Observations on Saprolegniæ

To the latter parasitic forms belong Saprolegnia de Baryi and S. Schachtii, found in the cells of Algæ and Hepaticæ respectively, according to Walz¹ and Frank,² and especially the Saprolegnia of the “salmon disease,” according to Professor Huxley.³

Generally described; the Saprolegniæ consist of a thallus,⁴ bearing reproductive organs of two kinds—zoosporangia and oogonia, with or without accompanying antheridia. The thallus consists of long, branched, tubular hyphæ, of which the main portions are free or “extramatrical;”⁵ shorter, rhizoid-like branches penetrating the tissues from which the fungus radiates.

De Bary has lately pointed out that the “intramatrica’ portion of the thallus does not spread far in the tissues, and no extension of the fungus occurs by outward developments from its internal branches; the hyphæ outside, however, continually send downward prolongations, which take root and spread slightly in the attacked tissues, and thus the area of “intra-matrical” hyphæ becomes extended.¹ Hence, if zoospores, &c., are prevented from again attacking the matrix from without, the Saprolegnia thallus does not extend far within the tissues —an important distinction between these fungi and those which, like Pythium, &c., attack a host at one point and send ramifications in all directions inside the tissues. The difference is, roughly speaking, analogous to that between a banyan tree and a bamboo, in so far that the former extends its area of feeding ground by sending down prolongations from its outer branches to root ‘afresh in the matrix or earth, while the latter extends itself under the surface by means of underground shoots, which protrude here and there further from the parent stock.

The tubular hyphæ forming the thallus vary much in diameter, partly according to position, main stems and branches being thicker than secondary and tertiary ones; their cellulose walls are very thin and transparent, and enclose protoplasmic and oily contents. These latter are, as a rule, very coarsely granular, causing the thallus branches to appear dense and opaque, usually with a yellowish hue.

Septa occur very rarely in the tubular branches, but are always found separating the zoosporangia and the sexual reproductive organs from the purely vegetative portions of the thallus.

The Saprolegniæ have usually been stated to be a-nucleate in common with other fungi². This, however, is not the case; publication is now the most important authority for the morphology of the group.

Schmitz has described nuclei in these and allied fungi, as well as in many others of the lower cryptogams-¹ I have lately also found nuclei of a very definite character in the mycelium of an allied fungus, and shall show that a perfectly definite division of nuclear masses occurs in the zoosporangia of Achlya and Saprolegnia. It is at least certain that the Saprolegniæ can no longer be regarded as devoid of nuclei, and the same probably holds good for all but the very lowest cryptogams; according to Schmitz the Phycochromaceæ and Schizomy cetes.

The reproduction of the Saprolegniæ takes place by means of asexual zoospores, produced in long zoosporangia; and sexual (at least morphologically they must be considered so) oospores, produced in oogonia, with or without accompanying antheridia. The details concerning both these kinds of structures may be deferred for the moment.

With this introduction, the immediate object of the present essay may be entered upon; that is, to describe some observations made during the past summer and autumn on species of Achlya and Saprolegnia, the two most important genera of the group. These observations are not all equally valuable or new (although some of the facts were observed before I was aware that others had discovered them),² but they have been made quite independently of the literature,³and are thus of some service as confirmatory evidence to those who wish to study the subject further.

I shall adopt the simple plan of describing what I have seen and drawn, together with methods employed, leaving more general conclusions until afterwards.

1. Achlya polyandra.—Masses of débris of “meal-worms” on which this species had been grown some months previously, and which had been kept in a cool cellar during the interval, were placed, together with a fresh meal-worm in a large deep glass beaker perfectly clean, with a considerable quantity of boiled and filtered water, and a glass plate over the top; the whole stood in a well-lighted room at the ordinary (summer) temperature. In the course of two or three days, during which the water was several times replaced, the floating grub was seen to be developing pale, cottony filaments in all directions, on and in the water around. These filaments proved to be slender, straight tubes, filled with hyaline protoplasm in which numerous large granules were scattered, especially in the larger specimens; the walls became distinctly coloured blue in Schultz’s solution, and in H₂SO₄, after treatment with iodine, the protoplasmic contents becoming yellow in the former reagent.

