Contribution to the Study of Gromia oviformisDujardin.

By

Margaret W. Jepps, M.A.,Lecturer in Zoology, Glasgow University.

With Plates 37-9 and 5 Text-figures.

FOE the last two years I have been making observations onGromia o v i f o r m i s in the hope of discovering somethingfurther of its life-history. Although I have not up to the presentsucceeded in continuing the life-cycle beyond the critical pointreached by preceding workers, I think it desirable to place onrecord a summary of my observations whether in confirmationof the results of my, predecessors or their extension in detail.

The organism I have studied is the Gromia o v i f o r m i sof Dujardin, found by him in 1835 on C o r a l l i n a in the Gulfof Lyons and along the French coast of the English Channel,the organism whose pseudopodia, ' filaments croissant et seramifiant comme des racines ' / are responsible for the newname, Ehizopoda, which he gave to those ' infusoria ' whichare amoeba-like animals with shells.

Max Schultze described the same animal from the AdriaticSea in 1854, but was misled by Dujardin's description of' granules ou des nodosites ' 2 in the pseudopodia of his Gromiao v i f o r m i s into believmg that he had discovered a new specieswith hyaline pseudopodia which he named Gromia du-j a r d i n i i. It seems clear, from a comparison of the figuresand descriptions, that Gromia o v i f o r m i s Dujardin is thesame creature as Gromia d u j a r d i n i i Schultze. This wasalso the opinion of Ehumbler (1904).

1 ' Ann. Soi. Nat.', s6r. ii, vol. iii, p. 314.2 Ibid., vol. iv, p. 343.

702 MARGARET W. JEPPS

Gruber (1884) found an organism which he identified asGromia d u j a r d i n i i of Schultze in the Gulf of Genoa, anddescribed besides four new species of Gromia from the samecoast, one of which he named Gromia d u b i a . Some at anyrate of the individuals comprising these new species areexamples of the small Gromia described below as the d u b i aform. The Gromia o v i f o r m i s Dujardin of Mobius (1888)from Kiel is also probably to be included here (as are thespecimens figured by M. Schultze in 1854).

Gromia o v i f o r m i s Dujardin was reported from Warne-munde, in the Baltic Sea, by P. E. Schulze in 1875.

In 1894 Schaudinn gave a new generic name to Gromiad u j a r d i n i i of Schultze. He considered that the characterof the pseudopodia (the absence of granules, the rare anasto-moses), and the presence of ' brown bodies ' in the cytoplasm,sufficed to separate this organism, not only from Gromia( = Gromia o v i f o r m i s Schultze), but from all the Foramini-fera, leaving it isolated among the Ehizopoda. Schaudinn pro-posed to call it H y a l o p u s d u j a r d i n i i .

GENERAL DESCRIPTION.

Gromia o v i f o r m i s occurs in large numbers on the sea-shores of Britain. Most of my specimens come from Millportin the isle of Cumbrae, in the Firth of Clyde. It is found onthe weed in coralline pools, on C l a d o p h o r a , on the wallsof rock crevices, and on other solid objects between tide-marksand to depths of a few fathoms. I have never found it amongstprofuse growths of E n t e r o m o r p h a .

Dujardin likened Gromia to ' un oeuf de zoophyte, ou unepetite graine de plante ' (' Infusoires ', p. 252). A young friend,to whom I showed some of my specimens, said that they lookedlike minute potatoes, an excellent simile. Individuals vary in sizefrom 8 mm. in diameter downwards, the smallest I have measuredbeing about 0-15 mm. across. They may be almost spherical,often flattened at two opposite poles into the shape of anorange. Lobed forms are very common, especially in thicktangles of C o r a l l i n a , and much depressed on flat rocky

GROMIA OVIFOBMIS 703

surfaces (figs. 1-8, pi. 37). I have never seen the long, beadedforms described by Schaudinn (1899, Text-figs. I-IV), nor anyas large as his 5 mm. specimens from the Adriatic Sea. Amongthe Gromias collected from a coralline pool there may be afew small ellipsoidal individuals of a fairly constant shape(figs. 9-11, PI. 37). These .are usually found in the debriswashed off the weed, since they are never as firmly attachedas are the spherical or flattened forms. They never reach alarge size, being rarely as much as 1 mm. long. Their breadthis usually about four-fifths of their length. A fair number ofGromias were collected in a D-net from a Z o s t e r a bed inCawsand Bay, near Plymouth, at a depth of 2-4 fathoms.These were all, with one exception, of the small oval type.It is possible that this type is specifically distinct from the largerGromias of the rock pools. No other differences in structureor behaviour between the two types haA'e been detected, sothat if they are to be separated as two species these pointsmust serve to distinguish them :

(1) The smaller average size of the ellipsoidal form.(2) Its constant shape.(3) Its weaker hold on the substratum, due to its being taller

than the spherical type and not sharing its habit ofmore or less burying itself in mud and debris.

