The Histology, Cytology, and Embryologyof Sponges.

By

D. A. Webb.

Scholar of Trinity College, Dublin.

THE classical investigations which form the foundation of ourknowledge of the minute structure of sponges were mainly car-ried out in the latter half of the nineteenth century by Haeckel,Schulze, Sollas, Polejaeff, Topsent, Minchin, Maas, and otherworkers. In the earlier years of this century while many paperswere published dealing with taxonomy, skeletal structure,physiology and descriptions of new species, the study of thesoft parts was somewhat neglected, though the work of Minchinforms an important exception. More recently, however, a revivalof interest in these aspects, assisted by modern fixing and stainingmethods, has resulted in a considerable increase in our know-ledge. It is the aim of this paper to give a r e s u m e of theliterature published during the last twenty year which deals withthose aspects of sponges indicated in the title. 1914 has beenchosen as the beginning of the period since all previous refer-ences are available in Vosmaer's bibliography.1

HISTOLOGY.

The most recent, and perhaps the most complete histologicalstudy is that of E e n i e r a e l egans (Bwk.) and E e n i e r as i m u l a n s (Johnst.), two well-known members of the Haplo-scleridae, by Tuzet (1932). She describes the following types ofcell:

(1) E x o p i n a c o c y t e s , which form an epithelium coveringthe entire outer surface of the sponge except where it is piercedby pores. (Strictly speaking, according to the terminology em-ployed by Minchin the word ' pore' is applicable to the super-ficial orifices only in Ascons. In all higher types the superficial

1 Vosmaer, G. C. J., 'A Bibliography of Sponges, 1551-1913'. Cam-bridge, 1928.

52 D. A. WEBB

orifice is an ostium, while the homologue of the pore is the' chamber-pore' or prosopyle. Th,e word ' pore' is, however, sowidely used by French and American authors to denote ostiathat one must be content merely to call attention to the am-biguity.) The pores are holes between these cells and not withina single cell. The exopinacocytes are flattened cells about14X4/LI, and on their inner surface are produced into bluntpseudopodia which project into the 'mesenchyme' layer. Thegranular nucleus has no nucleolus.

(2) E n d o p i n a c o c y t e s , which line all the inhalant andexhalant canals, i.e. form all the gastral layer with the exceptionof the lining of the flagellated chambers. They are similar to theexopinacocytes except that they are about half their length,possess no pseudopodia, and the nucleus contains a nucleolusfrom which radiate bands of chromatin.

(3) C h o a n o c y t e s , of typical structure, confined to smallspherical flagellated chambers. Each consists of a body, 5 x 3/M,collar and flagellum. The nucleus is at the base of the cell andcontains a nucleolus. The structures at the base of the flagellumwill be discussed in connexion with cytoplasmic inclusions.

(4) S i l i c o b l a s t s , which are elongated cells closely appliedto the spicules. No cases were found of a spicule being includedin a single cell, and the general consensus of opinion now is thatthe growth of the spicule is almost entirely extracellular. Thesilicoblasts have a reticulate nucleus with a nucleolus. These andall the following types are confined to the skeletogenous layeror 'mesenchyme'.

(5) S t e l l a t e ce l l s , consisting of a round body about 10/x.in diameter from which arise long fine pseudopodia which freelyanastomose with the pseudopodia of other stellate cells and ofexopinacocytes. The nucleus is granular, containing a nucleolus,and the cytoplasm has a finely granular structure in contradis-tinction to the previous types in which it is more or less hyaline.

(6) A m o e b o c y t e s , including the archaeocytes and phago-cytes of many authors. They are amoeboid cells with many in-clusions which presumably have been ingested. Their nucleusresembles that of the silicoblasts.

(7) G r a n u l a r ce l l s , which are of two kinds: (i) Those

SPONGES 58

with small, very numerous granules, which stain with silver,osmium, haematoxylin and fuchsin, but not with eosin, and donot stain blue with iodine. It is conjectured that they are lipo-proteid. (ii) Those with larger granules (about 1/x in diameter).These granules stain with eosin after fixation in Bouin's fluid.In other respects the two types of cell are similar. In both thereis a reticulate and nucleolate nucleus.

(8) Globofe rous cells, which are characterized by a singlelarge albuminoid inclusion that stains with fuchsin.

(9) F i b r o b 1 a s t s, the cells that secrete the spongin fibres,which in E e n i e r a form a considerable part of the skeleton.In their early stages they resemble amoebocytes; during sponginformation they become vaeuolated and eventually disintegrate,liberating the fibre. In R e n i e r a e l egans several cellsbecome arranged in a row to form a single large fibre: inR e n i e r a s i m u l a n s each cell forms a small fibre indepen-dently. This distinction is interesting in view of the fact thatBurton (1926), who has been attempting to restore order intothe hopelessly confused taxonomy of the Haploscleridae, hasdecided to include both these as well as several others of Bower-bank's species within the single species R e n i e r a c ine rea(Bwk.).

