on the reproductive processes of earthworms: part i. the ... · and these penes, during copulation,...

On the Reproductive Processes of Earthworms:Part I. The Process of Copulation and Exchangeof Sperms in Eutyphoeus waltoni Mich.

By

Kami Narayan Bahl, D.Sc, B.Phil.,Professor of Zoology, University of Luoknow, India.

With Plate 37 and 3 Text-figures.

CONTENTS.

PAGE

1 . I N T R O D U C T I O N . . . . . . . . . 4 7 9

2 . M A T E R I A L A N D M E T H O D S . . . . . . . 4 8 1

3 . T H E G E N E R A T I V E O R G A N S . . . . . . . 4 8 3

4 . D I R E C T O B S E R V A T I O N S O N C O P U L A T I N G P A I R S . . . . 4 8 8

5 . T H E A T T A C H M E N T O F T H E C O P U L A T I N G W O R M S . . . . 4 9 0

6 . T H E P E N I S A N D T H E S P E R M A T H E C A E . . . . . 4 9 6

7 . S U M M A R Y . . . . . . . . . . 4 9 9

8 . R E F E R E N C E S T O L I T E R A T U R E . . . . . . 5 0 0

E X P L A N A T I O N O F P L A T E . . . . . . . 5 0 0

1. INTRODUCTION.

IN a recent paper in this journal, Grove (3) has given a very-careful and detailed account of the reproductive processes ofL u m b r i c u s and has finally disposed of the discrepancies anddifferences in the accounts of these phenomena as given indifferent text-books. He has shown that the process of copula-tion and transference of sperms in this worm is very elaborate.' The seminal fluid issues from the apertures of the vasa-deferentia in segment 15, and is conducted beneath the slime-tube in pit-Kke depressions in the seminal grooves (two on eachworm) to the clitellum, where it accumulates in the space be-tween the lateral surfaces of segments 9 to 11 of one worm andthe clitellum of the other. Eventually it becomes aggregated

480 KARM NARAYAN BAHL

into masses in the groove between segments 9 and 10, and 10and 11, and passes thence into the spermathecae ' (3). It isclear from this description that in Lumbricus , besides thegenerative apertures, the clitellum and the two pairs of tem-porary seminal grooves play an important part in the passageof the seminal fluid from one worm to another.

While making observations on the reproductive processes ofan Indian earthworm Butyphoeus , I discovered that, inthis form, the method of exchange of the seminal fluid betweentwo worms was entirely different from that described by Grovein Lumbricus . In fact, the process is very simple and direct,and no intermediate structures like the clitellum and the seminalgrooves take part in it. In this genus the male pores open atthe distal ends of two ' penial lobes ' or ' penes ' on segment 17,and these penes, during copulation, are inserted into the sperma-thecal pores of the co-operating worm, so that the penes act astrue intromittent organs and the transference of sperms takesplace without any loss of spermatic fluid.

O'Donoghue (5) was under the mistaken belief that this simpleand direct method Avas the natural process in Lumbricus ,for he writes :

' During the act of copulation, two worms lie together inhead-to-tail directions in such a way that the openings of thevasa deferentia of one come to lie opposite to the openings of thespermathecae in the other and vice versa. The sperms are thentransferred from each worm to the spermathecae of the other,where they can live for some time until required ; after this theworms separate.' Grove has conclusively shown in his paperthat this account cannot apply in the case of Lumbricusbecause of ' the anatomical impossibility of the mutual apposi-tion of the apertures of the vasa deferentia with those of thespermathecae of the co-operating worm'. But O'Donoghue'sdescription, as we shall see presently, holds good for the processoccurring in Eu typhoeus .

Since the anatomy of Eutyphoeus is not so well knownas that of Lumbr icus , I have given an account of thecharacteristic features of the reproductive system before de-

REPRODUCTION OF EUTYPHOEUS 481

scribing the process of conjugation as observed by me for thefirst time in this worm.

E u t y p h o e u s is a genus of worms entirely confined toIndia, belonging to the sub-family Octochaetinae in the familyMegascolecidae. The arrangement of setae is lumbricine andthe worm is a surface-feeder,1 spending a great part of its timeoutside the burrow.

2. MATERIAL AND METHODS.

During the monsoon (July, August, and September) E u t y -p h o e u s occurs in very large numbers in my garden, and I havethus been able to observe and study its habits closely in itsnatural habitat. Two factors make conditions specially con-venient for a study of its reproductive processes : firstly, it isa surface-feeder and comes out on the surface of the ground forfeeding and reproduction ; secondly, it comes out not only atnight but at all hours of the day. While it was not actuallyraining, I could see scores of pairs every morning in sexualcongress on my lawn ; many a time I saw conjugating pairsbetween four and six in the afternoon (sunset being at about6.30 p.m.). In the morning conjugating pairs were found rightup to 9 o'clock, two hours and a half to three hours after sunrise.

In order to obtain preserved material for section-cutting andmicroscopic examination, I fixed several conjugating pairs inGrove's modification of Bouin's fluid (3). E u t y p h o e u s seemsto be much more active and sensitive than L u m b r i c u s , sothat the slightest vibration in the ground causes the conjugatingworms to separate. As a general rule, two worms from adjoiningburrows come out on the surface of the ground and copulate bytheir anterior regions keeping their tails in their burrows. Butin many cases only one of the two worms has its tail in the bur-row, while the other lies entirely free on the ground, having noconnexion with any burrow. As pointed out by Grove, the bestmethod of preventing the separation of two conjugating worms

1 This habit of life is in strong contrast with that of P h e r e t i m a ,which lives almost entirely underground and is very seldom seen outsideits burrow.

