Maupas_1900

MODES AND FORMS OF REPRODUCTION OF NEMATODES. 51

caudal tip is thin, awl-shaped and rather short. The 9 papillae are arranged in groups of three: 1° a rather tight posterior group; 2° an equally tight median group; 3° a preanal group with first two neighboring papillae, then the last one, which is rather far apart in the front. Ventral and dorsal anal glands (pl. XVIII, fig. 7, B) are found at the junction point between the intestine and the vas deferens.

The spicules (pl. XVIII, fig. 7 and 8), of 55 to 60 μ in length, are colorless and vigorous. They end into a rather fine tip and thicken strongly towards the front. The gubernaculum (accessory piece), seen from the side, has the shape of a rod. It is relatively long, its length slightly exceeding half of that of the spicules. The latter are completely free and independent of each other.

The testis, built according to the usual type, does not offer anything noteworthy. The spermatozoa, whether from the females or from the males, are absolutely identical in their shape, structure and volume. Of spherical and slightly oval shape (pl. XVIII, fig. 10), they measure 8 μ. Their finely granular cytoplasm envelops a very apparent small nucleus of a somewhat irregular shape. I never observed the least sign of motility of these spermatozoa.

Rhabditis Marionis is essentially oviparous. It lays its eggs as they mature, often before their first cleavage, at the latest after the third or the fourth. Therefore, never more than five to six eggs can be seen at any time within each uterus.

In order to have an idea of the number of fertilized eggs that this species could lay, I isolated a female, which I am going to tell the whole story of, because of the important fact that she gave me the opportunity to witness for the first time.

This female was obtained from an egg that was isolated as it was just laid. The temperature during the course of its culture was of 20° C. Hatching occurred 20 hours after the laying. The well-fed young larva took three days and a half to grow until the laying of its first egg. Then, on the first day it laid 61 fertilized eggs, on the second 48 and on the third 20, total 129; afterwards it only laid sterile eggs that fell apart rapidly. It had completely exhausted its stock of sperm.

These numbers of 61, 48, 20 and this total of 129 were very low, compared to those that I had obtained in the other hermaphrodites. The explanation came to me with the following observation. Already on the first day, by observing the mother when she had just laid its three to four first fertile eggs, I noticed besides these the presence of an equal number of shell-less eggs that were falling apart, which had obviously not been fertilized. Moreover, on the next day, I found about thirty of these unfertilized eggs next to the 48 that were developing regularly. From then on, there was no possible doubt: I had a female of which one of the ovaries had functioned according to the hermaphroditic mode, first producing spermatozoa, then ova, whereas her other ovary had functioned according to the dioicious mode, only producing ova. We shall see below that this case is rather frequent in this species.

By doubling the number of fertilized eggs, an average figure of 250 to 260 is reached, which quite precisely corresponds to those obtained in the previous species.

After having laid her 20 last fertilized eggs, the mother still lived for 24 days, laying unfertilized eggs almost until the end. If the incubation period of the egg is taken into account, this female had lived for 31 days. This length is probably one of the longest that a Rhabditis Marionis can attain. For in another culture where I had

This proportion is an average figure for the whole set; but in some of the cultures it rose to 13 for 1,000 and in others to only 2 for 1,000. In any case, this proportion is three to four times larger than in the two previous species.

These males, like those of the two already described species, were of very good constitution in all of their parts. Nimble and agile, they circulated rapidly in the middle of the swarming of the females. Their seminal reservoir was full of spermatozoa that were absolutely identical to those of the females.

On these cultures, I also ascertained the frequency in this species of the partial and incomplete hermaphroditism mentioned above regarding the female that was isolated and reared apart. Indeed, in five of these cultures, I found from the first day on a rather large number of unfertilized eggs. Since all the animals in these abundantly provided cultures were very vigorous, these layings of sterile eggs could not be attributed to a withering state of some individuals. A partial hermaphroditism of some females must therefore be admitted, as we have demonstrated above for the isolated female. I even observed one in which both genital glands had come back to be producers of only ova. These facts are very interesting. They prove us that, in Rhabditis Marionis, hermaphroditism is not yet fixed in an absolute fashion and that it can totally disappear under the influence of unknown causes. We will actually have to return more than once to this point, regarding species in which we again will find this incomplete hermaphroditism under a more marked form.