Another full histological account, in this case of M i c r o c i o n ap r o 1 i f e r a, has been given by Wilson and Penney (1930). Theirdescription is based on the study both of sections and of theliving dissociated cells. They classify the tissue elements asfollows:

(1) E p i t h e l i a , which they claim are syncytial—both thatcovering the external surface of the sponge, and that lining thecanal system. The former is pierced by ' pores' which are some-times partly occluded by a pore membrane. The nuclei areirregularly distributed, granular, without a nucleolus. Theexternal syncytium or epidermis is divided up into polygonalareas by ' epidermal lines' which are interpreted as thickeningsof the cytoplasm. That they are not cell-boundaries is shown bythe fact that they are not correlated with the distribution ofnuclei, and that they do not stain selectively with silver nitrate.

This remarkable result is borne out by Penney (1931), who

54 D. A. WEBB

found that in four fresh-water species (unnamed) investigatedby him both epithelia were syncytial. In this case epidermallines were not seen. There seems no reason to believe thateither he or Tuzet are making a mistake, but on the other handsuch an important difference between two members of the sameorder appears p r i m a facie unlikely. The question clearlyrequires further investigation.

(2) C h o a n o c y t e s , which may always be recognized bytheir containing a few small, bright red granules.

(3) N u c l e o l a t e ce l l s . Sluggish amoeboid cells with alarge conspicuous nucleolus and several large orange inclusions.These may be safely homologized with the larger granular cellsof Tuzet—(7) (ii) in the list given above.

(4) Grey ce l l s , in which the cytoplasm is filled withnumerous small grey granules, staining deeply with methyleneblue. In spite of the fact that they possess no nucleolus Tuzethomologizes these with her first type of granular cell.

(5) G lobofe rous ce l l s , almost exactly similar to thosedescribed by Tuzet.

(6) R h a b d i f e r o u s ce l l s , which are particularly abundantnear the epithelia. They are very elongated and contain numerousrod-like or fibre-like inclusions which stain very readily.

(7) F i b r e ce l l s , which are even more narrow and elongatedand are only found immediately beneath the epithelia.

They also describe scleroblasts, fibroblasts and sexual cells,whose nature and homology present no difficulty.

They maintain that all the mesenchyme cells are connectedtogether by fine protoplasmic processes, a view that is sup-ported by de Laubenfels (1932). This author in the course of ahistological investigation of I o t r o c h o t a b i r o t u l a t a (amember of the same family as Mic roc iona ) lays great stresson the importance of the hyaline intercellular ground substance,whose consistency varies from that of water to that of cartilage,and which acts as a scaffolding for the mesenchyme tissueswhose cells are widely separated from each other as in P r o -t e r o s p o n g i a . The different cell types of I o t r o c h o t a areeasily distinguished by the colour of their inclusions. Hedescribes:

SPONGES 55

(1) A m o e b o c y t e s ' A ' , with large dark purple granulesand no nucleolus. Homologized with nucleolate cells of Wilsonand Penney.

(2) A m o e b o c y t e s ' B ' , exactly similar except that thegranules are pale green. Perhaps correspond to grey cells.

(3) A m o e b o c y t e s ' C ' , with emerald granules, confinedto the superficial layer. Perhaps equivalent to rhabdiferouscells. Also globoferous cells and choanocytes.

Galtsoff (1925) has described the cell elements in disso-ciated preparations of M i c r o c i o n a , but his account has beensuperseded by the later and fuller one given by Wilson andPenney. According to these authors Galtsoff's pinacocytes areshreds of epithelial syncytium, his choanocytes are -wronglyidentified, and his archaeocytes, collencytes and desmacytesare respectively their nucleolate, grey, and fibre cells.

To sum up the above results, it appears that the cells of thesesponges fall into three categories. Firstly, those whose functionand homology is obvious and which are recognizable in allspecies examined, e.g. scleroblasts, fibroblasts, globoferous cells,choanocytes, germ-cells. Secondly, there are cells that have beenonly once described and whose equivalents cannot be recognizedin other species, such as stellate cells, rhabdiferous cells, &c.Finally, there are the granular mesenchyme cells, which in allthe species seem to be of two types, but about whose preciseequivalence there is some doubt. It would also appear that inthe opinion of the above authors the presence or absence ofa nucleolus is a point of no great significance.

Our knowledge of the porocytes has always been far fromsatisfactory. It had previously been held that, once differenti-ated, they were incapable of division, but Volkonsky (1930 b)has shown that in C l a t h r i n a they are capable of dividingwith a normal mitosis, and Prenant (1925), also working onC l a t h r i n a , claims that they arise, not as Minchin supposedfrom exopinacocytes, but from amoebocytes. The question as towhether non-calcareous sponges possess true porocytes is still anopen one, the minute size of the prosopyles in most formsrendering observation difficult.

With regard to the question of the identification of the

56 D. A. WEBB

Metazoan germ-layers in sponges, the only recent pronounce-ment is by Lameere (1926). He refuses to accept the 'inside-out' theory propounded by Balfour and elaborated by Maas andDelage, which supposes that the parenchymula is comparable toa Coelenterate planula, so that after metamorphosis the ecto-derm is internal and consists solely of choanocytes, while all theother tissue elements are endodermal. The main, but not theonly reason, according to Lameere, for rejecting this explanationis that the endoderm is without exception throughout theCoelenterata a single layer of cells. In spite of the reversalat metamorphosis he prefers the view of Haeckel, Leuckart, andSchulze that the adult sponge is directly comparable to aCoelenterate, the choanocytes being endoderm and the remainingtissues ectoderm.