NO. 283 I i

482 KARM NAEAYAN BAHL

is to cut off their tails quickly, so as to prevent them gettingback into their burrows. The fixation was most successful inthose cases in which only one worm had to be cut off its burrow,the other being already entirely free ; or in those cases in whicha large portion of the bodies of the two worms had come outsidethe burrows and consequently only a very small portion of thetail had to be cut off. As a general rule, the greater the distanceof the cut from the clitellum, the smaller the attempt on thepart of the worms to separate. In several cases the burrows ofthe copulating worms are very near and they put out only theiranterior seventeen to twenty segments on the surface of theground ; in such cases, cutting the worms off their burrows,necessarily very near the clitellum, almost invariably results intheir separation.

After cutting off the tails, Grove successfully removed theconnected anterior portions of L u m b r i c u s to the fixative.But it is impossible to handle E u t y p h o e u s in that way,since the slightest delay, even after cutting off the tails, resultsin the separation of the two worms. I therefore cut off theirtails with a pair of scissors in my right hand, and at once pouredthe fixative on the pairing worms from the bottle in my lefthand. The worms wriggle a bit under the fixative but soonquiet down. They are then removed from the ground by meansof a section-lifter and placed in the fixative in a glass vessel.The whole process of cutting off the tails and pouring the fixa-tive must be completed in a few seconds or else there is likelyto be a partial or complete separation.

The presence of earth and grit in the alimentary canal of theworms presented a difficulty in cutting serial sections, but thiswas overcome by opening the worms by a mid-dorsal incisionand wholly removing the alimentary canal. But a further andmore serious difficulty was the presence of grit in the ' pegand socket ' joints (vide infra) formed at the places where thepenis is inserted into the spermathecal pore. Eepeated cleaning'with a camel-hair brush and blow-pipe helped to remove thisearth, but some sand particles still remained which could notbe removed without separating the worms—hence the sections


in some places were damaged by the grit. Serial sections, bothtransverse and longitudinal, were taken through the regions ofcontact of the two worms, and were stained either with haema-lum or iron haematoxylin and mucihaematein, or iron haerna-toxylin and picro-indigo carmine.

Whole mounts stained with borax carmine were made of theexternal male genitalia after treatment with caustic potash.These preparations, together with serial sections, were veryhelpful in determining the relative positions of the active andreserve penial setae, the relations of the setal sacs and the pros-tatic ducts and the disposition of the openings of the vasadeferentia.

Very helpful observations were also made by graduallyseparating several pairs of worms which had been fixed in thepairing condition. A very good indication of the disposition ofthe various structures in the act of copulation was obtained inthis way and these observations were confirmed by a study ofserial sections.

8. THE GENERATIVE ORGANS.

Since the generative organs of E u t y p h o e u s present cer-tain features hitherto unnoticed by systematic workers, andsince it is necessary to understand the disposition of the variousparts to appreciate the details of the process of copulation andsperm-exchange, I am giving a short description here of thegenerative organs of E u t y p h o e u s w a l t o n i , the speciescommonly found in Lucknow.

The clitellum (fig. 1, PI. 37) is ring-shaped, thinner ventrallythan dorsally, and occupies four and a half, segments (half13-17). The last segment of the clitellum, i. e. the seventeenth,bears on its ventral surface a pair of deep pits, often mistakenfor male pores. Deeply situated in each of these pits is a smallfleshy lobe, the ' penis ', bearing at its tip a couple of penialsetae and the male pore proper. The female generative aper-tures are generally inconspicuous, but can be identified on thefourteenth segment on either side of the mid-ventral line. Thereis a single pair of spermathecal pores situated on the inter-

i i 2

404 KARM NAKAYAN BAHL

segmental groove 7/8 in the centre of prominent eye-shapedareas. The copulatory papillae occur as ' transversely ovalareas or glandular slits between the lines of the ventral setalcouples, somewhat transgressing these limits, almost constantlyon 15/16 and 18/19 ; they are often present on 14/15 and 16/17and rarely on 19/20 and 20/21. Sometimes a pair of eye-shapedpapillae are found also on 9/10 ' (6).

The male generative organs consist of (1) a common testis sacin the e l e v e n t h segment enclosing a pair of testes and a pairof seminal funnels ; (2) a pair of seminal vesicles, connectedproximally with the testis sacs in the eleventh and twelfth seg-ments, but extending distally backwards and forwards so as tooccupy in the adult worm segments 11, 12, 13, and 14 ; (3) apair of vasa deferentia leading from the seminal funnels downto the seventeenth segment; (4) a pair of penes, which act ascopulatory organs ; and (5) a pair of tubular prostates occupyingsegments 17, 18, and 19. The disposition of all these structuresis illustrated in figs. 3 and 4, PI. 37.

