STRUCTURE AND LIFE-HISTORY OF CRITHIBIA MELOPHAGIA. 189

IThe Structure and Life-History of Crithidia

melophagia (Flu), an Endo-parasite of theSheep-Ked, Melophagus ovinus.

ByAnnie Porter, B.Sc.L.ond.,

Zoological Reseai-cli Laboratory, University College, London.

With Plates 12 and 13 and 15 Text-figures.

CONTENTS.PAGE

Int roduct ion . . . . . 1 9 0

Material and Methods . . . . 190Dist r ibut ion of Paras i tes in the H o s t . . 192

Movements . . . . . 194Morphology: Pre-flagellate S tage . . . 197

Flagel late Stage . . . 1 9 7Post-flagellate Stage in t he R e c t u m of

the Hos t . . . 2 0 0Longi tudinal Division . . . . 202Hered i ta ry Infect ion of M e l o p h a g u s o v i n u s by C .

n i e l o p h a g i a . . . . 204Casual Infec t ion . . . . . 2 0 7

Environmenta l Effects . . . . 208General Remarks . . . . 210

Summary . . . . . 211Appendix I .—On the Occurrence of a Spirochiste (S.

m e l o p h a g i , n. sp.) in M e l o p h a g u s o v i n u s . 213Appendix I I .—Note on a F u n g u s found in the Mal-

pighian Tubules and In tes t ine of M e l o p h a g u s

o v i n u s . . . . . 214Appendix I I I . — O n the Occurrence of an Anti-coagulin

in the Al imentary Canal of M e l o p h a g u s o v i n u s ,and i t s Significance in Rela t ion t o C r i t h i d i am e l o p h a g i a . . . . 216

References to Li te ra ture . . . . 218Explanat ion of P la tes . . . . 220

VOL. 5 5 , PART 2. NEW SERIES. 1 3

190 ANNIE POUTER.

INTRODUCTION.

THE part played by insects as agents in the transmission ofthe pathogenic organisms of sleeping sickness and other pro-tozoal diseases gives great importance to the investigation ofthe parasites found within them. It is necessary for any-one seeking developmental stages of pathogenic flagellateProtozoa to have also a first-hand working knowledge o£ thepossible flagellates that may be purely parasites of the insectinvolved, for certain stages of insect flagellates may resemblepossible developmental phases of such organisms as Trypano-somes. Much useful information regarding stages of flagel-lates can be gained from the study of such a parasite asCr i t h id i a melophagia (Flu), occurring in the alimentarytract, ovaries, and ova of the sheep-"ked," Melophagusovinus. This insect, which is blood-sucking, is also knownas the sheep-" t ick" or sheep-"louse." It. belongs really tothe Diptera (Hippoboscidse), possessing extremely reducedwings.

Cr i th id ia me lophag ia (Flu) was recorded by E. Pfeifferin 1905, but not named by him. The parasite is of peculiarinterest, for I am able to bring forward evidence of a doublemode of infection, both hereditary and casual. Swingle (1909)studied the flagellate stages and binefly described infection inthe egg of Melophagus . Flu (1908) found parasites in thegut, ovaries, and larva, but was not clear as to the mode ofinfection (see p. 211).

Owing to conditions of environment it was impossible toconduct the whole of this investigation in a large city. Con-sequently the work has entailed travelling, and I have tothank many friends in agricultural centres for their kindlyhelp.

MATERIAL AND METHODS.

Many specimens of Melophagus ovinus were examined3uring a long period of investigation, but owing to theaffective operation of the dip laws in England there was

STRUCTURE AND LIFE-HISTORY OF CRITH1DIA MELOPHAGIA. 191

much difficulty in obtaining the " keds."J Indeed, it seemsprobable that the sheep-ked may soon become almost extinctin England. Those obtained came in very small numbers frommany localities in the south of England, namely, Sussex,Hampshire, Kent, Middlesex, and Gloucestershire. I alsoreceived a number of keds from different parts of Scotland,but these never contained the Or i th id ia .

Many of the Melophagus , however, were infected by afungus (see Appendix II). Where fungus was presentCr i th id ia very rarely occurred. I shall show later, fromexperimental evidence, that this fungus was fatal to theCr i th id ia (p. 210).

Unlike Swingle (1909), who found that practically everyMelophagus he examined in Nebraska was infected withCr i th id ia , I found that this was very far from being thecase. Much depended on the locality from which the Melo-p hag us was obtained. The more heavily infected individualscame from the southern districts of England. Often entirestocks of keds from one locality proved to be uninfected.Again, it was impossible to keep keds alive more than threedays after their removal from the sheep.

Both young and adult Melophagus and many puparia inall stages of development were carefully examined. Eaisingpupai'ia naturally upon a sheep was tried, but was not an easymatter, and as one could not be sure of having infected keds,there was always a percentage of uninfected puparia.

For observations of the living organism two methods of pro-cedure were followed. The alimentary canal was isolated anddivided into oesophageal, crop, stomach, intestinal and rectalportions, which were separated one from another. Thesewere either teased with needles, mounted in 0'75 per cent,salt solution, and covered, the cover-slip being carefullyvaselined, or the contents of the isolated portions of the gutwere expelled by gentle pressure, and these only wereexamined, being mounted as before. Alkaline methylene

1 In this paper I shall frequently use the term " ked " to denoteMelophagus ovinus.

192 ANNIE PORTER.

blue and neutral red were occasionally used as in t ra-vi tamstains and were sometimes useful.

For fresh preparations used in work on hereditai'y infection,the ovaries and gut were dissected out very carefully, keptas far as possible relatively in situ, and mounted in 0-75 percent. NaCl solution. The behaviour of the Cr i th id ia visiblethrough the walls of the gut and their action when theypassed out from it were then most carefully watched.

I have attached very great importance to the study of theliving organism in all its phases.

For making permanent preparations the alimentary tractof the Dipteran host was carefully removed and divided intoportions as before. These isolated portions were usuallyteased very finely and fixed wet. Formalin vapour and osmica.cid vapour were chiefly used for instantaneous fixation of thehanging-drop preparations, which were then spread. Thepreparations were subsequently treated with methyl or ethylalcohol. Corrosive-acetic-alcohol (Schaudinn's fluid) andBourn's fluid (slightly modified and containing a little alcohol)were also used for fixation.

Various stains were employed. Giemsa's stain gave somepretty results; thionin acted rapidly and well; iron-hsema-toxylin, carefully differentiated with iron-alum, was veryserviceable; while gentian violet and Delafield's hsematoxylinwere of great use, particularly in obtaining details of themembrane and flagellum.

In the investigation of Cr i th id i a tnelophagia, as in allother flagellates on which I have worked, I found that pre-parations mounted in neutral Canada balsam were superiorto dry films or to films mounted in any other media.

Preparations of ovaries, eggs, and puparia were treatedsimilarly. Special methods adopted are detailed in the sectiondealing with hereditary infection (p. 204).

DISTRIBUTION OP THE PARASITE IN THE HOST.

The Cr i th id i a parasitic in the alimentary canal of Melo-

phagus are often mixed with the blood obtained by the ked

STRUCTURE AND LIFE-HISTORY OF CRITHIDIA MELOPHAGIA. 193

from the sheep. This blood from the sheep in the oesophagus,crop, and anterior part of the stomach of Melophagus isalways fluid, and of an extremely bright red colour. That inthe remaining part of the stomach is duller red but fluid, andin the intestine the blood, now semi-digested, is alwaysdarker in hue, sometimes brownish or greenish, while in theextreme rectum it is black. The enhanced red colour in theanterior portions of the alimentary canal has been shownexperimentally to be associated apparently with the presenceof an anti-coagulin in the digestive tract of the sheep-ked(see Appendix III).

Crithidia can be found throughout the length of thealimentary canal of Melophagus ovinus. In the anteriorparts of the canal they are small, rounded, non-flagellatedforms, which, when they come in contact with the blood,rapidly develop and divide, the products of division becomingthe typical flagellates found throughout the i-est of the canal.The parasites, after this rapid development, pass backwardstowards the partly digested blood, which would appear to bea medium more suited to their requirements. In the posteriorthird of the stomach there are large numbers of youngflagellates which form great-aggregation rosettes (PI. 12,fig. 43) and clumps, while true division rosettes are alsopresent (PI. 12, fig. 56).

In the intestine the same holds good. When manyCrithidia are present in a ked, they usually swarm in thefore-part of the intestine. Repeated division occurs in theintestine, so that small flagellates are found in the rectum.Most of these attach themselves to the gut-wall or to debrisand encyst, the resting (post-flagellate) stage of the parasitethen being found on the walls of the rectum and in thefaeces.

