I COMPARATIVE STUDIES ON CERTAIN FEATURES OF NEMATODES AND THEIR SIGNIFICANCE BY DUNCAN CHARTERIS HETHERINGTON A. B. t Colorado College, 1919 M. A., University of Illinois, 1920 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY IN THE GRADUATE SCHOOL OF THE UNIVERSITY OF ILLINOIS, 1922 URBANA, ILLINOIS \ □ Zu Ml UNIVERSITY OF ILLINOIS THE GRADUATE SCHOOL iftU ••l < 6 =. 192 - 2 - I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY ENTITLED ^T2k / K 1 O jy Recommendation concurred in* BE ACCEPTED AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE DEGREE OF t!d’j TABLE OF CONTENTS Page I. Introduction 1 II. Some New Methods in Nematode Technique 3 (a) Fixation 3 fb) Preparation of speciments for 7 1. Clearing. 2. Totomounts. 3. Sectioning. 4. Staining. (c) Differential clearing and mounting 16 (d) Restoration of dried specimens 19 III. Examination of the Symmetry and Structure of the Head 20 20 25 27 27 30 36 36 39 44 44 44 45 49 Region fa) General considerations 1. Type form of the primitive nematode ... 2. Alternations in its bilaterality 3. Primitive orientation 4. Definition of the "Primitive Nematode . (b) Cephalic structure in free-living nematodes • 1. The symmetrical factor of the oesoph- agus 2. Pharyngeal modifications 3. Oral structure fl) The structurally simple form ... (2) The primitive form (3) Oral modifications arising from primitive structure by division and fusion of structural ele- ments (4) Modifications by loss of parts . Digitized by the Internet Archive in 2015 https://archive.org/details/comparativestudiOOheth Page 4. Considerations of symmetry in the head region 52 (1) Primitive and fundamental symmetry 52 (2) Radial symmetry 52 (3) Di-symmetry 54 (4) Asymmetry 54 (c) Cephalic structure in parasitic forms 55 1. Symmetrical factor of the oesophagus .. 55 2. Cephalic modifications and relations to habitat 56 3. Pharyngeal modifications 58 4. Oral structure and symmetry 59 (1) The simple forms 60 (2) Di-symmetrical forms derived from three-lipped forms 60 (3) Other di-symmetrical forms simulating jaws 62 (4) Variations arising from number of lips and capsule 64 IV. Ciliation among Nematodes 70 (a) Former views and present data 70 (b) Significance of Ciliation 74 V. Discussion and Conclusions 76 VI. list of Species Mentioned 80 fa) Free-living forms 80 fb) Parasitic forms 82 VII. Bibliography 86 VIII. Vita 100 IX. Explanation of Plates 101 ; ... - 1 - I. INTRODUCTION . The members of the phylum Nematoda both the parasitic and free living forms are exceptionally interesting in view of the varieties of structure existing in the cephalic region and also in view of the changes of structural symmetry from the funda- mental bilaterality to pseudo-radial symmetry, true radial sym- metry, and asymmetry. In the following pages the author has directed his atten- tion to a comparative study of the symmetry and structural var- iety of the cephalic regions existing among the nematodes, para- sitic and free living, endeavoring throughout to determine the most primitive cephalic plan from which the most complicated forms were derived and the order of this derivation. In addi- tion, some new and valuable methods of nematode technique have been devised to lessen the tedium and numerous difficulties involved in preparing this material for microscopical examination either as sections or toto-mounts. Furthermore, the question of ciliation among nematodes is discussed and evidence presented for the undoubted existence of such structures. The comparative study was undertaken at the suggestion of Dr. Henry B. Ward, to whom the author at this time wishes to express his sincere thanks, not only for his interest in this - - , ' - 2 - study,but also for his kindness in permitting the author to use his private laboratory files on nematode literature , and for access to and use of his large collection of unnamed material. Further thanks are due to Dr. H. J. VanG leave for reading and criticising the section on technique, and to Dr. R. Kudo for the loan of reprints on ciliation by Prenant and also for an original preparation of that author of modified cilia from the intestine of Ascaris megalocephala. i i t 1 - 3 - II. SOME NEW METHODS I N NEMATODE TECHN I Q.UE __ (ji) Fixatio n The difficulties involved in the preparation of nema- todes for microscopical examination either in toto or sections can be appreciated fully only by those who have made any attempts whatsoever in that direction. Without doubt this fact has been one largely contributing to the scarcity of workers in the field of nematology as compared with other branches of zoology, and has at the same time been responsible in great measure for the confu- sion of literature on the systematic relations of nematodes and the piling up of countless stereotyped descriptions based only, many of them, on external appearances, gross anatomical examina- tions and measurements. Among the legion of nematodes known, deplorably few comprehensive studies exist on anatomy, gross and histological, or upon physiological systems such as may be found in the works of Looss on the anatomy and life history of the Egyptian hookwoim and of Martini on the anatomy of Qxyurls cur vula. Until much more is known of the organology and minute details of structure of a great number of the reported nematodes, the un- tangling of the systematic features cannot be accomplished with ease or with certainty. It is for this reason that in the following pages so much space has been allotted to a discussion of technique and methods, with the hope that the disagreeable features of general < . S , ' : _ ;■ -4- technique may be dismissed to a large extent by the worker and that his time may be spent in the perfecting of finer methods for the demonstration of nematode structures at present known only in a most general way gathered often by inference from similar structures examined carefully in the larger and more accessible parasitic forms. The greatest obstruction in nematode preparation is the almost impenetrable cuticula, in which these organisms are encased, as it were, offering a splendid barrier against the en- trance of any ordinary fixatives and clearing media, particularly resinous media in which one often desires to mount specimens. For killing nematodes Looss (1901) recommended 70$ alcohol heated to 80-90 degrees C. into which the worms were placed. For subsequent study they were transferred to 70$ al- cohol containing 2-3$ glycerin for delicate forms and as much as 20$ for tougher specimens, and in this allowed to evaporate slow- ly to pure glycerine. In 1905, the same author proposed killing the worms in 70$ alcohol with 5$ glycerine by volume at 50-60° C. - the heat permitting the animals to be fixed in a relaxed and ex- tended condition. Material so prepared is not beyond reproach for histological details and the following modification, used hot or cold, gives better fixation: 7 0$ Alcohol 50 cc . Glycerin 5 cc. Sat Aq. Picric acid . 5 cc. Glacial acetic ... 2.5 cc. For portions of worms almost any recognized fixative is good - best of all for cell inclusions, etc., Flemming's chrom-osmic with . J t . ■ , . ' . . - . . » . . -5- or without acetic acid. However, for most purposes, the following fluid, (used cold), and for convenience designated as Carnoy- phenol, is superior to all: Absolute alcohol .... 20 cc. Chloroform 15 cc. Glacial acetic 5 cc. Phenol crystals to raise volume 10 cc. 50 cc . Carnoy' s fluid and several of its modifications, each suited to a particular problem in technique, have long been recog' nized for the fixation of various tissues, particularly those difficult to penetrate with ordinary killing fluids. Most tis- sues prepared with Carnoy' s mixture show excellent preservation. Carnoy-phenol was formulated in an attempt to kill nematodes in the most rapid way possible and at the same time prevent col- lapsing and distortion which often follows fixatives intended to give greater clarity of detail than can be obtained by the use of Looss* mixtures. The acetic acid hastens the penetration of the reagent and counteracts the tendency which the strong alcohol has to shrink tissues. At the same time the phenol clears the spec- imens and with the acid keeps the tissues very pliable so that brittleness is entirely eliminated and yet dehydration is com- p leted. The mixture makes an excellent killing fluid as shown by sections of material so fixed and such sections will take numerous stains which, however, will be mentioned later. Por treatment with this fluid, the nematodes newly collected are freed of adhering mucus or dirt by washing and then dropped into , , « the killing agent. With small and delicate forms fixation is almost instantaneous hut with large thick ones more time is re- quired which is, however, no longer than required by Looss’ method where the killing agent is steaming hot. Small specimens are left in the liquid for a few minutes and larger ones propor- tionately longer - 12 hours in no way harming them. Several nematodes were left in the fluid 5 months and from external ap- pearances have suffered naught hut discoloration due to extracts from the cork stopper of the hottle. Should the fluid prove to he too strong for certain nematodes, it may he diluted very slightly, then drawn off the material so prepared and replaced hy the undiluted fixative. This brings to mind the following further hints: the * solution should he kept moisture-free in glass stoppered bottles and renewed after two weeks, for upon standing, the alcohol forms to some extent an ester with the acetic acid, changing consider- ably the efficiency of the fluid; all operations involving the use of this reagent are best carried out in Syracuse crystals where the progress of action at any time may he observed and con- trolled under a microscope or binocular; and in addition, as this fluid is very corrosive and has a tendency to creep over the edges of low containers, it is best to prevent this hy dipping the rims of the crystals in melted paraffin over which no creep- ing takes place. .. , t • -v ■. - ’• '■ • .. - . « ; . -7- (b) Preparation of S pec imens Now that the specimens have been killed the more vex- ing procedures are ahead; namely, preparing totomounts and serial sections. In general, previous workers to accomplish this end relied upon the slow passage of the specimen from one medium to the next accomplished in a variety of ways among which may be mentioned: the use of an alcohol and clearing series each step of which varies from the preceding by a one percent increase in strength or less; the string siphon recommended by Magath (1916); the differentiator of Cobb (1890); or the slightly altered form of Magath; or the evaporation method of Looss ( 1905) which he used in clearing nematodes in glycerin. Of all these no one is meritorious as a time saver nor always for results produced - often days and even weeks elapsing before the process is complete. With the object in view of eliminating much of this time consumption, a series of experiments was run to determine if a set of alcohols with a constant density maintained by adding such substances as camphor, salol and chloroform to bring density to approximately that of water, might lessen the difficulties. Success was met with only in the lower alcohols of the series. As more water was removed greater was the tendency of the nema- todes to collapse and shrink, but if acid were present, this ten- dency was lessened to quite an extent. Then an isosmotic series was attempted and abandoned with no results. Indications were not so much that isosmotic solutions might prevent collapsing, but . r ^ ; , . ■ 8 - rather that collapsing and shrinkage were due to diffusion pres- sures set up when the material was removed from one fluid to another. If a series of dehydrating and clearing agents could be established each member of which would have equal penetrabil- ities or dif fusibilities , the question of shrinkage and collaps- ing of material would be solved because no unbalanced diffusion pressures would exist when the material passed from one fluid to the next. The shrinkage is due perhaps more to this cause than any other met with in the preparation of material for microscop- ical study. Greatest crumpling and collapsing always occurred be- tween 85$ alcohol and clearing. The difficulty seemed to arise from decrease of per- meability of the tissues, due to the hardening as they became more and more water free. In support of this fact it was noted that in one of the experiments where lactic acid had been added to the alcohols in small amount, the tissues did not harden and penetration of the fluids was accellerated partly no doubt due merely to the presence of the acid itself. In fact worms could be mounted rather readily in a resinous medium of lactic acid gum sandarac, absolute alcohol and phenol, but the resulting mounts were yellow, so transparent and structureless, due to the alteration of the protoplasm by the acid, that they were only jelly-like models - of gravely doubtful significance. As a result of the series of experiments mentioned, Camoy-phenol was formulated thru trial and error until it exists -9- in the proportions given earlier in these pages. By its use in methods to follow, a simple, rapid, and quite reliable technique has been worked out for preparing nematodes for a variety of treatments, chiefly sectioning and to to mounting. As thiB fluid is water free, all the tedium of dehydration with numerous fluids and much glassware has been eliminated. If material is killed in the fluid only two operations are required to bring the objects into paraffin, or balsam, and one to clear them in glycerine; if killed in other media and stored in alcohol, three operations will attain the same end. Nematodes may be taken from water or from 70-80$ al- cohol, glycerine or lacto-phenol, in which they have been stored and placed directly in the fluid. Smaller worms are cleared al- most instantly (larger ones less quickly) so that a rapid survey if desired may be made of internal organization after which they may be removed to 95 or 80$ alcohol for storage or if necessary thence to water for maceration or teasing. If the larger speci- mens do not clear sufficiently at first, almost any degree of clearness may subsequently be obtained by allowing the fluid in which the worms are placed to evaporate. The degree of clearing is proportional to the amount of evaporation. With specimens now in the fluid they may be prepared either for sectioning or for mounting in balsam with equal ease. Oils of synthetic wintergreen, oleum Cidri ligni (Merk) or ChClj may be dropped slowly into the dish with the specimens and mixed thoroughly by agitation. The change must be gradual at first, for it is at this point that the tissues become hardened, but as , . . « - 10 - soon as the mixture is three quarters clearing fluid, the greater part may he drawn off and the pure wintergreen, as the case may he, added and allowed to remain for 10 to 15 minutes or longer. If now infiltration hy paraffin is desired the wax is shaved into the dish with the specimens in wintergreen and the whole set aside in a slightly warm place for two hours when the worms may he placed, in pure paraffin, melting at 58° C. Unless serial sections of the whole nematode are to he prepared, better infiltration and better sections are assured hy cutting the material into pieces. Two hours is usually satisfac- tory for length