Until we know more of the life-cycle of Gromia it would beabsurd to make new species ; so that for the present I proposeto speak of the small oval form as the d u b i a form, Gruber'sname being particularly appropriate during the present stageof uncertainty.

The colour of Gromia varies with the nature of the groundon which it lives, since it depends almost entirely on the foodand other particles ingested. The shell is transparent, andcolourless excepting for a variable number of reddish-brownexcrescences on its outer surface, which may be so numerousas to give the shell a tinge of colour of its own on the upper side.Occasionally the collar round the mouth is of a pale brownishcolour.

The shell usually has a single large mouth on a flattenedNO. 280 3 A

704 MARGARET W. JEPPS

surface ; in the d u b i a form the mouth is always at one endof the long axis. Out of hundreds of specimens I have onlyfound one with more than one mouth. (Schaudinn, 1894,described as many as twenty-five in some large specimens.)This was a spherical Gromia, 1-5 mm. in diameter, flattened atthe two poles, with a mouth in the middle of each flattened surface(fig. 8, PI. 37). The diameter of the mouth is roughly one-fifthof that of the whole Gromia. It is bordered by a thickenedrim of the shell, carrying a thick soft circular collar, throughwhich the pseudopodia are extruded (fig. 7, PI. 37 ; fig. 13,PI. 38 ; fig. 15, PI. 39). These may be very long—commonlyfour or five times the diameter of the shell and sometimes muchmore. They are also comparatively thick, and because of theirabundance have proved a useful object for physiological study(Eeichert, 1865 ; M. Schultze, 1866 ; Butschli, 1894 ; Verworn,1897 ; Zarnik, 1907). The pseudopodia are perfectly hyalineduring life, no granules being visible in them even under anoil-immersion lens. They emerge very slowly, as a little bunchof stiff slender spikes, from an area of the cytoplasm lying justinside the mouth of the shell, and free from ingesta. Theyextend in all directions, gently waving about as if in search ofsomething of which to take hold. When a pseudopodium comesup against a solid object it sticks to it and flows out over it.As the pseudopodia increase they branch, and become irregu-larly varicose and ridged so that they look somewhat like thegnarled roots of a tree. From swollen places or knots therearise fresh pseudopodia extending at wide angles and oftenanastomosing with others in the vicinity. Some may creepback over the surface of the shell: otherwise there is no extra-mural protoplasmic layer (vide Schultze's figures, 1854, Taf. I,figs. 1, 4, 5). All kinds of small particles are engulfed by thepseudopodia and carried back towards the mouth as theseretract. The tip suddenly lets go and springs back as if it hadbeen stretched, and the whole pseudopodium begins to shorten,becoming very knotty as the branches flow back into the mainstream. The whole animal is sticky, so that particles adhereto it at any point of its surface, and a mass of mucus is poured

GROMIA OVIFORMIS 705

out round the mouth. In this mucus collects a great varietyof foreign objects—sand grains and muddy particles, spiculesof sponges &c, setae, bits of algae, diatoms—which hide thecollar from view until they are scraped off. This mass of debrismay cover half the shell in the large Gromias, but in the d u b i aform never extends far from the opening. As the mucus helpsto hold the animal fast, this partly explains the greater easewith which the latter form is dislodged. The pseudopodia arealso covered with mucus, which often persists, forming a trackof their positions, for a time after they are withdrawn.

The protoplasm of Gromia moves very slowly, and thepseudopodia are not capable of swift retraction. Consequentlythey are very often torn away by an accident, after whichix fresh set may appear to replace them in as short a time as15 minutes. As might be expected, the pace of a Gromiatravelling over the substratum is comparatively slow, muchslower than that of a Polystomella or a Miliolina for example.Dujardin saw a Gromia move 2 mm. in an hour. The greatestspeed I have observed is much less—about 6 mm. in 26 hours.This may be due to a lower temperature. Dujardin (1835 b)noted a decrease of activity in the cold of winter.

