The Nature and Formation of the Spermatoplioreand Sperm Plug in Carolnus Maenas.

By

J. F. Spalding, M.Sc. '

With Plate 18 and 8 Test-figures.

CONTENTS.

1. INTRODUCTION 399

2. T H E M A L E REPRODUCTIVE SYSTEM 400

3. FORMATION AND N A T U R E O F T H E SPEBMATOFHORE . . . 405

4. T H E EEMALE REPRODUCTIVE SYSTEM 410

5. T H E SPERM P L U G 413

6. DISCUSSION . . . . . . . . . . 416

7. SUMMABY - . 4 2 0

8. R E F E R E N C E S 421

1. INTRODUCTION.

A NUMBEB of workers have described the reproductive systemof the Decapod Crustacea, and the details of its anatomy andhistology are fairly well known. Brocchi (1875) described thestructures in the male Carc inus , whilst Cano (1889) madea general survey of the female system. Broekhuysen (1936) hassummarized this work and gives an account of the life-historyof Ca rc inus , and of the connexion between moulting andthe sexual cycle. The object of the present work was to in-vestigate the nature and formation of the spermatophore andsperm plug, both of which have been described by a number ofauthors, but never in great detail. The most exhaustive workon the spermatophore is that of Mouchet (1931) who showedthat the spermatophore of the Anomura was not composed ofchitin, as had been suggested by Jackson (1913) in the case ofE u p a g u r u s . The sperm plug has not received much atten-tion. Williamson (1894) described it in Cancer p a g u r u sbut did not investigate the histology of the spermatheea, so

400 J. F. SPALDING

that his account of glands surrounding it, which he supposedformed the substance of the plug, is unsatisfactory.

Carc inus maenas was selected as being easy to keep inthe laboratory. The animals were kept in porcekm sinks andfed regularly on mussels. As a number of them moulted it waspossible to study all stages from the freshly moulted crab upto those with fully hardened shells. The reproductive systemwas studied both by dissection and in serial sections of fixedmaterial. All the tissues were taken from living animals andfixed immediately. Susa's fixative gave the most satisfactoryresults. The most useful stain for general histological structurewas Mallory's triple connective-tissue stain, though for quickexaminations Mann's methyl-blue-eosin was very useful.Cytological detail was best seen with Heidenhain's haematoxylinfollowed with Biebrich scarlet; carbol-thionin, without acounterstain, was very good for testis tissue.

In addition simple chemical tests were made on both thespermatophores and the sperm plug to confirm deductions madefrom the specific staining reactions of the substances.

This work was carried out in the Department of Zoology,University of Bristol. The author wishes to acknowledge bisthanks to Professor C. M. Yonge for suggesting the line ofresearch and for his continued help and advice.

2. THE MALE EEPEODUCTIVE SYSTEM.

The testes are small paired tubular organs lying laterally inthe anterior part of the body cavity and closely applied to thehepatopancreas. Structurally they are almost identical withthose of Cancer as described by Pearson (1908). Each iscomposed of closely packed follicles in which the germ cellsmay be seen in different stages of development. Each follicleis surrounded by a thin epithelial wall. An unripe follicle showsa densely packed mass of developing cells with no centrallumen, whilst a ripe follicle shows a central cavity in which theripe spermatozoa lie free. The sperms closely resemble those ofthe crab, Cancer m a g i s t e r , as described by Fasten (1918).The development of what he terms the ' nuclear-mitoehondrialcup' makes the sperms very much larger than- the spermatids

SPERMATOPHOBB OP CABCINUS 401

from which they have developed. The unripe sex cells, sperma-togonia and spermatids, stain red with Mallory's stain, but thesperms stain a bright blue and have a translucent appearance.The testes are continuous with the vasa deferentia which beginas narrow tubes running in from the testes to form two coiledmasses, one on either side of the stomach. In this region theyare white, opaque, and greatly swollen. They are continued on.each side of the gut as straight tubes which open to the exteriorat the tips of two small papillae on the coxae of the last pairof walking legs. The swollen portion of the tubes has anextremely thin wall, easily broken in dissection. Euptureimmediately releases the milky white seminal fluid which is fullof spermatophores.

The genital papillae are no t intromittent organs servingto introduce the seminal fluid into the spermatheeae of thefemale. They are short, soft structures, which, even in a largecrab, will hardly be more than 5 mm. long. They are insertedinto a groove in the base of the true intromittent organ whichis a complex structure consisting of the highly modified firstand second pleopods. The first pleopod (Text-fig. 1, Px) isa hollow sickle-shaped appendage, grooved at its base, buttubular towards the tip which is outwardly directed and can beinserted into the vagina of the female crab. Its cavity is en-larged at the base to receive the rod-like second pleopod (P2)which moves as a piston inside the first. The expanded baseof the pleopod also contains large numbers of rosette glands(Text-figs. 2 and 3, G), exactly comparable to those describedfor H o m a r u s by Yonge (1932), who showed that they areresponsible for secreting the general body cuticle, while others,structurally identical, secrete the cement which binds the eggsto the pleopods of the female (Yonge, 1938). The ducts of theglands (D) all open into the central cavity of the pleopod.

