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HARVARD UNIVERSITY.
LIBRARY
OF THE
MUSEUM OF COMPARATIVE ZOOLOGY. M1524 GIFT OF i
ALEX. AGASSIZ.
aes au 396 Noanehe tt Isq¢
QUARTERLY JOURNAL
OF
MICROSCOPICAL SCIENCE.
EDITED BY
E. RAY LANKESTER, M.A., LL.D., F.R.S.,
DIRECTOR OF THE NATURAL HISTORY DEPARTMENTS OF THE BRITISH MUSEUM; HONORARY FELLOW OF EXETER COLLEGE OXFORD ; CORRESPONDING MEMBER OF THE IMPERIAL ACADEMY OF SCIENCES OF ST. PETERSBURG, AND OF THE ACADEMY OF SCIENCES OF PHILADELPHIA; FOREIGN MEMBER OF THE ROYAL BOHEMIAN SOCIFRTY OF SCIENCES, AND UF THE ACADEMY OF THE LINCEI OF ROME; ASSOCIATE OF THE ROYAL ACADEMY Or BELGIUM; HONORARY MEMBER OF THE NEW YORK ACADEMY OF SCIENCES, AND OF THE CAMBRIDGE PHILOSOPHICAL SOCIETY AND OF THE ROYAL PHYSICAL SOCIETY OF EDINBURGH} ASSOCIATE MEMBER OF THE BIOLOGICAL SOCIETY OF PARIS} FULLERIAN PROFESSOR OF PHYSIOLOGY IN THE ROYAL INSTITUTION OF LONDON.
WITH THE CO-OPERATION OF
ADAM SEDGWICK, M.A., F.R.S.,
FELLOW AND TUTOR OF TRINITY COLLEGE, CAMBRIDGE 3
AND
Wer Re WEEDON, MA, PRS;
JODRELL PROFESSOR OF ZOOLOGY AND COMPARATIVE ANATOMY IN UNIVERSITY COLLEGE, LONDON; LATE ’ FELLOW OF ST. JOHN S COLLEGE, CAMBRIDGE.
VOLUME 41.—New Sertes. With Aithographic Plates and Engrabings on Wood.
LONDON: J. & A. CHURCHILL, 7, GREAT MARLBOROUGH STREET. 1899,
CONTENTS.
CONTENTS OF No. 161, N.S., MARCH, 1898. MEMOIRS :
The Habits and Structure of Arenicola marina. By F. W. GamBLE, M.Sc., and J. H. Asuwortu, B.Sc., Demonstrators and Assistant Lecturers in Zoology, Owens College, Manchester. (With Plates 1—5)
The Aseptic Cultivation of Mycetozoa. By Casper O. Mituzr, M.D. (With Plates 6 and 7)
On the Development of Tubulipora, and on some British and Northern Species of thisGenus. By Stpnsny F. Harmer, Se.D., Fellow of King’s College, Cambridge ; Superintendent of the University Museum of Zoology. (With Plates 8—10) .
The Molluses of the Great African Lakes.—I. Distribution. By J. E.8. Moore . : ;
The Molluses of the Great African Lakes.—II. The Anatomy of the Typhobias, with a description of the New Genus Bathanalia. By J. E.S. Moorz. (With Plates 11—14)
CONTENTS OF No. 162, N.S., JUNE, 1898. MEMOIRS:
The Segmentation of the Ovum of the Sheep, with Observations on the Hypothesis of a Hypoblastie Origin for the Trophoblast. By Ricuarp AssHEeTon, M.A. (With Plates 15—18)
On the Heart-body and Ceelomic Fluid of Certain Polychaeta. By Lioness James Picton, B.A. (With Plates 19—22)
PAGE
43
73
159
181
205
263
iv CONTEN''S.
On the Hypothesis that, Lake Tanganyika represents an Old Jurassic Sea. By J. HE. S. Moors. (With Plate 23)
On the Reno-pericardial Canals in Patella. By Epwin 8. Goop- ricH, B.A., Aldrichian Demonstrator of Comparative Anatomy, Oxford. (With Plate 24)
CONTENTS OF No. 163, N.S., NOVEMBER, 1898.
MEMOIRS:
The Development of the Pig during the First Ten Days. By Ricuarp AssHeton, M.A. (With Plates 25—28) ‘
The Structure of the Mammalian Gastric Glands. By R. R. Buns ey, B.A., M.B., Assistant Demonstrator in ee Uni- versity of Mornin, (With Plate 29) ‘
On Certain Green (Chlorophylloid) Pigments in Invertebrates. By Marion I. Newsiein, D.Se., Lecturer on Zoology in the Kdinburgh College of Medicine for Women. (Krom the Laboratory of the Royal College of ee meu (With Plates 30 and 31) x ; :
Note on a (? Stomatopod) ee Larva. _ J. J. Lister, M.A., Demonstrator of Comparative Anatomy in the University of Cambridge
On the Nephridia of the Polychata.—Part II. Glycera and Goniada. By Epwiy 8. Goopricu, B.A., Aldrichian Demonstrator of Comparative Anatomy, Oxford. (With Plates 32—35) .
CONTENTS OF No. 164, N.S., JANUARY, 1899. MEMOIRS :
On Differences in the Histological Structure of Teeth occurring within a Single Family—the Gadide. By Cuartes 8. Tomes, M.A., F.R.S. (With Plate 36) .
A Description of ''wo New Species of Spongilla from Lake Tanganyika. By Ricuarp Evans, B.A. (With Plates 37 and 388) . 3 ; i . :
PAGE
303
323
329
361
391
433
439
459
471
CONTENTS.
On Tetracotyle petromyzontis, a Parasite of the Brain of Ammoccetes. By Atzert W. Browy, B.A., F.L.S., formerly Exhibitioner of Christ Church, Oxford. (With Plate 39)
Studies on the Structure and Formation of the Caleareous Skeleton of the Anthozoa. By Gitzert C. Bourne, M.A., F.L.S., Fellow and Tutor of New College, Oxford; University Lecturer in Comparative Anatomy. (With Plates 40—43)
The Structure and Development of the Hairs of Monotremes and Marsupials.—Part I. Monotremes. By Batpwin Spencer, M.A., Professor of Biology in the University of Melbourne, and Grorcina Sweet, M.Sc., University of Melbourne. (With Plates 44—46)
Trophoblast and Serosa; a contribution to the Morphology of the Embryonic Membranes of Insects. By ArtHur WILLEY, D.Sc.Lond., Hon. M.A. Cantab., Balfour Student of the Uni- versity of Cambridge
TirLe, INDEX, AND ConvTENTS.
Vv
PAGE
489
499
549
589
APR 27 1898
The Habits and Structure of Arenicola marina.
By
F. W. Gamble, M.Sc., and
J. H. Ashworth, B.Sc.,
Demonstrators and Assistant Lecturers in Zoology, Owens College, Manchester.
With Plates 1—5.
ConTENTS. - PAGE PAGE 1. Distribution: Varieties : 7. Gills : ; 3 wee Habits . : : : 1 | 8. Nervous System and Sense- 2. External Features: Seg- | organs . : : x Ok mentation: Skin: Sete. 5 9. Nephridia MAD ae ek: 3. General Anatomy of the | 10. Celom. : : 2. 26 Internal Organs. ; 9 | 11. Reproductive Organs -; =O 4. Musculature . : . 10 12. General Summary . . 34 5. Alimentary Canal . . 12 = 18. Literature pee 27 6. Vascular System. a a le |
1. Distribution: Varieties: Habits.
Tue common lugworm and its coiled castings of sand are familiar objects on almost all the sandy and muddy shores of Western Europe, but the exact geographical range of the species is doubtful. It has been recorded from the shores of North Siberia, Spitzbergen, Iceland, and Greenland (Wirén, 1883; Levinsen, 1883). On the north-east coast of America it has been found from the Bay of Fundy to Long Island
vou. 41, PART 1.—NEW SER. A
2 F. W. GAMBLE AND J. H. ASHWORTH.
(Verrill, 1881). On both sides of the Atlantic, latitude 40° N. marks approximately the southern limit of Arenicola marina. South of this it is replaced in the Mediterranean by A. Claparédii, Lev., and by A. cristata, Stimps., the latter also ranging on the west side of the Atlantic from Cape May (N. J.) to the Caribbean Sea. Its reputed occurrence on the north coast of Alaska (Murdoch!), at Vancouver Island (Marenzeller, 1887), Coquimbo, and South Africa requires confirmation.
An abundant, widely ranging, and undoubtedly old form such as Arenicola, might be expected to vary considerably in its habits and structure, though it has not hitherto been ascertained how far this is the case. Having paid special attention to this point, we have found that there are (at least on the Lancashire coast) two varieties of A. marina, differ- ing in habits, structure, and times of maturity, and that there is, in addition, considerable individual variability.
(1) From high-water mark down to the beginning of the Laminarian zone, the common shore lugworms (or “ lugs,” as fishermen call them, in contradistinction to the second variety, or “‘ worms”) sink their U-shaped burrows to a depth of from one to two feet below the surface. One end of the burrow is marked by a casting, the other by a “countersunk”’ hole, through which the head of the lugworm is protruded when the tide comes in. The size and colour of the animal vary with the amount of muddy organic matter in the sand. Where there is comparatively little mud, the Arenicola average about seven inches in length and are somewhat transparent, so that the superficial blood-vessels can be clearly seen through the thin body-wall. The gills, which are not very strongly developed, are composed of nine to eleven branches, each pro- vided with three to five pairs of short lateral twigs (Pl. 1, fig. 3). The proboscis and prostomium are only slightly pigmented, and being very vascular, appear red in colour.
Where, however, the amount of organic matter is consider- able, the worms are usually about ten inches long, and their
1 * Proc. U. S. Nat. Museum,’ Washington, vol. vii, 1884, p. 522.
HABITS AND STRUCTURE OF ARENICOLA MARINA. 3
prostomium, proboscis, gills, and epidermis are black. The gills are better developed than those of worms living in purer sand. These differences are probably due to more abundant nutrition. The time of maturity of both these forms of the littoral variety on the Lancashire coast is the summer, while at St. Andrews they are found mature from February to September.
(2) The second variety occurs on the Lancashire coast at the upper part of the Laminarian zone. Almost all the Arenicola from this zone (which can accordingly be obtained only at low spring-tides) are of this kind, which when fully mature, as it is from February to May, is probably one of the largest Polychets of our shores, measuring as much as fifteen inches in length and three in girth. It is almost black, the prostomium, proboscis, and the base of the gills being markedly so. The tail is shorter in proportion to the length of the body than in the littoral variety. The burrows are of con- siderable length, three feet or more, and are not U-shaped, but simply vertical. Like those of the littoral variety, they are lined by a greenish coating of mucus. The dark ‘‘ worms” appear to keep nearer the surface of the sand in cold weather than in summer,—at least, during the winter of 1893-4 large numbers were thrown up on the beach at Blackpool.
The most distinctive character, however, of this ‘‘ Lami- narian” variety is the gill (Pl. 1, fig. 2), which presents a structure hitherto only known in Arenicola cristata, Stimps. Instead of the somewhat simple gill seen in the shore lugworms, there is in the ‘‘ Laminarian” variety a highly developed pinnate structure, consisting of about twelve branches united by a connecting membrane at their bases, and bearing ten or more pinnules on each side of the main axis. Such a gill is undoubtedly a much more efficient respiratory organ than the gill of a shore lugworm, though it does not appear to possess the same power of contractility as the latter, and hence probably does not contribute so much to the move- ment of the blood. In some old specimens the gills lose many of their finer branches, perhaps owing to friction or to the
A F. W. GAMBLE AND J. H. ASHWORTH.
attacks of enemies,! and in such cases there is an approxima- tion to the type of gill seen in the littoral variety, though a certain amount of difference is always observable.
Thus there appear to be two varieties of the common lugworm on the Lancashire coast, distinguished by their habits, external features, and periods of maturity, but there are no important structural points of difference.
The habits of Arenicola marina at the breeding season are still to a large extent unknown, and developing eggs have not hitherto been obtained. It has been stated that, when mature, the animal is in the habit of swimming freely (Ehlers, 1892, a), but we are unable to confirm this. The post-larval stage, however, appears to be, for a short time, pelagic (Benham, 1893).
The curved burrow of the shore lugworm is formed by the combined action of the proboscis, the swollen anterior region of the body, and the waves of muscular contraction which pass along the body from behind forwards. When the proboscis is | everted and pressed into the sand, the prostomium is slightly retracted into the body. The proboscis is withdrawn full of sand, again everted, and the body is thrust forward, partly by contraction of the longitudinal muscles, partly by a peristaltic wave produced by the circular ones. The anterior end is in this way rendered swollen and tense, and is able to enlarge the burrow, and thus a passage is gradually eaten through the sand, smoothened by contact with the skin, and lined by the mucous secretion of the epidermis. The gill region being narrower than that which precedes it, is thus, to a certain extent, pro- tected from friction, while, as if to ensure this, the notopodial pencils of bristles are directed so as to protect the gills. After burrowing vertically downwards for a depth of from one to two feet, the worm forms a horizontal or oblique gallery, and then a second vertical one which ends at the ‘‘ countersunk ”’ hole, through which the anterior part of the worm may pro- trude, and so bathe the gills in fresh sea water.
* See the curious account of the ravages of Corophium longicorne, by d’Orbigny, ‘Journal de Physique,’ 1821.
HABITS AND STRUCTURE OF ARENICOLA MARINA. D
The amount and value of the work done by lugworms has been estimated on the shore of Holy Island by Mr. Davison (1891), and has also been adverted to by Mr. Hornell under the name of “ cleansing of the littoral.” Mr. Davison finds that the castings are larger and more numerous above than below half-tide; and as the result of several estimates and measure- ments he calculates that on the Holy Island Sands, the entire layer of sand, to a depth of two feet, passes through the bodies of the lugworms which live in it, once in twenty-two months, and that in a year the average volume of sand per acre, which is brought to the surface in the form of castings, is 1911 tons, representing, when spread out, a layer of thirteen inches in thickness over the surface of the sands.
2. External Features.
Segmentation.—The body is divided into an anterior chetigerous portion, a middle branchial one, and a posterior caudal region or tail. The first region begins with the pros- tomium, and is followed by a short achetous portion (fig. 1, MET), which in many specimens appears to be composed of four annuli, divided, however, by secondary circular markings. The first chetigerous annulus is produced into a strongly marked ridge, just behind which the notopodial sete (Chn.') are inserted, the corresponding neuropodia (Nm.') being very short and containing only a few sete. The intervals between the chetigerous annuli are subdivided into rings, of which there are, in the ‘ Laminarian” variety,22444..., and in the littoral variety 23444... respectively.
The cheetigerous annuli do not mark the true somites into which the body is divided. From a consideration of the internal anatomy (see p. 10) we have reasons for believing that, in the middle region of the body, the second groove behind each chetigerous annulus marks the boundary between the somites. A somite is, therefore, composed of a chetigerous annulus together with three annuli in front of, and one behind, it. The parapodia are not situated at the beginning, but slightly behind, the middle of the somites to which they
6 F. W. GAMBLE AND J. H. ASHWORTH.
belong, thus confirming Benham’s observations on the post- larval stage (1893).
The anterior region of the body is thus composed of the prostomium, six chetigerous somites, and a region between these, made up probably of two somites, but the exact number is somewhat doubtful. (See Plate 1, fig. 1, and explanation, p- 39.)
The second or branchial region of the body is composed of thirteen somites, and is distinguished by the presence of gills, a pair of which are attached to a slight fold of the skin just behind the notopodia. ‘The first gill is variable, usually fairly well developed, but always smaller than the rest and sometimes absent. The gills about the middle of the branchial region are frequently, but not always, the largest. Both the gills and notopodia are very sensitive, and are retracted from time to time on the application of stimuli, such as a strong light. This contraction of the gills proceeds sometimes as a wave down the body, and as Milne Edwards (1888) pointed out in his classical paper, considerably assists the circulation of the blood. The neuropodia in the branchial region extend towards the mid-ventral line, so as almost to meet, and are only separated by a groove which marks the line of the nerve-cord. This groove is continued on to the prostomium by a pair of diverging arms (‘‘ Metastomial grooves”) underlying the cir- cum-cesophageal nerve connectives (Pl. 4, fig. 19, C. MZ.).
The tail, which is devoid of setz and gills, is marked by a large number of secondary annuli, crowded together at first, but arranged in distinct somites of about five each, towards the hinder end. The caudal region varies much in length; some specimens have about thirty somites, but the number is not constant, possibly owing to the tendency of the worm to throw off the last few segments when irritated.
There is no change in the internal organs to mark the somite which bears the first gill, but the transition from the branchial to the caudal region is accompanied by the loss of parapodia, oblique muscles, and branchial vessels.
External apertures,—The mouth (Pl. 4, fig. 19, C, 70.),
HABITS AND STRUCTURE OF ARENICOLA MARINA. fi
when the proboscis is withdrawn, is a slightly crescentic trans- verse slit, bordered by papille and somewhat overhung by an upper lip. The anus, which is terminal, is often protruded, and the thin vascular swollen lips of the aperture project behind the last caudal segment.
The opening of the ‘ nuchal organ” is a fairly wide slit on the upper and hinder border of the prostomium (PI. 4, fig. 19, aands, NV.). Through this aperture, sea water (or a mixture of sea water and the secretion of the surrounding glandular cells) is probably introduced.
The openings of the otocysts are difficult to see. They lie behind the prostomium on each side of the anterior end in the position marked OT’. (Pl. 4, fig. 19, a and B). Each is placed at the point of intersection of the first transverse groove following the prostomium, with the oblique ‘‘ metastomial ” groove which marks the position of the nerve commis- sure.
The nephridial openings (fig. 1, NO), six in number on each side, though not so distinct as in some species (e.g. A. Claparédii), are not difficult to find. The first is placed behind and at the upper edge of the fourth neuropodium, and the other five in corresponding positions on the succeeding somites. They are minute slightly oblique slits, sometimes exhibiting tumid lips.
Skin.—The skin is subdivided into raised polygonal areas separated by corresponding shallow grooves, and is noteworthy in being devoid of special glands. Wirén (1887) has shown that the grooves are composed of columnar cells containing pigment granules, the raised areas being made up partly of larger cells containing still greater quantities of pigment granules and partly of clavate mucus-forming cells, which produce the slimy covering of the animal with which the burrow is lined.
The 5 per cent. formalin solution of the epidermal pigment is fluorescent, but does not yield any absorption bands, merely cutting off the rays at the blue end of the spectrum. In suc- cessively thicker layers of this solution, first the violet, then
8 F. W. GAMBLE AND J. H. ASHWORTH.
the blue, and lastly the green portions of the spectrum were cut off.
MacMunn (1889), however, has shown that the alcoholic extract of the integumental pigment shows a band in the blue and green (A 503—468) ; that the residue of this solution if dissolved in ether or chloroform yields two bands, X 503—474, and A 465—446; and that the residue of this solution again being dissolved in nitric acid gives two bands, A 500—468, and A 472—443, so that a chlorophan-lke lipochrome is present. It is probable that the pigment (melanin) of the skin is derived from the lipochrome of the yellow “glandular” tissue of the stomach, since the alcoholic extract of the latter yields a similar absorption spectrum.
Further investigation will be required to show in what way the transference of the pigment from the yellow peritoneal cells to the epidermis is brought about, and whether the dark- coloured, hairy-looking investment of the ventral vessel and its branches (Pl. 2, fig. 5) contributes to the melanin of the skin. In this connection the intermuscular extension of the ccelom, bringing it almost into contact with the epidermis at certain points, must be borne in mind (see p. 29).
Setz.—The notopodial sete are long capillary structures averaging 6 mm. in length, and bearing several rows of minute free and pointed hair-like processes (Pl. 3, fig. 10). The neuropodia in the anterior somites, which at first contain few sete, gradually extend by addition of new ones at their ventral edge, so as to almost reach the mid-ventral line (Pl. 1, fig. 1). By isolating the entire band of the sete the different stages in their development may be seen. The youngest sete are always at the lower end of the series ; the point of each seta is formed first, then the toothed ridge, and lastly the shaft. The fully-developed ventral seta is frequently almost smooth, owing to the wearing down of the teeth behind the apex. The middle of the shaft is straight, the inner end bent ven- trally, and the outer end bent slightly dorsally, ending with a finger-shaped process bordered on the convex side by a toothed ridge, while on the concave side it is slightly produced at one
HABITS AND STRUCTURE OF ARENICOLA MARINA. 9
point into a minute process (Pl. 3, fig. 12, proc.). This process is more constant in the Laminarian than in the littoral variety. It appears to correspond, in position, to the eharac- teristic tuft of hairs on the ventral setz of the Maldanide.
According to the age of the specimen the ventral setz differ in shape, and in the development of the toothed ridge. In sete from a small specimen (17 mm. long) the apex was bent more sharply on the shaft than in old examples, and the teeth were very prominent (Pl. 3, fig. 9). Apparently the production of fresh ventral sete goes on slowly throughout life, and the form which they assume before being cast out of the body, varies at different ages. Their size of course varies with the age of the worm to which they belong (see Pl. 3), but in a worm of average size their length is about ‘5 to ‘8 mm.
3. General Anatomy of the Internal Organs (Pl. 2).
In opening the body-cavity by a dorsal incision, the middle part of the alimentary canal is usually forced out through the cut by the pressure of the somewhat viscous ccelomic fluid. Normally this portion of the canal, being longer than the section of the celom in which it les, is swung to and fro by the movements of the body. This freedom of motion is ensured by the absence of mesenteries, by the absence of any vessels running from the body-wall into the dorsal vessel, and by the length and flexibility of the branchial and nephridial vessels, which are the only connection between the stomach and the body-wall.
The ccelom is exceedingly spacious, and continuous from one end of the body to the other. In front it is divided trans- versely by the origins of the buccal retractors (B. Sh.), which form a sheath round the proboscis, and by three septa or diaphragms (Pls. 2 and 3, figs. 5 and 6). The first of these septa (Dphm.) is placed obliquely, arising below behind the level of the first neuropodium, and being inserted dorsally in front of the first notopodial sacs. The result of this arrange- ment is that between the first and second diaphragms two pairs
10 F. W. GAMBLE AND J. H. ASHWORTH.
of setal sacs occur, caused by the forward shifting of the upper edge of the first diaphragm (fig. 5). The second and third are inserted both above and below, opposite the second groove behind the second and third chetigerous annuli. Between the first and second diaphragms, dorsal and ventral mesenteries occur, supporting the corresponding vessels; and it will be noticed that the dorsal mesentery ends in front, exactly where the first diaphragm would be inserted if it corresponded with the other two. The third diaphragm is perforated by the funnels of the first nephridia. There are, then, three diaphragms and not, as so often stated, four, and, while affording valuable evidence of the extent of the first and second chetigerous somites, they do not help in determining the number of seg- ments which compose the achetous portion following the prostomium.
Behind the last diaphragm the body-cavity is unsegmented up to the base of the tail. The segmental arrangement of the organs, however, can be recognised by taking the funnels of the nephridia as marking the anterior ends of the somites. The slight amount of connective tissue supporting the long afferent and efferent vessels (segmental vessels) (Pl. 2, fig. 5) of the nephridia and gills, may be regarded as the remains of the septa. Allied species of Arenicola fully confirm this view.
At the level of the thirteenth pair of notopodial sacs, the segmental afferent and efferent blood-vessels, which have hitherto run nearly parallel across the coelom, diverge. At the base of the tail, the connective tissue between them in- creases slightly in amount, septa forming which are continued down to the end of the body (fig. 5, C. Sp.).
4, Musculature.