CYTOPLASMIC INCLUSIONS.

During the period under review several workers have in-vestigated the cells of sponges by means of special cytologicaltechniques with considerable success. The first description ofcytoplasmic inclusions in sponges was given by Hirschler (1914),working on S p o n g i l l a fluviatilis. He observed in thechoanocytes a box-like Golgi apparatus in the distal part of thecell (i.e. at the opposite end from the nucleus); the basal granuleof the flagellum appeared to be inside it. It consisted of severalscales or plates which stained black after prolonged osmicationand were not bleached by turpentine. A somewhat similarGolgi apparatus was seen in the amoebocytes: it consisted ofseveral straight or curved rods distributed round the nucleus.According to Hirschler these represent the ' chromidia' describedby Jorgensen. By Sjovall's method he was able to detectmitochondria. In both choanocytes and amoebocytes theywere scattered throughout the cytoplasm: in the former theywere seen in the flagellum but not in the collar. Gatenby (1920a)gives a very similar account of both these structures in thechoanocytes o f G r a n t i a c o m p r e s s a . This author in a laterpaper on the same species (1927) mentions a peculiar processwhereby in certain regions of the sponge the comparatively largemitochondrial granules of the choanocytes undergo fragmenta-

SPONGES 57

tion, being broken up into about fifty minute granules. Thesignificence of this remains obscure.

Tuzet (1932) in the course of her histological description ofE e n i e r a deals fully with the cytoplasmic inclusions of thevarious cells. (The observations of this and other authors onthe cytoplasmic inclusions of the germ-cells will be more con-veniently dealt with below, in connexion with gametogenesis.)In almost all the cells there is a single annular dictyosome,usually situated close to the nucleus, and consisting of achromophil cortex surrounding an internal chromophobe mass.The only exceptions to this rule are the granular cells which havetwo such dictyosomes closely united together, and some of thestellate cells which have two, in this ease widely separated. Shediffers from Hirschler in describing the Golgi apparatus of thechoanocytes as being situated beneath and not around the basalgranule. Mitochondria were recognized in all the cells: in somecases they are in a single group, in others in two groups ordispersed. In the amoebocytes the mitochondria are at firstsomewhat scanty, but become plentiful as the cell ages; thisphenomenon has also been recorded in G r a n t i a by Gatenby.

It has been held by many authors that one of the functions ofthe mitochondria is intracellular digestion, and two authorsclaim to show evidence of this in sponges. Volkonsky (1930a)describes digestion in the choanocytes of Calcarea as follows.The particle is ingested in the upper part of the cell, just out-side the base of the collar, and is received into the substance ofthe cytoplasm. (The old theory that particles are ingested insidethe collar is now generally discredited.) A vacuole is formedround it which is at first neutral or slightly acid and later alka-line. At the latter stage mitochondria condense on it and thewhole region becomes rich in lipoids. If the food particle is onethat contains much lipoid matter—e.g. when a sponge is fed onmilk—the mitochondria become very swollen and take sometime to recover their normal form. Pourbaix (1933), in studyingthe digestion of bacteria • by the amoebocytes of R e n i e r as i m u l a n s , notes that the bacterium lies in an alkaline vacuolewhich is surrounded by a more or less complete ring of mito-chondria.

58 D. A. WEBB

The structures at the apical end of the choanocytes have givenrise to some discussion, the real issue at stake being whether orno the Golgi apparatus and the parabasal body are in this casesynonymous. The same question has of course been muchdebated with reference to the flagellate Protozoa. Volkonsky(1929a) describes a juxta-flagellar structure in the choanocytesof C l a t h r i n a , Sycon , and L e u c a n d r a which he calls the'corps apical'. Its form is somewhat variable but always ap-proximates to a chromophil cap resting on a chromophobe hemi-sphere. It is destroyed by alcohol or acetic fixatives, but hemaintains that it does not stain with Janus green and thereforecannot be the Golgi apparatus. It divides at or before prophase.He somewhat tentatively suggests that it is the homologue of theparabasal of flagellates. Elsewhere (1929&) he maintains thatit was this 'corps apical' and not the Golgi apparatus thatHirschler saw, and that the latter is represented by a ' zone deGolgi' in the middle part of the cell. In a later paper (1930&) hedefinitely states that the corps apical is equivalent to the para-basal. According to this account which is based on a study ofC l a t h r i n a all the cells have a 'cinetide', consisting of centro-some and parabasal, the choanocytes possessing a flagellum inaddition. The parabasal consists of a chromophil plate restingon a chromophobe mass, the former being always attached tothe centrosome which, in the case of the choanocytes, may bedifferentiated into two regions, the mastigosome and the para-basosome. In certain cells such as scleroblasts, amoebocytes andporocytes the parabasal divides precociously so that there arenormally two in each cell.