The second pleopods have never been adequately described.Williamson (1904) mentions the fact that those of Care inusdiffer considerably from those of Cancer* Pearson (1908)describes the latter as consisting of two parts, a horizontal rod,probably representing the protopodite, and a vertical roddivided into two parts by a transverse groove, which is the

402 J . F . SPALDING

endopodite. This description of the general relationships of thestructure is not very different from that given by Shen (1935)for' Ca rc inus . He describes the development up to the sixthyoung crab stage. In the mature male crab, howev ?r, there is

TEXT-FIG. 1.

The intromittent organ of the left side, lateral view. x6. A,abdominal segment; B, basal joint of fifth walking leg; O, genitalpapilla; Pv first pleopod; P2, second pleopod, inserted in cavityof first.

a small blade-like process arising from the tip of the endopodite(Text-fig. 4, E). Dr. I. Gordon (personal communication)considers that this blade may be the much reduced distalportion of the pleopod, thus making the condition entirelycomparable with that in Cancer , where the basal and distalparts are approximately equal in length. She adds that shewould hesitate to homologize such a structure as this bladewith either the a p p e n d i x i n t e r n a or the a p p e n d i x

SPEKMATOPHORE OP CABCINUS 403

m a s c u l i n a found in Macrura. It is not known at what stagein development this transverse division appears. It is difficultto assign any definite function to the process which may varyin shape, being long and whip-like in Da i r a , blunt and

TEXT-ITS. 2.

The intromittent organ of the left side, ventral view. x 7 | . C,cavity of first pleopod; D, duct through which the sperms areconveyed to the female; E, distal portion of second pleopod;O, mass of rosette glands. Other letters as before.

grooved in P i l u m n u s , or blade-like in Careinus . Probablythe 'intromittent organ complex' of the modified first andsecond pleopods functions in the same way in all the Brachyura,but modifications can occur in the shape of the second pleopodwithout impairing its function.

KOS. 331, 332 E e

404 J. F. SPALDING

The genital papilla is covered with chitin continuous withthat of the body surface and bears small backwardly pointingspines at its tip which hold it in place when it is inserted intlm pleopod. Pearson suggests that in Cancer the papilla is

TEXT-FIG. 3.

Transverse section through basal region of first pleopod. x30.C, cavity of pleopod; D, ducts of glands; G, mass of rosetteglands; M, muscle blocks.

inserted into the pleopod during copulation; but in all theindividuals of Care inus examined the papilla was already inplace, and, in fact, no amount of bending of the abdomen wouldcause its removal. Undoubtedly the spines are important forits retention. The seminal fluid is discharged into a large cavityat the base of the pleopod, and into the same cavity open theducts of the rosette glands. Any movement of the abdomen

SPBBMATOPHOBE OP CARCINUS 405

causes the second pleopod to move up and down inside the firstand this movement forces the sperms, together with the secre-tion of the rosette glands, up the narrow tube of the pleopodinto the vagina of the female.

, EN

TEXT-FIG. 4.A, ventral view of second pleopod of the left side, x 18. B, enlarged

view of tip of second pleopod. x 70. B, basal joint of pleopod;E, distal portion of endopodite; EN, endopodite; S, spines.

3. FORMATION AND NATURE OF THE SPERMATOPHORE.

Unlike the spermatophores of the Anomura, which arecomplex, pedunculated structures, those of Carc inus aresimple ellipsoidal capsules of uniform thickness, with no externalirregularities. They vary in size, but most are about 45/* long

406 J. F. SPALDING

and 35JU, broad. The capsule is fairly easily broken, but thefracture is quite irregular and there is no evidence of a definiteline of dehiscence as described by Mouchet (1931) for those ofthe Anomura.

Once the male crab has attained sexual maturity the gonoductalways seems to be fully charged with a milky seminal fluidwhich contains large numbers of spermatophores and alsodroplets of a clear fluid. This observation agrees with thestatement of Broekhuysen (1936) who remarks: 'the state ofswollenness and the whitish colour of the reproductive organsvary, it is true, a little, but there is strong evidence thatpractically all animals, as regards the development of theirsexual organs, could copulate all the year round.' No relation-ship could be found between the moulting cycle and the stateof the gonads in male crabs. The testes and vasa deferentiamight be equally mature, both in macroscopic and microscopicappearance, in freshly moulted crabs with completely softshells, as in those with a thick, red, barnacle-encrusted shellwhich had obviously not moulted for a long time.