The ovaries and ova serve as places in which a kind ofpost-flagellate development occurs, the ova being penetratedby flagellate forms of Crithidia, which rapidly lose theirflagella and ultimately round themselves off, and pass througha resting stage (PI. 13, tigs. 57-94).

194 ANNIE POBTEK.

The Malpighian tubules of Melophagus ovinus avesometimes invaded by Cr i th id ia melophagia , but this isnot common.

Parasites were more numerous in female than in malekeds.Repeated investigation of sheep's blood failed to show the

presence of any flagellate therein. Flu and Swingle obtainedsimilar results. C. melophagia is, then, purely a parasiteof Melophagus ovinns.

MOVEMENTS.

The movements of C. melophagia are very vigorous.The parasites are even more nctive than C. g e r r i d i s (seePorter [1909], p. 352). As in C. ge r r id i s , the membranetakes an important share in locomotion, but the movementsof the body of C. melophagia are not so noticeable as inthe parasite of the water-bug.

When C. melophag ia was examined under the waterimmersion (2-5 mm.) objective, the movements of the lessactive organisms could be analysed. In progression theorganism moves with its flagellum foremost, and the latterexecutes vigorous, slightly spiral, boring movements. Thebody also aids in progression, for waves pass from theposterior end towards the flagellum, causing a series ofperistaltic-like swellings. The body of the parasite seems tobecome shorter during this period, and then by relaxing tomove forwards. The bead-like swellings due to undulatorymovements are more noticeable in certain areas, and in theliving organism myonemes could be sometimes seen both onthe body and in the membrane in these regions. Flu hasalso figured myonemes on some of the pai'asites he drew, andobservation of them in life confirms his work, but it was withthe greatest difficulty that I could find myonemes in stainedspecimens (PI. 12, figs. 17, 18, 40, 42, 45).

The body of C. melophagia , compared with that of C.ger r id i s , is relatively rigid, but slight twisting movements dooccur. The previous workers on C. me lophag ia are agreed

STRUCTURE AND L1EE-HIST0RY OE C1HTHIDIA MELOPEAGiA. 195

as to this rigidity. The anterior end, to which the flagellumand undulating membrane is attached, is naturally moreflexible than the posterior end, and its movements are moremarked.

Movements of contraction of the posterior end of the body

.of 0 . nielophagia result in a temporary concentration of theprotoplasm around the nucleus of the organism. The bodythen resembles a short, thick pear, drawn out at its anterior

end into a long, narrow stalk. Sometimes the body remains

in this condition, which is fairly common in forms about toencyst, aud in snch forms withdrawal or degeneration of theflagellum, followed by the secretion of a thin gelatinous wall,

completes the encystment. In other parasites from thestomach, where no encystment occurs, this concentration ofthe protoplasm in the nuclear region is not so marked, andwhen relaxation occurs the organism is propelled forward witha very slight jerk, and repetition of the contraction follows,

as has been before described. The jerking is never so

marked as in Herpe tomonas , for the membrane has theeffect of producing smoothness of motion.

Reversal of the direction of motion occurs and is veryrapid. The flagellum swings out, describing a semi-circle, of

which the body acts as the diameter for an instant, but the

force of the movement of the flagellum is so great that the

body also swings outwards in a line with the flagellum, and

the organism moves away, not exactly in the same course as

before, but in one at a very small angle to it. The path of

the organism is frequently parabolic in nature.Many peculiar movements can be observed when C. rnelo-

phag ia is endeavouring to free itself from deb r i s in the

lumen of the gut. Much writhing, both of the flagellum and

body of such a parasite, is then seen, and the organism often

swings round and round, the point of attachment serving as

the centre of rotation. If the posterior end should be

attached, the flagellum executes violent lashings and spiral

movements, these latter not being, as a rule, very noticeable

in the normal organism.

196 ANNIE POKl'EK.

Occasionally I have seen the flagellum and membrane ofspecimens of C. melophag ia torn away from the body, andfor a few seconds after, the flagellum executed intermittentflickers or lashing movements before it finally became still.

Aggregation-rosettes (PL 12, figs. 41, 43; PI. 13, figs. 95,96) are common in C. melophagia . Rosettes seem to movefairly as a whole, and I have watched them rotate ratherquickly. Each individual of such a rosette is attached byits flagellum to debris, usually epithelial in nature, andmoves up and down in a slightly inclined plane.

In division the movements of the daughter organisms arevery noticeable. I will defer the description of their motionuntil division is discussed.

During encystment in the rectum of the host, which occurswith some of the parasites, movement of the nucleus towardsthe flagellar end oh the organism occurred. I have also seenthe migration of the nucleus from the mid-region of the bodyto near the flagellum during periods of violent movement ofthe latter organella. I have never seen migration of theblepharoplast in liviag organisms under similar conditions,though it may occur at times, since blepharoplasts cau occa-sionally be found in the post-nuclear region (PI. 12, figs. 40,42), as well as by the side of the nucleus (PI. 12, fig. 33) indifferent stained specimens. By far the commonest positionfor the blepharoplast is the pre-nuclear one. The othermovements occurring during encystment will be described inthe section of the paper dealing with that subject (see p. 200and text-figures 1-10).

The life-cycle of Cr i th id i a me lophag ia may be con-veniently divided into three stages, which gradually mergeinto one another. They are—the pre-flagellate, flagellate, andpost-flagellate stages. The morphology of these forms maynow be described.

STRUCTURE AND LIl'E-HISTORY OP CRITHIDIA MELOrHAGIA. 197

The Pre-flagellafce Stage.The early pre-flagellate stages of C. melophagia are

more or less oval or rounded bodies (PI. 12,-figs. 1-6), varyingfrom 4-5fj. to 6> long, and from 1 fi to 4'5ju broad. Theyare most abundant in the fore-gut of young Melopliagus,but the pre-flagellate stage is passed through with greatrapidity and is easily missed. This probably accounts for thevery brief references to these small forms by Flu and Swingle.

The protoplasm of the pre-flagellate forms is very finelygranular (PI. 12, figs. 1-5). The nucleus is usually roundand not quite central in position (PL 12, figs. 1, 9-12). Thebar-like blepharoplast (kinetoiincieus) is very deeply staining,and lies either below (PI. 12, figs. 2, 10) or to one side of thenucleus (PL 12, figs. 1, 6). A chromatophile area with itschromatin in a very diffuse condition is sometimes fairlyprominent, and from this a fine thread arises, which growsoutwards, forming the flagellum (PI. 12, figs. 9, 10), andappearing to draw out the end of the body with it (PL 12,figs. 11-13), while the periplast of the body forms the mem-brane (PI. 12, figs. 14-20). The posterior end elongates atthe same time (figs. 16-18) and the flagellate form (PL 12,figs. 19, 20) is assumed. This development is in accord withthat of 0. gerr idisand C. tabani, and I have watched theseprocesses in living specimens of both C. gerr idis and C.melophagia.

Division of pre-flagellate forms can occur before the develop-ment of the nagella (PL 12, figs. 3, 4). This will be describedin the section dealing with division.

The F lage l la te Stage.The mature flagellates vary very much in size, the variation

being due to division and growth. Very large forms (PI. 12,fig;°. 44,45) may be as much as 50 /i to 75 fi long, this measure-ment including the flagellum,1 while short forms just flagel-

1 I t is almost impossible to differentiate between tlie limiting areasof the body, the membrane and the free flagellum of C. melophagia,as so much variation occurs in different specimens.

198 ANNIE PORTER.

lated (PL 12, figs. 18, 19) in the crop, or the small formsproduced by division prior to encystment (PL 12, figs. 20,21 j 99) are very, much smaller (12/.i to 20 ji long). Thebreadth of the flagellates varies from 1'5/t to 2"8ju.

The protoplasm of C. melophagia is very slightly alveolaror almost hyaline, differing therein from the more alveolarprotoplasm of C. ge r r id i s . There is no suggestion of largepermanent vacuoles or of a cyto-pharynx. Occasionally theprotoplasm is more granular at the posterior end (PL 12, figs.SO, 34) and slight alveolatiou occurs there. At the anteriorend, near the origin of the flagellum, the remains of thechromatic area, from which the flagellum arose, sometimespersist.