Tuzet (1931), on the other hand, in describing the parabasalsof the ehoanocytes and other cells of E e n i e r a and H y m e n i -a c i d o n, holds that the chromophil region does stain with Janusgreen and that the parabasal is undoubtedly the Golgi apparatus.There is no doubt that both authors are discussing the samestructure, since both give identical accounts of its behaviourduring oogenesis. The position is, therefore, that the body thatHirschler and Gatenby identified as the Golgi apparatus Tuzetaccepts as such, but claims that it is also the parabasal; Vol-konsky agrees that it is the parabasal, but denies that it is the

SPONGES 59

Golgi apparatus. Quite recently Duboscq and Tuzet (1934)have published a detail'ed study of the Golgi apparatus and para-basal in several calcareous forms. It would seem to add con-vincing support to the theory which supposes the two structuresto be homologous or even identical. These authors claim to havefollowed the history of the numerous dictyosomes seen in theovum and early blastomeres, which become differentiated in theamphiblastula into the typical Golgi apparatus of the granularcells and the typical parabasal of the flagellated columnar cells.Furthermore, during the development of a nagellum by the cellsof the placental membrane, its Golgi apparatus became graduallytransformed into a structure closely resembling the parabasalof the choanocytes. Cell organs of a nature intermediate betweenparabasal and Golgi apparatus were also seen in scleroblasts andstellate cells.

It is worth noticing that de Saedeleer (1930) records a para-basal in Codosiga and other Craspedomonadina (Choano-flagellates) precisely similar to that observed in the choanocytes

Volkonsky (19306) describes an interesting phenomenon withreference to the parabasal of the choanocytes of C l a t h r i n a .It appears to undergo a regular cycle in which it leaves theeentrosome and travels up to the proximal end of the cell nearthe nucleus, where it swells up and disappears. A new parabasalis regenerated from the eentrosome: this also occurs when, assometimes happens, the parabasal fails to divide during celldivision. A very similar cycle has been observed by Lwoff andLwoff (1930) in the blepharoplast of L e p t o m o n a s c t e n o -c e p h a l i . Duboscq and Tuzet (1934), however, working onC l a t h r i n a and other Calcarea, find no evidence of the exis-tence of this periodical change.

The granules that are found in the cells of C l a t h r i n ac o r i a c e a, particularly in the porocytes and to a lesser extentin the amoebocytes, are described by Teissier and Volkonsky(1930). They regard them as cell inclusions sui g e n e r i s , be-ing neither vacuome nor ehondriome. These granules may beeither white or yellow, and the sponge as a whole is colouredaccordingly. They are soluble in alcohol except after fixation,

60 D. A. WEBB

which renders them insoluble and turns them a yellowish-brown colour. These authors consider them to be composed ofan oxylipoid material with a small amount of protein. Severalother authors have mentioned pigment granules in differentspecies, but in insufficient detail to be able to compare themwith those of O l a t h r i n a described by Teissier and Volkonsky.

GAMBTOGENESIS.

All forms in which gametogenesis has been thoroughly in-vestigated, viz. E e n i e r a , Sycon , G r a n t i a , and Spon-gi11 a, appear to be hermaphrodite. In all cases, however, stagesin spermatogenesis are much rarer than stages in oogenesis: intwenty-five individuals of G r a n t i a sectioned by Gatenby(1920«) only one showed spermatogenesis. And it appears thatit is only in littoral species that female elements are at all com-mon. According to Burton (1928) deep-sea forms reproducealmost entirely asexually. Among Hexactinellids there are onlytwo cases of ova recorded, four of embryos, and none of sperma-tozoa : reproduction seems to be effected mainly by buds. Amongdeep-water Tetraxonida ova, spermatozoa, and early embryosare quite unknown. Apparent highly developed 'embryos' arecommon, but there is strong reason to suppose that they areproduced asexually from aggregations of amoebocytes.

The origin of the germ-cells has given rise to considerablecontroversy. Haeckel held that they arose from choanocytes,but Schulze, Polejaeff, and Maas put forward evidence that theywere derived from amoebocytes, and under the influence ofWeismannian doctrines this view triumphed to such an extentthat Minchin, writing in 1900, does not even mention the othertheory. Dendy (1914), however, disinterred Haeckel's 'heresy'and stated his belief that in G r a n t i a the germ-cells arose fromchoanocytes. Gatenby (1920a) supported this view and broughtforward further convincing evidence. Tuzet (1930a and 19306)considered that in Cliona and R e n i e r a the germ mother-cell was an amoebocyte, but in a later paper on E e n i e r a (1932)she has changed her mind and holds that Haeckel's theory istrue for siliceous as well as calcareous sponges. There seems to belittle doubt that the germ-cells are derived from choanocytes

SPONGES 61

which sink down into the mesenchyme, though they passthrough a stage in which they bear a close enough resemblanceto amoebocytes to have deceived the earlier workers.

As already stated, stages in spermatogenesis are rare, and theprocess still remains rather a mystery. Quite recently Gatenby(1927) was able to write that 'except for Gorich's paper which iscytologically imperfect, the questions surrounding the appear-ance of sponge spermatozoa are largely unanswered'. The'sperm-morulae' described by Dendy (1914) in G r a n t i a bothGatenby (1920a) and Bidder (1920) believe to be a parasitic orendozoic alga. In this paper Gatenby describes nests of sper-matids lying between the flagellated chambers and surroundedby an envelope of pale cubical cells. He also mentions havingseen groups of cells which he thought were probably spermato-cytes. In a supplementary account (1927) he considers that inG r a n t i a spermatogenesis may take place in either of twoways—that previously described in which pockets of spermato-cytes are formed in the mesenchyme, or else by the rapid con-version of all the cells of a flagellated chamber into spermato-cytes whereby the collar and flagellum are lost, the nucleusbecomes reticulate, and the mitochondria fragment into smallergranules.

