STRUCTURE AND OVARIAN INCUBATION OF GAMBUSIA PATRUELIS, A TOP-MINNOW.1

BY JOHN A. RYDER.

INCE we have taken up our temporary residence at Cherrystone we have found this interesting genus of cyprinodonts in great abundance in fresh and brackish water streams, also in a fresh water pond in the vicinity, a few miles south of where our station is located. In the latter situation three forms have been collected all of which are in breeding condition-we will not say spawning condition, as they do not, as do most other fishes, commit their ova to the care of the element in which they live, but carry them about in the ovary, where they are impregnated and where they develop in a very remarkable manner.

Of the manner of impregnation we know little or nothing, except the evidence furnished by the conformation of the external genitalia of the two sexes. In the adult male, which measures one and one-eighth of an inch in length, the anal fin is strangely modified into an intromittent organ for the conveyance of the milt into the ovary of the female; a tubular organ appears to be formed by the three foremost anal rays, but one which is greatly prolonged and united by a membrane. At the apex these rays are somewhat curved toward each other, and thus form a blunt point, but the foremost one of the three rays is armed for its whole length with ridges at its base and with sharp recurved hooks at its tip, the other two at their tips similarly with hooks, and between their tips are two small fenestra or openings which possibly communicate directly with the sperm duct from the testes. The basal elements of the fin are aggregated into a cylindrical columnar truncated bony mass, which is prolonged upward into the cavity of the air-bladder for the distance of nearly the eighth of an inch; from it a series of fibrous bands pass to the dorsal and posterior wall of the air-bladder to be inserted in the median line. Whether this bony column serves to steady the fin in the act of copulation, or whether it serves to give passage to the sperm duct, is an unsettled question with the writer. The modified anal fin of the male measures a third of an inch in length. Other peculiarities of the male are noticeable—for instance, as the more abbreviated air-bladder or space which also occupies a more oblique

1 From the Forest and Stream, New York, Aug. 18, 1881, with notes and corrections.

position than in the female. The most remarkable difference presented by the male as compared with the female, however, is his inconsiderable weight, which is only 160 milligrammes, while that of the gravid female is 1030 milligrammes, or nearly six and onehalf times the weight of the male.

The female, as already stated, is larger than the male, and measures one inch and three-fourths in length. The liver lies for the most part on the left side. The intestine makes one turn upon itself in the fore part of the body cavity and passes back along the floor of the abdomen to the vent. The air-bladder occupies two-fifths of the abdominal cavity, and at its posterior end the wolffian duct traverses it vertically, to be enlarged near its outlet into a fusiform urinary bladder of very much the same form as in many embryo fishes. The ovary is a simple, unpaired organ which lies somewhat to the right and extends from the anterior portion of the body cavity to its hinder end, and serves to fill up its lower moiety when fully developed. The ova, when full grown, are each enveloped in a sac or follicle supplied with blood from a median vascular trunk which divides and subdivides as it traverses the ovary lengthwise in a manner similar to that of the stem to which grapes in the bunch are attached. In this way it happens that each egg or ovum has it own independent supply of blood from the general vascular system of the mother, from which the material for the growth and maturation of the egg is derived, and which afterward becomes specialized into a contrivance by which the life of the developing embryo is maintained while undergoing development in their respective follicles in the ovary or egg-bag. The ova develop along the course of the main vessel and its branches, as may be learned upon examining a hardened specimen, where the very immature ovarian eggs are seen to be involved in a meshwork of connective fibrous tissue, which serves not only to strengthen the vessels but also afterward enters into the structure of the walls of the ovarian sacs or follicles externally.

The very immature eggs measure from less than a hundredth of an inch up to a fiftieth, and on up to a twelfth of an inch, when they may be said to be mature. They develop along a nearly median rachis or stalk which extends backward and slightly downward, and which gets its blood supply very far forward from the dorsal aorta. The ova, after developing a little way, are each inclosed in a follicle, the Græfian follicle, ovisac, ovarian capsule,

membrana granulosa of Von Baer, or membrana cellulosa of Coste. As the egg is matured there is a space developed about it which is said to result from the breaking up of the granular layer of cells covering it. This space is filled with fluid, and in this liquid, which increases in quantity as development proceeds, the embryo top-minnow is constantly bathed. There is no trace whatever in the egg of this fish of an independent egg membrane, as is the case with all known forms which spawn directly into the water, and which is usually, if not in all cases, perforated by one or more micropylar openings or pores for the entrance of the spermatozoon. This fact raises the question whether the egg membrane or zona radiata usually present in the ova of waterspawning fishes is not entirely absent in all the viviparous species. Whether Rathke has recorded anything on this point in his account of the development of Zoarces, the viviparous blenny, I am not able to say at present, as I do not have access to his memoir.1 Suffice it to say, however, that with very cautious preparation, staining and dissection of the follicles inclosing the ova of Gambusia, I have completely failed to discover what I could regard as an egg membrane, although personally familiar with the appearance of the coverings of the ova of more than twenty species, embracing fifteen or more families. The zona radiata or covering of the egg in other bony fishes is said to be secreted from the cells lining the follicles and is composed of a gelatinoid substance, and it is often perforated all over by a vast number of extremely fine tubules, called pore canals by their discoverer, Johannes Mueller. No such structure existing as a covering for the egg of Gambusia, we are in a position to ask the question why such an unique condition of affairs should exist in this case? The answer, it would appear to us, is not far to seek. In the case of eggs which ordinarily hatch in water it is necessary that they should be supplied with a covering more or less firm and capable of protecting the contained embryo, which in the case of the top-minnow is not needed, because the embryo is developed so as to be quite competent to take care of itself as a very well organized little fish

