Sperm transfer in the genus Dolops involves the employment of spermatophores. In this respect the genus is unique within the Branchiura.

Proteinaceous wall material is secreted by arborescent spermatophore glands located in the carapace lobes of the male and is passed into a pair of long canals which terminate posteriorly as a pair of spermatophoric vesicles, part of whose walls are glandular. Spermatozoa from the vasa deferentia are injected into these vesicles and become surrounded by wall material. During the mating process the contents of each vesicle are squeezed out through the common genital orifice, where they become con- fluent and form a single, globular spermatophore. While its walls are still soft this is pressed against the base of the abdomen of the female, where it is pierced by two perforated spines at the end of the spermathecal ducts. The spermatophore hardens and remains attached to the female, thus giving the spermatozoa opportunity to migrate through the spermathecal ducts to the spermathecae. Not until after a moult of the female, however, during which the spermatophore is shed with the old cuticle, are the spermatozoa free to enter the eggs, each of which is believed to be pricked by the spermathecal spines during oviposition.

By virtue of the fact that it involves quite different organs and processes in the two groups, spermatophore formation indicates that the Branchiura and Copepoda are not closely related. On the other hand, the process is in no way suggestive of a close relationship of the Branchiura and Branchiopoda, and the isolated position of the Bran- chiura is emphasized.

The employment of spermatophores during sperm transfer in the branchiuran genus Dolops has recently been demonstrated (Fryer, 1958). That spermatophores are formed by members of this genus is of particu- lar interest in that hitherto the absence of these structures has been regarded as one of the diagnostic features of the Branchiura, and it is indeed well established that no such structures occur either in the well-known genus Argulus or in the less-known and entirely African genus Chonopeltis.

It is the purpose of the present paper to describe the structure and manner of formation of the spermatophores of Dolops ranarum (Stuhlmann), in which species they were first observed, and to give some account of the process of sperm transfer and associated aspects of the reproductive biology of this species.

Specimens of D. ranarum, which has a wide distribution in the inland waters of Africa, and which occurs ectoparasitically on several species of fishes, were collected for the present study first in Lake Bangweulu Northern Rhodesia), which produced the material on which the original observations on spermatophores were made, and later in Lake Victoria, where material for detailed study was obtained. If kept in dishes of clean water, specimens re- moved from the host survive for several days and not infrequently mate under such conditions. Observations on spermatophore extrusion and transfer were made on such specimens. Dissections were made of both fresh and preserved material, and sections were cut from specimens embedded in low-viscosity nitrocellulose. Ordinary neutral 4% formaldehyde was found to be a better general fixative and to cause less shrinkage than Bouin’s fluid, Heidenhain’s ‘Susa’ fixative, or alcohol.

The general appearance of a spermatophore is best understood by reference to figs, 1 and 2. A spermatophore in position on the female is shown in fig. 1, A. When thus attached it strikingly resembles a minute pearl in form, colour, and lustre. A fully formed spermatophore is a subspherical or ovoid structure. At one pole it is perforated by two small, close-set, circular apertures, which occasionally have somewhat raised, crater-likè rims, symmetrically arranged on each side of the meridional axis, and here too it gives rise to a projecting flange, whose exact shape varies slightly but which is basically a simple plate. The meridional axis of the spermatophore is not infrequently marked by a very shallow and indistinct furrow on its surface. The length of the longest axis of the spermatophore body varies from as little as 360 μ, to as much as 650 μ. The smaller specimens tend to approximate more closely to a sphere than do the larger ones. The apertures have diameters of about 50 to 60 μ.

In relation to the size of the spermatophore its walls are extremely thick (up to about 45 μ) and in sections appear perfectly homogeneous in structure. In newly formed specimens the central cavity is completely filled with spermatozoa.

FIG. 1.

A, spermatophore in position on female. B, detached spermatophore (lateral view), c, anterior end of a detached spermatophore from dorsal aspect, showing apertures, D, detached spermatophore (ventral view), as seen in position on a moulted cuticle.

FIG. 1.

A, spermatophore in position on female. B, detached spermatophore (lateral view), c, anterior end of a detached spermatophore from dorsal aspect, showing apertures, D, detached spermatophore (ventral view), as seen in position on a moulted cuticle.

FIG. 2.

Spermatophore in position. From a specimen embedded in L.V.N. and sliced to show the interior of the spermatophore and one of the spermathecae.

FIG. 2.

Spermatophore in position. From a specimen embedded in L.V.N. and sliced to show the interior of the spermatophore and one of the spermathecae.

FIG. 3.

Male reproductive system (semi-diagrammatic). The left spermatophoric canal is displaced and the spermatophore gland of that side is omitted.

FIG. 3.

Male reproductive system (semi-diagrammatic). The left spermatophoric canal is displaced and the spermatophore gland of that side is omitted.

Before the process of spermatophore production can be described some account of the male reproductive system and of certain accessory organs of the female system is necessary. Apart from casual statements, mostly by nineteenth- century investigators, usually referring to such details of the reproductive organs as could be discerned through the integument, the only account of the male system of any species of Dolops appears to be that of Maidl (1912) for D. longicauda (Heller). This is quite lucid, but his material was limited and as he was unaware that certain structures were concerned with the production of spermatophores he was unable to give a functional interpretation of his findings.

FIG. 4.

A, a spermatophoric vesicle as seen in ventral aspect in the living animal. (The ductus ejaculatorius cannot be seen.) B, semi-diagrammatic longitudinal section through a spermato- phoric vesicle and the posterior end of a spermatophoric canal.

FIG. 4.

A, a spermatophoric vesicle as seen in ventral aspect in the living animal. (The ductus ejaculatorius cannot be seen.) B, semi-diagrammatic longitudinal section through a spermato- phoric vesicle and the posterior end of a spermatophoric canal.