Tuzet (1930a) describes spermatogenesis in E e n i e r a . Theprimordial germ-cell undergoes amitotic division whereby oneof the daughter nuclei receives the nucleolus, the other most ofthe chromatin network. Of the two cells thus formed, thatpossessing the nucleolus undergoes no further developmentexcept that its cytoplasm stretches so as to embrace the othercell: it is alluded to as the cover cell. The second cell dividesrepeatedly, forming sixteen spermatogonia which undergo anormal maturation including a reduction division (from sixteento eight chromosomes). In the spermatid the mitochondria firstaggregate into four spherules: these subsequently adhere to-gether to form the middle piece of the spermatozoon. Elsewhere(1931 and 1932) she follows the behaviour of the dictyosome(parabasal) in the spermatogenesis of R e n i e r a and H y m e n i -a c i d o n. It divides with each cell division so that there is one ineach spermatid: precocious division in the primary spermatocy te?,

62 D. A. WEBB

however, usually means that at this stage there are two dic-tyosomes in each cell.

Del Bio Hortega and Ferrer (1917) published a very curiousaccount of the spermatogenesis of E e n i e r a p e r m o l l i s andR e n i e r a r o s e a . Weill (1926) has shown that it was basedon a misconception, and that the so-called spermatozoa werereally the nematocysts of a parasitic Scyphozoan, N a u s i t h o e( = S t e p h a n o s c y p h u s m i r a b i l i s Allman and S p o n g i -cola f i s tu l a r i s Schulze), that has been recorded from severalspecies of sponges. Half-digested nematocysts were describedas stages in spermatogenesis, while the ' immature spermatozoa'seem to have been genuine spermatozoa of this or some othersponge.

All authors are now agreed that sponge spermatozoa are ofan ordinary filiform type, like those of most other animals.

The accounts of oogenesis—mainly by the same authors—are more complete. Dendy's (1914) description of the processin G r a n t i a has been confirmed and amplified by Gatenby(1920a) and Duboscq and Tuzet (19336) except for the inter-pretation of certain granules in the cytoplasm that have givenrise to some discussion and will be dealt with later. The followingaccount is derived from these three papers. A choanoeyte losesits collar and sinks down into the mesenehyme, where it proceedsto grow into an oogonium, the flagellum being retained for awhile and then lost. There are at least two generations of oogo-nia, the first large (12-15/i in diameter), the second small (under8/x). Jorgensen had described these two generations but hadtransposed their order. When the first generation reaches thespireme stage prior to division it migrates back towards theflagellated chamber and undergoes division among the choano-cytes. The oogonia of the second generation then migrate backinto the mesenehyme to become oocytes. There appears to be anordinary reduction division, but there is some disagreement as tothe chromosome number in G r a n t i a . Duboscq and Tuzetregard twenty-six for the diploid number as most likely. In theoocyte, which grows to a considerable size, Golgi bodies, mito-chondria, and very delicate yolk-granules can be recognized.It is clearly differentiated into a hyaline non-vacuolated ecto-

SPONGES 63

plasm and a frothy and granular endoplasm: this can be observedeven in quite young oocytes. It possesses no apparent polaritynor anything that can be interpreted as organ-forming sub-stances. During its growth not only is yolk pushed into it by thesurrounding cells, but it appears to ingest whole amoebocytescontaining yolk, a process that is apparently unique in theanimal kingdom. Most authors imply, and Dendy (1914) andDuboscq and Tuzet (1932) explicitly state, that all oogonia oroocytes in one individual are always at practically the samestage in development.

In R e n i e r a , according to Tuzet (1932), the process issimpler. The choanocyte, having been transformed into a germmother cell, grows directly into an oocyte without any inter-vening oogonial generations.

Several authors have described chromophil and osmiophilbodies in the cytoplasm of the oocyte, but they disagree widelyas to their nature and origin. Both Dendy and Jorgensen haddescribed the extrusion of chromidia into the cytoplasm duringoogenesis. Gatenby, in his study of G r a n t i a (1920a and1920b), accepted this explanation first with hesitation, but laterwhole-heartedly. In these papers he expressed the view that theextruded granules were probably mitochondria, although theirfixing reactions were not quite the same as those of normalmetazoan mitochondria. In his later paper (1927) he felt moreconfident as to their mitochondrial nature, since he had beenable to follow their history and detect them in the flagellatedcells of the amphiblastula. But the situation was complicatedby the fact that he found very similar granules in the oocytesof Sycon without any signs of nucleolar extrusion. Thesebodies can be traced back to a single juxtanuclear structure inthe young oocyte which undergoes repeated division. Now threelater descriptions have been published of a body which is singlein the young oocyte and multiplies during its growth—inC l a t h r i n a by Volkonsky (1930b), who states that it is theparabasal, and in E e n i e r a and H y m e n i a c i d o n by Tuzet(1931 and 1932), who calls it the dictyosome which, it will beremembered, she (but not Volkonsky) regards as being identicalwith the parabasal. Finally, Duboscq and Tuzet (19336) describe

64 D. A. WEBB

nucleolar emission in S y c o n but declare that the granules thusformed are not mitochondrial.

