LIFE HISTORY OF THE LIVER-FLUKE. 99

The Life History of the Liver-Fluke (Fasciolahepatica).

A. P. Thomas, M.A., F.t>.S.,Ballinl College, Professor of Natural Science in University College, Auckland,

Kew Zealand late Demonstrator in the Anatomical Department, Uni-versity Museum, Oxford.

With Plates II and III.

IT has been known from very early times that the liver-rotof various herbivorous mammals is a parasitic disease due tothe presence of very numerous flukes in the liver of the affectedanimals. A.mongst our domesticated animals the sheep is byfar the most frequent victim. The fluke disease is alwayscommon in certain districts in England and in many parts ofthe world; but in consequence of a succession of wet seasonsthere was a serious outbreak of it in the winter 1879-80, andit is estimated that in the United Kingdom 3,000,000 sheepwere then destroyed by it. Hence special attention was calledto the subject, and the research summarised in the followingpaper was undertaken on behalf of the Royal AgriculturalSociety of England.

For the sake of convenience the subject is divided into thefollowing sections:—T. HISTORICAL. II. METHODS OF IN-VESTIGATION. III. LIFE-HISTORY.

I. HISTORICAL.

From what was known of the course of development amongstthe digenetic Trematodes, the nearest allies of Fasciola he-

100 A. P. THOMAS.

patica, there was reason to believe that here also an alternationof generations existed, and that one or more molluscs served asintermediate host for the asexual forms. Many attempts hadbeen made to discover the intermediate host by various eminentbiologists, including Professor Leuckart, von Linsto'w, Ercolani,&c, but all had proved fruitless, and notwithstanding its im-portant practical bearing the problem remained unsolved.1

Very many suggestions had been made as to the nature of theintermediate host. Moulinie'2 had found in Limax cinereusand Arion rufus (ater) sporocysts containing cercarise with arudimentary tail, and suggested that these might have someconnection with the liver-fluke. I met with this species inArion ater early in the course of my investigations, and wasable to disprove experimentally the conjecture that this wasthe cercaria of Fasciola hepat ica . Willemoes-Suhm3 haddrawn attention to the fact that liver-rot was very prevalent in

• the Faroe Islands, the molluscan fauna of which was restrictedto eight species, viz. Ariou ater , A.cinctus, Limaxagres t i s ,L. marg ina tus , Vi t r ina pellucida, Hyal ina al l iar ia ,Limnseus pereger, and L. t runca tu lus . Of these Limaxagrest is , our common grey slug, was by far the commonestand most injurious, and he suggested that this slug might actas intermediate host. Von Linstow4 had mentioned Planorbisvorjex as being possibly the host. Weinland6 had found theliver of L. t r unca tu lus infested with nurse forms. The

• A statement lias been published in several text-books, English andAmerican, to the effect that Cercaria cystophora inhabiting Planorbismarginatus is the larva of Fasciola hepatica. This, of course, is erroneous,and the mistake appears to have been copied from an abstract in the' Zoological Record' for 1872 of a paper by Willemoes-Suhm. The suggestionreally made in the original paper was that C. oystophora is the%larval formof Distoma lanceolatum. This specie is known on the Continent as thesmall liver-fluke, and is far less formidable than the larger, P. hepatica, thetrue liver-fluke. It appears not to exist in England.

2 ' Me'moires de 1'Institut Genevois,' vol. iii, p. 267.3 ' Zeitschrift fur wissentschafdiche Zoologie,' 1873, vol. xxiii, p. 339.4 'Arch, fur Naturgeschichte,' 1875, p 194.6 Abstract in ' Archiv fur Naturgeschichte,' 1874, vol. ii, p. 423.

LIFE HISTORY OF THE LIVER-FLUKE. 101

cereariaa had the habit of throwing off their tails and crawlingabout by the aid of their suckers, and he thought that thelarvae of the liver-fluke might encyst upon plants. Kiichen-meister had suggested certain slugs as possible hosts ofFasciola hepat ica. On April 7th, 1880, the 'Times'published a letter written by Dr. Cobbold to contradict thestatements made by Dr. J. Harley, who denied the existenceof any intermediate host. The letter contained the followingsentence:—" The investigations of the lamented Willemoes-Suhm render it almost certain that Cercaria cys tophorainfesting Planorbis marg ina ta is the higher larval state ofthe small fluke (Distoma lanceolatum), and the still laterresearches of Leuckart point to the mollusc called L y m n a atruncatula as the bearer of the cercarian stage of Fasciolahepatica.5' Seven days later, on April 14th, 1880, the lateDr. Rolleston gave, in a letter to the 'Times/ reasons forlegarding the black slug, Arion ater, as the intermediate hostof Fasciola. Since that time Ercolani1 has made a similarsuggestion for certain terrestrial molluscs. He found that larvaltrematodes were of far more frequent occurrence in land-molluscs tfian had hitherto been supposed, and this circum-stance, together with the failure of the most illustrious hel-minthologists to discover the genesis of F. hepat ica , led himto think that it was in this direction that success would even-tually be obtained.

On the 2nd of the following June the Royal AgriculturalSociety of England offered Dr. Rolleston a grant for an inves-tigation into the life-history of the liver-fluke. Dr. Rollestonbeing unable himself to undertake the work, mentioned myname to the Society, and the research was begun by me onJune 7th.

On Dec. 22nd, 1880, I found in a Limnaeus truncatulus,captured on an infected field at Wytham, near Oxford, on the24th Sept., and since that time kept in an aquarium in the

1 " Dell' Adattamento delle specie all' ambiente," ' Memorie dell' Accademia "delle Scienze dell' Istituto di Bologna,' serie iv, tomo ii, 1881, pp. 211, 327.

102 A. P. THOMAS. .

laboratory, a cercaria, which has since been proved to be thelarva of the liver fluke.

The reasons which led me to regard this as the cercaria ofFasciola hepatica need not be explained here, as they aregiven in another part of this paper. In a report in the RoyalAgricultural Society's 'Journal' for April, 1881, I describedthis cercaria as a new species, and at the end of the description,wrote as follows :—" The structure and habits of this cercariarender it possible that it may prove to be the. larva of Fasciola.hepat ica, but want of material has prevented my testing thequestion by giving the cysts to rabbits. I intend, however, topursue this case further."

On Dec. 12th, 1881, a paper appeared in the ' ZoologischerAnzeiger,' in which Professor Leuckart announced that he hadsucceeded in infecting young specimens of L. pereger, but hadbeen unable to obtain the development of the expected cercaria.He also made it known, for the first time, that the statementmade by Dr. Cobbold in the 'Times' for April 7th, 1880, wasfounded on a private letter from himself, and that the announce-ment that his researches pointed to L. t runca tu lus hadproved to be premature and incorrect, for on further examina-tion of his snails he had found tiiem to be not L. t runcatu lusbut L. pereger juv.

In the first number of the ' Archiv fur Naturgeschichte' for1882, which, however, bears no further date, ihe same resultswere given at greater length? Professor Leuckart stated that in anumber of specimens of L. t runca tu lus sent him by a friendhe had found three kinds of redise. One of these containedtailless distomes, which, he held, probably belonged to thedevelopmental cycle of the liver-fluke. He considered thissupposition to be entirely justified until further results wereobtained. A second form was not absolutely excluded from allconnection with the liver-fluke, but no such statement could bemade with respect to the third form. But in this third form Iat once recognised the cercaria found by me at Wytham, ofwhich a description had been published some eight monthsearlier.

LIFE HISTORY OF THIS LIVER-FLUKE. 103

During the summer of 1882 I at length obtained L. t runca-tulus for myexperiments, and succeeded in infecting them withthe embryos of F. hepatica. Before the end of August thedevelopment of the species was obtained up to the time whenthe tailed cercarise were nearly mature, and, as I had by mewell-preserved specimens of the re'dioe and cercarise found atWytham, I was able to compare the two forms step by step,and see that they were identical. A paper was drawn up forthe October number of the 'Journal of the Royal AgriculturalSociety,' giving these results, and was sent to the printer onSept. 1st. A. fortnight later it received a revision, which wasmerely verbal, and the whole of the copies were printed off byOct.Shd.