The male reproductive system and the spermatophore-producing organs

The gross morphology of the reproductive organs is depicted in fig. 3. As in all Branchiura, the paired testes are located in the abdomen. In D. ranarum each testis has 3 lobes. From each there passes anteriorly a fine vas efferens, which quickly unites with its partner to form a much wider median duct which extends over the greater part of the length of the thorax and functions as a sperm reservoir or vesicula seminalis. At its anterior extremity this bifurcates and each branch doubles back on itself and, now somewhat greater in diameter than the vesicula seminalis, passes posteriorly as a vas deferens. Although morphologically vasa deferentia, these tubes in fact store far more sperms than does the vesicula seminalis. Extra space for sperm storage is provided by two short, anteriorly directed horns, located where the vesicula seminalis and vasa deferentia unite. In the region of the fourth pair of legs each vas deferens narrows and, in a manner described below, unites with a vesicular swelling which forms part of the spermatophore-producing apparatus. These swellings lie ventrally and can often be seen through the integument of the animal, particularly in living specimens (fig. 4, A). It was after observing them in this manner in several species of Dolops that Thiele (1904) named them from the tubes with which they are continuous anteriorly as ‘prostate ampullae’, a term adopted also by Maidl (1912). Because of their function they are best referred to as spermatophoric vesicles. From the dorsal surface of each arises a canal which runs forwards below and somewhat to the outside of the vas deferens of its side as a wide tube, here designated the spermatophoric canal (fig. 4, A, B). This extends to about the anterior limit of the vas deferens before turning outwards at a rather acute angle and rapidly narrowing. Superficial examination, even of living specimens, usually gives the impression that this is a blind tube, but in fact it terminates in an arborescent glandular mass which ramifies through the carapace lobes and for the most part overlies the digestive diverticula of the alimentary canal (fig. 5). This glandular structure produces the spermatophore wall material and therefore merits the name spermatophore gland. Each spermatophoric vesicle is also continuous with a short, inwardly directed, narrow, chitin-lined ductus ejaculatorius; both of these open at the single median genital aperture. This duct is usually closed and cannot be made out except in sections. The vesicula seminalis and vasa deferentia are usually packed with sperms, whose axes in general lie parallel to those of the ducts they occupy. The lumen of each spermatophoric canal is filled with a structureless, white secretion, which stains in a uniform manner with a variety of stains. This is the material of which the spermatophore wall is composed. In living specimens peristaltic movements can sometimes be observed in the vesicula seminalis and in the vasa deferentia, but have not been observed in the spermatophoric canals.

FIG. 5.

Topographic sketch of a transverse section of a male, showing the distribution of the lobes of the spermatophore gland in the carapace fold. The section is cut at the level of the horns of the vesicula seminalis.

FIG. 5.

Topographic sketch of a transverse section of a male, showing the distribution of the lobes of the spermatophore gland in the carapace fold. The section is cut at the level of the horns of the vesicula seminalis.

FIG. 6.

Sagittal section through the region where the spermatophore gland unites with the spermatophoric canal. The white areas represent blood-filled haemocoelic spaces. (The drop- lets present at the union of gland and canal are believed to be artifacts, but it is just possible that they represent a fluid stage in the secretion of wall material.)

FIG. 6.

Sagittal section through the region where the spermatophore gland unites with the spermatophoric canal. The white areas represent blood-filled haemocoelic spaces. (The drop- lets present at the union of gland and canal are believed to be artifacts, but it is just possible that they represent a fluid stage in the secretion of wall material.)

FIG. 7.

Details of part of the spermatophore gland.

FIG. 7.

Details of part of the spermatophore gland.

FIG. 8.

Transverse section of the male reproductive system a short distance anterior to the spermatophoric vesicles.

FIG. 8.

Transverse section of the male reproductive system a short distance anterior to the spermatophoric vesicles.

Histologically the spermatophore gland consists of a system of branched ducts surrounded by glandular cells, whose cytoplasm and large spherical nuclei both stain deeply with haemotoxylin (figs. 6, 7). There are no intracellular ducts. The intercellular ducts unite and finally discharge into the wide spermatophoric canal, with the cells of whose walls the gland cells are in passes, for its wall, particularly in its ventral and outer lateral regions, is thick and consists of darkly staining columnar cells, several of whose nuclei direct continuity. The transition from gland cells to canal wall-cells is gradual as can be seen from fig. 6. The cells gradually become flatter and less deeply staining, their nuclei become smaller and more compressed, and the cell boundaries become less and less distinct, so that the canal wall appears to be syncytial. Throughout most of its length the wall is so compressed that it is difficult even to detect the presence of nuclei. It is apparent, therefore, that the canal serves purely as a storage chamber for the secretion produced by the spermato- phore gland. The same cannot be said of the spermatophoric vesicle into which this secretion contain two nucleoli (fig. 10, A, B). These are very obviously glandular in function, as their structure suggests, for in the region of their occurrence, and between them and the secretion which fills the spermatophoric vesicle, occurs another secretion with somewhat different refractive and staining properties (fig. 10, B). This is discussed below.

The histological nature of the walls of the sperm-carrying canals differs from region to region. The wall of the vesicula seminalis is thick and nuclei are readily apparent, but cell-walls are not often distinct. Over much of their length the vasa deferentia have similar though rather thinner walls. Towards their posterior extremities, however, considerable changes take place over a very short distance. It is here that each vas deferens unites with the spermato- phoric vesicle of its side and in this region each is modified to form a mechan- ism for the injection of sperms into the mass of prospective spermatophore wall material contained within the spermatophoric vesicle. As the vas deferens approaches this region it rapidly narrows and its walls thicken somewhat (compare text-figs. 8 and 9, A). The narrowing of the lumen continues, the wall-cells, and particularly those located ventrally, enlarge and tend to become vacuolate, and their boundaries become distinct (fig. 9, B). At the level of the anterior extremity of the spermatophoric vesicle the lumen of the vas deferens is very narrow and the duct is somewhat compressed laterally, so that the enlarged ventral cells of its walls tend to reach downwards towards the region where the spermatophoric canal gives rise to the vesicle (fig. 10, A). This process continues, the lumen of the vas deferens becomes slit-like, and the ventral portion of its wall, now composed of vacuolate columnar cells, ultimately unites with the wall of the spermatophoric vesicle (figs, 10 B, n). The lumen of the vas deferens thus opens into that of the spermatophoric vesicle.