of infiltration, altho less time is more desirable because of the hardening effect of the heat upon the already hard enough worms. However Looss (1905) left worms in paraffin melt- ing at 56-58 degrees C. for two days, and in my own experience worms infiltrated for 12 hours cut quite well except in the head regions. In very refractory cases, Looss resorted to coating the paraffin block face before cutting each section with very dilute celloidin which assists considerably in preventing the sections from rolling when they show that tendency. To mount whole preparations in balsam, the procedure is the same, including the bringing of the worms into wintergreen - ] wintergreen is here to be preferred to the other clearing fluids in general use in any laboratory because of its rapid penetrating power; xylol shrinks tissues too readily and should be entirely avoided. How the Syracuse crystal bearing the worms in a small quantity of oil is tipped only slightly and a large drop of pure ■ . , . . , - 11 - paper filtered Canada balsam, unthinned, is placed on the sloping bottom of the dish and the whole covered. The resin will flow slowly down and diffuse thruout the oil and specimens in the course of two or three hours. Should the resulting resinous mix- ture be too thin to dry rapidly upon mounting the objects, more balsam may be added as before. It is important not to rush this process because the thinner medium within the worms will move thru to the exterior faster than the balsam can penetrate to the interior with the result that the pressure becomes less within than without and unless the cuticula is thick, collapsing will result, but in all cases the more volatile fluids will vaporize - under this reduced pressure - and fill the body cavity and inter- stices between the organs with gas so that the preparations are again valueless, being utterly opaque. If collapsing has not taken place, the difficulty may be remedied by thinning the balsam with chloroform or benzol until the bubbles have gone, then controlling evaporation until the thickness of the fluid is again suitable for mounting. However, should collapsing as well have occurred, and should the specimens be valuable enough to warrant saving, restoration may be accom- plished by running the worms back to Carnoy-pheno 1 and leaving them there until the collapsed portions have plumped out. If this does not occur spontaneously , a slight manipulation by rolling the worm gently will usually restore shape. "Where the specimens are large, it is desirable to puncture the cuticula in several places before balsam clearing, taking care not to injure the underlying o rgans. ■ , . . i < . ■■■• . . . . . , - 12 - Turning now to the question of sectioning, the greatest problem is to procure unbroken sections in an uninterrupted series. The paraffin adheres rather poorly to the very smooth cuticula so that one is often confronted by having sections fall out or roll up. Because of the great hardness of the imbedded tissue, a hard paraffin - 56-58 degrees C. melting temperature - must be used which at the same time contributes to the hardening of the tissues by the use of the high temperatures necessary for infil- tration. The loosening of sections may be avoided to quite an extent by placing the worms near the termination of infiltration into a medium thick solution of celloidin for a few moments until the paraffin on the cuticula has become dissolved - then replacing the specimens immediately in the wax. The heat will bubble off the ether and alcohol which shreds and roughens the celloidin, making a better holding surface for the paraffin. Imbedding is done in the customary way, and sectioning should follow shortly because the hard paraffin upon standing has a tendency to granu- late and thereafter cuts very poorly. If sections 3-5 u. are to be cut, the room must be very cool and the microtome knife exceptionally sharp, otherwise the sections will never come off in a ribbon. Ordinary sections of 8-10 or more micra may be cut with relative ease and mounted in the usual manner, but if sections refuse to remain in the ribbon, treatment with thin celloidin as mentioned before for each section must be undertaken. , • . • : = . ' . . . -15- Following Camoy-phenol fixation numerous stains work nicely, preferably those which do not require taking the sections to water because in such cases the cuticula is likely to swell and tear loose the sections or parts of them. Delafield's or Ehrlich’s hematoxylins in 50 or 70/ alcohol stain well followed by Orange ”G" or some other counterstain. .Among the iron-hema- toxylins Dabell' s works splendidly and is, for the reason men- tioned previously, preferable to He idenhain* s , and because it is more selective and does not require a watery mordant. Staining is accomplished in the following manner: Sections are freed of paraffin and run down to 10 % alcohol in the usual way. At this point they are mordanted in 1 or 2 % solution of iron alum (ammonium ferric sulphate) in 10 % alcohol for 1/2 to 1 hour (or longer), rinsed in 70/ alcohol and placed in a 1 % solution of hemateim in 70/ alcohol for a period as long as or longer than that of mordanting. The sec- tions after this treatment and rinsing in water are ready for destaining which may be done rapidly in 0.5/ acid (HC) in 70/ alcohol, but preferably, for more even results, in the mordant until satisfactory differentiation has taken place. The sections may be counterstained or not as one desires, cleared, and mounted in damar or in what is an excellent medium - cedar immersion oil. Safranin M 0” counterstained with Lichtgrtin produces a pleasing stain in which, however, the Lichtgrftn is dominant because of the few nucleii appearing in any one section of mater- ial. But the most pleasing stain of all for presenting differ- entiated pictures is obtained with Mallory's triple stain. ( Guyer 1917). . . , * -14- Solution I Acid fuchsin 0.5 gram Distilled water . . . 100.0 cc. Solution II Anilin "blue (Grubler’s water soluble) . . 0. 5 gram Orange "G" ( Grubler) 2.0 " Phosphomolybdic acid 1 % aqueous solution .... 100 cc. Sections are run to 50/£ alcohol and thence to solution I where the time of staining depends upon the intensity desired - 5 min- utes or more. Then they are removed, drained of surplus stain, and put into solution II and left until differentiation has taken place, 10 to 20 minutes. Row the sections are rinsed in water, run as rapidly as possible thru the alcohols, cleared and mounted as mentioned previously. Should not enough stain have been re- moved by passage thru the alcohols - further deetaining may be controlled in the 95 % alcohol before final treatment for mounting. Ey this stain all cuticular parts are colored in shades of blue to purple blue - with an occasional exception where it is orange red. Muscle tissue such as the contractile portions of the muscle cells of the body wall are brilliant red as also are the muscle fibres and bundles of the oesophagus and other portions. Protoplasm is pink with a suggestion of a bluish tint; nucleii are darker red with brilliant orange nucleoli. Material fixed in Flemming's reagent and stained with Mallory shows less red with more yellow and purple shades: differentiation being even greater. Staining totomounts is not as yet very satisfactory nor advisable at times because much of the detail of internal structure of the nematodes is masked by the uniform coloration taken on by the cuticula and by the muscle cells beneath, whose - , , ■ : . • • ' ' . . / . ■ ■ • ■ . L -16 contractile portions usually stain deeply. Best results along this line using Carnoy-phenol in the process were obtained by using Orange "G", Safranin, methyl green, Lichtgrtln acid fuchsin, methyl blue, Mayer's HCl Carmine and a modified Ehrlich's hema- toxylin. With the exception of HCl Carmine, Safranin, and the hematoxylin, all these stains are used by adding the dry powder in very small amounts to Carnoy-pheno 1 and the degree of staining controlled. Safranin is utilized to saturation in 70 % alcohol and allowed to strongly overstain the specimen. Then they are removed to Carnoy-phenol until destaining is sufficient when clearing is at once undertaken. Acid fuchsin is the most tena- ceous of the stains mentioned and colors very rapidly. The most presentable mounts are obtained by slightly overstaining the spec imens in the phenol reagent with small quantities of acid fuchsin and Lichtgrttn added in powder form to make a dark solution. Then the cuticula and body-wall musculature are destained by placing the w f orms in 95 % alcohol and passing into it a small quantity of dry ammonium gas. When all color is totally gone and the spec- imens are white showing no clouds of red coming off, they are returned to pure reagent which again restores the red color, most of which is now only in the internal organs. Clearing and mount- ing are done as described previously. The HCl Carmine is used in 70-85/^ alcohol. V/ith the following modified Ehrlich's stain, a good coloration may be accomplished: . . , , i ■„ . a -16- Hematoxylin crystals . . . gram Alcohol b0% cc . Glacial acetic acid . . . .... 6. cc . Sat. Aq. Sol. Ammonium alum .... 10 . cc . Glycerine cc . Phenol cc . Staining is done in the phenol reagent until a deep purple shows on the nematodes; following this the destaining is done by the addition of HC until the tissues are a faint pink when the pro- cess is stopped by transferrence of the worms to fresh reagent. Upon clearing and mounting the purple color reappears, but is limited more to the internal organs than to the cuticular struc- tures. (c) Differential Clearing and Mounting Except for low power v/ork with a microscope, to to staining is of little value in examination of relatively large specimens because of the marked tendency it has to mask the finer details of structure one is desirous of seeing when using higher magnifications. Much greater latitude for observation is better obtained by utilizing degrees of clearing rather than staining. Permanent mounts may be made of glycerine-prepared specimens in glycerine Jelly properly sealed against evaporation, or material may be mounted after suitable preparation either in camsal-balsam, cedar immersion oil, or Canada balsam. These four mounting med- iums will give a differential clearing respectively indicated by the following approximate indices of refraction: 1.476, 1.47 , 1.520, and 1.535 (Lee 1913). To prepare the specimens for passage ■ . .. .* . ‘ ■' . . . J . ■ ■ f o : . ... I ■ . • ■ .r.vr.,: ' .... - -17- in to these media, they are first placed in Carnoy-pheno 1 and then "brought into the clearing fluids most suitable for passage into the mounting medium. For glycerine jelly mounts, the phenol re- agent is replaced by pure glycerine; camsal-balsam is preceded by clearing the material in camsal, a liquid formed by the mutual solution of salol (phenyl-salicylate) and gum camphor; immersion oil, by thin cedar oil and Canada balsam by oil of wintergreen. The process of clearing is accomplished as explained previously. Another excellent medium for small, very transparent worms, is “Diaphane" a resinous medium employing gum Sandarac on the order of Gilson’s "Euparal" which, because of its low in- dex of refraction shows greater detain in the cleared specimen than balsam. The nematodes are cleared carefully from the phenol reagent by camsal and allowed to harden for a short time in this. Then they may be transferred to diaphane, diluted to about 1/2 strength by absolute iso-butyl alcohol, and allowed to clear by gnetle evaporation of the alcohol. A slightly greater degree of clearing may be obtained by preparing the worms in the same manner and transferring to dilute Canada balsam in iso-butyl alcohol v/ith 5-10/ camsal added. This last ingredient makes the balsam dry very slowly and form a tough elastic medium. As well as having numerous toto mounts and sections for study, it is frequently very advantageous to have a number of specimens of any species cleared but unmounted, so that they may be available for temporary mounts at any time should occasion demand it. Nematodes cleared and stored in pure glycerine are . , . -18- excellent for this purpose. They are usually cleared enough for satisfactory examination of most gross details and have the ad- vantage over oil-cleared material in that they are unhardened and may he easily manipulated and rolled about under the cover glass. However it sometimes occurs that the objects do not become suffi- ciently clear. In such instances and in most cases where greater transparency is required -Amann'B Lactophenol (Lee 1913) works very well. Specimens may be removed from storage in 70-80^ alcohol placed in with a small quantity of the alcohol in a dish to which the lactophenol is added gradually to purity. In stubborn cases of penetration slight warming may be beneficial. It is not, how- ever, at all advisable to store material in this fluid because it will in time swell, macerate, and completely dissolve the whole objects due to the action of the lactic acid on the proteins. Material cleared in lactophenol may be returned to storage in al- cohol or transferred instead to glycerine. Very intense clearing such as will make quite visible the openings of the ducts of the aesophageal glands into the lumen of that organ in some nematodes, and also cuticular markings may be produced by placing the worms in glacial acetic acid with an equal amount of phenol crystals. Other intensities of clearing may be had by adding phenol to specimens already in lactophenol - merely increasing the proportion of the substance with the highest index of refraction ( 1.549 phenol). : -19- ( d) Restoration o f Dried Sp e cimen s_ It sometimes happens that specimens will accidentally become dried out thru breakage of the glass container or neglect to cover them. Such objects may be very well and often complete ly restored by the following method, such that staining, section ing or mounting may be undertaken. The dried worms are soaked in 35-50$ alcohol to which an equal volume of lactic acid is added. If they do not soften and plump out to normal size, more acid may be added. When restoration is complete, the nematodes must be freed of the acid by thorough rinsing in 70$ alcohol when they are ready for further operations. This method may be applied to cestodes, trematodes, arthropodes and their larvae with equally remarkable results. ill - 20 - I II. EXAMINA TION OF THE SYMMETRY AND STRUCTU RE OF THE HEA D R EGION (a) General Considerations Ward (1917) has proposed for the purpose of grouping nematodes according to the structure of the anterior end, par- ticularly the oral armature, the three terms: lips, jaws, and capsule, each referring to a typical oral organization. As an example of true lips may be mentioned a member of the genus Ascaris ( Fi g.ia-plaj as showing when viewed en face three lobe- like projections of which a large one, bearing two papillae, is dorsal, while the other two each bearing a single papilla, are ventral. The genus Camallamus (Eig.ia^l?) illustrates true jaws. Here the armature is divided into dextral and sinistral halves which act as a vice for maintaining a hold on the host tissue. The capsule or third type may be found among the members of the Strongyles ( Fi g.4 jtU) . Here the vestibule is cup-shaped with a large roomy interior opening to the exterior by an oval or cir- cular orifice. Within the