Univ. OF Library BINnTTiTR TT^J'^OFC 1 5 1S24 %* '>V TRANSACTIONS OF THE American Microscopical Society Organized 1878 Incorporated 1891 PUBLISHED QUARTERLY BY THE SOCIETY EDITED BY THE SECRETARY PAUL S. WELCH ANN ARBOR, MICHIGAN VOLUME XLI Number One Entered as Second-class Matter August 1^, 1918, at the Poit-offiie at Menasha Wisconsin, under Act of March 3, 1879. Acceptance for mailing at the special rate of postage provided for in Section 1 103, of the Act of October ?, 1917. authorized Oct. 21, 101R (Tbr (Callrgiate ^reaa CiEOKOE Banta Publishing Company Menasha, Wisconsin 1922 QM aoi A3 V. Lf l-Lf^ TABLE OF CONTENTS For Volume XLI, Number 1, January, 1922 The External Morphology of Lachnosterna crassissima Blanch. (Scarabs idae, Coleop.), with nine plates, by Wm. P. Hayes 1 The Respiratory Mechanism in Certain Aquatic Lepidoptera, with two plates, by Paul S. Welch 29 Department of Methods, Reviews, Abstracts, and Briefer Articles Dichromatic Illumination for the Microscope, with two figures, by L. A. Hausman . 51 A Modified Barber Pipette, with one figure, by Bert Cunningham 55 Cleaning Slides and Covers for Dark-field Work, by S. H. Gage 56 Proceedings of the American Microscopical Society 57 TRANSACTIONS OF American Microscopical Society (Published in Quarterly Instalments) Vol. XLI JANUARY, 1922 No. 1 THE EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA BLANCH (Scarabseidae, Coleop.) ' By Wm. p. Hayes Assistant Entomologist, Kansas Agricultural Experiment Station Introduction The present discussion of the external features of Lachnosterna craisis- sima Blanch, is offered to supply the lack of an available treatise in English on the morphology of the beetles belonging to the family Scarabaeidae. The nearest approach to the subject is the famous historical French work of Straus-Durckheim "Considerations Generales sur L'Anatomie Comparee des Animaux Articules" in which is included a description and many fine drawings of the anatomy of Melolontha vulgaris, Hanneton. This work appeared in 1828 and is a masterpiece of its kind. However, much of the anatomical nomenclature is now antiquated and the work itself hard to secure, consequently the present paper, dealing with a species of a closely allied genus of the same family, is here presented. The nomenclature used by the writer is that which was deemed most useful. It follows no one system of the modern writers but is adopted from such writers as Snodgrass, Crampton, and others. This species of the genus Lachnosterna was chosen for study because of its abundance in the vicinity of Manhattan, Kansas. Of the 23 species found in this vicinity, L. crassissima ranks first in relative numbers. A total of 86945 specimens of this genus have been collected by Mr. J. W. ' Contribution No. 54 from the Entomological Laboratory, Kansas State Agricultural Col- lege. This paper embodies the results of some of the investigations undertaken by the writer in the prosecution of project No. 100 of the Kansas Agricultural Experiment Station. The writer wishes to acknowledge his indebtedness to Dr. P. S. Welch for many helpful criticisms during the course of preparation of this paper and to the American Microscopical Society for a grant from the Spencer-Tolles Fund to publish the accompanying drawings. 1 2 \VM. P. HAYES McColloch and the writer during the years 1916-1920, and of this number 30230 were L. crassissima. The specimens were preserved in 70% alcohol, boiled in potassium hydroxide when ready to use, and studied under the binocular. ^ „ General Considerations Size. — The adults of this species are among the most broadly ovate of the genus Lachnostcrna. They vary greatly in length, width, and maximum depth, and, on an average, the females are somewhat longer than the males, as well as much broader posteriorly. The greatest body depth varies in individuals of both sexes. Not only in size is this difference noticeable, but also in respect to the regions of the body in the two sexes. The ma^es have their region of greatest depth through the thorax, which was found to average 7.1 mm., while the females are deepest through the posterior end of the abdomen where they average 7 .6 mm. Some of the depth varia- tion in the females is due, in part, to the degree of flexibility of the inter- segmental grooves, especially when the abdomen is distended with eggs. Because of the deflexity of both the labrum and pygidium, the maximum length of the males and females was arbitrarily measured from the emargi- nation of the clypeus to the basal or proximal edge of the pygidium and not to the extreme ends as is the usual practice. The average length of 25 males, chosen at random, was 18.2 mm. and, for the same number of females, 18.7 mm. The maximum length of the males was 20 mm. and of the females 20.9 mm., while the minimum was 16.3 mm. for the males and 16.5 mm. for the females. Width measurements were made at seven different regions of the body to get a general notion of the width variation. These regions were chosen arbitrarily as follows: 1 — At anterior margin of the eyes 2 — At anterior margin of prothorax 3 — At lateral angles of prothorax 4 — At base of prothorax 5 — At base of elytra 6 — At bulge near middle of elytra (widest points) 7 — At declivity of elytra near the distal end. The table on the opposite page shows the average width, length, and depth measurements of 25 males and 25 females. In individual specimens, the length measurement is highly variable, depending on the character of the specimens at hand. Alcoholic and living specimens are extensile because of the telescopic nature of the union of head and thorax and to a lesser degree the thorax and abdomen, thus causing differences in length. Dried specimens will, on the contrary, i)er- mit of more constant measurements. Color. — The general mass color of this species is chestnut-brown or castaneous (dragon's blood plus a slight admixture of vermilion Smith's EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA color chart, 1906, p. 154). However, some specimens vary to a dark brown, almost black, in which case a grayish iridescence is vjery apparent. Dorsally, the head, thorax, and elytra are shining and in certain lights a gray color exists. This grayish tinge is a structural color caused by fine striae on the elytra. These striae are also present on the thorax but the iridescence is not so pronounced. The eyes are black and prominent. Ventrally the ground color is castaneous. The thorax is covered with dull, yellow hairs, 1.5-2 mm. long, somewhat sparsely scattered on the pro- thorax, but several times denser on the meso- and metathorax. The abdo- men frequently has a grayish super-color imparted by an adhering exuda- tion which can be scraped off. The legs and antennae are lighter brown, almost ferruginous in color. Table I. — Average Size of Various Body Regions Averape Width Sex Head Prothorax Abdomen Greatest Length Depth At At At At At At At Eyes Anterior Margin Lateral Angle Base Base Bulge Declivity mm. mm. mm. mm. mm. mm. mm. mm. mm. Males. . . 18.2 4.7 .5.2 7.2 7.8 8.4 9.8 7.6 7.1 At thorax Females. 18.7 4.3 5.1 7.8 8.1 8.6 10.. S 8.2 8.2 .\t abdomen Skxi'al Dimorphism }falc. — The clul) of the antennae (Plate I, tig. 4) is about equal in length to the scape (s.) and funicle (fu.) combined. At the middle of the ventral surface the al:)domen is longitudinally depressed and the penulti- mate ventral segment bears a faint transverse carina near its distal end. Frequently, this carina is nothing more than a slightly wrinkled convexity. Immediately behind this carina the last, or ultimate, ventral segment has a deep, rounded fovea whose distal margin is obtusely and angulately emarginate. On the posterior tibia, the inner spur is about one-half the length of the outer and somewhat more slender spur. The pygidium is not gibbous and is more noticeably truncate at the distal end than is this structure in the female. The hind tarsi are longer than those of the female. Female. — The club of the antenna (Plate I, Fig. 5) is about as long as the funicle. The ultimate ventral abdominal segment is somewhat broadly and rather deeply emarginate distally, and the fovea is absent. The pygi- dium is gibbous, smooth, and shining at the apex of the gibbosity and more 4 WM. p. HAYES exposed than in the male. Ventrally, the abdomen is more broadly rounded and shining than that of the male and the longitudinal depression is lacking. The inner spur of the hind tibia is about equal in length to the outer and about as wide as the same spur in the males, while the hind tarsi are shorter than in the male. The tooth of the tarsal claws is somewhat larger than this tooth in the male. Contrast of Body Surfaces Dorsal Surface. — The dorsum of the head, thorax, and abdomen, by a casual examination, appears smooth and shining, but closer scrutiny reveals minute punctures over the entire surface. These punctures are more numerous on the front than on any other part of the body. Here they are closely placed, and often confluent. The clypeus is less densely punctured and the punctures are about equal in density to those on the thorax. A setigerous canthus is found on the eye. On the thorax a faint, smooth, median, longitudinal line is formed by the absence of punctures. Laterally, the punctures are less dense. The lateral margin is serrate and hirsute, and at the base transverse channels extend mesad, failing to reach the median line. The scutellum is large, somewhat heart-shaped, irregularly and less densely punctured. The elytra are likewise irregularly punctured, and five indistinct costse occur on each elytron. Ventral Surface. — Ventrally, the head is dark-brown, sparsely hairy and, in part, concealed under the anterior ventral margin of the thorax. The thorax is thickly covered with pale, yellow liairs about 1 .5 mm. long, and the legs are sparsely covered with shorter hairs. The punctures of the ventral abdominal surface are, on the whole, smaller than the dorsal ones. Each bears a short, recumbent hair and is more widely separated. There are about the same number of punctures per square millimeter as on the upper surface where they are larger l)ut more closely placed. Contour The transverse contour of the body exhibits four distinct geometrical figures in three principal regions of the body. Through the head a narrow, elongated oval is apparent, through the thorax a broad oval, while the abdomen shows a somewhat different contour for each sex. In the female, it is broadly oval, almost circular, and in the male much the same, except for the ventral surface where the oval is .somewhat flattened, due to the fovea OP the lower surface of the abdomen. Body Divisions The three general regions of the body are to be recognized by definite sutures which separate them. The head is the smallest division, being less than one-half the width of the thorax. It is greatly depressed and deflected. From above only the clypeus, front and eyes can be seen, as EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA 5 the labrum is deflected on the under side of the head. The head is tele- scoped within the thorax and connected to it by a sclerite-bearing cervicum or the so-called microthorax. The thorax is next in size to the head, somewhat oblong, being nearly twice as wide as long. The sides are nearly parallel at the base, but con- verge anteriorly. The oval contour of the three segments of the thorax is nearly similar in each with perhaps a more flattened aspect of the pro- thorax. Dorsally, the meso- and metathorax each bear a single pair of wings, those of the mesothorax being modified to form the elytra which cover the metathoracic wings. Each segment of the thorax bears a pair of jointed legs. The abdomen, which is larger than the other two divisions combined, is completely covered by the elytra, except at the pygidium. The pygidium is more exposed from above in the female than in the male. On the lower surface are found only six abdominal segments, while eight are apparent en the upper surface after removing the elytra. This condition is appar- ently constant in both sexes. The sexual differences of the abdomen have already been described. The abdomen is telescoped into the thorax on the lower side, and united to the metacoxai. Dorsally, the connection is made by a membrane to the postscutellum, and the parts do not overlap. The intersegmental grooves on the dorsum are comparatively wide and the fusion of the segments is loose and flexible, while ventrally, the segments are closely fused, forming narrow, curved grooves. Seven pairs of spiracles are to be found on the abdomen, only one of which can be seen below the elytra. The others are mostly in the ridges formed in the pleura. The Structure of the Head Front and Vertex. — The size and general contour of the head (Plate I, figs. 1-2-3) have already been noted. It is partly withdrawn into the prothorax and the mouth parts are wholly on the inferior surface with but a small part of the labrum visible from above. The front (Plate I, fig. 1, fr.) is a large somewhat rectangular area lying between the eyes, and limited anteriorly by the suture separating it from the clypeus (cly.). Its outer angles are extended to form the canthus of the eye (en.). No epicranial suture is present and the region is not sharply separated from the vertex. The front is closely and strongly punctate. Near the vertex^ at the point where the prothorax overlaps the head, the punctures disappear rather abruptly, except near the eyes, leaving a strong line of demarkation between the punctured area and the smoother region of the vertex and occiput. A few scattered punctures are to be found in these regions. Each puncture bears a recumbent hair which is inclined anteriorly. The vertex (v.) merely consists of the upper region of the head having no definite 6 WM. p. HAVES boundaries but lying between the front and the occiput; the occiput being the posterior region of the head lying above the opening of the occipital foramen. No ocelli are present. The Clypeus and Canthus. — The clypeus (Plate I, fig. 1, cly.) is situated on the anterior margin of the front, and the suture separating them, which is strongly sinuate, is known as the clypeo-frontal suture. The clypeus is somewhat rectangular, being twice as wide as long. Numerous punctures are present but they are not as dense as on the front. The anterior margin is slightly emarginate and a strong upturning gives to the whole sclerite a deeply concave appearance. The postero-lateral corners of the sclerite are bordered by the eyes and at this point a chitinous process protrudes upon and partly divides the eyes. This process, known as the canthus (Plate I, figs. 1 and 2, en.), appears somewhat as an extension of the clypeus, but in reality is a continuation of the anterior corners of the front lying partly under the clypeus. Hairs are scattered over the surface of the canthus. The Lahrum and Epi pharynx. — The labrum (Plates I and II, hg. 1, labr.) is attached to the anterior border of the clypeus, being greatly deflected and nearly hidden by the clypeus. Dorsally it is somewhat semi- circular in outline and is depressed to form a deep fovea near the center. It is covered with long thinly placed hairs. On its inner surface are two convergent rows of mesading point hairs (Plate II, fig. 1). The epipharynx (Plate II, fig. 1, epi.) is greatly reduced in this species. It has almost disappeared because of the extension of the labrum over most of its entire surface. A somewhat triangular elevated clump of hairs, or spines, is the most conspicuous remnant of the epipharynx. The Eyes. — The eyes (Plate I, figs. 1-2-3, e.) are the most prominent part of the head. They are large, somewhat oval bodies on the dorsal, lateral and ventral regions of the head. They are nearly divided dorsally by the canthus. The facets (Plate I, fig. 6, fac.) which are about .021 mm. in diameter, average about vS80 to the square millimeter. In shape they are somewhat regularly hexagonal and each hexagon is the cornea of a completely dis- tinct eye. Gena and Gula. — The la,teral parts of the cpicranium form the genae (Plate I, figs. 2 and 3, g.) whose ventral limits are determined by the sutures separating the genae from the large head sclerite — the gula (Plate I, figs. 2 and 3, gu.). The gula occupies about one-third of the ventral surface of the head. It is somewhat quadrate in outline, being slightly wider anteriorly where it is separated from the submentum by a transverse suture. The lateral margins are limited by the gular sutures and posteriorly by the cervical membrane. EXTERNAL MORPHOLOGY OF LACHNOSTERXA CRASSISSIMA 7 The Occipital Foramen and Tentorium. — The occipital foramen, or foramen magnum, is the large opening in the