It seems impossible to form an impartial judgment as to therelative merits of these views, especially as the later authorswhen describing phenomena that are inconsistent with earlierresults do not offer any explanations of the inconsistency. It isalso very uncertain to what extent all these bodies belong to thesame type. One can only hope that future work on these andother genera may elucidate the problem.

FERTILIZATION AND EMBRYOLOGY.

The first adequate description of fertilization in a sponge wasgiven for G r a n t i a by Gatenby (1920a, 1920b, 1927). As hepoints out, however, previous workers had seen stages in ferti-lization without realizing their significance, Jorgensen havingmistaken it for the expulsion of nuclear material and Dendy forphagocytosis. Gatenby's account of the process is as follows.

The spermatozoon is carried into a flagellated chamberbeneath whose lining lies an oocyte. It does not find its waydirectly to the oocyte, however, but only by means of an inter-mediary carrier cell. Accordingly it enters one of the choano-cytes lining the flagellated chamber: as a rule the choanocyteselected is one that lies directly over the oocyte that is to befertilized. In fact, the spermatozoon usually gets so close to itsultimate objective that it is held that this must be a case ofchemotaxis. Occasionally it makes a 'bad shot' and enters achoanocyte some distance from the oocyte; this seems to happenmore often in Sycon than in G r a n t i a . After the entryof the sperm the choanocyte undergoes considerable changes,whereby it is converted into a carrier cell; the flagellum andcollar are lost, the nucleus sometimes takes on a reticulatestructure so as to resemble that of an amoebocyte, the Golgiapparatus and mitochondria are retained but become much moredifficult to stain, and finally the cell rounds itself off and sinksdown into the mesenchyme from its place in the general epithe-lium. In most cases this suffices to bring it into contact with theoocyte, otherwise it travels through the mesenchyme till itreaches it. A few cases were observed in which fertilization took

SPONGES 65

place on that side of the oocyte remote from the gastral epithe-lium: presumably the carrier cell had wandered right round.Meanwhile changes have taken place in the spermatozoon. Itfirst loses its tail, and then the nucleus and middle piece swellconsiderably till it becomes converted into what is called byFrench authors the 'spermiokyste'. This is a ' cottage-loaf'structure consisting of the nucleus and middle piece, both moreor less globular, and tipped by the crescentic acrosome whichhas, however, become much more chromophobe. In this stateit lies in a vacuole of the carrier cell. When the latter comes upagainst the oocyte protoplasmic continuity is established at thepoint of contact and the sperm flows passively into the oocyte, itspath of entry being visible for some time as a chromophobe,streak-like vacuole. Its entry provides the stimulus for matura-tion of the ovum and two polar bodies are given off. Meanwhilethe middle piece of the sperm breaks up, the male pronucleusapproaches that of the ovum and both swell to the same largesize prior to fusion. As regards the fate of the carrier cellGatenby at first thought that it wandered off through the mesen-chyme and eventually degenerated, but later (1927) he affirmedthat it returned to its original situation and took on once morethe form of a choanocyte.

Duboscq and Tuzet's (1932 and 1933b) account of fertilizationin G r a n t i a and S y c o n is mainly confirmation of the aboveresults. They did not, however, see in S y c o n the spermatozoawith the long middle piece described by Gatenby, and suggestthat they must be degenerate or belong to some other organism.They describe the spermatozoa as having a conical head and adisc-shaped middle piece. In both genera the spermatozoaappeared to enter the choanocyte within the base of the collar,whereas food particles are ingested outside its base: this impliesan actively swimming sperm. They disagree with Gatenby'sfinding that the carrier cell returns to the flagellated chamberto become a choanocyte, and hold instead that it hypertrophiesand then degenerates in the mesenchyme. During the earlystages of cleavage of the ovum these hypertrophied cells arefrequently to be seen in its vicinity.

Tuzet (1930& and 1932) has investigated the process in theNO. 309 F

66 D. A. WEBB

siliceous sponges Cliona and E e n i e r a . She finds it verysimilar to that described in the calcareous forms with one im-portant difference—the carrier cell is one of the amoebocytesin the neighbourhood of the ovum and not a choanocyte. Thisdistinction between the two classes is correlated with structureand is indirectly confirmed by some results obtained by Pour-baix (1934) who investigated the feeding methods of variousspecies. She confirmed and elaborated the distinction first sug-gested by Metschnikoff, that in Calcarea ingestion of foodparticles is accomplished by the choanocytes, in Silicea, parti-cularly those with a highly evolved type of canal system, by theamoebocytes. Since spermatozoa are up to a point comparableto food particles it is very probable that the difference betweenTuzet's and Gatenby's results is due to the anatomical differ-ences between the two classes of sponge.

The essential features of sponge embryology, particularlyamong the Calcarea, have been known for some time, so thatrecent work has been only concerned with points of detail.Gatenby (1920a) describes the cytology of the amphiblastulaof G r a n t i a . There is no segregation of mitochondria or yolkinto certain areas during cleavage, the difference between thetwo main types of cell in the amphiblastula being due to a dif-ference in the nature of the ground-cytoplasm itself. As hasalready been mentioned, there is no apparent polarity in theovum. In most cases the flagellated hemisphere of the larvafaces the nearest flagellated chamber, and this being the aspecton which fertilization usually takes place it is possible that thelatter process may be the sole determinant of the position of theembryonic axis, but there are just enough exceptions to renderthis causal connexion somewhat doubtful. The cells of theamphiblastula are described as follows: •

(1) Flagellated columnar cells which possess a dense mass ofyolk at their inner end and mitochondria scattered throughoutthe cytoplasm. The nucleus is small and deeply staining andincludes a karyosome.'