The muscles of the body-wall are arranged in (1) an outer circular sheath, subdivided in the anterior and middle regions of the body into hoops, which cause the annulation of the skin; and (2) an inner longitudinal sheath of considerable strength and thickness divided by the nerve-cord and lines of
HABITS AND STRUOTURE OF ARENIOCOLA MARINA. it
insertion of the notopodial sacs into three parts, two ventro- lateral and one dorsal (Pl. 4, fig. 23). The intermuscular spaces are filled by ccelomic fluid, and are probably lined by a delicate peritoneum.
In the anterior region of the body there are a few circular muscle-bands which are stronger and more obvious than the rest (fig. 5, Df. Cire.).
The oblique muscles, which divide the ccelom longitudinally into three compartments, commence behind the third dia- phragm, and disappear at the base of the tail. These muscles are arranged in thin broad bands, arising at the sides of the nerve-cord, and are inserted right and left into the body-wall at the level of the notopodial sacs. They partly cover the nephridia, and in some specimens a muscle-band is attached to each nephrostome.
The musculature of the buccal mass consists of a strong sheath of fibres derived from the longitudinal layer just behind the first diaphragm. This sheath, which is loosely attached to the proboscis by slips which run through the cceelomic space between the two structures (PI. 3, fig. 6, B. Sh.), is inserted into the anterior part of the proboscis. Pressure of the ccelomic fluid at this point causes eversion of the buccal mass, which is withdrawn by the contraction of its muscular sheath.
The prostomium is retracted by a small sheet of muscle which arises partly from the longitudinal layer dorsally, and partly from the muscular covering of the circumcsophageal connectives ventrally, and it is inserted into the ventral surface of the brain, and the ventral and hinder edge of the nuchal organ (Pl. 3, fig. 6, Nu. Tr.).
The parapodial muscles are modifications of the longitudinal layer. One, the retractor of each notopodium, is remarkably long, reaching to the side of the nerve-cord (Pl. 3, fig. 18, Rn.). The protractors (Pn.) of the notopodia are six to eight in number, three to four being placed in front of, and three to four behind, the setigerous sac. They arise from the body- wall just below the dorsal longitudinal vessel, and are inserted into the base of each sac,
12 F. W. GAMBLE AND J. H. ASHWORTH.
The position and relations of the three anterior septa or dia- phragms, of the dorsal and ventral mesenteries between the first two of these, and the presence of regularly arranged septa in the tail region, have already been noted. It may be added that a pair of outgrowths from the first diaphragm lie under the cesophagus, opening anteriorly into the coelomic space in front of the first septum. They are very vascular, and con- tract rhythmically every three or four seconds during life, and are doubtless of use in everting the proboscis (Pl. 2 and 8, figs. 5 and 6, Dph. Ph.).
In the caudal region the intestine is attached both above and below to the body-wall by mesenteries, in which the dorsal and ventral vessels lie.
5. Alimentary Canal (PI. 2).
This consists (1) of an eversible buccal mass (Bucc. M.), of a pinkish or greenish-brown colour, which lies in front of the first septum; (2) of an esophagns, of a light brown colour, provided with a pair of glandular pouches behind the third diaphragm ; (3) of a gastric region, with yellow glandular walls, extending from the level of the heart to about that of the twelfth or thirteenth notopodium ; and (4) of an intestine, of a dark brown or almost black colour, folded in a concertina-like manner by the caudal septa, and opening at the terminal anus.
During life the buccal mass (or “ proboscis”) is constantly being everted and withdrawn, carrying sand into the cesopha- gus. During eversion several rows of curved, pointed, vascular papille (B. Pap.) are first extruded. Thése papille (Pl. 3, fig. 7) in old specimens are tipped with chitin, and recall the armature of the proboscis in certain Sipunculids (e.g. Phas- colion collare!). ‘Then the more globular portion of the buccal mass, covered with minute rounded processes, is pro- truded. Finally, when fully everted, the buccal aperture is surrounded by a few pointed pigmented papillz, which are continuous with the lining of the first part of the cesophagus.
1 Selenka, ‘ Die Sipunculiden,’ 1883, pl. vi, fig. 74.
HABITS AND STRUCTURE OF ARENICOLA MARINA. 13
The csophagus! itself is slightly looped behind the second diaphragm. It is a thin-walled distensible tube, the first part of which is lined by non-ciliated mucus-forming cells. The middle portion is lined by a cuticle, and the posterior part by cells resembling those of the stomach in bearing cilia. The cesophageal pouches (Oe. Gl.) are somewhat flask-shaped, and open into the cavity of the csophagus by a short tubular stalk. They are usually greenish in colour, but have a slight reddish tinge on account of their very large blood-supply. Their blood-vessels are connected with the lateral cesophageal and dorsal vessels. The cavity of the pouch is subdivided by twenty-five to thirty incomplete partitions, produced by in- folding of the wall of the pouch, and therefore covered on each side by the epithelial lining of the pouch (Pl. 4, fig. 22). Between the epithelial lamelle is a blood-sinus, which is slightly enlarged at the inner end and slightly thickened at the edge of each partition. The csophageal pouches are lined by ciliated epithelium, covered with a fairly stout cuticle, and contain glandular cells. The walls of the cesophagus are marked by longitudinal and circular muscular impressions.
The stomach, marked out by the patches of yellow tissue on its walls, extends from the level of the heart to about the twelfth notopodial sete. As we have already stated (p. 9), the stomach is bent upon itself and loosely attached to the body-wall. The patches of “chlorogogenous” tissue are at first arranged in symmetrical oval areas right and left of the dorsal blood-vessel, while more ventrally they are placed in two or three less regular series, and are separated from one another by a network of blood-vessels.2 About the level of the tenth setz these yellow areas all become subequal and arranged in a spiral manner, ending at the level of the four- teenth setz.
Stomach and Intestine.—The muscular wall of the
* The histology of the alimentary canal has been carefully investigated by Wirén (1887, p. 31). Our results agree very closely with his.
? This network is considered by Wirén and others to be parts of a con- tinuous sinus. We are not convinced, however, that this is really the case, and our reasons will be found on p. 17 infra.
14 F. W. GAMBLE AND J. H. ASHWORTH.
gastric region is exceedingly thin, and composed purely of circular fibres, which appear to confer very slight powers of peristalsis upon the stomach.
The mucous lining is strongly folded, and is composed of several kinds of cells. Some of the cells in all parts of the stomach are ciliated, others are apparently digestive, and a large number appear to secrete a mucus similar to that of the cesophagus, the cells themselves being discharged into the mucus which they help to form.
Commencing about the middle of the stomach (that is between the ninth and tenth segments) is a ventral groove formed by a couple of folds of its inner and lower surface. This groove! (Pl. 4, fig. 238, Gv.) is provided with specially long cilia, which produce a current of mucus from before back- wards. There are other smaller grooves on the side walls of the stomach and the anterior part of the intestine, whose general direction is downwards and backwards, and which open into the median ventral groove. ‘The direction of the current in all these is from before backwards. The ventral groove is continued back to the anus. ‘The intestine is dark brown or nearly black in colour externally. Its mucous lining is somewhat similar to that of the stomach, but is covered by a thin cuticle, and is not ciliated.
The process of digestion in the lugworm has not been at all fully investigated, but the series of events appear to be some- what as follows. The sand or mud is mixed with the mucous secretion of the esophagus, and is slowly carried backwards by peristaltic contraction. At the junction of the stomach and cesophagus the secretion of the wsophageal pouches is poured upon the sand. Wirén regards the contents of these pouches as acid and digestive. In several cases we have found the fluid neutral. In the stomach several changes occur. ‘The secre- tion of the gastric cells proper is probably digestive, and this, together with a further amount of mucus, is mixed with the sand, and shaken together by the swing of the loose gastric loop. In this way the food, which apparently consists of the
1 This groove has only hitherto been noticed by Wirén (1887).
HABITS AND STRUCTURE OF ARENICOLA MARINA. 15
organic substances! in the sand, is brought into contact with the digestive secretion. The ciliary action of the lateral and ventral grooves probably separates the digested substances from the sand and carries them slowly downwards and _ back- wards. ‘The lining of the stomach is very thin, and the lateral and ventral grooves are in specially close contact with the blood-plexus, in which the flow is, probably, slowly forwards, more rapidly in the sub-intestinal vessels. It seems probable, therefore, that the blood in the visceral plexus conveys the nutritive material to the hearts, which pump it along the ventral vessel to the various parts of the body.
The action of the chlorogogenous tissue round the stomach, and particularly of that in the neighbourhood of the ventral vessel and its branches, is uncertain.
6. Vascular System (Pl. 2, fig. 5).
The blood-vascular system of Arenicola attains a high degree of perfection. The large size of the chief vessels, the great development of the capillary system (especially on the walls of the alimentary canal), and the mechanism for promoting the flow of the blood, are features that dis- tinguish it.
There are two chief vessels running, one above, and the other below, the alimentary tract from end to end,—the dorsal vessel, which contracts fairly rhythmically from behind for- wards; and the ventral vessel, which is feebly, if at all, con- tractile. The walls of the gastric and intestinal portions of the gut areenclosed in a blood-plexus, and the cesophageal region is supplied by lateral vessels. The gastric vessels are connected with the ventral vessel by a pair of “‘ hearts ”’ placed a short dis- tance behind the esophageal pouches (fig.5, V.). These hearts drive the blood from the gastric vessels into the ventral vessei.
The dorsal vessel (D V) arises near the anus, and as it runs along the intestine gives off in each somite a pair of branches
1 Saint Joseph found in an Arenicola a whole Nereis almost digested. ‘ Ann. Sci. Nat.,’ series vii, t. xvil, 1894, p. 127.
16 F, W. GAMBLE AND J. H. ASHWORTH.
which are attached to the anterior face of the caudal septa, and which run downwards and forwards to open into the ventral vessel (Pl. 2, fig. 5). Of these there may be twenty-seven to thirty pairs. In front of the caudal region each of the last seven pairs of gills returns an efferent branch to the dorsal vessel, and between these there are three or two pairs of smaller branches which run round the alimentary canal from the ventral vessel to open into the dorsal one. From the level of the twelfth sete to the csophageal pouches the dorsal vessel does not receive any segmental vessels from the gills or nephridia, nor does it open directly into the heart (fig. 5). It merely receives numerous branches from the gastric plexus. In front of the heart it receives on each side a branch from the third nephridium and the fifth setigerous sac; a branch from the cesophageal pouches ; and one from the second nephridium and fourth setigerous sac. It then runs on and, piercing the third diaphragm, receives a branch running on the anterior face of the diaphragm from the first nephridium and third setigerous sac. On reaching the second diaphragm it receives a branch from the second setigerous sac, and after piercing the first diaphragm receives a branch from the muscles forming the buccal sheath. Thence the dorsal vessel breaks up into capillaries around the buccal musculature, prostomium, and otocysts. From these capillaries the ventral vessel takes its origin. It gives off a small unpaired branch running in the first diaphragm and to its pouches; a paired branch arising about midway between the first and second diaphragms to the neural vessels and second setigerous sac; a single small vessel supplying the second diaphragm and the neural vessels; an unpaired vessel to the third diaphragm, to the neural vessels in that region, and to the first nephridia; a pair of branches to the neural vessels and second nephridia; and lastly, a pair to the neural vessels and third nephridia. From this point ouwards the ventral vessel supplies the setigerous sacs, body- wall, nephridia (if present), and gills by large segmental vessels. The ventral vessel is very large and turgid in the gastric region, and is surrounded by tufts of dark brown chlorogogenous tissue,
HABITS AND STRUCTURE OF ARENICOLA MARINA. 7
which are also found in older specimens on the vessels running to the body-wall. This chlorogogenous tissue is first seen 01 the ventral vessel about the level of the eighth pair of sete. In the tail the ventral vessel ends in the obliquely p'aced intestinal vessels which encircle the intestine, and which form, along with the capillaries from its median terminal portion, the commencement of the dorsal vessel.
Visceral Plexus.—Wirén (1887) maintains that the in- testine and stomach are enclosed in a blood-sinus, thickened along certain lines which have been called the dorsal, gastric, and subintestinal ‘vessels.’ We are, however, of the opinion that the so-called sinus is a close plexus of vessels, some of which appear to have a distinct cellular lining. The dorsal vessel is, at any rate, a perfectly distinct structure with proper walls.
The subintestinal vessels (fig. 5, S. V.), which commence just behind the heart and run backwards, are moderately large up to the level of the thirteenth sete, but then taper rapidly and gradually disappear. They each receive seven segmental vessels. The first of these comes from the fourth nephridium, the second from the fifth nephridium and the first gill, the third from the sixth nephridium and second gill, and the other four from the third, fourth, fifth, and sixth gills. The sub- intestinal vessels open.through the plexus into the lateral gastric ones, and so into the heart. The flow in these vessels is probably slowly forwards.
The gastric vessels give off from the “auricle,” into which they expand, a lateral cesophageal vessel (Oe. Lat.), which, after giving off a stout branch to the cesophageal pouches, runs forwards to the buccal mass, supplying the wall of the ceso- phagus, as it does so, with numerous small branches.
Neural Vessels.—These are a pair of small vessels lying one on each side of the ventral nerve-cord, and accompanying it from one end of the body to the other. They arise round the nerve-connectives from the brain from capillaries of the dorsal vessel, and receive several branches from the ventral vessel (1) midway between the first and second diaphragms, (2) from
vot. 41, part 1.—NEW SER. B
18 F. W. GAMBLE AND J. H. ASHWORTH.
the vessel running in the second diaphragm, (3) from a vessel just behind the third diaphragm, (4 and 5) from the vessels to the second and third nephridia. Near the middle of each somite the two neural vessels are united by cross connections, which also supply the nerve-cord (Pls. 2, 3, fig. 18, N. V., IN: CV);
Behind the third diaphragm the neural vessels supply the oblique muscles by branches which run the whole length of the bands, and are connected with the outer longitudinal parietal vessel (fig. 13).
Vessels of the Body-wall.—This parietal system of true vessels is highly developed in Arenicola marina. It con- sists of two longitudinal vessels, (1) the nephridial longitudinal vessel (fig. 22, N. LZ. V.) running just below the level of the nephridiopores, and (2) the more important dorsal longitudinal vessel (fig. 13, D. L. V.), which runs just above the level of the insertion of the notopodial setal sacs. Both arise just behind the first sete, and increase in size as they pass back- wards. The former receive vessels from the nephridia, just behind which they taper and disappear. The latter, which may be traced to the anus, and are largest in the branchial region, receive branches in each somite: (1) from the segmental vessels ; (2) from its fellow of the opposite side. The body-wall in the dorsal and lateral regions derives its blood-supply from the nephridial and dorsal longitudinal vessels, and in the ventral region from the neural vessels. These parietal vessels (Par. V.) run just within the layer of circular muscles in almost every groove between adjacent longitudinal muscle- bands of the body-wall, are chiefly longitudinal in direction, but at frequent intervals there are cross connections. Branches from these vessels ramify between the bases of the epidermal cells, and are accompanied by extensions of the coelom.
Hearts.—The hearts are a pair of muscular bulbous swell- ings connecting the visceral plexus with the ventral vessel on each side. Each commences with the thin-walled expansion of the gastric vessel (“auricle,” fig. 5, d.v.) which, after giving off the lateral cesophageal branch, opens into the ventricle
HABITS AND STRUCTURE OF ARENICOLA MARINA. 19
(V.). The cavity of the ventricle is small and broken up by a spongy mass of cells. The ventricular walls are muscular, and contract from above downwards, forcing the blood into the ventral vessel. (We have sometimes seen an apparent reversal of the heart’s action.) The spongy cardiac body arises by ingrowths from the wall of the ventricle, chiefly in the middle and ventral regions. It gradually encroaches on the blood space, so as to reduce it considerably (PI. 5, fig. 36, Card. B.) in an old specimen. The cardiac body in a young specimen (fig. 38) is much smaller, and extends obliquely across the heart, its general direction being downwards and back- wards. The cells of the cardiac body in an old specimen which we have examined are loosely arranged, so as to cause the formation of a large number of intercellular spaces, some of which are of considerable size, and which are in life filled with blood (Figs. 36—38, B.S.). Between the cells there are numerous fibres, which are probably muscular. The cells are apparently of two kinds, which, however, merge into each other: (1) cells whose protoplasm has a very vacuolated appear- ance, and which contain few or no granules ( Vac. C.); (2) cells which contain a large number of yellowish granules in the protoplasm (G.C.). These latter cells are possibly glandular, and correspond to those found in the cardiac body of other Polychets. The function of the cardiac body may be, as Schaeppi (1894) suggests, to prevent regurgitation of the blood from the ventral vessel into the heart when the diastole com- mences. The “cardiac body” of Polychets, as hitherto described, is an unpaired structure lying in the dorsal vessel. That of Arenicola, however, is paired and in no way con- nected with the dorsal vessel. Hence a strict homology is scarcely probable.
Blood.—As Professor Lankester was the first to point out, the blood of Arenicola is strongly impregnated with hzmo- globin, but there has been no thorough investigation of the constituents of the plasma. Krukenberg (1882), it is true, made some experiments which led him to believe that there were no coagulable albumens in the blood of his specimens ; but as they
20 F. WwW. GAMBLE AND J. H. ASHWORTH.
were in a starving condition, a fresh examination is very desirable. A large quantity of albumen is certainly present, which when the specimens are fixed becomes very hard and brittle.
We have seen small cells (4 4 in diameter) in the blood- vessels of the nephridia, but it is doubtful if these are the blood-corpuscles, which we have not been able to demonstrate.!
General Remarks on the Circulatory System.—No other system of organs shows the true segmentation of the body of Arenicola so well as this. The lines of demarca- tion between the somites from one end of the body to the other are marked by the segmental vessels passing from the ventral to the dorsal vessel and breaking up on their way in the body-wall, nephridia, or gills. Throughout the gastric region, however, this arrangement is somewhat disguised, owing to the loss of the connection with the dorsal vessel, an altera- tion caused probably by the necessity for leaving this part of the alimentary canal freely moveable.
Wirén evidently believes that there is no capillary system except in the gills and the alimentary canal. He suggests that the assimilation of food and oxygen by the tissues is effected chiefly through the mediation of the ceelom, which he points out is parcelled off in the intermuscular spaces, by a channelling out of the subepidermic connective tissue, into * perihzemal canals.” Though this suggestion isa valuable and correct one, we have found a very perfect system of capillaries in the skin in all parts of the body, and in the nephridia and septa the same is the case. The extension of the ccelom into the intermuscular and subdermal spaces has, however, all the appearance of acting as the equivalent of lymph-spaces of higher forms. The transformation of the constituents of the blood into coelomic fluid takes place in all probability with especial rapidity in the neighbourhood of the dark chlorogo- genous processes of the ventral vessel (cf. Cuénot, 1891).
1 Since writing this we have discovered that these small cells are the blood-corpuscles.
HABITS AND STRUCTURE OF ARENICOLA MARINA. 21
7. The Gills (Pl. 1, figs. 2—4).
The general characters of these organs have been mentioned in the introductory part of this paper, and little remains to be added.
There are thirteen pairs of gills from the seventh to the nineteenth cheetigerous somites inclusive. The shape varies from the short dendritic type of the littoral form to the delicate, richly- branched gill of the Laminarian variety. The gills are hollow, being outgrowths of the body-wall enclosing an exten- sion of the clom, and what little evidence we have of their development (see Benham, 1893) points to their being inde- pendent structures, and not modified dorsal cirri.
The walls of the gills, though thin, are muscular, and there are also muscular bands stretching across the cavity of the gill (fig. 23); and Milne Edwards has pointed out that the contrac- tion of the gills, which often proceeds like a wave from before backwards down the sides of the body, must exert a powerful influence in propelling the blood partly into the efferent vessels, and partly to the parietal capillaries.
The ventral vessel supplies all the gills with their afferent branches. The first seven pairs return the blood to the sub- intestinal vessels, and so to the heart; while the efferent branches of the remainder open into the dorsal vessel.
8. Nervous System and Sense-organs.
This system is composed of the brain, the cesophageal con- nectives, the ventral nerve-cord, and the nerves arising from these. We have not been able to demonstrate a visceral nervous system.
The brain (Pl. 5, figs. 25, 26) is placed in the prostomium, of which it forms the chief part, being only separated from the epidermis by blood-vessels lying in extensions of the ccelom. It is a small elongated structure, measuring ‘75 mm. in length in ordinary shore lugs, and 1 mm. in the large ‘‘ Laminarian”’ variety. At its anterior end the brain is divided into two stout cornua (A. Cr.), separated by a cleft containing blood-vessels. About the middle of the brain the cornua unite, but only for a
22 F. W. GAMBLE AND J. H. ASHWORTH.
very short distance, a second connective-tissue partition divid- ing the smaller posterior cornua (P. Cr.), which gradually taper off and end at the hinder edge of the nuchal organ (Pl. 5, fig. 25).
Sections of the prostomium of the littoral variety of Arenicola (immature specimens, 4” long) exhibit a thick covering of ganglion- and glia-cells, forming the dorsal surface of the brain (fig. 24) ; a central fibrous portion; and a strong ventral membrane, into which the greater part of the pro- stomial muscles are inserted, though a few fibres are attached in front of and between the anterior cornua (PI. 5, fig. 25). In older specimens, and particularly in mature examples of the “ Laminarian” variety, the ganglion-cells are more scattered, and in other ways the brain shows greater differentiation. The anterior cornua, for example, are not only deep and thick, but give off from their dorsal surface short stout branches, along which the ganglion-cells are scattered, and which supply the prostomium. The central fibrous part of the brain also grows out ventrally in these large examples, separating the hitherto compact layer of cells and carrying them outwards or leaving them in clumps, and not evenly arranged as in young Areni- cola.
From the anterior cornua a large nerve arises on each side, in front of the origin of the esophageal connectives. It passes out to the under surface of the epidermis, and supplies the papillz on the upper surface and the sides of the mouth. The epidermis of the prostomium itself is in close contact through- out its whole length with the ganglionic covering of the pro- cesses arising from the dorsal and lateral surfaces of the brain. The posterior cornua seem to be specially connected with the nuchal organ, against which they lie and terminate (Pl. 4, fig. 21).
The most remarkable histological feature of the brain is the close contact between the large ganglion-cells of its upper surface and the sensory epithelium of the prostomium (figs. 20 and 24). Racovitza (1896) has figured (PI. 5, figs. 48 and 49) a similar condition in Clymene. It is only at this point
HABITS AND STRUCTURE OF ARENICOLA MARINA. 23
that the nervous system of the adult Arenicola marina can be said to have an epidermal position. Elsewhere it is separated from the epidermis by the circular musculature.
The circum-cesophageal nerve-connectives arise from the large anterior cornua in the form of two thick cords, covered on their outer surfaces by ganglion-cells (figs. 20, 21, 25, Oe. Comm.). From them a pair of short nerves (fig. 26, OT. N.) arise supply- ing the otocysts, and several longer ones are distributed to the oral papille of the ventral region of the mouth. The line of the connectives is marked externally by the ‘ metastomial groove” (Pl. 4, fig. 19, C.), and the commencement of the ven- tral cord by the junction of these grooves, which occurs on the ventral surface just in front of the first chetigerous annulus. The nerve-cord is protected by a delicate connective-tissue sheath, a thin sheath of circular muscle, and a thin layer of epidermis. Though nearly circular in section it is somewhat flattened from above downwards, but exhibits scarcely a trace of segmentation externally or internally. The ganglion-cells are arranged in two veutral groups, while the fibrous portion of the cord is dorsal. In the tail the ganglionic masses in- crease in size, and are separated from the skin by a thicker layer of circular muscle-fibres. Two “giant-fibres” are present in the branchial region, a single one only in the anterior and tail region.
From the cord a paired series of nerves is given off with great regularity, one opposite each groove separating the annuli of the somites, so that there are five nerves on each side of the body in each somite. These lie in the body-wall just beneath the circular layer of muscle, and, in some places where this layer becomes obsolete, they lie just under the epidermis. Dorsally these nerves thin out and become very difficult to trace.