vestibule and on the walls are var- ious organs for cutting, piercing, gnawing, etc. A closer study of the cephalic region, its structure and symmetry may indicate the value of these suggested groupings or may indicate further groupings or means of relating the nematodes within any one cate' gory, or still further may serve to show which structure is the most evolved and of highest type indicating the evolutionary status of the species or genus among the members of its genus L- . ' ■ - — - - — — : = . , . • . < ■ «' ■ , . - 21 - or family respectively. In order to obtain the proper perspective of the value of the cephalic structure either from a taxonomic or evolutionary point of view, it is first necessary to consider what may he termed as the primitive nematode, and then in this light determine whether cephalic organization has kept pace with or lagged behind the specialization of the nematode body as a whole, by which specialization it is fitted to its environment. It is for this reason that the primitive nematode, as defined by the observations of trained nematolo gists is reviewed regarding its form and sym- metry before the anterior portions of the free living and para- sitic forms are discussed critically with regard to their symme- try and specialization in structure. The great difficulty in such a definition or delinea- tion arises in the determination of criteria for primitiveness. In general a primitive organism is believed to be one with the most generalized structure or in other words an organism with the most avenues along which it may specialize. However, when one is confronted by a nematode which has organs or a system of organs which are structurally very generalized and at the same time there are other systems very highly specialized, the ques- tion may be asked if the simple structures have devolved or if they are hold-overs of the primitive form which existed in the ancestral nematode. There is naturally no adequate nor absolute solution to such a query and if any explanations are offered they can at best be based only on a critical examination of details , . . , . ■ . - 22 - in numerous free and parasitic species, each detail being selected with careful consideration of its stability in the stress of en- vironmental factors. According to Steiner (1919) the type form of nematode body is a spindle where the principal axis is much elongated over the two similar dextro-sinistral and dorso-ventral axes. Any alterations in the relative proportions of these axes of the primitive form will necessarily alter profoundly the general out- line of the body. With extreme lengthening, for example, of the principal axis and only a slight shortening of the other axes, it is a very easy transition into such a filariform individual as an adult Dracunculus medinens is Velschi, measuring more than a meter and a half in length. On the other hand, lengthening of the two secondary axes in greater proportion than the principal axis would produce a form of adult such as Heterodera schachtii Schmidt, the common parasitic nematode of the sugar beet. This parasitic female becomes at maturity a swollen lemon-shaped indi- vidual. In cross section the primitive nematode is always cir- cular with no suggestions whatever of dorso-ventral or lateral flattening. Neither is there any evidence of metamerism, a fact which is borne out in forms today in none of which there is the slightest suggestion of septae. In this connection there is also no coetome, the existing body cavity remaining as a derivi- tive of the primary body cavity or blastocoele. Segmentation, if such it may be called, is present in the cephalic bristles, , '•v , ! , . i I - 23 - according to Cobb, of about thirty percent of the free living nematodes and in one form Scaptrella cincta Cobb, even the man- dibles are jointed. However, this segmentation is limited only to the cephalic appendages . (Ti$. u yA. i.). The mouth of the primitive form is terminal as in present forms, except a few genera in which it has become secon- darily dorsal, notably in the genera of the family, Ankylos tomidae. Embryo logically the mouth is subterminal ventrally and during the development it migrates to the terminal position. There is, how- ever, still a difference of opinion among investigators on this point; some believe that the blastopore as a slit-like opening closes completely from behind forward and that the mouth forms independently in front by an extodermal invagination; other work- ers believe that the definitive mouth arises from an incomplete closure of the blastopore giving here the ventral location of the mouth which shifts at an early stage to the terminal position. The anus, however, is ventral, posteriorly, which too is typical of present-day forms barring for example, a few individuals such as the adult female of Heterodera schactii with a dorsal anus; Tr ichosomoides crassicauda Bellingham, members of the genera Trichuris Roederer Eustrongylides J&egerskiold and Hystrichis Dujardin, in which the anus is terminal. In free living forms the anus is always posteriorly ventral and a tail is present through the tip of which three caudal glands pour their secre- tions. These glands fabricate a cement-like substance which har- dens in the presence of water and serves to hold the individual « ■ ■ . - 24 - to the substrate of its habitat. The lack of a tail and the presence of a terminal anus do not seem to fit into the concep- tion of the primitive nematode but they may be of significance in the conception of the ancestor of the primitive nematode, a discussion of which will follow later in this paper. The openings of the reproductive systems of existing f orals allow the sex products to reach the exterior differently in the two sexes; by way of the rectum and anus in the male nema- tode and by way of the vulva in the female worn - an opening quite separate, generally on the ventral surface in the mid- line. It is believed by Steiner that the primitive nematode, male and female alike, possessed only one ventral orifice which was a common opening for the discharge of alimentary waste pro- ducts and reproductive elements, as well as serving for the dis- charge of the excretory system. In reality this primitive worm possessed a cloaca, which is present now in no known forms. In- deed these three systems - alimentary, excretory, and repro due tive- terminate in a great variety of positions in extant forms. Contrary to the hypothetical condition, the excretory system with fev/ exceptions opens mid-ventrally far anteriorly in the neighborhood of the nerve ring. The vulvar opening may be found posteriorly terminal in the parasitic nematodes belonging to genera Trichuris Heterodera, Eus trongylides and Hystrichis, but more often it is near the middle of the worm in free living and parasitic forms alike. In Syphacia and some Oxyurids it resides far forward in the anterior half of the body - even close 1 -25- to the nerve ring. In the male organisms the gonads open by their ducts into the rectum in connection with the spicular appa- ratus. Eeside these points in the foregoing paragraphs, the primitive nematode has a simple digestive tract, paired gonads, and paired excretory canals. These with all the other elements of the ancestral form are arranged in such a manner that the body is wholly bilaterally symmetrical. 2. Alterations in bilaterality Among the legion of nematodes existing now, strict bilaterality in which each half of the individual is a mirror image of the other, does not exist as far as known, at least in the adult forms. Some of the immature forms prior to their last moults more nearly approach bilaterality than do any of the adults for in them the gonads are present only as ’’anlagen,” ly- ing in the mid ventral line. Changes in bilaterality are very easily brought about by any shifting of the relative proportions of the axes: for example, any lengthening of the principle axis without proportionate concomitant increase of the other two axes would for mechanical reasons alone produce a serial ordering of elements which had heretofore lain side by side in the body cavity. Evidence of the fact is to be found most clearly in the serially arranged caudal glands of many of the free living forms. ( Tex - t j>Vg> fig.i^ Similarly the testes of the male have become serially ordered and then there has followed the disappearance of one. The femals reproductive system exhibits the largest number of . , . - 26 - variations in arrangement of its parts. The ovaries and uteri are double but the uterine ducts unite so that there is usually only one vagina and always one vulvar opening. In general one ovary is reflected anteriorly and the other occupies the pos- terior portion of the body cavity, or in cases where the vulva lies far anteriorly or far posteriorly either the anterior or posterior ovary may suffer suppression or become entirely rudi- mentary. Such changes as these just mentioned alter the actual bilaterality but do not in any way change the fundamental bilater- ality of the organism. Whatever changes in symmetry which replace or become superimposed upon the bilaterality of the nematodes as a whole are secondary features having arisen during the evolution of the primitive form into the present forms of great complexity. Strict asymmetry is most noticeably present in the free living nematode, Bunonema inequale Cobb and in related species which possess on the dextral side a row of immense tubercles giving the individual a curious unbalanced appearance. Such striking asymnetry is not very often seen and in place of it radial sym- metry constructed on plans involving varying numbers of radii is much more general. In order to understand better, perhaps, the advent of this type of symmetry, it is necessary to consider the question of the orientation of the primitive nematode with respect to its surroundings. Steiner has discussed this question in considerable detail and in main it will be outlined in the following few paragraphs. ' - , r . . -27- 3. Primitive orientation Whoever has dealt with free living or parasitic nema- todes is aware of the fact that they always lie upon either the dextral or sinistral portion of the body, such that their loop- ing and twisting is in reality confined to the plane of their principal axis. The morphological ventral surface becomes a lateral surface so that the creeping surface is in no way com- parable for example to the creeping surface of the earthworm. The embryological evidence indicates that the actual adult and embryonic ventral surfaces are the same so that there can not have been any shifting of the anal, excretory, and reproductive openings to a lateral field. This fact proves that the mode of locomotion engaged in by most nematodes has been acquired as a secondary means of progression. The primitive orientation was probably of such a nature that the principal axis was perpendicular to the substrata - the nematode being held in position by the secretions of the caudal cement glands. In this position the nematode could have waved back and forth in a dorso-ventral plane simulating the waving movements of some of the tubisficid worms. In support of this orientation, are examples of some half sessile free-living nema- todes {from fresh and salt water) which live on algae and aquatic vegetation and which may or may not possess eye spots. These sense organs may be blackish or red pigment spots or pigment cups, each of the latter bearing over it a single transparent lens, for example: , ■* 1 , . ‘ - 28 - Thoracostoma antarc ti cum von Li n s t o w Thoraco stoma lobatum Steiner Nemella ocella ta Co Lb Ionema ocellatum Cobb Onchulella ocellata. Cobb ( Fi g.io-v- y\. a..) These ocelli with few exceptions are to be found far forward ly- ing laterally upon the oesophagus; only in a few cases do they lie slightly dorsal or ventral with respect to the oesophagus. The lenses are so directed that light coming from a vertical source will fall upon them when the worm is oriented vertically. Should the primitive worm have moved normally on a side, as many of them do today, one or other of the eye spots would have been turned toward the substrate and would have thus become tempor- arily useless. Light to have stimulated both spots through the medium of the lenses with the worm so oriented would necessarily have come from a horizontal source which is quite improbable. Other nematodes, members of the genus Echilidium, possess pigment surrounding the oesophagus and above this a circlet of ocelli set to collect vertically falling light. Furthermore some free living nematodes possess many long delicate cephalic bristles ( Monhystera pilosa Cobb, Pomponema mirabil e Cobb) and others bear on their bodies many fine bristles Sphaerolaimus hirsutus Bastian, No tochaetosoma ten ax Irwin-Smith) ( Fig. »a-pU.) . These delicate processes can not be reconciled with a creeping mode of locomotion through sand, mud, and debris, but are consistant with a half sessile form of existance. Still other , - 29 - species are parasitized by epizoa and epiphytes which cover all portions of the body. For instance, a delicate filiform alga often covers the body of Spira par asitifera Eastian while vorti- cella may attach itself to the tail of the same worm. Such ectoparasites could neither remain attached to the nematode nor stand the wear and tear if the host thrashed about among the de- bris. According to Erwin-Smith, some members of the family Chae tosomatidae hitch along the rocks and vegetation in the man- ner of measuring worms by means of special adhesive bristles ar- ranged in two rows on the ventral surface near the tail and by other adhesive bristles on the dorsal portion of the cephalic region. Seurat believes these bristles are a special adaptation. Some other free living nematodes according to Cobb’s observations move as many rotifers do, in a looping fashion, using the caudal glands and suction created by the muscular oesophagus as means of fixation. The points reviewed in the foregoing paragraphs seem to point rather strikingly to the fact that the primitive nema- tode leads a half sessile life, oriented in an upright or nearly upright position, as do many of the free living forms today, many of which are not as highly specialized as the debris and earth inhabiting forms. Another feature of interest in this connection of the sessile tendency is the prevalence of radial symmetry in the anterior regions of great numbers of non-paras itic and para- sitic forms. A characteristic of sessile animals as the Coelen- terata is their radial symmetry and of the Echino derma ta, their pseudo-radial symmetry, which has become superimposed secondarily ; ;3Bta | mmm . v , -30- upon their primary bilaterali ty. As a result of the sessile tendency among the free living nematodes, radial symmetry is ‘be- lieved to have arisen. 