The Development of Amphibola Crenata (Martyn)

The Development of Amphibola crenata (Martyn).

By Winifred Clieyne Faruie, M.A., Geraldine, New Zealand.

With 13 Text-figures.

IN 1919 I gave an account of the general anatomy of this interesting New Zealand littoral Pulmonate, and in the course of that account showed that the genital duct is single ; it extends from ovotestis to genital pore in one undivided canal; in other words the different structures identified by Quoy and Gaimard, and by Hutton respectively, as oviduct do not exist.1 At the time when that article was published I was not in a position to state how the eggs were conveyed to the exterior, and proposed to gather material month by month till the date of egg-laying was determined and their method of passage was ascertained. As a result of numerous observations extending over two years I am now able to satisfy myself on these two points : (1) That the egg-cells descend by way of the hermaphrodite duct through the coiled non-glandular portion of the common duct. x On p. 84 I wrote : ' Sections across the right side of the body show no trace of a duct between the rectum and the genital duct, whereas sections across the genital duct itself show the existence of a deep fold in its wall, which serves to divide the duct into two portions, presumably, during the passage of the ova and spermatozoa.' I cut serial sections again across the same region when the animal was engaged in laying eggs, but with the same negative result: I can, therefore, only come to the conclusion that a distinct and separate oviduct does not exist, though unfortunately I was not able to detect ova actually in the lower region of the common duct. 454 WINIFRED CHEYNE FARNIE

(2) That the eggs, embedded in an albuminous coat and an egg-membrane or shell, leave the body by the genital pore below the right tentacle. But unfortunately I have been unable to trace their descent along the lower portion of the common duct, or to find the place of formation of the egg-membrane or shell. The series of observations had to come to an end, for I left Dunedin in order to take up a position in an inland school. f The work was carried out in the Biological Laboratory of the University of Otago, and I owe my thanks to Professor Benham for the interest he took in my work and for many helpful suggestions. SUMMARY. The development was traced out day by day, though details of later steps of cleavage and gastrulation were not studied : the formation of the trochosphere and veliger were, however, carefully followed out. Unlike what occurs in the majority of Pulmonates, the veliger stage is typically and well developed ; the trochosphere phase, on the contrary, is somewhat abbre- viated. The shell-gland is not formed by an invagination, but is represented merely by a thickening of the epidermis. The veliger escapes from the egg-mass by the action of the operculmn, the edge of which is used to bore through the egg- shell and surrounding jelly. It leads a free life ; but I was unable to keep the larva in captivity for more than one day after hatching. During this time it swims actively, coming to rest from time to time with its velar lobes downwards.

1. THE EGG. So far as I can ascertain nothing is known as to the development of Amphibola. For two years I made a series of monthly observations to discover at what period the discharge of the eggs occurs and where development takes place. The ova are fully formed in the hermaphrodite gland at the beginning of November, two or three weeks after the first DEVELOPMENT OF AMPHIBOLA 455 appearance of fully developed spermatozoa. After being discharged into the hermaphrodite duct they remain a short while in the slight enlargement at the junction of the seminal vesicle and the main duct. Self-fertilization probably takes place here as the seminal vesicle at this period contains live spermatozoa in active motion. I have found the ova passing through the coiled glandular portion of the common duct into the non-glandular portion. In the pouch at the commencement of the non-glandular portion, which contains secretion from the albumen gland, albumen is deposited around each egg. Unfortunately I have been unable to trace the course of the ova any further in the animal, and it is only on two

TEXT-FIO. l.