Sense-organs.—There is no doubt that the prostomial lobes, the nuchal organ, and the otocysts are sense-organs ; but there are, in addition, certain other structures, such as the sete! of the notopodia and some of the buccal papille, which,
1 Retzius has described free nerve-endings on these sete. ‘ Biologiska Foreningens Forhandlingar,’ Bd. ii, Hefte 4—6, 1891, p. 85.
24 F. W. GAMBLE AND J. H. ASHWORTH.
on account of their position, movements, and the nerves ending in them, may be considered as probably belonging to this category.
Professor Ehlers’ (1892) account of the nuchal organ and otocysts is an almost exhaustive description of these organs in Arenicola. We have worked over the whole subject again, however, and are able to add a few points to this important paper.
The nuchal organ belongs to the prostomium, whereas the otocysts belong to the metastomium. The prostomium and the nuchal organ are found, in varying degrees of complexity, in nearly all Polychets; the otocysts, however, occur in few and widely separated families.
The general appearance of the prostomial lobes and the opening of the nuchal organs have already been described. Seen from the dorsal surface the former consists of a small median papilla and two larger lateral prominences (Pl. 4, fig. 19), which together correspond with the single prostomial papilla of allied forms (cf. Racovitza’s figure of Leiocephalus, 1896, pl. v, fig. 5). In young Arenicola these lobes are transparent, and therefore red from the underlying blood- vessels. In old specimens they become dark-coloured and opaque from the deposition of pigment in them. In no species of Arenicola have eyes been discovered, although they are known to occur on these lobes in many related genera.
The prostomial epithelium is a complex of several distinct kinds of cells,—unaltered columnar elements, fusiform sense- cells, each ending in a conical prominence, glandular cells, and apparently also ‘ wandering cells” from the body-cavity. Underneath the epithelium is a connective tissue continuous with the supporting tissue, the neuroglia of the brain, which binds together the large ganglion-cells of the cornua of the brain. The prostomial sensory structure thus formed is very sensitive to light, but what function it subserves has not been determined with accuracy.
Nuchal Organ.—To the outer side of the lateral prosto- mial lobes is a depression guarded externally by a fold (just
HABITS AND STRUCTURE OF ARENICOLA MARINA. 25
above Nu., Pl. 4, fig. 19, B.). These two pits form the beginning of the nuchal organ and indicate its paired origin. Further back they unite to form a transverse groove (bordered by the hinder edge of the prostomium), which is continued inwards as a deep pit to the hinder margin of the brain (PI. 5, fig. 25). From the posterior cornua of the latter the nuchal organ is innervated.
In its paired form and under the names ‘“‘ Wimperorgane,” “ Wimpergriibschen,” the nuchal organ is well known in almost all families of Polychets, and a similarly placed organ is found in Sipunculids,! not to mention other more distantly related groups. It is always associated with the posterior lobe of the brain, and arises as a pair of pits from the surface of the prostomium. Of its development in Arenicola, however, we have no evidence, but the two depressions in front of the main part of the organ, together with the paired nerve-supply, point to its double nature.
The epithelium of this deep, pigmented pit (PI. 4, fig. 21, Nw.) is composed of long columnar ciliated cells, glandular cells which secrete the mucus in which the cilia work, and slender sense-cells. It seems probable that the whole organ is olfac- tory in function.
Otocysts.—The otocysts of Arenicola marina are a pair of flask-shaped structures projecting into the body-cavity close to the outer edge of the csophageal nerve-commissures. They open externally by a couple of apertures (Pl. 4, figs. 19,4 and B, OT.), at that point on the “ metastomial groove ” where the latter is crossed by the first groove of the body following the prostomium. The body of the flask is placed at an angle with the ‘‘ neck,” and contains the otoliths. It is lined by non-ciliated columnar sense-cells and supporting cells, which are surrounded by the nerve-fibres and connective-tissue fibrils, figured by Ehlers (1892, pl. xii). The neck of the otocyst is made up of a columnar epithelium covered with a thick cuticle, which gradually merges into the epidermis of the external surface, and ciliated cells only occur in its lower portion. A
1 Ward, ‘ Bull. Mus. Harvard,’ vol. xxi, 1891, p. 148.
26 F. W. GAMBLE AND J. H. ASHWORTH.
short nerve from the csophageal commissure supplies the otocyst.
If the otocyst of a fresh shore lugworm be rapidly dissected out under sea water and mounted, the sand-grains will be seen to execute a most extraordinary movement. Each one is rotating slowly and jostling its fellows, so that the whole contents of the flask are in a state of commotion. ‘The fluid in which the otoliths move is slightly viscous, and is a secretion of the walls of the otocyst, mixed with a little sea water. The sand-grains are covered with a distinct layer of some chitinoid substance soluble in boiling potash. Acids have no appreciable effect upon these grains, and under the polariscope they react as quartz does. Hence it seems clear that the otoliths of Arenicola marina (the other species of the genus differ most remarkably in this respect, as well as amongst them- selves) are quartz grains covered by an organic film, and sur- rounded by a fluid which is not merely sea water.
Large specimens of the “‘ Laminarian”’ variety were examined without being opened under sea water, and the otocysts were mounted by us in celomic fluid. No movement of the oto- liths was observed even in specimens which were perfectly healthy in all respects. The otoliths sometimes filled the _ expanded part of the organ, and it is possible that they had no room to turn round. But it appears to us more likely that if we assume the cause of the rotation to be the diffusion caused by liquids so different as sea water, in which the preparation was first mounted, and the somewhat viscous, perhaps albu- minous fluid inside the otocyst; then if we mount the otocysts in the same kind of fluid which they contain, no movement should occur; and the experiment showed that in these cases no movement did occur. The whole matter is one of very great interest, especially in view of the probable functions of such an organ as the otocyst. Ehlers has suggested that the movement is due to the cilia at the bottom of the neck of the otocyst ; but the same extraordinary movements are seen in the otocyst of A. Grubii, which is closed and has no cilia. We quite agree with Ehlers that there are no cilia in the expanded part
HABITS AND STRUCTURE OF ARENICOLA MARINA. 27
of the otocyst where the movement has been noticed, but we are of the opinion that the quivering motion of the otoliths is not a normal phenomenon, but is due to diffusion currents.
9. Nephridia.
There are six pairs of nephridia, belonging to somites 4 to 9. Of these the first pair seems to be unrepresented in any other species of Arenicola, and its variation in A. marina points clearly to a gradual degeneration which it appears to be under- going at the present time. It is not only the smallest of the series, but is sometimes represented merely by a funuel or by the secretory and terminal portions. Very rarely both the first nephridia are mere funnels, and again one may be fully developed and the other rudimentary, but they are never abso- lutely wanting. Their small funnels, which are of a bright pink colour, are placed on the anterior face of the third diaphragm with the long axes vertical (Pl. 2, figs. 13 and 14). One lip (the outer) is produced into processes corresponding to the dorsal lip of the other nephridia. The secretory portion is elongated, narrow, and usually brownish in colour, and the terminal portion opens just above the fourth neuropodium (Pl. 1, fig. 1) at a decidedly lower level than is the case in the succeeding nephridiopores.
The remaining five pairs are always in adults fully de- veloped. They are attached to the body-wall partly by connec- tive tissue, partly by the broad bands of oblique muscle which obscure them at first sight (Pl. 2, fig. 5). The nephro- stomes are very long, and bent upon the rest of the organ. The narrow slit-like aperture has a dorsal vascular lip bearing finger-shaped or spatulate ciliated processes, and an entire ventral one. The cilia just within the mouth of the funnel are exceedingly long, and produce a current tending to carry celomic fluid and corpuscles into the cavity of the organ. The middle or secreting portion is brownish (in old worms almost black), owing to the excretory granules which are formed in its cells. ‘The terminal rosette-shaped bladder, which is slightly lighter in colour, opens by a minute slit-like aper-
28 F. W. GAMBLE AND J. H. ASHWORTH.
ture through the body-wall, which thins out at this point (Pls. 1 and 4, figs. 1 and 22, NO.).
The blood-supply to the nephridia (Pl. 4, fig. 18) is derived from the ventral segmented vessels, which divide, one branch going to the funnel of the nephridium and the other to the body-wall. The former traverses the funnel, sending a vessel into each of the ciliated processes, and giving off numerous small branches to the lips of the funnel. After traversing the funnel the vessel runs over the secreting portions of the nephri- dium, supplying the genital strand in its course, and finally ramifies on the terminal portion. The blood is collected again into small vessels, which open into the dorsal longitudinal or nephridial longitudinal vessels of the body-wall, from which it is returned largely to the dorsal or subintestinal vessels, but in part passes into the parietal vessels.
In young specimens the funnels are naturally simpler, but have similar positions and relations, as may be seen in figs. 16 —18, which show nephridia from worms 29°5 and 44 mm. long, in which the processes on the dorsal lip are being formed. In the post-larval stage (Benham, 1893) the nephridia have no funnels, the development of which has still to be investigated.
10. Celom.
The colom of Arenicola is well developed, and continuous in all its parts. Not only does it form the space between the alimentary tract and body-wall from one end of the body to the other, but it is carried along with the blood-vessels into the intermuscular spaces. Thus the blood-vessels of the pro- stomium, of the buccal sheath, and of the body-wall generally, are accompanied by ccelomic canals which very probably serve as lymphatic spaces from which nutritive matters can be absorbed by the surrounding tissue, and into which waste nitrogenous substances may be excreted.
The segmentation of the body-cavity is very faintly marked. Anteriorly three diaphragms, perforated just above the nerve- cord, are present, whose position and relations are indicated
HABITS AND STRUCTURE OF ARENICOLA MARINA. 29
on fig. 5, and Pl. 3, fig.6. The whole middle region of the body is devoid of septa, which, however, reappear on the last two somites of the branchial region, and are present through- out the tail in a complete form, though they are perforated to allow of the more thorough circulation of the celomie fluid.
Arenicola fresh from the sand exhibits a series of peri- staltic waves of the body-wall from behind forwards, which can be easily seen if the gonads are sufficiently developed to cause slight swellings, which each wave carries forwards. These waves of fluid are probably of considerable physio- logical value. They assist the circulation of the fluid, the celomic cells, and the developing reproductive cells. They inflate the anterior digging part of the worm, and thus assist in burrowing. By their action the contents of the gut will tend to travel slowly backwards, the weak visceral muscu- lature being probably insufficient by itself to cause the requisite amount of movement of the sticky sand: while in defeecation the main agent is doubtless the pressure of the ccelomic fluid on the intestine, brought about by violent con- tractions of the body-wall.
The ccelom is lined by a very thin layer of flattened cells, which undergo remarkable changes in certain parts of the body, resulting in the formation of (1) chlorogogenous tissue, (2) ova or spermatozoa, (3) coelomic corpuscles.
The ccelomic fluid is a mixture of sea-water and globulins, among which only paraglobulin has hitherto been detected (Krukenberg, 1882, p. 87). We find that the specific gravity of the fresh fluid (including corpuscles) varies slightly, but is on the average 1°0288.1
On exposure to air this fluid coagulates, and a delicate fibrous network is formed, binding the corpuscles together. If carmine is injected into the celom, it is removed by the coelomic corpuscles, by the cells lining the celom and by the
' It was found to be least (1:0270) in specimens which had been kept for some time in sea water, and greatest (1°0311) in those which had been kept
for thirty-six hours in moist seaweed only. The specific gravity of the sea water used was 1°0264,
30 F. W. GAMBLE AND J. H. ASHWORTH.
nephridia, and there is no trace of carmine in the ccelom after forty-eight hours.!
Celomic Corpuscles.—These abundant cells occur in two chief forms, which probably pass into one another. The first varies from 8 to 20 w in length, is ameeboid, and usually contains yellow or brown granules of a very highly refractive character. The pseudopodia are often grouped at the two ends of the cell (PI. 5, fig. 24). The longer forms of this kind of corpuscle pass into the second or spindle-shaped cells of the ccelom, which measure as much as 50 wu in length, and contain no coloured granules. These fusiform elements are most abun- dant, and constitute the most characteristic features of the coelomic contents.
The chlorogogenous tissue of the ventral vessel and its branches in the body-wall consist of groups of cells about 20 u in length, full of large slightly yellow or deep brown granules, which are not highly refractive. The tissue in old black worms is immensely developed, so as to completely cover the vessel by the masses of hair-like threads, each thread consist- ing of a small blind diverticulum of the vessel surrounded by the chlorogogenous cells.
11. Reproductive Organs.
Thanks to the researches of Cosmovici (1880), Cunningham (1887), Kyle (1896), and others, the true ovaries and testes of Arenicola marina are now known to arise by proliferation of the peritoneal covering of an extension of the blood-vessel supplying the funnels of the nephridia. It is not certain that there is a corresponding gonad on the first pair of nephridia, but on each of the following five pairs the gonads are present during the breeding season. In both sexes the organ is a mass of cells, from which the ova or spermatoblasts break away at a very early stage, to ripen in the celom. The rachis is con- tinuous with the posterior angle of the nephrostome, and is developed around a backwardly projecting process of the
1 Schneider, ‘Arbeit. Naturf. Gesellschaft,’ St. Petersburg, Bd. xxvii, Heft 1, 1890.
HABITS AND STRUCTURE OF ARENICOLA MARINA, 31
nephridial vessel which comes off segmentally from the ventral vessel (Pl. 4, fig. 18, G. V.).
In large Arenicola, at certain seasons, the vascular process has no gonad, and it is possible, as Cuénot (1891) suggests, that a formation of the ameeboid corpuscles of the ceelom takes place at this point when the animal is not breeding.
After passing through the earliest stages of their develop- ment in the genital rachis, the young reproductive cells may be found at the breeding season in all stages of development in thecelom. The ova do not exhibit any considerable changes except in size in attaining maturity. They are nourished either directly from the celomic fluid, or possibly (Cuénot, 1891) by the ameeboid cells acting as follicle-cells, though we have seen nothing to support this view. Extrusion of a polar cell (?) has been observed by us in an ovum only about half the definitive size (Pl. 5, fig.35, aands). In thespherical ripe ova (which measure ‘16 mm. in diameter) a distinct but very thin vitelline membrane is present, and a small quantity of food-yolk in the form of very small granules in the proto- plasm. The production of ova by the fertile vascular pro- cesses of the nephrostomes must be extraordinarily great, since the spacious body-cavity of a large worm is eventually filled to bursting with them by the end of February.
We have not followed the development of the spermatozoa in great detail. The youngest stage which we have found in the coelom contained eight spermatoblasts arranged round a vesicular-looking blastophore (PI. 5, fig.30). Further division and elongation of the outer ends of the cells to form the tails of the spermatozoa produces the stages seen in figs. 31 to 34. The masses of spermatids are not spherical, but disc-shaped, their thickness being only about one quarter of their long diameter. They contain a cavity, the remains of the blasto- phore, together with a small quantity of a slightly fibrous coagulum in the centre of the cavity. Curiously enough, perfectly ripe males were comparatively rare in March and May of this year, when mature females were abundant. In most cases the body-cavity was full of spermatids in great bundles,
32 F. W. GAMBLE AND J. H. ASHWORTH.
as in fig. 34, The ripe spermatozoa closely resemble those of A. Grubii, which have been accurately figured by Claparéde.t They measure ‘058 mm. in length, and possess a curiously shaped head,‘004 mm. in length, and an extremely long slender tail (054 mm. long). The head (figs. 28 and 29) is divisible into three regions,—a rounded disc-like cap (S.) at the anterior end, which is partially divided by a median groove ; the nucleus (N.), which is large and oval in shape; and the “ middle piece ” (M.), which bears posteriorly a depression into which the tail is inserted. This depression is formed only at the time when the spermatozoa are fully ripe. The tail (7.) in the specimens which we have been able to obtain appeared to be a somewhat stiff filament, which could only be bent to a comparatively small extent.
The breeding season of the “‘ Laminarian” variety of Are- nicola marina lasts from February to May on the Lancashire coast. The large black ‘‘ worms” which may be dug out during the great spring tides of these months are then dis- tended with ova or spermatozoa. Males and females are not distinguished by external characters, but owing to the slight discharge of gonads from the nephridiopores consequent on the tense condition of the body, it is often possible to distin- guish the sex of an example without dissection. It is at present impossible to state how long these Arenicola live and how many times they breed. :
The ordinary littoral lugworms of the Lancashire coast and of the Isle of Man are not mature in the spring, and contain at most a few very small eggs. In the summer (August) of 1896 we found mature specimens, and we believe that this variety breeds through the summer, commencing at about the time when the deeper water form has ceased.
Relation of the Nephridia to the Reproductive System.—As is well known, the ova and spermatozoa escape by the nephridiopores, but it does not seem to have been noticed before, that in both males and females the bladders of the last five pairs of nephridia are specially enlarged (Pl. 3, fig. 15, B/.),
1 « Annélides de Naples,’ 1868, pl. xix, fig. 2, C.
HABITS AND STRUCTURE OF ARENICOLA MARINA. 33
and contain mature ova or spermatozoa, so that upon irritation a simultaneous discharge through all these apertures may occur. In one worm only eight inches in length the bladder of the nephridium was swollen with ova so as to measure 14 mm. in length and 6 mm. in width. During the discharge of ova from the female the eggs are caught by the slimy mucus covering of the body, and, owing to the movements of the animal, collect in strings round the body. We have not observed the formation of gelatinous capsules in which the eggs may be laid, since we have not worked at the oviposition of this species, about which nothing is at present known. At certain times of the year, chiefly in the spring, the nets used by shrimpers on the sandy coast near Lytham are almost choked by the balls of eggs, each moored by two “ cables ” to the sand. Whether these eggs belong to Arenicola remains to be seen, but their form differs from that of Phyllodoce found so commonly in early spring.
It has generally been assumed that the number of nephridia and gonads occurring in Arenicola marina is typical or fairly typical of the genus, and it is usually stated that the number of both these organs is a small one (five or six), An investigation of several other species of Arenicola, the results of which we hope shortly to publish in full, have shown that A. Grubii and A. Claparédii have five pairs of nephridia, and apparently the same number of gonads, whereas A. ecaudata has no less than thirteen pairs of nephridia, twelve of which bear large and complicated gonads of a size and complexity which is scarcely equalled by any other Poly- chet. What relations exist between A. marina and the other species of the genus cannot be discussed here, but it may be stated generally that the genus exhibits greater variety in the development of several systems of organs than has been hitherto suspected, and that it is no longer possible to exemplify the characters of Arenicola as a genus by using their particular grade of development in A. marina as a type.
vol. 41, part 1.—NmW SER. Cc
34 F. W. GAMBLE AND J. H. ASHWORTH.
12. General Summary.
The following is a recapitulation of the new points which we have found in Arenicola marina.
1. On the Lancashire coast, and probably elsewhere, two well-marked varieties of Arenicola marina occur, differing, as the following table shows, in general appearance, in their habits, in the structure of their gills, and periods of maturity.
Colour.
Name. Habitat. ——— Gills. ran Adult. | Young. ;
“Shore lugs,””/The sandy and muddy/Greenish | Semi- | Moderately} July, or littoral | shores of bays, estu-/brown or) transpa- developed. | August. variety, 6—8") aries, and harbours,| reddish |rent, yel- Branches long, excep- | extending from high| black jlowish or with 3—5 tionally 10” | water mark to and brown | pairs of sometimes beyond _gill-plumes low tide level Burrow U-shaped
Worms,” or/The sandy shore ex- Black orDark red, Very well | January Laminarian | posedat extreme low very dark opaque developed. | to May.
variety, | spring tides, occa-| brown | Branches 8—15'in | sionally extending with usually length above this. | about 12 ‘Burrow a_ vertical, pairs of di- shaft | | chotomous- ly arranged
| plumes
2. The cilia lining the central or gastric region of the alimentary canal are specially arranged (1) on the sides of a ventral groove which is continued to the anus, and (2) on curved shallow grooves running downwards and backwards into the former. The current caused by the action of these cilia carries a stream of mucus and of digested food slowly backwards and away from contact with the mass of sand in the gut. As these grooves are in close connection with parts of the visceral plexus, absorption may take place from them.
While the ventral groove is morphologically equivalent to the similar structure of Oligognathus (described by Spengel?),
1“ QOligonathus Bonellie,” ‘ Mitt. Zool. Stat. Neapel,’ iii, 1882.
HABITS AND STRUOTURE OF ARENICOLA MARINA. 35
and probably to the ‘‘ siphon ” of Capitellids, we have seen no reason for regarding it or any other part of the alimentary canal as “‘ respiratory ”’ in function.
3. In the circulatory system the two hearts each contain a cardiac body. This structure is composed of masses of granular and vacuolated cells, projecting into the cavity of each ventricle. Functionally they may be regarded as glandular valves pre- venting the reflux of blood into the gastric sinuses. While previously unknown in Arenicola, the ‘cardiac body” has been long known in allied genera (Ophelia, Tyrophonia, Chlorhzema), but as an unpaired structure in the dorsal vessel (Schaeppi, 1894). Hence, though histologically similar, it is very doubtful whether the paired structure of Arenicola, which has no connection with the dorsal vessel directly, is homologous with the unpaired organ of other Polychets.
Contrary to Wirén (1896), we regard the dorsal vessel as a distinct structure, the gastric blood-system as a plexus, and we find that the nephridia and body-wall, as well as the gills, are well supplied with capillaries.
4. Both the large pinnately-branching, and the smaller dendritic, types of gill occurin A. marina. The usual state- ment that the latter type of gill characterises this species, and that the former type is characteristic of A. cristata, must therefore be modified.
5. The brain is divided by a narrow cleft throughout the greater part of its length. The anterior cornua supply the prostomium, the buccal papille, and give off the cesophageal nerve-connectives. ‘The middle region of the brain supplies the upper part of the prostomium, and the posterior cornua innervate the nuchal organ.
_ In young specimens the almost uniform covering of ganglion-
cells of the brain is in close contact with the peculiar and complex sensory epithelium of the prostomium, but in old specimens of the “ Laminarian” variety fibrous outgrowths from the dorsal and lateral surfaces of the brain scatter this ganglionated covering.
6. The nuchal organ, though apparently single, shows traces
36 F. W. GAMBLE AND J. H. ASHWORTH.
of a double origin. It is probably an olfactory organ, and is developed from the posterior region of the prostomium.
7. The otoliths consist of quartz grains surrounded by a deli- cate chitinoid film, as Ehlers stated. The peculiar commotion observed in otocysts mounted in sea water was not noticed in others examined in celomic fluid. Hence the motion is probably a result of diffusion currents.
8. The first pair of nephridia are in process of reduction. In the others the form of the funnel at an early stage is described and figured. In adult examples the terminal portions of the nephridia act as receptacles for the ripe ova or spermatozoa.
9. The specific gravity of the coelomic fluid varies slightly, but is on the average (including the corpuscles) 1:0288, thus being only very slightly denser than sea water (1°0264).
10. The general analogies of Arenicola with certain other limnivorous Cheetopods are very striking. With the Sipunculids the Arenicolidz agree in the chitinous spines tipping the pro- boscis papille, the buccal papillz, the strong retractors of the “ proboscis,” the capacious and largely unsegmented ceelom, the general character of the musculature, the thin-walled looped alimentary canal with its ciliated ventral groove, the action of the body-wall in producing waves of coelomic fluid auxiliary to the process of burrowing and defzecation, and lastly, the pig- meuted nuchal organ. If we acknowledge the many points of agreement, which have for the most part arisen indepen- dently, between these two distantly related families under similar conditions of life, the true relationship between Arenicola and other genera of Polychets can only be ascer- tained by exercising the greatest caution in not confusing convergent adaptational characters with true genetic resem- blances.
1838.
1880. 1881. 1882.
1883.
1883.
1887. 1887.
1887. 1888.
1889. 1891. 1891. 1892.