The nucleus (trophonucleus) of C. melophagia is oval(PL 12, figs. 21-24; or rounded (figs. 26, 30, 32) and some-what vesicular. There is a fair amount of chromutin present,which may consist of a number of very fine granules, evenlydistributed (fig. 32), or the chromatin may be concentratedinto about eight masses (fig. 44), or, as is ofteu the case, thechromatin is present in the form of bars (figs. 25-29), whichsometimes extend across the whole breadth of the nucleus(figs. 34-37), less frequently across part of its breadth(figs. 24, 42), or in an even more rare condition dots andbars occur in the nucleus of the same organism (figs. 30, 39).In certain cases the chromatin of the nucleus may be con-centrated into a central mass (fig. 23).

The nuclear membrane is fairly distinct in most of thespecimens I have examined. I think that such a membranemust be present to keep together the nuclear material duringthe migrations of the nucleus seen during life.

The b l e p h a r o p l a s t (kinetonucleus) of C. me lophag iais very evident in a stained preparation, for it colours deeplywhatever stain be employed. Like the nucleus, it can alsobe seen in life as a small bright retractile bar. In somecases it is slightly bowed or curved (PL 12, fig. 32), or oval(PL 12, fig. 34). It is dumb-bell-shaped in forms about todivide (PL 12, fig. 44). The blepharoplast, which is typically

STRUCTURE AND LIFE-HISTORY OF CRITHIDIA MELOPHAGIA. 199

vod-like, usually lies transversely across the organism (PI. 12,figs. 21-28). It is exceptional to find it in any positiou otherthan anterior to the nucleus, though on a few occasions theblepharoplast was at the posterior end of the body (PI. ]2,figs. 40, 42), but in these cases the flagellum originated in apre-nuclear position.

As a rule the blepharoplast shows no differentiation ofstructure (PI. 12, figs. 21-39), but sometimes in dividingforms, in which the blepharoplast is dumb-bell shaped, thereseems to be a concentration of chromatiu in the ends of thedumb-bell (PI. 12, figs. 40, 44, 45). A clear area (PI. 12,fig. 81) is often present around the blepharoplast.

C h r o m i d i a are present, scattered in the general proto-plasm (PL 12, figs. 25, 37, 39). They stain in the same wayas the nucleus, and less densely than the blepharoplast.The occurrence of such chromatoid granules at division(PI. 12, fig. 45) suggests that they have been given off fromthe nucleus into the general protoplasm, and exercise somecontrolling influence over the same.

The u n d u l a t i n g membrane and the flagellum.—The flagellum originates from a chromatic area in the pre-flagellate form, and is attached to the body by a narrow mem-brane (PI. 12, figs. 21-46), which is a periplastic outgrowthof the anterior end of the body. There is but one flagellumin any single, uudividing individual (PI. 12, figs. 21-39).The flagellum is thick, but gets thinner towards its free end(PI. 12, figs. 40, 45). At times it appears to show very finetransverse striations.

In stained specimens the membrane sometimes shows myo-nemes (PL 12, figs. 39, 42, 45), though, curiously enough, themyonemes were much more obvious in some of the livingspecimens that I examined. Flu described myonemes inC. melophagia , but figured the myonemes as accompanyinga central spindle. This latter feature I have never seen.

A basal g r a n u l e (blepharoplast of Minchin) is oftenpresent (PI. 12, figs. 17, 27, 33, 42, 45) between the point oforigin of the flagellum and the blepharoplast (kinetonucleus).

200 ANNIE PORTER.

The Post- f lagel la te S tage of C. melophagia in thRectum of Melophagus ovinus.

The preparation of Cr i th id ia melophagia for life outsidethe body of the host occurs in the rectum of the sheep-ked.Large numbers of small flagellates (PL 12, figs. 27-29) arepresent in the hind gut, also some forms in process of division(PI. 12, figs. 97, 98). The small forms attach themselves tothe wall of the rectum and encyst there, but encystment canbe watched when the rectal contents are expressed on to aslide and examined under the microscope. The flagellate(text-fig. 1) at first executes violent lashing movements withits flagellum, and during this motion migration of the nucleusnearer the flagellar end of the organism frequently occurs(text-fig. 2). At the same time the body of the Cr i th id iashortens and thickens (text-figs. 3, 4; PI. 13, fig. 100), wavesof contraction passing rhythmically down the body, whichgradually may become somewhat triangular (text-fig. 5; PI. 13,fig. 101). The flagellum meanwhile shortens (text-figs. 5, 6),and the organism may bend on itself (text-figs. 6, 7) duringthis period. Concentration of the protoplasm occurs, theflagellum becomes less wavy (text-fig. 7), and, little by little,it contracts nearer the body (text-figs. 8, 9; PI. 13, figs. 102—106) and is withdrawn, the parasite becoming oval (text-fig.10; PI. 13, figs. 109-112). The organism at this timebecomes surrounded by a thin layer of retractile, gelatinoussubstance, which rapidly hardens to form a closely adherentresistant cyst-wall. The oval bodies (PL 13, figs. 109-114)so produced are post-flagellate forms, which become detachedfrom the walls of the rectum, and pass out with the fasces ofthe ked, from which fasces they can be recovered. Thesecysts, which measure from 2-5yu to 5*5 ju by 1'5/x to 3/i, servefor the infection of other Melophagus ovinus.

All Cr i th id ia melophagia do not go through a post-flagellate stage in the gut of their host. Some, after passinga portion of their existence as flagellates in the gut of the ked,

STRUCTURE AND LIFE-HISTORY OF CRITHIDIA MELOPHAGIA. 201

pierce the walls of the alimentary tract and make their wayto the ovaries of the ked, where their development is con-tinued.

TEXT-FIGURES 1-10.

Encystment of Cri t l i idia melophagia in the rectum.

Text-figs. 1-5.—Parasite rounding off and flagellum disappearing.Text-figs. 6-7.—Show bending of parasite on itself.Text-figs. 8-10.—Final stages in loss of flagellum and assumption

of typical cyst form.

Swingle (1909, p. 104) has described thick-walled cysts.I have but rarely seen the thick-walled forms (PI. 13, fig. 114);most of the cysts found being thin-walled.

202 ANN! 10 POBTEE.

LONGITUDINAL DIVISION.

The longitudinal division of the living organism lias beenfrequently watched. While the movements of the dividingflagellates are noticeable, those of the smaller dividing pre-flagellates are far less marked.

When a flagellate is about to divide, the protoplasm of theposterior end concentrates somewhat in the nuclear region,and the organism appears to shorten. The protoplasmmigi-ates from the centre of the parasite towards the sides, sothat a comparatively clear area is left at the centre (PI. 12,fig. 46). The greatest change at this stage is seen in theblepharoplast and flagellum. The blepharoplast becomesslightly dumb-bell-shaped (PI. 12, figs. 44, 45) and graduallyconstricts into two (PL 12, fig. 46). The flagellurn splitsrapidly at the body end (PI. 12, fig. 46), and then, more slowly,the halves become free. The nucleus meanwhile becomesslightly indented in the median line (PL 12, fig. 46) and thengradually constricts into two, the halves migrating to theperiphery (PL 12, fig. 47). During this nuclear division thedaughter-flagella execute very vigorous lashing movements,and a constriction appears at the flagellar end of the parentorganism. A split appears at this end (PL 12, figs. 47-49),and, at the same time, vacuoles in the clear median area fuse,aud thus the extension of the split is facilitated. The daughter-organisms rapidly separate from one another, their appear-ance at times being suggestive of diverging curved calipers(PL 12, figs. 51, 52). At length the two are pi-actically in astraight line (PL 12, figs. 53-55), in which condition theyremain for a short time and then finally separate.

The division of the pre-flagellate forms is initiated by thedivision of the blepharoplast, and is followed by the divisionof the nucleus and the appearance of vacuoles. A slight splitappears at one end (PL 12, fig. 3), and the organism remainsin this condition until the flagellum of each half has partlygrown, when final separation is effected by their movements.

Sometimes repeated division of a pre-flagellate form occurs

STRUCTURE AND Lll'E-H[STORY OF CRITHIDIA MELOPHAGIA. 203

and a rosette (PI. 12, fig. 4) is produced, but tlie rapidity ofthe process of formation of flagella causes short duration ofthe rosette stage. OQ the other hand, repeated longitudinaldivision of flngellated individuals occurs, and as the individualsso produced do not separate immediately, rosettes (PI. 12,fig. 56) are fprmed. In division, the posterior ends of thedaughter-organisms are the last parts to separate. As thedaughter-forms remain in proximity and themselves proceedto divide with rapidit}', true division-rosettes are formed, inwhich the posterior ends of the organisms are central, whilethe flagella radiate out from the common centre. Suchdivision-rosettes (PL 12, fig. 56) differ from the aggregation-rosettes (PI. 12, figs. 41, 4 3 ; PL 13, figs. 95, 96) where theorganisms become attached by their flagella. The distinctionbetween the two forms of rosettes has not been shown byprevious workers on C. m e l o p h a g i a .