FIG. 9.

A, transverse section of the left side of the male reproductive system immediately anterior to the spermatophoric vesicle. B, the same, a little further posteriorly.

FIG. 9.

A, transverse section of the left side of the male reproductive system immediately anterior to the spermatophoric vesicle. B, the same, a little further posteriorly.

FIG. 10.

A, transverse section of the left side of the male reproductive system, passing through the extreme anterior portion of the spermatophoric vesicle. B, the same, a little further posteriorly, showing the region of confluence of the vas deferens and the spermatophoric vesicle

FIG. 10.

A, transverse section of the left side of the male reproductive system, passing through the extreme anterior portion of the spermatophoric vesicle. B, the same, a little further posteriorly, showing the region of confluence of the vas deferens and the spermatophoric vesicle

All the organs of the reproductive system including the spermatophore glands are encased in a fibrous connective-tissue sheath, apparently continuous with the basement membrane. This fulfils several functions. First it serves as a means of suspending the various organs from the tergites of the thorax. It also forms a canopy over these organs in which is deposited much dark pig- ment (figs. 4 B, 8-10). Such pigment develops, often in very definite patterns, above the uteri of many female Branchiura and over the testes of males—a distribution which suggests that it serves to protect the genital products from the effects of light. The rigidity of this fibrous tissue also enables it to function both as an ‘endoskeleton’ which gives support to delicate structures and as a tissue from which slips of muscle can take their origin. Both the latter functions are exhibited by the connective tissue in the region where the vas deferens forms the injector of sperms (fig. n). Here the sheath supports the delicate vas deferens and to the outside is continuous with a strip of muscular tissue whose contractions, by pulling down the overlying sheath, will deform the posterior extremity of the vas deferens and, assisted by the peristaltic move- ments of the more anterior regions of the duct, will result in the discharge of sperms, which are in effect injected into the spermatophoric vesicle. In only one of the sectioned specimens was injection actually in progress, but in this specimen, although histological details were poorly defined, the injection was very clearly shown (fig. 12). The injected sperms are arranged in a compact mass or cord. This will facilitate their penetration into the wall material and, by ensuring that they cohere for a time, will enable the wall material to flow around them. It may be that the vacuolated cells at the extreme posterior end of the vas deferens are secretory. If this is not so, then it is difficult to understand whence the fluid which is undoubtedly present in newly formed spermatophores is derived.

A rather similar mechanism is concerned with the discharge of the spermato- phoric vesicles. The dorsal wall of each of these is overlain by muscular tissue which takes its origin in a local accumulation of fibrous connective tissue, itself slung from the tergites of the thorax which overlie the posterior end of the spermatophoric canal. This muscle is inserted on the ductus ejaculatorius and its contraction will result in compression of the spermatophoric vesicle, the opening of the ductus ejaculatorius, and the consequent extrusion of the spermatophore mass.

FIG. 11.

Details of the injection apparatus at the region of confluence of the vas deferens and the spermatophoric vesicle.

FIG. 11.

Details of the injection apparatus at the region of confluence of the vas deferens and the spermatophoric vesicle.

FIG. 12.

The injection of spermatozoa into the spermatophoric vesicle.

FIG. 12.

The injection of spermatozoa into the spermatophoric vesicle.

The female reproductive system in relation to spermatophore transfer

Certain organs associated with the female reproductive system fulfil an important function during spermatophore transfer and require description before the mating process is described. Besides possessing a single functional ovary whose contents are discharged through a single median aperture, the female reproductive system includes two quite separate spermathecae. These take the form of ovoid sacs located at the base of the abdomen. They have no direct connexion with the female reproductive system proper and, as in Argulus, each communicates with the exterior by way of an extremely fine and somewhat coiled duct leading inwards towards the bottom of the cleft between the abdominal lobes, where each terminates in a minute but sharp and sclerotized, perforated spine (fig. 13, A). These ducts and spines are here referred to as spermathecal ducts and spines respectively. The ducts have an extremely narrow lumen lined with cuticle and this is surrounded by a thick and apparently glandular wall. About a third of the way between the sperma- thecal spine and the spermatheca is the suggestion of a blind diverticulum similar to but very much shorter than that to be seen in certain species of Argulus, and here the wall of the duct is somewhat thickened. Coiling of the duct, which permits considerable freedom of movement, is doubtless neces- sitated by the mobility of the abdomen, which here contains many loose parenchymatous cells, and by the need to allow movement of the spermathecal spines. Around the base of each spermathecal spine is a region of thickened cuticle, between which and the spine itself a region of flexible cuticle permits the spine to be pointed in various directions. Near the base of the spine and somewhat posterior to it the cuticle of the abdomen is roughened by the presence of a number of minute spinules (fig. 13, B), whose significance be- comes apparent when spermatophore attachment is studied.

FIG. 13.

A, one of the spermathecae of the female and its associated duct and spine. The inset shows cellular details of the spermathecal duct. B, a spermathecal spine. The inset shows a different (and somewhat enlarged) view of the extreme tip and the aperture of the duct.

FIG. 13.

A, one of the spermathecae of the female and its associated duct and spine. The inset shows cellular details of the spermathecal duct. B, a spermathecal spine. The inset shows a different (and somewhat enlarged) view of the extreme tip and the aperture of the duct.