HABITS AND STRUCTURE OF ARENICOLA MARINA. 37
13. LITERATURE. Mitne-Epwarps, A.—“ Recherches sur le circulation des Annélides,”’ * Ann. Sci. Nat.,’ sér. 2, t. x, 1838. Cosmovici.— Arch. Zool. Expt.,’ vol. viii, 1879-80. VeRRILL, A. H.—‘ Trans. Connecticut Academy,’ vol. iv, pt. 2, 1881.
KRuKENBERG, C. F. W.—‘ Vergleich. Anat. Studien,’ Zweite Reihe, Zweite Abtheil., Heidelberg, 1882, p. 87.
Wrireiw.— Chetopoder friin Sibiriska,” ‘ Vega-expeditionens Vetensk Takttag,’ Bd. ii, 1883.
Levinsen.—“ Systematisk-geograph. Oversigt over de Nordiska Annu- lata,” ‘ Vidensk. Meddels. Kjobenhavn,’ 1882-3.
CunnINGHAM.—‘ Quart. Journ. Micros. Sci.,’ xxviii, 1887-8, p. 239.
MaRENZELLER.—‘ Zoologische Jahrbiicher,’ Abth. f. Systematik, Bd. iii, 1887, p. 12.
Winty.—‘ Kong]. Vetenskamps-Akad. Handlinger,’ 1886-7.
CunNINGHAM AND Ramacr.—‘ Trans. Roy. Soc. Edinburgh,’ vol. xxxill, 1888.
MacMuny.—‘ Quart. Journ. Micros. Sci.,’ xxx, 1889-90, p. 74. Cutnwot.—‘ Arch. Zool. Expt.,’ sér. 2, vol. ix, 1891. Davison.—‘ Geol. Magazine,’ vol. viii, 1891, p. 489. Euters.—‘ Zeit. f. wiss. Zool.,’ vol. liti, Suppl., 1892.
18924, Huters.—‘ Gottingen Nachrichten,’ No. 12, July 27th, 1892.
1893. 1894. 1896. 1896.
Brenuam.—‘ Journ. Marine Biol. Assoc.,’ N.S., vol. iii, p. 48. ScHaEPri.— Jenaische Zeitschr.,’ Bd. xxviii, 1894, p. 247. Racovitza.—‘ Arch. Zool. Expt.,’ sér. 3, vol. iv, 1896. Kytz.—‘ Annals and Mag. Nat. Hist.,’ ser. 6, vol. xviii, 1896.
38 F. W. GAMBLE AND J. H. ASHWORTH.
EXPLANATION OF PLATES 1—5,
Illustrating Mr. F. W. Gamble’s and Mr. J. H. Ashworth’s paper on “The Habits and Structure of Arenicola marina.”
List or REFERENCE LETTERS.
A. Cr, Anterior cornua of the brain. dz. Anus. Az, “ Auricle’”’ of the heart. B/7. Bladder or terminal part of the nephridia. Blph. Blastophore of spermatoblast. 8B. Pap. Papille of the buccal mass. Br. Gills. Br. Af. Branchial afferent vessels. Br. Hf. Branchial efferent vessels. BR. Brain. B.S. Blood spaces in the heart. 2B. Sf. Sheath of retractor muscle en- closing the buccal mass. Bucc. M. Buccal mass. Card. B. Cardiac body. Chi. Tiss. Chlorogogenous tissue on the stomach and ventral vessel. Chu. Notopodial chete. C.F. Cardiac fibres. C.Sp. Caudal septa. D. L. V. Dorsal longitudinal vessel. D.Nph. Dorsal lip of the nephrostome. Dp. Ph. Diaphragmatic pouch. Dphm. }~* Diaphragms or anterior septa. Zp. Kpi- dermis. G. Refringent granules in ccelomic cells. Ga. Ganglion-cells of the brain. Gast. Lat. Lateral gastric vessel. Gast. V. Gastric vessels. G. F. “Giant fibres.” Gl. Op. Opening of the esophageal glands into the cesophagus. G.S. Granular cells of the heart. Gz. Ventral groove of ali- mentary canal. G.V. Gonidial vessel. Int. V. Intestinal vessels. MV. “Middle piece” of spermatozoon. JM. Circ. Circular muscles. Mes. D. and Mes. V. Mesenteries supporting the dorsal and ventral vessels between the first and second diaphragms. M27. “ Metastomium,” or achetous portion of the body immediately following the prostomium. J. Long. Longi- tudinal muscles. I/O. Mouth. J. Od. Oblique muscles. MM. Pav. Parapodial muscles. Jf, Pr. Retractors of the prostomium. WV. Nucleus. WV. 4f- Afferent vessel to the nephridia. 4. C. Ventral nerve-cord. V. Cap. Ne- phridial capillaries. WV. Hf Efferent vessel from the nephridia. NWZV. Ne- phridial longitudinal vessel. Nm.1!~!° Neuropodia. No. !-* Nephridiopores. NPH.** Nephridia. MPHM. Nephrostomes. WS. Nervous elements and connective tissue round otocyst. Nw. Nuchal organ. Ww. Tr. Retractor muscle of nuchal organ. JV. V. Neural vessels. Oe. @isophagus. Oc. Comm. Circumcesophageal nerve-connectives. Oe. G/, Hsophageal glands. Oe. Gi. V. Vessel of cesophageal glands. Oe. Lat. Lateral cesophageal vessel. O. Of. External opening of otocyst. O7' Otocysts. OZ", Neck of otocyst. Odh. Otolith. OZ. N. Nerve to otocyst. Par. V. Parietal vessels. P. Cr. Posterior cornua of brain. Px. Protractor of notopodium. Pr. Prostomial lobes. Rn. Retractor of notopodium. §. Cap of spermatozoon, 8S. VY. Sub- intestinal vessels, 7, Tail of spermatozoon, V. Ventricle of the heart,
HABITS AND STRUCTURE OF ARENICOLA MARINA. 39
Vac. Vacuole. Vac. C. Vacuolated cells of the heart. V. Nph. Ventral lip
of nephrostome. V.V. Ventral vessel. J. 7. ITI. IV. &c. Somites beginning with the first chetigerous.
PLATE 1.
Fic. 1.—The anterior end of a large specimen of the “ Laminarian ” variety seen from the left side, to show the external features, the seg- mentation of the body-wall in relation to the internal metamerism, the nephridial apertures, and the commencement of the branchial region. The acheetous region following the fully everted buccal mass (Buce. MW.) extends forwards as far as the groove indicating the insertion of the first diaphragm dorsally (Dphm.!). We have considered the first chetigerous annulus and the annulus behind this, as composing the first chetigerous somite (JZ), although we are fully aware that, owing to the obliquity of the first diaphragm, and the absence of landmarks in the achetous region in front of this septum, it is somewhat hazardous to delimit this first chetigerous somite. x 4%.
Fig. 2.—View from the right side of two somites from the anterior part of the branchial region of a specimen of the ‘‘ Laminarian ”’ variety 7 inches long. The fourth gill is shown in detail, while the third and fifth are cut down to the base of the main branches. The large size of the spreading branches and the somewhat pinnate arrangement of the lateral twigs distinguish the gill of this variety of A. marina from that of the ordinary shore lugworm seen in figs. 8 and 4. The webbing at the bases of the branches is generally much more marked in old black examples than in immature dark red specimens such as the present. x 14.
Fic. 3.—Fifth gill of the right side of a shore lugworm 8 inches long, to show the features characteristic ef the littoral variety of Arenicola marina. The branches are united by extensive connecting membranes, between which the blood-vessels of the gill are faintly visible. x 14.
Fic. 4.—The first gill of the right side from the same specimen as Fig. 38. The ventral branches are apparently the last to develop, and are only just budding off the secondary leaflets. x 14.
PLATE 2.
Fic. 5.—Dissection of a large “ Laminarian” variety, to show the general characters of the internal anatomy (conf. pp. 9 to 10). The body-wall has been cut along the mid-dorsal line, the flaps pinned back, and the alimentary canal turned over to the left side. The special features shown are the vascular system, the nephridia, the septa, and muscles. xX 2.
40 F. W. GAMBLE AND J. H. ASHWORTH.
PLATE 3.
Fic. 6.—View of a vertical longitudinal section of Arenicola marina taken somewhat to the left of the middle line. The thickness of the body- wall is exaggerated. The stomach has been cut away behind the heart, to show the oblique muscles and the second nephridium. The main blood- vessels only are indicated, the object of the figure being to show the exact position of the three diaphragms (Dphm. 1%), of the buccal or proboscidal sheath (B.Sh.), and the relations of these to the external segmentation. x 3.
Fie. 7.—Chitinoid spines covering the buccal papille of that part of the proboscis which is first protruded during eversion. ‘They may be compared with the figures of ‘ hooks” from the proboscis of Sipunculids (e.g. Phasco- lion) shown in Selenka, ‘Die Sipunculiden.’ Caustic potash preparation. x 50.
Fie, 8.—Papille in situ on the base of the proboscis of young worm. x 6.
Fie. 9.—A gronp of neuropodial sete from a very young Arenicola marina 16mm. long. ‘The shape and strongly-toothed ridge distinguish these sete from those of the adult (figs. 1l and 12). ‘The youngest sete are on the left side of the figure. x 300.
Fie. 10.—Notopodial seta 6 mm. long. x 16.
Fic. 10a.—The tip magnified. x 50.
Fre. 108.—The toothing on the notopodial seta highly magnified. x 450.
Fic. 11.—Neuropodial seta (x 20), and enlarged (x 120).
Fic. 12.—A group of developing neuropodial sete in situ in the neuro- podium (Vm.) of a “ Laminarian” specimen. x 70. Proc. is referred to on De ws
Fic. 13.—The fourth and fifth chetigerous segments of the left side of a large mature “‘ Laminarian ” specimen. The first two nephridia are shown. The figure is a study of the blood-vessels of the nerve-cord, of the oblique muscles, and of the connection between the nephrostomial and the dorsal longitudinal vessels (D. Z.V.). xX 3%.
Fie. 14.—The first nephridium from the specimen shown in fig. 13, seen from the dorsal surface, to show the gonidial vessel (G. V'.) bearing blind, vascular processes. The gonidial vessel on this nephridium is sterile. x 4.
Fic. 15.—Fifth right nephridium of an adult male, to show the bladder distended with spermatozoa. The nephrostome is widely open. Seen on February 24th, 1897. x 4.
Fic, 16.—The second nephridium of the right side of a specimen 29°5 mm.
HABITS AND STRUCTURE OF ARENICOLA MARINA. 41
long, seen from the dorsal surface, to show the gonidial vessel (G. V.), the commencing processes of the dorsal lip, and the position of the external opening (Wo.2) with regard to the neuropodium (Vm*.). The ventral lip of the nephrostome (V. Vph?.) is seen through the dorsal one. x 60.
Fre. 17.—Funnel of the first left nephridium from the same specimen as fig. 16, seen from the right side, to show the vertical position of the nephro- stome and the commencing processes on the anterior lip. x 90.
PLATE 4.
Fic. 18.—The second right nephridium from a specimen 44 mm. long. Dorsal view, to show the remarkably complete capillary circulation and the extension of the vessel of the dorsal lip to form the gonidial vessel (@. V.). The ventral lip (V. WpA?.) is seen by transparency. X 65.
Fic. 19.—Three views of the anterior end of a specimen 8 inches long (littoral variety), to show the prostomium, nuchal organ, openings of the otocysts, and the secondary annulation of the skin. a. From the left side. B. From above. c. From below. x 12.
Fre. 20.—Transverse section across the middle of the prostomium to show the brain, the three prostomial lobes, and the rich blood-supply of this region. The brain lies in the central prostomial lobe, and its covering of ganglion- cells is closely applied to the overlying sensory epithelium. The section is cut across in the region of the posterior cerebral cornua (P. Cr.). X 65.
Fic. 21.—Transverse section of the same series as Fig. 20, across the nuchal organ, Vw., the hinder cornua of the brain, and one otocyst, with its contained otoliths. x 65.
Fic. 22.—Transverse section of the body a short distance behind the third diaphragm at the level of the openings of the esophageal pouches (GZ. Op.). The external aperture of the second nephridium is shown on the right side. The subdivision of the body-cavity into three longitudinal portions, and the structure of the cesophageal pouches, are well seen. X 38.
Fic. 23.—Transverse section of the body in the branchial region at the level of a parapodium. ‘lhe neuropodium is cut through its entire length on the left side. On one side of the nerve-cord a retractor muscle from the notopodium arises, on the other an oblique muscle. The vascular supply of the body-wall, sete, and gills is well seen. x 38.
PLATE 5.
Fic. 24.—Ameeboid and spindle-shaped cells of the celom. x 1000.
Fig. 25.—Sagittal section of the brain slightly to the left of the middle line, from a young littoral form about 3 inches long. The mass of ganglion-
42 F. W. GAMBLE AND J. H. ASHWORTH.
and glia-cells underlying the epithelium of the prostomium is distinct ; some of the cells of the latter are shown bearing sensory processes. The nuchal organ, Vw., is cut at its full depth. x 85.
Fre. 26.—View of a dissection of the brain, cesophageal connectives, oto- cysts, and the buccal sheath. The commencement of the neural vessels from capillaries of the organs just mentioned, is shown. Seen from the dorsal surface. The buccal mass, cut transversely, lies in the centre of the figure. x 6.
Fic. 27.—An otocyst with the otoliths composed of quartz grains. The sensory epithelium and the surrounding nervous and supporting cells are seen. x 160.
Fig. 28.—Otoliths to show the chitinoid covering of the quartz grains. x 500.
Fre. 29.—Ripe spermatozoon seen on March 10th, 1897. Length of head 4p, length of tail 54 4. x 3000.
Fic. 294,—Head and portion of tail of an immature spermatozoon seen on February 22nd, 1897. x. 38000.
Fries. 30—34.—Stages in the development of the spermatozoa.
Fig. 30.—The 8-celled stage, in which the spermatoblasts leave the tesiis.. =< 500.
Figs. 31 and 32. Later stages. x 500.
Fig. 33.—Two cells from a stage much later than the preceding, showing the commencement of the tail. x 2000.
Fig. 34.—Discoidal mass of almost ripe spermatozoa. xX 500.
Fic. 35.—Developing ova. (a and 4) show a polar body (?) x 250. (c) is a ripe ovum enlarged 125 times.
Fic. 36.—Longitudinal section of the heart of an Arenicola ou mm, in length, to show the cardiac body. x 82.
Fre. 37.—Histology of a portion of the cardiac body of fig. 36. x 500.
Fic. 38.—Longitudinal section of the heart of a young Arenicola 65 mm. in length, to show the cardiac body at an early stage of development. x 50.
THE ASEPTIC CULTIVATION OF MYCKETOZOA. 43
The Aseptic Cultivation of Mycetozoa. By
Casper O. Miller, M.D.
With Plates 6 and 7.
OBSERVATIONS ON THE CULTIVATION OF MyYCETOZOA.
Untit the work of de Bary nothing was known about the development of Mycetozoa further than that they appeared as a slimy mass from which the sporangia were formed. He made a short report (1) on the development of the zoospores from the spores at the “ Naturforscherversammlung,” in Gottingen, in 1854, which was followed by his other publications (2, 3). He speaks (8) of keeping portions of plasmodia in glass dishes containing water, or on slides, but they died in a few days without forming sporangia. Sporesof Aithalium septicum, planted on moistened tan on the 2nd of May, showed at the beginning of July colourless plasmodia, which continued through July without further development. Another culture of spores of the same plasmodium, planted the 13th of August, developed many zoospores, and on the 8th of October plas- modia were seen. Spores of Lycogala, planted in a dish containing water and decaying pine-wood, developed zoospores within twenty-four hours ; about the fourteenth day there were plasmodia present, which at the end of a fortnight had died without forming sporangia. He also planted spores of Stemo- nitis obtusata on decaying pine-wood, and found plasmodia on the fourteenth day, but they did not develop further. De Bary was unable to determine whether the plasmodia de-
44, CASPER O. MILLER.
veloped from a single zoospore or by the fusion of a number of zoospores.
Cienkowski (6 and 7) planted spores of Licea pannorum, Wallr., on decomposing carrots, and obtained plasmodia. He also planted the spores in water placed on slides, and saw the zoospores fuse to form plasmodia. Spores of Physarum album=Chondrioderma difforme, planted on microscopic- ally small portions of vegetable fibre, developed plasmodia on the fourth day, and twenty-four hours later they fructified, so that under good conditions they completed their cycle of development in five days.
Lieberktihn (9) described a plasmodium which he found in the bottom of a glass vessel in which spongillia were being cultivated,
Cienkowski (16) cultivated Didymium libertianum in water. In one to two weeks plasmodia appeared in the water or creeping on the wall of the vessel.
He also found a plasmodium in fresh water containing alge. He studied it in hanging drop-cultures and on the slide. He thought it probably was the same species which Lieberkiihn had studied. Sporangia did not form in any of his cultures.
Stahl (22) cultivated Aithalium septicum on moist tan, and saw a species of Physarum form small-stalked sporangia on a filter-paper culture. He did not use any aseptic pre- cautions, and does not state how long it took the sporangia to form after planting the spores.
Ward (25) found a plasmodium which formed sporangia on the roots of hyacinths which he was cultivating in water con- taining a small percentage of salts of lime, magnesia, potash, and soda. He then made a decoction of hyacinth roots, which he boiled and used to make drop-cultures. By planting the spores he succeeded in getting the zoospores and plasmodia in drop-cultures and on slides without other forms than bacteria. The cover-glasses were heated, and the cardboard used in making the moist chambers was boiled.
Strasburger (26) obtained Chondrioderma diff. by placing macerated stalks of Vicia faba on moistened filter-
THE ASEPTIC CULTIVATION OF MYCETOZOA, A5
paper under a bell-jar ; the sporangia developed after a few days. He also made drop-cultures of the spores of Chond. diff. in a decoction of cabbage-leaves or bean-stalks, leaving frag- ments of vegetable fibre in the fluid. He heated the cover- glasses and needle used in making the inoculations, but added alge and bacteria to the cultures. In many of the cultures the development did not go further than the formation of microcysts, but in more favorable cultures plasmodia de- veloped which fused with each other, and on the fourth or fifth day they crawled out from beneath the cover-glass and formed sporangia.
Wingate (82), in describing Enteridium rozeanum, says that Roze (12) cultivated plasmodia in earthenware dishes filled with sphagnum and water, into which he thrust dead branches of trees, pieces of decayed stumps, &c., which were taken from the neighbourhood of Paris to America. He ob- tained various plasmodia, and studied them until they formed sporangia. I have unfortunately not been able to procure the original work by Roze.
Lister (84) cultivated Chond. diff., and obtained the sporangia in from ten to fourteen days after planting the spores. ‘The writer has only seen a short report of the paper in Just’s ‘ Jahresbericht,? so that he does not know what methods were employed.
Celakovski (88) used the method which Pfeffer (35) found useful for obtaining plasmodia. He placed dried stalks of Vicia faba, or the leaves and stalks of other plants, par- ticularly of Typha latiflora, in broad crystallising dishes, poured enough water in the dishes to cover the greater portion of the nutrient material, covering the dishes with suitable lids, and sterilised them at a boiling temperature. He then planted spores of Chondrioderma diff. and Didymium macro- carpon. In from six to fourteen days plasmodia of the former were found in the cultures. He frequently obtained the two plasmodia together by simply moistening the stalks of Vicia faba, and placing them in a covered dish. By repeatedly transplanting he obtained the Didymium alone, without the
46 CASPER O. MILLER.
Chond. diff. He fails to mention how long the interval was between the planting of the spores and the formation of the sporangia. He also planted the spores of Arcyria punicea, Pers., Trichia nutans, Libert, and Stemonitis dictyo- spora, Rostaf., on sterilised decayed beech-wood in flat crystallising dishes, containing water to the depth of ‘5 cm. He did not see the plasmodia of the first two in the water; they developed in the interior of the wood, and only appeared on the surface when the sporangia were formed. The Ste- monitis developed plasmodia in the water, and fourteen days after their first appearance the sporangia were formed. He fails to state how long it took for the plasmodia to develop after the spores were planted.
Although Celakovski sterilised his nutrient media and the vessels, no observations were made as to the presence or absence of contamination. It is very difficult to prevent the contamination of cultures in a large flat dish, when the lid is removed or lifted for the purpose of examining the culture.
My cultures were first made as controls for another series of experiments, but the results seem of sufficient interest to publish as a separate paper.
Ture MertTHopS EMPLOYED.
In the summer of 1890, while making some experiments at the pathological laboratory of the Johns Hopkins Hospital, to determine what Protozoa one finds in the air, a number of flasks, containing sterilised water with 2 per cent. of milk added, were left uncorked for a number of days. Of the flasks one showed zoospores of Mycetozoa, which were trans- planted a number of times. The zoospores and plasmodia developed, but no sporangia appeared.
The first systematic attempt of the writer to cultivate Myce- tozoa was made at the Zoological laboratory at Heidelberg in 1893.
A culture was prepared in the laboratory for the study of Infusoria, by simply placing unsterilised hay in a glass jar with
THE ASEPTIC CULTIVATION OF MYCETOZOA. 4.7
unsterilised hydrant water, the jar being covered by a glass plate. On examining the culture ten or twelve days later zoospores of plasmodia were found in the water, and sporangia of plasmodia developed on the hay a few days later.
A number of similar cultures without aseptic precaution were then made of hay gotten from different sources, and they all showed the presence of plasmodia.
Next a series of cultures were made in tall narrow beakers, they being first closed with a large plug of cotton and sterilised in a hot-air steriliser. The beakers were then filled about half full with unsterilised hay. Care was taken to first wash the hands and sterilise the scissors, so as to be moderately certain that no spores of plasmodia were introduced from the hands or instruments. Water which had been sterilised in flasks was then poured into the beakers, until most of the hay was submerged, care being taken not to cover it completely.
In a few days the hay projecting from the surface of the water was covered with mould fungi. A pair of sterilised forceps was then used to remove the stalks of hay covered by the fungi, care being used to loosen up the hay so as to have some of it projecting above the water. If the hay is entirely submerged plasmodia may not develop, but when prepared as above, all of the cultures prepared with hay, whether gotten in Heidelberg or Baltimore, developed plasmodia. It would appear that plasmodia are constantly present on hay in one form or another.
Cultures prepared in the same way with the stalks of wild carrot picked out from the hay did not develop plasmodia.
A series of cultures were made by putting dried chestnut and oak leaves in sterilised Erlenmeyer flasks with sterilised hydrant water. In a number of these cultures plasmodia de- veloped. —
Elsewhere (45) the writer has described the aseptic methods employed in the cultivation of Protozoa, but for Mycetozoa some modifications are necessary. They will grow in sterilised dilute hay infusion, or 2 per cent. of milk in water, but for the formation of sporangia it is in general advantageous, and for
48 CASPER 0. MILLER.
some forms essential, to furnish them a mechanical support as a means of getting out of the water.
The medium which has proven the most generally useful is prepared as follows. A handful of hay is placed in a jar and washed repeatedly until the water remains colourless. It is then covered with fresh water and allowed to soak overnight. The following day the water is poured off, filtered, diluted with fresh water until it is of a white-wine colour, and 2 per cent. of milk is added to the infusion. It is then filtered, put into a flask, and sterilised for future use. The macerated hay is cut and placed in Erlenmeyer flasks ; the first portion is cut short enough so as to form a tolerably compact layer in the bottom of the flask to the depth of 1 cm.; the rest is cut sufficiently long to form a very loose layer reaching about two thirds the way up the sides of the flask, care being taken not to allow any of the stems to reach the cotton. Sufficient water is placed in the flasks to cover the hay, and they are sterilised for fifteen minutes. On the following day fresh water is substituted, and they are again sterilised. The water is once more poured off, and enough of the hay infusion and milk previously prepared is added until it is about 1 cm. deep. ‘The flasks are then sterilised in a steam steriliser for ten minutes on three suc- cessive days. They are then ready for use.