Longitudinal division results in the formation of bothequal and sub-equal daughter forms.

While the occurrence of equal longitudinal fission is thecommoner (PL 12, figs. 50, 54, 55), I have seen cases ofmarked inequality in the size of the daughter-pai'asites, theone being very thin and narrow, the other considerablybroader aud thicker (PL 12, figs. 51, 53). As the entireprocess of sub-equal division has been watched in livingorganisms, there is no possibility of it being mistaken foranything else. The polymorphism resultnnt on division isstrongly against the idea that there are sexual forms ofCr i th id i a , and I have never seen the slightest indicationthat there is sexual dimorphism, in C. m e l o p h a g i a , C.ger r id is , Herpe tomonas jaculum, H. muscse domes-ticae, H. culicis , and a new Herpe tomonas from Vespacrabro , all of which I have examined in the living con-dition (see Porter [1909] on C. g e r r i d i s and H. jaculum).

Division, usually twice repeated, is found to occur inparasites destined to encyst, and the resultant forms are verysmall. The first division is of the usual flagellate type(PL 13, fig. 97). The process of the second division rather

204 ANNIE PORTER.

resembles that of the pre-flagellate stages, for before it isaccomplished the flagella have almost disappeared. Some-times no flagellum is visible at all, and the parasites looklike dividing cysts. ,

On rare occasions the posterior end of a flagellate hasdivided before the anterior end (PI. 13, fig. 98).

THE HEREDTTABY INFECTION OF MELOPHAGUS OVINUS BY

CRITHIDIA MELOPHAGIA.

Casual infection of Melophagus ovinus by the ingestionof post-flagellate cysts of Cr i t h id i a melophagia is fairlyeasily observed. The development of the parasite in the eggcan only be studied with difficulty. I now wish to give afuller account than exists up to the present of the processesleading up to the birth of Melophagus infected withCrithidia melophagia.

The first point to be determined was the way in which theCrithidia reached the egg. Infected Melophagus werecarefully dissected so that no rupture of the gut was made.The ovaries also were dissected out and kept as far aspossible in the position beside the gut that they occupied inlife. Crithidia could be seen through the gut-wall movingactively about. Suddenly they concentrated in one placeand soon began to pass through the wall, their posterior(blunt) end first. They rapidly swain direct to the ovariesand penetrated them in the same way, that is, with the non-flagellar end first. The flagellum was very rarely used as aboring organ to allow of the passage of the organism.

Penetration of the ovaries of their host by the pai-asitesoccurs in other cases, e. g. C. gerridis, H. jaculum, butthe ova are apparently unattacked and the flagellates simplydegenerate. But in the case of C. melophagia the organisms(PI. 13, figs. 57, 59) make their way rapidly to the ova, towhich they cling, whether the ova are mature or immature.In some cases one Crithidia only enters the egg (PL 13,fig. 58); at other times several penetrate it at once. In

STRUCTURE AND LIFE-HISTORY OF ORITHIDIA MELOPHAGIA. 205

penetration the blunt end of the flagellate enters the eggfirst. Occasionally the flagella are cast off as the Crithidiapass into the egg arid remain on the outside.

In the case of older ova, the parasites seem to penetratethe egg at a definite spot (PI. 13, fig. 58), which probablybecomes the mouth of the embryo. Parasites invading olderembryos enter by the embryonic mouth. Like Swingle I didnot find parasites in the milk-glands or milk of Me-lo-phagus.

In investigations of the stages of C. melophagia inthe egg and puparia I found that smear preparationswere preferable to sections. Greater rapidity of manipula-tion and thinner preparations could be obtained by thismeans.• The method adopted was to prick the egg or open theyoung puparium and express the contents .on to a slide. Thecontents were at once fixed and then were allowed to flowover the slide, so that no artificial spreading was required,and therefore no mechanical distortion or tearing of theparasites could occur. The preparations so made containedmuch fatty matter. The slides were treated with ether toremove the fat, and then after washing with absolute alcoholwere staiued and mounted in the usual manner.

Once1 within the egg the parasite gradually loses its flagel-lam (PI. 13, figs. 61-63). This may be cast off entire, forflagella are found floating freely in the vitellus of eggs thathad been treated with the utmost care -in the manner pre-viously detailed. In many cases the flagellum appears tobe gradually absorbed (PL 13, figs. 64, 66). Longitudinaldivision of the flagellates in the egg may occur, thoughrarely.. The protoplasm of the Cri thidia then concentrates round

the nucleus and blepharoplast (PI. 13, figs. 64-69) and the para-site gradually becomes more or less rounded (PI. 13, figs. 70-73). Multiple division of both nucleus and blepharoplastthen occurs (PI. 13, figs. 74-77), and the daughter-blepharo-plasts appear to pass outwards towards the periphery (PI. 13,

VOL. 55, PART 2.—NEW SERIES. 14

206 ANNIE PORTER.

figs. 76, 77). A " plasmodiaP'* form (PI. 13, figs. 75, 77) isthus assumed. The protoplasm collects around the nuclei,and gradually fragmentation of the " plasmodiuin" occurs,the result being the formation of a number of small bodies,which rapidly round off, forming definite resting bodies (PI. 13,figs. 78-81). Sometimes these resting bodies remain inproximity to one another, so forming groups (PI. 13, figs. 80,81). The parasites now measure only 1*5 ft to4ju long and1 fi to 2-5 fi broad. Sometimes one chromatic mass (PI. 13,fig. 82) only can be distinguished. Often both nucleus andblepharoplasb (PI. 13, figs. 80, 81, 83, 84) are present.

As the embryo grows the rounded forms of the parasite inthe stomach (which is the chief cavity within the young Melo-phagus) also grow (PL 13, figs. 82-84). The Or i th id i athen undergo multiple division, small rosettes (PI. 13, figs.85-88), analogous to pre-flagellate rosettes, being produced.The division clusters may separate, giving rise to small, pear-shaped or ovoid individuals (PI. 13, figs. 89-94), or they mayremain as a rosette (PI. 13, fig. 88) for some time. Whetherthe C r i t h i d i a remain as groups or become isolated as ovalnon-flagellated bodies, they undergo no further developmentfor a considerable period. In fact, when the }'oung Melo-phagus is hatched, a month after extrusion of the puparium,there is Still no further differentiation in the parasite.

Freshly hatched Melophagus do not contain the fullydeveloped flagellates, but the rounded or pear-shaped pre-flagellate forms (PI. 13, figs. 92-94) and rosettes (PI. 13, fig.88) may be present. The parasites appear to lie dormant fora day or two, during which time the young insect does notappear to suck blood. Soon after the first meal of blood istaken, rapid development of the pre-flagellate forms occursand the adult flagellate form of the Cr i th id ia is quicklyassumed.

1 A plasmodium is really a multinucleate mass of protoplasmformed by f\ision of small amoebae. However, the term is sometimesused, as in describing certain Haplospor id ia , for a multinucleatemass of protoplasm formed by division.

STRUCTURE AND LIFE-HISTORY OF CfiJTHIDIA MELOPHAGIA. 207

CASUAL INFECTION.

The method of cross-infection in many species of C r i t h i d i ahas not been demonstrated, but in the cases known the casualor eontaminative method seems to prevail. The post-flagellatestages of C r i t h i d i a g e r r i d i s and C. t a b a n i are known,and the cysts of these parasites are shed in the faeces of theinsectan hosts. The crithidian cysts are swallowed by newhosts when they feed on material accidentally contaminatedby the faeces of their neighbours. The cysts then developin the alimentary tracts of the new hosts. M e l o p h a g u sovinus also becomes infected with its C r i t h i d i a by thecasual method.

When studying C. m e l o p h a g i a I have noticed that thefaeces of Me lophagus ovinus are voided near spots on thesheep from which blood has recently been sucked (particularlyis this the case at times of extrusion of puparia); that thefaeces contain crithidian, post-flagellate cysts, and sometimesactive flagellates; and that other Me lophagus , feeding atthe same spot, have thrust their proboscides into the semi-fluid faeces to reach the blood of the sheep. Ingestion ofcysts under such circumstances is easy. The ingestion offasces has been seen particularly well when a number of kedshave been kept confined to a small area of the sheep's body.