The spermathecae have a cuticular lining. Obviously, however, there is no means whereby the animal can completely rid itself of this cuticle at times of ecdysis. At each moult, therefore, the old lining which, judging from the fact that it is very thin in comparison with the functional cuticle, must undergo a considerable amount of digestion during the pre-moulting period, apparently splits along its longitudinal axis and is shed into the cavity of the spermatheca. By some means, not ascertained, successively moulted cuticles adhere to each other to form a compound structure which, when a spermatheca is dissected, readily slips out from within the functional cuticle. When flattened this struc- ture exhibits distinct annuli, which presumably enable one to determine the age of the parasite, at least to the extent of ascertaining the number of ecdyses through which it has passed since the spermathecae were formed. Up to 8 annuli have been counted in large specimens.

Extrusion of a spermatophore does not take place until the mating process has begun. While absolute proof is lacking, the inference is that in nature mating takes place while the parasites are on the host. That mating sometimes takes place away from the host cannot, however, be ruled out, for all the pre- sent observations were made on specimens which had been removed from the host and which on several occasions produced and successfully transferred spermatophores under these conditions.

During the mating process the female is seized by the male, which, in the cases observed, was always smaller than the female. This is probably the most usual state of affairs in nature, for mature females are usually larger than males. The female is gripped by the maxillulary hooks normally used for attachment to the host. These at first grip whatever portion of the female is available, but very quickly the male works itself into such a position that it overlies the female, its ventral surface being in contact with the dorsal surface of the female, and its hooks grip convenient points of the carapace. By slewing sideways either to the right or left the male manœuvres itself into such a position that the abdomen of the female can be gripped between legs 2 and 3. On the anterior face of the basal segment of leg 3 is a roughened projection, which doubtless facilitates this part of the proceedings. (On one occasion the abdomen was held for a time between legs 3 and 4.) A female ready for mating assists in this by lifting the abdomen. The male then flexes its body so as to bring the ventral surface of the abdomen, and hence the genital orifice, face to face with the comparable structures of the female. Not until this state of affairs has been attained does extrusion of the spermatophore begin, though the spermatophoric vesicles are very conspicuous at the beginning of mating as though they are bulging with material ready for discharge. The bulging may be an indication that sperms have been injected into the spermatophoric vesicles as a result of the stimulus given by the preliminaries of the mating process. At all events, as a result of the injection process described above, at the beginning of mating such a spermatophoric vesicle contains a mass of sperm surrounded by prospective wall material. Nipping off of this mass from the rest of the wall material in the spermatophoric canal is probably assisted by contractions of the muscular sheath which is present at the extreme pos- terior end of the spermatophoric canal but not elsewhere (compare fig. 9, A, B with fig. 8). Extrusion is initiated simultaneously in each spermatophoric vesicle. From each a quantity of prospective wall material (fig. 14, B) is exuded along its ejaculatory duct. There is presumably a core of spermatozoa within this wall material. Extrusion is probably assisted by contraction of the dorsal muscular tissue described on p. 417, possibly with the assistance of a local increase in the pressure of the haemocoelic spaces. The two exudates reach the genital orifice at the same time and there become confluent and pass to the exterior as a small globule (fig. 14, c). This then increases in size (fig. 14, D). At first its dual origin is clearly marked by a meridional furrow, but as the size increases still more and the wall material of each side mingles this becomes less and less distinct (fig. 14, C-E). When the whole of the material from the spermatophoric vesicles has been exuded, a process which takes only 4 or 5 sec, the now more or less globular and completely sealed spermato- phore is kicked away from the genital orifice by one of the fourth pair of legs. The last part of the spermatophore to leave the male genital orifice is drawn out into a small papilla which can be seen to be still very plastic (fig. 14, E). The spermatophore is then kicked round by the fourth pair of legs and the last part to emerge is pushed against the female (fig. 14, F). Although this cannot be observed at the time, subsequent examination reveals that during this part of the process the soft portion of the spermatophore is pierced by the spermathecal spines of the female and that these remain embedded in the wall (fig. 15, A, B). It is by this piercing of the wall while it is still soft that the two apertures of the fully formed spermatophore are formed. It appears that the fourth pair of legs of the male hold the spermatophore in position against the female for some time after first attachment. It is presumably during this period that the still very plastic wall material in contact with the female cuticle moulds itself to the shape of that cuticle and, by the forces of surface attraction, flows out to form the attachment flange. This explains why a spermato- phore always fits exactly the female to which it is attached although there is considerable disparity in size both among females receiving spermatophores and among the spermatophores themselves. The roughened nature of the cuticle near the base of the spermathecal spines described above doubtless serves to assist in the firm anchoring of the spermatophore.

FIG. 14.

Diagrammatic representations of spermatophore extrusion. A, the situation at the outset. B, extrusion begins and the material flows along the ductus ejaculatorius. c, material passes to the exterior through the single genital orifice as a single globule. D, the spermatophore increases in size. E, extrusion is complete. The arrow indicates how the complete spermato- phore is rotated during transference to the female. F, the spermatophore is pushed against the spermathecal spines of the female.

FIG. 14.

Diagrammatic representations of spermatophore extrusion. A, the situation at the outset. B, extrusion begins and the material flows along the ductus ejaculatorius. c, material passes to the exterior through the single genital orifice as a single globule. D, the spermatophore increases in size. E, extrusion is complete. The arrow indicates how the complete spermato- phore is rotated during transference to the female. F, the spermatophore is pushed against the spermathecal spines of the female.

FIG. 15.

A, T.S. through a spermatophore in position on a female, showing how the sperma- thecal spines penetrate its thick wall. (Note how the wall material in this region moulds itself to the shape of the female and fills the gap between the spermathecal spines.) B, the same, to demonstrate further the moulding of the wall at points of contact with the female.

FIG. 15.

A, T.S. through a spermatophore in position on a female, showing how the sperma- thecal spines penetrate its thick wall. (Note how the wall material in this region moulds itself to the shape of the female and fills the gap between the spermathecal spines.) B, the same, to demonstrate further the moulding of the wall at points of contact with the female.