Separate copies of the paper were received for distribution onthe 24th Oct., but the journal was not published until nearlythe end of the month.

Inthemean time a paper by Professor Leuckart appeared in the'Zoologischer Anzeiger' for the 9th October. In this he statedthat he too had succeeded in rearing the cercaria of F. hepaticain L. t runcatulus , "the only true intermediate host," and thatit had proved to be not the tailless form, but, on the contrary,the third form mentioned above, which he had supposed earlyin 1882 could have no connection with the liver-fluke.

On the 19th Oct. a resume of my completed researcheswas published in 'Nature,' attention being called to the factthat the cercaria of the liver-fluke was the one already dis-covered by me in Dec, 1880, and described in April, 1881, asprobably belonging to F. hepatica, and that the necessaryproof had been furnished by myself, and also independently byProfessor Leuckart.

II. METHODS OF INVESTIGATION.

On commencing the investigation into the life-history of theliver-fluke it was felt that where so many different molluscshad been suggested as possible intermediate hosts, it was neces-sary to examine the question anew, and not to be guided by

104 A. P, THOMAS.

the numerous conjectures already expressed, some of whichhad very scanty evidence to support them. Work was begunin two directions, first, by endeavouring to infect many of ourcommoner molluscs, both terrestrial and fresh-water. In thesecond place, numerous localities in the country around Oxford,in which sheep were known to have incurred liver-rot, werevisited, the whole of the invertebrate fauna was carefullystudied, and many specimens brought home to be dissectedand searched for larval trematodes. Very numerous places wereexamined in this way, but mention will only be made here of theone which proves finally to have given the correct solution ofthe problem. At Wytham, near Oxford, was a clearly circum-scribed area of infection. The fields, five in number, weresituated on the side of a hill far above the reach of riverfloods, lying upon the Oxford clay. They were searchedthoroughly by day and by night, and the various invertebrateanimals found, including snails, slugs, aquatic insect larvae,Crustacea, worms, &c, .brought home and examined. Fresh-water snails were very scarce on the ground; all that werebrought to light were two small specimens of Physa fonti-nalis , a small Cyclas, and Limnaeus t runca tu lus inmoderate numbers. The last-named species was found in aboggy spot in one of the fields. -In one of them was discovered,on the 22nd December, 1880, the peculiar and interesting formof cercaria to which allusion has already been made. Its moststriking character was due to the presence of very coarselygranular cells arranged in lobed masses along each side of thebody. It was very active, but soon came to rest, encystingitself upon surrounding bodies. The cyst was snowy white,from the presence in its substance of the highly refractivegranules already seen in the granular cells forming the lateralmasses, which were thus shown to be cystogenous organs.

The other points in the structure of the cercaria were allfavorable to the supposition that I had here discovered thelong-sought cercaria of the liver-fluke. I had already foundin a lamb's liver exceedingly minute flukes, smaller than anyyet recorded, one of them being only l ' l mm. in length, i.e.

LIFE HISTORY OF THIS LIVEE-FLUKE. 105

only l-30th part as long.as the adult, and these immatureforms gave me valuable information as to the structures to beexpected in the cercaria, the relative size of its suckers, &c. Itwas usually supposed that the sheep when grazing picked up theparasites whilst they were still within the snail. This viewwas upheld by so eminent an authority as Professor Leuckartin a paper published in the beginning of 1882.1 But I hadalready collected evidence from independent sources, whichinclined me to the belief that the larvae were picked up inthe encysted condition attached to the.grass. Hence the pre-sence of a special cystogenous organ in the cercaria, and itshabit of encysting on grass were highly suspicious. Further,the suspicions raised by the structure and habits of thecercaria. were increased by the fact that its nurse-form wasthe only one found upon the ground, although there was everyreason for expecting to find the larva of the liver-fluke, as aninfected sheep had been seen a .few months earlier wanderingover the boggy spot from which the L. truncatulus was ob-tained. I can testify that the sheep harboured numerousflukes, for its liver was sent me for examination; and therecould be no doubt that large quantities of fluke eggs had beenscattered all over the fields. My suspicions were accordinglyexpressed in a report to the 'Royal Agricultural Society'sJournal' for April, 1881.

During the summer of 1881 I was anxious to try infectiveexperiments with LimnEeus t runcatulus , but was unfortu-nately unable to obtain any specimens; the localities nearOxford, where I had formerly found it, were searched in vain.I went out repeatedly in quest of this snail, having on severaloccasions the skilled assistance of my friend and colleague,Mr. W. Hatchett Jackson, but we never found any other traceof this species than the empty shells. It could not be dis-covered in the localities given for it by Whiteaves in hispaper on the " Mollusca inhabiting the Neighbourhood ofOxford."2 My friends at a distance were appealed to, but were

I ' Arcliiv fiir Naturgescliiohte,' 1882, p. SO,5 'Proceedings of the Ashmoleau Society/ 1857.

106 • A. P. THOMAS.

unable to assist me. The comparative freedom from rot ofsheep in the neighbourhood of Oxford last year was probablydue to the real scarcity of this snail.

In 1882, however, there were floods in July, and the watersof the Isis brought down vast multitudes, probably "from itsbreeding haunts in marshy places up the river. So numerouswas it, that a single sweep of a small hand-net repeatedlygave me more than 500 examples, and this was in a ditchwhere last year not a single L. truncatulus could be found.• On obtaining the snails I had so long been searching for, Iexposed a number to infection by placing fluke embryos inthe vessel with them. The snails were speedily found to haveafforded a suitable place for the further development of theembryos; indeed, infection was too successful, for very largenumbers of them died simply from exhaustion owing to theexcessive number of parasites they contained.

1 It may be well to mention here that infection experimentshave been tried upon the following species of molluscs, as wellas uponL. t runca tu lus , viz., Limn se us pereger, L. palus-t r is , L. aur icu la r ius , L. s tagnal i s , Physa fontinal is ,P lanorbis marginatus , P . car ina tus , P . vortex, P.spirorbis , Bythinia ten tacula ta , Paludina vivipara,Succinea amphibia , Li in ax agrestis, L. cinereus, Arionater , A. hortensis. None of these could be infected, withthe partial exception of Limnseus pereger. With regardto this last species I can corroborate Professor Leuckart'sstatement that the youngest specimens only of this species canbe infected, and that even here development does not proceedbeyond an early stage.

Although it appears that L. t runcatulus is the only Eng-lish mollusc which can serve as intermediate host to the liver-fluke, it is quite possible that elsewhere some other mollusc ofsimilar habits may be victimised. L. t runcatulus has a verywide geographical distribution, but so, too, has the liver-fluke;and if the latter has the wider distribution, there must of coursebe some 'Other intermediate host. Leuckart1 states that

1 'Die mensohlicheu ParasiteD,' p. 531.

LIFE HISTOBY OP THE HVEB- FLUKE. 107

Fasciola hepat ica is reported from Australia, and a similarassertion has been published in the ' Veterinarian.' Accordingto Huttonl and Wallace/ the genus Limnseus does not existin Australia. Hence if both these statements are correct theremust be another intermediate host. The liver-fluke is. alsofound.in North America, where the genus Limnseus occursindeed, but not the species L. t runca tu lus . Sheep-rot isalso found in the Shetland Islands, where, according to Forbes,3

the genus Limnseus is represented by the species L. pereger;alonei It is, however, quite possible that in the last case, L.t runcatulus has been overlooked on account of its minutesize.

III . LIFE-HISTORY,

, A. Fi rs t generat ion—1. Egg.—The eggs of the liver-fluke occur in very large'numbers in the contents of the bile*ducts and gall-bladder of the infected animal. They give a darkbrown colour and sandy appearance to the bile, and in some ofthe smaller terminal ducts often form a stiff brown mass, com-pletely plugging up the lumen. They pass with the bile intothe intestines, and may be found abundantly in the droppiugsof animals suffering from liver-rot.