At shearing a slight injury was caused to one sheep, andthe keds seemed to collect round the small bleeding patch.Their habits were carefully observed then, and were similarto those described above. I do not agree with Swingle thatcasual infection is only a remote possibility; to my mind it isa certainty,

A modified eontaminative cross-infection is rendered pos-sible by the cannibalistic habit of M e l o p h a g u s o v i n u s .The keds have been seen to attack one another, the point ofseizure invariably being at the end of the abdomen near theanus. When a ked so attacked has been freed from itsaggressor and then dissected, I have found that the abdominalcavity was almost empty, the viscera having been devoured

208 ANNIB PORTER.

by the attacking ked. By this cannibalistic habit it ispossible for Melophagus ovinus to acquire practicallyevery stage of Or i th id ia melophagia direct, and this isprobably a subsidiary method of spreading the parasite.

ENVIRONMENTAL EFFECTS.

Cr i th id i a me lophag ia shows less response to slightchanges of environment than does C. g e r r i d i s or Her -pe tomonas j acu lum, both of which I have studied. Never-theless, under certain conditions remarkable effects havebeen produced by relatively simple means, and these may nowbe recorded.

(1) Eesponse to light.—Increased intensity of whitelight produces increased velocity of movement of Cr i th id iamelophagia .

Green light somewhat retards the movements of theorganism. This is also the case with Herpe tomonasjaculutn.

Intense light causes aggregation-rosettes of C. melo-p h a g i a to separate.

C. melophagia lives very much longer in diffuse lightthan in bright light.

(2) Eesponse to changes of tempera ture .—C. melo-p h a g i a can live at a temperatm-e just below that of theblood of the sheep, but the flagellates are killed at. a tem-perature above 40° C.

At room temperature (15° G.) the parasites will live forseveral hours.

(3) Response to change of medium.—Though theflagellates normally live surrounded by fluid blood (a discus-sion of which will be given in Appendix III), yet they canlive in other media and can resist the effects of such mediato varying degrees.

(a) Tap-wate r when added to the parasites in the gut-liquid seemed to have little effect. Though the movementsof the flagellate became slightly more active, this was possibly

8TEUCTUEE AND LIFE-HISl'OBY OF 01UTH1DIA MELOPHAGIA. 209

due to the greater space in which the parasites could move,the debr is being distributed over a greater area thanbefore.

(b) 0 '75 per cent . NaCl so lu t ion increased theactivity of the parasites.

Five parts of tap-water added to one part of 0-75 per cent.NaCl solution containing C r i t h i d i a caused the flagellatesto move more rapidly, the spiral boring movements of theflagellum becoming more exaggerated.

(c) Caust ic potash.-^-Two per cent, solution killed allthe Cr i t h id i a within a minute; 1 per cent, potash solutionkilled them in from seven to twelve minutes, but their bodieswere not dissolved, this poiuting to the chitinoid nature ofthe thin periplast or ectoplasm.

(d) Ace t ic acid.—One third, per cent, aqueous solutionhad the effect of swelling the parasites, which then died.

(e) Grape-sugar .—A most remarkable effect was thatproduced on C . me lophag iaby a solution containing a verysmall amount of grape-sugar. When this was added to th#parasites they commenced to divide very rapidly, and manydivisions occurred. To ascertain if there were a connectionbetween this division and the occurrence of sugar iu thenatural medium of the parasites, some experiments weremade. The results were as follows :

(i) Sheep-serum contains a very small amount of grape-sugar.

(ii) The liquid obtained when wool cut from the sheep wasboiled with water and then concentrated also showed tracesof sugar. There were, then, these two sources from which theked probably could obtain minute quantities of sugar. It ispossible that the traces of sugar may take a small share instimulating;division of C. melophagia , which goes on morerapidly in the stomach of the ked than elsewhere.

(/) Fresh blood (human or sheep's) added to a pre-paration of living C r i t h i d i a caused the parasites to moveaway to areas where the blood was somewhat less concentrated,where they proceeded to divide rapidly.

210 ANNIE POETJSK.

(g) D i l u t e g l y c e r i n e killed C. m e l o p h a g i a almost atonce. Vaseline had the same effect after a very short time.

(4) Effect of a p a r a s i t i c fungus of Me lophagusov inus on C. melophagia .—The presence of a fungus inM e l o p h a g u s ov inus has already been mentioned. As Ivery rarely found the fungus and C r i t h i d i a co-existing in aked, it was deemed advisable to find out any possible inter-relation of the two parasites. The Malpighian tubules of theked—often blocked with fungus—were the most heavilyinfected organs. Fungus taken from the Malpighian tubeswas crushed with a little water. The emulsion, whichprobably contained an enzyme, was added to a preparation ofactively moving C. m e l o p h a g i a . The movements of the

•flagellates slowed at once, their protoplasm became muchmore vacuolated, and the parasites appeared to burst. Afterseven to nine minutes no living C r i t h i d i a were to be seen.

The fungus-infected M e l o p h a g u s ov inus seems widelydistributed. Specimens from Scotland were practicallyalways heavily infected with it, and some keds from eachlocality tried in England also were infected. These kedsvery rarely contained C r i t h i d i a . The fungus seems tohave a pathogenic action upon the flagellate, and I believethat the co-existence of the fungus and C r i t h i d i a for longtogether is almost impossible.

GENERAL REMARKS.

Regarding the previous work done on the genus Cr i th id i a ,I have already noted most of the memoirs dealing with the •subject in my paper on O r i t h i d i a g e r r i d i s (1909). Conse-quently the remarks now appended relate especially to theflagellate of M e l o p h a g u s ov inus .

E. Pfeiffer (1905) first briefly described a flagellate asoccurring in the gut of M e l o p h a g u s ov inus . He mentionsthat L. Pfeiffer had seen and recorded the parasite in 1895.The flagellate stage only was described, and no definite namewas given to the organism, which was stated to be "like atrypanosome."

STRUCTURE AND LIFE-HISTORY OF CRITHIDIA MELOPHAGIA. 211

P. C-. Flu (1908) published an account of the flagellateunder the name of C r i t h i d i a m e l o p h a g i a . Flu stated thatlie saw parasites in the ovary of M e l o p h a g u s , and smallforms in the larva, but was unable to determine the mode ofinfection of the host.

L. D. Swingle (1909), woi'king in Nebraska, wrote adescription of the parasite, calling it C. me lophag i . Froma private communication I learn that Swingle's work wascompleted, but not published, before Flu's paper appeared,thus accounting for the specific name m e l o p h a g i (describedas new), which cannot stand. The chief value of Swingle'swork lies in the fact that he described rounded and" plasmodial" stages of the parasite as occurring in the eggof the host.

While Swingle was working in Nebraska, I was investiga-ting the parasite independently in England. It gives megreat pleasure to be able to confirm Swingle's work, and toadd many more details concerning the modes of infection ofthe parasite and its general life-history^.

SUMMARY.

(1) C r i t h i d i a m e l o p h a g i a is a flagellate occurring inthe alimentary tract, ovaries, ova, and puparia of Melo-phagus ovinus .

(2) The parasite has three stages in its existence, a pre-flagellate stage (PL 12, figs. 1-20), passed chiefly in the cropand fore-gut of the insect host, a flagellate stage (PL 12, figs.21-44), occurring chiefly in the posterior two thirds of thegut, and a post- f lagel la te stage, occurring either in therectum and faeces (PL 13, figs. 97-114) or in the ova andpupee (PL 13, figs. 57-94).

(3) The pre-flagellate stage is passed through very rapidly.These parasites are small, usually oval bodies, lfi to 4"5 fx by4"5 fi to 6 /j, vvitli round nuclei and bar-like blepharoplasts.The flagellum arises near the blepharoplast from a chroinato-phile area. Division of pre-flagellates may occur (PL 12, fig. 4).

212 ANNIE PORTBE.

(4) The flagellate forms are from 12 fx to 75 n long,, and15 ix to 2-8 fi broad (iucluding the flagellum). The generalprotoplasm is slightly alveolar. The nucleus is vesicular.The blepharoplast is well marked, rod-like, usually anteriorto the nucleus, and generally homogeneous.

Chromidia may occur as isolated granules.(5) The undulating membrane and flagellum are well

marked. There are indications of myonemes (PL 12, figs. 40,45) in some stained specimens, but the myonemes are moreevident in some living specimens. The membrane is of greatuse in securing smoothness of motion. The flagellum is longand forms a chromatic edge to the membrane. A basalgranule may occur near the root of the flagellum.