After soaking the hay for twenty-four hours in water, and boiling it several times in fresh water, about all of the soluble substance has been extracted, and the diluted hay infusion with 2 per cent. of milk is added; we thus have a medium of tolerably uniform composition.
Of the cultures gotten from the air several contained mould fungi, which were eliminated by putting the cultures in the oven at a temperature of 37° C.
One culture contained chroococci, and these were eliminated by keeping a series of cultures in a dark closet. It is not possible in every case to eliminate other protozoic forms that may be present, but one may at times succeed by taking ad- vantage of the fact that the encysted forms withstand drying. In this way one may sometimes succeed in separating Myce-
THE ASEPTIC CULTIVATION OF MYCETOZOA. 49
tozoa from the Infusoria, Amcebe, and other protozoic forms found in hay infusions.
The cultures are usually transplanted by means of a sterilised pipette.
Bacteria are found in all the cultures, and studies have been made with the view of finding out what effect bacteria have on the growth of Mycetozoa, and what bacteria, if any, are more favorable to their growth.
It is not the writer’s purpose to discuss the influence of the bacteria in this connection, but he will leave it for a future communication.
THe Mycrtozoa cULTIVATED. Physarum cinereum.
This was the first plasmodium from the air which was culti- vated. It will grow and form plasmodia in water with 2 per cent. milk or in dilute hay infusion. The best cultures are obtained when the hay also is present as described above.
In all the cultures where sporangia are formed, the plas- modia grew in the fluid and crawled on the side of the flask above the fluid preparatory to the formation of the sporangia. Although the largest plasmodia form in cultures containing hay, yet the sporangia only form on the glass.
The plasmodia spread out on the glass in the form of a yellowish-white network, consisting of primary trunks from which run branches anastomosing with each other, the net- work becoming finer as the periphery is approached. At the periphery there is a more or less flattened perforated proto- plasmic plate with a scalloped border. In the cultures not containing hay the principal trunks extend to the water; in cultures containing hay the plasmodia spread out from stems of hay leaning against the side of the flask (fig. 2), and it cannot be determined whether branches extend to the water.
In the more vigorous cultures the plasmodia are large enough to cover the whole inner surface of the flask above the water, but do not pass to the cotton plug.
voL, 41, part 1.—NEW SERIES. D
50 CASPER O. MILLER.
After remaining on the glass above the water for from two to twelve days, the protoplasm collects at one or a number of points at the periphery of the network, and forms sporangia, leaving behind a so-called hypothallus, retaining the shape and outlines of the original network, but much paler in appear- ance. The sporangia vary in number according to the size and vigour of the plasmodia. In one culture there were only two sporangia; in other cultures the sporangia form groups, the larger of which may contain from seventy to eighty spo- rangia. In the first stage of the formation of the sporangia the protoplasm is of a more yellow colour than that of the net- work. As the sporangia assume their completed shape the colour becomes a brownish red, which changes to a greyish white when the development is completed.
The sporangia are sessile, resting on a broad base. When isolated they are round, oval, or kidney-shaped. At times they are united, forming a long drawn-out sporangium with con- strictions at irregular intervals. The small oval or round sporangia may measure as little as 0°5 mm. in diameter, the long drawn-out ones may measure as much as 7 mm. On examination with the low power by reflected light the surface shows irregularly shaped small white elevations, between which are darker areas. Under the high power these white areas are seen to consist of aggregations of coarse granules, which dissolve on the addition of hydrochloric acid with the forma- tion of gas bubbles. The sporangia have no columella, and the sporangium wall is colourless. The capillitium is made up of a network of thin, colourless fibres attached to the wall of the sporangium. At the point of communication of the fibres there is a more or less flattened triangular or polygonal thick- ening, containing granules of lime. The spores are smooth and of a brownish-violet colour, measuring 8°5—13°5 w in diameter. The majority of the spores are spherical, but occa- sionally there are oval or irregular forms. From a study of the structure and the arrangement of the sporangia of this plasmodium, it would appear that it is identical with Phy- sarum cinereum, Pers.
THE ASEPTIC CULTIVATION OF MYCETOZOA. 51
Stemonitis.
In July, 1892, another series of flasks containing sterilised milk, 2 per cent.in water, was exposed for a month to the air; they were then closed with cotton and examined. In three of the flasks were flagellate bodies which the writer thought corresponded to the description given by Bitschli (29) of Mastigameba. From these flasks cultures were made, and in one of the transplantations plasmodia developed. At that time the writer had not studied plasmodia sufficiently to recognise the relationship between the zoospores and the plasmodia, and inasmuch as there were similar flagellates in all three flasks, he concluded that the plasmodia and the flagellates were independent forms. Nothing further was done with the cultures until July, 1893, when they were again transplanted, and plasmodia developed in all three cultures. At that time they were cultivated with 2 per cent. of milk in water, and in hay infusion. The zoospores and the plasmodia grew, but there was no formation of sporangia. The cultures were then made in flasks containing hay, with the idea that the plasmodia would be enabled to get out of the water to form the sporangia. Upon placing hay in the flasks a number of cultures formed sporangia.
All three plasmodia belong to the genus Stemonitis.
From a study of the sporangia there is no difficulty in deciding that two of them are distinct, while it is not so evi- dent that the third one differs from one of the others, but the writer is inclined to the opinion that it is also a distinct species. The writer is not familiar with the American My- cetozoa, and has not been able to get all of the literature on the subject ; it is possible that they agree with species already named. It will be necessary to refer to each of these cultures, and it will be more convenient simply to designate them as Stemonitis A, B, and C.
At a variable period after the inoculation of the cultures there appears, rather suddenly, a large yellowish-white plas- modium lying on the hay at the surface of the water, which
52 OASPER O. MILLER.
may cover an area measuring about 1 by 2 cm. They are composed of a network of short, thick, anastomosing branches, from the periphery of which extend branching, sausage-, horn-, or club-shaped prolongations. There is not much change in the appearance of the plasmodium for forty-eight hours ; during this time it may change its location on the hay, but the motion is a slow one. At the end of this time the motion becomes more rapid. The plasmodium moves some distance from the surface of the water, and settles upon the hay or on the glass. In one of the cultures the whole plasmodium had moved 6 cm.in four hours. When it has found a suitable place the peripheral prolongations are drawn in, there is no longer any evidence of the presence of a network; it then appears as an oval or rounded, conical or flat, yellowish-white mass, the surface of which is covered by a number of closely crowded small hemispherical prominences. From each of these prominences is formed a cylindrical sporangium. Soon after the sporangia assume their permanent form, the yellow- ish-white colour begins at the base to change to a reddish colour, which gradually ascends to the apex, and finally becomes a reddish or dark brown colour.
It takes from twelve to eighteen hours from the time the plasmodium leaves the water until the sporangia are fully developed.
The well-developed sporangia are cylindrical, closely crowded, and placed more or less perpendicular to the mem- branous hypothallus, from which extends a branch going to the surface of the water, indicating the route which the plasmo- dium took. When the sporangia are formed on the glass the plasmodia take an oblique course up the side of the glass.
There are slight differences in the appearance of the plas- modia of the three cultures, but the difference is not alone sufficient to enable one to say that they are distinct species. The peripheral prolongations of Stemonitis B are usually longer and thicker than Stemonitis A. The network of Stemonitis C is a more open one than that of the other two.
THE ASEPTIC CULTIVATION OF MYOETOZOA. 53
In some cultures the sporangia are imperfectly developed.
A typical sporangium of Stemonitis A measures about 3 mm. in height, including the stalk, which is 0:167mm. The diameter of the sporangium is about 0°3 mm., and is usually uniform throughout. The sporangium may be thicker toward the apex or base, The apex is usually rounded, but at times is more acute ; the base may or may not be symmetrical. The measurement of the stalk given above is about the average, and applies to fig. 9. In a few instances the capillitium extends to the hypothallus; in other instances the stalk may be 0°5 mm. long. The columella tapers gradually from the base to near the apex, where it divides into several branches, becoming continuous with the capillitium. Occasionally one finds a spindle-shaped thickening of the columella. The primary branches of the capillitium usually come off at an acute angle from the columella, forming one series of anasto- moses, and then divide into smailer branches, which go obliquely to the surface network. The surface network usually extends over the entire sporangium. ‘The meshes of the net- work average from 8 to 33. On the surface network are distributed small wart-like thickenings. The colour of the capillitium is a brownish violet. The spores measure 7—18 1, and are of a violet-brown colour; the membrane is finely warted.
The sporangia of Stemonitis B (fig. 10) measures 3°5— 3°83 mm. in height, not including the stalk, which is about 1°37 mm. long. They are tolerably uniform in thickness, measuring about 0°27 mm. in diameter. The columella tapers gradually from the base to near the apex, dividing into branches which become continuous with the capillitium. The capillitium fibres come off at right angles to the columella, forming one series of anastomosing arches from which pass out secondary fibres placed perpendicular to the surface ; they break up into branches which become continuous with the surface network. The capillitium is of a dark violet-brown colour. At the point where the primary fibres anastomose one frequently finds membranous expansions which are more marked than in the sporangia of Stemonitis A, but these
54 CASPER O. MILLER.
membranous expansions vary a good deal in sporangia from the same culture. The spores are of a light violet colour ; they have a smooth membrane, and are tolerably uniform in diameter, measuring 7°9—8°5 mw.
The sporangia of Stemonitis C resemble those of Stemo- nitis B. The sporangia of Stemonitis C measure 3°3— 3'7 mm. in length, and 0°37 mm. in thickness. The columella is not infrequently bent on itself at about the upper four fifths. The secondary fibres of the capillitium are longer than in the sporangia of Stem. B. The stalk measures 0°68—1:16 mm. in length. The spores are smooth, of a brownish-violet colour, measuring 7'4—1] w in diameter.
It would therefore appear that the differences between the sporangia of Stemonitis B and C are not less than those which separate some of the forms which are described in works on the subject under different names. It is possible that further cultivation may show that they are the same.
Hay anp Lear Cu.ururges.
In cultures made with unsterilised hay in jars without aseptic precautions, or in flasks with aseptic precautions, one finds bacteria, fungi, monadina, infusoria, and plasmodia developing with uniform regularity. Chondrioderma dif- forme and some species of Didymium, usually micro- car pon, appear together orsingly, the Chondrioderma being most frequently present. As has been stated before, some plas- modium appears in every culture made with unsterilised hay.
By the drying method the Chondrioderma diff. and Didymium microcarpon have been separated and culti- vated aseptically in flasks. They both form sporangia on the hay, and on the glass above the hay.
In a culture of Chond. diff. made in dilute hay infusion with 2 per cent. milk added, which had been kept in the dark for several weeks and then placed in the light, sporangia formed under the surface of the water. The sporangia were small, round, or pear-shaped, and did not show the presence of
THE ASEPTIC CULTIVATION OF MYCETOZOA. 55
any granules of lime in the sporangium wall. In all the other cultures observed the sporangia formed on the hay or on the sides of the flask above the level of the fluid.
In speaking of the classification of his plasmodium, Ward (25) says, ‘It is, indeed, not improbable that we have here an aquatic form of Didymium difforme, one of the com- monest of our Myxomycetes; and if so, we have another proof of the all but uselessness of attempting to classify the lower organisms until we know more of their habits under vary- ing conditions.” From the writer’s experience, he questions whether Ward did not really have the ordinary form of Didy- mium diff.= Chondrioderma diff. in his cultures, and whether the character of the fluid in which they grew, and the other conditions surrounding them, did not cause the sporangia to form only on the roots under the water or on the moist roots above the water.
Didymium farinaceum was obtained from a culture made with unsterilised leaves taken from the forest.
In one flask containing leaves, and in two containing pine- needles, plasmodia developed and formed sclerotia above the water on the side of the flask, but no sporangia appeared, so it was not possible to determine what species they were.
Spores of Aithalium septicum obtained from a tan-pile were planted in flasks, and yellowish plasmodia developed, but no sporangia formed. Spores from several varieties of Stemonitis collected at Heidelberg were planted in flasks. The zoospores and plasmodia developed, but only one of them formed sporangia.
Spores of Ceratium porioides, gotten from a pine stump, dried and planted aseptically, developed zoospores which have been cultivated for about four years, and as yet the writer has failed to find any plasmodia or sporangia. So far as I have been able to discover, no one has succeeded in cultivating . plasmodia of any of the Ceratiomyxa.
56 CASPER O. MILLER.
The Time of the Appearance of the Large Plasmodia, and of the Formation of the Sporangia.
Plasmodia, as we usually find them in nature, appear rather suddenly on decaying wood, tan, or leaves, and within a short time they form sporangia. We know little about their pre- vious growth.
Some Mycetozoa may form sporangia during any of the warm mouths, while, according to de Bary (21), others are characterised by forming sporangia only during a short time in the year. As has already been mentioned, Cienkowski and Strasburger obtained sporangia on the fifth day after planting the spores of Chond. diff., and Lister obtained them in from ten to fourteen days. Celakovski (88) men- tions that the sporangia of Stemonitis dictyospora, Rostaf., developed fourteen days after the appearance of the plasmodia, but does not state how long it took the plasmodia to develop.
Rex (36) mentions having seen Stemonitis Bauerlinii form sporangia op a decayed log in the autumn, and the next summer the same species formed sporangia three times on the same log at intervals of a month. One cannot say that the spores fell back on the log, developed zoospores, and from these new plasmodia grew and formed sporangia.
In the cultures made with unsterilised hay in water the conditions are practically the same. The forms of the Myce- tozoa, whether microcyst, sclerotia, encysted zoospores, or spores, have been dried for months. The hay is placed in the water and kept at the room temperature. The sporangia of Chond. diff. appeared on the hay from the twenty-fourth to the twenty-ninth day. Crops of sporangia continue to be formed on the hay every few days for from two to four weeks.
Didymium microcarpon first show sporangia on the hay from the twenty-first to the twenty-fourth day, and continue to form sporangia for several weeks.
When there were only a few stalks of hay projecting above the surface of the water the sporangia appeared, but were less
THE ASEPTIC CULTIVATION OF MYCETOZOA. 57
numerous than when a good many of the stalks projected. The time from the planting of the cultures until the sporangia form varies considerably.
Cultures of Stemonitis A, B, and C formed sporangia as early as the thirtieth, and as late as the seventy-sixth day. Two cultures made from the same parent culture in the same media developed sporangia on the thirty-second and seventy- sixth day respectively.
As a rule but one set of sporangia developed in the same culture. Sporangia do not develop in all the cultures; at times large plasmodia form on the hay and degenerate without forming sporangia.
Physarum cinereum formed sporangia from the twenty- second to the sixty-fourth day.
Didymium farinaceum formed sporangia on the dried leaves on the fifty-seventh day.
AEthalium septicum formed large plasmodia about the fifty-fifth day, remained on the side of the flask for about ten days, and then degenerated without forming sporangia.
Plasmodia under natural conditions leave their moist or wet habitat, crawl to the surface when it is dry, aud they are ex- posed to the light. In some of my cultures the formation of the sporangia seems to have been delayed by keeping the culture in the dark; some of the cultures were kept in the dark six weeks, and after being in the light for several weeks formed sporangia.
The zoospores develop readily in the oven at 37° C., but no sporangia formed in any of the cultures. The absence of light may have had something to do with the result.
Time of the Day at which the Sporangia develop.
De Bary (8) studied the formation of the sporangia of Physarum sulphureum, Didymium serpula, Mthalium septicum, and Stemonitis ferruginea, and found that usually the sporangia began to form in the afternoon or late evening, and the development was completed in some cases by
58 CASPER O. MILLER.
the next morning ; in others not until near the middle of the day.
In one observation by the writer, the plasmodium lying on the hay at the surface of the water began about noon to crawl up the side of the flask. By 6 p.m. the plasmodium had collected at the point where the sporangia formed; by 7 p.m. the branches were drawn in, and the surface was covered by a number of hemispherical projections; and by 6 a.m. the following day the sporangia were fully formed. In other cultures observed the plasmodia were resting at the surface of the water at 6 p.m.; by 9 o’clock the next morning they were out of the water, and the sporangia had begun to assume a cylindrical shape. By 11 a.m. the shape of the sporangia was fully developed; the colour appeared first in the base of the columella, gradually going to the apex. By 2 p.m. the sporangia were of a brownish-red colour except at the apex, which was yet a yellowish-white on the surface. By 5 p.m. the colour was fully developed and the sporangia were completed.
The sporangia of Phys. cinereum, so far as observed, began to be developed at 3—6 p.m., and were completed by the next morning. The sporangia of Chond. diff., Didym. microcarpon, and Didym. farinaceum also developed for the most part at night.
Observations and Speculations concerning the Formation and Growth of the Plasmodia.
In his first studies De Bary failed to show how the plasmodia develop, whether by growth from a single zoospore or by the fusion of a number of zoospores.
Cienkowski (6, 7) described and pictured the fusion of the zoospores to form small plasmodia, and he saw plasmodia which had later taken in foreign particles, spores, and micro- cysts.
De Bary (8, 21) accepted Cienkowski’s results, although he never saw the zoospores fuse.
Ward (25), in speaking of the fusion of the zoospores to
THE ASEPTIC CULTIVATION OF MYCETOZOA. 59
form plasmodia, says, ‘‘ The inference becomes almost a cer- tainty after watching the specimens under cultivation ;” but he did not actually see them fuse.
Strasburger (26) also describes the fusion of the zoospores to form Myxameeba.
The writer has not been fortunate enough to observe the fusion of the zoospores, but the accuracy of the observations of such competent observers as Cienkowski, Strasburger, Lister, and others can hardly be doubted. In the cultures, as the writer has studied them, however, he does question whether the fusion of the zoospores is the chief mode by which the plasmodia grow.
If a few drops of a culture containing microcysts of Stemo- nitis, with suitable bacteria, be inoculated in a flask con- taining sterilised water, with milk 2 per cent., the bacteria multiply at the expense of the milk. Within two or three days the fluid loses the slight opalescent appearance which it had, and on microscopic examination there are no longer milk globules present. I think, from our knowledge of bacteria, we can conclude that at least a portion of the milk has been consumed by them. During this time the zoospores have nultiplied by division; they feed on the bacteria, and possibly some elements of the milk which the bacteria may not have appropriated. In a few days the zoospores begin to encyst, and by the end of the second week the majority of the zoospores are encysted, while a smaller number remain active. If control cultures are made from the flask, it will be found that there are not near so many bacteria present as there would be in a flask containing a similar medium inoculated with the bacteria alone which grow with the zoospores. In from ten to fourteen days small plasmodia may appear; they increase in numbers and in size, and later large plasmodia are present.
In cultures made in flasks containing hay, with milk 2 per cent.in hay infusion, essentially the same changes take place, but the hay interferes somewhat with the examination. If examined about the end of the second week one finds bacteria, encysted zoospores, active zoospores, and a few small plasmodia. The
60 CASPER O. MILLER.
plasmodia increase’ in number and in size, but they are not seen macroscopically, If the culture be one which forms sporangia on the thirtieth day, and it is examined about the twenty-sixth day, one finds more small plasmodia and a smaller number of microcysts present in the fluid than at the previous examina- tious. <A large plasmodium appears, rather suddenly, on the twenty-eighth day, lying on the hay at the surface of the fluid. It does not increase noticeably in size for two days, and then passes up the side of the flask to form sporangia. If the fluid is examined the small plasmodia have disappeared for the most part from the fluid.
One may have exainined the culture the previous day without having macroscopically observed the presence of a plasmodium. It must have originated by the fusion of a number of small plasmodia, or have grown as a large plasmodium in the interior of the stalks of hay. One stalk of hay is not large enough to accommodate the plasmodium, and no branches of plasmodia are seen connecting the various stalks. The writer is of the opinion that it originated by the fusion of a number of small plasmodia. Plasmodia large enough to be seen macro- scopically have been observed by the writer to fuse on the slide.
What takes place in the culture seems to be as follows :— the bacteria multiply at the expense of a portion of the nutrient material: the zoospores multiply at the expense of the bacteria, and possibly some nutrient material which was not consumed by the bacteria; the majority of the zoospores encyst; small plasmodia develop from a single zoospore or by the fusion of several zoopores ; the plasmodia take in and digest active and excysted zoospores and bacteria; finally, the small plas- modia fuse to form the large plasmodium.
Celakovski (38) studied the action of the plasmodia Chond. diff., Didymium microcarpon, and Aithalium septicum on various substances placed in the fluid with them. He saw them take in microcysts which, after ingestion, were not found in vacuoles, but were simply surrounded by protoplasm. After two days the microcysts were expelled unchanged ; if dried
THE ASEPTIC CULTIVATION OF MYCETOZOA. 61
and again moistened they gave origin to active zoospores. He thus reached the conclusion that the plasmodia did not digest the microcysts.
It is well to consider the condition under which he placed the plasmodia. He removed them from the fluid to which they were accustomed, washed them in fresh water, and placed the microcysts, spores, &c., on or near the plasmodia. In some instances he washed them several times. To the writer this seems harsh treatment. It cannot be wondered at that they were not in a condition to digest foreign substances, ana that under normal conditions he got peculiar results.
The writer has observed living plasmodia which had taken in microcysts and rounded off zoospores which had not yet formed a cyst wall. In these instances the zoospores were lying in vacuoles. Plasmodia placed on slides under a cover-glass (with active and encysted zoospores in the same fluid in which they grew) and allowed to spread out were killed with picric and acetic acid, and stained with picro-carmine. One finds in such specimens microcysts and rounded-off zoospores lying in vacuoles. They are in various stages of degenera- tion, and stained with varying degrees of intensity. From a study of the specimens the writer does not see how one can reach any other conclusion but that the microcysts and zoospores are digested. If one examines a culture after having developed sporangia, there are a smaller number of microcysts present than there was some time previously.
If one places a few drops of a culture containing zoospores of Stemonitis on a slide under a cover-glass, placing it in a moist chamber for somé hours, on examination he will find many of the zoospores creeping around on the slide, feeding on the bacteria. If, now, a point is examined at one side of the cover-glass, and a drop of sterilised water be added to the culture at the opposite side of the cover-glass, it will be observed that the zoospores instantly draw themselves together, many of the vacuoles will disappear, and the bacteria or undigested granules which were in the vacuoles will appear as granular particles enclosed in the protoplasm. It will be
62 CASPER O. MILLER.
some minutes before the vacuoles reappear and the zoospores begin to feed again.
It is not an infrequent experience that some protozoic forms are killed by simply placing them in fresh water. The plas- modia may not be as sensitive as the zoospores. They may have sufficient vitality not to be killed by the treatment to which Celakovski subjected them, but the writer questions whether the results obtained under the conditions in his experiment can be used as a basis for conclusions as to what plasmodia do in the fluids in which they thrive. His studies also showed that the plasmodia did not digest the encysted or active Colpoda which they had ingested. By the study of plasmodia taken from hay cultures and placed on a slide with a few drops of fluid containing Colpoda, the writer obtained results which showed that they do digest Colpoda.
The observations of Lister (44) also show that plasmodia do take in microcysts, enclose them in vacuoles, and digest them.
Observations on the Ingestion of Foreign Substances and the Multiplication of the Zoospores.
Lister (80) described the taking up of bacteria by the zoospores of Stemonitis fusca and Trichia fallax. The bacteria were drawn in by pseudopodial prolongations, which always came off from the posterior extremity, and were carried to vacuoles near the nucleus, where they remained until digested. He also gave them particles of carmine, which were ingested but not digested.