The egg is an oval body, with a smooth, transparent,yellowish-brown chitinous shell. The average size may besaid to be 0'13 nim. in length by 0'08 mm. in .breadth, butthe dimensions vary greatly, the length from 0105 to 0-145mm., and the breadth from 0066 to 009 mm. The ante-rior end is a little more rounded than the posterior, and aslightly.serrated line running around it marks off a circularsegment, forming an operculum 0-028 mm. in diameter.The opposite end is frequently a little thicker, and slightlyroughened. '

The number of eggs produced by a single fluke is

1 ' Transactions ' of the New Zealand Institute, vol. v, p. 18.2 ' Geographical Distribution of Animals,' vol. i.

. 3 ' Brit. Ass. Report,' 1859, p. 127-

108 A. P. THOMAS.

exceedingly large, and its fecundity has been underrated.In one case I obtained 7,400,000 from the gall-bladderof a sheep suffering from the rot, and, as the liver containedabout 200 flukes, this gives an average of 37,000 eggs to eachfluke. And these eggs were found in the gall-bladder alone ;the liver must have contained at least as many more, and eggshad been passed copiously by the sheep for several months.The number of eggs produced by a single fluke may be safelyestimated at several hundred thousands.

When first formed, the egg includes a single germinal cell,supplied by the germarium, and fertilised by a spermatozoon,and a considerable number of secondary yolk-cells supplied bythe vitellaria, which serve as food to the growing embryo.Segmentation of the ovum takes place during the descent ofthe egg through the oviduct, but no further development takesplace so long as the egg remains with the body of the host.

'Fig. 1 on Plate II represents a fluke egg in the condition inwhich it is found within the bile ducts of the sheep; theembryo is represented by a pale spherical mass of delicatenucleated cells, and is situated near the opercular end of theshell. It is surrounded by the secondary yolk-cells, which arefilled with refractive spherules, both large and small, so thatthe examination of the embryo is rendered very difficult.

The further development of the embryo can only take placeout of the body of the beai-er of the adult fluke and at a lowertemperature. Eggs kept in an incubator, at the temperature ofthe mammalian body, do not make any progress, whilst theeggs kept at a lower temperature complete their developmentin a few weeks. The conditions necessary for developmentare moisture and a certain moderate degree of warmth. Light-I have found to exert no influence; eggs taken from the gall-bladder and placed directly with water into au opaque vessel,develop as soon as similar eggs exposed to light but otherwisekept under the same conditions. A temperature of about 23°C. to 26° C. is most favorable, and with this degree ofwarmth the embryo is formed in about two or three weeks.At a lower temperature development. takes place much more

LIVE HISTORY OF THE LITEE-l'LDKE. 109

slowly, and with an average warmth of 16° C. occupies two orthree months. During the winter no progress is made unlessartificial heat is supplied.

All the eggs under the same conditions, however, do notproduce embryos in the same time, a certain number are hatchedout on every successive day for .some weeks or even months,and at the end of this time some of the eggs may remain inthe same condition as when just taken from the liver. Noexplanation can be discovered in the eggs themselves of thevery variable time required for the development of the embryo,but the fact is of much practical importance, for eggs scatteredover any damp ground may render it dangerous for a long period.

The granular character of the secondary yolk-cells render itvery difficult to follow in detail the growth, of the embryowhilst still within the egg, and as the matter is one of theo-retical importance only, the examination of the developmentby more elaborate methods has been postponed in favour of"matter of more practical interest. I hope, however, to havethe opportunity before long of observing the formation ofthe layers in the embryo. All that can be seen in the egg bydirect examination is as follows. The embryo increases insize, being nourished by the absorption of the secondary yolk.The outlines of the yolk-spheres become more distinct andthe granules less numerous, whilst some of them appear tocoalesce or disintegrate (fig. 2, PI. II). Within them theoutline of the embryo is visible, often showing one or moreannular constrictions. As the yolk-cells are gradually used upthe body of the embryo becomes larger and more plainlyvisible, and comes to occupy the whole length of the egg (v.fig. 3). Its surface is somewhat bossy owing to the projectionof the cells forming the outermost layer of the body. A papillaappears at the anterior end, which is always directed towardsthe opercular pole of the shell, and a little way behind aquantity of dark bro.wn pigment is produced, giving rise to adouble eye-spot, while the surface loses its bossy appearance.Wave-like peristaltic contractions pass along the body from theanterior towards the opposite end.

110 A. P. THOMAS.

• In the last stage, when the embryo is ready to emerge fromthe shell, it lies slightly curved upon itself at one side of theegg (v. fig. 4), the remainder of the space being occupied bythe fluid remains and refuse of the yolk-spheres. At theanterior end, just beneath the operculum, is' a quantity ofviscid mucus, which forms a sort of lining or cushion againstwhich the head-end of the embryo is pressed. Around thebody of the embryo may be distinguished a bright border,which is formed by the cilia covering its surface; these cilia,however, can only in exceptional cases be seen in motion beforethe animal quits the egg.

2. Free Embryo.—The embryo is now ready to come forth;its movements become more marked, and at length a vigorousextension of the body causes the operculum to fly open, as ifmoved by a spring. The cushion of mucus pours out, theembryo thrusts the fore part of its body out of the shell, thecilia begin to move instantly the water touches them, and theanimal, after a short struggle, succeeds in drawing the wholeof its body through the narrow opening of the shell, and glidesaway with ease and rapidity through the water. Althoughlight has no influence in accelerating development, the embryoitself is very sensitive to it. Thus they congregate especiallyon the light side of a vessel containing them ; and I haverepeatedly observed that, although on removing a vessel ofeggs from the darkened incubator in which they were beinghatched, not more than two or three embryos could be seen,yet after it had stood in a window for twenty minutes thewater was quite nebulous from their presence.• The form of the free-swimming embryo is an elongated cone,with rounded apex (fig. 5, Plate II) , its average length 0-13mm., its breadth at the anterior end 0-027. The broader endor base of the cone is directed forwards, and in its centre is ashort retractile head-papilla. The whole of the surface, withthe exception of the head-papilla, is covered with long cilia,which are borne by an outer layer of flattened ectoderm cells.These cells are arranged around the body in transverse rings,usually five in number, though occasionally six may be

LIFE HISTORY OF THE LIVER-FLUKE. I l l

counted; they vary in length from '025—0-35 mm.; each hasa very small nucleus, 0"003 mm. in diameter. The cilia are ofthe same length ('012 mm.) over the whole of the surface, buton the cells of the anterior ring they are more numerous, andhence more conspicuous. This first row is composed of fouror sometimes five cells, arranged round the papilla, and theseare thicker than the other cells belonging to the same layer,often forming ear-like projections at the side of the embryo,and resembling epaulets. The second ring contains five or sixcells, the next two rows, each four, as a"rule, whilst the lastring is formed by two cells only. In the last two rings thecells are of greater length than in the others. Seen in asurface view the cells of this outer layer are polygonal andsometimes hexagonal. They overlap one another at theiredges, and it is probably owing to this fact that the outlinesappear double in silver nitrate preparations. In the smallnumber of examples possessing six rows of cells, the secondand third rows are formed by smaller cells..

Beneath the ciliated cells the body wall is formed by agranular layer, the cellular nature of which is not easily madeout. In favorable preparations, however, nucleated cells canbe seen slightly projecting on the inner surface. In the outerparts of the layer are situated both transverse and .longitudinalmuscle-fibres. The longitudinal are more feebly developed thanthe transverse, and are only seen with difficulty. The doubleeye-spot belongs to this deeper layer; it has been figured ashaving a form of the sign of multiplication. This, however, isnot the case, for it is really double, and has commonly the formof two crescentic masses of dark pigment, placed with theirconvex sides turned towards each other, and in contact nearthe anterior horns. On closer examination it is seen that eacheye-spot is composed of a cell in which the pigment is arrangedat one side in a crescent, the hollow of which is filled up byrefractive material which will act as a rudimentary ]ens. Thebody wall also contains numerous yellowish refractive granules,especially just behind the eye-spots, and to it belong the twociliated funnel-shaped spaces of the excretory system. These

112 A. P. THOMAS.

are situated one on either side of the middle of the body, ineach is a large cilium carried by a nucleated cell, and usuallydirected forwards. The cilium is connected with the cell by adisc at its base, it is tongue- or flame-shaped, and is con-stantly in motion, waves passing along the cilium from thebase to the tip, and hence towards the apex of the rathernarrow infundibulum. Just behind the head-papilla is agranular mass, which reacts with staining fluids differently fromthe adjacent tissues. This, from comparison with other tre-matode embryos, would seem to be a rudimentary digestivetract. Behind, the rest of the body cavity is occupied bydelicate round nucleated cells—the germinal cells.