head, opposed to a like opening in the thorax. Through the occipital foramen can be seen, within the head, a chitinous structure, the tentorium (Plate V, figs. 1-2-3, tent.). A large arch-like structure represents the body of the tentorium, while a pair of small, short, posterior arms (post, a.) are present. The anterior arms (ant. a.) are broad structures extending cephalad from the body of the tentorium, and the dorsal arms (dor. a.) are represented by a pair of short pointed processes extending cephalad into the head cavity. The Cervicum and its Sclerites. — The membranous area between the head and thorax is known as the cervicum. It contains six cervical scler- ites. No attempt to homologize these sclerites has been made. The draw- ing (Plate V, fig. 4) shows the left side of the cervicum with the dorsal edge to the right and the anterior edge toward the top. It will be seen that near the dorso lateral margin is a small hair-bearing sclerite and near the ventro lateral margin are two sclerites, a large anterior one which overlaps a small posterior one. The Head Appendages The Antennce. — The antennas (Plate I, figs. 4 and 5) have been pre- viously mentioned under the discussion of sexual differences. The male (Plate I, fig. 4) has a much larger club than the female (Plate I, fig. 5). Normally both sexes have 10 segments, three of which go to make up the lamellate club (cl.). The others form the funicle (fu.) and scape (sc). The Mandibles. — The mandibles (Plate III, figs. 1 and 6) are large complicated structures bearing on their inner surface a large, oval, grinding, or molar surface (mo.). Extending over the molar surface are a number of transverse ridges which are used in the process of grinding food. The anterior end of the mandible is thought to be the homologue of the galea of the maxilla. It is modified into a sharp cutting edge with two blunt teeth (gal.). Near the anterior end of the molar surface is a membrane (memb.) bearing various shaped spines and setae which are shown in Plate III, figure 5. Immediately caudad of the molar surface is still another membrane (memb.) bearing short broad stiff spines. These are shown enlarged in Plate III, figure 3. A transverse section through the molar at the junction of the ridges (Plate III, fig. 4) shows a flat surface with small ridges extended ectad over half the surface. Two chitinous apodemes (Plate III, fig. 2) are attached to muscles controlling the movement of the mandible. The Maxillce. — Each maxilla (Plate II, fig. 3) is divided into five prin- cipal regions: the cardo, stipes, palpifer, galea and lacinia. There seem to be no sutures delimiting a subgalea or dividing the galea into two lobes. The cardo (cd.) is rather short and broadly club shaped, being constricted 8 WM. p. HAYES somewhat posteriorly. Across the center is an abrupt change in contour, making the anterior region of the cardo much thicker dorso ventrally. This change of level is represented in the drawing by the transverse dotted line. The stipes (st.) is the large median triangular sclerite, alongside of which is the long narrow palpifer (max. pf.) bearing a four-jointed palpus (p.). On the margin of the stipes opposite the palpifer is a large somewhat triangular area with one corner elongated to form a large spine-bearing lobe. This is the lacinia (lac). On the ectal margin of the lacinia is a large five-toothed heavily chitinized structure, the galea (gal.). The Labium and Hypopharynx. — The labium (Plate II, fig. 2) is sep- arated from the gula by a transverse suture which extends across the ventral surface of the head in the region where the cardo of the mandible is articulated. Following Kadic's (1902, pp. 207-228) interpretation of the labium of Coleoptera, we find the following regions: The submentum is divided transversely into two regions, the anterior plate (Ap. Sm.) and the posterior plate (pp. sm.). The posterior plate is attached to the gula and is much wider at the postero lateral margins, somewhat constricted at the middle and slightly broader anteriorly. The anterior plate is more nearly quadrate, broader than long, and with the lateral edges rounding out to form a bulge near the middle of the sclerite. The mentum is separated from the anterior plate of the submentum by a transverse suture which has a distinct emargination near its center. Simi- larly, it is somewhat broadly quadrate and bears a few mesad pointing hairs. The anterior marein is strongly biemarginate. The glossa and paraglossae (Plate IV, fig. 1) are not evident on the ventral surface but are bent within the buccal cavity. The glossa (gl.) is a single median sclerite, while the paraglossae (pig.) are found on either side of it. They bear the three-jointed labial palpi. These structures are not easily located without having well cleared specimens. Near the base of the palpus on the inner surface is a diagonal suture limiting an area termed the squama palpigera (sq. pi.). The hypopharynx (Plate IV, fig. 1-3 hyp.) is a V-shaped spiny struc- ture lying on a clump of spines or strong hairs, principally on the inner surface of the anterior plate of the submentum. Caudad and dorsad to the hypopharynx are two long, narrow, chitinous structures known as the fulcrum hypopharyngeum (ful. hyp.). At the dorso-posterior end are two small transverse sclerites constricted somewhat near their middle. These are the pharyngeal sclerites (phy. scl.). To these structures are attached the anterior margins of the pharynx (phar.) and just posterior to the hypopharyngeum, on each side, is a row of backward pointing hairs. An- teriorly, the two arms of the fulcrum hypopharyngeum unite under the pharynx to form a sort of strengthening apparatus for the spiny structure underlying the hypopharynx which extends forward to form three arms. EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA 9 These are shown on their ventral aspect in Plate IV, figure 2. A lateral view showing the relation of these parts to the pharynx (phar.) is shown in Plate III, figure 3. The Prothorax Protergum. — (Plate VI, fig. 1). The tergum of the prothorax or pro- notuni is convex, nearly twice as broad as long, with the sides somewhat narrowing from the base to the apical margin and constricting rather sud- denly anteriorly. The lateral margins are distinctly crenate and ciliate, but not so represented in the drawing. A deep emargination occurs on the anterior margin which overlaps the head, extending to the middle of the eyes. The posterior margin is broadly angulate and overlaps, as far as the elytra, the mesothorax to which it is connected by a membrane. Close, though not very dense, punctures cover the surface, and a smooth median caudo-cephalad line is faintly evident. On each side near the posterior margin is an incipient channel extending from the postero-lateral angle to within a short distance of the middle. Pleura. — At the lateral margins, the tergum is not inflexed to form the so-called prothoracic epipleurae which are strongly evident in some Coleoptera (e.g. PterosUchus calif ornicus Dej.). The prosternum (Plate VI, fig. 2, pro. ster.) and the pleural sclerites compose the ventral aspect of the prothorax. The episternum (Plate VI, fig. 2, eps.) and the epimeron (epm.) have no line of separation. Anteriorly, the episternum elongates mesally to fuse with the sternum whose anterior margin turns inwardly to form a phragma. Likewise, the epimeron extends mesally, tapering towards its extremity. Thus the two extensions form the anterior and posterior margins of the coxal cavities (cc). The junction of the epimeron with the posterior region of the sternum creates in other Coleoptera the closed coxa] cavities. These are partly open. This species has no suture, as in Melolontha vulgaris, separating the sternum from the episternum. Prosternum. — The prothoracic sternum (Plate VI, fig. 2, pro. ster.) occupies the inferior, median region of the prothorax. It is quite irregular in shape and has, as mentioned before, no distinct line of demarkation separating it from the episternum. Externally, a noticeable feature is a caudad-projecting tongue between the cavities of the coxae. At the anterior end of this tongue an irregular, circular ridge causes the formation of a somewhat rounded depression of the sternum. This tongue-like projection after attaining the posterior margins of the coxae is expanded at right angles and extends laterally to meet the epimeron of each side. Inter- nally, after the removal of the coxae, the sternum will be found to have enlarged into a somewhat rectangular piece with rounded postero-lateral corners. It tends to form a concavity in which a part of each coxa rests. On the sides of the anterior edge of the internal sternum are two prolonged 10 WM. P. HAYES entosterna] apophyses (Plate VI, fig 3, es. aph.) which extend dorso- laterally. The first pair of spiracles located ventrally are suspended in the mem- brane which unites the prothorax to the mesothorax. The Prothoracic Legs The Trochantin. — (Plate VI, fig. 6, tn.). The trochantin is a small piece hidden within the interior of the prothorax, which, when viewed from its caudal aspect, presents a depressed or cup-like structure articulat- ing with the anterior margin of the coxa. The latero-dorsal margin bears a small, somewhat sharpened corner that is loosely articulated with a small apodeme on the inner surface of the prothoracic episternum. The end of the coxa articulates with the lower end of this same apodeme. The coxa. — (Plate VI, fig. 6, ex.). The coxa of the anterior leg is cylindrical in form, and slightly over three time as long as its greatest diameter. It lies transversely in the coxal cavity of the prosternum and extends laterally under the edges of the pleura, thereby concealing the articulation with the trochantin. On the inner surface is a large opening extending from near its lateral extremity to nearly half its length. The cephalic edge of this opening articulates with the trochantin and the caudal edge is connected by a membrane to the arms of the epimeron lying immediately behind. The opening is partly closed by an overlapping of its edges which serve as places of attachment for several muscles. At the distal end the coxa likewise articulates with the sternum near the mid-ventral line, and is thus fixed at both ends so that it moves in a rotary manner on its axis. There is a second opening at the distal end which receives a prolongation of the trochanter and thus permits of articulation at this point. yiie Trochanter. — (Plate VI, fig. 6, tr.). The trochanter is a small triangular piece lying between the co.xa and femur, articulating with both the coxa and femur, being more firmly attached to the latter. The Femur. — (Plate VI, fig. 6, f.). The femur is about as long as the coxa, is somewhat flattened and bears on its inner surface a groove-like depression in which the tibia may rest when folded back on the femur. A socket is located in its distal end which receives a condyle from the tibia, forming a ball and socket articulation ])ctween the femur and tibia. The Tibia. — (Plate VI, fig. 6, t.). The tibia is remarkal)ly adapted for burrowing in the soil. It is somewhat ol)lifiuely truncate at ils aj^ex, about equal in length to the femur, and is strongly compressed, esj)ccially at its anterior edge wliich bears the three tibial teeth. Of these the ter- minal tooth is very strong and about as long as the first tarsal joint, while the other two are broader and not so long. Near the femur the tibia is rather cylindrical and bears a terminal condyle for articulation with the EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA 11 femur. On its external margin, opposite the three teeth, is a strong mova- able spine. The Tarsus. — (Plate VI, fig. 6, tar.). The tarsus is composed of five segments. The first four are about of equal length, the fifth slightly longer. They are cylindrical, and become enlarged distally. The terminal segment bears a pair of large claws (t. cl.) each of which bears an intra- median (male) or median (female) tooth. The tooth is slightly larger in the female. The Mesothorax The Mesotergum. — (Plate VII, figs. 1, 2, 3). The mesothorax is the smallest of the three divisions of the thorax; it bears the second pair of legs and the first pair of wings modified to form the elytra (Plate VI, fig. 4). The dorsal or tergal region of the mesothoracic segment is occupied largely by the somewhat triangularly shaped scutellum adjoined to which are the points of attachment of the elytra (Plate VI, fig. 5). Three regions of the four which comprise the typical thoracic tergum (Snodgrass, 1909, p. 523) are to be distinguished in this species, namely, the praescutum, scutum and scutellum. The postscutcllum, or pseudonotum of some writers is absent. The Praescutum (Plate VII, figs. 1 and 2, praes. ph.) is composed of a small meso-cephalad projecting phragma between the prothorax and the metathorax. It is slightly concave and is reinforced at its cephalic margin by a somewhat heavier deposition of chitin, which forms a rod-like brace extending from one edge of the praescutum to the other (Plate VII, fig. 3). The scutellum (Plate VII, figs. 1, 2, 3, scl.) is a large somewhat triangularly shaped sclerite part of which is covered by the elytra leaving the posterior half exposed externally. Its anterior or covered portion is somewhat closely punctate and covered with recumbent hairs while the exposed part is sparsely punctate, devoid of hairs and rather shining. The lateral margins each bear, on their anterior halves, two mesad-projecting pieces which represent the divided regions of the scutum (Plate VII, fig. 2, set.). These phragma-like pieces are nearly triangular in shape and each ter- minates in a pointed ventro-cephalad projecting process which rests on the underlying metathoracic praescutal phragma (Plate VII, fig. 2, prs. ph.), and articulates with its antero-lateral projecting corners. At the cephalic margin of the scutum is a small depression or cavity in which the third axillary of the elytra lies when in the state of rest. The caudal margins are extended backward and unite to form a semicircular redupUcation on the inferior surface of the exposed port'on of the scutellum (Plate VII, fig. 3, scl. red.). To this redupUcation is attached a small membrane which connects the mesothoracic tergum to the lateral and caudal margins of the metathoracic praescutal phragma. The cephalic margin of the scutellum bears a membrane which connects the prothorax and mesothorax. The 12 WM. p. HAYES cephalo-lateral angles of the scutellum articulate with a small, sharp process which projects ventrad and unites with the anterior margin of the mesoepisternum. The Elytra. — (Plate VI, figs. 4 and 5). The elytra which cover the meso- and metathorax and the greater portion of the abdomen are large, somewhat rectangular wing covers extending caudad to the middle of the penultimate abdominal segment, leaving the pygidium exposed. Their lateral and posterior margins are somewhat abruptly declivitous. The upper surface bears faint traces of the nervures and at each humeral angle there is a slight protuberance. The elytra are inserted on the mesothorax between the scutellum and the mesopleura. The base of the wing covers is somewhat truncated and curves ventrad. Near the middle of the basal margins on each elytra is a strong, bifurcated apophysis (Plate VI, fig. 5), which articulates with the wing process of the mesothorax, there are three principal wing axillaries (Plate VI, fig. 5, 1 ax., 2 ax., 3 ax.) in the membrane which are very irregular in shape and impart a different appearance from every aspect in which they are viewed. The interlocking mechanism of the elytra is similar to that described for Lachnosterna fusca by Breed and Ball (1908, p. 291) who found in Coleoptera four devices for fastening the elytra in place. These are described by these writers as follows: 1. By a co-adaptation of the elytra along the dorsal suture. 2. By means of a groove on the dorsal face of the metathorax into which the swollen inner edges of the elytra fit. 3. By slipping the anterior edges of the elytra under the scutellum and hooking them (a) on to the scutellum, or (b) on to the metathorax. Pressure derived from the retracted prothorax may aid in keeping these edges n position. 