Egg-nidus (natural size). occasions out of many scores of observations that I have been able to trace their course as far as the non-glandular portion of the common duct. On one of these occasions, from a microscopical preparation of a freshly dissected specimen in salt solution, I observed several ova passing along the glandular portion of the common duct, during which the ova were con- stricted as they were forced round the several coils. This duct is thickly ciliated, so that, though it is extremely con- voluted, the ova did not take long to pass along it. I have been unable to ascertain where the shell is deposited. On November 3, 1919, I picked up on the mud-flats a ribbon- shaped, concavo-convex mass of sand particles (Text-fig. 1) lying quite by itself, the concave surface being next to the ground. It was an egg-nidus, for examination proved it to 456 WINIFRED CHEYNE FARNIE

contain masses of eggs embedded amongst sand and particles of mud held together by gelatinous material. The band is about 80 mm. in length and about 10 mm. across ; it is curved in itself, so as to describe rather more than a circle, one end over-passing the other. In other cases the two ends just meet. It is similar in appearance and structure to that of Natica,

TEXT-PIG. 2.

View of an egg with its albumen, shell-membrane, and gelatinous envelope. (Much enlarged.)

EXPLANATION OF TEXT-FIGURES 2-13. The following lettering is used throughout, alb., albumen of egg ; b., blastopore ; b.c, bodj'-cavity of larva ; b.w., body-wall; e.g., cerebral ganglion ; d.gl., digestive gland ; ent., enteron; /., foot; g., groove in mantle indicating the probable position of anus ; g.e., gelatinous envelope ; int., intestine ; k., kidney; k.c, central cell of kidney ; k.d., kidney duct; m., mouth; mac, macromeres ; m.e., mantle edge ; roic, micromeres ; m.s., mesoblastic strands ; n., nucleus of ovum or its position ; o., ovum ; oe., oesophagus ; op., operculum ; ot., otocyst ; pig.c, pigment cells in velum ; pig.in., pigment mass, perhaps repre- senting the future tentacles ; p.g-, pedal ganglia ; sh., shell of larva ; sJi.gl., shell-gland ; sh.m., shell-membrane of egg ; St., stomach ; v., velum ; vis., visceral mass of larva ; v.m., velar muscle ; y., yolk-granules.

but that of the latter is considerably larger, and is found in clear sand facing the open sea. The only Gastropoda found on these rnud-flats were Arnphibola, Patella, Siphonaria, Oncidiuni, and Chiton. It is known that Patella lays its eggs in masses surrounded by jelly, during September and October. DEVELOPMENT OF AMPHIBOLA 457

Siphonaria forms clear, gelatinous, ribbon-like masses with several eggs in each capsule. Oncidium masses its eggs in numbers to form gelatinous balls, and Chiton lays its eggs freely in water and the egg- shell is spiny. Since the nidus agreed with none of these descriptions as- given by Korschelt and Heider (1900) I concluded it must be that of Amphibola. Up till this time I had not traced the course of the ova through the common duct, and had no idea what the fully formed ova were like. Later I found similar egg-ribbons in association with Amphibola in such positions as to lead to the conclusion that the animals had just formed them. The chances were that I might find ova still in the common duct of such specimens, and thus be able to discover where the shell was deposited ; but as no ova were visible in the duct on examination, I concluded that the animals had either not formed these nidi or that they had deposited all their eggs. On every visit made in the forenoon, during the month of November, crowds of Amphibola were seen each close to the end of such an egg-ribbon as if it had recently been formed by the gastropod. But during the afternoon I found no animal in such positions, and as, during the morning, they were associated with none but fully formed nidi, I concluded that the eggs must be deposited during the very early hours of the morning only. Judging from the length of the nidus we would expect the eggs at either end to be in different stages of cleavage, but in only a few cases was this so ; for instance some of the eggs exhibited division into two, but most were still unseg- mented. This seems to agree with Korschelt and Heider's (1900) statement for Pulmonates that cleavage begins two hours after the egg is laid. On November 23, three weeks after examining the first nidus, a microscopical preparation of the glandular portion of the common duct of one specimen showed ova passing along it as described above, and as these ova resembled those already NO. 271 H h 458 WINIFRED CHEYNE FARNIE