In studying the reproductive processes of this worm, the pairof ' penes ' or copulatory organs are of special interest and,curiously enough, very little is known so far about these struc-tures and their importance is little realized. Beddard, who wasthe first to describe the reproductive organs of E u t y p h o e u sg a m m i i (1), does not mention these structures at all. Hedescribed the penial setae and their sac but missed the presenceof the penis altogether. Similarly, Michaelsen (4), who de-scribes as many as twenty-one species of E u t y p h o e u s , nevermakes even a mention of the penis, although he carefully de-scribes and sketches the penial setae of each species. BothBeddard and Michaelsen are further mistaken in saying thatprostates and sperm-ducts open into the same pores. As amatter of fact, the term ' male pore ' has been used very looselyand often incorrectly with regard to this genus. Finally,Stephenson and Ham Earn (7), while studying the prostateglands of this genus, duly recognize a ' papilla, which is sunkin a depression of the ventral surface and on which both the vasdeferens and the prostatic duct of one side open'. T h i s


p a p i l l a is r e a l l y t h e p e n i s and the penial setae arelodged in it. I have examined all the three species of E u t y -p h o e u s found in Lucknow (viz. E . g i g a s , E . i n c o m -m o d u s , and E . w a l t o n i ) for this structure, and haveverified the presence of ' penes ' in each of them.

Fig. 4, PI. 37, shows the terminal portions of the vas deferensand the prostatic duct and their openings on the ' penis '. Thepenis and the penial setae are protruded at the time of copula-tion, and are inserted into the spermathecal duct of the otherworm through the spermathecal pores (figs. 7 and 8, PI. 37).Each vas deferens begins as a continuation of the seminal funnelon each side in the eleventh segment and runs as a straight tubealong the inner side of the body-wall up to the seventeenthsegment where it passes underneath the prostatic duct, curvesand swells up into a thick muscular sac-like structure. Thevasa deferentia are lined internally with ciliated epitheliumright up to the point where they pass into this muscular sac.In this sac the ciliated epithelium is replaced by short columnarepithelium, and this is surrounded by a double coat of muscles—the inner layer of longitudinal and the outer of circular fibres—the circular coat being more than twice the thickness of thelongitudinal. The lumen of this sac is much wider than that ofthe vas deferens, and is produced into several diverticula whichincrease its capacity for holding the spermatic fluid. This fact,taken together with the presence of thick surrounding muscles,makes it very probable that the sac acts as a temporary sperm-reservoir, in which the spermatic fluid accumulates and fromwhich the sperms are squeezed out and passed on through thepenis into the spermathecal duct of the co-operating worm.The short terminal portion * of the vas deferens following themuscular sac traverses the body, of the penis and opens verynear its tip : this is the true m a l e p o r e or the opening of thevas deferens (fig. 4, PI. 37).

Coming out of the setal pore at the tip of the penis are thepair of active penial setae, which in copulating individuals mayproject as much as 900JJ. (nearly a millimetre) from the tip of

1 Comparable to the urethra of higher animals.

486 KAEM NARAYAN BAHL

the penis. The functional penial setae in E u t y p h o e u sw a l t o n i are unusually long, about 5-5 mm.,1 and they ' havea gentle S-shaped curve, forming about a quarter of a circle '.They are embedded in setal sacs which are thickly covered overwith muscle-fibres. The two setal sacs are distinct from eachother along the greater part of the length of the setae in thebody-cavity, and it is only when the setae pierce the body-walland enter the base of the penis that the two setal sacs unite.In the body of the penis, therefore, both penial setae are con-tained in a common setal sac. Further, this common setal sacis joined in the penis by the prostatic duct (vide infra).

Closely associated with the two functional penial setae thereare eight to ten other setae of progressive lengths in E u t y -p h o e u s : these I have called 'reserve penial setae'. In adissection from the dorsal side, one can easily make out a bandof connective tissue passing across the inner surface of the pros-tate gland in an obliquely transverse direction : both the activeand reserve penial setae in their sheaths are contained in thisband. This setal band is attached to the inner side of the body-wall ventrally at the region of the male pore and dorsally nearthe mid-dorsal line. The ' reserve ' penial setae lie internal tothe active setae in the same membranous band, but they donot pierce the body-wall and do not enter the penis. Thesesetae have the same structure as the active penial setae travers-ing the penis, but are of different lengths : they increase inlength from the inner flank of the membrane to its outer margin ;the smallest seta being on the inner side, and the longest oneson the outer side being the functional pair of the penial setae(fig. 3, PL 37).

Michaelsen (4) and Stephenson (6) did not notice this distinc-tion between the two kinds of penial setae, functional andreserve, although they have paid great attention to the minutestructure of the active penial setae. The functional setae arevery long and fragile, and since there are many chances ofthe copulation being disturbed or interrupted, it is probable

1 Michaelsen says they are 3-5 mm. in length (4), while Stephenson givesthe length up to 4-7 mm. (6).


that the functional penial setae often get broken or lost duringthe copulatory act and that they are replaced from the stockof reserve setae. That the worm possesses an armoury of penialsetae to replace those that get broken or lost is a very interestingfact in connexion with its reproductive processes—a fact unfor-tunately missed out by previous observers.

The prostate gland, described fully by Stephenson and HaruEarn (7), occupies segments 17 to 19, and forms a complicatedcoiled tube, opaque white in appearance. The tubular gland iscontinued into a shiny duct, the prostatic duct, which has aninternal lining of short epithelial cells surrounded by a thickcoat of circular muscle-fibres. The duct pierces the body-wallin the seventeenth segment and enters the base of the penis,joining at the same time the common setal sac of the two penialsetae (fig. 3, PI. 37). The setal pore at the tip of the penistherefore discharges the prostatic fluid besides forming anopening for the two penial setae.