The zoospores of Stemonitis A, B, or C have not been observed to put out pseudopodial prolongations and draw in food particles, but the writer has seen the zoospores of Stemonitis A, B, C, and Ceratium porioides take up bacteria and par- ticles of carmine by means of a kind of vacuole which forms at the surface of the body (fig. 12). It is situated most frequently in the anterior half, but may form at any portion of the surface. The formation of these vacuoles can best be studied in a culture which has been on a slide for some hours, If a drop is taken
THE ASEPTIC CULTIVATION OF MYCETOZOA. 63
from a flask culture, placed on the slide, and immediately examined, the vacuoles are not always found. In favorable cultures the zoospores are found creeping on the slide and changing their shape; the flagellum is in active motion. At the posterior portion of the body a pseudopodial prolongation is put out, by means of which they adhere to the slide. Not far from the attachment of the flagellum there arises a pro- jection, which is placed more or less perpendicular to the surface of the body. At first sight it appears to be simply a conical or papillary projection from the surface layer of protoplasm. On closer examination a thin fold of protoplasm is seen to arise from the whole length of the projection, and extends forward toward the flagellum, where a second similar projec- tion arises. At this stage the two projections, with the thin fold-like connections, form a funnel-shaped depression. ‘The apices of the two projections approach each other and fuse, converting the funnel-shaped depression into a closed vacuole, which passes backward, and is lost among the vacuoles near the centre of the body. The flagellum is in active motion, and throws the bacteria or particles of carmine into the funnel- shaped depression, which then closes and forms the vacuole. The writer has seen bacteria and particles of carmine taken up in this way. When these vacuoles are located in the posterior region of the body, the flagellum has not been observed to throw the bacteria into them. Fig. 12, a—j, shows the different stages in the formation of these vacuoles. The zoospores of Phys. cinereum have not been observed to form these vacuoles.
The ameeboid stage of the zoospores of Phys. cinereum is much more pronounced than that of Stemonitis A, B, C, and Cerat. porioides. A large part of the active existence of the zoospores of Phys. cinereum is passed without the presence of flagella. They put out pseudopodia which are more or Jess angular, and their change of shape resembles more that of an amoeba. They may ingest their food after the manner of ameebe.
The zoospores of Stemonitis A, B, C, and of Ceratium porioides are characterised by rarely being without a fla-
64 CASPER O. MILLER.
gellum, and when they do change their shape the pseudopodia are more rounded.
The zoospores of plasmodia usually have a single flagellum, but they may have two or four. The writer has not seen zoospores of the Endosporia with more than one nucleus, whereas large zoospores of Cerat. porioides have occa- sionally been found which have two distinct nuclei (fig. 12, /.).
In all species of plasmodia which the writer has examined, one can distinguish two forms of microcysts in the cultures. In the simple form the cyst wall is made up of a single homo- geneous membrane, closely applied to the protoplasm, as indi- cated in fig. 12, m. In the second form the cyst wall is made up of an outer thick membrane, irregular or scalloped in outline, and an inner, thinner membrane, which is closely applied to the protoplasm (see fig. 12, ”., 0.). One finds cysts intermediate between these two varieties. The simple micro- cysts of Stemonitis A measure 5—7 uw in diameter, the thick- walled cysts measure 10—14 pm in diameter.
In cultures of Stemonitis A, B, and C, and Phys. cin- ereum made in hay infusion, the thin-walled microcysts remain unstained, whereas the membrane of the thick-walled cysts may be stained a brownish colour.
In old cultures made in hay infusion at times one sees microcysts with dark brownish, almost black pigmented granules in their interior.
Lister (44) speaks of the spores of Ceratiomyxa as having four “ nucleus-like ” bodies, and pictures them indistinctly in his plates. The writer’s observations show, from staining with picro-carmine, that these bodies are true nuclei.
Famintzin and Woronin (10) showed that after the proto- plasm escaped from the spores of Ceratium it remains at one place for some time, undergoing ameeboid changes. It then divides into four round segments, each of which divides and gives origin to two zoospores, Lister (44) describes the naked spore dividing into eight spherical segments which re- main attached to each other. These develop flagella, and then separate,
THE ASEPTIC CULTIVATION OF MYOETOZOA. 65
The writer has seen them divide after the manner described by Famintzin and Woronin (see fig. 13).
Microscopical Appearance of the Plasmodia and the Structure of the Nuclei.
Cultures made in fluids without the presence of hay offer the best facilities for studying the plasmodia. The smaller plasmodia are usually found lying in or upon a clump of microcysts and bacteria. The larger plasmodia can be seen spread out on the bottom or sides of the flask. The closeness of the network, the size of the branches, and the peripheral arrangement of the network can be studied.
For microscopical study, the plasmodia, with a few drops of the fluid in which they grow, are placed on a slide by means of a pipette. A cover-glass is carefully laid on, and is supported by small bits of wax at each corner to prevent injuring the plasmodia by pressure. The specimen can be immediately examined, and then placed in a moist chamber for twelve to twenty-four hours, after which it may again be examined.
If it is desired to preserve the specimen, the plasmodium can be fixed and hardened on the slide and stained by any of the usual methods. Hardening in picric and acetic acids, and then staining in picro-carmine, give good results.
Fig. 6 represents a segment of a plasmodium taken from a culture of Stemonitis A. Some of the clumps containing microcysts, bacteria, and plasmodia were placed on a slide. The specimen was examined at the expiration of an hour, and a number of rather long, finger-lhke, blunt, unbranched proto- plasmic processes were seen radiating from the periphery of the clumps. At the expiration of twenty-four hours the clumps were surrounded by a network of protoplasmic branches. The primary trunks are large, and extend from the clump toward the periphery of the network. They anastomose with each other, and are also connected by means of a finer network of secondary fibres. At the periphery of the network are irregular, angular, flattened, protoplasmic expansions, which at times unite and form irregular plates, as shown in the
vot. 41, part 1.—NEW SERIES. E
66 CASPER O. MILLER.
figure. Occasionally one gets specimens where the fusion of these expansions is more extensive.
When the plasmodia spread out as in the figure, one may see microcysts which have been taken up from the clump by the plasmodium and carried along the branches toward the periphery.
One frequently finds small plasmodia composed of only a few branches which do not form as close a network as in the figure, and the ends of the branches are not so angular.
The writer has not, as yet, sufficiently studied plasmodia from Stemonitis A, B, and C to be able to point out any con- stantly marked characteristics which distinguish them. They are all more transparent and not so granular as the other plasmodia which have been studied.
Fig. 7 represents a portion of the periphery of a small plas- modium of Phys. cinereum taken from a culture and allowed to spread out in the same way. It has more the appearance of a spread-out, perforated, protoplasmic plate than a network of branches, and one cannot distinguish between primary and secondary branches.
Fig. 8 represents the peripheral expansion of a plas- modium taken from a hay culture in which were Chon. diff. and Didym. microcarpon. Here there are several large trunks going to a broad peripheral expansion, less perforated than Phys. cin.
The nuclei of the plasmodia of Stem. A are distributed irregularly along the branches of the network and in the peripheral expansions. In some of the larger branches they may be collected in groups, where, as in other branches, they are situated at irregular intervals. In the larger branches the most of the nuclei lie in the peripheral layer of proto- plasma, often immediately under the surface. As a rule one does not see the nuclei in the living plasmodia, but occasion- ally, if the nucleus lies at the side of the branch, a nucleus can be seen as a spindle-shaped body just beneath the surface, and may cause a bulging at that point.
The shape of the nuclei is usually that of a flattened oval
THE ASEPTIC CULTIVATION OF MYCETOZOA. 67
disc. When seen on the edge they appear spindle-shaped. They contain from one to six or seven small bodies, which may provisionally be called nucleoli. One finds nuclei con- taining two nucleoli. These nuclei are at times constricted, and suggest stages of division.
Strasburger (23) studied the division of the nuclei of Trichia fallax during the formation of the sporangia. He succeeded in finding stages which showed karyokinesis. Rosen (89) studied the division of the nuclei in the forming ethalia of Aithalium septicum, but found the division of the nuclei simpler than that described by Strasburger.
Lister (44) describes the division of the nuclei of zoospores and of the nuclei of the plasmodia by karyokinesis, but also concludes that they divide by direct division. The writer has not been so fortunate as to find nuclei dividing by karyokinesis, although frequent search was made for them.
The zoospores can frequently be seen to divide and form two zoospores, and to the writer there seems to be evidence that at times the protoplasm of the microcysts breaks up into a number of small segments, and these segments enlarge and develop zoospores.
The nuclei of Phys. cinereum are smaller than those of Stemonitis A. They are spherical, containing one or more nucleoli, and are distributed in every part of the protoplasm, but are more abundant in some portions than in others.
Before concluding the writer wishes to acknowledge the invaluable assistance which he received from Professor Biitschli while pursuing these studies in Heidelberg, also to Professor Wladimir Schewiakoff, then assistant at the laboratory of Heidelberg, for his assistance in the preparation of the plates.
68
fo)
10.
11.
12.
13.
14.
15.
16.
if 18.
CASPER O. MILLER.
BIBLIOGRAPHY.
. DE Bary, L.—‘ Flora,’ 1854, p. 648. . DE Bary, L.—“‘ Ueber die Myxomyceten,” ‘ Botanische Zeitung,’
1858, p. 357.
. DE Bary, L.—“ Die Mycetozoen,” ‘ Zeitschrift fiir wiss. Zoolog.,’ 1860,
p. 88.
. Crenkowsk1, L.— Ueber parasitische Schlauche auf Crustaceen und
einigen Insectenlarven,” ‘ Botanische Zeitung,’ No. 25, 1861, p. 170.
. Wicanp, A.—“ Zur Morphologie und Systematik der Gattungen Trichia
und Arcyria Pringsheim,” ‘ Jahrbiicher fr. wiss. Botanik,’ Bd. iii, 1863,
. Crenxowsk1, L.—“ Zur Entwickelungsgeschichte der Myxomyceten”
(same Journal), p. 325.
. Crenkowsk1, L.—‘‘ Das Plasmodium ” (same Journal), p. 325. . DE Bary.—‘ Die Mycetozoen’ (Schleimpilze), 1864. . Lirperkiinn.—‘ Ueber Bewegenserscheinung der Zellen,’ p. 376, Tafel iv,
fig.38; ‘Schriften d. Gesellsch. ziir Bef. der ges. Naturw. zu Marburg,’ November, 1873.
Famintzin, A., und Woronin, M.—‘ Ceratium hydnoides und C.
porioides als zwei neue Formen von Schleimpilze,’ Botanische Zeitung, No. 34, 1872.
Famintzin, A.—‘‘ Ueber zwei neue Formen von Schleimpilze,” ‘ Mém. de l’Acad. Imp. des Se. de St. Pétersb.,’ ser. 7, tome xx, 1873.
Rozz, E.*—“Des Myxomycétes et de leurs place dans la systéme,” ‘ Bulletin de la Soc. Bot. de France,’ tome xx, 1873.
RostaFinskI, J. F.—‘ Versuch eines Systems der Mycetozoen’ (Inau- gural Dissertation), Strasburg, 1873.
RostarFinskI, J. F.*—‘ Sluzowce (Mycetozoa) Monografia,’ Paris, 1875; ‘ Dodatek I. do Monografii Sluzowcow,’ 1876.
Scuuuzg, F. E.—< Rhizopodenstudien V.,” ‘ Arch. fiir mikrosk. Anatom.,’ Bd. xi, 1875.
Cienkowsk1, L.—‘ Ueber einige Rhizopoden und verwandte Organis- men,” ‘ Arch. f. mikr. Anatomie,’ Bd. xii, 1876. Cooks, M. C.—‘ Myxomycetes of Great Britain,’ London, 1877.
Bitscu11, O.—“ Beitrige zur Kenntniss der Flagellaten und verwandter Organismen,”’ ‘ Zeitschr. f. wiss. Zoologie,’ Bd. xx, 1878.
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THE ASEPTIC CULTIVATION OF MYCETOZOA. 69
Gruser, A.—“Dimorpha mutans,” ‘Zeitschr. f. wiss. Zoologie,’ Bd. xxxvi, 1881.
Kent, 8.—‘ A Manual of Infusoria,’ London, 1880-82.
DE Bary.—‘ Vergleichende Morphologie und Biologie der Pilze, Myce- tozoen, und Bacterien,’ Leipzig, 1884.
Staut, E.—‘‘ Zur Biologie der Myxomyceten,’’ ‘ Botanische Zeitung,’ 1884.
SrrasBuRGER, H.—‘‘ Zur Entwicklungsgeschichte der Sporangien von Trichia fallax,” ‘ Botanische Zeitung, 1884.
Zorr, W.—“ Die Pilzthiere oder Schleimpilze,” ‘ Handbuch der Botanik,’ Schenk., Hefte 15 und 16, 1884.
Warp, H. M.—‘* The Morphology and Physiology of an Aquatic Myxo- mycete,” ‘Studies from the Biological Laboratories of the Owens College,’ vol. i, 1886.
STRASBURGER, E.—‘ Das Botanische Practicum,’ Jene, 1887.
Ravyxizr, C.*—“ Myxomycetes Danie,” ‘ Bot. Tidsskr.,’ 1888-89.
Lister, A.*—‘‘ Notes on the Plasmodia of Badhamia utricularis and Brefeldia maxima,” ‘ Annals of Botany,’ vol. ii, 1888 and 1889.
Birscuui, O.—‘‘ Protozoa,” in Bronn’s ‘Klassen und Ordnung des Thierreichs,’ 1889.
Lister, A.— Notes on the Ingestion of Food-materials by the Swarm- cells of Mycetozoa,” ‘Journ. Linn. Soc. London (Botany),’ vol. xxv 1889, p. 435.
Scuroter, J.—‘ Myxogasteres (eigentliche Myxomyceten) in die natiir- lichen Pflanzenfamilien von Engler und Prantl,’ 36 Lieferung, 1889. Wivneate, H.—‘‘ Notes on Enteridium Rozeanum,” ‘Proceedings
of the Acad. of Nat. Sci. of Philadelphia,’ 1889.
Zorr, W.—‘ Vorkommen von Fettfarbstoffen bei Pilzthieren (Myceto- zoen),” ‘Flora,’ 1889, p. 3538.
Lister, A.—‘“ Notes on Chondrioderma difforme and other Myce- tozoa,” ‘Annals of Botany,’ vol. iv, 1890, pp. 281—298.
Prerrer, W.*—‘ Ueber Aufnahme und Ausgabe ungeléster Korper,’ 1890, p. 154.
Rex, G. A.—‘‘ A Remarkable Variation of Stemonitis Bauerlinii, Mass.,” ‘ Proce. Acad. Nat. Sci. Philadelphia,’ 1890, p. 36.
Rex, G. A.—‘‘ New American Myxomycetes,” ‘Proc. Acad. Nat. Sci. Philadelphia,’ 1891, pp. 389—398.
CxrLakovskI, L., jun.—‘‘ Ueber die Aufnahme lebender und todter verdau-
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70 CASPER O. MILLER.
39. von Rosen, F.—-‘‘ Studien tiber die Kerne und die Membranbildung bei Myxomyceten und Pilzen,”’ ‘ Beitrage zur Biologie der Pflanzen,’ Bd. vi, Heft 2, 1892.
40. Hertwie, R.—‘ Lehrbuch der Zoologie,’ Jene, 1892.
41. Mass, G.—‘ A Monograph of the Myxogastres,’ London, 1892.
42. McBrips, T. H.—“ The Myxomycetes of Eastern Iowa,” ‘ Bulletin from the Laboratory of Nat. Hist. State Univ. of Iowa,’ vol. ii, 1892.
43. Ceaxovskl, L., jun.—‘ Die Myxomyceten Bohmen’s,’ Prag, 1893.
44. Lister, AA—‘ A Monograph of the Mycetozoa,’ London, 1894.
45. Mitirr, C. O.—< Ueber aseptische Protozoenkulturen und die dazu verwendeten Methoden,” ‘Centralb, f. Bak. u. Parasitenk.,’ Bd. xvi, 1894, No. 7.
EXPLANATION OF PLATES 6 and 7,
Illustrating Mr. Casper O. Miller’s paper on “ The Aseptic Cultivation of Mycetozoa.”
Fic. 1.—Sporangia of Chondrioderma diff. on the glass above the fluid, from a culture made with unsterilised hay.
Fic. 2.— Physarum cinereum, showing sporangia at the periphery of the plasmodial network which radiated from the end of a stalk of hay leaning against the glass.
Hic. 3.—Stemonitis A, with the sporangia in process of formation on the side of the flask at a point where a stalk of hay touched the glass. The colour had not fully developed. The course which the plasmodium took is shown by the thread which descends obliquely to the water.
Fic. 4.—Sporangia of Stemonitis B, fully developed on stalks of hay.
Fie. 5.—A plasmodium of Stemonitis lying on the hay at the surface of the water before it ascends to form sporangia.
Fic. 6.—A portion of plasmodium of Stemonitis A., which had spread out under a cover-glass. The trunks radiate from a clump of encysted zoo- spores, some of which were enclosed in the protoplasm. Drawn with a camera lucida. xX 180.
Fic. 7.—A portion of a plasmodium of Physarum cinereum spread out under a cover-glass. x 1380.
Fig. 8.—A portion of a plasmodium gotten from hay. x 130.
THE ASEPTIC CULTIVATION OF MYCETOZOA. fa
Fic. 9.—A sporangium of Stemonitis A, drawn with a camera lucida x 66.
Fic. 10.—A sporangium of Stemonitis C. x 66.
Fic. 11.—A sporangium gotten on a stump at Heidelberg. It has not been identified with any of the species described in works on Mycetozoa.
Fic. 12.—Zoospores of Stemonitis. 12 a—A, A zoospore in the act of forming a nutrient vacuole, and taking in a granule of carmine. 12 %, J, showing the location of the nutrient vacuoli posteriorly. 12 4. A zoospore with four flagella. 127. A zoospore of Ceratium porioides, with two flagella and two nuclei. 12m. An encysted zoospore, with a thin wall. 12 x, o. Encysted zoospores, with thick walls.
Fic. 13 shows different forms of spores of Ceratium porioides, with the four nuclei and four zoospores developing from one spore.
cf - - ‘ y c a 2
ON THE DEVELOPMENT OF TUBULIPORA. 73
On the Development of Tubulipora, and on some British and Northern Species of this Genus.
By
Sidney F. Harmer, Sc.D.,
Fellow of King’s College, Cambridge ; Superintendent of the University Museum of Zoology.
With Plates 8—10.
Introduction.
THE principal object of the observations described in the present paper was to test the conclusion, at which I formerly arrived, that a process of embryonic fission is of normal occur- rence in Cyclostomatous Polyzoa. The process had already been demonstrated in Crisia (15) and in Lichenopora (16), and I am now able to show that the development of Tubuli- pora! takes place on essentially the same lines. In the course of these observations it became apparent that the discrimina- tion of the British species of Tubulipora had not been characterised with sufficient precision.
The satisfactory determination of the species of Cyclo- stomatous Polyzoa is not an easy task. There are perhaps few cases, even in this group, in which the synonymy is in a more involved state than in the genus Tubulipora. Many of the specific diagnoses which have hitherto been given are based on immature specimens, or are for other reasons insuf- ficient for purposes of identification, and the compilation of unduly long lists of synonyms is for this reason undesirable.
1 A preliminary note (17) on this subject was published, in which the genus appears as Idmonea.
74, SIDNEY F. HARMER.
The question is further complicated by difficulties in deciding which names have the greatest claim to be assigned to parti- cular species.
Before proceeding to the accounts of the species the scope of the paper may be stated. I have given diagnoses of two Northern species, one of which I believe to be new, and of all the forms ordinarily recognised as British species of Tubuli- pora, with the exception of T. lobulata, Hassall.
I have not had a sufficient supply of this supposed species to enable me to come to any clear conclusion about it. Many of the colonies which I have found on shells in the deeper Ply- mouth dredgings (20—30 fathoms) agree well enough with Hincks’s description of this form. I have also seen similar specimens from the Liverpool district, kindly lent to me by Professor Herdman; but I have not been able to examine un- injured mature colonies. In the absence of perfect ovicells, it is possible that some of the specimens supposed to be T. lobulata may be worn examples of other species of Tubuli- pora. I prefer, therefore, to express no opinion with regard to this form.
The paper is divided in the following way :
I. Structure of the colony and of the ovicell. (The terms “ ogeciostome” and “ oceciopore”’ are here proposed.)
II. History of the species and genus.
III. Synonymy, diagnoses, and accounts of the species. (New species, T. aperta.)
IV. The nature of certain vesicles found in the tentacles and other parts, with a few statements relating to the budding and the structure of the adult zocecium.
V. Description of the development. (The terms “ axial lobe” and “ lateral lobes”’ are here proposed.)
VI. The morphology of the internal parts of the ovicell.
I. Structure of the Colony and of the Ovicell.
A colony of Tubulipora, as of other Cyclostomes, takes its origin in the well-known circular primitive disc (Pl. 8,
ON THE DEVELOPMENT OF TUBULIPORA. 75
fig. 2) formed by the calcification of the body-wall of the metamorphosed larva. The early zoarial development is well described by Barrois (1, p. 70, pl. iv, fig.), whose account perhaps refers partly to T. phalangea and partly to T. plumosa; and it results in all species in the formation of a colony which is at first pyriform or fan-shaped. The terms “proximal” and “ distal’? will be used with relation to the primitive disc. The distal curved margin of the colony is bounded by the ‘terminal membrane” (16, p. 93), which consists partly of an uncalcified cuticle (ectocyst) and partly of underlying protoplasmic structures. The terminal membrane does not remain as a continuous sheet, since portions of it are continually cut off by the upgrowth of the calcareous septa which form the lateral walls of the zocecia or ovicells. Each of these structures is thus closed by a derivative of the original terminal membrane, and this name may consequeutly be ap- plied also to the uncalcified distal wall of each unit of the colony (fig. 24). Both the zocecia and the ovicells are added to, after their first formation at the growing edge, only by the prolongation of parts of the calcareous tubes which have already been developed, though this is not altogether true of the Lichenoporide, in which the character of the colony is altered by the subsequent development of “cancelli.’ It thus follows that a young Tubulipora colony is identical, so far as its calcareous structures are concerned, with the proximal part of the same colony at a later stage of its existence; and that in order to understand what a particular colony or ovicell was like at an early period of its development, it is only neces- sary to imagine the distal parts of the colony suppressed.
The form of a particular colony is due to the behaviour of its terminal membrane, which is identical in its extent with the growing margin. Should this remain undivided, and con- tinue to grow symmetrically, a pyriform or flabelliform colony will result, according to the relative activity of growth in the longitudinal and transverse directions. Under certain circum- stances, probably due to unfavourable conditions, the colony becomes mature by developing an ovicell without losing its
76 SIDNEY F. HARMER.
pyriform shape. I have found mature colonies of this type commonly in at least three of the species described in the present paper, namely in T. phalangea, T. flabellaris (fig. 4), and T. aperta (fig. 2).
In other cases the transverse growth of the terminal mem- brane is more active, and the flabelliform character becomes more marked (figs. 1 and 5), its lateral edges commonly growing proximally so as more or less to encircle the primi- tive disc. This flabelliform shape typically occurs in well- grown colonies of T. flabellaris and T. aperta. In other species the terminal membrane commonly divides at an early stage, so that there is a cessation of growth between the two parts of the divided membrane. The colony then grows into two lobes, which usually diverge from one another. This form of growth is well indicated by Pallas (83, p. 248) in his original account of T. liliacea (= T. serpens, auctt.). Colonies of this species, as well as those of T. phalangea and T. plumosa, often become mature in this condition ; but in all three species further divisions of the terminal mem- brane! usually take place, the colony thus becoming variously lobed. In T. liliacea alone of the forms described in this paper, there is in many cases a marked tendency for the lobes to become free and erect; and an Idmonea-like form thus results.