4. By hooking the anterior lateral edges of the elytra over ridges or into grooves on the lateral faces of the metathorax. In Lachnosterna, the first three methods are used to interlock the elytra while the fourth is present but not functional. The Mesopleura. — (Plate VII, figs. 4 and 5). The mesopleuron consists externally of two sclerites, the episternum (eps.) and the epimeron (epm.). The episternum is a subrectangular plate with a strongly rounded dorsal margin, which adjoins the alar membranes. The anterior and posterior margins are nearly parallel, the former serving as a place of attachment for the intersegmental membrane, and the latter bordering on the epimeron. The ventral margin is attached to the mesosternum (ms. ster.). The epi- meron (epm.) is nearly trapezoidal in shape with the cephalic and caudal margins nearly parallel. The cephalic margin connects with the epister- num, the caudal one joins the metathorax, the dorsal margin gives attach- ment to the alar membrane and the ventral margin tapers to meet and EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA 13 connect with the coxa of the mesothoracic leg (ex.). Between the coxa and the episternum is a small, narrow sclerite not visible externally — the trochantin (Plate VII, fig. 4, tn.) which articulates by means of a small condyle with the coxa. This sclerite is not present in the corresponding region of the metathorax. An internal view of either of the mesopleura (Plate VII, fig. 5) shows a strong entopleural structure arising along the suture separating the epis- ternum and epimeron and forming a pleural ridge which tapers at its ventro-mesal angle into a pleural arm (pi. a.) extending into the body cavity and terminating in a cup-shaped disk which serves for the attach- ment of muscles. The caudal margin of the epimeron presents internally a strong reduplication which aids in concealing the spiracles of the second pair of respiratory organs. The spiracles are not visible externally but lie in the suture between the mesoepimeron and the metaepisternum. The dorsal margin of the episternum is modified into a strongly chitinized blunt process, constituting the wing process on which articulates the bifurcated apophysis of the elytron. The Mesosternum — (Plate VII, figs. 6 and 7, ms. ster.). The mesoster- num is a transverse quadrilateral plate whose anterior margin serves as a place of attachment for the intersegmental membrane lying between the pro- and meso thorax. Its lateral edges border on the ventral margin of the mesoepisternum, and the posterior margin presents a biemarginate appearance with a median, caudal projecting piece which extends between the coxae. The whole external posterior margin is bordered by the meso- coxal cavities. With the coxa removed, it can be observed that the pos- terior margin is extended into a concave process in each cavity in which the coxa lies at rest. The extension joins the metasternum caudad of the coxae. The internal surface of the mesosternum (Plate VII, fig. 7, ms. ster.) shows a furcate process arising from the anterior portion of the concavities occurring in the coxal cavities. It consists of two anterio-dorsal pointing arms forming the so-called entosternum, or mesoentosternum, of the mesothorax. These arms are supported near their middle by a chitinous of the cephalic margin of the mesosternum. The Metathorax Metatergum — (Plate VIII, figs. 1 and 2). The four typical tergal re- gions are present in the metathorax. The praescutum (prs. ph.) is repre- sented by three distinct pieces, a large semi-oval median prephragma or praescutal phragma separated from the scutum by a large membranous area and two lateral parts (praes.) which support the large praescutal phragma. The scutum (set.) is composed of two large lateral halves separated by the notal groove (n. g.) containing the scutellum. The scutal halves are divided diagonally into an anterior and posterior region. The 14 WM. p. HAYES anterior region bears laterally the anterior notal wing process (a. n. p.) and the posterior region (the so-called "scapulaire posterieure^' of Straus Durckheim) carries laterally the posterior notal wing process (p. n p.) and the axillary cords (ax. c). The diagonal line of demarkation causing the division of the scutal halves is the outer evidence of an internal ridge (Plate VIII, fig. 2, d. rd.) on the interior surface of the scutum. The meta- scutellum (Plate VIII, fig. 1, scl.) is a two-lobed piece at the posterior median angles of the scutum. It elongates cephalad to form a tongue-like process, which lies in the notal groove and is limited anteriorly and dorsaUy by the membrane separating it from the praescutal phragma and internally by the entodorsum or V-shaped ridge (ent. d.) on the internal aspect of the metatergum. The postscutellum (pss.) is a large, irregular piece lying immediately behind the scutellum and scutum. It bears the post-phragma (post, ph.), is inflexed mesad to furnish attachment for several muscles and also bears the membrane which connects the thorax with the abdomen. The lateral edges are inflexed caudad of the alar membranes and articulate with the epimera of the metathorax. The Wings. — (Plate VII, fig. 9). The second or metathoracic pair of wings, which are membranous, are borne on the metathorax and are inserted between the metatergum and the metapleura in the alar mem- branes. In a state of rest the wings are transversely folded under the elytra and in flight extend nearly at right angles to the body. The wings are articulated to the body by four axillary sclerites (Plate VII, fig. 10, 1 ax., 2 ax., 3 ax., 4 ax.) similar to those described in Mclolontha (Straus Durckheim p. 109), one of which (4ax) according to Snodgrass (1909, p. 545) is an accessory plate not corresponding to the fourth axillary in other forms. The first axillary lies laterad of the scutum and its anterior outer margin abuts the basal enlargement of the subcostal vein of the wing. Between the first axillary and the bases of the radius and medius lies the second axillary which is partly overlapped by the first. The third is larger than the second, and lies at the bases of the cubital and anal veins, while the fourth axillary is quite small and lies l)clwcen the first and third axil- laries. The McUiplciira.--{V\-AW VHl, figs. ?> and 4). The metapleuron is composed of two principal sclerites homologus to those of the mesopleuron — the metacpisternum and metaepimeron — each of which is subdivided into two regions. The lower division of the e])isternum or katepistcrnum (keps.) is an irregular semioval j^iece attached to the lateral margin of the metasternum. Its dorsal and posterior margins are connected to the lower edge of the epimeron. Dorso-anteriorly the episternum exhibits the second subdivision or anepisternum (aeps.). This is an irregular sha]ied piece bordering on the cephalic edge of the epimeron and to which is fused the lower jmrt of the preparapterum (pptm.) which is likewise fused with EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA 15 the base of the wing process (w. p.)- Internally the preparapterum bears a large muscle disc or pronator disc (pn. d.). The epimeron is divided into two parts, the katepimeron and the anepim- eron. The latter (aepm.) lies immediately above the katepisternum and is elongated anteriorly to form the wing process (w. p.). The katepimeron (kepm.) is a quadrilateral piece lying caudad of the anepimeron. Just above the epimeron is the alar membrane in which is located the wing axillaries. The epimeron is connected posteriorly by an articulation with the postscutellum on its lateral edge. On its inner surface the suture between the episternum and epimeron is extended to form the pleural ridge which elongates into an adf ureal process (pi. a.) that rests on the lateral arm of the mesoentosternal furca. The ventral end of the pleural ridge extends to the coxa. The Metasternum. — (Plate VII, figs. 6 and 7). The metasternum occupies the lower surface of the metathorax. It is much the same in shape as that of the mesothorax, but considerably larger and lies between the meso- and metacoxse. On the internal surface of the sternum (Plate VII, fig. 7) is the large endosternum projecting dorsally (Plate VII, fig. 8). It consists of two laterally projecting arms which furnish support for the adfurcal processes of the entopleura and a large and somewhat pointed cephalad projecting arm. A manifestation of this structure is discernible on the outer surface of the metasternum in the form of a faint mid-ventral line. The Metathoracic Legs The metathoracic legs are different in structure, especially in the form of the tibiae from the prothoracic legs which, as mentioned before, are remarkably adapted for burrowing. While this modification is not present in the hind tibiae, broadly speaking the metathoracic legs are quite similar to those of the mesothorax which for this reason are not treated in this discussion. In the metathoracic legs a well marked sexual difference, to which reference has been made, is apparent in the distinctly longer tarsi of the male. The trochantin of the metathoracic legs is absent, although it is to be found both in the pro- and mesothoracic pairs. The Coxa. — (Plate VIII, fig. 5, ex.). The coxa of the metathoracic leg is attached to the posterior margin of the ventral surface of the meta- thoracic segment, and likewise serves as a place of attachment for the intersegmental membrane lying between the thorax and abdomen. Ex- ternally it presents a flattened surface in the same plane as the metasternum and like the coxa of the prothoracic legs is more or less immobile, except in a semi-rotary manner. It lies at right angles to the longitudinal axis of the body and extends from the elytra at the lateral margin to the mid- ventral line. Internally it presents a hollow arrangement near the opening 16 WM. p. HAYES of which is a chitinous ridge or infolding that permits the attachment of the flexor muscles. The Trochanter. — (Plate VIII, fig. 5, tr.). The trochanter of the meta- thoracic leg is similar to that of the two other pairs of legs. It lies between the coxa and femur and is triangular in shape. The Femur. — (Plate VIII, fig. 5, f.). The femur is slightly longer than the coxa. It is somewhat flattened with rounded edges, tapering toward the distal end where it articulates with the tibia. The Tibia. — (Plate VIII, fig. 5, t.). The tibia of each meso- and meta- thoracic leg differs from that of the prothoracic leg in that there is no flattened modification for digging and burrowing as is present in the front leg. The proximal end articulates with the femur and the distal end with the tarsus, where it is slightly broadened and bears two sharp spurs vary- ing in size in the two sexes. These have been described in the paragraph on Sexual Dimorphism. The Tarsus. — (Plate VIII, fig. 5, tar.). The tarsus is similar to that of the other legs in having five segments. The terminal one has two sharp claws, each bearing a median tooth. The Abdomen The abdomen of Lachnosterna almost equals in volume the remaining portions of the body, and is directed in the horizontal plane. At its base it equals the thorax in size, to which it is attached throughout its complete circumference. Dorsally, it is connected by a membrane to the postscutel- lum and thereby conceals the postphragma. Ventrally, it is joined to the posterior edge of the internal opening in the metathoracic coxae. Concerning the number of evident (not actual) abdominal segments in this species, there are six ventrally and eight dorsally, while the actual number is perhaps eight ventrally and nine dorsally (Plate VIII, fig. 8). The ninth or terminal segment is reduced in size with only the ventral portion apparent externally, while the dorsal part is modified to form an infolding within the anal opening and is not visible from the exterior. Each segment, with the exception of the first, consists of two principal parts, a dorsal or tergal region, and a ventral or sternal area. The dorsal and ventral sclerites are united laterally by a membrane which permits dilation and contraction of the abdomen. Posteriorly, the membrane disappears, leaving no such separation between the terga and sterna of the terminal segments. The Terga. — Nine terga are present. The elytra in the state of rest cover the first six terga which are only slightly chitinized. There is evidently not the necessity of heavier chilinizalion which characterizes the remaining or unprotected parts of the body. These terga are united by comparatively wide membranous areas which are larger laterally than EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA 17 near the median region. The two remaining visible terga (seventh and eighth) are not protected by the elytra and are consequently densely chitin- ized as is also the ninth which has disappeared within the anal opening. The seventh and eighth are the widest tergites. They make up the pygi- dium and are more closely fused than the anterior terga. The Sterna. — The first sternite has disappeared and only a rudiment of the second is present which is covered by the metathoracic coxae. The remaining segments are more heavily chitinized than the second, are closely fused to each other, and permit of no movement as in the terga. The male sternites (Plate VIII, fig. 6) are somewhat flattened and the eighth sternite has a rounded fovea which is absent in the female (Plate VIII, fig. 7). These differences, due to sex, have been mentioned in the discussion of sexual dimorphism. The Spiracles. — There are nine pairs of spiracles, two thoracic and seven abdominal. The first pair of prothoracic spiracles is located ven- trally, and each spiracle is suspended in the membrane which unites the pro- thorax to the mesothorax. The second pair is not visible externally but is to be found in the suture between the mesoepimeron and the metaepister- num. The third pair, or first abdominal pair, lies dorsally in the mem- brane between the metathorax and the first abdominal segment. The next five pairs are found on the ridge formed between the tergites and ster- nites. These six pairs of abdominal spiracles are covered by the elytra. The last, or posterior pair, is exposed below the elytra on the seventh abdominal segment and lies in the suture between the sternum and tergum. The Genitalia. — The genitalia properly speaking are perhaps more con- cerned with internal anatomy and should be discussed in such a treatise. However, they possess chitinous structures which are tegumentary in nature and will, therefore, be briefly discussed here, chiefly because of their importance as specific characters in taxonomy. In the male (Plate IX, figs. 2 and 4) a heavily chitinized semicylindrical sheath or box, termed the telum (Plate IX, fig. 4, te.), surrounds the true membranous penis. The posterior end is shown in perspective in the drawings showing its relation to the claspers which surmount the telum and in this species are rather symmetrical. These claspers (Plate IX, fig. 2, els.) are of much taxonomic importance. Underlying the telum is a small Y-shaped chitinous structure (Plate IX, fig. 3). Posteriorly, the branching arms are bent dorsally and to them is attached the membrane which constitutes the anterior region of the cloaca. The membrane is also attached to the inner margin of the last ventral and abdominal segments. Anteriorly, this structure extends into the body as far as the sixth ventral abdominal segment. The female genitalia (Plate IX, fig. 1) are shown in three views. The organ consists of a pair of broad inferior plates (inf. pi.) which surround a 18 WM. p. HAYES smaller pair of superior plates (sup. pi.) somewhat cylindrical in shape and strongly divergent. Literature Consulted 1908 Breed, R. S.. and Ball, E. F. The interlocking mechanisms which are found in connection with the elytra of Coleoptera. Biol. Bull., 15:289-303. Abstract, Proc. of Seventh Int. Zool. Cong., Cambridge, Mass. 1912. 1902 CoMSTOCK, J. n., and Kochi, C. The skeleton of the head of insects. Amer. Nat. 36:13-45, 29 figs. 1909 Crampton, G. C. A contribution to the comparative morphology of the thoracic sclerites of insects. Pros. Acad. Nat. Sci., Phiia., 61:3-54, 4 pis. 1907 Hardenberg, C. B. A comparative study of the trophi of Scarabaeidae. Trans. Wis. Acad. Arts and Sci., 15:548-602, 4 pis. 1902 Kadic, O. Studien iiber das Labium der Coleoptern. Jena Zeitschrift fiir Nat. Wissenschaft, 36:207-228. 1901 KOLBE, H. J. Vergleichendmorphologische Untersuchungen an Coleoptern nebst Grundlagen zu einen System and zur Systematik derselben. Arch. Naturg. 67:89-150, 2 pis. 1892 Smith, J. B. The mouth parts of Coprls Carolina; with notes on the homologies of the mandibles. Trans. Amer. Ent. Soc. 19:83-87, 2 pis. 1906 Explanation of terms used in entomology. Brooklyn Ent. Soc. Separate, p. 154 and plate IV'. 