The male then releases its hold on the female and the mating individuals separate, the female carrying away the now firmly attached spermatophore. The spermatophoric vesicles of the male no longer bulge with material but the spermatophoric canals are by no means emptied.

No ‘former’ or moulding organ is involved in the formation of a spermato- phore and the form assumed by the latter is fairly readily explained by basic physical principles. Confluence of the wall material from each side and of the two sperm masses must take place in the same way as that in which two soap bubbles become confluent on contact. When this has taken place, the situation during subsequent extrusion is one in which an incompressible fluid (the sperm mass) is surrounded by a soft, deformable, and, to some extent at least, extensible sheath (the spermatophore wall) of homogeneous and isotropic material. Such a mass will, as a result of the mechanical forces (surface ten- sion) operating in such a system, tend to give rise to a spherical structure just as soap bubbles and drops of fluids tend to be spherical. An abnormal event greatly clarified this and other aspects of spermatophore structure. On one occasion a mating male and female were disturbed when an attempt was made to move them under a microscope, and the female turned on its back. Mating nevertheless continued and exceptionally clear observations on spermato- phore extrusion were made. The male, however, failed to attach the spermato- phore and the structure fell away as a completely sealed and almost perfect sphere, whose even contours were marred only by the faintest trace of a papilla at that portion of the surface which last left the male genital orifice.

Under normal conditions other mechanical forces operate and lead to deformation of the spermatophore from the spherical form. Differential hardening of the wall is probably unimportant because of the rapidity of extrusion, but attachment at one point brings into play forces other than sur- face tension and explains why the free surfaces of a spermatophore are not part of a perfect sphere.

The way in which the flange is formed has not been observed, but it is apparent that this structure is derived from the last, and therefore the most fluid, portion of the spermatophore to emerge from the genital aperture. Contact between this and the cuticle of the female will bring into play attractive forces such as can be simulated for instance by dipping part of a small ball-bearing into Canada balsam and then placing it on a hard surface. Extension in one direction of the broad ‘contact base’ thus formed can be achieved if there is opportunity for the balsam to flow between two closely apposed surfaces—as it does between a slide and slightly raised coverslip. (By such a simple set-up a state of affairs remarkably similar to the spermatophore and its flange can be produced.) Such a possibility also presents itself to the soft part of a spermatophore, as can be seen from fig. 2, in which, incidentally, the gap occupied by the flange does not necessarily represent the width at the time of its formation. It is possible that the secretion produced by the walls of the spermatophoric vesicles is responsible for the formation of the flange of the spermatophore. This material, whilst still contained within the vesicles, main- tains its own individuality and, as shown by this and its somewhat different staining reactions, must have at least slightly different properties from the rest of the wall material. The impression gained from sections is that this material is more fluid than the main secretion. On the other hand, apart from the fact that the flange sometimes becomes rather brown after pre- servation in formalin, which may merely reflect its thinness, it and the walls of the body of the spermatophore appear to be of the same composition. If the entire structure is composed of material produced in the spermatophore glands, then it can only be suggested that the secretion of the spermato- phoric vesicles either serves as a lubricant during spermatophore extrusion or assists in some unknown way in the hardening or waterproofing of the walls. No outer layer can, however, be detected in sections of the walls.

No dissolution of any part of the spermatophore on the part of the female is necessitated. Whether any female secretion facilitates flow of the flange material is not known, though there is some darkly staining glandular tissue in the region of the spermathecal spines which could conceivably produce such a secretion. Apart from this possibility the part played by the female is purely that of mechanically puncturing the spermatophore by the spermathecal spines.

Although, because of the opacity of their thick walls, it is quite impossible to observe the sperms within the spermatophores, it was found that, by sub- jecting the completely sealed spermatophore mentioned above to intense reflected illumination, changing patterns of reflection could be observed, showing that the sperms were active within and suggesting continued movement in one direction. Ruptured spermatophores also contain active spermatozoa. It appears, therefore, that activity of the sperms commences as soon as the spermatophores are discharged, and that some seminal fluid is injected into the spermatophoric vesicles with the spermatozoa. Vigorous and continuous activity takes place within the spermathecae, through the walls of which circulation of the sperms can be readily observed.

It is obvious that sperms migrate from the spermatophores through the spermathecal ducts to the spermathecae, but the mechanism whereby this is accomplished is open to debate. The cavity of the spermatophore contains nothing which could swell and push out sperms in the way in which they are pushed out of the spermatophores of certain crickets (Khalifa, 1949a), nor are any of the sperms used for this purpose as they are in at least some calanoid copepods (Heberer, Lowe, cited by Marshall and Orr, 1955). Chemical attraction of the sperms to the spermathecae seems the most plausible explanation, but it involves one difficulty. As the sperms arrive they must displace some of the presumed attractive fluid which will flow into the spermatophore, thereby to some extent reducing the relative attraction of the spermathecal fluid. Even in spermatophores cast off at ecdysis, however, some sperms remain, so it may well be that a sufficient number to ensure efficiency is attracted to the sperma- thecae before the attractive gradient becomes too small to be effective. Other theories, which demand pumping movements on the part of the sperma- thecae, are less satisfactory; for unless the two operate antagonistically, for which there is no evidence, the setting up of a negative pressure in the sper- matophore, even if only momentarily, is involved.

As the only exit from the spermathecae of the female is through the spermathecal ducts, it follows that the spermatophore must be shed before sperms become available for fertilization of the eggs. In other words, a female carrying a spermatophore can never deposit fertile eggs. Barring accidents, for which no evidence was obtained, the only way in which a female can rid itself of a spermatophore is by moulting. During this process the spermathecal spines and the linings of the spermathecal ducts are shed with the general cuticle, and the spermatophore, being firmly attached to the latter, is shed with them. A route to the exterior is now open to the sperms.