4. Definition of the "Primitive Nematode" Steiner (1919) has in short defined the primitive nema- tode as a bilaterally symmetrical, spindle-shaped animal affixed to its support by the secretions of three adhesive glands at its caudal extremity; possessing a simple digestive tract with a muscular and oesophagus paired gonads in the two sexes lying par- allel, one on each side of the intestine, throughout their length; their ducts opening with those of the paired excretory vessels and the intestine into a cloaca discharging by an anus to the exterior in the mid ventral line anterior a short way to the ter- mination of the tail. Seurat ( 1920) after a careful considera- tion of what he believes to be primitive characters still main- tained in some of the present day nematodes, avoiding characters induced by adaptation to environment (parasitic adaptations as complex ovejectors, organs of fixation, buccal cavities armed with teeth or free-living adaptations as long cephalic bristles, ventral adhesive setae of the Chaetosomatidae , buccal stylets of Xiphinema and Dorylaimus, etc.) defines the primitive nematode as follows: A "Etre vermiforme, de petite taille, detriticole ou saprozoite, vivant dans un milieu humide; symetrie bilaterale; bouche subterminale ventrale, limitee par trois levres, une » — . . . . . ’ ' . . ... . - 31 - dorsale et deux subventrales ; queue conique robuste, presen tant trois glandes caudales qui s’ouvrent a sa pointe. Cuticule lisse, couverte de papille sensoreilles eparses ; epiderme a cellules distinctes; quatre bandes dorsale, ventrale et laterales separant quatre champs musculaires; cellules musculaires de grande taille, peu nombreuses; bandes laterales presentant des glandes cutanees unicellulaires , eparses. Cavite buccale tubuliforme, courte; intestin anterieur allonge, a lumiere triquetre, tapisse interieurernent d’une mem- brane cuticulaire, differencie en un oesophage renfle en massue dans sa region terminale et en un bulbe a clapets ( proven tricule) ; intestin moyen d’origine endodermique , forme d’un petit nombre de grandes cellules, emettant parfois un caecum dorsal dans sa region anterieure; intestin terminal court, tapisse d’une mem- brane cuticulaire, en rapport a son origine avec trois glandes rectales unicellulaires. Appareil excreteur pair, comprenant de chaque cote du corps un canal anterieur et un canal posterieur que viennent s’ouvrir a un pore lateral, ou debouche egalement une glande unicellulaire ; parfois, cet appareil est double et comporte un second systeme de canaux s’ouvrant dans la moitie posterieure du corps (l). Sexes separes; dimorphi3me sexuel faible, le male etant s implement caracterise par un developpement plus riche de pa- pilles du voisignage de l’orifice sexuel. Glandes genitales paires; les deux tubes genitaux males, differencies en testi- cule, canal deferent et canal ejaculateur s’ouvrent a peu de distance en avant de l’anus et remontent parallelement vers . , , , . , ' , - 32 - l’avant; deux glandes a ciraent debouchent dans la region proxi- male du canal ejaculateur; organes d' accouplement representes par deux spicules egaux, glissant dans un gorgeret impair. L’appareil femelle est forme de deux tubes differencies en ovaire, oviducte, uterus et vagin, s’ouvrant au dela du milieu du corps et remontant parallelement vers l’avant; ovaires en massue, a ovocytes peu nombreau; uterus servant a 1' emmagasinement d’un tree petit nombre d’oeufs de grande taille, pondus a un etat devolution peu avancee. Le nombre des tubes genitaux s’eleve parfois a deux ou meme a trois paires. Oeuf mixtoleci the , a cytoplasme clair; segmentation totale, inegale. La larve mene une existence libre comparable a celle de l’adulte et subit quatre mues au cours de son evolu- tion, son principal accroissement se produisant au moment de ces mues ( caractere conserve chez le Cephalobus ciliatus). Ebauche genitale representee, chez la larve venant d’eclore, par un massif impair de deux cellules germinatives et de deux cellules somatiques; cette ebauche, qui reste impaire durant toute la vie, n'evolue que tres tardivement, au cours de la seconde moitie de la vie larvaire" (2). "Vermiform beings of small size living in detritus or decaying material in moist surroundings; bilaterally sym- metrical; mouth subterminal ventrally, limited by three lips, one dorsal and two subventral; tails stoutly conical, present- ing three caudal glands which open at its point. Cuticula smooth, covered by scattered sensory papillae; epidermis of distinct cells; four bands dorsal, ventral and lateral separat- t , I , « ' , ■ . . , - 33 - ing four muscular fields; muscle cells of large size, few in number; lateral bands present scattered unicellular cutaneous glands . Buccal cavity tubuliform, short; anterior intestine (oesophagus) elongated with tripartite lumen, lined interiorly by a cuticular membrane, differentiated into a clubshaped oesophagus swollen at its terminal portion and a bulb with valves ( proventricle) ; middle intestine of entodermal origin, formed of a small number of large cells, giving forth sometimes a dorsal coecum in its anterior region; terminal intestine short, lined by a cuticular membrane in connection at its origin with three unicellular rectal glands. Excretory apparatus paired, comprising^ from each side of the body^an anterior canal and a posterior canal which come to open by a lateral pore where there equally empties a unicellular gland. Sometimes this apparatus is double and admits of a second system of canals opening in the posterior half of the body. (This form is realized in some fe- males of the genus Rhabdites opening alone without any single gland by a small pore laterally situated in a band of muscles.) Sexes separated; sexual dimorphism faint, the male being characterized simply by a richer development of papillae in the presence of the sexual orifice. Genital glands paired; the two genital tubes of the male being differentiated into testicle, vas deferens and ejaculatory canal opening a short distance in front of the anus and extending in parallel toward the anterior portion of the body; two cement glands empty into the proximal region of the ejaculatory canal; copulatory organs - 34 - represen ted by two equal spicules sliding in an impaired groove ( guberusculum) . The female apparatus is formed of two tubes differentiated into ovary, oviduct, uterus and vagina, opening anterior to the middle of the body and extending in parallel toward the front; ovaries clublike, oocytes not very numerous; uterus serving for storage of a very small number of large sized eggs, borne only to a slight stage of development. The number of genital tubes may advance sometimes to two or even three pairs. Eggs mixtoleci thal , with clear cytoplasm; segmentation total, unequal. The larvae lead a free existance comparable to that of the adult and undergoes four moultB in the course of its evolution or growth, its principal increase in size occurring at the moments of these moults, (a character conserved in Cephalobus ciliatus). The genital anlage is represented in the hatching larva as an unpaired group of two germinative cells and of two somatic cells; this anlage which remains unpaired throughout life develops only very slowly, in the course of the second half of the larval life." There are no living forms today as yet known which possess all the primitive characters set forth either by Steiner or Seurat. The latter author makes no statements regarding prim- itive spacial orientation and further regards the three-lipped form - one dorsal and two ventral - as the probable early form. Among the free living nematodes members of the genus Rhabditis have conserved some of the ancestral characters in the structure of the digestive tube and genital organs but have gone far afield in the acquisition of radial symmetry of the mouth, in the re- . - 35 - duct ion of the male genital system to a simple tube and also in the structure of the lateral lines. On the other hand, of all the parasitic nematodes, those guarding the most numerous primitive features are the members of the Oxyurid group. They possess primitive musculature, primitive structure of the lateral bands, the excretory apparatus and digestive tube. Contrary to these ancestral features are the extreme modifications of the ovo- jector in the female and of the spicular organs and truncated tail of the male. ' ■ 36- (b) Cephalic Structure in Free-living Nematodes 1. The symmetrical factor of the oesophagus After the preceding discussion of the primitive nema- tode, its bilateral nature and orientation, the following sections will be limited to a consideration of the structure and symmet- rical content of the cephalic region, ( l) of free-living species, and ( 2 ) of parasitic species, in an effort to determine the prim- itive condition and the successive changes which evolution has imposed upon the early type. One element of the anterior region which is ever a possessor of triradial symmetry in all the members of the Myosy- ringata, Ward (1917), is the oesophagus. In cross section, this organ exhibits a triquetrous lumen, "sechseckig" as Schneider (1866) calls it, with three alternating obtuse angles directed apex lumen-ward, the other three, acute angles, apex outward. Of the three portions into which the muscular tube is divided, one-third is always dorsal and the remaining two-thirds are sub- ventral, so that one of the acute angles mentioned is always directed ventrally. The few exceptions existing to this type of oesophagus have been placed in the group Trichosyringata Ward, a group characterized by the possession of a capillary oesophagus. The morphology of such an oesophagus has not been carefully worked out so that as yet statements regarding its symmetry and structure are not on a substantial basis. It may be that some of the gen- - 37 - era in that category will have to he removed, such as, for example, Trichosomo ides crassicauda Bell, which, according to Rauther, shows a triquetrous oesophagus, at least for a considerable part of the length of that organ. The triradial nature of the oesophagus is such a dis- tinctive feature of the phylum Nematoda and is so universal throughout the group, that it may he accepted as one of the most stable factors in nematode organization. Tb r this reason, it may he considered a primitive feature - certainly, if not primitive, it is one of the earliest features to have been established, in the evolving ancestor. "When this triquetrous organ, which under- lies all the superficial structures of the cephalic region, is used as the basis of determining the symmetry of the head, the only possible symmetrical divisions involving all structures would be two in number; namely, one of bilaterality, and of tri- radiality, the latter of which by division of sectors might readily pass into conditions of multiple symmetry, among the more superficial structures as the lips. Exceptions to tri- radiality would, of course, occur in nematodes possessing cephal- ic branches to the lateral excretory canals, amphids and ocelli. Normally radiality merges progressively into bilaterality as the region of the nerve ring is approached in an antero-posterior direction. When, however, the more superficial structures of the nematode head and pharyngeal region are examined, these funda- mental di- and tri-radial symmetries give place to curious mix- tures of symmetrical patterns in one and the same nematode, I , ■ - 58 - involving plans "based on multiples of two and three radii. Lips, papillae, sensory hairs, cephalic "bristles, teeth, and cuticular processes are compounded in a variety of ways; for example, Oxyur is obvelata Rudolphi is owner of three lips arranged in correspondence with the three sectors of the oesophagus "but the six papillae are grouped in a dextral and sinistral row of three each. (Fig. tfA.i.) ; Protospirura mur is Gmelin carries a right and left row of three lips each and four papillae one at the base of each terminal lip of each row. ( Pi g. »o^>U) ; again, the ela- borately constructed Mononchus gerlachii de Man, a marine nema- tode, possesses six radially arranged lips each bearing, centrally placed, a single papilla and at a lower level each carrying two papillae save the two central lateral lips which have again only a single papilla each. Immediately below the lips on the walls of the vestibule are twelve rounded projections of unknown sig- nificance. Beneath these there is a chitnious skeletal structure hexagonal in optical section merging into the triangular lumen of the pharynx which itself passes into the ever present triquetrous oesophagus. ( Fig. 3|>U.) . These examples were chosen at random from countless other similar simple and still other beautifully intricate forms merely to show the variations possible, of which the last spec- imen ( Monochus gerlachii ) illustrates symmetry build on two, three, four, six, and twelve radii. Yet this last whole complex arrange- ment really becomes bilateral because of the unpaired median lateral papillae and a very large dorsal tooth not previously mentioned, situated on the roof of the buccal cavity about midway . . • • , ' c«; . . . . ,1 . . -39- be tween the oral aperture and the oesophageal region in the mid dorsal plane. 2. Pharyngeal modifications Turning now to a closer study of cephalic structure, we find among some of the marine nematodes a remarkably simple head region from the standpoint of structure, but from the point of view of their genesis they may not be perhaps termed simple in the sense of meaning primitive; however this point will be reconsidered in another section. In genera belonging to the order Litinia Cobb 1920, there are forms in which the head is devoid of lips; papillae are indistinct or minute; no pharynx is present; the oesophagus is simple with no bulb; and cephalic bristles may be absent. Litotes minuta Cobb is extremely simple for the mouth opens directly into the oesophagus; no lips or bristles are present, but papillae - six in number - exist; and the body as a whole is rather simple and the amphids are very in- distinct. In Alaimella cincta Cobb, the head possesses a simple mouth surrounded by six papillae and probably six flat amalgamated lips which can scarcely be comparable to lips as defined by an example of Ascaris in a previous portion of this paper. There are also foud cephalic bristles present in this species. A re- lated species, A. truncat a, the type for the genus Ailaimella, has similarly four bristles and six papillae and in the male two testes are present, indicative of a primitive nature. Ionema o cellatum Cobb possesses two ocelli - lenses directed anteriad, simple circular mouth, no pharynx, no lips, and four cephalic , . * , - ■ ' . - 40 - setae. Schistodera exilis Cobb and Tycnodora pachydermata Cobb similarly have circular mouths; however, the former bears four minute papillae around the mouth and the latter, two circlets of setae, the first and anteriormost composed of six and the second, of four. Each one of the nematodes just mentioned lives free upon algae and "seagrass" or upon the sand at the bases of this vegetation. Still other marine forms possessing no pharynx an d a simple mouth circular in outline, devoid of any form of lips, are members of the interesting family Chaetosomatidae. These nema- todes are not strikingly organized in structure except in the remarkable possession of ventral adhesive bristles on the pos- terior portion of the body and dorsal cephalic adhesive bristles or setae, which, according to observations by Irwin-Smith, are utilized in creeping. In this direction these forms have spec- ialized to quite a degree. Also the genera Ionema, Schistodera, Nemanema, Cobb, and Thoracostoma Marion have this simple mouth and lack a pharynx. Next in simplicity are nematodes which not only have a simple circular mouth with none or amalgamated lips, ( Terschel- lingia longicaudata. , Monhystera steno s oma ) but also possess a pharynx. The pharynx is one structure which is subject to the greatest diversification and is to a great extent indicative of height of specialization and adaptation in free living forms and also among parasitic species. Nemanema simplex Cobb, a marine algae- inhabiting nema- tode, exhibits a very simple cephalic region; bristles are L .. . — - - ' . . . . — — « . « . 