1906 Snodgrass, R. E. A comparative study of the thora.x in Orthoptera, Euplexoptera and Coleoptera. Proc. Ent. Soc. Wash., 9:95-108, 4 pis. 1909 The thorax of insects and the articulation of the wings. Proc. U. S. Nat. Mus., 36:511-595, 6 figs., 30 pis. 1909 The thoracic tergum of insects. Ent. News, 20:97-104, 1 pi. 1828 Straus-Durckiieim, H. Considerations generales sur I'anatomic comparec dcs aiiimaux articules. Paris, pp. 19 434, 10 pis. List of .Xbbkkviations a. anal opening a.n.p. anterior notal wing process a.a. anterior arm of endoslernum ap.sm. anterior plate of submentum la 2a 3a first, second and third anal veins ax.c axillary cord aepm. anepimeron c. costa aeps. ancpisternum c.c. coxal cavity ant. antenna cd. cardo ant. a. anterior arm of tentorium cdy. condyle EXTERNAL MORPHOLOGY OF LACHNOSTERXA CRASSISSIMA 19 cl. club n. notum els. claspers of male n.g. notal groove cly. clypeus oc.for. occipital foramen en. can thus P- palpus CUi CU2 first and second cubitus pgl. paraglossa ex. coxa phar. pharjnx cx.ph. coxal phragma phy.scl. phryngeal sclerite d.rd. diagonal ridge of scutum pl.a. pleural arm — entopleurum dor.a. dorsal arm of tentorium pn. pronotum e. eye pn.d. pronator disc. ely. elytra p.n.p. posterior notal wing process ent.d. entodorsum p.n.r. post notal ridge ent.ster. entostemum post.a. posterior arm of tentorium epi. epiphars-nx post. ph. postphragma epm. e])imeron pp.sm. ])osterior plate of submentum eps. episternum pptm. prcparapterum es.aph. entosternal apophysis praes. praescutum f. femur praes.ph. praescutal phragma-mesotergum fac. facet prs. ph. praescutal phragma-metatergum fc. furca pro. ster. presternum fr. front pss. postscutellum fu. funicle r. ridge ful.hyp. fulcrum hypopliaryngcum rd. radius g- gena s. scape gal. galea sc. subcosta gl. glossa scl. scutellum gu- gula set. scutum hyp. hypophar>nx scl.red. reduplication of .scutellum nf. pi. inferior plates sp. spiracle kepm. katepimeron sq. pi. squama palpigera kei)S. katepisternum St. stipes la. lateral arm of entosfernum sup.pl. superior plate labi. labium t. tibia labr. labrum tar. tarsus lac. lacina te. teluni post.a. posterior arm of tentorium t.cl. tarsal claw m. mentum tent. tentorium rad. medius tn. trochantin m.d. muscle disc. tr. trochanter mand. mandible V. vertex max. maxilla w.p. wing process max.pf. maxillary palpifer 19 abdominal segments memb. membrane 1 ax. first axillary mo. molar 2 ax. second axillary ms. ster. mesosternum 3 ax. third axillary mt.ster. metasternum 4 ax. fourth axillary. 20 WM. P. HAYES fac Pi.ATi: I Fig. 1. I'ront view of head. Fig. 2. Lateral view of head. Fig. 3. Ventral view of head. F"ig. 4. .Antenna of male. Fig. 5. Antenna of female. Fig. 6. F'acets of the compound eye. EXTERNAL MORPHOLOGY ^ LACHNOSTERNA CRASSISSIMA 21 DaJt.pC Plate II Fig. 1. Epipharynx and internal aspect of clypeus and labrum. Fig. 2. Gula and Labium. Fig. 3. Left maxilla. 22 WM. P. HAVES Plate III Fig. 1. Side view of right mandible. Fig. 2. Apodeme of mandible. Fig. 3. Membrane and hairs at base of mandible. Fig. 4. Portion of cross section thru molar. Fig. 5. Hairs and setx from upper membrane of mandible. Fig. 6. Inner surface of mandible. EXTERNAL MORPHOLOGY OF LACHNOSTERXA CRASSISSIMA 23 ^ _ CS^"-' ful .hyp nyp fMl.hyp Plate IV Fig. 1. Labium from within showing hypopharynx and fulcrum h)T3ophar>'ngeum. Fig. 2. Junction of arms of fulcrum hypopharyngeum, ventral aspect. Fig. 3. Lateral view showing relation of fulcrum hypopharyngeum to the pharynx. 24 \VM. P. HAYES oo.for tenti post .a Ua ant. a 3 Plate V Fig. 1. Tentorium — looking througli occipital foramen. Fig. 2. Anterior view of tentorium. Fig. 3. Dorsal view of tentorium. F'ig. 4. Left side of cervicum (dorsal margin to the right). EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA 25 pro.ster post.ph e 8 . aph cx.ph t.cl Plate VI Fig. 1. Dorsal view of prothorax. Fig. 2. Ventral view of prothorax — coxae removed. Fig. 3. Anterior aspect of prothorax. Fig. 4. Left elytron. Fig. 5. Articulation of elytron. Fig. 6. Right prothoracic leg. 26 WM. P. HAVES praes.ph pfaea.ph 10 Plate VII Fig. 1. Dorsal view of mesothorax with portion of right elytron. Fig. 2. Left lateral aspect of mesothoracic tergum. Fig. 3. Internal aspect of mesothoracic tergum. Fig. 4. Left mesopleuron, external aspect, coxa ex situ to show trochantin. Fig. 5. Left mesopleuron, internal aspect. Fig. 6. Meso — and metastcrna, external aspect. Fig. 7. Meso — and metastcrna, internal aspect. Fig. 8. Metathoracic endosternum. Fig. 9. Metathoracic wing. Fig. 10. Axillary sclerites of the metathoracic wing. EXTERNAL MORPHOLOGY OF LACHNOSTERNA CRASSISSIMA pi- s . ph P^s.ph 27 ppin Plate VIII Fig. 1. Metatergum, external aspect. Fig. 2. Metatergum, internal aspect. Fig. 3. Left metapleuron, external aspect. Fig. 4. Left metapleuron, internal aspect. Fig. 5. Right metathoracic leg. Fig. 6. Ventral aspect of abdomen, male. Fig. 7. Ventral aspect of abdomen, female. Fig. 8. Left lateral aspect of abdomen, male. 28 WM. P. HAYES ^J— sup.pl — Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Plate IX Female genitalia, dorsal, ventral and lateral aspects. Male genitalia, arranged perspectively. Y-shaped supporting structure of cloaca and telum. Male genital organ with telum in place. Dorsal aspect of Lachnosicrna crassissima. THE RESPIRATORY MECHANISM IN CERTAIN AQUATIC LEPIDOPTERAi By Paul S. Welch Introduction According to one of the current theories, insects arose from a terres- trial ancestry and the aquatic habit, wherever manifested, was secondarily acquired. The general appUcation of this theory to all aquatic insects is sometimes questioned but there seems to be an almost universal agreement that many of the higher orders, including the Lepidoptera, are preeminently terrestrial in organization and that their aquatic representatives display an evolution superimposed upon a terrestrial background. These insects, invading water, had certain vital problems to solve, respiration being among the first, and the diverse but successful adaptations offer interesting material for study. The writer has found the aquatic Lepidoptera favorable for the study of larval adaptations for the following reasons: 1. While the Lepidoptera constitute a large, well-defined order, the insignificant number of aquatic forms made it possible to examine most of the American species. 2. Although inconsequential numerically, they manifest adaptations to aquatic Kfe as perfect and as diverse as many of the more conspicuous groups. 3. Heterogeneity in the methods of solving aquatic problems may ap- pear even among species of the same genus. 4. The abundance of some species in certain environments has provided ample material for extensive observations and experiments. 5. This group of aquatic insects is practically unstudied from the morphological, physiological and ecological aspects. Excluding the semi-aquatic forms, aquatic Lepidoptera fall into two general classes when considered from the point of view of larval respira- tion: (1) Those which are devoid of any special respiratory organs, and secure the requisite oxygen from the atmosphere by some physiological adaptation, from the dissolved supply in the water, or through a combina- tion of both; and (2) those with special morphological devices in the form of gills. In the first class belong such forms as Bellura melanopyga, Nyniph- ' Contribution from the University of Michigan Biological Station, and the Zoological Laboratory, University of Michigan. 29 30 PAUL S. WELCH ula icciusalis, and Pyrausta pemtalis, while Nymphula maculalis, N, obscuralis, and Catadysta fulicalis are typical representatives of the second. Acknowledgments The writer wishes to express his indebtedness to Professor S. A. Forbes, Professor J. G. Needham and Mr. J. T. Lloyd for certain materials speci- fied on later pages. Use has been made of data accumulated by two of the writer's graduate students, Miss Ethelynn Hopkins and Mr. Jennings Hickman. During the summer of 1919, Miss Hopkins studied briefly the tracheation of certain instars in Nymphula maculalis and Nymphula icciusalis, using living material. Later, Mr. Hickman examined the gills of Nymphula maculalis, using serial sections of preserved specimens. The work of both of these students has been repeated and extended by the writer. Material The principal objects of the work on which this paper is based were to determine the structure and mode of function of the respiratory organs developed in this group; to study the degree of change from the original terrestrial organization of the larvae; to get some measure on the efficiency of 'Special organs; to examine the various instars with respect to the respira- tory problem; to test certain doubtful statements in the literature; and if possible to determine something as to the course of evolution in such a hmited group. In order to accomplish these ends it was necessary to study as many representatives of the two above mentioned classes of aquatic Lepidoptera as possible. Owing to the abundance of several species in the vicinity of the University of Michigan Biological Station, repeated observations and experiments have been made during the past four summers. Some of the necessary preliminary work on habits and life-history has been published already (Welch, 1914, 1915, 1916, 1919). The material used most exten- sively in the present work is as follows: Non-gilled Larvae Nymphula icciusalis Wlk. — This species occurs abundantly at Douglas Lake, Michigan, and several instars have been studied in both living and preserved form. Nymphula oblitcralis Wlk. — Preserved material of the larvae of this species was secured from the collections of the Illinois Natural History Survey through the courtesy of Professor S. A. Forbes. The life-history of this species was described by Hart (1895, pp. 176-180) and the speci- mens used were from Hart's collections and identified by him. Living material has not been available. RESPIRATORY MECI^NISM IN LEPIDOPTERA 31 y ymphula sp. — Larvae of a species of XympJiula occasionally appeared at Douglas Lake on the leaves of the yellow water-lily but it has not been bred to the adult stage and its specific identity is not known. Gilled Larvae N ymphula mactilalis Clem. — Living material in all stages of the life- history including larval instars is available in abundance at Douglas Lake, Michigan, during July and August. Observations were also made on living specimens at Lake Oneida, N. Y., during the summer of 1916. Liberal use was made of preserved material in connection with the morphological work on the spiracles and gills. The biology of this species (Welch, 1916) has already been described. Xympliula obscuralis Grt. — Preserved larvae were received from the Illinois Natural History Survey. Hart (1985, pp. 167-173) reported on the life-history of this form and the specimens sent to the writer were from Hart's collections and identified by him. No living material was available. Cataclysta fulicalis Clem. — Preserved larvae were sent to the author by Professor J. G. Needham and Mr. J. T. Lloyd, from collections made in the vicinity of Ithaca, New York. These gilled caterpillars have been described by Lloyd (1914, 1919) and are conspicuously different from the non-gilled Cataclysta forms in other parts of the world. Living material was not available. The Tracheal System Since the gilled caterpillars represent the greatest progress in the direction of special aquatic respiratory adaptation and since the gap between the gilled and non-gilled forms is without intergrades, it was of interest to compare the tracheal systems of species representing these two classes and to seek light on the following questions: (1) Do the non-gilled forms possess a system of tracheation built on the plan of terrestrial lepi- dopterous larvae? (2) Do the gilled forms have the same system of tra- cheation found in the non-gilled aquatic forms? or (3) Has the acquisition of gills been accompanied by noteworthy changes in the fundamental tracheation? These questions are significant in connection with certain facts of gen- eral habits and life-history. There is little or no doubt at present that the non-gilled forms once included in the genus Hydrocampa and the gilled forms once included in the genus Paraponyx really comprise one genus, N ymphula, and they are usually so treated. In Douglas Lake and other similar lakes studied by the writer, it is commonplace to find the gilled caterpillars and non-gilled caterpillars thriving side by side in the same vegetation beds, subject to identical environmental factors. In N . macula- lis the first instar is devoid of gills, these appearing only in the second instar, when forty gill-filaments come into existence. The gill-filaments 32 PAUL S. WELCH increase in number with each succeeding molt until the final larval instar has an equipment of over four hundred. Non-gilled Larvae A study was made, using living material, of the arrangement and dis- tribution of the tracheae in the different larval instars of .Y. icciusalis, giving special attention to the early instars and the full-grown larva. Taking into account only such tracheae as appear under the higher powers of the binocular microscope and the medium powers of the compound microscope, and using only fresh preparations from which none of the air had been lost, it was possible to diagram the fundamental structure of the tracheal system and to make comparisons not only in the different instars but also with other species. It has thus been found that the ground plan of arrangement and dis- tribution of tracheae is essentially that which characterizes the terrestrial larvae. Deviations and minor variations appear but none of them seem to bear any significant relation to the acquired habits of the larvae. No important changes of any kind appear as later instars are reached. Gilled Larvae Detailed studies of tracheation in the larval instars have also been carried on with ^Y. maculalis using fresh, living material. In the first instar the system is almost a dupUcate of that in the first instar of N . icciu- salis. In the second, the appearance of gills is accompanied by no conse- quential change, the gills being supplied by short direct branches from the main longitudinal tracheae. Also in the rapidly increasing gill complexity of the later instars there is no deviation which the writer can recognize as having any significance in relation to the aquatic habit or to the acquisition of gills. The results of this part of the work seem to show that fundamentally these aquatic caterpillars have retained the original terrestrial form of body tracheation and that the gill tracheation is a system superimposed upon the one already present in the whole group. Gills have therefore been developed with a minimal change of the original tracheation. It would appear, if these conclusions are well taken, that the larval type represented by that of N . icciusalis is the older one phylogenetically and that the gilled caterpillar has a more recent origin. Structure of the Gills A detailed study of the structure of the gills in all of the species avail- able was made by means of longitudinal and transverse sections of pre- served material and by the examination of living material, whenever the latter could be secured. The relative transparency of the living material, especially when submerged in dilute glycerine, or some of the oils used for RESPIRATORY MECHANISM IN LEPIDOPTERA 33 clearing made it possible to study certain features more readily than in sections, particularly the distribution of tracheoles. Sections cut 6 microns thick and double stained in haemotoxylin and eosin gave satisfactory re- sults. High magnification was often required for the examination of sec- tions, particularly for the study of the histological features, and for the more critical and difficult features a 1 .9 mm. oil immersion fluorite objec- tive was used in connection with a monobinocular microscope. The Gill-wall A study of the gill-wall shows that it is essentially a continuation of the body- wall, having the same set of layers. In order to determine whether any differences in thickness appear, a number of measurements were made of the various layers of the gill and of adjacent portions of the body- wall, the averages being given in the following table. All of these measurements were made on specimens in the last larval instar. Measurements in this and succeeding tables are expressed in fractions of a miUimeter. The terms epidermis and dermis are used instead of the primary cuticula and secondary cuticula of some authors. Epidermis Dermis Hypodermis Total N. maculalis Gill-wall 0.0004 0.0056 0.0017 0.0077 Body-wall 0.0004 0.0115 0.0027 0.0146 N. obscuralis GiU-wall 0.0012 0.0017 0.0009 0.0038 Body- wall 0.0005 0.0037 0.0010 0.0052 C. fulicalis Gill- wall 0.0037 0.0075 0,0025 0.0137 Body-wall 0.0037 0.0190 0.0033 0.0260 It thus appears that there is a distinct reduction of thickness in the walls of the gills as compared with the body- wall, this reduction occurring mainly