After a considerable mass of these radiating tubules had become developed, certain of them were found to bear zoosporangia. The development of a zoosporange was observed many times in the following manner:— A broad glass slip being placed in the water under the whole growth of Achlya, the attacked meal-worm was lifted up bodily, and transferred to the stage of the microscope; plenty of water being carefully added to the specimen, the upper, more or less floating branches, could be easily observed with a Zeiss D with a little care. The great advantage of this or a similar method is that the Achlya goes on growing almost undisturbed, and fresh water can be continually added as evaporation goes on. If a higher power is needed, it is very easy to place a small piece of very thin, perfectly dry and clean glass, so as to float on the flooded object, and remove it dexterously afterwards. These very delicate cryptogams will not grow in a normal manner under the pressure of an ordinary cover-slip, if continued, and the above method (or that, of suspending a small specimen grown on a fly’s leg in a drop of water under a cover-slip) is advantageous in many ways. I may add, however, that with care observations may be made with Zeiss E without any cover-slip at all on favorably situated portions.

The Zoosporangium is simply the terminal portion of a branch spreading freely in the water; this becomes slightly dilated into a club-shaped body, into which very granular protoplasm collects, giving the young zoosporange a dull grey appearance, easily detected with a good hand lens. The apex of this body remains blunt, and the walls are not thickened During the course of about an hour after the protoplasm has slowly accumulated, the following changes occur:—(1) A thin septum becomes formed at the base of the dilated portion, separating its dark grey, non-vacuolated, coarsely granular protoplasm from the more sparsely granular, vacuolated contents of the rest of the branch; and (2) an aggregation of the contents of the zoosporange around numerous centres takes place. To take an instance actually observed (Pl. XXII, fig. 1). The gradual swelling and filling of the zoosporange (a) was completed by about 11 a.m. -, at 11.15 (b) the septum had become formed, and the protoplasmic contents were already denser and showing signs of aggregation at many centres; at 11.45 this had proceeded so far that no doubt of the existence of a multitude of small semi-detached masses could be entertained (fig. 1 c).

And now followed a most remarkable phenomenon. At about twelve o’clock the appearance shown at c was replaced by one similar to that shown at b; this had occurred in many previous examples, and puzzled me exceedingly, and I was accordingly prepared to watch the exact sequence of events in this instance. The protoplasm at 11.50 was distinctly divided up into a large number of nearly globular independent masses, slightly compressing one another. No membrane could be detected around any of these, though, as subsequent investigations showed, a sort, of watery-looking, clear boundary stood between the masses. At one to two minutes after 12 the masses and their clear boundaries became indistinguishable; this fading away of the granular appearance took place so quickly that it might almost be termed sudden, and the protoplasmic mass was now in a condition apparently like that before the division, except that its granules were smaller and probably more numerous, and the mass seemed more translucent than before. During the next three minutes a large number of small, clear, equidistant areas (E) made their appearance in the now almost hyaline, fine-grained protoplasm, and a curious, pale, watery look had replaced the dark grey appearance of the earlier stages. These bright vacuole-like spots seemed approximately equal in number to the masses which preceded them (D), and one could well believe that the clear spaces form at points corresponding to the centres of the former bodies; this, however, could not be decided. In three or four minutes after the last condition was figured, the almost sudden reappearance, so to speak, of the rounded or polygonal solid masses occurred (F), as if the contents had gone back to the state figured at D. This time, however, the separation of the masses became more evident, and at ten minutes past 12 the apex of the sporangium gave way suddenly, and the whole mass of separated blocks of protoplasm suddenly flowed out into the surrounding water, and remained at the mouth of the zoosporangium as a spherical clump of protoplasmic globules (figs. 8 and 4).