It is not easy in dissection to say where the testis ends andthe vas deferens begins, but microscopical examination revealsa point where the cells round the central lumen begin to forma simple cubical epithelium. Taking this region as the beginningof the vas deferens it is then possible to divide the remainderof the tube into four distinct functional regions which, however,tend to merge into one another in general histological detail.These may be summarized as:

(1) Nearest to the testis, a region where sperms occur freein the lumen of the duct unaccompanied by any fluid havinga characteristic staining reaction.

(2) A region in which a fluid staining red or orange withMallory's triple stain is mixed with the sperms. This fluid willsubsequently be referred to as 'substance A'.

(3) A region in which a fluid is added staining blue with thesame stain. This will be referred to as 'substance B'.

(4) A region in which large masses of the substance B areto be seen, together with fully formed spermatophores.

The last region forms by far the largest part of the duct and

SPBRMATOPHOEE OF CABCINUS 407

reaches from the coiled mass lying on the digestive gland to theexternal genital opening. Though the tube may vary greatlyin diameter in this region, there is hardly any histologicaldifferentiation in the lining epithelium and it is probablymerely an elongated reservoir for the storage of the fully formedgenital products. It is by far the most conspicuous part of theinternal reproductive system and is immediately seen onopening the body cavity of the animal.

The separate regions and their products may now be describedin greater detail.

Region 1.—The wall of the vas deferens is here formed ofvery low columnar cells, which do not appear to be glandularin function, encircled by a very thin layer of muscle. Function-ally it is continuous with the lower part of the testis tubule,since the sperms lie free in its cavity with no accompanyingfluid.

Reg ion 2.:—Here the cells are rather shorter, forming acubical epithelium. In no preparation could definite signs ofsecretory activity be seen but throughout the region the spermsare mixed with the red-staining substance A. This at firstappears as small masses lying between the sperms, but lowerdown the tube these masses are seen to have coalesced so thatsmall groups of sperms are entirely surrounded by the fluid(Text-fig. 5, fig. 1, PL 18). This is the actual region of spermato-phore formation. Churning movements of the vas deferens bysome means mould the sperms into ellipsoidal masses roundwhich the capsule is then formed out of substance A. It wasimpossible to study this process except by examining the con-tents of different parts of the tube in fixed and sectionedmaterial. Attempts were made to obtain smears from this partof the genital duct, a method which had given good results inthe study of spermatogenesis by Fasten (1918). However, thesmears only showed fully formed spermatophores or completelyfree sperms, so that details of the actual process of capsuleformation are not known.

Reg ion 3.—This is sharply differentiated from the precedingregion by its very distinct histological features. The epitheliumconsists of tall cells of very uneven height with large bulbous

408 J. F. SPALDING

inner ends which project irregularly into the lumen of the tube(fig. 2, PI. 18). The nucleus (AT) is situated near the basementmembrane and there is usually a mass of secretion inside thecell which stains blue with Mallory's stain. In so ne cases thisblue secretion could be seen actually leaving the inner end of

TEXT-FIG. 5.

Diagram showing the contents of the vas deferens, A, in Region 2;B, in Region 3. x 500. A, red-staining substance A; B, separatedroplets of blue-staining substance B; C, spermatophore capsule;S, masses of sperms being formed into spermatophores; SP,spermatophores.

the cell, whilst large droplets of it (B) are always seen in thispart of the duct together with fully formed spermatophores(SP). In addition small globules of substance A, presumablythe surplus not used to form the capsules, could usually be seenlying amongst the droplets of substance B, from which they areclearly distinguishable by their different staining reaction.

Eegion 4.—As stated above, this constitutes by far the

SPEKMATOPHOBE OF CABCINUS 409

largest part of the vas deferens, forming an elongated seminalvesicle for the storage of the ripe genital products. At first itswalls have a cubical epithelium, but lower down, as the diameterof the tube increases, the cells are very flattened.

Jackson (1913) stated that the spermatophores of E u p a -gurus were 'chitinous', although he made no chemical tests,but Mouchet (1931) has shown that chitin is not present in thespermatophores of a large variety of Anomura. But she wasunable to decide of what the capsule was composed, obtainingnegative results with the Diaphanol-Naphthol reaction ofSchulze and Kunike (1923) which gives specific colour reactionsnot only for chitin but also for keratin, cellulose, tuniein,spongiolin, conchiolin, and cornein.

The integument and egg-membranes of the Crustacea consistof underlying chitin and outer cuticle (Yonge, 1932, 1938;Mawson and Yonge, 1938), the latter being formed by tegu-mental or cement glands respectively. When treated withMallory's stain the chitin stains blue and the cuticle red, a factwhich has been confirmed in the present work wherever sectionscontained these substances. This suggested that the red-staining substance A might be similar to the cuticle whichPryor (1940) has shown is probably a complex scleroprotemallied to the epicuticle of the Insecta. Unfortunately Pryor'spaper appeared after the writer left the University of Bristoland there has been no opportunity to employ his techniqueon the spermatophore capsule.