1 . . . - 41 - absent; the mouth is a round orifice; surrounded by possibly six exceptionally indefinite papillae, and the pharynx is the merest conoid suggestion. This almost unnoticeable pharynx as in the form above may become greatly elongated ( R hynchonema cine turn Cobb) and constant in width; short and narrow ( Li tonema nudum Cobb) ; or cavernous and greatly modified as in the genus Monomchus Bastian (Fig. 14 ^>U.) notably, and in many others. The very undifferentiated conoid pharynx and its derivatives attained chiefly by elongation are prominent in nematodes living on a more or less liquid diet. In general, too, the enlarged and widened pharynges are greatly modified by armatures of onchi derived from their walls. Such are to be found among nematodes living upon a solid diet demanding more or less maceration. A very special type of such a pharynx is found among the spear bearing nematodes obtaining food by piercing and then sucking the fluids, for example, from the roots and tender shoots of either water or land inhabiting plants. There are, of course, intergradations among the phar- ynges such that one may arrange a series beginning with nematodes possessing no pharynx (Litotes) followed by forms with larger but unarmed pharynx which in turn pass into other forms bearing teeth - the simpler ones bearing three teeth, one each in a posi- tion corresponding to the respective sectors of the oesophagus, ( Mesonchium poriferu m Cobb). The tooth bearing type, perhaps by further modification, passes into the spear bearing nematodes as Dorylaimus, Heterodera, Dorylium, Eutylenchus, Tylenchorhyncus , etc. Among the Mononchs the dorsal tooth is very large and works . , < , • . ' ' < ■ ’ - 42 - in opposition to the teeth belonging to the subventral sectors of the pharynx. The teeth on these last two portions may be very small and there may be only one per section as in Mononchus radia - tu8 ; small and accompanied by numerous denticles ( M. denta tus Cobb); or by two rasp-like structures approximated near the mid ventral line of the pharynx so that they may operate against the large dorsal tooth ( M. mus co rum Bast ian) ; or further there may be no ventral onchi at all ( M. zschokke i Mengel) . The phar- ynx is partially mobile in some forms by means of three seams or hinges, one ventral and two lateral and a bit dorsal. In other cases where the walls of the cavity appear immobile, the lips seem to force the food down upon the teeth and rasps by which means it is torn apart and made ready for swallowing. Another type of armature is that found in Synonchium obtusum Cobb. Here what is apparently the pharynx is highly muscular and forms a large muscular bulb. The mouth is really the large flaring, triangular opening to the pharynx located in a slightly shallow depression formed by the six double amalga- mated flaring lips. The sectors of the pharynx are equal and each is armed in its mid line by a mandible mounted by three inpointing teeth and flanked on each side by a small tooth. (Fig. Wien the pharyngeal bulb contracts, the mandibles are approximated, drawn inward and downward while the lips are slightly raised, partly covering the mandibles. Another nematode, Xyala s tricta Cobb, has three similar but less elaborate mandibles. Still another example of the open flaring and armed pharynx may be found in Gammanema fera x Cobb. Here the base of the pharynx I . . , • . _ . , * ■ - 43 - is armed with three ribs, each bearing an inward pointing ouchium. Jointed mandibles (Fig.T-fAv ) are present in Scaptrell a cincta Cobb, and inpointed ones are again present in Cheironchus vor ax Cobb and Selachinema. There are forms which have greatly developed dorsal ouchi which have become in many cases much elongated (Anaxonchia) and well buried in the oesophageal musculature (- Fig v K These spear-shaped ouchi are movable and perhaps they form, as Cobb has suggested, the transition from the pharynx armed with ouchi to the spear-bearing pharynx. There is still another spear- bearing group in which the so-called stylet is hollow, sallowing the fluids to be drawn through it by suction created in the oe- sophagus. Dorylaimus and Discolaimus are good examples of this construction. Some species indicate by the structure of their stylets that these organs may have arisen by the partial fusion of three ouchi. A few of them distinctly show construction from three portions ( Tylopharynx striata de Man). It may be possible then that by certain developments during the evolution of these trionchiate forms a partial fusion of the on chi has taken place with and accompanying elongation and narrowing of the pharyngeal cavity giving rise eventually to the hollow pharyngeal stylet. Each of these pharynges has its symmetrical content, but these relations will be considered in a following section in connection with oral structures. * . - ' . . - 44 - 3. Oral structure The oral structures of the free living nematodes are equally as complicated as the pharyngeal and show almost as numerous variations. The simple circular mouth , noted in connec- tion with some of the nematodes mentioned in a preceding para- graph as possessing no pharynx is, we may say, the simplest from a structural standpoint, hut whether it is the most primitive form cannot yet he said. In view of the fact that the oesophagus is triquetrous, it would appear that the most primitive form of mouth would not he a circular orifice hut rather a triquetrous or triangular opening agreeing in symmetry with the oesophagus, however, either view might he taken and some consideration which will follow later will support the former view - that of the prim itive round mouth opening. In Thoraco stoma setosum v. Lins tow a triangular mouth occurs hut in consideration of certain spec- ializations in the head region, it mig^it better he interpreted as a fusion of three lips. The next structurally simplest form and the one at present conceded most primitive is that found among members of the genera, Enoplus and Rhahditis. The most primitive type of free living nematode in the estimation of nematolo gists is found in this last genus, where the mouth is surrounded by three lips, definite well formed lips. One of these is dorsal and the other two are suhventral corresponding again with the divisions of the oesophagus. The lips may he entire or there may he signs of \ . . . , ■■ , - 45 - division as in Rhabditis pellio Butschli where each lip is divided incompletely into two portions by a shallow groove running longi- tudinally along its mid- region. Similarly Rhabditi s lambdiensis Maupas possesses three lips distinctly bilobed, each bearing a pair of priminent setifoim papillae, all equal and exhibiting as a whole perfect radial symmetry of the head region. Quite in contrast to division of lips may be mentioned the curious labial variations found arising from outgrowths of the lips in the genera Terato cephalus and Cephalobus. In the former genus the species Teratocephalus crassidens de Man, the six lips surrounding the shallow pharynx have cuticular wings on the edges, partially fused near the bases of these lips, such that a corolla-like structure results with a continuous edge. The apices of the four sublateral lips bear a fine bristle. (Figs . \ yU) . On the other hand Cephalobus ciliatus von Lins tow has arising from each of its three lips a thick column which bifur- cates and gives forth two rather long processes whose edges are beset with bristles at regular intervals. ( Fig. • In a position alternating with the lips, the cuticula near the peri- fery has become elongated into a stout hom-like process. The significance of such diverse outgrowths of lips as represented in the two genera above is not yet known. The radial symmetry of the heads in these two species is only superficial because just below the lip region on the lateral fields lie the amphids, one dextral, and one sinistral. They perforce shift the symmetry to bilaterality. , - 46 - Evidence gathered from an examination of numerous free living nematodes seems conclusively to show that forms having numerous lips acquired these labial organs by the subsequent divi- sion of the primitive three lips. Six lips is a very common num- ber among many genera and appears as suggested by Rhabditis to have arisen by the division of each of the three lips of the prim- itive form into two parts. Further, division, though incomplete, of these six lips gives rise to twelve lipped forms such as An axon chi urn litorium Cobb; complete division, to twelve small lips as Iotodorus punctulatus Cobb, and perhaps to the twelve highly modified lips or labial setae in Pomponema mirabile Cobb. Division of lips would then indicate a more evolved nature than the three lipped forms. This fact seems to be well borne out because many lipped forms occur among genera which have rather elaborate pharynges, (Monomchus) and which have specialized in other lines too - choice of habitat, loss of structures, as caudal glands and of one gonad by suppression. The Mononchs are to a large extent land inhabiting predatory nematodes rather more advanced in this last respect than their fresh and salt water relatives. The six lipped condition permits the oral aperture to open widely allowing the mononch a greater certainty in seiz- ing its prey and macerating it against the pharyngeal ouchi. While specialization may go in one direction, namely, division of lips, still further specialization even in advance of division results by the union or amalgamation of lips. Degrees of confluence or amalgamation may be easily seen after a survey of a large number of species. For instance, one may begin with a ■ . . s ■ ■ : ' , , 1 . < « - 47 - form possessing three distinct lips (Rhabditis) and these may then become confluent as in Monhystrium transitans Cobb ^ Fi g g. The mouth opening resulting in such a case is triangular or triquetrous. Monhystera stenosoma de Man seems to indicate this, even better than the preceding species. By far the most common number of lips is six, and they offer many interesting variations, finally becoming confluent and forming a mouth capsule generally in connection with a large cavernous pharynx or buccal cavity. Among the Mononchs as previously men tioned, there are six lips which in many species are very well defined and separated ( Mononchus regius Cobb) and in others the six lips exhibit various stages of union, for example, i n Monon' chus ma.ior Cobb, the lips have become more rounded and less dis tinct so that the mouth opening assumes a hexagonal outline - in another genus and species Bolbella tenuidens Cobb the mouth has become a perfect circle and the six lips have lost entirely their individuality externally, but internally the organization still indicates lips. An interesting feature, too, of this species is its asymmetry; the amphids, instead of being mid- lateral, have shifted slightly and occupy a dorso-lateral posi- tion and contrary to the general rule the dorsal onchus is not the one which has become specialized but instead the sub-medial dextral onchus has elongated and assumed a spear- like nature. A characteristic feature of the genus Anguillula is the entire lack of lips, however, the papillae and internal • - >. , .. ■ .. , . - 48 - arrangement of cuticular structures indicates very distinctly that the capsule is the result of completely fused lips, six in number (Fig. d" ). In longitudinal section the mouth cavity is definitely divided into two portions: an upper vestibule, thin walled, with the concave surface facing inward. This por- tion has probably arisen from the under surface of the fused lips. These organs in many lipped forms have a tendency to become thin- ner and less distinct so that one might easily expect them to become still less thickened after fusing and losing their iden- tity to a greater or less extent. Following this vestible (in Anguillula aceti Mttller) there arises the pharynx properly speak- ing, set off from the preceding structure by a distinct break in the cuticular wall of the buccal cavity. ( Fi g, I'b p\*) . The lining of the pharynx is much thicker and in cross sections of the phar- yngeal region, the lumen of the canal is triangular. The walls of the canal exhibit cuticular thickenings, one in the mid- line of each sub-ventral sector which are opposed to a small triangular tooth in the mid-line of the dorsal sector. A similar distinction between pharynx and vestibule may be seen in Monhystera stenoso ma. Here the capsule has arisen from the fusion of three lips. Cephalic papillae are not defin- itely known to exist but the head bears in addition to the two lateral amphids four pairs of submedian bristles, the anterior- most member of each pair appearing slightly shorter than the other. Turning now to a consideration of the oral organization among the spear bearing nematodes, one finds lips again, having undergone fusion either partial or complete. Complete fusion . , . , . , . ' - 49 - occurs in the genus Ty lencholaimus , hut evidence of labial struc- ture still remains in the presence of six papillae surrounding the mouth. Other genera, Tylenchus and Dorylaimus, exhibit the same general structure but some of the species among the numerous ones in the latter genus show distinctly the lip-like nature. The fusion is complete enough to produce a circular mouth open- ing but the peripheral outline of the crown of lips viewed en face is scalloped showing the external outline of six lips ( Do ry - laimus labiatus de Man). There seems to be in these spear bear- ing forms, derived from others possessing armed pharynges, an interesting correlation between extent of lip confluence and pharyngeal specialization. The mononchs with wider but less com- plex pharynges, from the standpoint of genesis, possess less con- fluent lips. In this connection one may ask whether the forms cited in an earlier portion of this section as possessing no trace of lips or pharynges have never acquired a pharynx or lips or whether these structures have been lost by devolution or even indicate a greater stage of evolution. It cannot be said defin- itely at present which view is the better one to accept. Another interesting oral and cephalic structure and one which might be interpreted as the forerunner of jaws is to be found in Synonchium obtusum Cobb, which was described in connec- tion with pharyngeal modifications. The six double lips have fused completely showing only a continuous crenate rim, as it were, surrounding the large flaring triquetrous mouth and pharynx. (Fig. 11^4.3). Incidentally this nematode possesses pure tri-radial symmetry as far as the level of the amphids which shift the total , d n . , . • , . ’ , . . , ' - 50 - symmetry of the head to bilaterality. In transition to jaws bilaterality becomes the only symmetry in the head region. The apparent change from bi-radial pharynx to jaws comes through the loss of the dorsal sector of the pharynx accompanied by a lateral shifting of the other two sections. Se la ch in ema f e rax Cobb il- lustrates this change very beautifully because there remains a vestigial dorsal sector which however is greatly overshadowed by the