The following peculiarities concerning these bodies and their exit were noticed. At the moment before the apex of the zoosporangium bursts the isolated, though closely packed, masses of protoplasm showed slight amoeboid movements, and during the rapid expulsion were actively changing their form; the instant they reached the exterior, however, they all became strictly spherical, if free, slightly polygonal if compressed by neighbouring ones (fig. 4). The whole process of exit and rounding off only occupies a few seconds, and in a well-grown mass of Achlya dozens of zoosporangia may be emptying their amoeboid contents at the same time; in such cases, also, the presence of this phenomenon can be detected with a hand-glass, the heads of globules being quite distinct in a good light.

The future behaviour of these zoospores—for such they must be considered—may be described, as before, from what was observed in a given example. Careful examination of one of the more loosely attached specimens on the outer portions of the spherical group (figs. 3 and 4) convinces the observer that an extremely delicate envelope becomes developed at the periphery of the resting globular zoospoore (fig. 5 a), and the body remains in this condition for some hours.

The specimen referred to was drawn at 1 p.m., and remained in this condition until about 4 p.m. Soon after this there were signs of change going on in the neighbouring specimens, and this particular globe was carefully watched. At 4.14 a slight protuberance made its appearance at one side, rapidly increased in size during the next minute or two (fig. 5 b—d), and a clear space (e) was then seen separating the delicate envelope from the granular, slightly amoeboid protoplasmic contents, which were, in fact, becoming withdrawn to pass through to the outside. A minute afterwards the whole of the protoplasm was outside, except a minute papilla, which slipped out forthwith (fig. 5f, g), and the mass commenced to writhe slowly in an amoeboid manner outside the very delicate empty envelope, in the side of which could be seen the minute pore through which the zoospore had slipped out. At 4.18, the moment of complete exit, a clear spherical vacuole was seen at one side of the zoospore (g); the latter then quickly acquired a reniform shape, and from the sinus (corresponding to the hilus of the kidney), two minute cilia with knobbed tips were observed to spring forth, quickly grow in length, apparently at the expense of the knobs at their ends, and begin to wave slowly about. The zoospore now (4.25) commenced to swing perceptibly as the lashing of the cilia became more vigorous; nevertheless it did not move away for some time; at 4.35, however, the zoospore was freely and rapidly moving about, and at once disappeared from the field.

This process, described as faithfully as possible in one case, was repeated by the contents of the rest of the spheres composing the globular mass at the mouth of the zoosporangium (fig. 4), and all were nearly in the same stage of development at the same instant; consequently the exit of the zoospores from the spherical envelopes can be readily observed when the critical time is carefully watched.

The mass of empty envelopes remain behind, appearing as an exceedingly delicate network (fig. 6), and even simulating parenchyma of great tenuity, mutual pressure causing the spheres to become polygonal. Here and there the minute pore can be observed as a dark spot in the side of the envelope, and sometimes a zoospore escapes much later than its companions.

There is little more to be said of the zoospores. Some time after their exit they come to rest, round off, and each at once commences to put forth a simple tube (fig. 7), having first lost its cilia and vacuole, and acquired several brilliant granules, which become arranged around the periphery. If the germination occurs on a proper matrix, such as a meal-worm, fly, &c., the tube enters and commences to grow into a rhizoid-like portion, a new thallus becoming developed from outside. On glass, &c., the tube soon reaches the end of a limited growth, its contents fade, and the whole dies.