' The embryo is exceedingly active, and with head-papillaretracted swims swiftly and restlessly through the water, notunlike some of the, larger infusoria, though more rapidly.Sometimes it goes directly forwards; and then rotates on itslongitudinal axis, just turning a little from side to side, as ifsearching for something. At other times, by curving its body,it sweeps round in circles, or, curving itself still more strongly,spins round and round without moving from the spot. Whenthe embryo, in moving through the water, comes in contactwilh any object, it pauses for a moment, and feels about as iftrying to test its nature, and, if not satisfied, darts off hastilyagain. But if the object be a Limnseus t runcatulus it atonce begins to bore. Prof. Leuckart has said of the head-papilla of the embryo, that " it seems to 'have the function ofa tactile organ." But I have no doubt that it has the functionassigned to it in my former papers, viz. that it is a bor ing-organ.1 The papilla is ordinarily short (about '006 m. inlength), and the end is quite blunt, or may have a slightdepression in the middle. A differentiation in the tissue ofthe head-papilla is visible in the form of a delicate rod-shapedstructure, occuping the axis, certainly not distinct enough tobe called a spine, though the papilla seems to possess consider-able rigidity. It is particularly evident in preparations ofembryos killed with osmic acid and stained with picro-carmine.

1 'Roy. Agrio. Soc. Journ.,' 1881, p. 7.

LIFE HESTORY OF THE LIVER-FLUKE. 113

As soon as the embryo begins to bore the head-papillabecomes longer, conical, and pointed. The embryo spins roundon its axis, the cilia working vigorously and pressing theembryo against the surface of the snail. This pressure is in-creased by the body of the embryo being alternately drawn upand then suddenly extended. As the papilla sinks further intothe tissues of the snail, it becomes longer and longer until itmay reach five times its original length (Plate II, fig. 6), andthe tissues of the snail are forced apart, as if by a wedge,leaving a gap through which the embryo squeezes its way intothe snail.

The embryo appears to exert an instinctive choice in select-ing the host into which it enters. It is conceivable that thetissues of Limnseus t runca tu lus are softer than those ofother molluscs, and that the embryo is able to bore its wayinto the former whilst it cannot do so into the latter. Butfrom the greater eagerness which the embryo exhibits whenplaced on a slide with Limnseus t runca tu lus I do not thinkthat this is the correct explanation. Moreover, the tissues ofsuch snails as young specimens of Physa fontinalis or Lim-naeus pa lus t r i s appear to be quite as soft as those of L.t runca tu lus , and yet if a quantity of embryos are placed in avessel containing equal sized examples of the three species justnamed, it is found, on subsequent examination, that whereaseach L. t runca tu lus may contain fifty or more intruders, theother snails are quite free from them. The most probableexplanation seems to be that there is some difference iu thenature of the secretion of the surface of the body in thesesnails, which is sufficient to serve as a guide to the instinct ofthe embryos.

But, although the embryo instinctively chooses the snail inwhich its further development is possible, it does not alwaysmake an equally happy selection of the part of the snail intowhich it enters. I have found as many as a dozen embryosembedded in the substance of the foot of a L. t runca tu lus ,such a place of course is unfavorable to further development,but they may remain alive there for two or three days. Once

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114 A. P. THOMAS.

only I found a full-grown sporocyst in the foot of a snail; thesurvival of this one in so unsuitable a place was probablyowing to its having accidently forced its way into a connectivetissue space or into a venous sinus. The most natural situa-tion for the development of the embryo seems to be the pul-monary chamber, and this organ is, of course, from its positionand the thinness of its walls, most easily accessible to theembryo. Other embryos, however, may be found in the bodycavity of the snail.

The average maximum duration of the embryo's free andactive life in water is only about eight hours, though occasionallyone may live over night. During the last portion of the time itsmovements become slower, and it will then in desperation oftenendeavour to bore into any object which presents itself, eveninto its own empty egg-shell. If an embryo has not succeededin finding a host, its motion becomes gradually feebler, and atlength ceasing the body assumes an oval or elliptical shape ;the outer ciliated cells absorb water and swell up into roundvesicles, and the whole body disintegrates. In a feebly alkalinesolution of peptone I have kept them alive for three days.The cilia were not lost until the third day, though theirmotion became very sluggish; the embryos increased a little insize and remained alive even after a number of the ciliated cellswere detached.

3. Sporocyst.—Arrived within the suitable snail the embryoundergoes a metamorphosis, loses its organs of locomotion, anddegenerates into an inactive sporocyst. The outer layer of cili-ated cells is lost, whilst the embryo changes in form. Theciliated cells absorb water and appear as round or hemisphericalvesicles with the cilia standing out perpendicularly from theirsurface (fig. 7). During the metamorphosis embryos mayhave various irregular shapes, but sometimes retain a lesselongated conical form, even after they have lost the ciliatedcells. The conical form is, however, soon lost, and the embryostake an elliptical shape such as is shown in fig. 8. The eye-spots of the embryo become detached from one another andlose their crescentic form; but they, as well as the head-

LIFE HISTORY OP THR LIVER-FLUKE. 115

papilla, persist, showing the identity of the young sporocystwith the embryo of the liver-fluke. After the change in formhas taken place the length is only about '07 mm. The rudi-mentary digestive tract remains for a time, but later on is nolonger distinguishable. The growth of the various larval formsof trematodes depends very much on the temperature to whichthey are exposed. During warm summer weather it is veryrapid, and in the case of Fasciola hepat ica, the sporocystmay reach its full size before the end of a fortnight; in autumndevelopment to the same stage takes a period of double thelength. The gporocyst commonly preserves the ellipticalshape until it reaches the length of '15 mm., after this time thegrowth is most rapid in the longitudinal direction and theform becomes sac-shaped. The contents of the sporocyst areformed by a number of very clear rounded cells, some of whichare the germinal cells of the embryo or cells derived fromthem by division, others are formed by a proliferation of theepithelium lining the cavity of the sporocyst. If the sporo-cyst be contracted, these cells seem to fill up the whole of thespace, and the cells which are still attached to the body wall,and form part of its inner surface, cannot be properly distin-guished from those which are lying free. But if a sporocystbe chosen for examination which is not in a state of contrac-tion, cells of various sizes, with very large nuclei, may be seenprojecting here and there from the inner surface; sometimes ina single layer, at other times in rounded heaps, two or threecells deep. It is very difficult to follow the earliest stages inthe formation of the spores within the sporocyst, but by the timethe sporocyst has reached the size of '2 mm., there are alwaysindications that the contents are becoming arranged in separateballs of cells—the germs of the next generation.

The sporocyst continues to increase in size, and ultimatelyreaches the length of -5—#7 mm. (Plate III , fig. 10). On theouter surface is a structureless cuticle, and beneath this is thethin layer in which the external circular and internal longitu-dinal muscle-fibres are often the only structural elements whichcan be distinctly observed; but in some cases, though not in

116 A. P. THOMAS.

all, there is visible beneath the cuticle a finely granular layerin which the muscle-fibres appear to be embedded.

It appears probable that some of the most superficial cells ofthe body, or at least portions of them, are converted intomuscle-fibres, whilst others undergo more or less degeneration.These muscle-fibres are more feebly developed in the sporo-cysts than in the redise which form the next asexual genera-tion, and in accordance with this feeble development the sporo-cysts are exceedingly inert, and rarely show any movements.In the redia active movements are necessary in order that thedigestive tract, which is present, may be filled with food, andthe muscular system accordingly reaches a greater development.In the sporocyst the digestive tract is altogether rudimentary,and as no exertion is required to procure the nourishment, thedegeneration of the muscle-fibres has to some extent followedthat of the enteron. In those sporocysts, however, in whichthe power of performing active movements is useful for someother reason, the muscles may retain a high degree of develop-ment; as is, for instance, the case in the sporocysts of Cer-caria l imacis, which, as soon as their included cercarise aresufficiently matured for transference to the ultimate host, boretheir way out, through the thick integument of the slugs (Arionater and Limax cinereus), which serve as intermediate hosts,and are then left behind in the mucous track ofthe slug.