The female generative organs consist of (1) a pair of ovariesin the thirteenth segment, (2) a pair of oviducal funnels in thesame segment leading into the oviducts, which open to theexterior on the fourteenth segment by two pores lying anteriorto the zone of setae, and (3) a pair of spermathecae in the eighthsegment. Of these, the spermathecae are the only structuresof interest in studying the process of copulation in the worm.There is a single pair of them and each consists of a thick sac-like ampulla and a short muscular duct about as long as theampulla.1 A pair of diverticula, each consisting of about fouralmost globular seminal chambers, open into the proximal endof the duct. The seminal chambers are arranged in a fan-likemanner, and the diverticula, which lie abreast of and notopposite to each other, are externally apposed to the base ofthe ampulla. The minute structure of the spermathecae and therole of the spermathecal duct and aperture are described in asubsequent section of the paper.

1 Both Michaelsen (4) and Stephenson (6) say that the duct is thin andis about half as long as the ampulla. But I find that the duct is thick andmuscular and is about as long as the ampulla.

400 KABM NARAYAN BAHL

The copulatory papillae form an important feature of theexternal genitalia of this worm, and, as we shall see presently,play an important part in the act of copulation. The numberand position of these papillae is very variable and are best seenin copulating worms. Some of these papillae are permanent,while others are temporary and are formed only at the timeof copulation. The permanent papillae are circular cup-shaped shallow depressions, found always in the interseg-mental grooves on the ventral surface. Temporary papillae, onthe other hand, are formed as conical outgrowths of the body-wall and are found not in the intersegmental grooves but in themiddle line of the segments themselves. At the time of copula-tion, the temporary papillae generally fit into the permanentcup-shaped depressions of the other worm.

4. DIRECT OBSERVATIONS ON COPULATING PAIRS.

Two worms coming out of adjacent burrows meet and in lessthan five minutes get apposed to each other in the typical head-to-tail position as shown in fig. 5, PI. 37. The male genital pitson the seventeenth segment of each worm come to lie oppositethe spermathecal pores (7/8) of the other and the interveningareas of the two worms are closely adpressed against each other.While the ventral surfaces of the worms are in such close con-tact, a very intimate connexion is established between the regionof the male pores of one worm and the spermathecal pores of theother. The area of skin surrounding each spermathecal apertureis drawn out to form an elongated papilla-like structure (fig. 2,PI. 37), so that the apertures of the spermathecae instead oflying flush with the surface are now raised up on the top ofthese papillae. At the same time, the two deep pits on theseventeenth segment, at the bottom of which a penial papillais situated, open out to form shallow cups, each with a thickcircular rim around it and a protruding penis in its centre(fig. 2, PI. 37). Each spermathecal papilla now closely fits intoeach of these cups, and the penis with its two penial setae isinserted completely into the spermathecal aperture. Thus a' peg and socket ' joint is formed at four places in a con-

REPRODUCTION OF BUTYPHOBUS 489

jugating pair, and one can easily see the male genital cupsclosely embracing the spermathecal papillae on examining acopulating pair from the side (fig. 5, PL 37). The changes inshape and size of the genital areas of the conjugating worms canbe recognized by comparing figs. 1 and 2, PI. 37, which showformation of the ' s p e r m a t h e c a l p a p i l l a e ' and the' m a l e g e n i t a l c u p s ' i n a copulating worm in place of the' s p e r m a t h e c a l p o r e s ' and the ' m a l e g e n i t a l p i t s 'of a non-copulating individual.

It is a remarkable fact that -while the worms are intimatelyconnected at their spermathecal and penial regions and are alsoclosely applied to each other in the intervening area, the anteriorseven segments of both worms keep free. Both the wormskeep exploring the ground by their anterior segments and pick-ing up earth particles with their mouths while still in congress.Feeding and copulation therefore go on side by side at thesame time.

In L u m b r i c u s , during the process of adjustment of thetwo worms, there is a copious effusion of mucus which leads tothe formation of two mucus tubes, enveloping the worms. InE u t y p h o e u s , on the other hand, no tubular mucus envelopesare formed, and the only mucus secreted is at the ' peg andsocket ' joints and the apposed ventral surfaces presumably tomake the attachment at these places secure. In fact, the mucustubes are necessary in L u m b r i c u s when we consider the factthat the seminal fluid is conveyed along an external groove andtherefore needs protection ; but in E u t y p h o e u s , where thetransference of sperms takes place through an intrornittentorgan and the fluid is not exposed to the exterior at all, a tubularenvelope of mucus is unnecessary.

Grove confirms Hering's estimate of two to three hours as theperiod of duration of the sexual congress in L u m b r i c u s ; inE u t y p h o e u s , on the other hand, observations on severalpairs, from the beginning to the end of the process, have shownthat the period never exceeds one hour. The shorter period inthe case of E u t y p h o e u s is easily accounted for, when weremember the fact that the time taken for preliminary adjust-


merits is very small—less than five minutes—and that the timetaken in L u m b r i c u s for the conduction of the spermatic fluidalong the seminal grooves and its collection between the clitel-lum of one worm and segments 9-11 of the other—about 30minutes—is saved in the case of E u t y p h o e u s . In E u t y -p h o e u s the sperms are injected, so to speak, directly into thespermathecae by the penis, while in L u m b r i c u s the trans-ference is effected through the intermediation of the seminalgrooves and the clitellum, and this involves about half an hour.