'Rathke's description accords pretty closely with my account of the egg follicles of Gambusia given farther on. The narrow, elongate stigma, devoid of vessels, on the follicle, spoken of on page 4 of his memoir on Zoarces, probably corresponds to what I have called the follicular foramen. He has described a vascular network in the follicle, a stalk joining it to the vascular rachis and a space around the yelk much as in Gambusia.

when it leaves the body of its parent. Nature will not waste her powers in an effort to make useless clothes for such of her children as do not need them; on the contrary, she is constantly utilizing structures economically, and often so as to serve more than one purpose. This is the apparent answer to the query with which we started.

The follicles or sacs containing the ova are built up internally of flat, polygonal cells of pavement epithelium, and externally of a network of multipolar, fibrous, connective tissue cells and minute capillary blood vessels, with cellular walls, which radiate in all directions over the follicle from the point where the main arterial vessel joins the follicle, and which, together with its accompanying veins and investment of fibrous tissue, constitutes the stalk by which the follicle and its contained naked ovum is suspended to the main arterial trunk and vein. The capillary system ends in a larger venous trunk, which also follows the course of the main median arterial trunk back to the heart by way of the Cuvierian ducts. The very intricate mesh-work of fine vessels which covers the follicle supplies the developing fish with fresh oxygen, and also serves to carry off the carbonic dioxide in much the same way as the placenta or after-birth performs a similar duty for the young mammal developing in the uterus of its parent. There is this great difference, however, between the fish and the mammal. In the former there is no uterus; the development takes place in the follicle in which the eggs have grown and matured; there is no true placenta, but respiration is effected by a follicular meshwork of blood vessels, and the interchange of oxygen and carbonic dioxide gases takes place through the intermediation at first of the fluid by which the embryo is surrounded in its follicle, and later when blood vessels and gills have developed in the embryo they, too, become accessories to aid in the oxygenation of its blood. In the mammal there is a uterus; the egg must leave its ovarian follicle; be conveyed to the uterine cavity before a perfectly normal development can begin; there is a fully developed richly vascular placenta joined to the foetus, the villi or vascular loops of which are insinuated between those developed on the maternal surface of the uterine cavity. In both fish and mammal, however, this general likeness remains; that there is no immediate vascular connection between mother and embryo. In both the respiration of the embryo is effected by the transpiration of

gases through the intermediation of membranes and fluids, oxygen being constantly supplied and carbonic dioxide carried off by means of a specialized portion of the blood system of the maternal organism.

There is still another difference which distinguishes the developing fish from the mammal, which has not been noticed. The body of the former is built up by a gradual transformation or conversion of the substance of the yelk into the various structures which make up its organization. In other words, the young fish obtains no nutrition from its parent; there is merely an incorporation of the stored protoplasm of the yelk sack. In the mammal, on the other hand, the embryo receives nourishment through the placental structures, the largest proportion of the embryo is built up from the protoplasm supplied from the blood system of the parent. Judging from the large size of the young of some viviparous fishes, such as in Embiotoca, it is possible that there may be some exceptions to the rule indicated above.

Besides the very intricate network of capillary vessels which covers the follicles of the ovary of Gambusia, a large opening of a circular or oval form makes its appearance in the wall of each one at or near the point of attachment of the vascular stalk by which they are supported. This opening appears to increase in size as the young fish develops; whether it is present during the earliest stages of the intrafollicular development of the embryo I do not know, as I did not have an opportunity to see those phases. A branch from the main nutritive vessel frequently lies near the margin of the opening, curving around it. Whether this opening serves the same purpose as the micropyle of ova provided with a membrane, would appear very probable, as it is difficult to see in what other manner the milt, which is probably introduced into the ovarian cavity by the male, could reach the ovum through the wall of its follicle. The opening into the follicle may be named the follicular foramen. Through it the cavity in which the embryo lies is brought into direct communication with the general ovarian space.

We found ourselves unable to determine the species of the form, the structure of which is described above; none of those described in Jordan's Manual appear to agree with our species." It may be, as some of us have surmised, that the isolation of the form on the eastern peninsula of Virginia, for a great length of