two powerful submedian chitinous jaws. Another species yet undescribed by Cobb shows no remnant of this dorsal sector. Chieronchus vorax Cobb by a similar elimination of the dorsal sector has two jaws or mandibles. Pseudonchus ro tundicephalus Cobb again supports the formation at least of bilateral mouth parts from the loss of the dorsal pharyngeal sector. So far there is no evidence which supports the view that submedian sec- tors ever fuse giving rise to jaws which work in apposition dorso ventrally. Perfect disymmetry in a dextro-sinistral sense is found in the following three extraordinary genera, Diploscap ter , Wilsonema and Heth, of which D. coronatus (Maupas), W. cap i ta turn Cobb, and H. juli Cobb are respective examples. The first possesses four strong outwardly directed hooks, two dorsal and two ventral and between them laterally are two serrated flaps or lips one right and one left. Cobb interprets in his illus- tration of this species the two flaps as lips as well as the four hooks, each a lip. If this is true, the nematode possesses six lips; the two laterals having flattened out and become flaplike; the two dorsals and tv/o ventrals having fused and formed a single Ij •_< • . ■ . . - 51 - dorsal and a ventral double hooked structure. The second species above is distinguished by dorsal and ventral double "combs'* and two lateral columns, a dextral and a sinistral one, tipped each by a finer process. Here the disyrame try is most prominent dorso- ventrally. The last species of the three evinces again lateral disymmetry. ■ -52- 4. Considerations of symmetry in the head region After the considerations of oesophageal, pharyngeal, and oral structure in the preceding paragraphs, the following ones will he devoted to an examination of the symmetry of the nematode head as a whole, considering the parts played by these structures in determining this symmetry. In view of the fact that the out- standing symmetry of the nematode body as a whole is bilateral, the same relation must have applied to the cephalic region in the primitive form. This seems to be borne out by what is known of nematode embryology, and by features which nema to lo gists have come to accept as primitive. The primitive mouth was ventral and the oesophagus arose from three rows of cells, the dorsal one of which was the equivalent of the other two. These features alone would establish bilaterality in the embryo and in the primitive adult. By an equalization of the three oesophageal sectors, the triquetrous and the tri symmetrical nature of this organ be- came apparent. The primitive nematode had three lips correspond- ing to the symmetry of the oesophageal sectors giving rise ul- timately to a purely trisymmetrical structure. Only very few radially symmetrical cephalic regions exist among nematodes today, because there are in the free living species organs such as am- phids, ocelli, and with or without these structures, the cephalic bristles and sensory papillae which in a great number of forms fall into a bilateral arrangement, despite radial ordering of . . ■ . , ■ ' , - 53 - other structural elements. The difference usually arises from a lack of the lateral median bristles and papillae being the equiva- lents of the others. In an otherwise radially symmetrical head, one often finds only four cephalic bristles instead of six, as if the median lateral ones had been lost and in cases of duplication of bristles or papillae on the lips the lateral median lips are the ones which lag behind the others in this respect. This peculiarity is well illustrated by Cobb as exist- ing among the mononchs in relation to the labial and cephalic papillae. The arrangement of papillae in these forms follows the law for the arrangement of tactile cephalic setae of nemas in general, namely: ”... When six are present one is found on each of the two lateral lines and one on each of the four submedian lines; when more than six are present, the increase occurs first on the submedian lines, the commonest number being ten, -- one on each lateral line and two on each of the four submedian lines; when the number is in excess of ten, the increase is again more commonly found on the submedian lines.” Obviously structures following this order of arrangement shift apparent radial symme- try into bilaterality again. Radial symmetry with few exceptions is actually attainable only if lips alone are concerned, as has already been shown. The pharyngeal region is frequently non-radially sym- metrical, rarely so when it becomes armed with ouchi because the dorsal ones usually have a tendency to surpass in size the other ouchi. The small, smooth, prismatic or cylindrical, and unspec- ialized pharynx readily falls in line with any symmetry which the -54- lips impose upon it. Disymmetry either do rso-ven tral or dextro- sinistral exists, as we have seen, in a few free living forms. Its origin is explicable in a few cases as the result of loss of the dorsal lip and pharyngeal sectors. In such cases the ceph- alic symmetry shifts undeniably into the fundamental bilaterality. Asymmetry occurs least of the other types. In summary it appears then that true radial symmetry is not as general a condition among nematodes as a superficial examination would lead one to expect. Radial symmetry, however, is common, and a striking feature if sensory organs pharyngeal onchi and other armatures are neglected or considered secondary in importance to the basic plan of the head region. The apparent order of symmetrical succession in the nematode body beginning with the primitive worm is most probably the following, applied, of course, only to the cephalic region: 1. Primitive bilaterality 2. Radial symmetry 3. Disymmetry 4. Asymmetry . . , , . 1 . - 55 - (c) Cephalic structure in parasitic forms 1. Symmetrical factor of the oesophagus What has previously been said regarding the symmetri- cal factor of the oesophagus in free-living nematodes is true of the parasitic forms also, at least those possessing the char- acteristic triquetrous muscular oesophagus. Those nematodes hav- ing a capillary oesophagus referred to as belonging to the group Trichosyringata comprise a restricted number of genera, among them being Trichina, Trichuris, Trichosomoides, Capillaria, etc. Species belonging to these genera have no free-living larval forms and depend for distribution not upon any migratory effort on their own part but rather upon direct transmission of the embryos or eggs into the new host. Trichina, as is well known, is spread by the ingestion of the uncooked flesh of the host con- taining the encysted immature worms. The gravid female of Hepaticola hepatica Hall dies in the liver tissue of the host, leaving there a mass of eggs which have no way of reaching the exterior or attaining a new host. In such a case, cannibalism seems to be the only agent upon which the species can depend for propagation. From the point of view of such extreme parasitism in this respect and further from the loss of one or both spicules of the male, the absence of one testis and one ovary in the respective sexes and the oviporous or ovoviviparous condition of the female one might be justified in suspecting that the capillary oesophagus is a degenerating ' ‘ * ' . -56- one, departing in this respect from the normal form. With regard to the symmetry, thie type is bilateral, if the row of large nucleated cells of the structure is dorsal and the capillary tube remains in a ventral median position. 2. Cephalic modifications and relations to habitat The transition from a free to a parasitic mode of life brings with it profound modifications in the organism. These changes are most admirably seen when a comparison is made of the fascinating and intricate structure of free living nema- todes with the gross and rather monotonous anatomy of the para- sitic species. Sensory bristles, cephalic setae, ocelli, and other sensory structures as well as the amphids found in free forms are among the first organs to disappear after the assump- tion of parasitism. Then the digestive system is progressively altered most noticeably in the cephalic region - the oral and pharyngeal structures. Concommitant with the general simplifica- tion and loss of structures, there is a rather progressive hyper- trophy and complication of the reproductive systems, because as parasitism increases the chances for favorable propagation are lessened. Further adaptations are seen in the production of cuticular cephalic expansions, as the lateral alae of Oxyuris tet rapt era von Linstow, the cuticular bosses of Gongylonema musculi Newman, the ventral cuticular combs or spines of Rictu- laria or the hook beset head of Echinocephalus, etc., which are . . ■ . ' . ■ , ■ . - 57 - only few of the possible variations. In a general way, these outgrowths are adaptations which form excellent hold-fast or- gans, as they are found only among those species which inhabit the alimentary canal. At this point it might be well to consider the possi- bilities of specialization of the nematode parasite with respect to its habitat. Specialization among the parasitic species is usually the opposite of that among free living forms, for with ♦ few exceptions (hold-fast organs and reproductive organs) the term implies a simplification or a loss of existing structures. The roundworms living in the posterior and anterior portions of the alimentary system of their host more nearly approach the free living forms than any others. For example, those living in the caecae and large intestine of animals (Oxyuris, Ascaris) live largely upon the bacterial flora because most of the split proteins and carbohydrates of the digested food have been re- moved from the intestinal contents by absorption long before these regions are reached. In the stomach little actual hydrol- ysis of the food is accomplished so that nematodes inhabiting this organ must use and digest to a large extent the food pre- sented them by the host. This fact is demonstrable in Proto- spirura muris inhabiting the stomach of the common mouse, for in this species, the intestine is usually filled by minute frag- ments and starch granules derived from the hosts diet of grains. Other species such as those of Ancylostoma actually feed upon the intestinal papillae according to the observations of Loose. ■ . ' ' 2 -58- From a parasitic point of view, the most highly spec- ialized nematodes are those inhabiting the circulatory system (Filaria), the body cavity and the connective tissues (Gongy- lonema and Dracunculus) . They must needs depend for their nour- ishment upon the absorbed food products circulating in the blood and lymph with which they are bathed and from which they osmoti- cally acquire the necessary elements for their own metabolism. One would expect in such a case to find a correlation between extent of parasitism in connection with cephalic structure, and such is indeed true as will come out in following discussions. 3. Pharyngeal modifications Pharynges among the parasitic round worms are not as diverse in form nor as complicated in structure as many of those in the free living nematodes. There are no integradat ions from a simple conoid pharynx, through the various changes of size and armature to the fusion of parts and formation of spears which one finds among the nonparasitic genera. Indeed the phar- ynges are few and are prominent only among the Strongyles where one finds them exceptionally well developed. The spear bearing forms exist only among the phytoparas i tic nematodes as Heterodera and Tylenchus but, as these round worms are to a large extent free living, they cannot be adequately classed with the more parasitic animal forms. The simplest pharynx is really little less than a circular vestibule in many species; For example, Protosoirura * ■ ' ' -59- muris (Fig io^U). illustrates this quite well as a short cylindrical or faintly prismatic passage leading from the lips into the oesophagus. Other instances of such a simple pharynx may be found in such nematodes as Eustrongylides ignotus JAgerski$ld and related species. The most elaborate pharynges are, however, found among the Strongyles where they are often large and capa- cious, almost smooth, or else armed by teeth and cutting plates. These structures are prominent among the characteristic features of the tribes Strongyleae, Bunostomeae, Ransomeae, Cylicostomeae, and a few genera of undetermined tribal relations. But since this capsular formation is so intimately connected with oral structure, its nature will be described in the following para- graphs in connection with modifications of the lip region. 4. Oral structure When the oral armatures of the parasitic nematodes of the alimentary tract are examined, one finds as a common thing various modifications of lips unarmed or armed with teeth or cuticular thickenings. Three lips are present in the most prim- itive of parasites, namely genera of the Oxyuridae, Heterakidae and Ascaridae,of which the members of the last family show a great number of modifications. In Heterakis papjllosa Bloch the three lips are small and equal. A similar equality and tri-radiality exists in Falcau 3 tra siamensis Baylis; the lips are somewhat flattened antero-posteriorly except for two pro- minent papilla bearing projections on each (Fig. t • - 70 - IV. Ciliation Among Nematodes. (a) Form er vie ws on ciliation and present data. It has been a generally held view that nematodes in oompany with arthropods form the two animal groups totally devoid of cilia at any stage in their existence. Fasten, however, disproved the opinion for arthropods by demonstrating cilia as being present in the reproductive ducts. Shipley in remarking upon the absence of cilia in these two groups believes the con- dition is correlated with the tendency to form cuticula among nematodes and in arthropods with the great proclivity for chitin- ization. This view is possibly correct when one examines the variations of intestinal linings among different species of nematodes. Prenant, who has made a special study of cilia and ciliary modifications has recently placed in the category of "bordure en brosse" the characteristic intestinal lining of Ascaris megalocerhala. An examination of one of his original preparations of the sectioned intestine of this ascarid, shows the lumen-ward end of the cells covered by a thick, rather finely striated border. The elements, however, composing this border are not separately distinguishable for the whole lining is a unit. Just beneath this border is a distinct row of darkly staining . -71- basal granules which, too, are more or less confluent and followed by a relatively deep homogeneous zone. This latter portion gives way to the subcentral granular zone through the middle of which is a relatively clear space. The nuclei are basal and lie in a re- gion containing numerous filaments running the long way of the cell. Other authors in treating of this border have pictured the same condition but called it either a cuticular border or a "stabchensaum" . Looss found a similar striated border upon the intes- tinal cells of Aucylostoma duodenale but in the cases where indi- vidual rods or elements were visible and separate he attributed to a degeneration of the border as such were usually seen in adult worms, the younger specimens exhibiting a more united and homogeneous appearance. Such a feature is, I think, due partly to the fixation of the material as I will mention later. Martini