(2) Granular cells, with rather less yolk and about the sameamount of mitochondria as the flagellated cells. Their nucleusis larger and paler, but also contains a karyosome.

SPONGES 67

(3) Mesenchyme cells, of which there are only two or three.They contain very little yolk but are crammed with mitochon-dria. They are derived from flagellated cells that sink in to thecentre of the larva.

(4) Duboscq and Tuzet (1933a) describe in the larva ofS y c o n four peculiar cells situated in the third row of flagellatedcells counting from the equator. They are symmetricallyarranged so as to form a Greek cross when the larva is viewedin transverse section. They are considerably larger than theflagellated cells, the nucleus is central with a chromatic ringimmediately distal to it, and there are large granules at the base.It is conjectured that they may be light-perceptive cells. Theirultimate fate is unknown.

Gatenby and King (1929) state that the embryo of G r a n t i ais surrounded by a definite placental membrane composed offlattened cells containing osmiophil granules. They are appar-ently derived from amoebocytes. Those cells adjacent to thegranular hemisphere of the larva are more granular than thosenext the flagellated hemisphere. Gatenby (1920a) had pre-viously noticed that the surrounding maternal cells often pene-trate in among the granular cells of the larva so that the lineof demarcation between the two tissues may be ill defined.Duboscq and Tuzet (1933a) interpret this as a means of feedingthe larva: whole cells filled with yolk penetrate into it, much asin the growth of the oocyte. According to these authors thedischarge of the larva is heralded by the acquisition of flagellaby the cells of the placental membrane. The cavity containingthe larva then becomes confluent with the nearest flagellatedchamber, and the larva passes out. Usually a great number oflarvae are released simultaneously, and according to Orton(1929) this is followed by the break-up of the sponge, so thatG r a n t i a normally dies from over-reproduction. 0. Jorgensen(1918) has followed the history of the larvae. They swim freelyfor twenty-four hours, then settle to the bottom and overgrowthof the granular cells begins. This lasts about two days, by whichtime fixation is well in progress. This author states that alllarvae are ripe at the same time—early September—but Dendy(1914) quotes Orton as saying that there are two breeding

00 D. A. WEBB

seasons, June and October, while according to his own observa-tions embryos are released throughout the summer. It seemsrather doubtful that G r a n t i a has a fixed breeding season.

Only two recent papers have been published dealing with theembryology of non-calcareous sponges. Vaney and Allemand-Martin (1918) describe the larva of H i p p o s p o n g i a which isreleased at Tunis in the early summer. It is ovoid, the broader(posterior) end being marked by a dark ring. The surface isciliated all over, but the cilia in the area circumscribed by thisring are extremely long. The cells of the ring are loaded withpigment granules that are periodically discharged. Under theepithelium lies a syncytial layer and beneath this a solid core oflarge fusiform cells. Wilson (1932) has investigated the 'inver-sion of the germ-layers' in the Monaxonid Mycale and findsthat, although there is an outward migration of the amoebocytesso that the larval epithelium (now internal) develops into thechoanocytes, there is no evidence for the process described byDelage whereby, in order to effect this inversion, the epithelialcells were supposed to be engulfed and subsequently liberatedby the amoebocytes.

REFERENCES

Bidder, G. P. (1920).—"Syncrypta spongiarum, nova", ' Journ. Linn. Soc.Zool.', 34, p. 305.

Burton, M. (1926).—"Observations on some British species of Spongesbelonging to the genus Eeniera", 'Ann. and Mag. of Nat. Hist.', 17, p. 415.

(1928).—"A comparative study of the characteristics of shallow-waterand deep-sea sponges, with notes on their external form and reproduction'','Journ. Quekett Micro. Club', 16, p. 49.

Dendy, A. (1914).—"Observations on the gametogenesis of Grantia com-pressa", 'Quart. Journ. Micr. So.', 60, p. 313.

Duboscq, 0., and Tuzet, 0. (1932). "Sur la fecondation de Sycon ciliatumLieberkiihn", 'Compt. Rend. Soc. Biol. Paris', 109, p. 829.

(1933a).—"Quelques structures des amphiblastules d'eponges cal-caires", 'Compt. Rend. Acad. Sci. Paris', 197, p. 561.

(19336).—"Sur l'ovogenese et la fecondation des eponges calcaires:Grantia compressa pennigera Haeckel et Sycon ciliatum Lieberkiihn",'Arch, de Zool. Exp. et Gen.', 73, p. 45.

(1934).—"Sur le parabasal ou corps de Golgi des eponges calcaires",'Arch, de Zool. Exp. et Gen.', 76, p. 78.

Galstoff, P. S. (1925).—"Regeneration after Dissociation (an experimental

SPONGES 69

study on sponges). I. Behaviour of dissociated cells of Microciona pro-lifera under normal and altered conditions", 'Journ. Exp. Zool.', 42,p. 183.