(6) The post-flagellate stage in the host's rectum (PI. 13,figs. 97-114) gives rise to resistant (resting) bodies that arepassed out as cysts with the faeces and serve to infect newhosts. The cysts measure, on the average, 4 /j. by 2'5 /u. Theflagellates divide, usually twice, and the four small formsthus produced lose their flagella, become round, and theninvested with a thin gelatinous wall.

(7) The post-flagellate stages in the ova and puparia ofMelophagus (PI. 13, figs. 57-94) serve for the hereditarytransmission of C. melophagia . The flagellates passthrough the wall of the gut near the anterior ends of theovaries, swarm towards and enter the ovaries and penetratethe ova—the posterior (aflagellar) end of the -parasite beingused in penetration. Within the ova each parasite loses icsflagellum and becomes ovoid or rounded (PJ. 13, figs. 64-73).Nuclear multiplication follows and " plasmodial" forms areproduced (PI. 13, figs. 74-77). These give rise to small,rounded bodies (PI. 13, figs, 83, 84) about 3 M by 2 M whichundergo multiple fission to form rosettes (PJ. 13, fig. 88),which give rise to the typical pre-flagellates.

(8) The young Melophagus do not show flagellates untilafter their first feed of blood, the blood stimulating the pre-flagellates to form flagella.

(9) Multiplication of 0 . melophagia by longitudinal

STRUCTURE AND LIFE-HISTORY OF CRITH1DIA MELOPHAGIA. 213

division occurs in both the pre-flagellate and the flagellatestages of the parasite.

(10) Infection of M e l o p h a g u s ov inus with C. melo-p h a g i a is either hereditary or casual. In the case of casualinfection the insects ingest the post-flagellates voided withthe faeces of other M e l o p h a g u s .

(11) A very dilute solution of grape-sugar causes theparasites to divide. There are only traces of sugar in bothsheep-serum and wool extract.

(12) Sheep's blood or human blood added to t h e C r i t h i d i aalso increased the rapidity of their growth and division.

(13) A fungus present in the Malpighian tubules and gutof the ked (see Appendix II) has a rapid, fatal effect on theCrithidia.

(14) An anti-coagulin is present in the salivary glands,stomach and intestine of Melophagus ovinus (see Appen-dix III).

(15) A new spirochaste, S. melophagi, was found inthe gut, ovaries and puparia of a few of the Melophagusexamined (see Appendix I).

APPENDIX I.

On the Occurrence of a Spirochaste, S. melophagi,n. sp., in Melophagus ovinus.

I wish to record the occurrence of a rare spirochaste in thegut, ovaries and puparia of Melophagus ovinus. Thespirochaste was observed in life in the above-mentionedorgans of a very few of the Melophagus examined, and atvery different periods of the year (February, September,October). Very few spirochastes occurred, and consequently itis impossible to give full details regarding size and structure.The organisms seen were from 10 fi to 30 ft long and werenarrow. They vary in length, some being practically halfthe length of others, indicating the probability of transversedivision. As some parasites were thicker than others there

214 ANNIE PORT Jill.

is the inference that longitudinal division takes place.This would be in accordance with the behaviour of otherspirochsetes, for Fantham (1907-8-9) has shown that bothforms of division occur in S. balbiani i and S. anodontas.I (1909) also have observed the same, while the jointresearches of Fantham and myself (1909) have demonstratedthat both directions of division occur in S. recurrenfcis andS. duttoni, and that there is a periodicity in the directionof division.

The movements of S. melophagi are fairly active, andare of the typical spirochEete nature, namely, of forwardprogression accompanied by spiral or corkscrew rotation,onits course.

The occurrence of S. melophagi in the ovaries, ova andpuparia of the ked is of much interest, for it indicates thatthe spirochtete is transmitted hereditarily. Hence Melo-phagus ovinus can transmit both Cri th idia melophagiaand Spirochreta melophagi to its offspring.

APPENDIX II.

Note on a F u n g u s found in the Malpighian Tubulesand In t e s t i ne of Melophagus ovinus.

A fungus was present in many specimens of Melophagusovinus examined, especially those obtained from Scotland.Cri thidia were not seen in the "keds" received from

• Scotland, and I have shown experimentally that the actionof the fungus is fatal to the flagellate.

The fungus occurred chiefly in the Malpighian tubules ofthe insect, and to a lesser extent in the intestine. TheMalpighian tubules were frequently blocked by the fungus..A brief description of the fungus may now be given.

The hyphse were long and filamentous with few septa.Many spores were produced. At the extremity of somehyphas globular heads were formed, possibly due to sexualprocesses. The globular bodies contained many nuclei (text-fig. 11) fairly evenly distributed through the protoplasm.

STBTTOTURE AND LIFE-HISTORY OF CRITHIDIA MELOPHA.GIA. 2 1 5

Nuclei and protoplasm then shrank away from the wall ofthe rounded body—provisionally called a sporangium (text-fig. 12)—so that a space intervened. Segregation of theprotoplasm round the nuelei followed (text-fig. 13), and amorula-like body resulted. The morula differentiated into amass of rounded spores (text-fig. 14), each of which formed aspore coat for itself. The sporangium ultimately ruptured

TEXT-FIGURES 11-15.

Fungus parasitic in Melophagus ovinus.

Text-fig. 11.—Hypha with globular head.Text-fig. 12.—Differentiation of nuclei within the head (sporan-

gium).Text-fig. 13.—Spores forming in sporangium.Text-fig. 14.—Mature sporangium.Text-fig. 15.—Dehiscing sporangium.

(text-fig. 15), and the numerous small spores were set free.Some spores remained in the Malpigliian tubes, others passedout into the intestine and were voided with the faeces.

Parasitic fungi have been previously recorded in insects,for example, in the house-fly, caterpillar, mosquito. Thefungus mentioned by Schaudinu in Culex was probably amember of the Entomophthore te , or related thereto. The

216 ANNIE P011TEK.

fungus infesting M e l o p h a g u s ovinus seems to be morenearly allied to the Peronosporeaa .

I learn from a private communication that a similar funguswas found last year by Dr. H. B. Fautharn, of Cambridge, inthe alimentary tract and Malpighiau tubes of the grouse-fly,O r n i t h o m y i a l a g o p o d i s . From examination of a pre-paration of the fungus of O r n i t h o m y i a , kindly lent to me,I believe that the fungi of the grouse-fly aud the sheep-kedare very closely related.

APPENDIX III .

On t h e Occur rence of an Ant i -coagul in in the Ali-m e n t a r y Canal of M e l o p h a g u s ovinus, and i t sSignificance in Relat ion to Cri thidia melo-phagia.

The pronounced and peculiar brightness of the blood in thecrop and fore-part of the stomach of the keds examined wasnoticed very early in the investigatiou. The blood of thesheep in the stomach of keds that had not fed for as long asthree days was still practically fluid and had not coagulatedmuch, while twelve to twenty-four hours after feeding theblood had not coagulated at all. This led me to suspect thatan anti-coagulin, such as bad been described in a tick (Argaspersicus) by Nuttalland Strickland (1908), was present herealso, and a series of tests were performed at different timeswhich verified this inference. Every test that I performedhad the same result—coagulation was delayed.

The method of testing was simple. Separate emulsions ofthe salivary glands, stomach, and intestine of Melophagusovinus were made with O75 per cent. NaCl solution. Aknown quantity—about 0*5 c.c. of human blood from a prickedfinger—was then mixed with the same quantity of organ-emulsion, while for control purposes the same quantity ofblood mixed with 075 per cent. NaCl solution was used.The test fluid and the control fluid were taken up in smallglass capillaries, and the test was applied by blowing out the

STRUCTURE AND LLFE-K[STORY OP CRITH1DU MELOPHAGIA. 217

liquid at stated times and noting when coagulation occurredin each. Typical results of these experiments are tabulatedbelow:

A. Adul t Melophagus .Coagulation period Coagulation period.

Experiment. of blood and organ- of blood and -75emulsion. NaCl solution.

(1) Salivary gland . 20 min. . 7-8 min.' (2) „ „ • 22 „ . 8 ,,

(3) Intestine . . 14 „ . 8 „(4) „ . 14 „ 30sec. . 8 „Obviously an anti-coagalin was pi'esent, for considerable

delay of clotting occurred.B. Young Melophagus.—Here the interval between the

clotting of the test and control preparations was noted. Afew typical results are given :

(1) Blood mixed with emulsion of the salivary glandsclotted nine minutes after the control.

(2) Emulsions of intestine added to blood caused the latterto take three times as long to clot as the control preparationstook.