Among other abnormalities in the course of phenomena such as the above, mention should be made of one which is not uncommonly met with, and which I have drawn at fig. 8. In certain of the zoosporangia the completely separated zoospores remain behind, rounded off, and form their delicate membranous envelopes while still in the cavity; not only so, they germinate in this position, each pushing a short tube through the sporangium wall (fig 8, B) before emptying its contents on the exterior. In the example figured, the apex of the sporangium became open in the usual manner at length. The figure B represents part of A under a higher power and twenty-three hours later. These “Dictyuchus “forms were obtained from specimens of Achlya polyandra which had remained about six hours in the same water on a slip of glass; towards the end of the period the fungus was obviously passing into a state of inanition. As fig. 8 B shows, the sporangium becomes filled with an apparent tissue of extreme delicacy—the empty membranes of the zoospores—and the name “Dictyuchus” was given to express the net-like structure thus produced. Whether the genus “Dictyuchus” exists on a firmer basis than this I do not know.¹

With respect to the sexual reproductive organs of this Achlya, my observations cover a considerable field; as before, the description applies strictly to what I have seen. The oogonia and antheridium branches become produced in large quantities when the cultivated Achlya is allowed to remain quite still, floating on the surface of abundance of water; their presence is soon detected with a good hand lens, and further examination gives the following information concerning them.

The oogonia arise as globular or nearly pear-shaped swellings of the ends of very short branchlets, developed at nearly equal intervals along the course of a vigorous branch (fig. 9); the short branchlet is usually much smaller in diameter than the parent twig, but resembles it in possessing thin walls and coarsely granular protoplasmic contents, and in its cylindrical shape. The balloon-like terminal swelling receives a large supply of protoplasm, which accumulates in it as a yellowish-grey dense mass, and then becomes shut off from the pedicle by a thin, sharply-marked septum (fig. 10). In the pedicle, which is about as long as the longer diameter of the oogonium, the remaining protoplasm is much more watery and poorer in granules; the latter is inserted sharply, as it were, into the parent branch, and there is no septum at the base—the cavity of the two remains continuous throughout. In vigorous specimens (fig. 10) the granules often seem to be arranged in rows, embedded in the layer of transparent protoplasm lining the cylindrical cell walls. The groups of Oogonia, marked by their yellowish-grey contents while young, present a striking object (fig. 9), like groups of berries developed in racemose order; the pedicles are usually slightly curved in various directions. As well shown in fig. 9, the oogonium-bearing branches may be of various orders, very commonly secondary and tertiary. Since the thallus has accumulated much material, and the asexual reproductive organs have been for the most part emptied when the oogonia arise, it is usual to find empty zoosporangia terminating the main twigs. The production of lateral branches from beneath the sporangia is characteristic of Achlya, and that such may bear oogonia is sufficiently demonstrated by fig.9 B. Such is the typical mode of development of the oogonium. Before proceeding to describe the changes which its contents undergo, we may examine the mode of origin and growth of the so-called “antheridial “branches.

These are longslender tubes, springing from the main branches from points either close to the oogonia (fig. 10) or at greater distances apart, or even from separate branches. The diameter of the tube is commonly less than that of the pedicel, but may equal it: within its thin walls are finely granular, watery protoplasmic contents, not always easily distinguished. As seen in fig. 12, the “antheridial branch” arises as a simple tube; it often begins to form branches soon after its origin, and these spread in all directions, curling and waving as they do so. In this manner they become wrapped or coiled around objects, such as neighbouring branches or oogonia, with which they come in contact (fig. 9 B). It is in this coiling, of the antheridium branch about an oogonium that the first stage of a proper sexual process has been recognised by earlier observers.

During the development of the coiling antheridial tubes above described, the granular, yellowish-grey contents of the oogonium, undergo certain changes, which result in their complete transformation into the egg-cells or oospheres. A clear, almost watery spot appears in the centre of the mass (fig. 11 a), and slowly increase in bulk as the dense grey granular protoplasm recedes to the walls; in this latter are large, fatty-looking granules, which seen from the surface (fig. 11 b) are in slow but evident motion. This retirement of the protoplasm to the sides is followed by another process; a collection of the whole mass into two or more clumps, which then slowly round off as naked oospheres (fig. 12), consisting of the fatty protoplasm only, suspended in the oogonium cavity, which appears otherwise empty. This collecting of the protoplasm to form the eggs, or oospheres, is a remarkable process in more respects than one; it takes place slowly, and occupies several hours altogether. I will confine my description to one case observed.