seen in the nidi I believed that I had conclusive evidence for my original opinion. This was confirmed on November 24, when I actually saw one Amphibola form a nidus in the laboratory in a dish of sea-water and sand. I noticed the animal with the right side of the head-region curved under, and clear jelly-like material issuing from beneath the right tentacle. Closer examination showed that the animal was laying eggs, but it had not yet begun to mould sand particles round them. The animal had the foot bent to form a channel, and was moving it as if to keep the sand together. The eggs appeared as numerous white specks about the size of a pin- point, and were visible only on account of their numbers. Examined under the microscope, the ova, which measured Ts mm. in diameter, appeared almost wholly black except for a small clear patch at one end. This I take to be the position of the nucleus (n, Text-fig. 2), as it is conspicuous in egg-cells still in the hermaphrodite gland. The clear albumen round the egg was enclosed in a thin oval shell-membrane (sh.m., Text- fig. 2), which in its turn was surrounded by a spherical gelatinous envelope (g.e., Text-fig. 2). It is not easy to release the eggs, as, usually, when they are teased out of the nidi, the gelatinous envelope is ruptured by coming into contact with the particles of sand. On killing this specimen I found the hermaphrodite gland to contain both ova and ripe spermatozoa. The hermaphrodite duct was filled with ova to the commencement of the common duct, but there were none in the non-glandular portion of the common duct nor in the penis. Thus it appears as if batches of eggs pass down the duct at intervals. I have been unable to obtain sections of the eggs ; for in the first place the eggs are so minute that it is impossible to remove the albumen, and, secondly, embedded as they are amongst particles of sand and mud, it is quite impossible to get microtome sections. I have tried killing with corrosive sublimate, glacial acetic acid, and with Carnoy's acetic alcohol, and staining with DEVELOPMENT OF AMPHIfiOLA 459 borax carmine. But in all eases too much shrinkage takes place, and the albumen stains too deeply to allow any structures to be studied. I have also tried osmic acid solution, 1 per cent, as tried by Lankester (1874), but without good results. I have tried Macbride's (1914) method of killing with corrosive sublimate, and treating with drops of glycerine, but though occasionally useful it usually made the specimens too transparent.

TEXT-FIG. 3. TEXT-FIG. 4. TEXT-FIG. 5.

Fig. 3.—Gastrula stage of the sixth day, from below. Kg. 4.—Gastrula, a few hours later than that figured above. Fig. 5.—Gastrula of the ninth day, showing the closure of the blastopore. Surface view.

Most of the illustrations are made from living specimens examined in salt solution. By keeping the nidi sometimes wholly immersed in, and sometimes just damp with, sea-water I was able to keep embryos alive from the time of laying up till the free-swimming stage, but of course on account of their minute size I could not examine the same embryos from day to day. However, as nearly all embryos are in the same stage in the same nidi, the stages of development could be watched daily. I was unable to keep them alive more than one clay after they became free swimming, so that I do not know how long it is before the larvae settle down and assume the final form.

Hh2 460 WINIFRED CHBYNE FARNIE

2. CLEAVAGE. The egg divides into two, and then into four by the end of the second day. The following day the egg shows four small micromeres cut off from four large macromeres. On only one occasion did I see any further stages of division. This was a sixteen-celled stage viewed from the vegetative pole. It showed eight dark macromeres and eight light-coloured micromeres at the back. Usually division proceeded rapidly during the night from the eight-celled stage, so that on the following

TEXT-FIG. 6. TEXT-PIG. 7.

sh.gl Fig. 6.—Early trochosphere at the ninth day. The blastopore is closing, the foot and velum are differentiated. Pig. 7.—Trochosphere on the tenth day, in side view. Cilia have appeared on the velum and on the foot; the shell has been formed over the shell-gland ; the enteron is seen surrounding a mass of yolk-granules. day the gastrula stage was reached in specimens under observa- tion. Text-figs. 3 and 4 show the gastrula stage, which has evidently been formed by the overgrowth of clear, granular micromeres (mic). The macromeres (mac.) appear as dark granular cells surrounding the blastopore (b.), which by focusing can be seen to lead into the archenteric cavity. This stage is reached by the sixth day. On the ninth day a further change is visible. The blastopore closes up as is shown in surface view (Text-fig. 5). The embryo undergoes a change of shape (Text-fig. 6) due to the develop- DEVELOPMENT OF AMPHIBOLA 461 ment of two swellings which are the first signs of the foot (/, Text-fig. 6) and the velum (v, Text-fig. 6). No details of internal anatomy can be seen, for the interior appears as a dark visceral mass (vis.) with a space all round. This is the early trochosphere stage.