Comparing the behaviour of the emulsions of the salivaryglands of young and of older keds, the anti-coagulin seems tobe more strongly developed iu the salivary glands of theolder keds, while a similar comparison between the intestinalemulsions would tend to show that the anti-coagulin wasmore abundant in the intestines of young keds.

The temperature at which the anti-coagulin was destroyedwas also investigated. It was found that below 50° 0. the

.anti-coagulin would act. At about 55° C. its action waschecked. When 60° C. was reached it was destroyed.

Human blood mixed with emulsions of any part of thealimentary canal at once assumed the vivid red hue so notice-able in the blood removed from the gut of the keds..

The red blood-corpuscles of the sheep, seen en masse,appear far brighter on adding emulsions of the gut of theked containing the anti-coagulin. When much water wasadded to normal blood, haemolysis occurred, and the colour

218 ANNIE PORTER.

of the solution so obtained was made much brighter when anemulsion of crushed salivary glands of the ked was added toit. The leucocytes of the sheep's blood occurring in the gutof the ked do not appear to be affected in any way by theanti-coagulin.

Anti-coagulin appears to be found in all parts of the alimen-tary canal of the ked and to decrease in amount from beforebackwards. As before mentioned, I determined experi-mentally that freshly shed, and therefore fluid, blood actedas a stimulant to division of the Cr i th id ia . This artificialcondition is the counterpart of the natural condition of theblood within the fore-gut of the ked. There, owing to theaction of the anti-coagulin, the freshly ingested sheep'sblood does not clot, but remains fluid. It is probable thatCr i th id ia within the gut are stimulated by this fluid blood,and divide rapidly. I obtained similar results in the case ofHerpe tomonas jaculum, where "division of the flagellateHerpetomonad takes place rapidly under natural conditionsafter ingestion of blood by the host" (Porter [1909], p. 382),If the Cr i th id ia are in the pre-flagellate condition the rapidmultiplication is followed by the outgrowth of flagella, afterwhich the organisms separate and pass further down thealimentary canal. The presence of anti-coagulin, from thesalivary glands, in the contents of the fore-gut of the kedma.y be the cause of the rapidity with which the pre-flagellatestage of C r i t h i d i a melophagia is passed through, theblood, kept fluid by the anti-coagulin, acting as a stimulusto further development.

REFERENCES TO LITERATURE.

Further references will be found at the ends of some of the papersquoted.

Bruce, Sir David, Hamerton, A. E., Bateman, H. B., and Mackie, F. P.(x, 1909).—"The Development of Trypanosoma gambiensein Grlossina palpalis ," 'Proc. Roy. Soc.,' ser. B, lxxxi,pp. 405-414, pis. 10, 11.

STRUCTURE AND LIFE-HISTORY OF OEITHIDIA MELOPHAGIA. 219

Fantham.H. B. (i, 1908).—" Spirochasta (Trypanosonia) ba lb ian i i(Oertes) and Spirochaata anodontee (Keysselitz): their Move-ments, Structure and Affinities," ' Quart. Joum. Micr. Sci.,' 52,pp. 1-73, 3 pis.and Porter, Annie (1909).—" The Modes of Division of Spiro-

chaeta r ecu r ren t i s and S. du t ton i as observed in the LivingOrganisms," 'Proc. Roy. Soc.,' ser. B, lxxxi, pp. 500-505.

Flu, P. C. (1908).—" Ueber die Flagellaten im Darm von Melophagusovinua," ' Archiv f. Protistenkunde,' xii, pp. 147-153, 1 pi.

Leger, L. (1902).—" Sur un flagelle parasite de l 'Anopheles maculi-pennis," ' O.B. Soc. Biol.,' liv, pp. 354-6, 10 figs.

Mackinnon, D. L. (1909).—" Note on two New Flagellate Parasites inFleas—Herpetomonas c t enoph tha lmi , n. sp., and Cri-th id ia hystricliopsylla3, n. sp.," 'Parasitology,' ii, pp. 288-296,1 pi.

Minchin, E. A. (1908).—"Investigations on the Development of Try-panosomes in Tsetse Flies and other Diptera," ' Quart. Joum.Micr. Sci.,' 52, pp. 159-260, 6 pis.

Novy, F. G., MacNeal, W. J., and Torrey, H. N. (1907).—"The Try-panosomes of Mosquitoes and other Insects," ' Joum. Infect.Diseases,' iv, pp. 223-276, 7 pis.

Nuttall, G. H. F., and Strickland, 0. (1908).—"On the Presence of anAnti-coagulin in the Salivary Glands and Intestines of Ar g u spersicus," 'Pai-asitology,' i, pp. 302-310.

Pattern, W. S. (1908).—"The Life-Cycle of a Species of Cr th id iaParasitic in the Intestinal Tract of Ger r i s fossarum Fabr.,"' Archiv f. Protistenkunde,' xii, pp. 131-146, 1 pi.

(1909).—"The Life-Cycle of a Species of C r i t h i d i a Parasiticin the Intestinal Tracts of Tabanus h i la r ius and Tabanussp.," 'Archiv f. Protistenkunde,' xv, pp. 333-362, lp l .(1909).—" A Critical Review of our Present Knowledge of the

Hsemoflagellates and Allied Forms," ' Parasitology,' ii, pp. 91-143.• and Strickland, C. (1908).—"A Critical Review of the Relationof Blood-sucking Invertebrates to the Life-Cycles of the Try-panosomes of Vertebrates, etc.," ' Parasitology,' i, pp. 322-346.

Pfeiffer, E. (1905).—"Ueber Trypanosonienahnliche Flagellaten imDarm von Melophagus ovinus," ' Zeitschr. f. Hyg.,' 1,pp. 324-29,1 pi.

Porter, Annie (1909).—" The Morphology and Life-History of Cri-t h i d i a gerr id is , as found in the British Water-Bug, Ger r i spaludum," ' Parasitology,' ii, pp. 348-366, 1 pi.

220 ANNIE POUTER.

Porter, Annie (1909).—"The Life-Cycle of Herpe tomonas jaculum(Leger), Parasitic in the Alimentary Tract of N epa cinerea,"' Parasitology,' ii, pp. 367-391, 1 pi.

Pratt, H. S. (1893).—"Beitriige znr Kenntnis der Pupiparen (DieLarvevon Melopliagus ovinus)," 'Archiv f. Naturgesch.,' liii, pp.151-200, 1 pi.

(1899).—"The Anatomy of the Female Genital Tract of thePupipara as observed in Melopliagus ovinus," ' Zeitschr. f.wiss. Zool.,' lxvi, pp. 16-42, 2 pis.

Robertson, Muriel (1909).—" Studies on Ceylon Heematozoa: I, TheLife-Cycle of Trypanosoma vittatse," ' Qtiart. Journ. Micr.Sci.,' 53, pp. 665-695, 2 pis.

Scliaudinn, F. (1904).—"Generations- nnd Wirtswechsel beiTrypano-soma und Sp i rocha t e (Vorl. Mitt.)," 'Arbeit, a. d. Kaiserl.Gesundheitsamte," xx, pp. 387-430, 20 figs.

Swingle, L. D. (1909).—"A Study on the Life-History of a Flagellate(Cri thidia melophagi , n. sp.) in the Alimentary Tract of theSheep-Tick (Melophagus ovinus)," 'Joui-n. Infect. Diseases,'vi, pp. 98-121, 3 pis.

Woodcock, H. M. (i, 1909).—" The Hsemoflagellates and Allied Forms,"article in ' Treatise on Zoology,' edited by Sir Ray Lankester,pt. i, fasc. i, sect. G., pp. 193-273.

EXPLANATION OP PLATES 12 AND 13,

Illustrating Miss Annie Porter's paper on "Crithidiamelophagia."

[All figures were outlined with an Abbe-Zeiss camera-lucida, using a2 mm. apochrornatic (Zeiss), or T \ inch achromatic (Zeiss) objective,,and compensating oculars 8 and 12 of Zeiss. The magnification is inall cases approximately 1500 diameters, except where otherwise stated.]

PLATE 12.

F i g s . 1-20.—Pre-flagellate Stages.Pig x.—Pre-flagellate with round miclens, bar-like blepharoplast.

No flagellum. Crop. Giemsa.Fig. 2.—Oval pre-flagellate. Blepharoplast slightly constricted.

Crop. Delafield's hsematoxylin.Fig. 3.—Dividing pre-flagellate. Crop. Delafield's htcmatoxylin.