The capsule of the spermatophore was not dissolved in coldconcentrated hydrochloric acid, proving that it is not chitinous,while it rapidly disintegrated in 40 per cent, caustic soda at70° C. therein agreeing with the cuticle. Yonge (1932) showedthat the isoelectric point of the chitin of Ho mar us is atpH 3-5 whereas that of the cuticle is at pH 5-1. Employingsimilar technique a figure of between pH 4-8 and 5-2 wasobtained for the capsule of the spermatophore, indicating afurther resemblance to the cuticle. In addition the stainingreactions with Mallory's triple stain were very consistent. Theorange-red colour of substance A was given only by the bodycuticle and by a part of the sperm plug in the female (see below).

410 J. P. SPALDING

Similarly the translucent blue stain was seen only in the bodychitin, substance B and, as noted below, in another portion ofthe sperm plug.

It has already been shown by Yonge (1938) thaf the oviductin Homarus and other Decapoda secretes a chitinous mem-brane around the eggs as they pass down and it is not unreason-able to assume, especially in view of what will be stated belowabout the sperm plug, that substance B is chitin or its fluidprecursor. Substance A is unlikely to be identical with cuticlebecause the rosette glands which normally form this are absent,but it is probably a protein of somewhat similar properties.

Mouchet produced dehiscence of the stalked spermatophoresof the Anomura by a variety of treatments. Dilute sea-water,solutions of dyes such as methylene blue or Congo red, anddistilled water caused immediate dehiscence of the capsulesof B u p a g u r u s , Por cell ana , and G a l a t h e a . It seemspossible that in each case the rupture was produced by theosmotic uptake of water, though this is not explicitly stated.The capsules of Carc inus , however, did not dehisce underany of these treatments. They were nevertheless very easilybroken by the slightest pressure on the cover-slip, so that themechanism of dehiscence may be mechanical rupture producedby squeezing as the spermatophores are forced through thevery narrow tube of the intromittent organ. This is confirmedby the fact that in a female which had very recently copulatedmost of the sperms were already free and only a few intactspermatophores remained.

4. THE FEMALE REPRODUCTIVE SYSTEM.

The ovaries are paired tubular organs which in the ripefemale are bright orange in colour and occupy more than halfthe general body cavity. Their general structure closelyresembles that of Homarus as described by Herrick (1911).The oviducts are very short and open into an internal sperma-theca which in turn communicates with the exterior by a shortvagina. The oviduct cannot be seen in a general dissection sinceit is surrounded by the ovary right up to its junction with thespermatheca, but longitudinal sections through this region show

SPERMATOPHORE OF CARCINUS 411

that here the ovary has a central lumen in which eggs lie freewithout any surrounding follicular epithelium, so that it maybe regarded as a functional oviduct despite the absence of anydefinite epithelial wall.

The spermatheca varies very much in size with the conditionof the animal. Text-fig. 6 A shows the condition in a hard-

BTEXT-FIG. 6.

The spermatheca in longitudinal section; A, of a hard-shelled crahwith no sperm plug; B, of a soft-shelled crah with sperm plugpresent. x2. A, upper part of sperm plug staining red; B,lower part staining blue; EP, stratified epithelium lining upperpart of spermatheca; F, external female opening; L, limit ofchitin lining spermatheca and vagina; OV, oviduct; P, spermplug; S, sperms; V, vagina.

shelled female that has not recently copulated. The sperma-theca is merely a small dilation of the oviduct in which canusually be found a few free sperms. In a soft-shelled femalethat has recently copulated the spermatheca is a large almostspherical chamber (Text-fig. 6 B) in which lies the sperm plug(P) and the surrounding spermatozoa (S). Its external appear-ance is shown in Text-fig. 7. The oviduct enters it at its sideand the vagina leads from the base. The dorsal half of thechamber is lined with a very thick stratified epithelium (EP)which may be as many as twenty cells thick. The cells forming

412 . J. F. SPALDING

this epithelium are uniform in size, and the general appearanceis very similar to that of a thick transitional epithelium in thevertebrates. It is obviously highly elastic. The lower half ofthe spermatheca, together with the vagina (V), is lined withchitin (Text-fig. 8, C) which is continuous with that of thegeneral exoskeleton. It is secreted by a very prominent tallcolumnar epithelium (CEP). The cuticle covering the general

TEXT-JIG. 7.

Lateral view of the spermatheca. x 2. / , part of internal skeleton;M, body muscles; O, ovary surrounding the oviduct; SP, sperma-theca. Other lettering as in Text-fig. 6.

body surface does not extend over all this internal chitin,reaching only a little way into the vaginal opening.