It is generally assumed that in argulids generally, the spermathecal spines are used for the penetration of the eggs as, one by one, they are deposited, and that by this means each egg receives an injection of sperms. There seems to be no reason to doubt this, for although the actual injection does not appear to have been observed, there are no other apparent means whereby the eggs, which have very thick shells and lack a micropyle, can be fertilized. In D. ranarum oviposition takes place in dishes with some reluctance, but has been observed at least in its gross aspects, though it has not been possible to make out the part played by the spermathecal spines during this process, as these are very small and inconveniently situated for observation. However, the observations made it clear that during oviposition each egg must pass beneath that portion of the abdomen on which the spermathecal spines are located and that it would be difficult for the latter to avoid touching the egg surface. As the study of spermatophores reveals, the spermathecal spines are capable of penetrating quite thick objects and it is easy to believe that some of the con- siderable muscular convulsions which accompany oviposition assist in driving them through the egg-shell. Very slight compression of the spermathecae would be sufficient to inject a few sperms into each egg.

The female reproductive cycle therefore consists of mating, ecdysis, and oviposition, in that order. Further moults can take place without interfering with oviposition, provided that sperms are still available, but after each mating the cycle must be repeated and follow the above sequence.

Certain of the properties of the spermatophore wall material are evident from the above account, and are supplemented here by brief notes on other physical and chemical properties. Precise quantitative terms cannot be applied to the physical properties, but it is hoped that the purely descriptive terms used here will convey a general impression of the nature of the wall material.

If a full spermatophoric canal is removed from a fresh specimen and com- pressed between a slide and cover slip, wall material flows slowly out and exhibits the fluidity to be seen at spermatophore extrusion. From fresh material of D. ranarum it is possible also to remove the secretion contained in the spermatophoric canals by use of a pair of very fine forceps. The entire contents of a canal can be extracted through the posterior end of the canal by this means, and it is then possible to observe the material from which sper- matophores are constructed. Whether removed ‘dry’ or under water the material is usually sufficiently viscous to permit extraction in a single piece and it emerges like an elongated white worm. It is very viscous, somewhat sticky, ductile, and fairly elastic. If a little of this material is stretched into a long thin strand, say 50 to 70 p in diameter, without coming into contact with water, it appears almost colourless by both reflected and transmitted light. If water is added a surface reaction takes place and, within a minute or so, the material becomes grey by transmitted and silvery by reflected light. This change in optical properties seems to be due to a very fine puckering of the surface layer of the material, which can be followed under the microscope. Presumably the same reaction takes place during spermatophore formation and is the first process in the hardening of the wall.

Although extracted wall material kept immersed in water remains quite soft for several hours, its property of ductility is almost completely lost in less than 30 min. Old spermatophores are still ‘soft’ in so far as the surface admits of permanent indentation if pressed with the point of a fine needle. If teased by fine needles, old spermatophores rupture in an irregular manner but are not strikingly brittle nor do they have the ‘toughness’ which one associates with uncalcified cuticular structures in arthropods. In specimens fixed in formalin (which seems to toughen them) the walls are, however, sufficiently strong and rigid to permit clean bisection with a sharp, fine scalpel.

Spermatophores are not chitinous but proteinaceous in composition. This is shown by their complete dissolution in a hot saturated aqueous solution of potassium hydroxide and by the strongly positive result which they give to the xanthoproteic test. They also give a positive result to the argentaffin test and dissolve in a solution of sodium hypochlorite. Only a 1 % commercial pre- paration of the latter was available (Brown (1952) recommends a 10% solution), but even in this they dissolved within a few hours with the evolution of gas bubbles. These tests, and the failure of the spermatophores to be dis- persed in boiling water, dilute hydrochloric acid, dilute caustic alkalis, cold saturated aqueous solutions of caustic alkalis, or a solution of calcium chloride, indicates that they are composed of quinone-tanned protein, though their colour differs from that of most such tanned proteins. They dissolve com- pletely and within a few minutes in cold concentrated hydrochloric acid but are not dispersed completely by cold concentrated nitric and sulphuric acids. In nitric acid they become brown and translucent but do not dissolve even after several hours’ immersion, while in sulphuric acid they rapidly collapse and fragment and become a rich red brown, but again persist in this state even after several hours’ immersion. The application of heat, however, causes rapid solution in these acids. In the case of sulphuric acid the resulting solution is wine red in colour.

A test for lipids (Sudan black) gave a positive result even after the spermato- phore had been immersed in xylene for a few hours. The presence of lipids, apparently firmly bonded to the protein, may assist in waterproofing the spermatophore. In spermatophores of the terrestrial cricket Gryllus (Acheta) domesticas L., Khalifa (1949a) found no trace of lipids.

Change of the wall material from the fluid to the solid state is reflected by a change in staining properties, for it was observed that while the secretion in the spermatophoric canals stained easily with haematoxylin, similarly treated sections of the spermatophore wall stained much less readily.

After it had been discovered that the spermatophore wall material gives the reactions of a quinone-tanned protein (p. 425), it was thought that the glandular tissue of the spermatophoric vesicle might secrete the aromatic material which tanned the protein secreted by the spermatophoric gland, much as one gland secretes protein and the other the tanning orthodihydroxy- phenol during the formation of structures as diverse as the ootheca of cock- roaches (Pryor, 1940) and the byssus of the lamellibranch Mytilus (Brown, 1952). However, this appears not to be the case, for material that had been carefully removed from the spermatophoric canal and had never been in con- tact with or even in proximity to the glandular wall of the spermatophoric vesicles, gave just as positive a result to the argentaffin test as did material from the vesicles themselves. ‘

Quinone-tanned proteins tend to be dark, but the spermatophores of D. ranarum have a pearly lustre. This may indicate a fine lamination of the wall—a lamination in which layers of one refractive index are separated by layers of different refractive index, so that pearliness arises from multiple re- flections from thin films, as in real pearl. (I am indebted to Dr. L. E. R. Picken for this suggestion.) The transmitted colour of the wall is thus obscured. When a piece of the wall is immersed in water and examined by transmitted light under the microscope, however, surface reflection is abolished and the wall is seen to be very pale amber.