STRUCTURE AND LIFE-HISTORY O.l<' C1UTH1D1A MELOPHAGIA. 221

Fig. 4.—Division i-osette of pre-flagellates. Two individuals again•dividing. Crop. Delafield's hEematoxylin.

Figs. 5-8.—Elongating pre-flagellates. Crop. Thionin.

Fig. 9.—Large preflagellate, with round nucleus, rod-like blepharo-plast, flagellum just differentiating. Crop. Gieinsa.

Fig. 10.—Rounded form. Flagellnm longer than in fig. 9. - Crop.

•Giemsa.

Fig. 11.—Smaller parasite with large nucleus and long flagellum.

•Crop. Delafield's haamatoxylin.

Fig. 12.—Parasite, showing elongation of flagellai1 (anterior) end ofthe body. Crop. Giemsa.

Figs. 13 and 14.—Crithidia with elongated posterior ends. Anteriorpart of stomach. Giemsa.

Fig. 15.—Pre-flagellate with posterior blepharoplast. Crop. Giemsa,

Fig. 16.—Parasite with anterior end more developed. Crop. Giemsa.

Figs. 17 and 18.—Almost mature flagellates, membranes showingmyonemes. Crop. Giesma.

Figs. 19 and 20.—Practically adult flagellates. Fore-part of stomach.Thionin.

F igs . 21-43.—Flagellate Stage.

Fig. 21.;—Small flagellate. Nucleus with chromatin in granules•extending part way across the body. Rod-like blepliaroplasti Intestine.Giemsa.

Fig. 22.—Flagellate, with well-marked myonemes on the body.Stomach. Gentian violet, x 2250 approximately.

Fig. 23.—Parasite, with flagellum almost continuous with, theblepharopUtst. Nucleus with central chromatin. Stomach. Delafield'shamiatoxylin.

Fig. 24.—Crithidia showing blephavoplast posterior to the nucleus—an uncommon condition. Stomach. Giemsa.

Figs. 25, 26.—Flagellates showing chromidia in their posterior ends.Chromatin of nucleus in bars. Stomach. Giemsa.

Figs. 27-29.—Parasites with somewhat pointed posterior ends.•Chromidia present in fig. 29. Intestine. Thionin.

Figs. 30, 31.—Crithidia showing somewhat alveolar protoplasm.Stomach. Thionin. x 2250 approximately.

Fig.. 32.—Flagellate with blunt posterior end, round nucleus contain-ing large chromatin granules, and extending across complete breadth•of body; blepharoplast curved. Stomach. Thionin. x 2250 approxi-mately.

VOL. 55, PAliT 2.—NEW SEIUES. 15

222 ANNIE PORTER.

Fig. 33.—Parasite with scattered «hroinidia. Blepharoplast slightlyposterior to and to the side of the nucleus. End of crop. G-iemsa.

Fig. 34.—Crithidia with large oval blepharoplast. Stomach.Giemsa.

Fig. 35.—Narrow parasite. Intestine. Giemsa.

Figs. 36, 37.—Longer parasites with many chromidia. Stomach.Iron-hsematoxylin.

Fig. 38.—Flagellate showing alveolar protoplasm, nucleus andblepharoplast almost in contact. Intestine. Thionin. x 2250 approxi-mately.

Fig. 39.—Long form. Nucleus with chromatin arranged in bars.Oval blepharoplast. Membrane distinct. Intestine. Giemsa.

Fig. 40.—Long parasite with thick flagellum. Myonemes presenton body. Blepharoplast showing constriction, so about to divide.Ohromatin of nucleus in large masses. Stomach. Delafield's ha;ma-toxylin.

Fig. 41.—Small aggregation-rosette, showing entanglement of largeand small flagellates. Stomach. Giemsa.

Fig. 42.—Flagellate with rounded nucleus and posterior blepharoplast.Basal granule near root of flagellum. Myonemes in membrane.Intestine. Iron-hsematoxylin.

Fig. 43.—Large rosette. Many parasites shown aggregated arounda piece of debris. The flagella serve as points of attachment, thereindiffering from a division-rosette. Common in stomach and intestine.Delafield's liEematoxylin.

Figs. 44-56.-—Stages in Division.

Fig. 44.—Parasite showing constricted blepharoplast with clear ai-ea.around it. Cliromatin in nucleus arranged in masses at peripheiy.Intestine. Thionin. X 2250 approximately.

Fig. 45.—Stage similar to fig. 44. Well-marked myonemes on bodyand membrane. Giemsa. x 2250 approximately.

Fig. 46.—Parasite with both nucleus and blepharoplast constricted.Flagellum beginning to split at base. Stomach. Delafield's hsematoxylin.

Fig. 47.—Flagellate with anterior end of body, nucleus and blepharo-plast all divided. Stomach. Delafield's hseniatoxylin.

Figs. 48,49.—Somewhat rounded parasites; bodies of daughter-formsnot yet diverging from one another. Stomach. Thionin.

Fig. 50.—Daughter-organisms forming a V. Stomach. Giemsa.Figs. 51, 52.—Further stages in the divergence of the bodies of the-

STRUCTURE AND LtVK-HISTORY OF CKITHIDIA MELOPHAGIA. 223

daughter-forms. The flagella have interlocked. Intestine. Delafield'shsematoxylin. The parasites represented in fig. 51 divided sub-equally.

Fig. 53.—Sub-equal division. Daughter - organisms are almostseparated. Intestine. Delafield's hsematoxylin,

Tigs. 54, 55.—Parasites about to separate. Stomach. Giemsa.

Fig. 56.—True division-rosette. The separation of the daughter-individuals takes place from the flagellar end backwards, so that ina rosette the posterior ends of the organisms are centrally directed.Stomach. Thionin.

PLATE 13.

Figs. 57-94.—Stages of the Paras i t e in the Ovary, Eggs,and Pupar ia .

(The eggs in figs. 58, 64, 65 are represented diagranimatically.)Fig. 57.—The flagellate as it penetrates the ovary. Delafield's

hEBmatoxylin.Fig. 58.—Flagellate in the act of penetrating a young egg, the blunt

end of the parasite being iised. Thionin. The egg of Melophagusovinus is represented diagranimatically.

Figs. 59, 60.—Flagellates from ovary, Flagella somewhat reduced.Giemsa.

Figs. 61-63.—Flagellates from within the egg. Giemsa.Figs. 64, 65.—Rounding-up forms of 0. meloplagia within eggs.

Delafield's hsematoxylin and fresh preparations. Eggs of Melophagusrepresented diagraniinatically.

Figs. 66-72.—Series of parasites showing successive stages in shorten-ing and rounding-up of flagellates when within the eggs. Delafield'shsematoxylin.

Figs. 73, 74.—Parasites showing nuclear division. Very youngpupai-ium. Giemsa.

Figs. 75-77.—"Plasmodial" stages of C. melophagia in developingpuparia. Peripheral blepharoplasts seen. Giemsa and fresh prepara-tions.

Figs. 78-81.—Rounded parasites resulting from plasmodial forms.Delafield's hcematoxylin.

Figs. 82-84.—Parasites produced by growth of forms similar to thoseshown in fig. 81. Giemsa.

Figs. 85-87.—Rosettes of somewhat oval parasites from youngpuparium. Delafield's hsematoxylin.

Fig. 88. — Well-defined division-rosette from mature piiparium.Giemsa.

224 ANNIE PORTEK..

Pigs. 89-91.—Dividing forms. Mature puparium. Giemsa.Figs. 92-94.—Parasites, resembling pre-flagellates produced from cyst.

Mature puparium. Delafield's haematoxylin.Figs. 95, 96.—Small aggregation-rosettes. Intestine. Thionin.

F ig s . 97-114—Post-flagellate Stages in Rec tum.Fig. 97.—Parasite dividing.prior to encystment. Intestine. Thionin.Fig. 98.—Uncommon form of division, occasionally seen in living

specimens. Rectum. Giemsa.

Fig. 99.—Small fomi. Flagellum in process of absorption. Rectum.Giemsa.

Fig. 100.—Parasite snowing concentration of protoplasm in the regionof the nucleus. Rectum. Giemsa.

Fig. 101.—Form common in rectum. Body much flattened. Flagellumdisappearing. Delafield's huematoxylin.

Figs. 102-108. — Parasites showing progressive disappearance offlagellum. Rectum. Thionin.

Figs. 109-112.—Post-flagellate cysts from rectum. Giemsa.Fig. 113.—Post-flagellate cyst from faeces of Melophagus ovinus.

Giemea.Fig. 114.—Thick-walled cyst. Rectum. Giemsa.

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