The common basal part of a colony consists of a mass of pyramidal tubes with pointed proximal ends, the arrangement being comparable with that of a honeycomb, all the “ cells” of which look in one direction, represented in Tubulipora by the growing margin of the lobe. The base of the colony, or basal lamina, is the sum of the lower walls of the more proximal parts of the zocecia. It is completely adherent to the substratum in many species of Tubulipora, but it may grow out freely from it, and the colony thus becomes erect. Each zocecium is developed at the growing edge by the forma- tion of a new radial septum in connection with the basal
1 A commencing division of the terminal membrane is indicated on’ the right side of fig. lL.
ON THE DEVELOPMENT OF TYBULIPORA. 77
lamina, and grows at first in a more or less horizontal, centri- fugal direction. As its length is added to at the growing edge, its upper wall gradually rises above the general level of the colony, and some of the zoccia thus form projecting ridges (fig. 1). As the zocecium rises up in this way, its basal wall splits off from the basal lamina, between which and itself a cavity thus originates. This cavity is the beginning of a new zocecium, which continues to grow in the same manner.
It follows from this description that each zocecium proxi- mally reaches the basal lamina, as in Lichenopora (16, wood- cut, p. 84); a younger zoccium always originating from the basal side of an older one, and making its appearance distally to it after a certain time.
As the zoccium continues to grow longer, its distal part be- comes free from the common basal mass of the colony. It may become completely free on all sides, and it then, by the activity of its own terminal membrane, develops into a curved tube, which is generally cylindrical, though in some cases with an oval transverse section and orifice. Very commonly in Tu- bulipora the zoccium does not become free on its basal side, where it remains connected with one or more of its younger neighbours. In this way it forms the beginning of a row, usually uniserial, of connate zoecia, as show in figs. 1, 5, and 9. The adjacent zocecia of a series are separated by a flat septum, corresponding with the intersecting plane of two cylinders. Any one of the middle zoccia of a simple series will thus have four flat walls (fig. 1).
The shape of the orifice corresponds with that of the zocecium, being at first angular in the case of serial zoccia. If, however, growth of these goes on actively, the series may be resolved above into its constituent units, each of which then becomes cylindrical with a round orifice.
The ovicell of Tubulipora is an enlarged zocecium. This is shown by several facts, the most striking of which is that when young it has a polypide, and then differs in no way from an ordinary zoecium. The proximal end of the ovicell thus takes part in the formation of the basal lamina. On looking
78 SIDNEY F. HARMER.
down into the mouth of the open calcareous funnel (fig. 4.7), which is the condition of the young ovicell at the time when it commences to expand distally, a tubular cavity can be seen, which passes down to the basal lamina and represents the ovi- cell in its zocecium stage.
The upper wall of the expanded distal part of the ovicell is thickly perforated by pores (fig. 1). These pass through the calcareous layer; they are wider internally and constricted ex- ternally, where they are closed by a cuticle, the pore being entirely filled with a few cells. The pores are always much more numerous in the upper wall of the ovicell than in the walls of the zoccia, as Waters has pointed out (45, p. 277, and in other papers). It is perhaps probable that the function of these pores is mainly respiratory, to provide for the gas- exchange which must be necessary for the development of the great mass of larve which are formed inside the ovicell. The definitive assumption of the ovicell-character by a zocecium is thus marked by a great increase of the porosity of its upper wall,
In some species, and notably in T. flabellaris (fig. 4, ovicell 2) and T. aperta, the more porous part can in some cases be seen to be separated from the less porous part by a sharp line. ‘The proximal part has the appearance of a zocecium ; and we thus have an external indication of the time at which the fertile zocecium became definitely an ovicell.
An ordinary zocecium was seen to become, sooner or later, free from the common basal part of the colony, unless it remained connected with it by forming one of a connate series or of a fasciculus of zocecia. The corresponding process in the ovicell is the formation of its tubular portion ending in the orifice. This part, in all respects comparable with the up- standing free part of an ordinary zocecium, is, however, developed late, and can only be seen in ovicells which are nearly mature. The ovicell, in fact, remains a part of the common basal mass of the colony for a long period, during which its distal end is expanding and increasing the space available for the growth of the embryos. Before the distal
ON THE DEVELOPMENT OF TUBULIPORA. 79
expansion begins the ovicell has become separated by younger zocecia from the basal lamina, and its floor is thus formed by their proximal parts. The upper part or roof of the ovicell spreads out horizontally as the conspicuous, porous, calcareous film usually described as the ovicell. If no fresh zocecia were formed at the growing edge, the roof of the ovicell wouid be a simple fan-shaped film. But new zoccia continue to grow in the same way as if no ovicell were present (Pl. 10, fig. 32). The floor of the ovicell, formed by the upper walls of the younger zoecia, or really by the septa dividing its own body- cavity from that of these zocecia, thus rises into radial ridges which encroach on its cavity. As these become more vertical they meet the growing horizontal roof of the ovicell, and with further prolongation stand up from it at a right or obtuse angle.
In most of the species investigated the zocecia which are younger than the ovicell are arranged in connate series. But whereas the proximal parts of the fertile lobes have their zocecia in obliquely transverse, alternate rows (fig. 5), these series usually become radial in the region of the ovicell. In other words, a young zoccium which has reached the roof of the ovicell in the manner just indicated does not become frée, but remains connected with the expanding growing edge by a still younger zoecium. A radial series of connate zoccia thus results, and the roof of the ovicell is divided into two lobes, one on each side of the zocecial series. After a time the series may become completely free from the growing edge, and the two adjacent lobes of the roof of the ovicell may then unite distally to the series.
In my preliminary note (17, p. 212) I have stated that the zocecia may thus be left as columns passing freely through the cavity of the ovicell. I believe that this statement is not correct for Tubulipora. The cavity of the ovicell may indeed surround the zocecium, or series of zocecia, on the proximal, lateral, and to some extent on the distal sides; but I have found no evidence that two lobes which have become con- tiguous on the distal side of a zocecium ever really unite, as
80 SIDNEY F. HARMER. :
they certainly doin Lichenopora. In transparent prepara- tions it can be seen that the cavities of the two lobes remain separated by a vertical radial septum, the upper edge of which is generally indicated as a line on the roof of the ovicell even in dried colonies (fig. 5.s.).
As the radial series of zocecia are formed all round the peri- phery, the ovicell itself consists of a cavity which branches dichotomously at the proximal end of each series. In large, actively growing colonies (fig. 5) the ovicell usually branches in a palmate manner, and the primary lobes may undergo further dichotomy once or more. The extent to which the ovicell branches is not a specific character, but is probably dependent partly on nutrition and partly on temperature. Figs. 2 and 4 represent mature fertile colonies of T. aperta and T. flabellaris respectively, and fig. 1 a fertile lobe of T. plumosa. ‘The ovicell shown in the last figure is much more complicated than that shown in the former two; but it must be distinctly understood that the ovicell of T. flabel- laris and T. aperta may be large and much branched in vigorous colonies.
After the ovicell has reached a certain size it develops its tubular portion, ending in the orifice through which the larve make their escape. Here, however, there is some modification in the course of the development as compared with an ordinary zocecium. While the orifice of the latter is merely the part which, for the time being, forms the distal end of the zocecium, the lobes of the ovicell undergo a considerable amount of growth, in most cases, after the orifice is fully formed. Sooner or later, however, the peripheral ends of the lobes, closed merely by a soft terminal membrane during growth, become completely calcified, except in the abnormal specimens of T. aperta (fig. 2), described under the heading of that species, in which accessory openings may be formed by the outgrowth of the lobes into tubular passages which more or less resemble the real orifice of the ovicell. The complete calcification of the peripheral ends! the lobes of the ovicell takes place, as in other Cyclostomes, by the encroachment of the calcareous
ON THE DEVELOPMENT OF TUBULIPORA. 81
roof on the terminal membrane, which is, so to speak, gra- dually constricted until it completely disappears.
My study of Tubulipora has fully confirmed the conclusion stated in my paper on Crisia (18, p. 128), that the form of the orifice of the ovicell is of great importance for the discrimination of the species. As I am convinced that this structure will become increasingly important in the systematic study of the Cyclo- stomata, I venture to think that a special terminology may be convenient for descriptive purposes; and I therefore propose to term the passage by which the larve escape from the ovicell the “oceciostome,”’ and its actual external orifice the “oeciopore.” The oceciostome is usually a tubular or funnel- shaped structure, as in the species of Tubulipora here de- scribed, and in many other Cyclostomes. The part connecting the cavity of the ovicell with the oceciopore may be termed the ‘‘ tube” of the oceciostome, whatever its shape. The tube is not a necessary part of the occiostome, since it is absent in Crisia aculeata, Hassall, in which the oeciopore represents the entire oceciostome.
The structure of the wall of the tube is commonly different at its two ends. The proximal portion is often pierced by pores identical with those in the roof of the ovicell, of which it is a direct continuation (fig. 1), while the distal portion is imperforate. Valuable specific characters are afforded by the shape, size, and relations of the oceciostome, and by the size and position of its tube and oeciopore.
The constancy of the characters of the oceciostome has been verified by the examination of numerous specimens of several species from various localities. While the shape and size of the entire colony, and therefore of the ovicells, has been shown to be highly variable, the oceciostome retains its character, whatever the condition of the colony. I do not, of course, mean to assert that there is no variation in the oeciostome. Variation does occur, sometimes within fairly wide limits; but it is usually possible to decide the species at once by an inspection of its occiostome. In order to do this it may be necessary to have a fully developed oceciostome, since the
vou. 41, parr 1.—NEW SER. Fr
82 SIDNEY F. HARMER.
specific characters are not completely shown in the immature condition of this structure. There are occasional cases in which the occiostome of a given colony may appear inter- mediate between two species. In most of these cases com- parison with other specimens will, however, usually leave no doubt of the existence of a definite character for the oceciostome of each species, in spite of the occasional occurrence of difficult cases.
The value of the characters of the oceciostome in diagnosing a species is strikingly illustrated by the specimens of T. flabellaris, Fabr., which have come into my hands. Fig. 4 is an Arctic specimen of this species, dredged by Colonel H. W. Feilden on July Ist, 1895, in Barents Sea, and presented by him to the Cambridge Museum of Zoology. Although the colony is very much smaller than the form of this species figured by Smitt (40, pl. ix, fig. 6),1t possesses no less than six complete ovicells, each with a fully formed oceciostome (1—6), five of which are visible in the position in which the colony lies, besides one immature ovicell (7). The ovicells are crowded and are very small, exhibiting hardly any of the lobing which may occur in this species as in others. The principal characteristic of the species, the flattened shape of the oceciostome, is nevertheless as well marked in each ovicell as it is in the lobed ovicells of a larger colony, sent to me by Dr. Nordgaard from Hammerfest.
The above case can hardly be regarded as a mere alee abnormality. I have examined four specimens (including the above) from Colonel Feilden’s collection, and all of them showed the same peculiarities. ‘Two other colonies (about the same size as the above) had each four small, contiguous ovi- cells with fully formed oceciostomes, and an immature fifth ovicell. The last consisted (unlike the others) of two diverg- ing lobes, which may, however, have been formed from two larve. One lobe had two ovicells with occiostomes, and at least one immature ovicell, and the other lobe had one ovicell with a fully developed oceciostome. In every ovicell the long flattened oceciostome, which I regard as the distinguishing
ON THE DEVELOPMENT OF TUBULIPORA. 83
feature of this species, was developed on precisely the same type.
A case of the abnormal development of four contiguous ovi- cells has been described by me (18, p. 166, pl. xii, fig. 13) in Crisia ramosa. These cases are interesting as showing the essential similarity of the ovicell and the zocecium. The pro- duction of an ovicell is probably induced by the development of an embryo from one of the eggs which so commonly occur in young zocecia, most of which do not ordinarily become fertile.
The simple ovicells of the specimens of T. flabellaris from Barents Sea are similar to those which were figured by Savigny (87, pl. vi, figs. 4.2 and 5.2), and described by Audouin under the names of Proboscina Boryi, and P. Lamou- rouxii. Ovicells of a similar type are known in Diasto- pora suborbicularis, Hincks, and in many fossil forms. They are figured, for example, by Gregory (12, pl. 1, fig. 6; pl. iii, fig. 8), and this type of ovicell is given by Walford (41, p. 78) as a characteristic of his genus Pergensia, although I cannot agree with him in thinking that the character shows any approach to the Cheilostome genera Lekythopora and Pecilopora. It appears to me probable that the stunted specimens of T. flabellaris above described have reverted to a more primitive condition (a conclusion borne out by the evidence of fossils) in developing a less complicated form of ovicell than is usually produced in other recent species, and even in T. flabellaris itself. This reversion may be re- garded as due to the small size of the colony, in conjunction with the successful development of primary embryos in several contiguous zoccia. It is probable that this points to a time when the ovicells were ordinary zocecia, and that the restric- tion in the number of fertile zocecia now so characteristic of Cyclostomes is not a primitive feature of the group.
References to the oceciostome in any form hitherto described as Tubulipora or Idmonea are curiously rare; and most authors do not seem to have considered the possibility of the occurrence of this essential part of the ovicell. Smutt (40)
84 SIDNEY F. HARMER.
has described it in Idmonea atlantica (p. 443), in I. ser- pens [= T. liliacea of the present paper] (p. 446), in T. fimbria [= 'T. aperta] (p. 455), in T. flabellaris (pp: 456, 457), and in T. lobulata (p. 457). The descriptions there given are not, however, sufficiently precise to be used in discriminating the species, nor are they illustrated by figures which really show their character. Waters (44, pp. 256—259, and 48, p. 339) has described the occiostome in one or two species of Tubulipora or Idmonea; and Kirkpatrick (22, p. 22, pl. iv, figs. 6 a, 6 6) has described and figured the ocecio- stome of I. pulcherrima. These are almost the only refer- ences I have been able to find to the occiostome of recent species of Tubulipora. It appears to me that a Tubuli- pora colony reaches its fully adult condition when the ovicell, with its oceciostome, is fully formed. If this view be correct, the importance of the oceciostome as a specific character is intelligible. Colonies in which this structure is not yet deve- loped are in a state of immaturity. It is perhaps as reason- able to characterise a species of Tubulipora without taking account of its oceciostome as it would be to describe a new species of Cervus without describing the form of the antlers. It is unfortunately impossible to describe the oceciostome of a Cyclostome in the majority of cases, and particularly in fossil specimens; but I think the description of this structure should be an essential part of a diagnosis wherever it can be given.
The absence of the oceciostome in a given case may depend on the season of the year at which the specimen was obtained. It is, for instance, somewhat difficult to find the oceciostomes of the British species in colonies collected in the spring, in which the formation of the ovicell is beginning; but it is easy to find them in uninjured healthy colonies collected in the summer.
The following table illustrates the use which can be made of the oceciostome in distinguishing the species treated of in this paper. The “lower” surface of the colony is that by which it is fixed, so that “ upwards” means away from the basal surface :
ON THE DEVELOPMENT OF TUBULIPORA. 85
at least in well-developed colonies 5 ne
[te more or less obviously arranged in connate series, Zocecia not in connate series, even in the fertile lobes.
: Oceciostome with a well-developed tube, usually more
{
| or less free, the oceciopore being larger than an orifice
and looking upwards T. aperta (Pl. 8, fig. 2)
Oceciopore much larger than an orifice, opening upwards or obliquely horizontally
T. plumosa, W. Thomps. (fig. 1)
Oceciostome or occiopore not larger or only slightly
larger than an orifice ; : tno
Tube of the occiostome recumbent on a zoccium or
series of zowcia, the oceciopore opening horizontally
or downwards : . 4
Tube of the occiostome free, eaecile ciranieeneeal the oceciopore being slit-like
T. flabellaris, Fabr. (fig. 4)
Oceciopore larger than an orifice, opening horizontally
T. liliacea, Pall. (=T. serpens, auctt.) (figs. 7, 8)
Oceciopore concealed, smaller than an orifice, looking
downwards T. phalangea, Couch (figs. 5, 6)
The average size (in thousandths of a millimetre = ph) of the oceciopores and of the orifices is stated in the following table, the greater diameter of the oceciostome being added in T, phalangea, the only species in which the occiopore is narrower than the occiostome:
Oceciopores. Orifices. T.phalangea. - 120 Shits aie . 160 T.flabellaris . . 165 2 75 T. liliacea . ‘ . 260 ; : : G5 Taperta, <9 . 280 : : 5 . 170 T, plumosa. : . 93855 185
It should be expressly noted that the ree of the oldest zocecia of a colony may be much smaller than that of the younger zocecia. The above averages are taken from zocecia which have reached their full size.
86 SIDNEY F. HARMER.
II. History of the Species and Genus.
The tenth edition of the ‘Systema Nature’ gives the diagnosis of certain species referred to the genus Tubulipora, of which the first or type species is T. musica, the organ- pipe coral. Amongst these (p. 790) occurs T. serpens.
The twelfth edition contains (p. 1271) a diagnosis of T. serpens, as “T. tubulis cylindricis erectis brevissimis dis- tautibus axillaribus, basi repente dichotoma divaricata.” This is practically identical with that given in the tenth edition, to which a reference is given, with the further reference ‘ Ameen. Acad.,’ i, p. 105, t. 4, f. 26.
The species is thrown up on the shores of the Baltic, while a similar but smaller form occurs in the Mediterranean, the only locality mentioned in the former edition.
A reference to the first figure which is quoted by Linneus, namely, to’ that contained in his ‘ Amecenitates Academic’ (vol. i, 1749), leads to the conclusion that the original descrip- tion did not refer to the species which is now usually known as Idmonea serpens. The figure is on a plate headed “», 812,” and the description is on p. 209 [not 105]. The figure, with which the description agrees, represents a stone bearing a closely adherent species, consisting of an open network of tubes with single pores at considerable intervals, usually at the angles of the meshes. It is hardly possible to recognise any similarity to any species of Tubulipora or Idmonea; but, on the contrary, the figure is strikingly sug- gestive of the Alcyonarian Sarcodictyon catenatum, Forbes, and closely resembles the figure of that species given by Herdman in the ‘ Proc. Liverpool Biol. Soc.,’ vol. ix, 1895, pl. viii, fig. 2.
Whether the description of Linneus referred to Sarco- dictyon or to an Alecto-like form must be left an open question,! but I think that it can have had no connection with any species of Tubulipora or Idmonea.
In 1755 (9, p. 74) Ellis described, under the name of the
1 Milne Edwards (29, p. 331) believed that it referred to an “ Aulopore,”
ON THE DEVELOPMENT OF TUBULIPORA. 87
‘small purple Eschara,” the form which is now com- monly known as Idmonea serpens. The purple colour and the parallel arrangement of the zoccia are expressly men- tioned. The figures e and £ on pl. xxvii represent the species as growing on a substance which is doubtless the stem of the “sickle coralline”? (Hydrallmania falcata), as appears from the description, on p. 75, of the Cellepora shown on the same stem. The occurrence on this species of Hydroid is eminently characteristic of the ‘small purple Eschara.”
In 1766 (33, pp. 248, 249) Pallas described the same species under the name of Millepora liliacea, referring to Ellis’s description and figures; and it is given as Millepora tubu- losa in the well-known work of Ellis and Solander (10, p. 136).
In the enlarged thirteenth edition of the ‘ Systema Nature ’ (1788) Gmelin complicates the question by describing the species under no less than three different names.! The first of these (tom. 1, part 6, p. 3754) is Tubulipora serpens. Linneus’s diagnosis is repeated, but a reference to former editions of the ‘Systema’ is omitted, while the small purple Eschara of Ellis, and Millepora liliacea of Pallas are given as synonyms. The second (p. 3790) is Millepora tubulosa, Ell. and Sol., the small purple Eschara of Ellis appearing a second time as a synonym. The third (p. 3790) is Millepora liliacea, Pall., and Tubulipora serpens, Linn., is given as a synonym.
It appears to me that the Linnzan name must be rejected, and, following the ordinary laws of priority, that the choice must lie between Tubulipora liliacea, Pall. (1766), and T. tubulosa, Ell. and Sol. (1788). Ifthe tenth edition of the ‘Systema Na- ture’ is adopted as the commencement of the binomial system, Pallas’s name has the right to be accepted ; while the adoption of the twelfth edition as the starting-point would necessitate the employment of T. tubulosa, Ell. and Sol. I shall follow the example of Mr. Hincks® in regarding as valid Pallas’s
1 As has already been pointed out by Lamouroux (25), p. 66. 2 See his remarks on Flustra securifrons, Pall., on p. 122 of the ‘ British Marine Polyzoa’ (1880),
88 SIDNEY F. HARMER.
names, published in the year before the part of the twelfth edi- tion of the ‘ Systema Nature’ which referred to Zoophytes.
I have come to the conclusion, from my study of the de- velopment, that it is not possible to separate ‘‘Idmonea” liliacea generically from the British forms recognised as “Tubulipora,” although I claim no novelty in that con- clusion. It thus becomes necessary to consider whether the genus which includes the two species should be called Idmo- nea or Tubulipora.
The genus Tubulipora was founded by Lamarck (23) in 1816, while Idmonea is due to Lamouroux (25), and dates from 1821. Lamarck’s type-species is T. transversa, said to be found on Fucus in the Mediterranean. Thesmall purple Eschara of Ellis, and Millepora tubulosa of Ellis and Solander, are given as synonyms, and from this, with the diagnosis, it might be concluded that T. liliacea, Pall., is the type-species of the genus Tubulipora. H. Milne Edwards has, however, figured (80, pl. ix, figs. 3 and 3a) a specimen from the Paris Museum with the statement (p. 218, note) that it is the one from which Lamarck’s description was taken. He regarded the species as an Idmonea (29, p. 382), a course which is hardly justifiable considering that it was the type-species of the earlier genus Tubulipora. If, then, we are to accept Milne Edwards’ figures as a correct representa- tion of Lamarck’s species, [dmonea becomes, on his showing, a synonym of Tubulipora.
If a generic distinction between Tubulipora and Id- monea, in the ordinarily understood sense, can really be maintained, this is a regrettable conclusion, since it results in the substitution of Tubulipora for Idmonea, and would necessitate the use of some other generic name for the species usually understood to belong to Tubulipora. If Lamarck’s type-specimen is still in existence, and the evidence that it is the type-specimen is satisfactory, I suppose there is no option but to regard the synonyms which he himself gave for T. transversa as erroneous. But as the evidence is perhaps not quite certain, and as, moreover, it is not clear that any
ON THE DEVELOPMENT OF TUBULIPORA. 89
generic difference between Tubulipora and Idmonea can be maintained, I shall regard the species described in this paper as members of the genus Tubulipora.
The type-species of Lamouroux’ genus is Idmonea tri- quetra, a fossil form from the “terrain a polypiers” (Ba- thonian) of Caen. The description might lead to the inference that the species is erect, but Gregory (12, p. 134) states that it is always an encrusting form, and has justly remarked that it is therefore impossible to define Idmonea as consisting only of erect species. If this is so, it becomes very difficult to draw any line between 'Tubulipora and Idmonea. Dr. Gregory’s catalogue includes no species of Tubulipora, but he dis- tinguishes (p. 134) a family Idmoniidz from the Tubuliporide mainly by the existence of regular transverse rows of zowcia in the former. I do not think that this distinction can be maintained, either as the character of a family or even of a genus. Lamarck’s type-species of Tubulipora was defined as having its zoccia in transverse series, and this feature is strongly marked in other recent species which are ordinarily included in that genus. The only character in Dr. Gregory’s diagnosis of _Idmonea (p. 184) which is not applicable to many species of Tubulipora is the ridged or triangular cross- section of the branches, and it is very doubtful if this is really a valid generic difference.
From a superficial examination of the ovicells of Idmonea atlantica, and from a consideration of Smitt’s description and figures (40, p. 443, pl. iv, figs. 5, 7), it appears to me that this form at least is closely allied to T. liliacea.