Stuhlmann (1891) referred to the spermatozoa of D. ranarum as being fadenformig. This is an apt description of a fully mature spermatozoon. Com- plete maturity, however, seems usually, but by no means invariably, to be delayed at least until the sperms arrive in the spermathecae of the female, and even there incompletely mature sperms are often to be found.

A fully developed sperm has the general appearance of a minute nematode worm and has a length of about 138 to 165 p.. There is absolutely no head. The diameter of the sperm body (the term ‘flagellum’ seems inappropriate in this case) is about 0-7 p, but it is difficult to measure this object accurately. In many specimens almost all the sperms present in spermatophores and many of those in the spermathecae of the female appear at first sight to have a dis- tinct head. In some this appears to take the form of a simple flattened swelling at the tip of the sperm, while in others a spiral thickening seems to be present on its surface. Both these conditions are, however, due to a coiling of the distal end of the sperm body and each represents a stage in the maturation of the sperm. The head of a sperm showing the more complex of these two conditions is shown in fig. 16, c. Of the two this represents the least mature condition. For convenience this is called stage 1. Stage 2 (the apparently headed sperm without a spiral) results from a partial uncoiling of the tip of the sperm (fig. 16, B, D) and a consequent elongation of the sperm body. Further un- coiling of the sperm tip gives rise to the fully mature thread-like sperm. The distribution of these stages is difficult to understand, for completely uncoiled spermatozoa have been found in some specimens to be more abundant than the other stages even in the vas deferens of the male, and have also been found even in the testes. Apart from suggesting that the time and place at which sperms become fully mature is unimportant, this inconsistency remains un- explained. It is, however, presumed that complete uncoiling is necessary before a sperm can penetrate an egg.

FIG. 16.

A, spermatozoa at stage i of maturity. For explanation see text. B, spermatozoa at stage z of maturity. For explanation see text, c, the tip of a spermatozoon at stage r of maturity, showing coiling. (Owing to the difficulty of measuring the diameter of the sperm the scale is only approximate.) D, diagrams illustrating the uncoiling of the tip of a spermatozoon. The same region is denoted by the same number in each case.

FIG. 16.

A, spermatozoa at stage i of maturity. For explanation see text. B, spermatozoa at stage z of maturity. For explanation see text, c, the tip of a spermatozoon at stage r of maturity, showing coiling. (Owing to the difficulty of measuring the diameter of the sperm the scale is only approximate.) D, diagrams illustrating the uncoiling of the tip of a spermatozoon. The same region is denoted by the same number in each case.

The very tightly packed sperms of the vasa deferentia (p. 412) are, in this situation, immobile. Feeble movement is initiated in a small proportion of these when they are released into a 0 · 6% solution of sodium chloride.

Sperms contained in spermatophores are motile (see above, p. 423), and those in the spermathecae of the female can be seen through the integument to be in constant and active motion, circling round and round within these receptacles. Dissection reveals that these may be in any of the three stages described above. Activity within the spermathecae is maintained for at least 7 days and, if activity and viability are in this case synonymous, probably for much longer.

Sperms are inactivated and presumably killed within a few seconds of being released into fresh water—the medium in which Dolops habitually lives. This stands in marked contrast to the behaviour of the sperms of many marine invertebrates when released into sea-water and recalls the maladaptation of the sperms of many freshwater fishes to the medium into which they are re- leased (Huxley, 1930;Rothschild, 1958). One very cogent reason for the always complete protection of sperms from the surrounding water during their transference is thus obvious. Survival of released sperms is greatly prolonged if a 0-6% solution of sodium chloride is used instead of tap-water. Death of sperms released into water is therefore presumably due to the sudden change in osmotic pressure and electrolyte concentration to which they are subjected.

This constant activity of sperms within the seminal fluid stands in marked contrast to the behaviour of those of certain other invertebrates such as sea- urchins in which the spermatozoa are motionless in undiluted semen and only acquire motility, which is intense and of short duration, when shed into water. As the sperms lack any organ of food storage, this constant activity indicates that they are bathed in nutrient fluid.

In the spermatophore-producing Crustacea for which functional accounts are available, the wall material is secreted by part of the vas deferens and comes to surround the sperm mass already present. This is so, for instance, in the copepod Calanus finmarchicus (Gunnerus) (summary of various papers in Marshall and Orr, 1955), and in various decapods (Spalding, 1942; Mathews, 1954, 1956, and other papers). In Dolops the situation is quite different, for spermatophore wall material is produced by glands which are independent of the main reproductive tract, and the only active part played by the vas deferens in spermatophore formation is the injection of sperms into the mass of prospective wall material, which accumulates in special reservoirs. In this respect the process in Dolops bears greater similarities to that taking place in certain caddis flies (Khalifa, 19496), cockroaches (Khalifa, 1950), and scor- pions (Abd-el-Wahab, 1957; Alexander, 1957, 1959), in which accessory glands are responsible for the secretion of spermatophores, than to that taking place in other Crustacea. (In the light of Alexander’s work it is apparent from the detailed account of Abd-el-Wahab that the scorpion with which he dealt produces spermatophores, though he does not specifically state this.) The similarity of the process in Dolops and the insects mentioned above is, how- ever, by no means complete even in its grosser aspects, for in most of the insects the wall material (often accompanied by extra protein masses) is ejected before the sperms, which only penetrate it after it has filled the bursa copulatrix of the receiving female. Particularly interesting similarities to the process taking place in Dolops are found in certain cockroaches (Khalifa, 1950) in which wall material is secreted into a single sac, into which spermato- zoa are injected before spermatophore ejection takes place. The outstanding differences in spermatophore formation here, other than those necessitated by differences in the complexity of the wall, are that only one sac is present in Blatella (as compared with two vesicles in Dolops) and that the two masses of spermatozoa injected into it maintain their individuality throughout instead of becoming confluent as in Dolops.