3. THE LARVA. On the tenth day the trochosphere is seen to be moving slowly within the shell-membrane, the movement to and fro

TEXT-FIG. 8.

Trochosphere of the twelfth day : side view. The velum is now heavily ciliated. being due to the development on the foot of cilia (Text-fig. 7) which are plainly visible. Closer examination shows a patch of four long cilia on each side of the rounded edge of the velum. The whole of the outer layer of the trochosphere consists of numerous small, clear cells. The visceral mass (vis.) appears to be filled with yolk-granules (y.), which gives it a darker appearance than the rest. In only one specimen was I able to detect a trace of an alimentary canal. This consists of a curved tube (ent.) which appears to commence between the velum and the foot, and runs backwards to the hinder end of the body, where it takes an abrupt turn and ends near the hinder portion of the foot. Between these two limbs of the alimentary canal is a mass of yolk-granules. In all trochospheres examined the body-cavity (b.c.) was visible as the embryo rotated. Sometimes 462 WINIFRED CHEYNE FARNIE only a small space was visible between the outer layer and visceral mass, but at other times the space extended completely round the visceral mass (as shown in this Text-fig. 7). Neither mouth nor otocyst was visible at this stage. What I take to be the shell-gland is a thickening of ectoderm opposite the velum (sh.gl., Text-fig. 7), outside which is the larval shell. Korschelt and Heider (1900) say that in Pulmonata the shell-gland arises on an ectodermic invagination on the dorsal surface, and that it sinks in very deeply but; later flattens out again. Lankester observed the same in the development of Limnaea. I have looked carefully at all stages between the first appearance of the velum and the fully developed trochosphere for any such invagination but I have failed to find any; and have always seen the thickening as described above. On the twelfth day the trochosphere is fully developed, and, instead of moving only to and fro, it rotates rapidly with a rolling movement upon its long axis. This movement is due to the great increase in the size of the velum and of the velar cilia (Text-fig. 8). The velum is now a large prominent structure, larger than the foot. The velar.cilia which are present all round the margin are plainly visible under the lower power of the microscope, those at the sides being long and in constant motion with a whip-like action (v.c, Text-fig. 8). The shell (sh., Text-fig. 8) has appeared as a small, thin, transparent cap over the posterior end of the embryo opposite to the velum, in the same position as the shell-gland appeared to be in the early trochosphere stages. The velum does not appear as a ridge, as described by Lankester (1874) in Limnaea. Prom the first it is always a prominent swelling. There is no trace at this stage of a mantle fold. On the fourteenth day the embryo assumes the true veliger stage (Text-fig. 9). The velum is fully developed, is distinctly bilobed, and is interrupted ventrally. The foot occupies the same relative position, but the cilia have increased in length. DEVELOPMENT OF AMPHIBOLY 463

As the embryo rotates a ciliated opening (m.) with a V-shaped lower lip is apparent between the velum and the foot. This is

Early veliger, of the fourteenth day, viewed from above. The velum is now bilobed and groups of pigment have appeared on the edge ; the mouth, cerebral ganglia, and other internal organs have become differentiated.

TEXT-FIG. 10.

Veliger of the fifteenth day, in side view. The mantle has grown down and the shell is.well developed. the mouth. On either side of the mouth, lying deep within the foot, is a pair of refringent circular patches, the otocysts (ot.). 464 WINIFRED CHEYNE FAENIE Along the margin of the velum pigmented cells have become differentiated (pig.c). They may represent the ectodermal excretory cells of Oncidium as described by Korschelt and Heider after Joyeux Laffuie, but I have found no evidence of their excretory functions. The cerebral ganglion (e.g.) is a transversely oval mass, just

TEXT-FIG. 11.