An oogonium was favorably situated for observation from above, and at about 12 noon had attained the stage figured at fig. 11 a. The coarsely granular protoplasm aggregating on the walls, was in a state of continuous slow-flowing motion, quite distinct to one observing a given granule from the upper side (fig. 11 b) this specimen was watched carefully from this time forward till nearly 5 p.m., and underwent changes which were figured as follows (fig. 14):

For a long time the mass on the walls slowly heaved and flowed, without its lateral continuity becoming broken. At about 1.30 to 2 o’clock, however, the surface view showed that the dense layer was breaking up into more distinct masses; and at 2.35 oblique, broad bands of fatty granules represented the connection between two large masses aggregated at the sides (fig. 14 f). On watching the uppermost of these bands, the slow breaking up and passage over to either side of the granular protoplasm was distinctly observed (fig. 14 g, h). About 5 or 10 minutes before 4 the whole of the protoplasm was thus collected into two equal lumps, still somewhat flattened on one surface to the walls of the oogonium, and standing on opposite sides (fig. 14 i, k). The next five minutes were occupied in the collection of a few scattered granules, the raising up of the centre of each lump from the wall, and its ultimate withdrawal altogether towards the centre of the oogonium (fig. 14 l). During the latter process, the eggmasses were distinctly amoeboid; each had its surface alternately raised into lumps and smoothed off again, and in.some cases small particles of the protoplasm became detached and taken up again.¹ There is not the slightest doubt as to the accuracy of these observations; the amoeboid motion continued for some time, then slowly ceased, and at 10 minutes past 4, the two perfectly spherical oospheres lay obliquely in the oogonium, mutually in contact, as shown in fig. 14. The oospheres in this condition are apparently ready for “fertilization,” and the following phenomena occur.

One or more of the antheridial tubes, coiled closely about the oogonium (figs. 10, 11, &c.), while the above described processes have been going on, begins to send a tubular process through the oogonium wall, at or about the time when the oospheres are smooth and rounded off; the tubular process thus sent into the cavity of the oogonium (fig. 12) has been termed the “fertilising-tube.” It is a direct prolongation of the “antheridial branch,” and contains finely granular protoplasm; it grows for some time in the cavity of the oogonium, coming in contact with the oospheres—even running on their surfaces. I have never seen it enter an oosphere, nor have I seen it open at the end or emptied of contents. Whether anything passes from it to the oospheres cannot be decided; but De Bary gives such strong reasons for doubting that any fertilising process whatever occurs, and supports his conclusions by so many examples and so much observation that it would be presumptuous to attempt to decide the question without devoting at least equal energies and time to the task. So far as my observations go, they decidedly fail to supply evidence for the view that anything is emptied from the tube into the oogonium or oosphere. Before offering any further remarks on this subject, it will be convenient to describe the remaining observations made on other species.

Achlya apiculata is the name by which Professor De Bary designates a species not yet (I believe) described; my observations on this form are not yet sufficient to enable me to do more than depict the formation of the zoospores. I have never seen the Oogonia or Antheridia.