Gatenby, J. B. (1920a).—"The germ-cells, fertilization and early develop-ment of Grantia (Sycon) compressa", 'Journ. Linn. Soc. Zool.', 34,p. 261.

(19206).—"Further notes on the oogenesis and fertilization of Grantiacompressa", Journ. Boy. Micr. Soc.', 40, p. 277.

(1927).—"Further notes on the gametogenesis and fertilization ofsponges", 'Quart. Journ. Micr. Sci.', 71, p. 173.

Gatenby, J. B., and King, S. D. (1929).—"Note on the nutrient membraneof Grantia amphiblastula", 'Journ. Boy. Micr. Soc.', 49, p. 319.

Hirschler, J. (1914).—"tlberPlasmastrukturenindenTunicaten-, Spongien-und Protozoenzellen",' Anat. Anz.', 47, p. 289.

Jorgensen, O. (1918).—"Note on the larvae of Grantia compressa", 'Rep.Dove. Mar. Lab. Cullercoats', 7 (N.S.), p. 60.

Lameere, A. (1926).—"Sur la morphologie des spongiaires", 'Ann. Soc.R. Zool. Belg.', 56, p. 103.

Laubenfels, M. W. de (1932).—"Physiology and morphology of Poriferaexemplified by Iotrochota birotulata Higgin", 'Carnegie Inst. Publica-tions' (Washington), 435, p. 37.

Lwoff, M., and Lwofl, A. (1930).—"Les constituants de l'appareil parabasalchez Leptomonas ctenocephali Fanth. Le cycle du corps parabasal",'Compt. Rend. Soc. Biol. Paris', 103, p. 16.

Orton, J. H. (1929).—"Reproduction and death in Invertebrates andfishes", 'Nature', 123, p. 14.

Penney, J. T. (1931).—"Notes on fresh-water sponges and their epithelioidmembranes", 'Journ. Elisha Mitchell Sci. Soc.', 46, p. 240.

Pourbaix, N. (1933).—"Note sur la nutrition bacterienne des eponges",'Ann. Soc. R. Zool. Belg.', 63, p. 11.

(1934).—"Recherches sur la nutrition des spongiaires", 'Nat. y Res.Inst. Espan. Oceanogr.' (Madrid), Ser. II, No. 69.

Prenant (1925).—"Observations sur les porocytes de Clathrina coriaceaMont.", "Trav. Stat. Zool. Wimereux', 9, p. 198.

Rio Hortega, P. del., and Ferrer, F. (1917). 'Bol. Soc. Esp. Hist. Nat.', 17.Saedeleer, H. de (1930).—"L'appareil parabasal des craspedomonadines

et des choanocytes des eponges", 'Compt. Rend. Soc. Biol. Paris', 103,p. 160.

Teissier, G., and Volkonsky, M. (1930).—"Les granulations pigmentairesde Sertularella (Hydraire) et de Clathrina (6ponge), etude comparee",'Bull. Soc. Zool. Frang.', 54, p. 380.

Tuzet, O. (1930a).—" Sur la spermatogenese de Feponge Reniera simulans",'Compt. Rend. Soc. Biol. Paris', 103, p. 970.

(19306).—"Sur la fecondation de Feponge siliceuse Cliona viridisSchmidt", 'Compt. Rend. Acad. Sci. Paris', 191, p. 1095.

70 D. A. WEBB

Tuzet, 0. (1931).—"L'appareil parabasal et les dictyosomes chez Renierasimulans Johnston et Hymeniacidon sanguinea Grant", 'Compt. Rend.Acad. Soi. Paris', 192, p. 698.

(1932).—"Recherches sur l'histologie des eponges Reniera elegans etR. simulans", 'Arch. Zool. Exp. et Gen.', 74, p. 169.

Vaney, C, and Allemand-Martin, A. (1918).—"Contribution a 1'etude dela larve de l'Hippospongia equina des cotes de Tunisie", 'Compt. Rend.Acad. Sci. Paris', 166, p. 82.

Volkonsky, M. (1929a).—"Les choanocytes des eponges calcaires. Le corpsapical, organite cellulaire nouveau " , ' Compt. Rend. Soc. Biol. Paris', 102,p. 664.

(19296).—"Les choanocytes des eponges calcaires. Resultats de quel-ques techniques cytologiques et particulierement des impregnationsme'talliques", 'Compt. Rend. Soc. Biol. Paris', 102, p. 759.

(1930a).—"Les choanocytes des 6ponges calcaires. Les ph&iomenescytologiques au cours de la digestion intracellulaire", 'Compt. Rend.Soc. Biol. Paris', 103, p. 668.

(19306).—"Eponges calcaires: la cinetide des cellules de Clathrinacoriacea Mont.", 'Bull. Soc. Zool. Franc.', 55, p. 183.

Weill, R. (1926).—"Les cleptocnides des Sponges a propos des spermato-zoides de Reniera decrits par P. Del Rio Hortega et F. Ferrer ", ' Bull. Soc.Zool. Frang.', 51, p. 61.

Wilson, H. V. (1932).—"The larval metamorphosis in monaxonid sponges",' Journ. Elisha Mitchell Sci. Soc.', 47, p. 27.

Wilson, H. V., and Penney, J. T. (1930).—"The regeneration of sponges(Microciona) from dissociated cells", 'Journ. Exp. Zool.', 56, p. 73.