shows in his studies upon Oxyuris curvula iden- tical structures in the intestinal cells (figs. 2 and 3, pi. 4) and calls the lining a "stabchenbesatz" . In an alcohol prepar- ation the stabchen are not clearly separated but in another a gold chloride one, the lumen ends are apparently free. Following the border is an indistinct layer of basal granules from which fibrils may be traced rather indistinctly into the body of the cell proper. Rauther also in working upon Enophus describes the intestinal cells as covered by a "stabchensaum" (Fig. 1, pi. 4) . . . . -72- In his illustration the distinctness of the striae is unmistakable and a splendid basal granule layer is present where each granule is identifiable as well as the fibres running from them into the cell itself. Another example of apparent ciliation is to be found in Ichthyonema pellucidum in the intestinal canal (Pig. 5, pi. 4). Jagerskiold in describing it states that the lining of this mater- ial did not resemble the "stabchenlage" of most nematodes because the little rods were widely separated and quite long. He did not, however, examine any fresh material and concludes, "so glaube ich nicht fehlzugreifen, wenn ich es als eine eigenthumlich ausgebildete stabchenlange und nicht als ein Wimperkleid betrachte." An interesting feature of these "bordures en brosse" is that there is no vibratile motion in the cilia. Cobb, 1898, makes this statement, "Toward the end of the seminal vessel, near the ejaculatory duct, the epithelium bears projections having am- oeboid movements or cilia having active vibratile motions. This latter interesting fact, first made known by the author, is of special importance as being the first discovery of a ciliated eipthelium among nematodes, a tissue which had been supposed not to exist in the group, and the supposed absence of which had given rise to phylogenetic speculations." He does not, however, present any drawings or name the species in which such a condition occurs. During some observations upon Protospirura muri s. the author was particularly struck by the apparent beautiful ciliation ■ * , ' -73- of the intestinal cells as they appeared in sections prepared from material killed in Carnoy- phenol. Previous examination of sections from specimens killed by Looss* method, showed a more or less hyaline structurless intestinal lining broken into bristle-like portions here and there which, since they were found in mature specimens, were interpreted as degenerating portions of the cuti- cular lining. More material was killed in Flemming's mixture with- out acetic acid and sectioned. The cells exhibited the same ciliation which, too, was present in a very young specimen fixed previous to its last moult. For further proof of the ciliary nature some fresh, living nematodes were collected and the intestine examined immed- iately in normal salt solution under dark field illumination and oil immersion. The individual cilia could very plainly be seen in both cases but no motion of their own was ever noted. They would, however, wave back and forth in response to currents of water flowing through the intestine when the slightest pressure shifted the coverglass. In sections (Fig. 4, pi. 4) stained with Dobell's iron hematein one may distinguish the long cilia, an indistinct, rather fused row of basal granules and the fibrils extending into the cell body. Usually the middle portion of the cell is very granular and at times alveoler in appearance so the fibrils disappear but sometimes reappear in the region of the nucleus. Such cells are structurally identical with other ciliated cells and differ physiologically only from lack of . . * . , . ■ . . . . , . 1 - r s • * . - ' - 74 - mot ile cilia. (b) Sign ificance of ciliation . From a morphological point of view this loss of motion and graded fusion of cilia indicates a retrogression and an atrophy because the divers parts constituting the vibratile apparatus become less and less evident until they are finally obliterated. But from a physiological point of view, this regression, when it is a case of differentiation, is a step in ad- vance, for there results the formation of new organs with new func- tions. In the case of nematodes the possession of cilia, though immotile, is, I think, to be construed as a hang-over from a more primitive condition of active ciliation. This being the case, the way is open for phylogenetic speculations and a still closer possible relationship can exist then between the nematodes and the rotifers and gastrobricha to which the roundworms at present seem most related. With a ciliated alimentary tract throughout , as roti- fers have at present, the ancestral nematode would necessarily have had no need of a muscular sucking oesophagus nor lips nor pharynges. A simple circular mouth would be the most logical one, such as many of the rotifers possess. Perhaps then the very structureless mouth region of some of the simpler marine nematodes mentioned in the fore part of this paper, possess the most prim- itive form of oral structure — circular mouth, no lips, indis- ■ . , . " V 1 W ? ; . — ^ .. -’V - 75 - tinct papillae and no pharynx — in contradistinction to the three lipped form described by Seurat as the most primitive con- dition of oral structure. Loss of ciliation may have arisen by the propensity for cuticularization and by some other unknown change or cause, the simple ciliated oesophagus became a muscular sucking organ to carry on the process of acquiring food after ciliary motility had given place to non-motility as evinced by the "bordures en brosse" of species today. . ' - 76 - V. DISCUS SION AMP C ONCLUSIONS . Regarding orientation of the primitive nematode with respect to its surroundings Steiner* s view maintaining a position perpendicular to the substrate and the half-sessile mode of life seems to be tenable and is well borne out by many of the free- living forms living on and about marine algae and particularly by those worms possessing eye spots with the lenses vertically oriented. The crawling mode of travel engaged, in by many nema- todes as they lie upon a lateral surface is a secondarily ac- quired mode of locomotion. Furthermore, the vertical orientation suggests that possibly the ancestor of the nematode, in vi ew of cilia being present, was a free swimming pelagic elongate animal which, after assuming the tendency to cuticularize , settled down to a half-sessile life. The ancestral mouth, if the digestive tract were ciliated was possibly ventral and circular, opening into a ciliated oesophagus, only slightly muscular or not at all so, and in all probability the anus of such an individual would be terminal as well as the openings of the excretory system. This is in accord with single openings of these systems spoken of by Seurat in the definition of the primitive nematode. Such an ancestor might easily be derived from a trachophore form by extensise elongation and a partial migration of the mouth anteriad. The symmetry of such an individual would be bilateral which is in accord with the fundamental bilaterality of the nematode. -77- The structural units, lips, jaws, and capsule, proposed by Ward primarily for the parasitic nematodes, are equally applicable to free-living forms, but here there are intergradations from one form to the other so that as a means of grouping the free-living roundworms, these terms are too concise and do not permit of placing many intermediate conditions. From the foregoing discussion and the data in the pre- ceeding sections, the following conclusions may be drawn: 1. Cilia are present in nematodes in a non-vibratile form but structurally identical with vibratile cil- iated cells. They indicate specialization. 2. The nematode, ancestor was probably ciliated through- out its digestive tract, possessed perhaps external cilia, a ventral, simple mouth, and terminal anus. 3. Loss of ciliation was succeeded by a half-sessile life and tendency toward cuticularization . The mus- cular oesophagus arose as a pumping organ. 4. The fundamental symmetry is bilateral. True radial symmetry is very noticeably in the cephalic region and is a secondary condition resulting, perhaps , from the sessile tendency. 5. Li- symmetry is tertiary as in asymmetry. 6. The simplest and perhaps most primitive cephalic organization was and is in some forms today, a round -7 8- or triquetrous mouth opening directly into the triquetrous oesophagus, with small or indistinct oral papillae. Successive complication and evo- lution of structure w as probably in the following order: (a) Three lipped forms with no pharynx. fb) Three lipped forms with developing pharynx. fl) Two-lipped forms with no pharynx arising from loss of the dorsal lip. (2) JawH arising from loss of upper or dorsal cephalic sector and mi- gration laterally of the two sub- ventral sectors. (c) Multiple lips by division of the primitive number. (d) Partial fusion of multiple lips and develop- ment of large armed pharynges. (e) Capsule formed by susion of lips in connection with a large pharynx armed generally by strong onchi. fl) Spear-bearing forms from fusion of lips, elongation of pharynx, fusion and separation of onchi from the phar- yngeal walls to form the buccal sty- let moved by special muscles. 7. Di-symmetry is more noticeable among parasitic forms than among free-living forms. 8. Cephalic organization has kept pace with other special- * - 70 - izations and bears a relation to habitat and is indi- cative in a general way of the evolutionary status of a genus. - 80 - List of Free-living Species Cited 1. Alaimella cincta Cobb 2. Alaimella truncata Cobb 3. Anaxonchium litorium Cobb 4. Bunonema inequale Cobb 5. Cephalobus oiliatus von Linstow 6. Cheironchus vorax Cobb 7. Diploscapter coronatus Cobb 8. Dorylaimus labiatus de Man 9. Gammonema ferax Cobb 10. Heth juli Cobb 11. Ionema ocellatum Cobb 12. Iotodorus punctatus Cobb 13. Litotes minuta Cobb 14. Mesonchium poriferum Cobb 15. Monhystera pilosa Cobb IS. Monhystera stenosoma de Man 17. Mononchus gerlachei de Man 18. Mononchus muscorum Bastian 19. Mononchus radiatus Cobb 20. Mononchus zschokkei Menzel 21. Nemella ocellata Cobb 22. Notochaetosoma tenax Irwin- Smith 23. Onchullela ocellata Cobb 24. Pomponema mirabile Cobb - 81 - 25. Rhabditis lambdiensis Maupas 36. Rhabditis pellio Btitschli 27. Scaptrella cinota Cobb 28. Selachinema ferax Cobb 29. Sphaerolaimus hirsutus Bastian 30. Spira parasitifera Bastian 31. Synonchium obtusum Cobb 32. Teratocephalus crassidens de Man 33. Terschellingia longicaudata de Man 34. Thoracostoma antarcticum von Linstow 35. Thoracostoma lobatum Cobb 36. Thoracostoma setosum von Linstow 37. Tycnodora pachydermata Cobb 38. Tylopharynx stricta de Man 39. Wilsonema capitatum Cobb 40. Xyala striata Cobb ' . . ■ - 82 - List of Parasitic Species Mentioned Aoanthocheilonema diacantha Molin Syn. Tilaria diacantha Molin Anoylostoma duadenale Dub ini Ascaris holoptera Rudolphi Ascaris nasuta Schneider Ascaris osculata Rudolphi Diaphanocephalus costatus Diesing Diplotriaena diuce Boulenger Dracunculus medinensis Velsch Euatrongylides elegans vonOlfers S yn. Strongylus pappillosus Rudolphi Strongylus mergorum Rudolphi Strongylus elegans Rudolphi Strongylus tubifex Rudolphi Eustrongylus tubifex Die sing Hystrichis tubifex Molin Hystrichis elegans Railliet Hystrichis pappULosus von Linstow Tropidocerca paradoxa von Linstow Eustrongylides ignotus Jagerskiold Syn. Eustrongylus papillosus Diesing Eustrongylus tubifex Schneider Hystrichis papillosus Molin : . ... - 83 - P ilaria bancrafti Cobbold Syn. Trichina cystica Salisbury Tilaria sanquinis hominis Lewis Tilaria sanquinis hominis aegyptiaca Sonsino Tilaria sanquinis hominum Hall Tilaria sanquinis hominis nocturna Manson Tilaria nocturna Manson Tilaria Wuchereri da Silva Lima Gnathostoma spinigerum Owen Syn. Chieracanthus robustus Liesing Chieracanthus robustus Lie sing Chieracanthus socialis Leidy Chieracanthus siamensis Levinsen Tilaria radula Schneider Gnathostoma paronai Porta Gnathostoma spinigerum Mitter Gongylonema musouli Neuman Syn. Gongylonema minimum Molin Tilaria musculi Rudolphi Hystriohis acanthocephalicus Molin Heterodera schachtii Schmidt Kalicephus inermis Molin Syn. Strongylus colubri jararaca M.C.V. Strongylus crotali M.C.V. ■ % - . - 84 - Ophidasoaris mombasica Baylis Oxyuris equi Schrank Syn. Oxyuris curvula Rudolphi Oxyuris hydro! Galeb Oxyuris monhystera von Linstow Oxyuris obesa Diesing ft Oxyuris obvelata Syn. Ascaris vermicularis muris e Froelich Ascaris obvelata Rudolphi Eusaria obvelata Zeder Asoaris oxyura Nitzsch Syphacia obvelata Seurat Oxyuris tetraptera von Linstow Syn. Ascaris dipodis Rudolphi Ascaris tetroptera Nitzach Oxyuris semi lance data Molin Oxyuris obvelata Dujardin Polydelphis quadricornis Wedle Syn. Ascaris quadricornis Wedl Ascaris quadricornis Stossich Ascaris quadrangularis Schneider Ascaris quadrangularis Stossich Ascaris quadrilobata von Linstow Protospirura ascaroidea Hall 1 - 85 - Protospirura labiodentata Hall Syn. Spiroptera labiodentata von Linstow Protospirura muris Gmelin Syn. Lumbrici muris Werner Ascaris muris Gmelin Ascaris obtusa Proelich Pusaria muris Zeder Spiroptera obtusa R. of Parona Tilaria muris Stossich Spiroptera brauni von linstow Solerostoma equinum Mueller Set aria equina Abildgaard Spirocera subaequalis Molin Tanqua tiara von Linstow Syn. Ascaris tiara von linstow Ctenocephalus tiara von linstow Tetradonas tiara von Linstow Triohosomoides crassicanda Bellingham Syn. Trichosoma crassicanda Eberth Trichocephalus crassicanda Eberth Trichodes crassicanda von linstow Trichosoma muris decumani Bayer of Stossich Triodontophorus minor looss *. - 86 - Bibliography. Bastian, H. C. 1864. Monograph on the Anquillulidae , £6:73-184., 5 pi. Trans. Linn. Soc., Bastian, H. G. 1866. On the Anatomy and Physiology of the Hematoids, Para- sitic and Free, Trans. Roy. Soc., 1866., 545-638., 7 pi. Baylis, B. A. 1916. On Crassicanda crassicanda (Grepl. ) and its Hosts. Ann. and Mag. Hat. Hist. (8), 17:144-148. 1916a. Some Ascarids in the British Museum. Paras. 8: 360-378., 4 pi. 1919 . Crossophorus eollaris, Hemprich and Ehrenberg, a lit- tle known Hematode Parasite of the Hyrax. Ann. and Mag. Hat. Hist. (9 ) :4 :343-349 . 1980. Observations on the Genus Crassicanda. Ann. and Mag. Hat. Hist., (9) :5:410-419. Baylis, H. A. and Lane, C. 1980. 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Ueber die lippen einiger Oxyurisarten,Zeits. fur Wissen. Zool., 19:234-243, 1 taf. Organisation et developpement des Oxyurides., Arch, de Zool. Exp. et Gen., 7:1-107, 9 pi. Notes sur la faune parasitaire du Congo Beige., Renne Zool. Africaine., 5:1-90. -90- Gehuchten, 1892. A. Van Contribution a L'Etude du Mecanisme de L* Excretion Cellulaire. La Cellule. 9:95-116, 1 pi. Gilson. G. 1906. Un nouveau medium solidifiable pour le montage des preparations microscopiques. Goldschmidt, R. 1908. Das Hervensystem von Ascaris lumbricoides und megalo cephala. , I., Zeit. f. wiss. Zool., 90:73-156, 3 taf. Guyer, M. F. Animal Micrology. 