While the pseudopodia are well extended the collar is pushedout by the emergent protoplasm so that there is an appearanceof a short neck. In the retracted condition the collar sinks in,partly or quite closing the aperture. It may even be inverted,perhaps pulled in by pseudopodia attached to its base, so thatit hangs inside the shell.

THE SHELL.

The minute structure of the shell was roughly described byBiitschli (1894, p. 101). He gives an account of the closingmechanism of the collar and rim of the shell, ascribing to thelatter a degree of elasticity which allows the mouth to bestretched by the issuing pseudopodia. Awerinzew (1903)describes the shell as composed of an outer perforated layerand an inner structureless membrane.

An empty shell, from which the contents have been squeezed3 A 2

706 MARGARET W. JEPPS

out, is easily studied under the microscope. It consists of twolayers. The outer layer is composed of short irregularly prismaticrods of various forms and sizes, each of which is perforated byone or more canals. An inner layer, closely applied below this,is a structureless membrane (Text-figs. 1 and 2). The shellvaries considerably in thickness in different specimens, andsometimes in different parts of the same shell, this variationdepending on the length of the rods. These are always longertowards the rim than over the rest of the shell, Avhere they maybe as little as 2-3 ft especially in small individuals, or in largeGromias as much as 15-20ft in length. In the majority ofshells they are between 5/x and 10/x long. The structurelessinner layer extends beyond the rim, which it helps to form(vide Text-fig. 3, and fig. 15, PI. 39), to support the thick collar.This is a soft structure, often showing wrinkle's on its outersurface. Each of these parts of the shell varies somewhat in itschemical reactions and solubilities ; some of their propertiesare shown in the following table : *

Reagent.Tap-water3 per cent, acetic

acidCone, hydrochloric

acid (cold) 2

Cone, hydrochloricacid (boiling)3

50 per cent, caus-tic potash (cold)

50 per cent.caustic

Time.3 months3 months

2 months

1 hour

2 months

10 minutes

.Boats.InsolubleInsoluble

Insoluble

Deformed, in-soluble

Fell apart , anddissolved inone to severalweeks

Dissolved

Basal membrane.InsolubleInsoluble

Insoluble

Insoluble

Usually re-mained ;sometimesdisintegratedafter severalweeks

? 5

Collar.InsolubleInsoluble

? 4

v

? 4

•>potash (boiling)1 I t was found desirable to colour the shell when making these tes ts .

Bethe's chitiii stain was used. I t gives a very resistant colour (green inhydrochloric acid, and violet in potash), and does not appear to alter thesolubilities of t he shell.

2 Unstained rods and collar turn a beautiful pale violet colour in2-3 days, and then brown. 3 The shell turned brown.

4 I n a few hours the collar was reduced to a smaller ring surrounding t h eaperture, and disappeared after several weeks.

" The membrane broke up owing to the violence of the boiling.

GROMIA OVIFOBMIS 707

No part of the shell gave any cellulose reaction with iodine,and none stained with trypan blue.1 The collar stains ratherdifferently from the mass of the shell, taking up acid stainsmore readily (eosin, picronigrosin) and basic stains less freely(haematoxylin, picrocarmine). Bethe's stain, not too intense,makes good permanent preparations.

The properties of the outer layer of the shell indicate that itis in the same class as the fresh-water Ehizopod shells investi-gated by Awerinzew (1907), which he concluded were composed

TEXT-FIGS. 1 AXD 2.

0 5 10P.Fig. 1.—Surface view of shell. Fig. 2.—Shell in optical section.

of a nitrogenous organic substance (to which he gave the namep s e u d o c h i t i n ) impregnated with silica.

THE PROTOPLASMIC BODY.

The granular protoplasm fills the shell as a rule. Whenspecimens are found in which this is not the case it is probablethat they have been crushed so as to cause part of their contentsto escape by the mouth, or that they have exuded part of theirprotoplasm apparently voluntarily, perhaps as an act ofdefaecation. For the protoplasm is crowded with all kinds ofdebris—food residues, diatom shells, sand grains, pieces of algaltissue, various spicules and spines of other marine animals, &c.Many of the smaller particles are made up into brown, usuallyoval, masses, the stercomata (Schaudinn, 1899), and these are

1 As Dr. M. Lebour discovered, this is a good stain for the tests ofDinoftagellates (vide ' DinoflageJlates of the Northern Seas !, Plymouth,1925, p. 162).