No secreting cells could be found in connexion with thespermatheca with the exception of the chitinogenous epitheliumin its lower part. There is very little muscle surrounding thespermatheca," but blocks of circularly and diagonally runningmuscles are found in the vaginal wall (fig. 3, PL 18, M).

• • A large number of females in different stages of the moultingcycle were dissected and, as a result, the description given byBroekhuysen (1936) can be amplified. Copulation always occursimmediately after the female has moulted. In the aquarium

SPBRMATOPHOEE OF CABCIHCS 413

females and males were kept together and any female whichmoulted was immediately fertilized. In one case a female wasfound with fully distended spermathecae, showing that copula-tion had occurred, although the animal had only half freeditself from its old carapace. The soft-shelled female, therefore,always shows very conspicuous spermathecae in which lie thehard gelatinous sperm plugs. Before the shell has fully hardened,i.e. whilst the carapace is still pale green in colour and can beeasily indented by the finger, the sperm plug has noticeablydecreased in size. By the time the shell is first really hard, thespermatheca appears only as a small opaque swelling at thetop of the vagina, the opacity being due to the contained massof sperm. Fortunately one female was fixed in the act of egg-laying ; sections of the base of the oviduct show a tube of almostuniform lumen from the ovary to the exterior, the spermathecabeing indistinguishable (fig. 3, PI. 18).

It is still not definitely known where the actual fertilizationof the egg takes place in the Brachyura. Fig. 3, PL 18, showsthe eggs actually in the oviduct already surrounded by the firstegg membrane, which Yonge (1938) has shown to be chitinousin H o m a r u s , as it is also in Carc inus (personal com-munication). The eggs are in close contact with a mass ofspermatozoa (S) which under the high power were found to beunexploded. Possibly direct contact with the eggs in thisregion causes explosion of some of the sperms and consequentfertilization of the eggs.

After egg-laying the female cannot moult until the eggs arehatched. An ovigerous female usually has a very small ovaryand a soft flabby oviduct. Sperms were often seen in the oviductat this stage, but it was not possible to tell whether they werestill viable. When the eggs are shed, moulting follows, and, ifcopulation occurs, the cycle is repeated.

5. THE SPEEM PLUG.

The sperm plug is a structure which is found in the sperma-thecae of females which have recently copulated. It is formedduring, or very soon after, copulation and does not persist verylong as it is sometimes absent from still soft-shelled females

414 J. F. SPALDING

which must have moulted, and therefore copulated, only a fewdays previously. It is relatively large; in a female of carapacewidth 8 cm. the plug in each spermatheca was about 1 cm. longand 0-7 cm. broad. It fits the spermatheca very closely so thatits shape is roughly spherical, with a deep equatorial groove(Text-fig. 8, G) which corresponds with the ridge at the edge ofthe chitin lining the lower part of the spermatheca. The groove

TEXT-FIG. 8.

Longitudinal section through spermatheca and vagina with spermplug present. x 5 . C, chitin lining vagina and spermatheca;CEP, chitinogenous epithelium; G, groove separating the twoparts of the sperm plug; S', sperms lying on top of the plug;8", sperms held in the folds of chitin in the lower part of thespermatheca. Other letters as in Text-fig. 6.

divides the plug into two parts which show different stainingreactions with Mallory's stain, and which can therefore beassumed to be chemically distinct. The upper portion stainsorange and is probably allied in nature to the cuticle; the lowerstains bright blue and is probably chitin. A few spermatophoreswere usually found embedded in the lower portion of the plug,

SPEBMATOPHOEE OF CAJtCINUS 415

together with a number of free sperms. The upper portionrarely showed more than a few scattered spermatophores.

The following table summarizes the properties of the twosubstances:

Upper Substance.Tough, and homo-

geneous. Hyaline inappearance.

General properties.

Treated with hydro-chloric acid.

No effect in the coldafter two hours. Onheating — disinte-grates leaving aresidue.

Dissolves completely.

Lower Substance.

Rather stringy consis-tency. Not homo-geneous. Easilyteased into separateportions.

No effect in the cold.On heating — dis-solves completely.

Leaves bulky residue.Treated with 40 percent, caustic potash,

10 mins. at 100° C.Staining reaction with Yellow to red. Blue.

Mallory's triple stain.

These tests suggest that the upper part of the plug is composedof a substance similar to the general body cuticle, whilst thelower part is made of chitin.

Williamson (1894) considered that after the moult the glandsof the spermatheca secreted a fluid which hardened on contactwith sea-water to form the plug which, in Cancer , lies partlyin the vagina and partly in the spermatheca. Despite carefulexamination, no secreting cells of any sort could be found inthe neighbourhood of the spermatheca in Carc inus with theexception of those of the chitinogenous epithelium in its lowerregion. In a crab dissected within twelve hours of moultingthe chitinous lining of this region was almost fully formed.It seems unlikely that this glandular epithelium could makefirst a loose mass of chitin to form the lower part of the spermplug, and then a normal layer of chitin over the genera! epithelialsurface.