Immediately following the layer of muscle-fibres is an epi-thelium, which lines the cavity of the sporocyst, and forms thegreater part of the thickness of the body wall. It is composedof cells of very various sizes, round or polygonal in form, andcontaining large nuclei (fig. 11). The layer in most places isonly one cell deep, though adjacent cells may overlap oneanother ; but in places, and especially in the less mature sporo-cysts, it is two or three cells deep. The excretory system islodged in the body wall of the sporocyst; on each side may bedistinguished an irregular group of about half-a-dozen ciliatedinfundibula. They have the same structure as the two describedabove as being present in the embryo, and are always found inthe middle third of the length of the body. No clearly defined

LIFE HISTORY OP THE. LIVER-FLUKE. 117

or regular canals can be distinguished, but the ciliated infundi-bula appear to communicate with an extensive system ofirregular lacunae 'between the cells of the body wall.Numerous yellowish refractive granules occur in thetissues of the sporocyst. or in the body cavity; they are foundwithin the cells, but are especially numerous between them, oron their surface. They are also present in large numbers inthe lacunae, and there frequently exhibit molecular motion,thus showing that the lacunae contain a fluid of some tenuity ;occasionally a whole group of them may be seen to move enmasse, or by careful pressure on the cover-glass may be madeto travel for a short distance along the lacunar passages. Noexternal opening of this system of passages can be seen, norany communication with the body cavity be clearly proved.In the ciliated infundibula, however, there is present in thelower wall of the space and close to the base of the flame-shaped cilium, an elliptical structure, closely resembling thatwhich Fraipont has described in the ciliated infundibula ofcertain other trematodes, as an opening into the body cavity.This interpretation has more recently been called in question,but whatever the real nature of this structure may be, and itis difficult to see what else it can be, there can be no doubtof its presence in the sporocysts. The yellowish granulesdescribed appear to be excretory products formed within thecells of the sporocyst and then ejected. They are partiallysoluble in acids, leaving an organic basis.

Wagener1 found in the sporocysts of Cercaria macrocerca(the larva of Distoma cygnoides) vibratile lobules (Flim-merliippchen) or ciliated spots, which he did not describe indetail. Thiry3 described in the same sporocysts a system ofvessels, the branches of which had ciliated terminations open-ing into the body-cavity. The vessels, however, were verypale and difficult to follow, and only in one instance, where theanimal was exceptionally transparent, did he plainly see thewhole system with its branches. The ciliated ends, on the

1 'Beitrage z. Entw. d. Eingeweidewiirmer,' p. 65.2 ' Zeitschrift fur wissentscbaftliche Zoologie/ x, p. 272.

118 A. P. THOMAS.

other hand, were present in almost all fairly developed ex-amples; in form they most closely resembled the ciliated open-ings in Clepsine complanata, as figured by Leydig. Thevessel was opened on one side and expanded into a two-hornedlobe, covered on its inner surface with cilia. In short, theciliated opening was described as similar to the inner ends ofthe segmental organs of various annelids. I have never foundthe sporocysts which Thiry studied, but it seems improbablethat there should be any great difference in the structure of theciliated ends in question in the various species of sporocysts.The isolated cilia really present are very large and their motionpeculiar, so that it is not difficult to understand that any onewho had before his mind the segmental organ of the earthworm,and was not prepared to see a large isolated cilium with.fTr aninfundibulum, might take the waves passing along the ciliumfor waves travelling over a series of small cilia. There can beno doubt, therefore, that the ciliated infundibula have essen-tially the same structure in the asexual generations (sporocystsand rediee) as in the adult sexual trematodes.

Amongst the digenetic trematodes the reproduction of sporo-cysts by sporocysts takes place, either by transverse fission,which may be continued through several generations, as in thecase of Cercaria l imacis, or by the formation of sporocystswithin the parent, or both methods may occur in the samespecies (e. g. Cercaria chlorotica, &c). But the only way inwhich the sporocysts are multiplied in the case of Fasciolahepatica is by transverse division, and this is of far lessfrequent occurrence than in some other species of trematodes.There appears to be a great and invariable increase of thenurse-forms amongst the Distomidee, and in Fasciolahepatica the multiplication is effected by the production ofnumerous broods of the more highly organised redise. Fissiondoes, however, sometimes occur, and usually at an early stagein the growth of the original sporocyst. A constrictionappears about the middle of the body, and becomes deeperand deeper (fig. 9), and finally the two halves are completelysevered. One of these contains the remains of the head-

LIFE HISTORY OP THE LIVEJi FLUKB. 119

papilla and the two separated eye-spots, whilst the other is, ofcourse, without the signs of any such structures. Hence sporo-cysts can be found, which even at an early stage show no traceof head-papilla or eye-spots; and in the majority of adultsporocysts these organs have degenerated entirely.

B. 1. Development ofRedia within Sporocyst.—It hasbeen mentioned above that the germinal cells which give rise tothe redise are in part already present in the embryo, but thatthey gain an increase in their numbers by the proliferation ofcells lining the body cavity. The earliest stages in the develop-ment of the spores cannot be so well distinguished in the sporo-cyst as in the redia. The cells within a sporocyst having thelength of about '2 mm., begin to show an arrangement intorounded masses or solid morulse. One side of the morula thenbecomes flattened, and the cells here then appear to be invagi-nated, producing a gastrula, whilst the surface becomes smooth,and its outline first round and then oval. The cells forming theopposite sides of the archenteron are in contact, so that there isno archenteric cavity. Each spore may now be seen to be sur-rounded by a delicate membrane, and as it increases in size itsform becomes more nearly quadrate. At one end a number ofcells are separated to form a spherical pharynx leading into theblind digestive tract, which now extends a little beyond themiddle of the body. A little behind the pharynx, the bodyshows a slightly raised annular ridge, whilst more posteriorlytwo short blunt processes are formed. Germs, as describedabove, and in various stages of development, are found in eachmature sporocyst; there is usually one redia (or less frequentlytwo), nearly ready to leave the sporocyst, with two or threegerms of medium size, and several small ones. Owing to thevarying size and shape of the included germs, the .sporocystshave frequently a very irregular outline.

As soon as the redia is ready to issue from the sporocyst,which is usually the case by the time it has reached the lengthof "26 mm., it shows active movements, which increase instrength until at length it succeeds in rupturing the wall of thesporocyst, and as this state of contraction is continued, the

120 A. P. THOMAS.

wound produced by the forcible exit of the redia is kept closeduntil it has healed up. Meanwhile the development of theremaining germs proceeds. Many of the nurse-forms oftrematodes are known to possess a special birth opening forthe escape of the brood, and even amongst the sporocysts suclian opening is present in those possessing-a filiform shape; andI have myself observed this structure in the sporocysts ofCercaria gracilis. But in F. hepat ica no such definiteopening can be detected in the sporocyst.

%. The Free Redia.—The sporocysts of the liver-fluke arefound in the pulmonary chamber of the snail, or less abundantlyin the body cavity. But the free rediae force their way throughthe tissues of the host, and wander into the other organs, andespecially into the liver. They are usually found, with thedigestive tract quite yellow from the remains of the snail'sliver-cells, with which it is filled. In thus forcing their waythrough the tissues they necessarily inflict much injury ontheir host, so much, in fact, that comparatively few snails survivethree weeks from artificial infection, and the majority, even ofthese, die before the time when the cercariae are completelymature. Thus, in the laboratory at any rate, the fluke-diseaseis more fatal to the snail than it is subsequently to the sheep.