708 MARGARET W. JEPPS

so numerous that they are responsible for the general browncolour and the opacity of the animal. Lying amongst thestercomata are numerous yellow refringent bodies, the xantho-somes of Ehumbler (1894). These are usually 1-2/*. in diameter,and irregularly spheroidal or ellipsoidal in form; they oftenappear double and sometimes in clusters of up to ten or twelve(Text-fig. 3). Nothing definite appears to be known as to theirchemical nature or function, although they are referred to againby Schaudinn (1899, p. 178), Zarnik (1907), and Awerinzew(1910). They are soluble in strong acids, insoluble in alkalies,water, alcohol, and xylol. They would appear to be some formof waste product, as they remain behind in the shell with thestercomata at sporulation. They are heavier than the latter,since they tend to sink to the lowest part of the shell, wherea concentration of them results in the orange patches spokenof below. No fat or glycogen has been observed in the cyto-plasm.

On its surface the protoplasm forms a very flexible pellicle,beneath which it is comparatively free from detritus to a smalldepth ; Awerinzew's inner layer of the shell, closely applied tothe surface of the protoplasm, seems to correspond with thispellicle rather than with the actual inner shell-layer described'below. There is a mass of similarly clear protoplasm justwithin the mouth. Pseudopodia may arise from any point ofthe surface not closely covered by the shell, and may extendinside the shell or issue from the mouth. In either case theyare perfectly free from granules (in the living state), and maybe considered as concentrations of the ectoplasm which is other-wise scarcely to be visibly distinguished. With thionin theouter cytoplasm stains somewhat metachromatically, and ittakes on a bright colour after a few minutes in mucicarmine ;it seems likely that it is impregnated with the mucus alreadymentioned. Pseudopodia, fixed with corrosive sublimate, andstained with thionin, show a distinct skin which stains meta-chromatically, enclosing protoplasm faintly seen to have afine-meshed alveolar structure, stained pale blue. (This tube-like structure led Zarnik to propose the name Solenopoda for

GROMIA OVIFOEMIS 709

a new order of protozoa, in which Gromia was to be iso-lated, and the new specific name s o 1 e n o p u s for the Gromiahe studied.)

Besides the solid bodies already mentioned there are embeddedin the cytoplasm a large number of nuclei. These are spherical,

TEXT-FIG. 3.

Small part of a section of Gromia (vide fig. 15, PI. 39). Senii-diagrammatio. c , collar ; ect., clear ectoplasm ; n., nucleus ;ols., outer layer of shell; rs., rim of shell; s., stercomata; w.,vegetable remaias ; x., xanthosomes.

about 3-5-5-Oju. in diameter irrespective of the size of theGromiaj and evenly distributed throughout the whole animal.The nuclear membrane is not always to be made out. Thechromatin is in the form of a large karyosome and a varyingamount in granules scattered through the rest of the nucleus(figs. 16-20, PI. 39).

7 1 0 MARGARET W. JEPPS

NUCLEAR DIVISION.

Nuclear division in Gromia has not yet been described.Zarnik (1907) merely remarks that he finds various appearancesindicating that it is amitotic. It is not at all easy to observe.After long search through numerous smears and sections a fewnuclei were seen in a recognizable stage of division, and thesegave a clue to the interpretation of other appearances whichhad already been noted. In sections of two Gromias 1 all thenuclei were in the condition shown in figs. 37-42, PL 39. Thekaryosome seems to be budding off small portions of its sub-stance in a fairly regular manner, portions which are sometimesas large as the remainder. This Avould appear to be an earlystage of division since the nuclei are of the full-grown size ; allthe nuclei of each Gromia are almost at the same stage. Thenext stage to be recognized is shoAvn in figs. 21 and 22, PL 39.This is a much later one, when the nuclei are almost dividedinto two. Their internal structure is obscured by the diffusedark stain they take on at this stage ; it is only possible insome of the less deeply stained nuclei to make out several moreor less definite masses of chromatin inside them. Nuclei in thisstage occurred here and there in preparations in which most ofthe nuclei were either in advance of it or behind it. It waspossible, after making these observations, to pick out kineticnuclear forms in any smear of Gromia protoplasm, and alsoin sections, where they are seen to occur especially in the morecentral regions of the protoplasm. It seems then that there arealways some nuclei undergoing division, small groups of neigh-bouring nuclei tending to be at approximately the same stage.A series from such smears is shown in figs. 26-31 of PL 39.The reconstruction of the interkinetic nucleus is shown infigs. 23, 25, and 32-6, PL 39. The nuclei in the latter prepara-tion tend to be rather smaller than usual.