Veliger of the nineteenth day, in side view : showing all that can be seen of its anatomy by transparency in the living larva. The various regions of the enteron are defined; the kidney has appeared and other structures. above the gap in the velum, and on either side is a patch of pigment which may represent the tentacles (pig.m.). These two structures are visible only when the animal is in such a position as to be examined directly from above. A side view of the veliger is shown on Text-fig. 10. On the hinder upper surface of the foot the operculum has developed (op.), and as the animal rotates this is seen as a flat plate pro- jecting beyond the margin of the foot posteriorly. The presence of this operculum again emphasizes the fact that these eggs cannot be those of Oncidium, for Joyeux DEVELOPMENT OF AMPHIBOLA 465 Laffuie (Korschelt and Heider, 1900) says that no operculum is present in the larval stages of that mollusc. The visceral mass (vis.) appears as a lobed structure filled with yolk-granules, but no details as to stomach and intestine can yet be determined. A thin shell (sh.) is present surrounding the embryo as far as the velum, and both shell and visceral portion of the embryo show beginnings of a coil. The body- wall is seen as a thin membrane lining the inside of the shell, and is separated from the visceral mass of the body-cavity which is traversed by strands of tissue (m.s.). Lang (1900) calls these strands ' mesodermal strands ' in his figure of 0 n c i d i u m. He also figures muscles in the velar lobes, but I have seen none in Amphibola at this stage. The mantle edge (m.e.) can be seen lining the anterior margin of the shell. I have been unsuccessful in ascertaining when the mantle fold develops. Lankester (1874) traces its development in L i m n a e a to the early trochosphere stage, but I have seen nothing to correspond to this description, probably on account of the opacity of Amphibola embryo. Lankester finds that the foot in Limnaea arises first as an unpaired swelling and later assumes a distinctly bilobed form. Korschelt and Heider (1900) affirm that this is the usual mode of development in Pulmonata. In Amphibola, however, the foot does not become bilobed at any period of development. By the nineteenth day the larva is fully grown and almost fills its case (Text-fig. 11). The velum is very flexible and con- tractile, its contractility being due to the presence of a stout velar muscle (v.m.) which stretches from the velar lobes to its attachment to the shell posteriorly. The mouth leads into a long oesophagus (oe., Text-fig. 11) which about half-way down the visceral portion of the embryo turns on itself to form a U-shaped tube, probably the stomach (si.). Lying below this, and, as far as I can ascertain, in com- munication with it, is a large sac-like structure filled with granules of albumen. This portion probably represents the digestive gland (d.gl.), as in one specimen I later observed 466 WINIFRED CHEYNE FARN1E a similar structure becoming lobed (Text-fig. 12). Leading away from the liver, close to the entrance of the stomach, is

TEXT-FICI. 12.

dig.gl.

Veliger of the same date in a latero-ventral view. This is drawn from a larva mounted in glycerine and shows fewer details than the previous figure, but the course of the kidney is better seen.

TEXT-MG. 13.