The possibility, therefore, remains that the plug is made bythe male and not by the female crab. It has already been shownthat the seminal fluid contains numerous droplets of the blue-staining substance B (fig. 2, D, PI. 18) which, it is suggested,

416 J. F. SPALDING

is a fluid precursor of chitin. This must pass into the vaginaof the female in the process of impregnation and would certainlybe present in sufficiently large quantity to form the lower partof the plug. Now although the seminal fluid also contains smalldroplets of the red-staining substance A, which are probablythe unwanted surplus after the formation of the spermatophorecapsules, the total quantity of this substance is very small incomparison with that of substance B. The two halves of thesperm plug are almost exactly equal, so there must be a furthersource of the red-staining substance. This can only be in theglands surrounding the cavity of the first pleopod of the malewhich are structurally identical with the tegumental glandsknown to be responsible for the secretion of the cuticle.

6. DISCUSSION.

The first question to be considered concerns the function ofthe spermatophore. The sperms are surrounded with a sub-stance resembling in many respects that known to be responsiblefor the body cuticle and for attachment of eggs in the DecapodCrustacea. It is interesting to note that Manton (1938) saysthat the external layer of the spermatophore of P e r i p a t o p s i sSedgwicki stains red with Mallory's stain, suggesting thatthe same substance may be used for the same purpose in theOnychophora. It is possible that this substance also resemblesthe cuticle in the possession of low surface tension which isindicated by the ease with which it spreads itself round themasses of sperms in the vas deferens, never forming the compactglobules characteristic of the fluid precursor of chitin, whichmust have a much higher surface tension.

It is possible that in the Brachyura the spermatophores aredegenerate structures which have lost their original functionand now serve merely to keep the sperms together during theirtransmission to the female. In the Anomura the pedunculatespermatophores are attached to the external surface of the bodyof the female and are thus exposed to sea-water from the timeof copulation to that of egg-laying (Jackson, 1913; Mouchet,1931). The same is true of the single spermatophore of Luc i fe r(Brooks, 1882) and a similar condition is found in some of the

SPERMATOPHOEE OF CABCINUS 417

Macrura, e.g. A s t a c u s . It seems possible, therefore, that theBrachyura may be descended from an ancestral stock in whichfertilization was external and in which the sperms needed someprotective capsule to protect them from the action of sea-water.

The phylogeny of the Brachyura is still rather obscure, butBouvier (1896) suggested their origin from the Nephropsidea.Dr. W. T. Caiman (personal communication) says that this viewstill, broadly speaking, holds the field. The Anomura, thoughmore or less intermediate between the Maerura and theBrachyura, are not to be regarded as transitional. Spermtransference in the Macrura has not been very thoroughlyinvestigated, but probably the condition found in Homarusanuer icanus , described by Herrick (1911), is more or lesstrue for the Macrura generally. Here the spermatophores ' havethe appearance of semi-transparent rods of vermicelli about aninch long, and consist only of opaque masses of sperms and thegelatinous medium described'. Normally in the Lobster onlyone spermatophore is formed in each vas deferens, but two maysometimes be present. A grouping of the Decapoda based onthe type of spermatophore, the place of fertilization, and thenature of the spermatheca might be represented thus:

ANOMURA.External fertilization.Pedunculate spermatophores.

/MACRURA.External fertilization.Large, non-pedunculate spermatophores.

-BRACHYURA.Internal fertilization.Non-pedunculate spermatophores.

ANCESTRAL NEPHROPSID STOCK. (Bouvier.)

Hamon (1937) attempted to discover the mechanism ofdehiscence of the spermatophore of E u p a g u r u s . He con-cluded that a number of factors might be effective, but did notdetermine which was actually concerned in nature. The threechief factors are: (i) mechanical pressure from -without, (ii)osmotic pressure from within, (iii) imbibition of water by the

418 J. F. SPALDING

internal substances, producing the same effect as the osmoticuptake of water. It is not possible to state definitely thatthe last two factors are not concerned in the dehiscence ofthe capsule of Carc inus , but the experiments made on theseminal fluid suggest that the osmotic dehiscence is not of greatimportance. Hamon showed also that liver extract would causedehiscence in E u p a g u r u s and suggested that this was dueto the digestion of the substance joining the two valves of. thepedunculate spermatophore together. Liver extract of Car-c inus did not produce dehiscence of the spermatophore aftermore than two hours. Extracts of the tissue of the spermathecawere also without effect. Such a result does not, of course,disprove the presence of an enzyme capable of producingdehiscence, since such an enzyme might only be present fora very short period immediately after copulation. It was notpossible to get enough recently copulated female crabs to cheekthis. The general impression gained from this work is thatmechanical pressure causes the dehiscence of most of thespermatophores as they are forced through the duct of thefirst plgopod, any that survive this process being possiblydissolved by a cuticle-digesting enzyme the main function ofwhich is to dissolve the upper part of the sperm plug.