If these observations give the essentials of nuclear division in

1 One of these was known to have withdrawn its pseudopodia a weekearlier. Both were in aquaria, and were probably about to prepare forsporulation.

GROMIA OVIFOEMIS 711

firomia, it is seen to be a very simple process; not howevera pure amitosis, since there is some rearrangement of the•chromatin. It is true that there is no spindle, but the ratherindefinite chromatin blocks may be regarded as a kind of pro-chromosome.

BPORULAXION.

Gromia, brought into the laboratory and kept in smallaquaria, has always sooner or later undergone a form of sporula-tion, sometimes within a ieAv days, sometimes after weeks or evenmonths. (This was also the experience of Zarnik, 1907.)Sporulation occurs whether the sea-water is left unchanged ornot, and whether or not aeration is supplied ; feeding on impurecultures of diatoms seems to make no difference. No growthin size of individuals was observed in any of my cultures, andno change of form, oven when lobed individuals were taken offwee d and put on the smooth bottom of the aquarium (cf., however,Schaudinn, 1894). Large and small specimens of both formshave a precisely similar fate. The pseudopodia are withdrawn,leaving the animal attached by the circumoral mucus. Awhitish film appears on the surface of the brown protoplasmicbody, and gradually increases in opacity until sometimes ina few hours, sometimes in a week or more, the Gromia becomesmilky white in its upper half; if it be reversed the lower halfis seen to be brown as before, but in the centre of the brownarea, i. e. at the lowest point, is an orange patch. At this stageit may be possible to arrange the illumination under the micro-scope so as to see that the contents of the white area are smallparticles in rapid motion, just like a crowd of active spermato-zoa. Then the milkiness disappears, sometimes gradually, sothat it is only under a lens that one sees this disappearance tobe due to the escape of the small active bodies from the shell,sometimes suddenly so that their escape is visible to the nakedeye like small puffs of smoke emitted from one or more pointsof the shell. When this discharge is over the upper part of theshell remains apparently empty, and the brown contents of thelower half have a ruffled surface which is very different from

712 MARGARET W. JBPPS

the smooth outline of the protoplasm containing stercomataof the living animal (fig. 14, PL 38). When examined under themicroscope the residue is seen to consist of stercomata, xantho-somes, and particles of debris which have fallen in layers (pre-sumably according to their weight) to the bottom of the shell—all the cytoplasm having been withdrawn. Such a Gromia hasof course no further life, only the shell and the food residuesremaining.

Sporulation has also been observed to take place on the sea-shore, large numbers of Gromias growing on C l a d o p h o r abeing found in this condition together in April 1925 besideLoch Sween, in Kintyre.

The process was observed long ago by Strethill Wright (1861)., He observed a Gromia in an aquarium to be ' filled as to itsupper part with a milky matter, which, when pressed out,proved to be a congeries of cells and large active molecules,such as are obtained from the sperm-sacs of H y d r a v i r i d i s ' .These, he says, he is ' persuaded are the spermatozoa ofGromia '. Schaudinn (1894) briefly describes, without figures,the whole process of sporulation as the formation of oval orpear-shaped gametes (5-8/u, in diameter), each with a longnagellum (30-38n long) and a nucleus (3-6/n across). These fusein pairs inside the shell of the parent organism. He was notable to follow the fate of the zygote. In 1899 Schaudinn pub-lished some figures of Gromia and these gametes in his paperon T r i c h o s p h a e r i u m . Zarnik in 1907 described the pro-duction of swarm spores in Gromias in the laboratory ; he givesdetails of their formation by rapid divisions of the nuclei andof their structure, some of which I have not been able to con-firm. He did not see any ' copulation '. I was interested tonotice, when I read his paper after writing the foregoingaccount of sporulation as seen by the naked eye, that he alsocompares the escape of the swarm spores to a cloud of smoke.I cannot find the fuller account which he says he intended topublish elsewhere. Awerinzew (1910), in a preliminary note,again described these changes as gamete formation, in thecourse of which is a chromidial stage followed by differentiation

GROMIA OVIFOEMIS 713

of secondary nuclei from the chromidial mass. The sperm-likegametes, which are slightly anisogamous, leave the shell andfuse in pairs. He also observed the formation of isogametes asdescribed by Schaudinn. Awerinzew promised a fuller accountwith figures later, but I have failed to discover that this promisehas ever been fulfilled.