in the dermis. The basement membrane is so delicate that it has been left out of account in all measurements of body-wall and gill-walls. The Gill-cavity The interior of each gill is merely a cavity (Pi. X, fig. 6) enclosed by the walls described above and containing certain structures to be discussed in another connection. This cavity extends continuously from base to tip and lacks completely, in the species examined, the alveolar type of tissue which appears within the gills of some insects. The size of this 34 PAUL S. WELCH cavity depends entirely upon the dimensions of the gill as a whole and has a direct connection with the haemocoele, in fact, it is a continuation of the haemocoele. In the Nymphula group, the gills are relatively large, have a spacious gill-cavity, and the passage from the haemocoele to the gill-cavity is broad, while in Catadysta fnlicalis the gill as a whole is small and slender, the wall thick, the gill-cavity much reduced, and the passage from the haemocoele to the gill-cavity often smaller in diameter than that of the gill-cavity. For example, in one series of eighteen measurements the aver- age diameter of the gill-cavity was 0.0054 mm. while the average diameter of the opening into the haemocoele was 0.0039 mm. Contents of the Gill-cavity Tracheae Nymphula maciilalis. — As previously mentioned, the gills in this species are supplied with tracheal branches arising directly from the adjacent main longitudinal tracheae. Since the gills become branched in later instars, the supplying tracheae branch correspondingly. Each filament, therefore, has one main trachea, axial in position, which extends from the base almost to the tip, gradually decreasing in diameter distad. A very few instances of two tracheal branches entering a gill-filament were observed, both extending well towards the tip of the filament and both giving rise to tracheoles. The origin and distribution of the tracheoles were best studied in hving material, although certain data were confirmed by means of serial sections. At frequent but irregular intervals (PL XI, figs. 9, 10, 11) along the sup- plying trachea short tracheoles arise singly, extending ectad to the inner surface of the gill-wall and giving off numerous fine branches, all of which break up into very minute tracheoles and have a rather definite arrange- ment as follows. These tiny tubes all extend longitudinally, proximad and distad, very near or in contact with the ental surface of the gill-wall, and approximately parallel to each other, so that the periphery of the gill- cavity is bounded by a thin zone composed of countless, minute, parallel tracheoles. The terminal tracheoles of each individual tuft intermingle with those of the adjacent tufts but also in an approximately parallel fashion. All attempts to determine the character of the terminations of these tracheole endings, using the best preparations and the highest mag- nifications, hav/' thus far been futile. In living material and in whole mounts they appear to unite with the basement membrane and serial sections confirm this conclusion, but nothing further can be said as to the exact relation to the gill-wall. No tracheoles were found lying free in the gill-cavity. The profusion of these tracheoles, intimately related to the entire inner surface of the gill-wall, points definitely to the principal func- tion of these body projections. RESPIRATORY MECHANISM IN LEPIDOPTERA 35 Nymphula obscuralis. — Since it has not been possible to study living material of this species, the tracheation of the gills cannot be so definitely described. However, serial sections show a type of structure closely resem- bling that of N . maculalis. It is probable that both species have systems which are very similar. Catadysta fulicalis. — Serial sections including all parts of the body of the larva show no tracheation (PL X, fig. 5) of the gills. No branches of the body tracheal system approach the bases of these organs and in no sense are they to be regarded as tracheal gills. Body Fluids In living specimens of N . maculalis, it is easy to observe the movement of fluid, not only within but also into and out of the gill-cavity, thus giving added proof of the continuity of this cavity with the haemocoele. Blood corpuscles can be detected in this fluid. Sections confirm the observations on living specimens, showing that the gill-cavities invariably contain haemocoele fluids. Preserved material of .V. obscuralis yielded similar results. The small attenuated gill-cavities of Catadysta fulicalis contain only the remains of fluid originating from the haemocoele. Discussion It thus appears, from the point of view of structure alone, that two distinct gill types occur in aquatic larvae of Lepidoptera: (1) combination tracheal-blood gills, and (2) blood gills. As already pointed out, the profusion of tracheoles in each gill-filament in N. maculalis indicates the respiratory nature of these organs. With its equipment of over four hundred gill-filaments the mature larva appar- ently has more than ample provision for respiration, especially since these larvae live in surface water rich in dissolved oxygen, and often in vegeta- tion beds which contribute additional oxygen. This gill equipment is also striking in view of the fact that certain non-gilled Nymphula larvae thrive in identically the same external conditions. Nymphula obscuralis, accord- ing to Hart (1895, p. 170), has an average of four hundred and eighty-four gill-filaments. In Catadysta fulicalis, the number is smaller, the full- grown larva having about one hundred and twenty unbranched gills. Both blood gills and tracheal-blood gills are known to occur in limited numbers in other orders of insects. There is no special difficulty in under- standing the mode of functioning of the ordinary tracheal gill, but in the combination described above and in the blood gill of the Catadysta type, certain problems arise, first of which is the nature of the relation, if any, of the blood (the body fluid which circulates in the gill-cavities) to the transportation of oxygen. In the blood gills of certain chironomids, the 36 PAUL S. WELCH blood contains haemoglobin and with this carrier present the gills have definite significance. In the larvae of N. maculalis and in certain other insects having similar gills, some carrier other than haemoglobin seems necessary to enable these structures to function as gills. The actual dis- covery of invisible carriers has not yet occurred. Muttkowski (1920, 1921a, 1921b, 1921c) suggests that possibly haemocyanin may constitute such a carrier. Rose and Bodansky (1920) demonstrated the presence of copper in a number of marine organismiS and Muttkowski (1921a) found it in a large number of animals representing six phyla. The last named in- vestigator holds that "Copper is found in insect blood in quantities com- parable to that of crayfish blood. Its role is therefore interpreted as being identical, — namely that it serves as the nucleus of a respiratory protein,— hemocyanin. Insects, therefore, have two sources of oxygen,— atmospheric air led directly to the tissues by way of the tracheae, and fixed oxygen ar- ried by the respiratory protein of the blood." Possibly this is a hint in the right direction and invisible oxygen carriers in insect blood may soon be identified. Since the circumstances seem to demand the presence of some oxygen carrier, the question arises concerning the mode of functioning of the com- bination gill. Does such a gill have two separate and distinct methods of securing and distributing oxygen? The position and distribution of the tracheoles are such that there seems to be no ground for assuming any rela- tion to the blood as an intermediary between them and incoming oxygen. Perhaps the tracheal system might function as completely if the gill-cavity were filled with alveolar tissue instead of blood. On structural grounds alone, it appears possible that two distinct methods could exist side by side. It might be suggested that in N. maculalis and N. obscuralis the gills are really tracheal gills and that the presence of blood in the gill cavity is entirely incidental, but such a suggestion loses weight when C. fulicalis is considered since its gills, if they function at all, must do so through the intermediation of the blood. It has not been proven absolutely that these lateral outgrowths in C. fulicalis arc functional gills and as respiratory organs they might be questioned completely. Such a view would render similar organs in other orders of insects devoid of respiratory significance and, pending further investigation, it would seem that circum- stantial evidence points rather definitely to the conclusion that these organs do function in res])irati()n Judging entirely from the structure of these gills, a contrast appears between the Nymphula group and Cataclysta which may indicate difference in degree of efficiency. In the former, the larger number of gills, the rather spacious gill-cavities, the thin gill-walls, and the profuse tracheation all suggest an efficient equipment. In the latter, however, with only about one hundred and twenty gills, with the very small gill-cavities connected RESPIRATORY MECHANISM IN LEPIDOPTERA 37 with the haemocoele by still smaller lumina, with no traces of tracheation, and with the conspicuously thick gill- walls, the effectiveness of the system seems much smaller. The presence and absence of gills within the genera Xymphula and Cataclysla and the existence of distinctly different gill types in these two closely related genera give added support to the theory of the independent origin of the various aquatic insects, emphasizing the fact that in these animals types of adaptation and genetic relationship may have no close correlation. The Spiracles and Connecting Tr.aciieae The secondarily acquired nature of aquatic habits and structures naturally directs attention to the character of the spiracles. In the gilled forms, is the gill system superimposed upon an unmodified holopneustic tracheation, or have modifications occurred leading towards suppression of the spiracular equipment? In the non-gilled forms which lead a sub- merged existence, has the characteristically terrestrial holopneustic trachea- tion been modified? A common statement appears in the literature to the effect that many nymphs and larvae living in water have apneustic trachea- tion, breathing directly through the skin or by means of gills. It is also pointed out that between the completely apneustic and the typical holop- neustic tracheation a variety of intermediate stages exists. The gilled larvae of certain Xymphula species have been described as having apneustic tracheation in which the spiracles are closed, and the spiracular branches (stigmatal branches) have become solid cords. The writer has searched in vain for any thoroughgoing morphological work bearing on this subject. Among workers who have studied Old World species of Nymphula three of the most recent might be mentioned. Rebel (1899) made some studies on Nymphula iParaponyx) stratiotata and states that the tracheation is apneustic. Portier (1911) in a voluminous paper dealing with several aquatic insects, carried on some physiological experiments with larvae of XympJmla stratiotata and claims to have shown that (a) larvae sub- merged for five minutes in olive oil colored with alcanine showed no oil in the tracheae; (b) that under the binocular microscope the spiracular trunks did not have the aspect characteristic of air filled tracheae but looked like heavy cords; (c) that larvae suffered no effects from submer- gence in oil, but a small geometrid larva so treated became inert and oil was found in its tracheae; (d) that larvae were perfectly normal after fifteen minutes submergence in soapy water, but the geometrid larva so treated became apparently dead in three minutes; (e) that larvae immersed in olive oil, ether, and alcanine became aenethetized after one minute, but microscopic examination showed no penetration of the colored oil, recovery occurring when returned to water; (f) that larva treated as in (e) for 3S PAUL S. WELCH twenty hours did not show the tracheal system invaded; (g) that a larva placed under reduced air pressure showed bubbles of gas gradually form on the surface of the body where the integument is thinnest but none formed about any of the spiracles. From these experiments and without morphological confirmation Portier concluded that the spiracles of the larva of N. stratiotata are closed and functionless. Wesenberg-Lund (1913, p. 126) states that in N . ("Paraponyx'^) stratiotata the spiracles are func- tionless but no evidence is given in support of this conclusion. Observations and Experiments of Nympiiula Maculalis Attention was first directed to the spiracles by evidence that these larvae can exist out of water for considerable periods of time. While it is common for the pupa to be formed on the lower, submerged surface of a water-lily leaf, the full-grown larva sometimes crawls out of the water, onto the upper leaf-surface and there forms the pupa. In order to con- struct the silken covering, tie down the case to the leaf, and transform into a pupa, a considerable period of time must be spent out of water. Mature larvae placed in containers without food and with little or no moisture often lived from four to eight days. Larvae, in containers with food material and just enough water to keep the surrounding air moist, were allowed to gradually dry up. In such cases the gills became dried and black, but the larvae lived about fourteen days. It is thus evident that these caterpillars can exist for days apart from water and respire by means other than gills. Furthermore, it seems unlikely that this can be accounted for on the basis of cutaneous respiration. These results led to a critical morphological examination of the spiracles and their connecting tracheae in A', maculalis, a study which was later extended to include all of the strictly aquatic caterpillars available for examination. Morphology of the Spiracles and Connecting Tracheae A detailed morphological study of the spiracles and their connecting tracheae was made on serial sections, cut six to seven microns thick, and double stained. Both transverse and longitudinal sections were used and all critical points determined with a modern monobinocular micro- scope equipped with a 1.9 mm. fluorite oil immersion objective. Since the spiracular aperture may be narrower in one dimension than the other, the long dimension being transverse to the long axis of the body, measure- ments on transverse sections might lead to error if not checked on longi- tudinal sections. In all such cases the measurements were made from the edges of the opening, not including accessory structures, and in such a way as to give an average of the two principal diameters of the aperture. On the other hand, the connecting tracheae arc practically cylindrical, thus making it possible to record measurements from any section passing RESPIRATORY MECHANISM IN LEPIDOPTERA 39 through the center of the lumina. Mature larvae were usually used, al- though sections of earlier instars were examined from time to time. In this connection it should be pointed out that, as usual in lepidopter- ous larvae, the spiracles on the meso- and metathorax are absent in all of the species examined. In the following tables percentage of decrease, wherever expressed, is calculated by using as the standard of comparison the dimensions of the largest spiracle and connecting trachea (usually those of the second abdominal segment) of the series. It must also be understood that this is merely a convenient way of comparing the degree of reduction of the other spiracles and tracheae and is not intended to imply that even the largest may not have undergone some reduction themselves. Since there is no way of determining the diminution of the largest spiracle, if it has been reduced, it would not be possible to express the true amount of reduction of the other spiracles on the same individual. Nymphula maculalis Examined externally, under magnification, nine pairs of spiracles are observable on segments 1, and 4-11. All are minute and inconspicuous except those on 5, 6, and 7 which are distinctly larger. The following table comprises one set of diameter measurements which is representative of all others made in this work: Segments Spiracles Lumen of Spirac- ular Tracheae Percentage of Decrease Spiracles Tracheae 1 2 0.0050 0.0011 85.5 96.0 3 4 0.0051 0.0011 85.2 96.0 5 0.0345 0.0276 0. 0. 