It is somewhat remarkable that so conspicuous an object asthe sperm plug has received so little attention. Williamson(1894, 1904) has given the only detailed description of thesperm plug in Cancer , though it is briefly referred to byWitten (1903), whilst the similar structure in H o m a r u s isdescribed by Bumpus (1891). Broekhuysen (1936) states thatin the females of Carc inus which have just moulted the'copulatory pouches' are filled with the male sexual products.' The latter form in a relatively short time a cheese-like sub-stance which becomes visible on pressing on the copulatorypouches.' In animals recently moulted, but in which the shellhad already become hard, 'the cheese-like substance had givenway to a more liquid substance'. This is the only suggestionin the literature that the sperm plug originates from the malecrab. Williamson, followed, by Witten, assumed that thespermatheca ""itself produced the necessary fluid which then

SPERMATQPHOEE OF CAKCINTTS 419

hardened on contact with sea-water. As the present work hasshown, there is no evidence for this, glands capable of producingsuch a secretion being nowhere found in the region of thespermatheca.

None of those authors have commented on the dual nature ofthe sperm plug, though Williamson (1894) shows the constrictionof the plug by the inwardly projecting fold of chitin in thespermatheca. In Cancer the plug lies partly in the sperma-theca and partly in the vagina, and would be much moreefficient in preventing the loss of sperms through the vaginathan that of C a r c i n u s . He suggests that when the shell ofthe crab hardens and the plug disappears, the vulva, or terminalportion of the vagina, then closes by means of sphincter musclesand thus retains the sperms in place. No such effect was noticedin Ca rc inus , and indeed it seems rather remarkable thatsperms are not lost even when the plug is in place. As shownin Text-fig. 8, some of the sperms lie on top of the plug and areheld in place by it, but a large number lie below it in folds ofchitin from which there is nothing to prevent their loss to theexterior. The vagina always seemed to remain open. It is noteasy to be certain of this because there is always the possibilityof shrinkage in the fixation of so large a structure as thespermatheca, but in any case, once the plug has gone thereseems little to prevent the loss of sperms.

The plug is possibly destroyed by the action of enzymesproduced by the spermatheca, though no confirmation of thishas been obtained. Williamson speaks of an amber-colouredfluid which surrounds the sperms in a hard-shelled crab, andfrom one of his drawings it looks as if the chitin is first dis-solved leaving the upper part of the plug intact for a certaintime. Broekhuysen also refers to the more fluid nature of thespermathecal contents in the hard-shelled crab.

No light can be thrown at present on the significance of thedual nature of the plug. It is known that the product of thetegumental glands hardens relatively slowly on contact withsea-water, whilst fluid chitin hardens fairly rapidly. Thismeans that the lower part of the plug is probably hard whilstthe upper part is still fluid. This may account for the fact that

NOS. 331, 332 P f

420 ' J. F. SPAL0ING

spermatophores are always found trapped in the chitin, but onlyrarely in the red-staining part of the plug, suggesting that themore fluid nature of this region is of value in allowing thesperms to free themselves from the plug.

Possibly the whole question of sperm transference in theDecapoda is connected with the explosive nature of the sperms.Although the process of explosion has been described by anumber of authors (Koltzoff, 1906; Herrick, 1911; Fasten,1926) yet the question of what stimulus is actually responsiblefor explosion is still unsettled. The present work, besidessuggesting that fertilization is internal in Carc inus , alsopoints to the probability that the actual stimulus of contactwith an egg is necessary to produce explosion, since spermslying free in the oviduct were not exploded. These sperms mustbe in contact with sea-water which cannot therefore, by itself,be responsible for their explosion. If this is so, the mainfunction of the spermatophore is not likely to be to protect thesperms from sea-water, and it seems likely that its mainpurpose is simply to keep the sperms in place until they areneeded for fertilization. This adds weight to the theory thatthe primitive spermatophore is the stalked type which isattached to the external surface of the female, and that in theBrachyura this has degenerated in correlation with the develop-ment of the internal spermatheca.

7. SUMMARY.

1. The structure of the male reproductive system of Car-c inus is described with special reference to the region offormation of the spermatophores and the anatomy of theintromittent organ.

2. The vas deferens produces two fluids, one of these isprobably chitin in a fluid form, whilst the other is similar in somerespects to the substance forming the general body cuticle, i.e.protein. This substance forms the capsule of the spermatophore.

3. The intromittent organ consists of the modified first and•second pleopods and is richly supplied with rosette glandssimilar to those producing the body cuticle. The ducts of theseglands open into the cavity of the first pleopod and their

SPEBMATOPHOEB OP CABCINUS 421

secretion could easily pass with the seminal fluid into thefemale crab.