During the period of copulation the clitellar segments showmuscular contractions, as a result of which distinct longitudinaldepressions are formed on the lateral surfaces of the clitellumand a few neighbouring segments. There seems to be no doubtthat these contractions exert some pressure on the prostateglands and the muscular sac at the end of the vas deferens, andthus aid in the transference of the prostatic and spermatic fluidsfrom one worm into the other. These muscular contractionsleading to the formation of longitudinal depressions take the placeof peristaltic movements described by Grove in L u m b r i c u s .

When the transference of sperms is over, the separation takesplace by one worm withdrawing into its burrow after releasingits hold at both places one after the other. The other wormremains on the surface for a time but eventually it also getsback into its burrow.

By means of these observations it is established that a ' pegand socket ' joint is formed at four places in the two copulatingworms, and that mutual exchange of spermatozoa takes placeby the penetration of a pair of protruding penes of one worminto the spermathecal ducts of the other. The details of thewhole process can be best studied by a microscopic examinationof sections, and these are described in the following sections.

5. THE ATTACHMENT OF THE COPULATING WORMS.

We have already had a general indication of the mode ofattachment of copulating worms from our direct observations,but there are several details which need to be considered fullyhere.

REPRODUCTION IN EUTYPHOEUS 491

There is no doubt that the most intimate connexion betweenthe worms is established at four places, where ' peg and socket 'joints are formed. Examination of figs. 6, 7, and 8 clearly showsthat the spermathecal papilla is closely fitted into the genitalcup of the co-operating worm so much so that, in sections, onecan demarcate the body of one worm from that of the otheronly after a close scrutiny. The attachment is specially in-timate between the tip of the spermathecal papilla and the baseof the penis of the other worm, the epidermis of one being closelycontiguous with that of the other. The attachment is made stillmore secure by the secretion of mucus on both sides by thegoblet-cells of the epidermis, and it is found that when theworms are fixed and an attempt is made to separate the copulat-ing individuals, the opposing surfaces of the two worms at thesejoints are so firmly connected together that it needs a greatdeal of care to separate them.

Looking at a copulating pair either in the living conditionor in the preserved state (fig. 5, PI. 37), one cannot help noticingthat there is a strong suction at the four ' peg and socket ' joints,whereby the male genital cup in one worm adheres to the surfaceof the other worm and leads to the formation of a spermathecalpapilla in the latter. Figs. 8 and 9, PL 37, show the male genitalcup closely embracing the spermathecal papilla and the penispenetrating the spermathecal duct. This change from thenormal disposition of these structures (fig. 1, PI. 37) to theircopulatory disposition (fig. 2, PL 37) is brought about by theaction of the muscles surrounding the genital pit. These musclesby their contraction change the deep genital pit into a thick-rimmed shallow cup which closely surrounds the spermathecalpore of the other worm at the time of copulation. The wall ofthe cup contracts, expels the air, and produces a partial vacuum.This causes suction on the apposed surface of the other wormand thus leads to the formation of a papilla round the sperma-thecal pore, which closely fits into the cup.

The disposition of the muscles surrounding the male genitalcups and the spermathecal papillae has been determined by astudy of serial sections through these structures, and also by


a dissection of these muscles in a hardened specimen. Portionsof the body-wall including the male genital areas were hardenedin chromic acid, and the muscles were dissected out by a pairof needles under a binocular dissecting microscope to make outtheir arrangement. It has been found that besides the circularand longitudinal muscles of the body-wall, there are threespecial sets of muscles in the genital area of E u t y p h o e u s

TEXT-FIG. 1.

rph m

A semi-diagrammatic representation of the double loop of the longi-tudinal muscles which surround the male genital cups. Thesphincter muscles and a few radial muscles of the genital cups arealso shown, cl, clitellum ; Im, longitudinal muscles which runstraight in an ordinary worm but contract in a copulating wormto form a double loop ; sphm, sphincter muscles which effect theprotrusion of the penis by their contraction ; vnc, ventral nerve-cord.

which aid in copulation. Firstly, there are special ventro-lateral muscles in each of the segments 14 to 19; secondly,there are radial muscle-fibres, which radiate in all directionsfrom the epidermis of the genital cup, lie at right anglesto this epidermis, and traverse through both the circularand longitudinal layers ; thirdly, a thick sphincter muscle liesat the bottom of the cup surrounding the base of the penis.


The disposition of all these muscles is shown diagrammaticallyin Text-figs. 1 and 2.

The longitudinal muscles of the body-wall ran in straight linesin an ordinary earthworm, but in a copulating specimen theypartially surround the genital cup and show a double loop intheir course, as shown in the accompanying text-figure. Thereis no doubt that these muscles by their own contraction and

TEXT-FIG. 2.