The redia increases in size until it may reach the lengthof 13 mm. to 1*6 mm. It has an elongated cylindricalform (PI. I l l , figs. 12, 13), and at a little distance behind thepharynx there is present an annular ridge or collar projectingfrom the surface, the use of which will be explained below.From this ring or collar the body tapers gently towards theanterior end, which is abruptly truncated, and includes in itscentre the mouth. Behind the collar the body becomes a littlenarrower, but then swells out gently again until it reaches themiddle of its length, from which point it tapers, at first almostinsensibly, and then more rapidly, the extremity being conicalwith rounded apex. At a distance from the posterior end,equal to about one fourth of the total length of the body, aresituated two short and bluntly conical processes, which serveas rudimentary feet, and are no doubt of much service insteadying the redia and preventing it from slipping backwards

LIKE HISTORY OF THE LIVER-FLUKE. 121

•whilst wandering through, the tissues of the host. They arenot situated on opposite sides of the body, but are closetogether on the same surface, and their bases may even beconnected by a low transverse ridge. They are directed out-wards and somewhat backwards, their axes being usuallyinclined at an angle equal to or rather less than a right angle.

The body-wall has a similar structure in both redia andsporocyst, so that it will only be necessary to describe thepoints in which a difference exists. The muscle-fibres are farmore strongly developed, especially in the anterior part of thebody, so that the redise show considerable activity as comparedwith the sporocyst. When the body is fully extended it mayhave a length twice as great as when in a state of contraction.If an example of the host be chosen, which has a clear andtransparent shell, and has had the greater part of its liverconsumed by the parasites, the redise may be observed perform-ing movements of elongation and contraction whilst still withinthe living snail.

In the collar or ring mentioned above the muscle-fibres arestrongly marked, and have a peculiar arrangement. The trans-verse muscle-fibres appear to lie directly under the cuticle, andare closer together at the sides of the ridge then near its mostconvex part. The longitudinal muscle-fibres, however, do notfollow the curve of the surface, but stretch across from one tothe other side of the base of the ridge. Sometimes, when thering is strongly marked, the longitudinal muscle-fibres as theypass forwards may spread out in a fan-shaped fashion beforethey are finally inserted in the cuticle (fig. 16). The extent towhich the ring projects above the rest of the surface of thebody varies very greatly according to the size and condition ofthe redia, and may be altered from time to time by the con-traction of the muscle-fibres. It is greatest in those whichshow the most active movements, least in those which are themost passive. The smaller or half-grown redias commonlyshow the greatest activity, and in one of these I have observedthe ring so enormously developed that the diameter of the bodywas almost doubled at this point. Those redise in which fully-

122 A. P. THOMAS.

developed cercarise are present are frequently very sluggish intheir behaviour, and the ring may then be relatively incon-spicuous. In very young redise the outline of the body appearsto present a slight process on each side anteriorly, and withoutthe most careful focussing it is often impossible to see thatthese are simply the optical expression of the collar, the tissuesof which are still so delicate that the ridge is flattened above,and therefore, owing to the transparence of its substance, notreadily recognised.1 The function of the collar is to maintainthe shape of the body and to produce a firm basis upon whichthe neck of the redia can be moved. I have observed a redia,whilst the whole of its body behind the ring was at rest, stretchforth its neck in such a way as to sweep a considerable area iufront, and thus be enabled to reach conveniently the tissues ofthe snail upon which it was browsing. When disturbed theneck was retracted and the pharynx drawn back close to thecollar. But although the collar has thus a supporting function,there is no thickening of the structureless cuticle in it, suchas could be termed a definite skeletal structure.

The excretory system is better marked in the redia than inthe sporocyst, and definite canals can be distinguished in thebody-wall. Sinuous longitudinal vessels, one on each side, havebeen described in the redise of several other Trematodes, and

' Dicsing (' Wien. Sitzungsberichte,' vol. xxxi, p. 248) Las described theredia of Cercaria fallax as having two short processes situated auteriorly,and two, pf thrice the length, posteriorly. De Filippi (' Memorie della RealeAccademia delle Scienze di Torino/ Ser. ii, tomo xviii, p, 207) has describedthe redia of Cercaria tuberculata as having four lateral processes, twoanteriorly and two posteriorly. I have met with a species which appears to beidentical with Cercaria tuberculata, and in the redia recognised a collar.The same writer has figured (ibid., vol. xvi, pi. i, fig. 13) in the young rediaof Cercaria coronata four processes; the two placed in front are slightlysmaller than the two posterior, but otherwise they are drawn as if exactlyalike. There can be no doubt that in all these cases the structures describedas anterior lateral processes are simply the projecting borders of the trans-parent collar, seen perhaps in the flattened redia. From comparison with thedescriptions given by these distinguished observers 1 was led in my first paper('Roy. Agricult. Soc. Journ.,' 1881, p. 19) to similarly misinterpret thecorresponding projections in the young rediaj of Pasciola hepatica.

LIFE HISTORY OF THE LIVEIi-FLUKE. 123

may also be distinguished here, though the main trunks areless distinctly visible than their ramifications, and can rarelybe followed for any great distance. Hence it is impossible todiscover whether the system of vessels opens externally. Thebranches begin with a long narrow infundibulum, in which aflame-shaped cilium is constantly working, as described in thesporocyst. The ciliated infundibula are arranged in two groupson each side of the body; the anterior group on each side liesa short distance behind the collar, the posterior close to theprocesses which serve as feet (fig. 15). The ciliated cells donot all lie at the same level beneath the surface, so that occa-sionally two of the infundibula may be seen lying across oneanother, and sometimes the cells may lie free within the body-cavity, the end of the cell opposite the cilium being connectedwith the wall by one or more processes (fig. 14).

The digestive tract is the characteristic structure of theredia, and at once differentiates it from the simple sporocyst.Quite at the anterior end of the body is the mouth surroundedby projecting folds, which may be termed the lips. The trans-verse muscle-fibres are especially well developed in the lips,and assisted by the transverse muscles of the following part ofthe body-wall, serve as a sphincter muscle in closing the orificeof the mouth. The space within the lips is very small, andleads almost directly into the pharynx, an elliptical muscularorgan by means of which the animal draws in and crushes thetissues which serve as food. Its outer surface is formed by aclearly marked limiting membrane, so that it is everywheredistinguished with readiness from the mass of ill-defined cellsin which it is embedded, and its cavity is lined by a thickenedcuticle. To the pharynx immediately succeeds the digestivesac, a blind tube of very simple structure. Its wall is com-posed of a single layer of clear nucleated cells (fig. 12), sup-ported by a basement membrane, and when it is distended thecells are flattened out till they are little more than discs, inwhich the nucleus causes a distinct swelling. The digestive sacis seldom more than -3—'4 mm. long, and may be less than this,but its length differs a good deal, not only in different indivi-

124 A. P. THOMAS.

duals, but also according to the amount of food contained init. It reaches its full size early; indeed, in a redia not halfgrown it may be as long as in a full-grown example.

The body-cavity is traversed in different directions by bridgesor trabeculse of tissue, in which cells of various shapes, some ofthem with long processes, can be distinguished. This tissue ismost abundant in the anterior part of the redia around thepharynx, and digestive tract, and here often contains fibres,probably contractile. Its amount varies very greatly in differ-ent specimens, and behind the digestive tract is sometimes alto-gether absent. At other times it is so extensively developedthat the cavity of the redia appears to be divided up into anumber of imperfect compartments, in which the germs lieloosely. De Filippi appears to have observed similar trabeculsein the redia of Cercaria coronata, of which he says,1 " L acavite du corps est traversee sans ordre par des brides."

There is always a good deal of tissue around the pharynxand beginning of the digestive tract, aud embedded in it maybe seen, in favorable specimens, a few large round cells withclear protoplasm and large nucleus; each has a process orduct (?) passing towards the angle formed by the junction ofthe digestive sac with the pharynx. They are probably glan-dular in function. At the side of the redia, a little behind thecollar, there is present a birth-opening (PI. I l l , fig. 13, v), whichpermits the exit of the brood when ready to leave the parent.Such an opening has been seen in a number of rediae of differenttypes, and probably exists in all.