Lwoff (1925) gives the fullest account of the sporulation ofGromia and some figures of the ' spores ', which, however, hesays are not gametes. He has not observed any fusion in pairs,even when ' spores ' from different Gromias are mixed. Themeasurements given by Lwoff for the ' spores ' are 3/x xl-5^,and for the length of the flagellum 20/J.. They are formed b3r

rapidly succeeding divisions of the nuclei, as the protoplasmwithdraws to the surface of the stercomata (vide his figs. 12-15).There is no chromidial stage. The cytoplasm then divides upround the small nuclei.

To the account given by Lwoff I have nothing of importanceto add. My observations coincide with his, and it is thereforeonly necessary to summarize them here.

As the pseudopodia are finally withdrawn all the nuclei beginto divide more or less together. The subsequent divisions followso rapidly that the nuclei of succeeding generations are progres-sively smaller (figs. 37-52, PI. 39). When there is any noticeabledifference in the stages reached by the nuclei it is observedthat those of small areas or zones are all at the same stage.No certain opinion as to the number of these divisions wasobtained,1 nuclei of all sizes between 5/x and 2/x being observed.I have seen appearances in sections suggesting the formationof a chromidial stage as described by Awerinzew, but withLwoff I attribute them to defects in fixation.2 While thesenuclear divisions are in progress the cytoplasm is graduallyfreeing itself of debris ; these are left behind in a large central

1 Possibly there are three.2 Cytological fixation of whole Gromias is very uncertain, irregular

penetration and the presence of so many foreign bodies of unknown com-position sometimes causing much distortion. I find Bouin's picro-formo]-acetic fairly satisfactory for fixing whole animals. Sections 5-6 p thickhave proved the best.

714 MARGARET W. JEPPS

vacuole formed by the centrifugal streaming of the protoplasmout in all directions towards the shell (Text-fig. 4). Kg. 53,PL 39, shows a part of the dense layer of small nuclei (2/t)lying close under the shell before the cytoplasm segments roundthem. In fig. 54 segmentation of the cytoplasm has taken place,each nucleus being surrounded by a small portion. A fewshreds and granules are not included, and these are probably thepabulum which brings the crowd of bacteria, flagellates,

TEXT-FIG. 4.

jsig. 4.—(section ot a Uromia.n, nuclei (2 ft); si, stercomata, &c.; x, mass of xanthosomes.

amoebae, and ciliates into the shell after the escape of theswarm spores. Figs. 55-7, PI. 39, illustrate stages in thedifferentiation of the flagellula from the simple cytoplasmicmass by its condensation round the nucleus, leaving a clearcap of cytoplasm at one side. The flagellum arises usually,but not always, immediately behind this. The fully formedflagellula measures about 3/x x 2/x, and the flagellum 20/x ormore. As Lwoff points out it swims with the clear cap leading,the flagellum trailing behind. Text-fig. 5 was drawn from livingspecimens ; they show a small number of shining granulesround the base of the cap, and a refractive body in its apex.Figs. 58-62, PL 39, represent fixed and stained flagellulae.After leaving the shell the swarm spores swim off very actively

OEOMIA OVIFORMIS 715

in all directions, spreading in a little cloud a centimetre or morefrom the remains of the parent Gromia. They may swim aboutfor some hours if in running water, but die very soon in a smalldrop which is not renewed. I have not been able to cause themto undergo any fusion, either among themselves or with theflagellulae from other Gromias. Excepting for small and incon-stant differences in size, all the flagellulae are alike. In mysmall aquaria they are devoured in large numbers immediately

TEXT-FIG. 5.

0, 5 (I-Kg. 5.—Flagellulae.

after liberation by other protozoa, especially ciliates and smallflagellates ; and this may possibly account for my failure upto the present to rear a new generation in aquaria kept formany months after sporulation has occurred in them.