A semi-diagrammatic representation of the special ventro-lateralmuscles in each of the segments 14 to 19. cl, clitellum ; mgc, malegenital cup ; vim, ventro-lateral muscles ; vne, ventral nerve-cord.

with the aid of the sphincter muscles surrounding the base ofthe penis and the ventro-lateral muscles lying on the innercoelomic side of the body-wall, bring about the protrusion of thepenis, and change the male genital pit into a cup. This cupproduces a spermathecal papilla on the body of the other wormby a process of suction, and that is how a very intimate con-nexion is established between the two worms at these ' pegand socket' joints. After copulation these three kinds ofmuscles relax, and the radiating muscle-fibres (figs. 7 and 8,PI. 37) contract and pull the penis inwards, the wall of the


TEXT-FIG. 3.

A diagrammatic representation of two copulating worms showingthat their segments do not lie parallel to each other but alternate,


cup being also drawn in to form the male genital pit again.The process of suction can easily be noticed by a reference tofigs. 7 and 8, PL 37.

An attachment, not perhaps as intimate as that at the ' pegand socket ' joints, but still quite effective, takes place at severalplaces on the ventral surface. But before considering the detailsof this attachment it is well to emphasize the fact that, as ageneral rule, the attachment of the two worms is not parallelbut oblique, that is to say, a segment of one worm does not lieopposite to a segment of the other, but, as shown in theaccompanying text-figure, the segments of the two wormsalternate. Consequently, the intersegmental grooves of oneare opposed to the middle of the segments of the other. Cor-related with this alternate arrangement of the segments in thetwo copulating worms is the fact that permanent papillae(glandular areas) which are always intersegmental in positionget apposed to temporary integumentary outgrowths which arealways situated about the middle line of a segment. Beginningwith the anterior end, the first pair of temporary conical papillaeare found in the middle line of the sixth segment : these out-growths are fitted into the glandular areas in the intersegmentalline 18/19 of the other worm. Similar paired papillae on theninth segment fit into the glandular depressions on the inter-segmentum 15/16. Immediately following these temporaryoutgrowths are a pair of permanent papillae on the inter-segmental line 9/10 : these papillae project on the surface andare received into depressions on the fifteenth segment. Again,there is, as a rule, a single or paired integumentary papilla onthe tenth segment which fits into the permanent papillae on theintersegmental line 14/15. In the case of those worms whichhave copulatory depressions on the intersegmental lines 19/20

the intersegmental lines of one lying opposite the middle linesof the segments of the other. The permanent papillae which areintersegmental are shown as circles with black dots, while thetemporary papillae which are present only on some of the segmentsare shown as circles only, cl, cliteUum ; mgp, male genital pit;spa, spermathecal aperture ; tp, temporary papillae ; pp, per-manent papillae.


and 20/21, it is- probable that the co-operating worm formscorresponding conical papillae at the time of copulation on thefifth and fourth segments respectively. I have never seen theglandular areas on 19/20 and 20/21 in action, but, as statedbefore, have always found the first six segments free.

Since all systematic accounts are naturally based on non-copulating individuals, only the permanent glandular areas havebeen noticed by previous observers, and no mention has everbeen made of the temporary conical papillae which occur in thesixth, ninth, and tenth segments. So far as I am aware, theformation of these temporary conical papillae for purposes ofcopulation has never been observed before, and it is an interest-ing fact that these papillae closely fit into the glandular areaswhich are permanent structures in the worm. I need only addthat while the permanent copulatory papillae form cup-shapeddepressions with special gland-cells beneath the surface, thetemporary conical projections which fit into these depressionshave no special gland-cells besides the ordinary mucus-secretinggoblet-cells of the epidermis.

6. THE PENIS AND THE SPERMATHEOAE.

The penis is a muscular structure, the outer surface of whichis covered by a single layer of flat epithelial cells and the bodyof which is tunnelled through by two tubes—one, the terminalportion of the vas deferens corresponding to the urethra ofhigher animals, and the other, the tube conveying the prostaticsecretion and at the same time lodging the two penial setae(fig. 4, PI. 37). The muscles which form the greater part of thethickness of the penis are the radiating muscles which run at rightangles to the epidermis of the genital cup (figs. 7 and 8, PI. 37);at the base of the penis, however, these muscles converge andtraverse the penis along its length forming almost entirely thesubstance of the penis. The penis may thus be compared toa double-barrelled gun—the body of the gun being formed ofmuscles running along its length and the two barrels corre-sponding to the vas deferens on the one hand and the combinedprostatic tube and setal sac on the other.

REPEODUCTION OF EUTYPHOBUS 497

The protruded penis is 840/x in length and, at the time ofcopulation, the whole of it is inserted into the spermathecal ductof the other Avorm through its spermathecal pore. The twopenial setae project about 900fi—almost a millimetre—beyondthe tip of the penis, and these also are inserted into the sperma-thecal duct. The spermathecal pore in a copulating worm isfunnel-shaped, having a wide mouth leading into a narrow duct.The opening is about 375 ̂ in diameter, while the spermathecalduct into which it leads is only about 90 fx. The penis being a longtapering structure, its thick base fits into the wide spermathecalopening, while the body of the penis penetrates the narrow sper-mathecal duct and fits so closely into it as to make it difficult insections to demarcate the outer covering of the penis from the innerepithelial lining of the spermathecal duct (figs. 7 and 8, PI. 37).