Veliger on the twenty-first day, ready to break through the shell- membrane and albumen by means of its operculum and thus to enter on its free-living career. DEVELOPMENT OF AMPHIBOLA 467 a long tube (int.) which crosses to left side, turns on itself, crosses above the rest of the alimentary canal to the right side, and ends near the bottom of a depression (g.) at the edge of the mantle. This depression probably corresponds to the triangular groove as figured by me in the adult animal (Parnie, 1919). I have not observed any anus in this depression. In fresh specimens examined in salt solution, the alimentary canal was seen to be lined by cilia, and globules of albumen were driven along by their movement. In all accounts of development of the alimentary canal in Pulmonata, the larger cells of the nutritive masses have been observed to be dorsal and the smaller cells ventral. In Amphibola I have observed the reverse to be the case, though the relative positions of stomach and liver seem to follow the position in typical forms. The only representative of a kidney I can find is a pair of tubes situated just behind the mantle edge on either side of the oesophagus (Text-fig. 11, k.). In fresh specimens it seems to consist of a central rounded body at the posterior end, clear in the centre and with granules lining the edge. Leading away from this body and passing forwards to the velum is a ciliated duct (k.d.) in which the movement of the cilia is plainly visible under the high power. When the specimen was killed with acetic acid, and made transparent in glycerine, and the larva viewed rather more from the ventral surface than laterally (Text-fig. 12), the kidney appeared as a U-shaped tube of cells with one big curved cell (k.c.) at the base and two cells in either limb. The portion at the base appears round in the fresh state, probably on account of the tube turning on itself to form the other limb, which is not visible as the animal rotates rapidly. Neither in the fresh specimen nor in the transparent one can I find any opening into the body-cavity. The external aperture appears to be on the velum. The primitive kidney, it is stated, is visible in early trochosphere forms, but the first appearance in Amphibola is in the fully developed veliger. The form of kidney presented by Amphibola agrees 468 WINIFRED CHEYNE FARNIB with the description in Korschelt and Heider, except that they find the inner end opening by ciliated aperture into the body-cavity. I can still find no trace of tentacles or eyes unless the pigmented masses (pig.m., Text-fig. 9) mentioned above could be regarded as such. The late development of tentacles is not surprising as they are very feebly developed in the adult, and the eye is practically functionless. In Text-fig. 12 I have shown anterior to the otocysts two thickenings which may represent the pedal ganglia. I have seen these only in specimens made transparent in glycerine. Korschelt and Heider (1910) figure the pedal ganglia in this position. A day or two after the fully developed veliger stage the larva rotates rapidly within its membrane (Text-fig. 13). The velum has decreased in size, while the foot has increased. The albumen shows signs of shrinkage and becomes crumpled (alb., Text-fig. 13). The operculum extends to the outer margin of the albumen, and as the animal rotates rapidly the operculum breaks through the shell-membrane and the larva is set free about one-quarter of an hour later. In all cases where the freeing of the larva was observed the gelatinous envelope had been ruptured in mounting the specimens. When free the larvae swim rapidly, but occasionally come to rest with the two velar lobes downward. Lankester (1874) says that the full development of Limnaea takes from twenty days upwards. Amphibola agrees with this, for I have found that from twenty-one to twenty-four days are necessary before the embryo is ready to become free swimming. As a general rule the trochosphere stage of Pulmonates seems to be feebly developed, but as will be seen from the fore- going description this stage in Amphibola is well developed and remarkably active. In this respect it resembles the trochosphere of 0 n c i d i u m. The trochosphere of Limnaea as figured by Lankester (1874) and MacBride (1914) resembles that of Amphibola except that Limnaea shows the DEVELOPMENT OF AMPHIBOLA 469 presence of a primitive kidney, an invaginated shell-gland, and an anus. The veliger stage in Pulmonates is usually passed through quickly, but that of Amphibola is very persistent. Although it resembles that of Limnaea it seems to be more closely allied to that of an Opisthobranch, the relationship being strengthened by the presence of an operculum. Auricula (as described by Korschelt and Heider, 1900) seems to have a somewhat similar veliger stage with an operculum, and this larva swims freely in the sea. From the comparison of the anatomy of Auricula with that of Amphibola, I came to the conclusion that the two are closely allied, and the study of the embryology seems to strengthen this view.

BIBLIOGRAPHY. Farnie, W. C. (1919).—"The Anatomy of Amphibola crenata ", ' Trans. N.Z. Inst.', vol. 51. Korschelt and Heider (1900).—' Text-book of Embryology', Part IV. Invertebrata. Lankester, E. R. (1874).—" Development of Limnaea stagnalis ", ' Quart. Journ. Micr. Soi.', vol. 14. MoBride (1914).—' Text-book of Embryology ', vol. 1. Pelseneer (1906).—" Mollusca " in Lankester's ' Treatise on Zoology'.