In conclusion I have great pleasure in recording my indebted-ness to the Zoological Society of London for hospitality at theirPlymouth table in August 1924, when some of the foregoingobservations were made ; to Mr. E. Elmhirst, of the MarineStation at Millport, who first drew my attention to the profu-sion of Gromia there, and has kept me supplied with materialin Glasgow ; and to Mr. P. Jamieson, who has cut most of mysections for me.

Professor Graham Kerr has been good enough to read mypaper, and I offer him my best thanks for his helpful criticism.

7 1 6 MARGARET W. JEPPS

SUMMARY.

1. Gromia o v i f o r m is Dujardin is a common Britishmarine Khizopod, which frequently reaches a diameter of 2 mm.

2. There appear to be two distinct forms of Gromia ov i -f or mis ; a smaller oval variety is provisionally distinguishedfrom the type as the d u b i a form.

3. The apparently homogeneous pseudochitinous shell hasa complex microscopic structure, of which some description isgiven.

4. The protoplasm, which fills the shell, is crowded withstercomata, xanthosomes, and a heterogeneous collection ofingested debris, all of which take a part in giving its colour tothe animal.

5. There are numerous nuclei scattered throughout the pro-toplasm. Some of them are always undergoing a simple kindof division, which, however, involves some rearrangement ofthe chromatin.

6. A process of sporulation has been seen, occasionally innature, and frequently in aquarium specimens; repeateddivisions of the nuclei results in the formation of very numerousuniflagellate swarm spofes, 2-3/J, in diameter. These swim outof the shell. Their further development has not so far beenobserved.

R E F E R E N C E S .

Awerinzew, S. (1903).—' Mitth. zool. Stat. Neapel', vol. xvi, p. 361.(1907).—' Arch. f. Protistenk.', vol. viii, p. 95.(1910).—' Zool. Anz.', vol. xxxv, p. 425.

Biitschli, O. (1894).—' Microscopic Foams and Protoplasm.' Translated byMinchin, E. A.

Dujardin, F. (1835 a).—' Ann. Sci. Nat.', se>. ii, vol. iii, p. 312.(1835 b).—Ibid., vol. iv, p. 343.(1841).—' Infusoires.'

Gruber, A. (1884).—' Verhandl. der Akad. der Naturf.', vol. xlvi, p. 475.Lwoff, A. (1925).—' Trav. de la Stat. de Wimereux ', vol. ix, p. 140.Mobius, K. (1888).—' Abhandl. der Akad. zu Berlin.'Reichert, C. B. (1865).—' Arch. f. Anat. u. Physiol.'Rhumbler, L. (1894).—' Zeitschr. f. wiss. Zool.', vol. Ivii, p. 566.

GROMIA OVIFORMIS 717

Schaudinn, F. (1894).—' Sitz.-Ber. d. Ges. naturf. Freunde Berlin'. p. 14(also in ' Arbeiten ', 1911, p. 45).

(1899).—' Arch. z. d. Abh. d. kgl. preuss. Akad. d. Wiss. Berlin',p. 14 (also in ' Arbeiten ', 1911, p. 150).

Schultze, M. (1854).—' Uber den Organismus der Polythalamien.'(1866).—' Arch. f. mikr. Anat.', vol. ii, p. 140.

Schulze, F. E. (1875).—Ibid., p. 116.Wright, T. Strethill (1861).—' Ann. and Mag. Nat. Hist.', ser. 3, vol. vii,

p. 360 (also in ' Proc. Roy. Phys. Soc. Edinburgh', vol. ii, p. 273).Zarnik, B. (1907).—' Sitz.-Ber. d. phys.-med. Ges. z. Wiirzburg', p. 72.

EXPLANATION OP PLATES

PLATE 37.

Figs. 1-11 were drawn from living G r o m i a ov i fo rm is under aGreenough binocular microscope.

Figs. 1-3.—Depressed lobed individuals from a flat rocky surface.Figs. 1 and 2 represent two views of the same individual.

Figs. 4 and 5.—Irregularly lobed forms from among the branches of analga.

Figs. 6 and 7.—More regular forms not so closely attached to any object.In fig. 6 the brown excrescences are conspicuous, and stumps of the cut-offpseudopodia are visible below.