The zoosporangium (fig. 15) differs somewhat in shape from that of A. polyandra, the apex especially being more pointed. In a specimen carefully watched for some hours, the sporangium was at first filled with very finely granular grey protoplasm, and two or three large vacuoles remained below, abutting on the somewhat swollen-looking septum; the tube below the septum contained many and large vacuoles, the nets or bridles between which were slowly streaming. Such being the condition of affairs at 9.30, the only observed difference at 9.50 was that the vacuoles had disappeared from the zoosporangium, and the fine-grained protoplasm reached close up to the now more sharply-marked septum. About 10 o’clock the tip of the sporange appeared brighter and marked by faint longitudinal striæ (fig. 15 c), and a slight tendency to the formation of brighter areolæ seemed evident in the protoplasm. At 10.10 this was distinctly marked (fig. 15 D); the protoplasm arranged itself slowly into polygonal masses, each with a brighter central part. This stage lasted for nearly ten minutes, the division lines becoming brighter and sharper, until the blocks stood nearly isolated, and then, quite suddenly, at 10.20, the separation lines disappeared and the blocks fused together, and a uniform grey, granular mass (E) resulted as before. This particular sporange was not observed further, but in fig. 16 are drawings of what was seen in another specimen from the same cultivation. At 10.35 the breaking up into the preliminary blocks was nearly complete (a, fig. 16) and very distinct; the hard, sharp division lines disappeared quite suddenly about two to three minutes later, and then the evenly granular protoplasm became marked out into bright areas (fig. 16 b) by small vacuole-like points. These increased slowly in size, and at 10.45 the sporange presented a peculiar lustrous aspect, the granules appearing remarkably sharp and black in the bright, watery-looking matrix. It seemed also that there were relatively more vacuoles than preliminary divisions: this difficult point could not be decided. At 10.50 the second series of division planes were established (fig. 16 c). I could not satisfy myself that each vacuole occupied the centre of one of the blocks, though such was un-doubtedly true sometimes; it seemed that in some cases a large block became further cut up into smaller ones. This process proceeded very rapidly, and by 10.53 the zoospore masses (fig. 16 d) were finally isolated, and slipped out in the next two or three minutes. The further fate, &c., of the zoospores need not be described in detail -, they behave essentially as before.

The observations on this genus will be confined to the forms of Saprolegnia ferax (Pringsheim), and the following descriptions, &c., will refer particularly to that called S. monoica in the sense of the above author.

Through the kindness of Prof. De Bary, I was enabled to infect “meal-worms” and house-flies with S. monoica, and in two or three days had excellent cultivations floating in abundant water as before. The methods of observation, &c., need not be detailed; they are practically the same as those described for Achlya.

Fig. 17 shows the various stages of development of the zoosporangium and zoospores; the segmentation of the protoplasm takes place as before, and need not be further described. At the completion of the second segmentation, the masses of protoplasm behave in a manner quite different from those of Achlya, however, since, instead of simply slipping out of the apex of the zoosporangium and then rounding off, they acquire two terminal cilia at once, and pass off as actively moving zoospores (fig. 17 f). Each zoospore is a top-shaped mass of finely granular protoplasm, with two very long cilia actively waving at its pointed (forward) end, and with a sort of zone of three small vacuoles around its broader part (figs. 17 g, 19 a). In this condition it moves rapidly from the point of exit, coming to rest (h) after some minutes. With care it is quite possible to watch a zoospore through all its changes. Fig. 19 shows the phases actually seen in the case of a zoospore emitted from the zoosporangium in fig. 17. It became free about 9 a.m., and moved actively for ten minutes, rounding off and losing its cilia and vacuoles in an instant (c f. fig. 19 a, b). In this quiescent condition it remained for some hours unchanged, excepting that an envelope was gradually formed on its surface. At 2.30 p.m. the contents of the little sphere came out (c and d) as an amoeboid, naked mass, which gradually acquired a kidney-shape and a large vacuole, and developed two lateral cilia; these latter, as before, arose as two minute knobbed processes, which slowly increased in length and began to wave, causing the body of the zoospore to swing more and more, and at length (about 3 p.m.) to move away. This particular specimen was then lost. But I observed another (f to i) for nearly an hour and a half; it was just coming to rest (g) about 4 o’clock, and had commenced to germinate before 5 p.m., growing very rapidly (i) and then dying. In another-case (fig. 18) I followed the second more closely. It escaped from the envelope (a, b) about 2.10 p.m., and swarmed as a kidney-shaped spore (c) for nearly half an hour; it then lost its cilia, writhed two or three times in an amoeboid manner (d), and suddenly became rounded off (e) as a naked spore. This was at 2.55 p.m. At 3.15 (f) it began to germinate, by throwing out a slender tube, which had reached a considerable length by 4 o’clock (g), when the whole was dying.