6 0.0322 0.0253 0.66 0.8 7 0.0322 0.0184 0.66 33.3 8 0.0119 0.0011 65.5 96.0 9 0.0085 0.0017 75.3 93.8 10 0.0068 0.0017 80.2 93.8 11 0.0051 0.0013 85.2 95.2 The Larger Spiracles and Connecting Tracheae In the larger spiracles the outer margin of the aperture bears a com- plete set of elongated, closely set, chitinous spines (PI. X, fig. 1) which have a rich yellow color when viewed under magnification. These spines form an almost continuous marginal guarding device, the free ends con- verging so that the form of the whole is that of a truncated cone. The free ends of the spines mark the periphery of an aperture which is much 40 PAUL S. WELCH smaller than the spiracle itself. The external cuticula extends into the lumen of the spiracular trachea, Uning it for the entire length. However, certain modifications appear chief among which are the distinct reduction in thickness and the numerous filiform chitinous projections which extend into the lumen. A continuation of the hypodermis of the body-wall con- stitutes the major part of the wall of the spiracular trachea and shows no significant changes in structure. The short connecting trunk is terminated at its ental end by a well- developed closing apparatus (PI. X, fig. 4), composed essentially of a closing bow, a closing band, a closing lever, and an occlusor muscle. The closing bow is a chitinous, crescentic band (PL XI, fig. 7) lying in the lining of the lumen and extending through one-half of the total circumference. From one end of the closing bow a similar band continues around to a point about opposite the middle of the closing bow where it meets the end of the closing lever. The closing lever is located at right angles to the lumen and projects radially for its entire length, covered by an extension of the hypodermal wall. A short, broad occlusor muscle extends from the end of the closing lever diagonally to the free end of the closing bow. It thus appears that the chitinous band formed in this way is absent for about one fourth of the circumference of the lumen, thus failing to form a com- plete ring. Thus far it has been impossible to determine the exact relation of the chitinous parts of the closing apparatus to each other. Studies were made using thin serial sections in all the principal planes; also by dissecting out a portion of the body of the larva containing a large spiracle and its related tracheal parts, placing them on a slide in strong potassium hydrox- ide solution and boiling by holding the slide over a small flame. By the use of this last named method the soft parts were all removed leaving only the chitinous portions. Difficulties in tracing out certain minute portions of this closing apparatus have not been entirely overcome either by the kind of preparation or by high magnification. It appears, however, that the chitinous band, forming the closing lever and the closing bow, is one continuous structure. From the outermost end of the closing lever a long muscle band extends diagonally to the body-wall. On the opposite side a similar muscle ex- tends from a point near the origin of the occlusor muscle diagonally to the body- wall. Beyond the closing apparatus the lumen oi)ens directly into that of the main longitudinal tracheal trunk. The Smaller Spiracles and Connecting Tracheae. — The spiracles on seg- ments 1, 4, 8, 9, 10, and 11 show a marked reduction in size, in fact, they are so small that magnification is necessary to locate them definitely. Structurally, these spiracles and their connecting tracheae difTer markedly from those on 5, 6, and 7. At the margin of each spiracle there appears, instead of a thick set crown of chitinous spines, a solid, continuous, chiti- RESPIRATORY MECHANISM IN LEPIDOPTERA 41 nous rim (PI. X, fig. 3) which projects distad from the body-surface. It would appear that in the process of reduction the spiny crown of the origi- nal large spiracles became fused into one continuous margin. At the base of the tiny cup thus formed the lumen becomes reduced to an extremely fine canal which extends without change in diameter to the closing appar- atus located well within the haemocoele. This canal is lined throughout by a uniform, thin extension of the external cuticula, but the hair-like projec- tions characteristic of the large spiracular trunks are here entirely wanting. As will appear in the table, the lumen is very minute, but by means of thin, serial sections and high magnification it has been possible to demon- strate that it is open throughout its course. The bulk of the wall of the spiracular trachea is composed of an extension of the hypodermis of the body-wall, but it also has become reduced, being about one-half the thickness of the same layer in the larger spiracular tracheae. The connect- ing trachea has not changed in length and terminates in a closing apparatus similar to that described for the larger spiracular tracheae, except that it also has become considerably reduced in size. All of the parts are repre- sented, however, and the whole closing contrivance has every appearance of being completely functional. It also appears that reduction in the small spiracles is not uniform, but gradually increases posteriorad. This, however, does not seem to hold for the connecting tracheae. Xy mph ula obscuralis An examination of Xymp/iula obscuralis showed a condition very simi- lar to that in X. maculalis. While the following diameter measurements were taken from a single mature larva, they are representative of those for other larvae. The structural features of the spiracles, the connecting Spiracles Lumen of Spiracular Tracheae Percentage of Decrease Segment In Spiracles In Tracheae 1 2 0.0074 0.0005 66.6 97.3 3 4 0.0074 0.0005 66.6 97.3 5 0.0222 0.0185 0.0 0.0 6 0.0222 0.0185 0.0 0.0 7 0.0222 0.0185 0.0 0.0 8 0.0074 0.0024 66.6 87.0 9 0.0074 0.0017 66.6 90.8 . 10 0.0074 0.0017 66.6 90.8 11 0.0074 0.0005 66.6 97.3 42 PAUL S. WELCH tracheae, and the closing apparatus, are also so similar that no description is necessary here. Cataclysta fulicalis In Cataclysta fulicalis the spiracles and the connecting tracheae differ from those of the gilled Nymphula caterpillars in that no reduction of any kind appears, all being of practically uniform size and structure. Some variation occurs but it is slight and apparently insignificant. The circle of guarding spines at the outer periphery of the spiracle is less con- vergent (PI. X, fig. 2) than in the gilled Nymphula larvae, thus forming a wider aperture. Structurally, the spiracles, connecting tracheae, and the closing apparatus are similar to those of the gilled Nymphula group, slight but inconsequential deviations being present. The following table includes a typical set of diameter measurements made on one specimen: Segment Spiracle . Lumen of trachea 1 0.026 0.03 2 3 4 0.029 0.033 5 0.028 0.043 6 0.028 0.035 7 0.03 0.035 8 0.035 0.033 9 0.031 0.040 10 0.031 0.038 11 0.031 0.038 When all measurements were averaged, the spiracles and their connect- ing trunks were found virtually uniform in size. The larvae of Cataclysta fulicalis have thus acquired a system of gills without accompanying changes in the spiracular system, the latter being as completely open morphologically as any terrestrial caterpillar. Nymphula ohliteralis The spiracles and connecting tracheae are distinctly open and show no evidence of definite reduction. A certain variation appears, as will be noted in the following table, but even the smallest found is far above the corresponding reduced structures in N . maculalis and N. ohscuralis. In the following representative diameter measurements taken from the record of one mature specimen, nothing is especially noteworthy except the large spiracle and connecting trachea on the fourth segment. Whether the difference in size is an evidence of a slight reduction of spiracles and connecting tracheae in the posterior segments is uncertain. The principal features of the closing apparatus are shown in figure 8. Segment. Spiracle. . Lumen of trachea 1 0.032 2 3 4 0.044 5 0.04 6 0.04 7 0.04 8 0.032 9 0.032 10 0.036 0.028 0.064 0.028 0.028 0.024 0.020 0.020 0 020 11 0.04 0.0.^6 RESPIRATORY MECHANISM IN LEPIDOPTERA 43 Xympliula sp. A non-gilled form, distinctly different from any other used in this work but whose specific identity is unknown^ was examined in this connec- tion. Sections showed all of the spiracles to be distinct, well-developed, open, and approximately uniform in structure and size, the same being true of the connecting tracheae. Average measurements for the whole series are as follows, the extreme variations deviating very little from the average: Diameter of the spiracles, 0.05569; diameter of the lumina of the tracheae, 0.0527. Expcrifuoits In order to demonstrate experimentally the open condition of the spiracles and connecting tracheae and check the morphological results, certain experiments were made on the caterpillars N. maculalis. Larvae dropped into hot water invariably give off one or more bubbles of gas from the large spiracles on segments 5-7, thus showing definitely the open condition of these spiracles and their connecting tubes. No gas was given off from the reduced spiracles, but it does not follow that such failure is due to complete closure since the amount of gas in the tiny lumina is extremely slight and the expansion of the heated gas in the longi tudinal tracheal trunks would be more likely to be released at the larger and more open spiracles on segments 5-7. It was found that the larvae of N. maculalis could live in commercial kerosene for 6-7 hours. They were then submerged in kerosene colored with Sudan III. Since the translucency of the body-wall made it possible to trace much of tracheal system, it was easy to examine the spiracular connections at any time and to follow the entrance of the colored liquid into the larger spiracles. Positive evidence that these spiracles are mor- phologically open was thus repeatedly secured. This penetration, into the large spiracles, occurred within an hour, but entrance into the smaller ones was much slower although ultimately the colored liquid was observed in some of the connecting tracheae. DlSCUSSION It thus appears, at least in Xyviphula maculalis, X. obscuralis, and Cataclysta fiilicalis, that in spite of the gill development, the tracheal sys- tem of the larvae is morphologically open. The reduction of certain spiracles and their connecting tracheae in the first two is definite and striking but has not progressed to the place where the lumina and apertures are completely closed. Since the previous work on this subject has been done on unavailable foreign species it is not possible, on the basis of the present work, to absolutely refute the statements made in the literature, but the writer is inclined to suspect strongly that what has been found 44 PAUL S. WELCH in N . niaculalis and .Y. obscuralis is likewise Irue of N . stratiotata and other foreign gilled representatives of that genus. Portier's results (1911), secured as they were without any attempt at critical morphological work, cannot be regarded as conclusive. However, it is not inconceivable or impossible that certain species might have progressed to the point of closing the spiracular system but if such a condition does exist it should be demon- strated more convincingly than has heretofore been done. In regard to the functioning of these reduced spiracles and tracheae, no serious question can be raised concerning the larger ones on segments 5-7 in N . maculaUs and lY. obscuralis since their size, open character of the connecting tracheae, and structure of the closing apparatus all indicate the possibility of normal activity. In spite of the small diameter of the more reduced spiracles and tracheae, the writer has thus far found nothing which would prohibit at least a limited functioning of these organs. That air will pass through pores and tubes of smaller diameters than those of the organs under discussion is now known, and many of the very minute insects known to have a typical holopneustic type of tracheation have openings and tracheae no larger than the reduced ones of the gilled Nympli- ula caterpillars. Likewise if the minute tracheoles of the tracheal system which are less than one micron in diameter can transport atmospheric gases, failure of the reduced spiracles and connecting tracheae to function would have to be due to some feature other than the structure of the tubes themselves. That there would no difficulty in the ventilation of such a system has recently been shown by Krogh (1920a; 1920b) since in small forms diffusion alone will provide the necessary oxygen transportation, although it may be assisted by respiratory movements of the animal, if the latter are manifested. In certain insects having apneustic tracheation the spiracles and con- necting tracheae are said to be temporarily open at the time of molting. This, however, does not account for the open character of the gilled larvae of Nymphula since sections of specimens in various parts of the stadia involved were studied and all yielded the same result. There seem to be no reasons for assuming that open spiracles and open connecting tracheae are necessarily inimical to larvae existing in water since certain well known forms, as for example, Bellura melanopyga, have complete sets of open spiracles, yet are related to the water in such a way that most or all of these organs are submerged for long periods of time. It is not unlikely that still other aquatic larvae, thought to have true apneustic tracheation, will be found to possess morphologically open spiracles. What part these oj)en spiracles ])lay in the life ol the forms involved is difficult, at present, to specify. As has been pointed out, the gilled Nymphula larvae can pass extended periods of time out of water, at least RESPIRATORY MfXHANISM IX LEPIDOPTERA 45 during the last larval stadium. This indicates functioning of the spiracles, since it is unlikely that the requisite amount of oxygen could be secured by cutaneous means alone, especially after the surface of the body became dry. In regard to submergence, it is possible that a provision against penetration of water into the tracheal system is afforded in the combina- tion of structures present. The marginal crown of guarding spines or their derivatives, if hydrofuge in character, may constitute an efficient protec- tion. It is also possible that the well developed closing apparatus plays some part in this connection. General Considerations From the point of view of the respiratory mechanisms involved, the true aquatic Lepidoptera comprise a heterogeneous assemblage, including those, on the one hand, which have made no morphological advance to- wards the aquatic life, and those on the other hand, which manifest highly developed morphological adaptations of an aquatic sort. These adapta- tions involve, in the most complex type, the addition of structurally com- plex gills, and the marked reduction in size of spiracles and connecting tracheae. It should be noted that apparently no advantage has accrued to the possessors of the complex adaptation since in all of the situations examined by the writer the non-gilled larvae, having the unmodified tracheal system of a terrestrial type, have had every appearance of being as successful in the aquatic medium as their more specialized relatives, often existing in identically the same environment and offering an interest- ing parallel in the solution of the same problems by very different means. There would seem to be a considerable advantage in the possession of over 400 gill-filaments and a set of reduced spiracles, particularly in those cases where the size of the body proper is virtually that of the associated non- gilled forms. As previously mentioned, the structure of the gills in Nymph- ula is such that it seems almost inconceivable that they do not function as true respiratory organs. In a recent paper. Fox (1921) claims to have demonstrated that in a certain chironomid larva, oxygen is not taken up by the ventral blood gills; that the anal gills take up less than the corre- sponding area of the body-surface; and that most of the oxygen is received through the body-wall in general. These surprising results require con- firmation and at present need not be regarded as serious ground for ques- tioning the function of the gills in other insects. On the basis of structure alone it might appear that the small blood gills of Cataclysta represent a more primitive stage in the development of aquatic respiratory adaptation than that represented in the gilled larvae of Nymphula. However, there is no indication that any of the Nymphula species have ever had blood gills only. The evolution of these larval 46 PAUL S. WELCH adaptations has apparently been a sporadic phenomenon with the extremes sometimes occurring within the confines of a single genus. Summary 1. Fundamentally, aquatic larvae of the genus Nymphula have retained the original terrestrial type of body tracheation in practically unmodified form. The tracheation of the gills has been superimposed upon the terres- trial type with minimal change to the latter, and the non-gilled larval type is doubtless the older one phylogenetically. 