III. Synonymy, Diagnoses, and Accounts of the Species.
The material on which the following account was based was collected mainly in the Salcombe Estuary, in South Devon, in March and April. I have to thank Dr. A. M. Norman for having recommended me to choose that place as a base of operations. Other specimens were collected by myself while working at the Plymouth Laboratory and on other parts of
90 SIDNEY F. HARMER.
the English coast, and in Norway. I have to express my great indebtedness to my friends who have kindly given or lent me specimens from other localities ; and particularly to Professor Herdman and Miss Thornely for specimens from the Liverpool district; to Professor M‘Intosh for material from Scotland; to Dr.O. Nordgaard for specimens from Norway; and to Dr. F. M. Turner for material from Guernsey. I must also express my obligation to Mr. A. H. Church for determining one or two seaweeds on which my specimens were found, and for some observations on the amount of annual growth of the colony; and to Mr. S. D. Scott for some observations on the excretory vesicles.
Tubulipora, Lamarck.
Zoarium with a distinct basal lamina, adnate or erect, beginning as a pyri- form or flabelliform colony, which may become lobed by the division of the terminal membrane. Lobes short and adherent, or longer and dichotomously divided once or more often, sometimes becoming erect. Zocecia with a free, cylindrical, terminal portion ; or connate in obliquely transverse series, in which they are separated by flat septa corresponding with the intersection of two cylindrical zocecia. The series are arranged alternately on opposite sides of the axial line of the lobe, but the transverse arrangement usually becomes radial in the distal part of the fertile lobes. Ovicell an enlarged zocecium, which extends into the intervals between the parallel or radial series.
The number of the tentacles is usually eleven or twelve in the three species I have studied by means of sections. Of these, T. phalangea and T. plumosa seem to have eleven tentacles in most cases, Milne Edwards (80, p. 195, note), giving the number as twelve for the former. In T. liliacea I have counted twelve tentacles in most cases, a number agree- ing with Dalyell’s statement (6, p. 86); but one polypide had thirteen, and several had eleven.
T. liliacea, Pallas (figs. 7—9).
Tubulipora and Idmonea serpens, auctt. (not Tubipora serpens, Linn. [27, p. 1271], nor Fabr. [11, p. 428).
Small purple Eschara, Ellis (9, p. 74, pl. xxvii, figs. e, ).
ON THE DEVELOPMENT OF TUBULIPORA. 91
Millepora liliacea, Pallas (33, p. 248).
Millepora tubulosa, Ell. and Sol. (10, p. 136).
Millepora tubulosa and M. liliacea, Linn. Gmel. (28, p. 3790). Tubipora serpens, Dalyell (6, p. 85, pl. xviii, figs. 11—15).
Tubulipora serpens, Johnst. (19, pl. xxxi, figs. 4—6; and 20, pl. xlvii, figs. 4—6). Couch (5, pp. 105 [part], 106). Smitt (40, pp. 399, 444, [part], pl. iii, figs. 4a—4e, 5a, 54; pl. ix, figs. 1, 2a, 26). Busk (2, pp. 25, 26 [part], pl. xxii, figs. 1—3).
Tdmonea serpens, Hincks (18, p. 453, pl. Ixi, figs. 2, 3). Levinsen (26, p. 76, pl. vii, figs. 6—10).
Zoarium adnate or erect, its form being greatly influenced by the sub- stance on which it is growing; commonly dividing several times dichoto- mously. Zocecia curved for the most part in one plane, with the serially connate, alternate arrangement strongly marked, though sometimes obscured in small or irregular colonies. In well-branched colonies the inner zoccia are much longer than the outer ones, so that the height of the transverse series diminishes greatly in passing from the inner to the outer side. Ectocyst usually vitreous and hyaline. Oceciostome about 260 » in diameter, slightly larger than the orifice of a zocecium, opening horizontally.
Common on Hydroids (especially Hydrallmania falcata), from 20 to 40 fathoms ; but also found on Cellaria from the same depth, and on red sea- weeds from shallower water.
This is the “small purple Eschara” of Ellis, and the Tubulipora or Idmonea serpens of most writers. I have explained on p. 86 the reasons for rejecting the familiar specific name.
The distinctive feature of this species is the form of the oceciostome (figs. 7 and 8). In size it is intermediate between the corresponding structure of T. plumosa and that of T. phalangea, and is somewhat larger than the orifice of an ordinary zoccium. The difference between the oceciostome of T. liliacea and that of T. phalangea (figs. 5 and 6) is not always apparent at first sight, the oceciopore being often con- cealed in both cases. But whereas in the latter species it is seldom possible to see the oceciopore in any position in which the uninjured colony may be placed, it is nearly always
92 SIDNEY F. HARMER.
possible in T. liliacea to see it by inclining the colony in a suitable direction. The oceciopore typically opens hori- zoutally ; or, in other words, its plane is vertical to the upper or exposed surface of the ovicell. The edge of the oceciopore may be slightly everted, so as to form a narrow brim, or there may be no eversion; and the upper lip may be horizontal and more prolonged than the lower lip.
Tn all cases the occiopore is relatively large, varying from 230 w to 270 pu, that of an ordinary zowcium being 145 p to 190 u. When the structure is placed in a suitable position it is possible to see some way down the tube of the oceciostome (fig. 7). This cannot be done in T. phalangea.
The tube is moderately long, and is recumbent on a zocecium or series of zocecia. It may occur on the proximal side of the series, and look towards the oldest part of the colony, or it may be placed on the distal side and look towards the growing edge.
T. liliacea is very common on certain Hydroids, parti- cularly on Hydrallmania falcata, a fact familiar to many of the older naturalists, and on Sertularella. It is easily obtained from the masses of Hydroids brought up by trawlers in water of twenty fathoms or more. The form of the entire colony varies a good deal. It may remain closely attached to the narrow stem of the Hydroid, its basal lamina curving round the stem and thus giving rise to very irregular colonies ; or it may remain attached merely by a small central area, and grow out into free, erect branches, as in the var. radiata of Hincks. In this condition it assumes a typical _ Idmonea- form, having a very strongly marked alternate arrangement of connate plates of zocecia.
In a particularly fine specimen of this form, from the Trondhjem Fjord, which I owe to the kindness of Dr. Nord- gaard, a single series consists in some cases of as many as eight zoccia, the inner ones being very much taller than the outer ones. The tip of the longest branch is about 11 mm. from the centre of the colony. The occiostome belonging to this branch is on the distal side of the seventh series (of one
ON THE DEVELOPMENT OF TUBULIPORA. 93
side of the branch) from the bifurcation preceding the ovicell ; whereas in another branch the oeciostome is on the proximal side of the eighth series of one side, the ovicell itself beginning immediately after the fifth series.
The ovicells of this colony extend through a region of four or five transverse series of zocecia on each side of the fertile lobe. The shape of the ovicell is of course affected by the strongly marked alternate arrangement of the series, and its roof is thus a comparatively narrow, curvedly zigzag band, running along the middle of the lobe, and giving off an inter- serial lobe on the convex side of each bend. The ovicell may thus be described as consisting of a regularly undulating axis, with an alternately pinnate arrangement of simple lobes ex- tending between the series of zoccia. The ends of the branches are bifurcated, but the same ovicell extends into both halves of the fork by division of its main axis. The same arrangement is figured by Smitt (40, pl. iv, figs. 5 and 7) in Idmonea atlantica.
A great contrast to this Idmonea-like colony was afforded by a fine specimen from the Liverpool district kindly lent to me by Professor Herdman. A narrow branch suddenly ex- panded into a nearly semicircular fertile lobe, 6 mm. in trans- verse diameter. The zoccia in this lobe had a Tubulipora- like arrangement, consisting of radial series, which showed a distinct tendency to become frayed out into separate zocecia at their upper borders. The occiostome had the typical form, and other colonies from the same locality were in no way dif- ferent from the more ordinary type of T. liliacea.
T think there can be no doubt that the form of oceciostome which I have described is quite characteristic of this species. Although I first noticed it in a number of specimens from the Plymouth district, it is not a local peculiarity, since I have found precisely the same form in the specimens which have just been alluded to from Trondhjem and Liverpool, as well as in a series of colonies from Hydroids dredged in St. Andrews Bay (20 to 27 fathoms), kindly given to me by Pro- fessor M‘Intosh. The variations in the size of the ocwciopore
94 SIDNEY F. HARMER.
are indicated by the following list of measurements of the transverse diameter :
Plymouth (on red seaweed) : : . 220 p. St. Andrews (on Hydroid) ‘ ‘ . 230 p. Plymouth (on Hydroid) ' ; . 260 pz. Trondhjem . : ; - 265 p. Plymouth (on Hiydroid) . . 280 p Liverpool (probably on Hydroidy : - 300 x.
The average of this series of measurements is 260 yu.
T. phalangea, Couch (figs. 5, 6).
Tubulipora phalangea, Couch (5, p. 106, pl. xix, fig. 7 [figure bad]). Johnston (20, p. 273 [part], pl. xlvi, figs. 1—4). Busk (2, p. 25, pl. xxiii, fig. 2).
Tubulipora flabellaris, Hincks (18, p. 446, pl. Ixiv, figs. 1—3).
Tubulipora verrucaria and T. verrucosa, Milne Edwards (29, pp. 337, 328, 323, pl. xii, fig. 1).
Zoarium entirely adnate, variously lobed, sometimes consisting of a series of divaricated lobes, sometimes almost circular in outline, and then reaching a maximum diameter of at least 15 mm. In stunted specimens the terminal membrane may not divide, but gives rise to a single small fertile lobe, the
whole colony being pear-shaped. Zocecia serially connate, the series alternate near the base of elongated branches, but becoming radial in fertile lobes. The series are commonly resolved above into their component elements, the zocecia having a longer or shorter free cylindrical portion; but they may remain entirely connate to their ends. Zocecia narrow and long compared with those of most other species. Oceciostome (fig. 5) about as large, at its widest point, as an orifice, averaging 150 p in diameter, the tube bent com- pletely round, so that the oceciopore (fig. 6), which averages only 120 y in its longer diameter, looks down on to the roof of the ovicell, and can rarely be seen without dissection of the colony. The tube of the oceciostome is adnate to a series of zocecia, and its upper exposed surface is convex. The primary zoccium diverges from the plane of attachment to a greater extent than in most species, and the proximal part of the colony is usually rather deep and narrow.
Common (in Devonshire) on red seaweeds, shells, and stones from about three fathoms to moderately deep water. I have seen one specimen from the Outer Hebrides.
The reasons for maintaining that this species is distinct
ON THE DEVELOPMENT OF TUBULIPORA. 95
from T. flabellaris, Fabr., are given below, under the ac- count of that species.
The occiostome of T. phalangea is shown in figs. 5, 6. The convex upper surface is really the outer part of the wall of the tube, which has the form of a NM, one limb of which rises vertically from the roof of the ovicell, the other being shortened and opening downwards. It results from this arrangement that it is quite impossible to see the adult ocecio- pore in the great majority of cases without breaking off the series of zocecia which bears the tube, and turning it over until the occiopore becomes visible. It is then seen to have the form shown in fig. 6, being conspicuously smaller than that of T. liliacea. The diameter of the occiopore varied from 110 to 135 yw in five specimens measured (average 118 yw), the widest part of the entire tube varying from 110 to 180 pu (average 139 mw) in the same specimens. The occiostome may be quite symmetrical, or it may be distorted so that the occio- pore looks obliquely downwards. When the occiostome is typically developed (as in the great majority of cases), it differs to a striking extent from that of any other species described in this paper.
In its typically developed form this species is distinguish- able by its very long and slender zoecia, the ends of which are more commonly dissociated from their neighbours (and are therefore completely cylindrical) than in T. plumosa, with which it commonly occurs. The character of the oldest zocecia is of some value in distinguishing the species. While T. plumosa is usually depressed in the oldest part of the colony, this species has rather the opposite tendency, and the primary zocecium usually grows upwards at a considerable angle from the plane of support, the interval being occupied by the proximal ends of the next zoecia.
The general characters are well described by Couch (5, pp. 106, 107), though his fig. 7, pl. xix (wrongly given as fig. 8 in the text), is excessively bad. H. Milne Edwards (29, pl. xii, figs. 1, 16, &c.) gives excellent figures of this species, accom- panied by some anatomical details (pp. 323, &c.), under the
96 SIDNEY F. HARMER.
name of T. verrucaria, Fabr., accidentally given as T. ver- rucosa in one place on p. 328. The name employed by Milne Edwards cannot be retained, since the Madrepora verru- caria of Fabricius is a Lichenopora. From an inspection of the original description and figures I can see no sufficient reason for believing that the fossil Diastopora plumula, Reuss, is identical with the present species or with T. flabellaris, Fabr., although Pergens (34, p. 9) considers the specific name given by Reuss to be the correct name of one of the forms to which the name T. flabellaris has been ascribed.
T. phalangea is common in the Salcombe Estuary, at a depth of 3 to 5 fathoms, on red seaweeds, where it occurs in company with T. plumosa, and on dead shells. It is equally common at Plymouth from 3 to 15 fathoms; and I believe" that the greater number of specimens of Tubulipora found on shells in the shallower parts of the Plymouth district belong to this species. In the deeper water (20 to 30 fathoms) a considerable proportion of the specimens may belong to the form identified by Mr. Hincks as T. lobulata, Hassall; but I am at present unable to express any positive opinion with regard to Hassall’s species.
I have seen a typical specimen of T. phalangea, kindly lent to me by Professor M‘Intosh, from the Outer Hebrides ; but the rest of my material has been obtained from Devonshire. T. phalangea is very variable in the form assumed by the colony. It may consist merely of a single small, fertile lobe, the whole colony being then pear-shaped, and closely resembling the Obelia tubulifera of Lamouroux (26, p. 81, pl. lxxx, figs. 7,8), a Mediterranean form with which it may be identical. It may consist of a small number of well-separated lobes, or it may have an almost completely circular outline. Colonies of the last type may reach a diameter of nearly an inch. The complexity of the ovicell varies greatly with the size of the colony, the large colonies having very complicated ovicells with numerous palmately arranged lobes extending between the radial series of zocecia; fig. 5 being by no means
ON THE DEVELOPMENT OF TUBULIPORA. 97
an extreme case of this kind. The smaller colonies have simpler ovicells, but this is also the case in other species.
My Salcombe specimens were dredged in March and April ; and an examination of this material gives some hints with regard to the meaning of the differences in size. A very iarge number of colonies found on shells were small and pyriform, although in many cases possessing a mature ovicell; while other small colonies consisted of only two or three lobes. These colonies were nearly all brown, and more or less en- crusted with foreign matter. Here and there an old lobe had recommenced to grow, and had given rise to a fresh and clean lobe, whose brilliant white colour (in spirit specimens) forms the most striking contrast with the older lobes. The colonies of Tubulipora have in fact the power, which is probably common to all Cyclostomes, of regenerating new zowcia from various parts of the old colony (cf. 18, p. 141). In the species now under consideration a small part of the edge of the old colony here and there becomes active, so that a series of fresh lobes, with narrow bases, may be seen growing out from various parts. These lobes have in many cases acquired a considerable size by the beginning of April, and have developed mature ovicells. A few quite young, healthy colonies were found amongst these specimens. It is probable that these last were colonies which had recently commenced their ex- istence, that the brown specimens belonged to a previous year, and that the fresh lobes proceeding from them were entirely recent growths. If this is a correct inference, it may be suggested that the small brown colonies were produced late in the year when the temperature was becoming low, so that although they became mature so far as the external characters of their ovicells were concerned, they were unable to grow large. There was of course no doubt that the finely developed speci- mens were actively growing when they were dredged.
I think it probable, therefore, that the difference in the development of the entire colony may, in some cases at least, be of a seasonal nature.
I may here refer to some interesting remarks which have
vou. 41, part 1.—NEW SER. G
98 SIDNEY F. HARMER.
been made to me by Mr. A. H. Church, whom I had consulted on the growth of Tubulipora. Mr. Church informs me that Rhodymenia ciliata, a red seaweed on which I obtained nearly all the material (T. phalangea and T. plumosa) which I have used for sections, is an annual. It follows, therefore, that even the largest colonies (some 12—15 mm.) found on this seaweed must represent the growth of one year. Mr. Church informs me that R. ciliata usually dies in the winter, the middle of which period may be regarded (for Algz) as February, but that the specimens I dredged (in a sheltered estuary) at the beginning of April must have grown in the preceding year. I have not noticed processes of regeneration in colonies growing on seaweeds, the evidence from which is not entirely concordant with that from shells. The specimens growing on Rhodymenia (and the same is true of specimens of T. plumosa collected at the same season on Saccorhiza bulbosa) show no apparent discontinuity of growth, nor were any stunted mature colonies observed in this situation, The colonies were probably far too large to have grown in the year in which they were collected; and although the growth may have been less active or dormant during the winter, there was no interruption sufficient to give rise to a marked discon- tinuity, as in the specimens growing on shells. It may, how- ever, be noticed that there is no reason for assuming that these latter were the growth of the year immediately preced- ing. They may be evidence of unfavorable conditions more than one year before, in which case the absence of similar colonies on the Rhodymenia growing in the same locality would be due to the fact that this plant is an annual. I think it follows from the observed facts that growth may start at any time when the species is breeding, and that a colony which begins existence in the summer continues to live through the winter, and produces ovicells in the spring.
Mr. Church informs me that in some cases of regeneration which he has observed (on a glass bottle) the central part of the colony had decayed, leaving the new growths with a vacant
ON THE DEVELOPMENT OF TUBULIPORA. 99
space in the centre. I have had no opportunity of examining cases of this kind.
Hincks (18, p. 447) has referred to a curious lobed Tubuli- pora, about an inch in diameter, which he has met with in Salcombe Bay. I have obtained some specimens which appear to correspond with Mr. Hincks’s description. One or two of these colonies were from the Salcombe Estuary, and had the oceciostomes of T.,phalangea. Two others were sent to me from Plymouth by Mr. Church, and had the occiostomes of T. plumosa. The variety is a very curious one, and is characterised by its nearly circular outline and by the great crowding of the zoccia, the series being placed very close together, and the lobes of the well-branched ovicells being correspondingly narrow. On closer examination a consider- able difference (no doubt specific) between the Plymouth and the Salcombe specimens becomes apparent. A perfect colony of the former measures about 15 mm. in diameter ; it is com- posed of twelve well-marked lobes, and closely resembles the form of T. plumosa which is found on Saccorhiza. These lobes can be readily made out without any magnification, whereas the Salcombe specimens do not appear obviously lobed when examined with the naked eye.
I regard this variety as due to an excessive growth of the edge of the colony, resulting, by the mutual pressure of the lobes, in a crowding of the zocecia, and in the acquirement of a circular form. This appears to me to be a further instance of the tendency of different species of Tubulipora to assume the same general form as the result of some unknown factors in the environment.
T. flabellaris, Fabricius (fig. 4, described on p. 82).
Tubipora flabellaris, Fabr. (11, p. 430). Tubulipora flabellaris, Smitt (40, p. 401, pl. ix, figs. 6—8). ? T. flabellaris, Levinsen (26, p. 76, pl. vii, figs. 1—38). Zoarium entirely adnate, more or less fan-shaped in form in well-developed
specimens. Stunted colonies may occur, as in the preceding species. Some of the zocecia are free, others are in connate series, which are more or less
100 SIDNEY F. HARMER.
developed, and become radial in fertile lobes. Ovceciostome consisting of a greatly compressed tube, whose oceciopore is slit-like. The longer diameter of the slit averages about 165 » (130—180 pz), being equal to or somewhat less than the diameter of an orifice. The tube is not recumbent on a series of zocecia, but stands up freely from the roof of the ovicell, its two narrow edges being placed in a radius of the colony.
This seems to be an essentially Northern species, and I have no evidence of its occurrence in British waters. I have examined specimens from Greenland (the locality from which the type-specimens came), Barents Sea (50 fathoms, growing on Cellularia peachii), and Hammerfest ; and I have also obtained what I believe to be a young form of this species from Godésund, Bjorne Fjord, Norway. The specimen figured (fig. 4) is a stunted, somewhat abnormal form of this species from Barents Sea (see the description on p. 82), but it well illustrates its characteristics by showing five perfectly typical oceciostomes.
Professor Smitt (40, pp. 400—402, 454, 458), who is fol- lowed in this respect by Mr. Hincks, regards T. phalangea of Johnston as identical with T. flabellaris, although he believes the original T. phalangea of Couch to be identical with T. lobulata, Hassall. It appears to me that the characters of the oceciostome are amply sufficient to separate T. pha- langea from T. flabellaris. I first became acquainted with the oceciostome of the latter in two colonies from Hammer- fest, kindly sent to me by Dr. Nordgaard. The collection of Polyzoa given to the University Museum of Zoology by Miss E. C. Jelly contained a moderate number of specimens on sea- weed from Greenland. The form and size of the oceciostome were identical in these and in the Hammerfest specimens. The colonies from Greenland, although very small and stunted (one of them, with only twenty-seven zocecia, possess- ing a complete ovicell), did not resemble the Barents Sea specimens from deeper water in possessing a multiplicity of ovicells.
It can hardly be doubted that the Greenland specimens, sent by Miss Jelly, belong to Fabricius’ species. The flabelliform shape, the connate series of zocecia, the occurrence on sea- weed, and the small size (given by Fabricius as 13 lin. in trans- verse diameter), all agree closely with the original description. The species has been more fully described by Smitt, who gives excellent figures. In two of these (40, pl. ix, figs. 6, 8), re-
ON THE DEVELOPMENT OF TUBULIPORA. 101
presenting specimens from Spitzbergen, the peculiar form of the flattened oceciostomes, with their radially arranged flat sides, is indicated, while the flattened form is expressly men- tioned on p. 457. I have not seen specimens so finely de- veloped as that shown by Smitt in his fig. 6, in which the radial serial arrangement of the zocecia is strongly marked (as many as twenty being stated to occur in one series). The difference between Smitt’s specimens and those examined by me may, however, be seasonal, as suggested under the last species.
It may be remarked that Smitt’s conclusion that T. pha- langea is asynonym, partly of T. lobulata, Hass., and partly of T. flabellaris, Fabr., does not appear to have been based on an examination of actual specimens of the first-named species.
Tubulipora aperta, n.sp. (figs. 2, 3).
Tubulipora fimbria, Smitt (40, pp. 401, 452, pl. ix, fig. 5). ? Tubulipora fimbria, Levinsen (26, p. 75, pl. vi, figs. 45—50).
Zoarium entirely adnate, pyriform, flabelliform, or lobed. Zocecia not serially connate, or only exceptionally united in very short series. Ectocyst with few pores. Oceciostome about 280 » in diameter, larger than an orifice, more or less funnel-shaped ; the oceciopore opens upwards, and is circular or oval. Tube of the oceciostome usually more or less free, and diverging from the zocecium on which its base is recumbent, the edge of the oceciopore often resting on the wall of another zoccium. Accessory openings sometimes present at the ends of the lobes of the ovicell.
Common on the fronds of Laminaria saccharina in Norway. My largest colony is 5°25 mm. in transverse diameter.
This species, which I believe to have hitherto received no distinctive specific name, has been described and figured by Smitt under the name of T. fimbria. This name, applied by Hincks to T. plumosa, Thomps., was given by Lamarck to an immature specimen of Tubulipora, of which the locality is not recorded, and I give my reasons for not accepting it on page 107. The name aperta is suggested in reference to the wide occiopore, which is usuall clearly visible from above,
102 SIDNEY F. HARMER.
and is not concealed either by the zocecia or by other parts of the oceciostome. The specimens on which my account of this species is based were found principally at Godésund, a small island off the north of Tysnas6, at the entrance to the Bjorne Fjord in Norway. They were not uncommon on the fronds of Laminaria saccharina, where they occurred in company with Lichenopora verrucaria, Fabr. Most of the speci- mens collected at the end of June had fully developed ovicells. Specimens collected at Lervik, in the Hardanger Fjord, at the same period in a previous year were, however, not provided with ovicells. Smitt describes the species as occurring