As with the two previous species, I have tried to obtain heterogamous refertilizations, by gathering and isolating together males and females that had exhausted their own stock of sperm. Five

MODES AND FORMS OF REPRODUCTION OF NEMATODES. 55

experiments were thus instituted with a total of 28 females and 42 males. A single one, composed of 5 females and 2 males, gave an entirely negative result, none of the females having been refertilized, even though they had lived for seven days with their males. On the four other preparations, on the contrary, a great part, the majority I believe, of the females were refertilized and laid numerous wellconstituted eggs during the five to six days that they spent in the company of males. The number of unfertilized eggs, the presence of which was ascertained every day, indicated however that some of the females were never visited by the males, even though the latter were in almost two-fold number. In any case, the higher proportion of refertilizations proves that the reproductive sense of these males was less atrophied than in the previous species. This agrees with the also higher proportional number of males, and these two facts, with the frequency of monoic females, tend to clearly demonstrate that the hermaphroditism of Rhabditis Marionis is less developed and less complete than that of R. elegans and Caussaneli.

Juveniles that came from 150 to 200 eggs originating from heterogamous refertilization were kept and raised until the adult state. All became protandrous hermaphroditic females, which started to lay regularly developing eggs. The heterogamous fertilization thus had, in this Rhabditis, no arrhenotokous influence such as that ascertained in Rhabditis elegans.

RHABDITIS DUTHIERSI mihi

I found this species once, in a blackish soil sampled in the forests of the Edough, next to Bône. I brought it into culture on depression slides and let it multiply by feeding it rotten flesh. I dedicate it to my

illustrious teacher, Mr. de Lacaze-Duthiers, professor at the Sorbonne.

Measurements:

The body of the females (pl. XIX, fig. 1), cylindrical in its median part, narrows at both extremities. In the front, it is truncated by the mouth; in the rear, it thins out in a fine tip. The body of the males, as usual, is always shorter and thinner.

The cuticle is colorless, thick, highly birefringent and bears fine transverse striations. These striations, on living material, require a high magnification to be seen; but after incubation in 1 % acetic acid they become easily visible. I did not see lateral membranes. The cuticle is composed of two layers, which clearly separate under the action of 1 % acetic acid.

The mouth (pl. XIX, fig. 2) is not differentiated from the rest of the body by any narrowing nor protuberance of any sort. It is lined with three scarcely protruding lips, slightly serrated into two littledistinct lobes, each bearing a papilla; the latter thus numbering six around the full circumference. The buccal cavity has the shape of a perfect cylinder with thick walls. The posterior constriction and bulge are clearly outlined.

The esophagus (pl. XIX, fig. 3), modeled according to the usual type, is remarkable by the exceptional bulge of its two bulbs. The valves (teeth) of the posterior bulb are well developed and vigorous.

MODES AND FORMS OF REPRODUCTION OF NEMATODES. 57

In the front, the esophagus sheath envelops the buccal cavity, reaching upwards slightly beyond the posterior extremity of the first third of its length.

The tail of the females (pl. XIX, fig. 4) thins out rapidly and ends in a long and very thin tip. It is slightly flexuous and undulating. It bears a pair of fine lateral papillae (pl. XIX, fig. 4, p), inserted behind the anus at a distance slightly shorter than the length of the rectum.

The intestine, seen with transmitted light, has an opaque blackish aspect, caused by the many granulations stored in these cells. The length of the rectum equals the diameter of the body at the level of its anterior end. It is endowed, at this end, with dorsal and ventral glands, within which a large nucleolated nucleus can be seen.

The nerve ring (pl. XIX, fig. 3, c), of fibrous structure, collars the esophagus at the constricted neck between the two bulbs, bending over obliquely towards the ventral side and sending extensions into the direction of the excretory pore. The latter is located at the level (pl. XIX, fig. 3, p) of the posterior bulb. The unpaired canal is strongly chitinized. I have no observations concerning the lateral branches.

The vulva (pl. XIX, fig. 1, v), located slightly anteriorly to the middle of the whole body length, bears scarcely protruding lips. The vagina is very short. The paired genital tube is little developed. Its anterior and posterior bends terminate far away, on one side at the back of the esophagus, on the other side in front of the rectum. The very short uteri do not ever contain more than five or six eggs each. The oviduct or tuba also serves as a seminal receptacle. The germigen, which extends until the bend, contains 7 to 8 ovules. The

ovary, folded in the other direction, always ends slightly anteriorly to the level of the vulva.

The eggs (pl. XIX, fig . 5), of oblong shape and rounded at the extremities, measure 60 μ in length and 33 in width.