2. Gills in the aquatic Lepidoptera are all hollow outgrowths of the body-wall, the cavity being in direct communication with the haemocoele. All of the layers of the body-wall are represented but in reduced thickness, maximum diminution appearing in the dermis. 3. In all of the gilled larvae of Nymphula examined, the gill-cavity con- tains both an elaborate set of tracheae and tracheoles and a considerable quantity of body fluid, thus constituting a combination tracheal-blood gill. In the larvae of Cataclysta fulicalis the gills have no traces of tracheae and are thus blood gills only. 4. In the non-gilled larvae of Nymphula and the gilled larvae of Cataclysta fulicalis the tracheation is typically holopneustic, no reduction of any significance appearing either in the spiracles or their connecting tracheae. 5. In gilled larvae of Nymphula, a distinct reduction appears in the spiracles and their connecting tracheae on segments 1, 4, 8, 9, 10, and 11, those on segments 5, 6, and 7 being much larger and having undergone less reduction. 6. Morphological and experimental studies on gilled Nymphula larvae have shown that in spite of the striking reduction of some of the spiracles and connecting tracheae, the tracheation is still holopneustic, all spiracles and tracheae being morphologically open with nothing to indicate that they are functionless. While gilled representatives of foreign species of this genus have not been available, it is very probable that the statements in the literature to the effect that they have a closed tracheal and spiracu- lar system are in error, due to insufficient study. 7. The gilled larvae of Nymphula macidalis may live for extended periods of time outside of water, even after the outer surface becomes dry and the gill-filaments shriveled, indicating that respiration through the spiracles is being accomplished. 8. Reduction of spiracles and possession of gills do not seem to be necessarily correlated or coexistent since in Cataclysta fulicalis both gills and an unreduced tracheal system are present. 9. In spite of the contrast between the gilled and the non-gilled species, the former seem to have no advantage over the latter, at least in those cases where both forms exist side by side in the same habitat. RESPIRATORY MECHANISM IN LEPIDOPTERA 47 Literature Cited Fox, H. M. 1921 Methods of Studying the Respiratory Exchange in Small Aquatic Organisms, with Particular Reference to the Use of Flagellates as an Indicator for Oxygen Con- sumption. Journ. Gen. Phj's., 3:565-573. 5 fig. Hart, C. A. 1895 On the Entomology of the lUinios River and Adjacent Waters. Bull. 111. State Lab. Nat. Hist., 4:149-273. 15 pi. Krogh, A. 1920a Studien iiber Tracheenrespiration. II. liber Gasdiffusion in den Tracheen. Pfliiger's Archiv ges. Phys. d. Menschen u. d. Tiere, 179:95-112. 5 fig. 1920b Studien iiber Tracheenrespiration. III. Die Kombination von mechanischer Ventilation mit Gasdiffusion nach Versuchen an Dytiscuslar\'en. Pfliiger's Archiv. ges. Phys. d. Menschen u. d. Tiere, 179:113-120. 2 fig. Lloyd, J. T. 1914 Lepidopterous Larvae from Rapid Streams. Journ. N. Y. Ent. Soc, 22:145-152. 2 pi. 1919 An Aquatic Dipterous Parasite, Ginglymyia acrirostris Towns., and .Additional Notes on its Lepidopterous Host, Elophila fulicalis. Journ. N. Y. Ent. Soc, 27:263-265. 1 pi. Muttkowski, R. a. 1920. The Respiration of Aquatic Insects. A Collective Review. Bull. Brook. Ent. Soc, 15:89-96, 131-141. 1921a Copper in Animals and Plants. Science (N.S.), 53:453-454. 1921b Studies on the Respiration of Insects. I. The Gases and Respiratory Proteins of Insect Blood. Ann. Ent. Soc. Am., 14:150-156. 1921c Copper: Its Occurrence and Role in Insects and Other Animals. Trans. Am. Micr. Soc, 40:144-157. Portier, p. 191 1 Recherches Physiologiques sur les Insectes Aquatiques. Arch. d. Zool. Exp., (5), 8:89-379. 4 pi. 67 fig. Rebel, H. 1899 Zur Kenntnis der Respirationsorgane Wasserbewohnender Lepidopteren Larven. Zool. Jahrb., abt. f. Syst., 18:1-26. 1 pi. Rose, W. C. and Bodansky, M. 1920 Biochemical Studies on Marine Organisms. I. The Occurrence of Copper. Journ. Biol. Chem., 44:99-112. Welch, P. S. 1914 Habits of the Larva of Bellura melanopyga Grote (Lepidoptera) Biol. Bull., 27:97-114. 1 pi. 1915 The Lepidoptera of the Douglas Lake Region, Northern Michigan. Ent. News, 26:115-119. 1916 Contribution to the Biology of Certain Aquatic Lepidoptera. Ann. Ant. Soc Am., 9:159-187. 3 pi. 1919. The Aquatic Adaptations of Pyrausta penitalis Grt. (Lepidoptera.) Ann. Ent. Soc Am., 12:213-226. Wesenberg-Lund, C. 1913 Wohnungen und Gehausebau der Siisswasserinsekten. Fortschr. d. Naturwissensch. Forsch., 9:55-132. 59 fig. 48 PAUL S. WELCH Abbreviations Used in Plates bl. blood cl. closing apparatus c.t. connecting trachea d. dermis ep. epidermis glc. gill cavity hyp. hypodermis l.t. longitudinal trachea mil. muscle sp. spiracle t. trachea RESPIRATORY MECHANISM IN LEPIDOPTERA 49 4 (t 6 Plate X Fig. 1. Longitudinal section through large spiracle and connecting trachea in larva of Nymphula maculalis. Fig. 2. Longitudinal section through spiracle and connecting trachea in larva of Cata- clysta fulicalis. Fig. 3. Longitudinal section through reduced spiracle and connecting trachea in larva of Nymphula maculalis. Fig. 4. Drawing of caustic potash preparation of large spiracle and connecting trachea in larva of Nymphula maculalis. Fig. 5. Transverse section of gill-filament in larva of Cataclysta fulicalis. Fig. 6. Transverse section of gill-filament in larva of Nymphula maculalis. 50 PAUL S. WELCH Plate XI Fig. 7. Transverse section at ental end of connecting trachea for large spiracle in larva of Nymphula maadalis, showing structure of closing apparatus. Fig. 8. Transverse section at ental end of connecting trachea for spiracle in larva of Nymphula oblikralis, showing structure of closing apparatus. Fig. 9. Camera lucida drawing from living larva of N. maadalis showing tracheation of gill-filament as it appears under low magnification. _ Fig. 10-11. Camera lucida drawings from living larva of N. macidalis showing distribu- tion and arrangement of finer tracheoles of gill-filament as they appear under high magnifica- tion. DEPARTMENT OF METHODS, REVIEWS, ABSTRACTS, AND BRIEFER ARTICLES DICHROMATIC ILLUMINATION FOR THE MICROSCOPE By Leon Augustus Hausman, Ph.D. The practise of using monochromatic light in photomicrographic work is widespread. The advantages in the use of such light in conjunction with ocular microscopic examination seem to be less appreciated; while the use of dichromatic illumination, such as that described in this paper, the writer believes to be new. In using monochromatic light, i.e., light of a given color or wave- length for microscopic examination, the purpose is three-fold: (1) to increase the resolving power of the objective, (2) to secure greater con- trast between different parts of the specimen, and (3) to afford relaxation for the eyes. Abbe's formula for ascertaining the resolving power of an objective is to multiply the numerical aperture of the objective by twice the number per inch of the waves of the light employed. Hence it follows that the shorter the wave length of the light, the greater the resolution of the objective. This has an application of appreciable value in connec- tion with the visibility and sharpness of focus of minute objects which seem to lie just upon the border-line of vision. Moreover after working long with white hght, green light affords a grateful relaxation to the eyes. The microscope screen (S, Fig. 1) devised by the writer, has been used with success both for monochromatic and dichromatic illumination. With the former type of illumination the color filter is inserted in the slide (CI, Figs. 1 and 2) and the light obtained from the arc-lamp (Al, Figs. 1 and 2). For the examination of minute structures in the protozoan cell, and of the pigment granules in the cortex of mammalian hairs, green or blue illumination was found to be excellent. The former is more restful to the eyes, especially when making protracted examinations. It is, moreover, of greater luminosity, and hence permits of greater ease in focussing the specimen. In the examination of protozoa the writer's practise is to focus the object by the green light, and then exchange this for the blue. Violet light was found unsatisfactory, because of its lack of luminosity. Certain 51 52 LEON AUGUSTUS HAUSMAN, PH.D. ■ M £ f ^J'^m j^M V^^^^^^B ^uSS' ^^s i^^*^\^L . ^. Fig. 1 Assemblage of apparatus for dichromatic illumination. Microscope (M) in position behind the screen (S), which bears slides (CiandC2) for supporting the color filters. Illumina- tion of the object above and below is secured from the two arc lamps (Ai and A2). Fig. 2 Diagrammatic view of apparatus for dichromatic illumination, from above. Ai and A2 arc lamps, for transmitted and reflected light respectively; Bi and B2, plano-convex condensers; the former to deliver practically parallel rays to mirror, the latter to illumine object from above; Ci and C2, apertures for color filters; M, microscope. The screen (S) is stippled. DEPARTMENT OF METHODS 53 stained structures show up well with monochromatic light, particularly if the color used be complementary to the one used as stain. By employing a new type of illumination, i.e., dichromatic, it is possible by means of the illumination alone to invest certain portions of the speci- men with one color, and other portions with another, for the purpose of bringing out thus by contrast the forms or relationships of the two. The principle of dichromatic illumination is to illuminate the microscope field and the transparent portions of the specimen with one color, by means of transmitted light, and the more dense, or opaque portions of the speci- men with another color, by means of reflected light, and to secure the maximum contrast between the structures so illuminated by using comple- mentary colors. Such a result has been secured by means of the apparatus shewn in Figs. 1 and 2 (whose letterings are similar). The microscope (M) is suffi- ciently protected from all rays of light save those desired, by the screen (S), which bear two slides (CI and C2). In these slides are apertures wherein can be placed color filters. Filter CI delivers to the microscope mirror light of one color for illuminating the field and the transparent portions of the specimen, and filter C2 delivers light of another color which is focussed upon the specimen from above. The transparent portions of the specimen are therefore viewed by transmitted light of one color, the opaque portions by reflected light of another color, and the translucent portions by a combination of these two. Slide CI bearing its filter can be moved laterally, and slide C2 vertically. The positions which the filters can be made to occupy, together with the various positions of the micro- scope within the screen, make it possible to secure light from any of the angles useful in microscopic work. Illumination is furnished by two mov- able arc-lamps, Al for delivering light to the mirror, and A2 for lighting the specimen from above. Within the microscope screen two movable plano-convex condensers are used; one of long focus (Bl), for delivering to the mirror virtually parallel rays; the other (B2) of short focus for condensing the light upon the specimen from above. With such an apparatus the writer has secured good results in the examination of mammalian hairs, for the detection of delicate scalation. With further experimentation the use of such lighting may possibly be widened. Color filters for use in photomicrographic work can be purchased and used in the screen. Good filters can be made, however, in the laboratory by developing out unexposed lantern slides, or dry plates, and then staining the clear gelatin film with the desired colors. Care must be taken to secure color filters that will allow only one color of light to pass through them, i.e., they must furnish, as nearly as possible, monochromatic light. Some 54 LEON AUGUSTUS HAUSiMAK, PH.D. of the stains, in aqueous solution, which have been recommended for this purpose are: (1) Yellow — a saturated solution of picric acid. This absorbs almost completely the blue and violet end of the spectrum. (2) Green — methyl green. The absorption spectra varies with the depth of the color. (3) Copper sulphate. This absorbs the red almost entirely. Methyl blue is fairly good. (4) Red — Safranin. The procedure in making color filters from lantern slide plates is to develop and fix the unexposed plates and then to allow the gelatin film to soften by placing Ihe plates in a bath of slightly warm water, say 80 degrees F. for a few minutes. They may then be removed to baths of the different stains, and allowed to soak for an hour or so, or until the gelatin is evenlv stained. It was found best to make up a series of saturated aqueous solutions of the stains, and from these gradually to increase the depth of color of the various baths until the desired depth was secured in the gelatin film. The optimum for color filters is the greatest depth of color which one can use and still secure sufficient luminosity for good focus. The depth of the color of the filter giving the best results will depend upon the brightness of the illuminant. This latter should give a strong, white light. Such a light, with a spectrum very much like that of sunlight, can be obtained from the electric arc. A MODIFIED BARBER PIPETTE The writer has been much inlerested in the various moditications of the Barber pipette. One, which seems to be more simple than any here- to-fore described, has been in use in these laboratories since 1916. It seems worth while to describe it. The device consists of a bar A (side view — figure 1, top view — figure 2) fashioned to fit partly around the objective and fastened to it by the thumb screw B. The rod C passes thru this bar and is held in position by the thumb-screw D. The bar E is fastened to the rod C by a screw which also passes thru the spring F. The part G is composed of a ring thru which a glass rod passes (the latter held in position by the thumb screw H) and an angular piece with pivots at the angle. There is a thumb screw I which controls the upward and downward movement of the point of the pipette J. The camera bulb K completes the apparatus. This device, minus the hollow tube and the camera bulb, was devised l^y the late Dr. J. J. Wolfe for use in the picking up of diatoms by adhesion. The hollow tube and camera bulb were added by the writer for picking up copepods. This device later became very useful in the segregation of living diatoms. A mechanical stage may be used with the pipette. Altho the laboratory possesses a real Barber pipette, this simpler apparatus is preferable when diatoms or larger or- ganisms are to be segregated. Bert Cunningilxm. Trinity College, Durham, N. C. y::) CLEANING SLIDES AND COVERS FOR DARK-FIELD WORK As particles of dirt, show with the same brilliancy as the bodies one wishes to study, the slides and covers must be made very clean. One of the most satisfactory ways of cleaning the slides is that suggested by Stitt in his book on the blood etc., p. 235, 5th Ed. near the top. I have found the modification here given very satisfactory: (1) 5 grams of powdered Bon Ami are mixed with 100 cc of water and thoroughly shaken up. (2) New slides and covers are put into this mixture and well wetted, then they are taken out and stood up on blotting paper to drain and dry. The method answers well also for the final polishing of cover-glasses that have been cleaned in acid-dichromate mixture, and for used slides that have been cleaned in any approved method. (3) Whenever one wants a slide or a cover one of those on which is the dried Bon Ami is taken and wiped with a clean piece of gauze. It is astonishing how quickly and well the cleaning can be done in this way. Very few of them show any particles of dirt with the dark-field miscroscope. (4) Cleaning the used slides and covers. — Hot water is allowed to flow on the slide to wash off the oil, then the cover is removed and put into cleaning mixture (Sulfuric acid and dichromate). The slide can usually be well cleaned with the Bon Ami. S. H. G.