1917. Revised Ed. 289 pp. Hall, M. C. 1916. Uematode parasites of mammals of the orders Rodentia, Lagomorpha, and Hyracoidia, Proc. U.S. Hat. Mus., 50:1-258, 1 pi. Halle z, R. 1885. Recherche s sur L'Embryogenie et sur les conditions du developpement de quelques Hematodes., Mem. de la Soc. des Sci. de Lille, (4):15:5-71, 4 pi. 1887. Anatomie de L*Atractis Dactylura (Orig.), Ibid., 5-20, 1 pi. Hofmanner, B. 1913. Contribution a l 1 etude des Hematodes libres du lac Leman., Rev. Suisse de Zool. 21:589-658, 2 pi. Hofmanner, B. and Mezel, R. 1915. Die freilehenden Hematoden der Schweiz, de Zool., 23:109-243, 3 pi. Rev. Swisse -91- Ihle, J. E. W., and VanOordt, G. J. 1920. On the larval development of Oxyuris equi (Schrank), Konin. Akad. van Weten. Te Amsterdam, 23:1-10. Irwin-Smith, Vera A. 1917. On the Chaetosomatidae . , Proc. linn. Soc. N.S.W., 42:757-814, 7 pi. Jagerskiold, L. A. 1894. Beitrage zur Kenntnis der Nematoden, 2ool. Jahr. , 7:449-532, 5 taf. 1897a. Ueber den Oesophagus der Nematoden., Kongl. Svenska Vet. - Akad. Handlingar, 23:3-26, 2 pi. 1901a. Weitere Seitrage zur Kenntnis der Nematoden, Eongl. Svenska Vet. - Akad. Handlingar, 35:3-80, 6 taf. 1909. Nematoden aus Agypten und dem Sudan, Results of the Swedish Zool. Exped. to Egypt and the White Nile, 1901, No. 25;l-66 , 4 taf. 1909a. Zur Kenntnis der Nematoden - Gattinngen Eustrongylides und Hystrichis, Nova Acta Regiae Soc. Upsaliensis, (4 ) :2 :l-48 , 5 taf. Jammes, 1. 1894. Recherches sur 1* organisation et le developpement des Nematodes, 205 pp., 11 pi. Johnson, G. E. 1913. On the Nematode of the Common Earthv/orm, Quat. Jour. Micro. Sci., 58:605-652, 1 pi. 1895. Eorschelt und Heider, Textbook of Embryology. , Part I., 484 pp. lee, A. B 1913 The Microtomist 1 s Vade-Mecum., 7th. ed -92- Leuckart R. 1852. Ueber das Vorkommen und die Verbreitung des Chitins bei den wirbellosen Thieren., Archiv. fur Raturge- schichte, 18:22-28. 1887. Reue Beitrage zur Kenntnis des Baues und der Lebens- geschichte der Rematoden, Abhand. der mathemat. - phys. Classe der Konigl. Sach. Gesell. der Wissen. , 13:567-704, 3 taf. Leydig, F. 1885, Zelle und Gewebe, 219 pp. , 6 taf. Linstow, 0. v. 1897. Rema the Imin then, Archiv. f. Raturgesch. , 1897: 281-291, 2 taf. 1899. Rematoden aus der Berliner Zoologischen Sammlung, Mitteil. Zoolog. Samml. Berlin, 1:5-28, 6 taf. 1902. Atractis cruciata und Oxyuris monhystera, zwei neue Rematoden aus Metopooeros comutus, Gentralblatt f. Bakteriologie. , Abt. 1:31:28-32, 1 taf. 1904. Rematoda in the Collection of the Colombo Museum Spolia Zeylamica of Ceylon, 1:1-14, 2 pi. 1906d. Parasites from the Gharial Garialis gangetious, Geoffr., Jour, and Proc. Asiatic Soc. of Bengal n.s. 2:269-271, 1 pi. 1906e . Ostpruussische Rematoden, Schriften der Physik. okonom. Gesellschaft zu Konigscherg. i, Pr. 47:111-114, 1 pi, 1906f. Rematoden des zoologischen Museums in Konigsberg, Archiv. fur Raturgeschichte , 1:248-258, 3 taf! 1907c. Rematoden aus den Koniglichen Zoologischen Museum in Berlin, Mitteil. aus d. Zoolog. Museum in Berlin. 3:251-259, 2 pi. 1909a. Parasitische Rematoden, Die Susswasser fauna Deutschlancfe 15 :47-83 . looss, A. 1901. The Sclerostomidae of Horses and Donkeys in Egypt. , Rec. of Egyptian Gov*t School of Med., 1901, 27-138 13 pi. , * . . . . -93- Looss, A. (continued) 1901. Zur Sammel - und Gonser vie rungs technik von Helmin- then, Zool. Anz., 24:302-304; 309-318 Review, Jour. Appl. Micros., 1900-01:1580-1582. 1905. The Anatomy and Life History of Agchylostoma duo- denale, Dub., A Monograph, Part I. Ibid. 3:11-158, 10 pi. 1906. Ibid. Part II. Ibid. 4:163-613, 9 pi. luk^anow, S. M. 1888. Notizen uber das Darmepithel bei Ascaris mystax. , Archiv. f. Mikros. Anat. , 31:293-302. MacCallum, G. A. 1918. Rotes on the genus Camallanus and other Nematodes from various hosts., Zoopath., 1:125-134. Magath , T . B . 1916. Nematode Technique, Trans. Am. Micro. Soc., 35: 245-256. 1919. Camallanus americanus nov. spec.. Ibid., 38:49-170, 10 pi. Man , J . G . 1886. de Anatomische Untersuchungen Uber Preilebende Nordsee - Nematoden. , 82 pp., 13 taf. 1895. Description of three Species of Anguillulidae , ob- served in Diseased Psuudo-Bulbs of Tropical Orchids, Trans. I* pool. Biol. Soc., 9:76-94, 3 pi. 1904. Ein neuer freilebender Rundwurm aus Patagonien, Bericht der Senchenberg. Naturf. Gesell. in Frankfurt a. m. 41-46 pp 1904a. Resultats du Voyage du S. Y. Belgica en 1897-1898- 1899, Zoologie, Nematodes Libres, 3-51 p. 11 pi. -94- Man, J. G. de (continued) 1 1907a. Sur Omelques Especes Nouvelles ou Peu Connues de Nematodes Litres Habitant les Cotes de la Zelande, Mem. Soc. Zool. d. Prance, 20:33-90, 4 pi. 1907-08. Contribution a la Connaissance des Nematodes Libres de la Seine et des Environs de Paris, Ann. d. Biol. Lac., 2:9-29, 3 pi. 1910. Beitrage zur Kenntnis der in dem weissen Schleim- fluss der Eichen lebenden Anguilluliden, Zool. Jahrb. , 29:359-394, 3 taf. 1912. Odontopharyns longicaudata n. g. n. sp., Zool. Jahrb., 33:637-642, 1 taf. 1917. Beitrag zur Kenntnis der in Norvegen Prei in der Reinen Erde Lebenden Nematoden, Tijdschr. d. Ned. Dierk. Vereen, (2) 16:103-118, 1 taf. 1919. Die Prei in der Reinen Erde und im Sussen Wasser Lebenden Nematoden der Niederlandischen Fauna, Ge- kurzte Ausgabe, 176 pp. 1919a. Atlas for above ref. , 34 tafeln. Marchi, P. 1871. Monographia sulla storia genetica e sulla anatomia della Spiroptera obtusa Rud., Mem. r. Accad. Sci. Torino., (2) 25:1-30, 2 pi. Mart in , 0 . 1910. Beitrage zur Kenntnis der Verbreitung und Entwick- lung des Sklerostomum edentatum Looss., Archiv. f. wissensch. u. prakt. Tierheilkunde , 37:5-50, 1 pi. Martin, E. 1906. Uber Subenticula und Seitenfelder einiger Nematoden, I., Zeits. fur Wissensch. Zool., 81:699-766, 3 pi. 1907. Ibid. II,, Ibid, 86:1-54, 3 pi. -95- Martin, E. 1908a. (continued ) Ibid. III., Bemerkungen uber determinierte Ent- wicklung. Ibid. 91:191-235. 1909e . Ibid. IV. Tatsachliches. Ibid. V. Zusammenfassende und theoretische Betrach- tungen. Ibid. 93:535-624, 2 pi. 1913. Uber die Stellung der Nematoden im System. , Verhand. der Deut. Zool. Gesell., 23:233-248. 1916. 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Etude des cellules a membranelles dans les brenchies et les tentacules de quelques groupes d 1 Invertebrates , Archiv. d'anat. micro., 16:305 344, 2 pi. Railliet, A. 1900. Observations sur les Uncinaires Des Canides et Des Felides, Archiv. d. Paras., 3:82-95. Railliet, A. and Henry, A. 1903. Une Forme Larvaire de L'Oxyure du Cheval, Ibid. 7:133-137. 1909. Sur la Classification des Strongylidae : I. Metastrongylinae , C. R. Soc. Biol., 66:85-88. 1909. Sur la Classification des Strongylidae: II. Ankylostominae, Ibid. 66:168-171. 1912. Les Oesophagostomiens Parasites de I'Hoipne, Archiv. d. Paras., 14:562-583, 3 pi. 1913. Sur les Oesophagostomiens des Ruminants, Bull. Soc. Path. Exotique, 6:506-511. 1915. Sur les Nematodes du Genre Camallanus Raill. et. Henry, 1915, Ibid. 8:446-452. 1916a. Sur les Oxyarides, C. R. Soc. Biol., 79:113-115. 1916b. Nouvelles Remarques sur des Oxyurides, Ibid. 79:247-250. Rauther, Max. 1905. 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Sur devolution des Nematodes parasites, IXe. Congres internat. de Zoologie tenu a Monaco., 623-643. 1916e. Contribution a l'Etude des Formes Larvaires des Nematodes Parasites Heteroxines, Bull. Sci. de la France et de la Belgique, (7) 49:297-377. 1917a. Filaires des Reptiles et des Batraciens, Bull, de la Soc. d*Histoire Naturelle, 18:236-242. 1919. Contributions Nouvelles a 1* Etude des Formes Larvaires des Nematodes Parasites Heteroxines, Bull. Biol, de la France et de la Belgique, 52:344-378. • Considerations sur la Geonemie des Nematodes, Comptes rendus des seances de la Societe de Biologie, 82:986-990. 1919a , > . * ■ c • \ r . . , . * r . 1 1 ■> -98- Seurat, L. G. (continued) 1920. Eistoire Naturelle des Nematodes de la Berberie, Premiere Partie, 221 pp. Shaw, H. B. 1916. The Sugar Beet Nematode and its Control, Sugar, 1916, 3-55. Shipley, A. 1910. Nemathelminthes, Cambridge Natural History, 2. Steiner, G. 1919c. Untersuchungen uber den allgemeinen Bauplan des Nematodenkorpers , Zoolog. Jahrbucher, 43:1-96, 3 taf . 1921. Beitrage zur Kenntnis mariner Nematoden, Ibid. 44:1-68, 4 taf. 1921a. Ost-asiatische marine Nematoden, Ibid. 44:195-226, 3 taf. Stossich, M. 1889. II Genere Physaloptera Rudolphi, Bollettino della Societa Adriatica di sci. nat. in Triesta, 9:1-24, 3 tav. 1890. II Genere Trichosoma Rudolphi, Ibid. 12:3-38. 1891. II Genere Disoharagus Dujardin, Ibid. 13:1-28, 3 tav. 1896. II Genere Ascaris linne , Ibid, 17:3-114. 1897a. Pilaire e Spiroptere, Ibid. 18:13-162. 1899a. Strongylidae . Ibid. 19:55-152. -99- Travassos, 1. 1921. Contribuiooes para o conheciraento da fauna helmint- olojica brasileira, Mem. do Instit. Osv/aldo Groz. 13:5-140., 58 pi. Ward, H. B. and Magath, T. B. 1916. Notes on Some Nematodes from Fresh-Water Fishes, Joum. Parasitol. , 3:57-64, 1 pi. Ward, H. B. 1916a. Nematoda, Ref. Handbook of Med. Sci., 676-704. 1917. On the Structure and Glassification of North American Parasitic Worms, Journ. Parasitol., 4:1-12, 1 pi. Ward, H. B. and Whipple, G. C. I3ie>. Fresh-Water Biology, 1111 pp. Washburn, E. W. 1921. Principles of Physical Chemistry, 511 pp. Willows, R. S. and Hatschek, E. 1915. Surface Tension and Surface Energy, 80 pp. - 100 - VITA Duncan C. Hetherington was born August 8, 1895, in Denver, Colorado, but his elementary training was received in the public schools of Colorado Springs, Colorado. He graduated from high school with high honors in June, 1915, and in the fall of the same year entered Colorado College, where all his under- graduate collegiate work was taken. During his junior year he was laboratory assistant in elementary zoology and in the fourth year had full charge of experimental physiology. As a senior he was elected to Phi Beta Kappa, and obtained in June, 1919, the degree of B.A. cum laude . The following year he entered the University of Illinois as a scholar in zoology, re- ceiving in June, 1920, the degree of Master of Arts. The same year he received election to associate membership in Sigma Xi . For each of the two succeeding years he was granted a fellowship in zoology, permitting application of full time to graduate work. The summer of 1921 was spent in attendance at the Puget Sound Biological Station fulfilling a requirement for the degree of Doctor of Philosophy which was granted in June, 1923. Publications: Notes on a Microsporidian Parasite of a Nematode, R. Kudo and D. C. Hetherington, in press. Jour. Paras., March, 1922. ♦ - 101 - Explanation of Plates Plate 1 Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10. Oxyuris obvelata. Head of a female en face showing the 3 lips and the 6 papillae disymmetrically arranged. After Fldgel. x 720. Teratocephalus crassidens. Lateral view showing the peculiar flanged lips. After de Man. x 240. Mononchus gerlachei. Head en face showing the 6 par- tially fused lips, the pharyngeal armature, the large dorsal tooth and the superficial radial symmetry. After de Man. x 690. Same as Fig. 2, dorsal view. After de Man. Anguillula aceti. Head of female en face, showing fused lips and hexagonal mouth opening. After de Man. x 1440. Cephalobus ciliatus. Showing the peculiar prolongations of the lip3. After de Man. x 240. Jointed mandibles of Scaptreila cincta inflexed and ex- tended. After Cobb, x 600. Optical cross section of the posterior portion of buccal cavity of a growing female, Anguillula aceti. After de Man. x 2160. Cross section of pharynx of Mononchus muscorum showing the sutures and the dorsal tooth. After Cobb, x 450. Head of Protospirura muri3 showing the di symmetry of the two tri-partite lips. Original red. x 0.6. ' ' -103- Plate 2 Fig. 1. Tanqua tiara, showing the collar and the two lateral lips Dorsal or ventral view. After Baylis and Lane x 117 ± Fig. 2. Crossophorus collaris. Diagrammatic representation of lip and fimbriae relations and symmetry. After Baylis and Lane . Fig. 3. Crossophorus collaris. Dorsal view showing the fimbriae and the two symmetrically placed papillae of the dorsal lip. After Baylis and Lane, x 58 i Fig. 4. Agchylostoma caninum, showing the ventral teeth and cap- sule. After Looss. x 100. Fig. 5. Kathleena arcuata. Head en face showing apparent tri- radial symmetry and large intsrlabia. After Godoelst. Reduc . x 0.5. Figs. 6 and 9. Kathleena tricuspis. Head en face showing similar features as above. Also dorsal view of head. After Godoelst. Reduc. x 0.5. Fig. 7. Lateral lip of Ascaris ferax (Crossophoris collaris) showing the asymmetrical arrangement of papillae. After Schneider, x 45. Figs. 8 and 11. Spirocera subaequalis, showing the radial symme- try of the oral region and the buccal armature. After Seurat, x 115. Figs. 10 and 14. Thoracostoma chilensis. (10) Lateral view of head showing location of an ocellus. (14) Dorsal view of oesophagus showing the two ocelli with vertically dir- ected lenses. After Steiner x 188- and x 375 respectively . ■ - 104 - Fig, 12. Ascaris rosmari. Lips viewed from the front. After Baylis, x 90. Fig. 13. Sclerostoraum equinum. Dorsal view showing the leaf- crown, dorsal gutter and large buccal cavity. After Looes. x 29. Fig. 15. Falcaustra siamensis showing true triradial symmetry. After Baylis. x 130. . . ■ — -105- Plate 3 Fig. 1. Gnath03toma spinigerum. Dorsal view showing the two lateral lips and the spine beset head. After Baylis and Lane x 48. Figs. 2 and 6. Triodontophorus minor showing the terminal mouth and three pharyngeal teeth. Front and lateral views. After Loess x 94 ± . Fig. 3. Hystrichis acanthocephalicus, showing the spine beset head and simple triquetrous mouth and six clrcumoral papillae. After J&gerski8id x 90. Fig. 4. Protospirura labiodentata, view en face showing the two tripartite lateral lips and di symmetrically arranged papillae. After Von Linstow. Figs. 5, 7, and 9. Heads of larval Agchylostoma duodenale. (5 and 9) Dorsal and lateral views showing the pharynx, x 372. (7) Head en face showing the triquetrous mouth opening, the 3 fused lips and 6 papillae, x 570. After Loose. Fig. 8. Eu3trongylides elegans, showing the prominent papillae and slight tendency toward disymmetrical arrangement. After J&gerskidld x 44 ± . Fig. 10. Tail region of Thoracostoma acuticaudatum, showing the serial arrangement of the caudal glands. After JAger- skiflld x 87. Fig. 11. Synonchium obtusum, showing the six double confluent lips, the flaring triquetrous pharynx, and its armature of man- dibles. After Cobb x 450. .... . -106- Fig. 13. Fig. 13. Fig. A. Fig. B. Fig. C. Fig. D. Carnal lanus americanus, dorsal view of the oral appara- tus of a female, showing the two lateral valves and the dorsal trident. After Magath x 90. Kalicephalus willeyi. Front view of head showing the lateral approximation of the capsular edges. After von Lins tow. Diagram of planes of triradial symmetry in the nematode oesophagus. Same in cross section. Diagram of cross section showing bilateral symmetry. Diagram showing plane of bilateral symmetry of the nematode oesophagus. i! | 1 1 . ' i -107- Plate 4 Fig. 1. Enoplus sp. Section of the intestine wall showing ciliated border with the basal granules and the fibrillar continuations. After Rauther. x 1440. Figs. 2 and 3. Intestinal cells of Qxyuris curvula. (2) Alcohol prep, showing the "stabchensaum . " (3) Gold chloride prep, showing ciliary structures. After Martini x 1.4 orig. Fig. 4. Intestinal cells of Protospirura muris showing the long separated cilia, the rather indistinct basal granules and the fibrillar extensions within the cell body. The dark bodies within the clear spaces are infections of Thelohania reniformis. Kudo and Hetherington, a micro- sporidian parasite. Original x 2100. Fig. 5. Section of intestinal wall of Ithyonema pellucidum showing separated ciliary structures. After J&gerskiftld x 1.2 orig. ■ * . — * • ••'■ , PLATE II PLATE III