Fig. 8.—Subspherical Gromia with two mouths.Figs. 9-11.—Specimens of the d u b i a form.

PLATE 38.

Figs. 12-14 are from unpreserved Grornias ; figs. 12 and 14 drawn undera Greenough binocular and fig. 13 under a low-powered microscope.

Fig. 12.—A Gromia crawling under a coverslip. Oral view. Thepseudopodia emerge through the mouth of the shell, but their bases arenot visible above the body of the Gromia.

Fig. 13.—Side view of a d u b i a form. The protoplasm does not com-pletely fill the shell, which is slightly crumpled at A.

Fig. 14.—Remains of a Gromia after the escape of the swarm spores.

PLATE 39.

Fig. 15.—Part of a section of a Gromja with widely open mouth (f obj.8 comp. eye-piece). The dotted circle shows the position of the smallportion shown in Text-fig. 3. C, collar ; N., nuclei; Ps., pseudopodia ;U.S., rim of shell.

Figs. 16—62 were drawn from preparations fixed either with Bouin'spicro-formol-acetic or with a modified Schaudinn's sublimate alcohol (con-

718 MARGARET W. JEPPS

taining 4 per cent, of acetic acid), and stained with Heidenhain's ironhaematoxylin, under a 2 mm. oil-imm. objective and a no. 12 comp. ocular.Figs. 16-36, 50 and 51, 55-62 from smears, and figs. 37^19, 52-4 fromsections.

Figs. 16 and 17.—Nuclei from a Gromia with extended pseudopodia,.1 day in an aquarium.

Fig. 18.—Nucleus from a Gromia with pseudopodia, 3 months inaquarium.

Figs. 19 and 20.—Nucleus from a Gromia fresh from the sea-shore-..Fig. 20 is probably an early stage in division.

Figs. 21-5.—Nuclei very strongly differentiated, from a Gromia a daysin aquarium. Fig. 24 probably an early stage in division, figs. 23 and 25late stages.

Figs. 26-8.— Nuclei in which the karyosome is breaking up, early stagesof division.

Fig. 29.—Nucleus from Gromia with pseudopodia, 1 day in aquarium.The karyosome has broken up.' Figs. 30 and 31.—Nuclei in later stages of division from Gromias freshfrom the shore. In fig. 31 the diffuse stain is becoming less in the daughtermicleus.

Figs. 32-6.—Nuclei from Gromia 1 day in aquarium. Reconstitution ofinterkinetic nucleus.

Figs. 37—53 show a series of nuclei illustrating the decrease in size by therapid divisions leading to sporulation. These nuclei are of course onlya few out of hundreds from which complete series of stages can be made ateach division.

Figs. 37-42.—From a Gromia 3 months in aquarium. Figs. 37 and 38 fromnear the edge of the section, figs. 39-42 from farther in, showing differencesin fixation (cf. figs. 26-8 from smears). The pseudopodia had been with-drawn for several days.

Figs. 43, 45, and 46.—From a Gromia some days in aquarium, pseudo-podia withdrawn. Fig. 43 showing ? first sporulation division.

Figs. 44 and 47-9.—From a Gromia 1 or 2 days off the shore. Pseudo-podia withdrawn. Fig. 49 ? early second sporulation division.

Figs. 50 and 51.—From a Gromia which had been 9 days in an aquarium.The pseudopodia had been withdrawn at least 2 days.

Figs. 52 and 54.—From a Gromia 1 day off the beach, with a whitefilm, and an orange patch below (vide p. 711). Fig. 52 shows the last,? third, sporulation division, and fig. 54 the segmentation of the cyto-plasm.

Fig. 53.—From a similar Gromia to the last, but after 3 weeks in anaquarium. The pseudopodia had been withdrawn for 2 to 5 days. Sporu-lation divisions complete, cytoplasm not yet segmented.

GEOMIA OVIFORMIS 719

Figs. 55-7.—From a Gromia which had been 3 weeks in an aquarium,no food being supplied. It was described as ' beginning to go milky'.The differentiation of the flagellulae.

Figs. 58-62.—From a Gromia, 3 weeks in an aquarium, which had gone' milky ' ; the contents were ruffled, and the upper part contained activeflagellulae, some of which are shown in the figures. Fig. 62 shows onewhich has been strongly differentiated, and in which the granules haveretained the stain.

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