the fine microscopic structure of this organ gives us a right to make a supposition that will better explain its functions than the supposition made by the great naturalist. The projecting bone of the third segment of the last branchial arch serves as support or basis to the labyrinthine apparatus (see PL XLI, fig. 8, os.).
o06 NICHOLAS This bone basis is very fine and consists of a typical (partly described by Kolliker) 1 bone tissue, in which star-like bone elements are not seen ; when the section is made in a direction parallel with the surface of the lamellae one can distinguish in the thickness of the bone ramous canals (described by Kostler), which make it look something like the dentine of teeth. The bony basis is covered by a periosteum, which consists of very fine long and flat cells, and passes into a connecting adipose tissue, which, on its external surface, becomes a connective tissue (cutis) ; the cutis is covered by an epithelium; among the cells of this can be seen numerous goblet-like mucous cells. From the above one sees that the labyrinthine apparatus is not a very complicated organ, and by its structure approaches the general typical structure of the skin coverings of fishes; but, as we shall see directly, special elements of the labyrinthine apparatus appear to be strongly modified. Let us begin with the layer of connective tissue which lies next to the periosteum. This connective tissue consists of star-like cells with very long processes, and with a small cell-body and a little nucleus. These cellules, connected by their outshoots, form a netted tissue (fig. 8, ctj. ad.); between the meshes of this tissue are big, fat, adipose cells, that make a fresh tissue look very odd; but generally there remain no traces of fat in the sections, because the preliminary preparation in spirit, chloroform, &c, dissolves the fat. It is only in objects prepared in osmic acid that the fat is retained where it is when sections are made (fig. 8); but meanwhile an organ without fat differs very much from one with it, as the fat in a tissue does not lie in mass, but in small round groups (cj't. ad.). The skin cohering over these groups of fat is lifted or raised, whereas between them the coverings are much thinner, and consequently the surface of 1 A. Kolliker, " Ueber verscliiedene Typen in der microscopischeu Structur des SkeleUes der Knockeufisclie," ' Verhandluugeu der physicalischincdiciiiisclier Gesellschaft zu Wiirzburg,' Band viii.
LABYRINTHINE APPARATUS OF LABYRINTHIC FISHES. 507 the labyrinthine apparatus under a microscope looks very much like a counterpane or a stitched soft lining with edged risings (fig. 10). The meshes of the netting with fat in them get narrower and narrower the nearer they are to the cutis, and in the cutis the star-like connective tissue becomes fibrous. In this fibrous tissue over the groups of adipose cells one sees numerous capillary vessels (figs. 8 and 9, cp.), which form very pretty and characteristic braids or interweavings. These braids are in relation with the risings of the adipose masses or accumulations ; between these accumulations capillaries are not observable. All the above-described accumulations or gatherings of adipose tissue have their separate arterial and venous branches; these branches rise to the surface of the accumulation, ramify severally, and partly pass into the capillary network of the cutis. Thus every part of the capillary network, with its separate adipose accumulation, has its special arterial and venous branches, and is connected with the nettings of other risings only by means of these branches. The capillary net of every rising has the form of a very pretty rosette (figs. 11, 12). This rose looks as if composed of separate petals, shaped out by the bendings of the meshes of the capillaries. The capillary windings of every petal are of three and seldom of four rows, wherefore the petal consists of an external, middle, and interior vessel. These vessels communicate on one side with the arterial branches, and on the other with the venous branch, so that in every petal can be observed an arterial and a venous half, or, to be more precise, one half that brings in the blood, and the other which lets it out from the interweavings. The connective tissue that lies next to the cutis somewhat changes its appearance at the edges of the lamellae of the labyrinthine apparatus. Here it is so swollen, and has such an appearance, that it can hardly be taken for a connective tissue (figs. 7 and 8, cjt. mrg.). Here it has no adipose matter; its cells become much thicker, more juicy, but their offshoots get so small that in sections they can be observed only wbeij
508 NICHOLAS ZOGRAFP. the tissue has been split asunder into its component parts by means of fine needles, after it has been macerated for thirtysix hours in weak alcohol. On its outside the cutis is covered with an epidermis of many layers (figs. 7, 8 and 9). This epidermis consists of numerous very small cells, scattered among which there is a quantity of mucous goblet-like cellules. The latter are not seen in sections kept in alcohol and prepared in paraffine or soap, but they are well observed in sections prepared in osmic acid, and embedded in the white of egg according to the method of Kalberla. In such sections we observe that the number of goblet-like cells in the epidermis is very great, and that consequently these cellules can let out a considerable quantity of slime or mucus which covers the labyrinthine apparatus. The bottom of such cells is fast set in between other cells of the epidermis, and that is the reason why when teazed they come out with irregular edges (fig. 3); one can observe in their goblet-like cavity a fine irregular protoplasm network and also a slime or mucus, the traces of which are seen outside the cell. Having described the construction of this apparatus, so complicated and entangled externally, but so simple in its microscopic structure, I now return to its functions. The first view of the strongly-developed capillary networks which are separated from the exterior by a fine epithelium of two or three layers and have arterial and venous vessels, and let only one or two corpuscles of blood through their cavities, makes us suppose that these capillary nets are the principal part of the organ. If, on the other hand, we remember that the organism of labyrinthine fishes must have adaptations for the characteristic, partly terrestrial or amphibious mode of life which is described by all authors, we shall have to seek in the organism of these fishes for such adaptations, and we must hope to meet with organs which will admit oxygen to the blood, not only from the air dissolved in water, but also from the atmosphere. The labyrinthine apparatus is such an organ. To establish a decisive view as to the physiological