4. The female reproductive system is described with specialreference to the spermatheca and the cyclical changes occurringin this organ.

5. The sperm plug is composed of two parts distinguishableby their staining reactions. One is similar to the cuticle in itsgeneral properties and is probably the product of the rosetteglands in the male pleopod. The other is probably chitinformed from its fluid precursor in the seminal fluid of the malecrab.

6. It is suggested that the spermatophore of the Brachyurais a degenerate structure having lost its main function, theprotection of the sperms during the interval between copulationand egg-laying, in correlation with the development of theinternal spermatheca and internal fertilization.

8. EEFBBENCES.

Bouvier, E. L., 1896.—"L'origine homarienne des Crabes", 'Bull. Soc.Philomath. Paris', 8.

Brocohi, M., 1875.—"Organes genitaux males des Crustaces decapodes",'Ann. Sci. Nat., Zool.' (6), 2.

Broekhuysen, G. J., 1936.—"Development, Growth and Distribution ofCaroinides maenas", 'Arch, neerl. Zool.', 2.

Brooks, W. K., 1882.—"Lucifer, a study in Morphology", 'Phil. Trans.',B, 173.

Bumpus, H. C , 1891.—"Embryology of the American Lobster", 'Journ.Morph.', 5.

Cano, G., 1891.—"Morfol. dell' appar. sess. femminile . . . nei CrustaceiDecapodi", 'Mitt. Zool. Stat. NeapeP, 9.

Fasten, N., 1918.—"Spermatogenesis of Cancer magister", 'Biol. Bull.', 34.1921.—"Explosion of spermatozoa of Lophopanopeus belius",

ibid., 41.Hamon, M., 1937.—^"Debiseence des spermatophores d'Eupagurus

prideauxi", 'C.R. Acad.^Sci. Paris', 204.Herrick, F. H., 1911.—"Natural History of the American Lobster",

'Bull. U.S. Bur. Fish.', 29.Jackson, H. G., 1913.—'Eupagurus', L.M.B.C. Mem. No. 21.Koltzoff, N. K., 1906.—"Gestalt der Zelle.—I", 'Arch. mikr. Anat.', 67.Manton, S. M., 1938.—"Reproduction in the Onyehophora", 'Phil. Trans.',

B.208.

422 3. F.

Mawson, M. L., and Yonge, C. M., 1938.—"Egg Membranes in Chiro-eephalus diaphanus", 'Quart. Journ. Mior. Soi.', 80.

Mouohet, S., 1931.—"Spermatophores des Crustaces decapodes", 'Ann.Sta. Ocean. Salammbo', No. 6.

Pearson, J., 1908.—'Cancer', L.M.B.C. Mem., No. 16.Pryor, M. G. M., 1940.—"Hardening of Cuticle of Insects", 'Proc. Roy.

Soc. Lond.', B, 128.Sohuke, P., and Kunike, Q., 1923.—"Mikroohemie tierischer Skelett-

sutotanzen", 'Biol. Centrbl.Y48.Shen, 0. J., 1935.—"Post-larval Development of Caroinus maenas",

'Proo. Zool. Boo. Lond.', 1935.Willianwon, H. C, 1894.—"Life-history of Cancer pagurus", 'Kpt. Fish.

Bd. Scot.1,18.—-1804.—"Life-history of Cancer pagurus", ibid., 22.Wifcten, B. P., 1008.—" Changes accompanying Eodysis of Cancer pagurus ",

'Northumberland Sea-Fish. Comm., Bpt. Soi. Invest.', 1903.Yonge, 0. M., 1932.—"Nature and Permeability of Chitin.—I", 'Proe.

Roy. Soo. Lond.', B, 111.—— 1988.—"Membranes surrounding Eggs of Homarus vulgaris and

other Deoapodft", 'Proo. Zool. Soo. Lond.', A, 107.

9. DESCRIPTION OF PLATE 18.LBTTBEING.

A, red-staining substance A; B, blue-staining substance B; C, chitin;CEP, ohitinogenous epithelium; OU, cuticle; E, epithelium of vas deferens;F, external female aperture; M, muscle; N, cell nuclei; O' eggs in oviduct;O", eggs in vagina passing to exterior; OV, oviduct; 8, spermatozoa;8P, spermatophores.

Fig. L—Section through upper part of vas deferens, showing sperma-tozoa collecting in masses which are being surrounded by substance A.X275.

Fig, 2.—Section through vas deferens a little lower down than fig. 1,showing the columnar epithelial cells responsible for producing substance B,droplets of which can be seen together with fully formed spermatophores.xl25.

Fig. 3.—Longitudinal section through the oviduct and vagina of afemale at the moment of egg-laying. Eggs already surrounded by theinner egg membrane seen in the oviduct surrounded by spermatozoa:two others in the vagina passing to the exterior, x 12.

Quart. Journ. Micr. Sci. Vol. 83, PI. 18

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