The male tail (pl. XIX, fig. 6, A, B) opens up in a wide bursa, opened in the front and with an elliptical contour at the back. It belongs to the leptoderan type, that is with a free caudal extension at the back. This extension has the shape of a thin awl-shaped tip of a length equal to approximately half that of the spicules. The nine papillae are arranged in four groups: 1° a rather tight posterior group; 2° a median group, also of three, which are less tight; 3° a group of two that are not very close; 4° finally a last isolated single papilla, far apart anteriorly, at the level of the anterior extremity of the spicules. Only the last three papillae are preanal.

The spicules (pl. XIX, fig. 6 and 7), of a slightly smoky color, are very strong, very thick and quite arched. They end with a rounded thick tip, bilobed, very characteristic for the species. The gubernaculum (accessory piece) is also vigorous and thick; its length almost equals half of that of the spicules. The latter are free and independent of each other.

The testis, built according to the ordinary type, does not offer anything peculiarly noteworthy. In all observed males, it appeared to me well constituted and the seminal reservoir was full of spermatozoa. These (pl. XVIII, fig. 12) are absolutely identical to each other, whether of masculine or of feminine origin. Of spherical shape, they are quite voluminous and measure 12 to 14 μ in diameter. Their finely granular cytoplasm envelops a small opaque nucleus, of a slightly irregular shape. When treated with acetic acid, this nucleus appeared to me composed of five small spherical

MODES AND FORMS OF REPRODUCTION OF NEMATODES. 59

chromosomes (pl. XVIII, fig. 13). I did not observe any sign of motility in the spermatozoa.

Rhabditis Duthiersi is oviparous. Its little developed uteri never contain more than 5 to 6 eggs and these are always laid at a rather early stage of development. It lays 200 to 210 fertilized eggs, after what, its stock of sperm being exhausted, it only lays unfertilized eggs. These sterile layings can last for 6 to 8 days and thus emit two to three times more sterile eggs than fertilized eggs. At a temperature of 20° C., it lays a daily average of 100 eggs.

The existence of this nematode is slightly slower and longer than that of Rhabditis elegans and R. Caussaneli. At a temperature of 20° C., its eggs take 24 to 26 hours to develop until hatching. The juveniles then grow for three days until the laying of the first egg. The fertile laying lasts two days to two and half days. Thereafter, the sterile layings can last for six to eight days, still followed by three to four days of absolute sterility, leading to death. The total length is thus of twenty to twenty-three days.

The movements of Rhabditis Duthiersi are slow and heavy. It wriggles and moves very little.

This Rhabditis is close to Rhabditis elegans and R. Marionis. It can be distinguished from the former by its longer esophagus and tail, by its cylindrical buccal cavity, by its oviparity and above all by its leptoderan bursa, with longer and stronger spicules. The similarity with the latter is closer. It can be however easily distinguished by the much longer female tail, by the male caudal papillae, the last of which is moved further forwards, and above all by its spicules, shorter but more thickset and ending in a round bilobed tip.

As for the previous species, I wanted to ascertain the frequency of males using cultures that were organized and studied with the method described for Rhabditis elegans. These four cultures gave me a total of 1,279 females and 37 males; thus 20 males for 1,000 females. This proportion is an average drawn from the total; but in one of the cultures, it rose to 35 for 1,000, whereas in another, composed of 461 individuals, I did not find a single male. The general proportion is much greater than those found in the previous species.

I observed three females, which, from the beginning of their layings, simultaneously laid fertilized eggs that developed normally, and sterile shell-less eggs that were falling apart. They continued in this manner for three days, then only laid sterile eggs. It is obvious that these three females had formed spermatozoa in one of their genital glands only and that I had there cases of partial or incomplete hermaphroditism, similar to those that we already observed in Rhabditis Marionis.

I tried to obtain in Rhabditis Duthiersi heterogamous fertilizations with females that had exhausted their own stock of sperm. Three series were organized, in which 62 females and 41 males were held together during 3, 4 and 6 days, respectively. Only in the first series did a useful and fertilizing mating occur. Indeed, for three days I found in this preparation layings of fertilized eggs that hatched and gave birth to regularly developing juveniles. When at the adult stage, these juveniles gave 70 females and 1 male. It must be concluded that the fertilization through heterogamy did not have any determining action on the sexuality of its products, in contrast to what we had observed in Rhabditis elegans.