It has already been mentioned that a spermatheca ofE u t y p h o e u s consists of an ampulla and a pair of diverticula,each diverticulum consisting of about four almost globularseminal chambers. Beddard (2) points out that in the case ofB e n h a m i a and P e r i c h a e t a , the spermatozoa are alwaysfound in the diverticula and n o t in the ampulla. The same isthe case in E u t y p h o e u s . The question naturally arises thatif sperms are contained only in the diverticula, what is the originand nature of the fluid in the ampulla ? The inner epitheliallining of the ampulla is made up of tall columnar cells which aresecretory in nature. The nucleus is situated in the middle ofeach cell, while its distal half projecting into the lumen of theampulla is found full of secretory granules and vacuoles. Com-pared with these columnar secretory cells of the ampulla, thediverticula are lined with a low quadrangular epithelium withno obvious suggestion of a secretory nature. The lumen of theseminal chambers of each diverticulum is choked full of spermswith their heads adpressed against the lining epithelium (fig. 9,PI. 37). •

Grove (3) has established by means oJ his observations onthe contents of the spermathecae of L u m b r i c u s that massesof mucus are present in the spermathecae besides a thin mucousenvelope round each spermatozoon. Grove believes that mucin

NO. 283 K k


is secreted by the cells of the lining epithelium and is pouredinto the cavity of the spermatheca, where it is used probably asfood for the spermatozoa stored in it.

In Eutyphoeus , on the other hand, the question is com-plicated by two factors which are absent in the case of L u m -br icus . The first is that Eutyphoeus possesses a pair ofprostates and at the time of copulation the spermathecae receiveboth the prostatic and spermatic fluids from the co-operatingworm. Secondly, a spermatheca has two distinct chambers, theampulla and the diverticulum, and of these it is the ampullathat has a secretory epithelial lining while the sperms are con-tained in the diverticula, the walls of which are apparentlynon-secretory. The prostatic fluid must either go into theampulla or into the diverticula. The ampulla presumably con-tains its own secretion and it is likely that the prostatic fluidgoes into the diverticula along with the spermatic fluid andserves as a nourishment for the sperms as long as they arestored in the spermathecae.

As a preliminary to a piece of a work on the physiology of thegenerative organs of earthworms, the litmus test was appliedto the prostates and spermathecae of Pheret ima and Euty-phoeus with the following results :

Ampulla. Diverticulum. Prostates.1. P h e r e t i m a Markedly acid Markedly alka- Slightly alka-

line line.2. E u t y p h o e u s Markedly acid Markedly alka- Slightly alka-

line line.

The surprising fact comes out that the contents of the ampullaand the diverticulum are markedly opposed in their reaction tolitmus while the prostates and the diverticula agree. Of course,the evidence of acid or alkaline reaction is only prognostic, andfurther evidence is necessary to establish the fact that prostaticfluid serves as food for the stored sperms in the diverticula ofthe spermatheca. The prostate glands are absent in Lum-bricus, the best-studied form amongst earthworms, and hencevery little attention has so far been paid to the elucidation ofthe function of the prostatic secretion in earthworms.


7. SUMMARY.

1. The method of exchange of the seminal fluid in Euty-p hoe us is very simple and direct as compared with theelaborate process in Lumbricus . No intermediate struc-tures like the clitellum and temporary seminal grooves takepart in the process in Eu typhoeus .

2. During sexual congress, the co-operating worms becomeattached to one another in a head-to-tail position in such a waythat the spermathecal apertures (7/8) of one are apposed to thepenial segment (seventeenth) of the other and vice versa,

3. The male ' genital pits' are everted to form ' genital cups'and the penis is protruded. The genital cups produce a suctionon the area of skin surrounding the spermathecal pores of theco-operating worm, and thus cause the formation of sperma-thecal papillae. In this way a ' peg and socket ' joint is formedat four places in a copulating pair and, at each joint, the attach-ment is intimate, the genital cup closely embracing the sperma-thecal papilla and the penis penetrating the spermathecal duct.

4. There is a further attachment between the ventral sur-faces of the two worms by means of permanent copulatingpapillae and temporary integumentary outgrowths.

5. The function of the penis as an intromittent organ inEutyphoeus has been elucidated for the first time and adistinction has been made between ' functional' and ' reserve 'penial setae.

6. The exchange of sperms is mutual. The penes inject bothspermatic and prostatic fluids into the spermathecae. Thesperms are invariably found in the diverticula and n o t in theampulla, which probably contains a secretion of its own epithe-lium. There is some evidence to believe that the prostaticfluid serves a nutrient medium for the sperms in the seminalchambers of the diverticula.

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8. EEFERBNCBS TO LITERATURE.

1. Beddard, F. E.—" On the Structure of Three New Species of Earth-worms, with Remarks on Certain Points in the Morphology of theOligochaeta ", ' Quart. Journ. Micr. Sci.', vol. 29, 1889.

2. ' A Monograph of the order Oligochaeta.' Oxford, 1895.3. Grove, A. J.—" On the Reproductive Processes of the Earthworm,

Lumbricus terrestris ", ' Quart. Journ. Micr. Sci.', vol. 69, 1925.4. Michaelsen, W.—" The Oligochaeta of India, Nepal, Ceylon, Burma,

and the Andaman Islands ", ' Mem. Ind. Mus.', vol. 1, no. 3, 1909.5. O'Donoghue, C. H.—' An Introduction to Zoology.' London, 1920.6. Stephenson, J.—' Oligochaeta.' In the Fauna of British India series,

1923.7. Stephenson, J., and Haru Ram.—" The Prostate Glands of the Earth-

worms of the Family Megascolecidae ", ' Trans. Roy. Soc, Edin.',vol. 52, 1919.