The germs produced within the redia develop either intodaughter-rediae or into cercarise, and it appears to me that slightdifferences exist between the individuals giving rise to one or theother of these generations. A redia producing rediae is usuallysmaller, but its pharynx and digestive sac are larger; for ex-ample, two rediae were taken from the same snail, one producingrediae measured less than 1 mm. long, with a pharynx '117 mm.and an intestine '44! mm. in length, whereas in the slightly largerredia containing cercariae the pharynx measured "078 mm. and

1 Ibid., vol. xvi, p. 426.

LIFE HISTOEY OF THE LTVEK-FLUKE. 125

the digestive sac "24 mm. A further distinction lies in the numberof the progeny ; a mother-redia may contain from one to threewell-formed daughter-rediee with a few germs in various stagesof growth;' the highest total observed was ten. On the otherhand, in a well-grown redia producing cercarise, I have counteda total of twenty-three.

The early stages in the development of the spores is thesame, whether they are destined to become redise or cercarise.Some of them may be formed from the cells which fill the body-cavity in the very young rediae, but the majority seem to beformed in the following way:—Some of the cells lining thebody-cavity of the parent, especially those at the posterior end,are greatly enlarged, and each of these germinal cells undergoessegmentation, giving rise to a morula. Fig. 18 represents alarge number of germinal cells in the hind end of a young redia,in which no morulse were yet present. Similar cells may befound in the mature redise (fig. 13 k'), for they retain thepower of producing more spores as the older ones reach theirfull development and quit the parent. Hence we find in theadult redia germs in all the successive stages of growth. Eachmorula or germ is enclosed by a delicate membrane forming aloose envelope. The germs are usually detached from the body-wall whilst still small and lie free in the cavity of the parent,but occasionally they may remain in si tu in the body-walluntil they have attained a considerable size (fig. 13 w'). Themorula soon becomes flattened on one side (fig. 12 s), and thecells of this area are then invaginated, giving rise to a gastrula(fig. 12 m)} whilst the germ again becomes round. The oppo-site sides of the archenteron are in contact, so that there israrely any archenteric cavity, and as growth proceeds and thecells become more numerous it is no longer possible to distin-guish the cells of the endoderm, for the cells have the samesize and appearance. Nevertheless it appears to me probablethat the cells invaginated form the digestive tract, whichbecomes visible at a later period in the development, rather thanany other cells in the germ. As the germ continues to increasein size the surface becomes smooth and the outline oval.

126 A. P. THOMAS.

Further growth in size is accompanied by a change in shape,and it then becomes possible to distinguish between the germsdestined to become rediae or cercariae. The growth of theyoung redia within the redia agrees in every respect with thedevelopment of the mother-redia within the sporocyst. Thegrowth of the cercaria follows a different line.

It may be asked what determines the character of theprogeny, whether the germ shall become redia or cercaria.My observations are not sufficiently extensive to definitivelydecide the question, but it appears to me that the season ofthe year is one of the principal determining causes. Rediaeproducing rediae were only found during warm weather, in thecold months cercarise were always produced directly. Further,it is a noteworthy fact that I found at the beginning of theautumn a redia, containing a single daughter-redia in additionto numerous cercarise and their germs (fig. 13). I am inclinedto think that the redia was producing redise but that a fall oftemperature induced the formation of cercarise instead. Theexplanation suggested is the more likely to be correct, sincesuch an arrangement would be highly advantageous to thespecies.

C. 1. The developmentof the cercaria within the redia.—The earliest stages of the development have already beendescribed up to the time when the germ is an oval mass ofcells. As this continues to increase in size it assumes a moreelongated shape, whilst one end becomes rather more attenuatedthan the other. The more slender end becomes slightly con-stricted off to form the rudiment of the tail, which as yet isvery stumpy. The remainder of the germ forms the body ofthe cercaria, it becomes more depressed in shape, whilst cellsare separated at the anterior end to form an oral sucker, in themidst of which opens the mouth, and in the centre of theinferior surface to form a ventral sucker equal in size to the oral.The digestive tract is now visible as a solid mass of cells.Immediately following the oral sucker is the rounded pharyn-geal bulb. Then comes a narrow oesophagus ascending slightlytowards the dorsal surface, and at a short distance in front of

LIFE HISTOEY OP THE LIVEE.-FLUKE. 127

the ventral sucker it bifurcates to form the two limbs of theintestine, which reach, one on each side of the ventral sucker,to nearly the end of the body. The limbs of the digestivetract are solid, bein'g formed for the most part by single rowsof thick disc-shaped cells (fig. 13). The cells are finelygranular at this stage, and show out distinctly against theclear spheroidal cells which surround the limbs and produceconcave impressions on the surface. At the sides of the bodyrefractive granules begin to collect in certain of the cells, whichare destined to assist in the formation of the cyst of thecercaria, and may conveniently be termed cystogenous. At firstthe granules are few and inconspicuous, but gradually becomemore and more numerous until at length they may obscure thenuclei, and render the cells opaque. Many of the cells in thebody of the cercaria are crowded with most remarkable rod-shaped bodies closely resembling bacteria in size and shape(fig. 20). They reach the length of "006 mm., and are oftenarranged in rows side by side, whilst the long axes of nearlyall the rods in each cell have approximately the same direction.Both Wagener and De Filippi appear to have observed similarstructures in the cercaria of Amphistoma subclavatum.The former speaks of them as "rod-shaped corpuscles," andthe latter says that " their form may not inaptly be comparedto that of a shuttle or spindle, with thick walls, and truncatedat both ends. They are destined to disappear later." Thesebodies are not precisely like the narrower ones found in thecercaria of the liver-fluke, but they are probably correspond-ing structures.1

An adult redia generally contains a brood numbering abouta score; amongst these there will be one, two, or three cercarise

1 Prof. Leuckart (' Zool. Anz.,' Oct. 9th, 1882) has also found these curiousbodies (which had already been described by me m the ' Jouro. Roy. Agric.Soc.,' for April, 1881), and as he was unable to find any spines on the cuticleof his cercarise, he suggests that the rod-like bodies are subsequently arrangedin bundles to form the spines of the adult fluke. But I have found thespines in the most mature cercarise, and can say that these rod-shaped bodieshave no connection with them, though I am unable to suggest any probableexplanation as to their nature.

128 A. P. THOMAS.

approaching complete development. On one occasion I countedas many as six.

2. Free Cercaria.—As soon as the cercaria has reachedthe limit of development within the redia, it escapes from theparent by the birth-opening (fig. 13, v r) and then by the aid ofsuckers and tail, crawls or wriggles its way out of the host.The free cercaria is very active, and its tissues so contractilethat the form and dimensions of the body are constantlychanging. When in a relatively quiescent condition, the bodyhas a depressed oval form (Plate III , fig. 19), its average sizeis "28 mm. long and #23 mm. broad, though the largest maymeasure over "3 mm. in length. The tail is more than doublethe length of the body, and is exceedingly contractile. Theoral sucker is subterminal, the opening of the mouth beingdirected downwards and forwards, and has a diameter of '06mm.; the pharynx is •034 mm. in diameter. The ventral suckeris situated slightly behind the centre of the ventral surface, andis equal in size to the oral, or is sometimes a little larger. Asis the case with all the cercarise produced in redise (with thepartial exception of Disto ma Paludinse impurse armatum)the cercaria has no head spine. In the most mature specimens,and especially in such as have left the redia in the naturalcourse, and have not been disturbed by the dissection of theirhost, the surface of the body is beset anteriorly with exceedinglyminute spines. But the most striking character is due to thepresence of the cystogenous cells, large nucleated cells socrowded with coarse, highly refractive granules as to berendered quite opaque. They are arranged in two lobed massesextending along each side of the body (Plate III , figs. 19 and 21),from the level of the pharynx to the posterior end of the body.Just in front of the ventral sucker is another group of thesecells, which is often large enough to connect the two lateralmasses, and behind the ventral sucker others are scattered.Cells of the same kind, and showing a similar arrangement, arefound in Cercaria tubercula ta (inhabiting Bythin iatentaculata), a species which shows at first sight a remarkableresemblance to the cercaria of Fasciola hepatica. I have.

LIFE HISTORY OF THE LIVEE-FLUKE. 129

however, myself met with C. tuberculata, and from com-parative measurements, as well as the difference in the host, canstate with confidence that the species are quite distinct. Andeven in an armed cercaria, recently found in Liinnseuspereger, I found cells, showing a similar arrangement, dis-tinguished from the remaining cells of the body, not, indeed,by coarsely granular contents, but by the possession of a pro-toplasm of a finely granular nature. In this case also the moregranular cells are probably cystogenous.