In the normal condition of affairs such a germinal tube enters the body of the insect, and continues the life-cycle. In some cultivations one often finds bright white clumps of germinating zoospores (fig. 20) lying at the bottom of the water; these result from numerous zoospores coming to rest about the same time, falling quietly through the still water, and, again, germinating almost simultaneously.

There is little more to be said concerning these processes. The zoosporangia of this Saprolegnia vary in shape within wonderfully wide limits; some are almost as broad as long, others nearly tubular, while pyriform, top-shaped, and irregular specimens of all kinds occur. In cultivations, allowed to starve from want of renewed water, &c., imperfect and distorted sporangia reach a certain stage of development, and then, acquiring very thick walls, remain in a resting condition, springing into activity again when the conditions of the environment improve. I have drawn one or two specimens of such dormant branches of the thallus of Sapr. monoica at fig. 21. I have not yet obtained oogonia of Saprolegnia monoica, and must refer to the literature;¹ drawings of the ripe oospores are given at fig. 22, but no attempt to produce them by cultivation on my part have yet succeeded.

The immediate object of this paper, to describe accurately a few careful observations in the hope that they may help to stimulate others to pursue the subject, is now ended; but it may be well, before concluding, to call attention to some general conclusions which have been drawn lately, and on which such observations as the above throw light.

Apart from the question as to whether the Saprolegnia be regarded as true Fungi or not, they may certainly be considered as forming a distinct group of parasitic and saprophytic organisms inhabiting water, and multiplying by means of zoospores and oospores as above described.

As respects the asexual mode of reproduction, by means of zoospores, all observers are now fairly in accord as to the main facts. With respect to the processes of incomplete segmentation preceding the formation and escape of the zoospores from the sporangium, it appears to be best explained as a phenomenon of nuclear division, in which the cell plate first formed becomes used up again. Büsgen,² who observed a similar process in several other cases, draws attention to Strasburger’s discovery,³ that in the development of pollen grains and spores it sometimes happens that a “primary cell plate” is first formed, and then disappears, as if its materials were used up again. This certainly appears to explain the phenomena of the division, &c., inside the sporangium of Saprolegnia; but why should the protoplasm make so many tentative efforts, so to speak, before once more growing out as a thallus ? Why should the protoplasmic masses, once having become zoospores, still hesitate (if the word may be permittted) before growing on, and, having rested awhile, again become zoospores, but of a different kind ?

It might be suggested that the entire series of phenomena should be connected and looked at in some such way as the following:

1st. The zoospore masses are formed, excreting a clear intercalary substance (primary cell wall of Strasburger), which they then take up again.

2nd. A more energetic separation follows, resulting in complete isolation, passage out, and removal to a distance. This active phase, though more energetic and lasting than the preceding, is in its turn superseded by a resting state, and the protoplasm excretes the substances for a membrane.

3rd. After the period of rest the protoplasm once more moves actively (having left its membrane behind) as a still more energetic zoospore—at least it moves for a longer period—.which in its turn comes to rest, but only for a short time prior to germination.

4th. It then, having formed certain brilliant granules and a cell wall, throws out a germinal tube at the expense of its contents; this soon dies if no proper matrix be at hand.

Unfortunately this restatement of the matter does not seem to help us. One can dimly see that the little protoplasmic zoospore undergoes processes of activity and rest—possibly partial exhaustion—and it is not absurd to conceive that something is gained by an active vacuolated stage.

In Achlya—the above applies to Saprolegnia¹—the first and second stages occur as before, only the second stage seems to be less energetic, and the amoeboid bodies only succeed in reaching the mouth of the sporangium. The third and fourth stages are much the same.