The spermatophore of Dolops is definitely a single structure. In most other spermatophore-producing arthropods in which a double set of organs is involved two spermatophores (as in cyclopoid copepods) or a spermatophoric mass (as in certain decapods) are produced. Spermatophores so produced may fuse, without, however, fusion of the two sperm masses, as in the prawn Penaeus japonicus Bate (Tirmizi, 1958), or the alder fly Sialis lutaria L. (Khalifa, 1949b).

In scorpions, and apparently in pseudoscorpions, a single spermatophore is produced, as in Dolops, from paired organs, and its formation in many ways resembles the process which takes place in Dolops. Here, however, at least in scorpions, two completed halves are secreted and these do not become con- fluent but are cemented together by the secretions of special glands (Alex- ander, 1959).

The discovery of spermatophores in the Branchiura, from which their absence had hitherto been regarded as diagnostic, raises questions regarding the relationships of this enigmatical group. The occurrence of such structures in one genus of the group, however, does little to clarify these relationships, for spermatophores are capricious in occurrence within the animal kingdom and seem to have arisen many times independently in compliance with neces- sity. For example, while the true fishes as a whole do not employ them they are found in a single isolated case, namely, in Horaichthys setnai Kulkharni, and Khalifa (19496) has pointed out that in insects spermatophores have arisen several times in a phylogenetically disconnected manner as a means of solving the problem of sperm transfer. One thing is clear, however : there is no justification whatsoever for suggesting a relationship between the Branchiura and the Copepoda (a group to which the former has frequently been assigned) on the basis of the common possession of spermatophores. Had the two been at all closely related one would expect to find at least some common features relating to spermatophore formation; for although, as Mathews (1954) has shown in the Decapoda, even species of otherwise closely related genera do not necessarily produce similar spermatophores, the process is basically the same within any particular group. In the Copepoda and Branchiura the process is fundamentally different. Thus in the Copepoda spermatophores are formed within modified portions of the main genital tract: in Dolops accessory organs are developed. In the Copepoda each spermatophore is from the time of its inception a single structure : in Dolops it is of dual origin. In the Copepoda spermatophores are complete before transference : in Dolops they are completed only at the time of transfer. After transference they may remain attached to the female throughout life in the Copepoda and in no way interfere with egglaying, whereas in Dolops ecdysis is essential before the laying of fertile eggs is possible. The latter point emphasizes an important distinction between the Copepoda and the Branchiura and one which only Gurney (1949) appears to have stressed: namely, that adult copepods never (or perhaps only as an abnormal and as yet unrecorded event) moult, whereas members of the Branchiura continue to moult throughout life. That an aspect of reproductive biology bound up with the use of spermatophores should involve a relationship to such a fundamental distinction emphasizes the differences between the two groups.

One superficial similarity between the process of spermatophore transfer in Dolops and the Copepoda (in so far as one can generalize from the limited observations made on the latter group) is the use by the male in both cases of the legs to assist in spermatophore transfer. In cyclopoid copepods in particular (Hill and Coker, 1930) the process resembles that seen in Dolops, and the more precise and well-known method of spermatophore transfer in at least some calanoids presumably had a similar origin. Such similarities are, however, of as little value as the possession of spermatophores in deciding affinities, for it is apparent that whatever appendages are most conveniently situated for this purpose will be used to guide the spermatophore into position. Thus in cyclopoid copepods several pairs of legs play a part in spermatophore transfer, whereas in calanoids only the fifth pair, which is rudimentary in cyclopoids, is concerned in spermatophore transfer. Similarly in certain decapods, pre- sumably because of the location of the male genital aperture, the abdominal appendages assist in spermatophore transfer.

On the other hand, the possession of spermatophores by Dolops gives no indication of any affinities between the Branchiura and the Branchiopoda, in which spermatophores are completely unknown. The evidence therefore tends to emphasize the isolated nature of the Branchiura rather than to indicate affinity with any other group.

Similarly a study of spermatophores, beyond emphasizing the isolated position of the genus Dolops, throws little light on the relationship to each other of the various branchiuran genera.

As to how the evolution of spermatophores could have taken place, the following suggestions are offered. In at least some species of Argulus, and probably in all, accessory glands homologous with but much less extensive than the spermatophore glands and spermatophoric canals of Dolops are present, and presumably represent an anatomical feature of long standing in the Branchiura. According to the figure given by Wilson (1902) for A. americanas Wilson, and to my own observations on A. africanus Thiele, these organs take the form of rather short, blind ducts with no great glandular efflorescence distally. It may be that in the primitive argulids, which would include the ancestors of Dolops, they secreted some kind of cement which formed a temporary ‘seal’ around the opposed genital apertures of the male and female during copulation. On the evolutionary line taken by Argulus, leading to the development of complex claspers on the legs of the males, in which, in certain species at least (personal observations), a conical papilla with a wide aperture has been developed to serve as a primitive penis, there would be no need for the production of this secretion to be copious and therefore no tendency towards spermatophore formation. The more copious production of such a secretion in forms less specialized in these directions would tend towards the formation of a bubble at times of copulation and this would, during its transient existence, afford protection to the sperms during trans- ference. It would require but a small step to make the bubble a more permanent feature of the reproductive process. Its gradual modification and the utilization of the spermathecal spines already present (as must have been the case in order to allow of perforation of the egg-membranes) would lead to the kind of structure seen today. Throughout this process selection would favour the production of more abundant and more suitable cement, and the spermatophore gland and its associated canal and reservoir would gradually increase in size and specialization towards this end. The carapace lobes, filled as they are with parenchymatous cells and having abundant haemocoelic spaces, would readily permit the incursion within them of glandular tissue, in much the same way as they came to house the digestive diverticula of the alimentary canal.

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