The other organs of the body are much obscured by thepresence of the opaque cystogenous cells, but the contractilevesicle of the excretory system, together with the principallateral vessels, one on each side, which contain small highlyrefractive concretions, can be made out.

3. The Cyst.-«-When the snails infested with the larvalforms of Fasciola hepatica are kept in an aquarium, thecercariae may occasionally be found swimming about in thewater, for the granular cells which render the body nearlyopaque when viewed under the microscope by transmitted light,give it a snow-white appearance by reflected light, and it isthus rendered conspicuous for its size. The life as a free-swimming animal, however, never seems to last long, for, oncoming in contact with the side of the aquarium or the water-plants contained in it, the cercaria proceeds to encyst itself.Numbers of minute snow-white cysts may thus be seen adher-ing to the walls of the aquarium or to the dark-green leaves ofthe water-plants. The way in which the cyst is formed can bereadily observed under the microscope, for when examined onthe glass slide the cercaria soon comes to rest, and assumes arounded form, whilst a mucous substance is poured forth allover the body, together with the granules forming the contentsof the cystogenous cells already mentioned. The tail issometimes shaken off before the encystation begins, but, as arule, the tail remains in connection with the body during theprocess, and continues to be energetically lashed from side toside, until finally a more vigorous movement detaches it. Thewhole process of forming the cyst is very rapid, and in a few

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130 A. P. THOMAS.

minutes a layer of considerable thickness is formed, whilst itssubstance begins to harden. The cysts, as already remarked,are snowy-white, but the body of the included Fasciola isquite transparent.

The habits of the intermediate host (Limnaeus trunca-tulus) are of much importance, as showing the manner inwhich the cysts are distributed in places where they are likelyto be picked up by some herbivorous mammal, within whichthey can attain the adult state. Limnseus t runcatulusbelongs to the group of fresh-water Pulmonata; it is a commonsnail, but one which is often very difficult to find on accountof its small size and peculiar habits. It has a very widegeographical distribution, being found, according to Dr. GwynJeffreys, throughout Europe, in North Asia, Morocco, Algeria,Madeira, and (doubtfully) in Guatemala. Several species be-longing to the genus Limnaeus occasionally crawl for shortdistances out of the water, but in. L. t runcatulus this habitis so much more strongly developed that the snail should betermed amphibious. Indeed, it is oftener found out of thewater than in it. When kept iu an aquarium it quits thewater, and as often as it is put back crawls forth again so longas the necessary strength remains. It is said to breed onthe mud at the sides of ditches. To show how much it livesout of the water I may briefly relate my own experience.There were floods on the Isis in July last, and the watersbrought it down in vast multitudes, probably from its breedinghaunts in marshy places far up the river. It was extremelyabundant, and a single sweep of a small hand-net repeatedlygave me more than 500 examples, and this was in a ditch wherepreviously I could not obtain a single L. truncatulus.

All along the margins of the ditches the ground was coveredby them, and they were found in numbers on the flooded groundwhen the flood waters had retired. On returning a monthlater to the same ditches I was unable to find a single examplealive in the water. There had been dry weather since the flood,but early that morning heavy rain had fallen, and I foundnumbers of specimens of L. t runcatulus out on the gravel of

LIFE HISTORY OP THE LIVER-FLUKE. 131

a path near the ditch, and these seemed to have crawled outof the grass when revived by the rain. At the roots of thegrass, along the margin of the ditch, others were found inabundance. Some few shells were quite empty, but the ma-jority contained the dried remains of the snail, which hadshrunk far back into the spire of the shell. Most of theseappeared to be quite dead, but were, however, merely dormant,for on placing them in water the tissues imbibed moistureand assumed their normal bulk, and after a few hours thesnails had regained their full activity, and were seemingly nonethe worse for their prolonged desiccation. To test their powerof resisting drought I collected specimens of L. t runcatulusand placed them in an open vessel on a shelf in a dry labora-tory, iu a position where the sunshine fell on them for an houror so daily. I found that rather more than 50 per cent, with-stood twenty-six days of this treatment, and some few revivedafter more than six weeks. That the snails can live on moistground quite away from any quantity of water for considerableperiods, is sufficiently proved by the fact that I have kept themalive for eleven weeks on moist grass and moss, even wheninfested with Fasciola hepatica.1

It is clear, therefore, that the species of snail under con-sideration, when left on the fields by the passing away of aflood, continues to wander and feed so long as the bottom ofthe grass remains moist. It is equally clear that the numbersso left are recruited from surrounding ditches and streams. Adrought may render the snail dormant, but, unless too longcontinued, it revives at the first shower of rain. If thereare fluke-eggs on the ground and water in puddles or ditchesfor them to develop in, the L. t runcatulus will most certainly

1 Sir Charles Lyell ('Life,' vol. ii, p. 212), in speaking of Madeira, saysthat Limnaeus truncatulus was unintentionally introduced by the Por-tuguese thirty years before, and has spread so widely that it is now found evenin the pools and ruts in the roads, so that it must have a mode of distributionwhich needs investigation. It will be seen from the above account that theterrestrial habits of this snail, and its power of withstanding drought, areamply sufficient to explain its spread in Madeira.

132 A. P. THOMAS.

be infected with the larval forms of the liver-fluke; and owingto the habit this particular snail has of living so much out ofwater, either on the banks of ditches or further away towardsthe centre of the fields, if they are damp enough, the cercarisewill, on leaving their host, encyst on the grass in the places wherethey have the best chance of being transferred to the herbivorousmammals grazing on the ground. Having thus gained a suit-able home they will attain the mature sexual condition, andreproduce their species by means of ova, thus completing thedevelopmental cycle.

Man himself sometimes serves as host to the liver-fluke, andin this case the cysts are probably eaten with water-cress.

4. Growth of Sexual Fluke.—From observations,1 whichI need not describe here, it appears probable that six weekselapse from the time of the entrance into the ultimate hostbefore the fluke begins to produce eggs. During growth thebody undergoes a very great change in form ; the posterior part,which contains the reproductive organs, far outstrips the ante-rior part (figs. 24—26). The ventral sucker shares in somedegree the greater growth of the hinder portion of the body;in the cercaria the suckers are of nearly equal size, and thesame was the case in a young fluke 11 mm. in length. Butin specimens 2—3 mm. long, the diameters of the oral andventral suckers have usually the ratio of 1: l ' l , and in stilllarger examples 6—8 mm. long, the ratio is 1 : 1-2, whilst inthe adult the ratio is 1: 1*35, though there is much individualvariety.

The smallest fluke I have yet found in the liver of a sheep isrepresented in fig. 23; the digestive tract, which in the cercariawas simply forked, has already acquired a large number ofbranches, though they are comparatively simple as yet. Theysubsequently attain a much more complex form, owing to thenumber of secondary branches. This branched intestine ishighly characteristic, and affords the principal reason for sepa-rating the three species which constitute the genus Fasciolafrom the species forming the distinct genus Distoma, none of

1 ' Journ. R. A. S.,1 1881, p. 25.

LIFE HISTORY OF THE LIVER-FLUKE. 133

which have a branched digestive tract. I t is usually supposedthat the liver-fluke passes out of the sheep at the beginning ofthe summer, i.e., life lasts only about three quarters of a year.But I have shown elsewhere1 that the life of the liver-flukemay extend beyond one year, and have found both digestive andreproductive organ in full functional vigour in flukes at leastthirteen months old; the oviduct was filled with eggs, andthere was no indication of any exhaustion of the supply.

For an account of the economic aspects of the subject, in-cluding the discussion of preventive measures, I may refer to apaper in the forthcoming number of the ' Journal of the RoyalAgricultural Society.'

It gives me much pleasure to take this opportunity of thank-ing Dr. Acland for kindly permitting me to use the SanitaryLaboratory of the Oxford Museum for my experiments, andProfessor Moseley for kindly placing apparatus, &c, in theAnatomical Department at my disposal.

1 Ibid., p. 26.