In the “Dictyuchus” form the second stage is still more abbreviated; it consists merely in complete isolation. The resting globules germinate in sitûin the zoosporangium.

That we are brought face to face here with a profound problem in its simpler forms is obvious. Perhaps the only light it affords us as yet is the suggestion once more of the exceedingly complex nature of the changes proceeding in the simplest piece of protoplasm. It appears somewhat significant that the second form of zoospore—the reniform one with lateral cilia—is that most constant. This is the only form in the nearest fungoid allies of the Saproleginæ, and must probably be regarded as the most ancestral form; nevertheless it is not easy to suggest how or why the other zoospore was acquired.

With respect to the “sexual reproductive organs” of this group, much has been written and many theories advanced since Alexander Brown and Pringsheim first described them and their relations. The antheridia were first believed to pour granular matter into the oogonium amongst the ova (oospheres). Then Pringsheim discovered that the oospheres in certain cases become normal oospores without the appearance of antheridia. Certain small antherozoid-like bodies were then believed to be set free and find their way into the oogonia amongst the oospheres. Meanwhile other observers denied that the “antheridia” either formed antherozoids, or that the tubes sent into the oogonium emptied anything into its cavity.

The discussion seems to have been somewhat in this state when Cornu,¹ in 1872, described the process of fertilisation, &c., as consisting neither in the formation and entry ot antherozoids, nor the emptying of granules between the oospheres, &c., but in the passage of protoplasmic contents from the antheridium through the fertilising tube and into the substance of the oospheres. This view has been accepted somewhat widely.

De Bary seems to have maintained for some years that, in some cases at least, no passage of material takes place through the tube—at any rate, not as protoplasm; but that the tube remains closed at the end, and never enters the substance of the oosphere; on the contrary, the antheridium either remains coiled round the oogonium, or the tube which it sends into the cavity simply touches or pushes the oospheres. He thus thought that the fertilising influence must pass through the closed walls of the tube which remains closed.

Pringsheim, in 1874,¹ again examined the question, and came to the conclusion that Cornu was wrong, and that where the tube comes in contact with the oosphere it remains quite distinct from it, however closely applied. Thus no slow passage over of protoplasm into the substance of the oosphere occurs, and hardly any, if any, contents of the antheridium disappear. Pringsheim further came to the conclusion that in some cases, since the oospheres become ripe oospores without any antheridial branches coming near them, the phenomenon must be considered one of parthenogenesis.

De Bary’s lately published views have been already referred to. He finds, after prolonged and exact researches, that not only does no observable passage of anything take place through the fertilisation tubes; not only does the naked oosphere clothe itself with a membrane (thus indicating that it no longer requires fertilisation) without the contact of the tube, but that normal, ripe oospores are produced habitually in some forms without an antheridium branch ever being formed at all. Such cases De Bary considers not “parthenogenetic,” in Pringsheim’s sense, but apogamous.

One more point may be shortly adverted to. It appears as said to be a constant phenomenon in certain forms, perhaps in all, that the masses of protoplasm forming the oospheres throw off smaller or larger portions of their substance during their amoeboid movements preceding their final rounding off as smooth oospheres; if these detached masses of protoplasm are to be regarded as of the nature of the “polar cell “observed to be thrown off by animal ova prior to fertilisation,¹may not the hypothesis thrown out by Balfour apply also to the cases observed by De Bary ?

De Bary shows that these protoplasmic bodies are taken up again, and that such oospheres as have again absorbed the thrown-off bodies, become ripe oospores, capable of germination after a period of rest without being fertilised. Balfour suggested that the “polar cells “are thrown off to prevent parthenogenesis, i. e. to prevent the egg dividing up and developing an embryo which has not benefited (in Darwin’s sense) by receiving protoplasm from a distance; the further development of the non-fertilised oospores (ova) of Saprolegniæ may be possible because the “polar cells” are again absorbed? Here, however, the proper limits of the present essay have been passed.