EXPLANATION OF PLATE 37.

Illustrating Professor K. N. Bahl's paper on ' The Process ofCopulation and Exchange of Sperms in Eutyphoeuswal ton i ' .

Fig. 1.—A photograph of the external genitalia and their disposition inanon-conjugating E u t y p h o e u s . g.p-, genital papillae situated on theintersegmental grooves 9/10. Genital papillae are also present on inter-segmental grooves 14/15,15/16, and 18/19, and on the nineteenth segment;m.g.p., male genital pits with the penis retracted ; sp.a., spermathecalaperture.

Fig. 2.—A photograph of the external genitalia and their disposition ina conjugating earthworm, gp., genital papillae ; m.g.c, male genital cupaformed from the genital pits, with the penis protruded out; sp.p., sperma-thecal papillae, formed from the spermathecal apertures of fig. 1.

Fig. 3.—A dissection of E u t y p h o e u s w a l t o n i showing the disposi-tion of the reproductive organs, e.g., cerebral ganglia ; ov., ovary ; ov.d.,oviduct; pr.gl., tubular prostate gland ; pr.d., prostatic duct; p.s., penialsetae, active and reserve ; s.m.b., membranous band lodging the active andreserve penial setae ; sp.amp., spermathecal ampulla with diverticula ;sp.d., spermathecal duct; s.f., seminal funnel; s.v., seminal vesicle of the leftside, that of the right side having been cut off to show the seminal funnel;t.s., the common testis sac ; t.v.d., the terminal portion of the vas deferensswollen out to form a muscular sac; v.d., vas deferens; v.n.c, ventral nerve-cord.

Fig. 4.—The penis together with the terminal portions of the vas deferens


and the prostatic duct, showing also the disposition of the active and reservepenial setae, a.p.s., active penial setae; b.l., branching lumen of the muscu-lar sac ; c.s.s., common setal sac of the two penial setae; m,b., membranousband lodging penial setae ; m.s., muscular sac with its lumen produced intodiverticula presumably to increase the capacity for holding the sperms ;o., opening of the prostatic duct into the common setal sac of the activepenial setae ; o.v.d., opening of the vas deferens on the penis, the real malepore ; o.s.s.pr., common opening of the setal sac and the prostatic duct;p., penis; pr.d., prostatic duct surrounded by circular muscle-fibres; r.p.s.,reserve penial setae; t.v.d., terminal non-ciliated portion.of the vas deferenstraversing the penis and corresponding to the urethra of mammals ; s.s.,setal sac of one of the two penial setae ; v.d., ciliated vas deferens before itswells up to form the muscular sac.

Fig. 5.—A photograph of two worms in sexual congress (enlarged). Thegenital cup of one is seen embracing the spermathecal papilla of the otherand vice versa, thus producing the ' cup and socket' joints, g.c, genitalcup ; s.p., spermathecal papilla inserted into the genital cup.

Fig. 6.—A longitudinal section through one of the ' peg and socket'joints showing the penis of one worm inserted into the spermathecal ductof the other. The muscle-fibres radiating from the epidermis of the genitalcup are also seen. These radiating muscles produce a suction leading tothe formation of the spermathecal papillae. The body-wall of the upperworm is produced to form a spermathecal papilla, which fits into the genitalcup formed by the lower worm. AA', the genital cup embracing thespermathecal papilla ; amp., ampulla of the spermatheca ; i.e., bloodcapillaries ; b.w.1, body-wall of one worm ; b.w.2, body-wall of the otherworm ; m.s., muscular sac during the course of the vas deferens with itsbranched lumen; p., penis traversed by the vas deferens on the left and thecommon prostatic duct and setal sac on the right; pr.d., prostatio duct;pr.gl., prostatic gland ; r.m.f., radial muscle-fibres; s.p., spermathecalpapilla ; sp.d., spermathecal duct; v.d., vas deferens.

Fig. 7.—A microphotograph of a transverse section through a ' peg andsocket' joint. The penis of the lower worm is fully inserted into thespermathecal duct of the upper, and the prostatic duct of the lower wormis seen entering the common setal sac of the penial setae. The radialmuscles, which produce a suction leading to the formation of spermathecalpapillae, are very clearly seen in this photograph, m.g.c, rim of the malegenital cup ; pr.d., prostatic duct; pr.d'., prostatic duct joining one of thesetal sacs, s.s.; s.s'., setal sac of the other penial seta ; pr.d.ss., the commonsetal sac and prostatic duct traversing the whole length of the penis ;«., broken bits of penial setae in the upper region of the spermathecal duct;r.m., radial muscles ; sp.amp., spermathecal ampulla.

Fig. 8.—A microphotograph of the section next to the one shown infig. 7 in the same series. In this section the urethra or the terminal portion


of the vas deferens traversing the penis is well seen, ur, urethra. Otherletters as in fig. 7.

Kg. 9.—A reconstruction of six consecutive sections to show the sperma-thecal ampulla and its diverticula with their seminal chambers in longi-tudinal section, amp., ampulla containing its own secretion ; b.w., body-wall ; div., seminal chambers of the diverticula filled with spermatozoa ;sp.c, muscular spermathecal canal.

on the reproductive processes of earthworms: part i. the ... · and these penes, during copulation,...

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