LABYRINTHINE APPARATUS OP LABYJRINTHIO FISHES. 509 functions of any organ would be possible either after exact or precise physiological experiments, or after having 1 functionally studied the anatomy of the' organ by comparing its structure with the structure of already well-known organs^ the physiological functions of which are indisputable. The first method we cannot adopt. Notwithstanding my trying to introduce a very fine bent glass tube under the gill-operculum into the cavity of its labyrinthine pouch, it seemed impossible to do so without making ruptures through which little bladders of gas do not enter in the pipe but pass out. Perhaps if I had examined a greater amount of living material I could have accumulated and analysed a certain quantity of gas from the labyrinthine pouch, but the material I had was insufficient^ and therefore I could not solve the question by mere experimenting. I then applied myself to the study of facts got by anatomy. Could the capillary net that has its arteries' and veins be an organ for oxygeilising blood? Did it let in venous blood and let out arterial, or vice v'erss? To solve the question I applied injections, injections through the arteria branchialis, which is very fine and slender, gave no results; injections through the spinal aorta almost the same; I say " almost/ because once after having made some unsuccessful injections I happened to inject some of the fluid through the aorta into the labyrinthine apparatus, and into a part" of'the branchial arches iu a direction opposite to the course of the blood. But this not quite successful experiment, together with the results obtained on another occasion, convinced me that the labyrinthine apparatus is ill reality a supplementary respiratory apparatus, helping the fish to breathe while in the open air or in the damp atmosphere of drying' water-basins! The causes of my supposition areas follows : Cuvier had made a supposition that the vessels of the labyrinthine apparatus are supplied with blood from the gill-artery which passes' through a separate branch.' But for all the preciseness of Cuvier's experiments this great naturalist did not succeed in seeing the supposed connection, arid had to limit himself to supposition. All my efforts seemed to fail till I happened to fall upon a
510 NICHOLAS ZOGRAFF. very simple and convincing method. For long I took great pains to find a means of injecting one way or the other some fluid into the vessels conducting blood to the labryinthine apparatus, but unfortunately I did not succeed. But two or three times I thought the fine light blue mass prepared according to Beal did penetrate through the gill-arteries from the heart into the apparatus, and that is the reason why in my remarks published in 1886 I ventured to say that Cuvier's hypothesis was very probable. 1 Operating with chloroform upon Macropodus I observed that both the vessels of the labyrinthine apparatus and the gillvessels of the fishes thus operated upon were overfilled with blood. Then I tried to rapidly curdle the blood by pouring some boiling water over the organ, and then making it hard with alcohol. My experiment proved to be successful. Not only the labyrinthine apparatus and the gill-vessels, but even the " bulbus arteriosus " were filled with coagulated blood, wherein one could very well distinguish the red corpuscles. After that I began to prepare sections of the part of Macropodus which lies next to the gills and its labyrinthine apparatus, and found that the blood with its red elements could be tinted with boracic carmine so as to enable one to discern the finest bloodvessels. Several sections thus prepared convinced me that the artery leading to the labyrinthine apparatus (fig. 7, ar. Ib.) is a branch of the artery of the fourth branchial arch (ar. br), and consequently furnishes the apparatus with venous blood, the blood returning from the apparatus to the spinal aorta through the junction which I had already observed before, and through which the injected mass passed into the apparatus. So the labyrinthine apparatus is a supplementary apparatus of the fourth branchial arch, and the circulation of the blood of the gill-apparatus of Labyrinthici differs from the circulation of other Teleostei in the presence of an arterial branch (and 1 " Ueber den sogenanntess Labyrintliapparat der Labyrinthfische (Labyrinthici)," von Nicolaua Zograff, ' Biologisohes Centralblatt,' Band i. No. 22, 1886, p. 687.
LABYBINTHINE APPARATUS OF LABTBINTHIO FISHES. 511 probably of a vein too) from the fourth arch to the labyrinthine apparatus (fig. 13). In certain other fishes we meet with the same branches of gill-arteries. Johann Miiller, in his famous work on Ganoid fishes, describes the artery of the undeveloped extra gill as an arterial branch of the first branchial arch. 1 Therefore, that the artery leads to the supplementary gill-apparatus from the gill-artery is not an anatomical rarity, but a consequence of the greater development of capillaries, and their particular disposition in the labyrinthine apparatus. If we remember that Peters proved the labyrinthine apparatus to be only a part of the branchial arch irregularly developed, we must, in my opinion, have expected that the labyrinthine apparatus would receive its artery from the artery of its arch, and that its function would be the same as that of the other branchial arch, but adapted to the special wants of these semiterrestrial fishes. 1 J. Miiller, * Ueber den Bau und die grenzen der Ganoiden und ueber das natiirliche System der Tische,' Berlin, 1876. VOL. XXVIII, PAHT 4. NEW SER.