-^GE. 56 PROCEEDINGS OF THE AMERICAN MICROSCOPICAL SOCIETY Minutes of the Toronto Meeting The 40th annual meeting of the American ^Microscopical Society was held in affiliation with the American Association for the Advancement of Science at Toronto, Canada, Decem- ber 29, 1921. In the absence of the President, Frank Smith, and both Vice-Presidents, Professor Albert M. Reese acted as Chairman. The report of the Treasurer for the year 1921 was read by the Secretary and referred to an Auditing Committee composed of Profs. R. J. Pool and R. H. Wolcott. The report of the Custodian was read by the Secretary and referred to an .Auditing Com- mittee composed of Messrs. Edw. Pennock and F. E. Ives. The meeting voted to send congratulations to the Custodian, Mr. Magnus Pflaum on the growth of the Spencer- Tolles Fund. The Secretary presented a general report on the affairs of his office. The following officers were nominated and elected for the constitutional periods: Presi- dent, Dr. N. A. Cobb, Bureau of Plant Industry, Washington, D. C; Urst Vice-President, Professor E. M. Gilbert, University of Wisconsin; Second Vice-President, Professor Z. P. Metcalf, North Carolina State College of Agriculture and Engineering; Custodian, Mr. Magnus Pflaum. Philadelphia, Pa. Dr. B. H. Ransom, Bureau of Animal Industry, Professor Chancey Juday, University of Wisconsin, and Professor George R. La Rue, University of Michigan, were chosen as the elective members of the Executive Committee for 1922. Adjourned. P. S. Wfi.ch, Secretary. Reports of the Treasurer and Custodian Because of unavoidable delays, the reports of the Treasurer and the Custodian can not appear until the April number. 57 >-^ TRANSACTIONS OF THE American Microscopical Society Organized 1878 Incorporated 1891 PUBLISHED QUARTERLY BY THE SOCIETY EDITED BY THE SECRETARY PAUL S. WELCH ANN ARBOR, MICHIGAN VOLUME XLI Number Two Entered as Second-class Matter August 1?, 1918, at the Post-office at Menasha Wisconsin, under Act of March 3, 1879. Acceptance for mailing at the special rate of postage provided for in Section 11 03, of the Act of October 3, 1917, authorized Oct. 21, 1918 ullip dnllpgiatp l^reaa Geokge Banta Publishing Company Menasha, Wisconsin 1922 TABLE OF CONTENTS For Volume XLI, Number 2, April, 1922 On the Protozoa Parasitic in Frogs, with thirty-six figures, by R. Kudo 59 A New Suctorian from Woods Hole, with one plate, by F. M. Root 77 Department of Summaries Ten Years of Heredity, with eight figures, by A. Franklin Shull 82 Department of Methods, Reviews, Abstracts, and Briefer Articles A New Micro-slip, with one figure, by F. J. Myers 101 Killing, Staining and Mounting Parasitic Nematodes, by H. G. May 103 A New Locality for Spongilla wagneri Potts, by Frank Smith 106 Some Interesting Studies on Spider Anatomy, with one plate, bj' E. W. Roberts. . 107 Annual Report of the Treasurer 110 Custodian's Report for the Year 1921 Ill ^'1 TRANSACTIONS OF American Microscopical Society (Published in Quarterly Instalments) Vol. XLI APRIL, 1922 No. 2 ON THE PROTOZOA PARASITIC IN FROGS* By R. Kudo Universitj' of Illinois Probably no other animals have been for many year.s more favorite objects of studies by zoologists than the frogs. The amphibians have been examined by several Protozoologists and we know at present a consider- able number of Protozoa of a great variety parasitic in frogs from various parts of the world. Numerous publications dealing with the protozoan parasites of frogs have been issued by authors of several nationalities. Aside from the papers by North American workers such as Ohlmacher (1893), Whinery (1893), Gurley (1894), Stebbins (1904, 1905), Lewis and Williams (1905), Metcalf (1909) and Swezy (1915, 1915a), a large majority are widely scattered in various periodicals, and are not always easily referred to. Undoubtedly this hardship concerning literature prevented the students in Zoology from taking advantage of the material. If one possesses there- fore brief accounts of the Protozoa commonly found in frogs, hundreds of which are sacrificed yearly by students in Zoology and by special investi- gators, one can utilize both material and time in carrying out observations upon these interesting Protozoa. The present paper is an attempt to meet this need. It deals with my observations on the Protozoa parasitic in North American frogs which I have examined during the last two years, together with the description of methods of observation, and with brief review of and reference to the works of the previous investigators on the subject. The following six species are described in order: 1. Entamoeba ranarum from the intestine 2. Leptotheca ohlmacheri from the kidney 3. Haemogregarina sp. from the blood *Contributions from the Zoological Laboratory of University of Illinois. No. 199. 59 60 R. KUDO 4. Trypanosoma rotatorium from the blood 5. Trypanosoma parvum nov. spec, from the blood 6. Opalina sp. from the intestine I Entamoeba ranarum (Grassi) Dobell 1908 Habitat. — In the large intestine of Rana temporaria, R. esculent a, R. clamitans and Bufo vulgaris. Dobell (1909) saw that about 23% of Rana temporaria in Cambridge and Munich, were infected. I have seen a number of amoebae whose characters agree on the whole with those de- scribed by Dobell for Entamoeba ranarum m one out of 14 individuals of Rana clamitans from New York in August of 1920. In Rana pipiens which I have studied in 1920 and 1921 at Urbana, Illinois, I did not observe any host individual that harbored the organism. This of course does not mean its absence in a frog of this species, since I have not ex- amined them as thoroughly as I did in the case of Rana clamitans. Historical. — Lieberkiihn (1854) probably noticed the Amoeba in the intestine of the frog which he studied. Grassi (1879) examined and named it Amoeba ranarum. Dobell (1909) found an Amoeba in the frogs of England and Germany, and studied them in detail. Quite recently, the same author (Dobell, 1918) states that although the Amoeba resembles closely morphologically to Entamoeba histolytica of human intestine, they are distinct species. I have met with apparently the same Protozoon but once, and could not carr}^ observation concerning its development. Distribution. — Europe and North America. Methods of observation. — A portion of the large intestine of a frog is cut into small pieces in physiological salt solution on a cover-glass, made an ordinary fresh preparation and observed. The organism may live for several hours. The general appearance, changes in form of the body through the formation of pseudopodia and the structure of the protoplasm can be studied. To make permanent preparations, make smears on slides or cover-glasses and fix them with hot sublimate-alcohol-acetic mixture (2 parts of saturated aqueous solution of corrosive sublimate, 1 part of absolute alcohol and 5% of glacial acetic acid) for about 20 minutes. The smears are then immersed for about 15 minutes in a weak iodine alcohol (50%) and then transferred into a plain alcohol to remove the iodine. Staining with Delafield's haematoxylin, Heidenhain's iron haematoxylin or Dobell's alcoholic haematcin, brings out satisfactory results. Morphology. — Amoeba of moderate size. When alive, the cytoplasin is poorly differentiated into ectoplasm and endoplasm. Lobose pseudopodia are actively formed at one time from any part of the body. The peripheral portion of the cytoplasm is somewhat hyaline, while the main part of the body is granulated and contains bacteria, yeasts and other particles ON THE PROTOZOA IN FROGS 61 present in the host intestine. The nucleus is spherical and faintly visible in living condition with an oil immersion objective. No contractile vacuole is present. Dimensions vary from 15 to 40/i in the largest diameter. When stained, the cytoplasm becomes highly vacuolated or reticulated. The nucleus is spherical and usually contains a distinct karyosome. Figs. 1 and 2. Entamocha ranarum. Fig. 1, a living individual. Fig. 2, an individual stained with Delafield. .\ 1500. Development. — According to Dobell, the cysts are found in the host intestine in winter months. They are spherical, and measure 10 to 16/i in diameter with a large nucleus. The nucleus divides twice producing four daughter nuclei. Further changes are not known. Dobell suggests that the cysts serve for the dissemination of the organism. The same author (Dobell, 1918) recently found that although Entamoeba ranarum and E. histolytica can hardly be distinguished morphologically from each other, the cysts of the latter species when introduced into the intestine of tad- poles did not undergo changes which take place in their proper habitat, and concluded that these two forms should be held as different species. II Leptotheca ohlmacheri (Gurley) Labbe 1899 Synonyms. — Chloromyxum (Sphaerospora) ohlmacheri Gurley 1893, Leptotheca ranae Thelohan 1895 and Wardia ohlmacheri Kudo 1920. Habitat. — In the kidney of Rana clamitans, R. pipiens and Bufo lentiginosus. Out of 14 individuals of Rana clafnitans examined in New York from July to September, 1920, six were infected by the parasite. Out of 24 Rana pipiens bought from a Chicago biological supply store and examined between November and December, 1920, ten were found to be infected by the Myxosporidian. Thelohan (1895) named a Myxo- sporidian which he saw in the kidneys of Rana esculenta and R. temporaria, Leptotheca ranae. He has not given description nor figure for it, but I am inclined to think this is probably identical with the American species. 62 R. KUDO Historical. — The spores of the Myxosporidian were first found by Ohlmacher (1893). Whinery (1893) also worked on them. Gurley (1894) summarized the observations of the two authors. Thelohan (1895) found Leptotheca ranae in France (?). No body seemed to have worked on the organism until 1920 when I found the vegetative stages and spores of a species what appeared to be identical with the present form. I have studied its morphology and development, the result of which will be stated elsewhere (Kudo, 1922). Distribution. — North America and Europe (?). Methods of observation. — When the infection is heavy, isolated spores may be found in the cloaca of the host, but the kidney must be examined for both spores and trophozoites. A small part of the kidney is cut into small pieces on a slide in a drop of physiological salt solution and made fresh preparation. In order to remove the fat globules that are usually present in smears of kidneys, one drop of weak potassium hydrate solution may be added to it. If any spores are present, they will be easily recog- nized under a low power due to their peculiar appearance. If the infection of the kidney is detected, hanging drop preparations or fresh preparations with physiological solution should be made immediately and sealed with melted parafilin. By using oil immersion objective, one can distinctly see the detailed structures of the spores and trophozoites of various develop- mental phases. To make permanent preparations, smears of variable thickness should be made. In thinly made portion, one can see the number and structure of the nuclei in well stretched trophozoites, while in thickly made part, the shape, general appearance and arrangement of nuclei and cytoplasm around them may be studied. Smears are well fixed with sublimate-alcohol-acetic mixture. For staining, besides the three methods stated for Entamoeba ranarum, Giemsa's method brings out beautiful results. Section preparations should also be made in order to observe the location of various developmental stages of parasites in the host organ and the relation between the parasite and host body. Morphology. — Fully grown trophozoites are usually rounded or oval in form. Long conical pseudopodia are actively formed. Frequently the trophozoites are completely rounded without any pseudopodia. The body is colorless, granulated and extremely h}'aline. The cyto]>lasm is indistinctly differentiated into endoplasm and ectoplasm. The cndo- plasm is finely granualted and contains a vegetative nucleus, two develop- ing spores and fat globules of variable size and number. The ectoplasm is only distinctly visible where the pseudopodia are formed, the latter being usually entirely composed of ecto])lasm. Before starting spore formation the trophozoites multi])ly by active gemmation. In almost all cases, disporous, rarely monosporous and more rarely trisporous. ON THE PROTOZOA IN FROGS 63 Figs. 3 to 6. Trophozoites of Leplotheca ohlmacheri. Fig. 3, a trinucleate trophozoite with a vegetative nucleus and two generative nuclei. A thin smear stained with Delafield. Fig. 4, a young trophozoite in which four polar capsules are beine formed: fresh preparation. Fig. 5, a thinly spread trophozoite with a vegetative nucleus and two developing spores. Giemsa. Fig. 6, a rounded trophozoite with two mature spores: fresh preparation. All X 2350. Dimensions of fully grown disporous trophozoites are 30 by 20^t, 38 by 25/i, 40 by 20/z, etc. Each spore develops independently. Development. — With regard to the interesting development of the Myxosporidian the reader is referred to one of my papers (Kudo, 1922). Morphology of the spore. — Oblong with its largest diameter standing at right angles to the sutural plane. Anterior end is conspicuously attenuated due to the thickening of the spore membrance at this point while the posterior end is rounded. In lateral view, it is nearly circular with a pointed anterior end. In an anterior end view, it is regularly oblong. The spore membrane is moderately thick. Sutural ridge is well marked, especially at the anterior end. The membrane is somewhat irregularly striated. Three to seven fine striae run parallel to the sutural 64 R. KUDO line on each valve and the remaining striae make somewhat similar angles with the sutural line. The striae in lateral view run parallel to one another except those on the posterior margin where a few make angles with the former. The striae on each valve vary from 25 to 35 in number. Figs. 7 to 13. Spores of Leptotheca ohlmacheri. Figs. 7, 8 and 9, the upper surface, optical section and lower surface views of a normal fresh spore. Fig. 10, anterior end view of a fresh spore. Fig. 11, an optical section of a fresh spore with two large sporoplasms. Fig. 12, a fresh abnormal spore showing two sporoplasms and two capsulogenous cells, each con- taining a polar capsule. Fig. 13, a section through a spore showing the deepi)' stained polar capsules and two uninucleate sporoplasms. Section: Delafield. Figs. 7 to 11, x 1500; Figs. 12 and 13, x 2350. Two polar capsules usually equal in size, occupy the anterior portion of the spore. The polar filament is coiled four to six times and is distinctly visible in fresh condition. It can be extruded under the action of potas- sium hydrate or mechanical pressure as I stated elsewhere (Kudo, 1918, 1921). Without staining the filament can be seen under a low magnifica- tion. Two independent sporoplasms occupy the extracapsular cavity of the spore, which condition is very rarely seen in Myxosporidia. They appear homogeneous in fresh state. Staining reveals that each sporo- plasm contains a single nucleus. Dimensions of fresh spores: sutural diameter and thickness, 9.5 to 12/i, breadth, 13 to 14.5^i, diameter of polar capsules 3.5 to 4.5 fx, length of extruded polar filament, 42 to 62 (x. Those of stained spores: sutural diameter and thickness, 8.5 to 10 ju, l)readth, 9 to 12 /z, diameter of polar capsules 3 to 4 ^t. Ill Ilaemogregarina .sp. Our knowledge of haemogregarines is still in great confusion because their development has not been studied except species occurring in reptiles. The haemogregarine described here seems to agree with the following ON THE PROTOZOA IN FROGS 65 species: Drepanidium magnum Grassi et Feletti 1891, Drepanidium krusei Labbe 1892 and Karyolysus clamatae Stebbins 1905. Habitat. — In the blood cell and plasma of Rana clamitans and Rana pipiens. I have observed it quite frequently in the last named host species. Quite frequently the frogs were found to harbor trypanpsomes at the same time. Historical. — While Lankesterella minima seems to have repeatedly been studied by European authors (see for instance, Hintze, 1902), the present form has been seen rarely. It seems to be this form that attracted the attention of some North American investigators such as Langmann (1898- 1899) and Stebbins (1905). I have seen quite frequently the haemogre- garine in frogs of New York and Illinois, but so far have not seen Lankes- terella minima, the common European form. Methods of observation. — Same as those stated for trypanosomes. Morphology. — The haemogregarines found in the blood of frogs may be spoken of under two types: intracorpuscular and extracellular. The intracellular stage is cylindrical in shape, usually lying on one side of the erythrocyte and displacing the nucleus to the other side. The posterior end is usually folded up. In fresh preparation, the oblong nucleus with a distinct membrane and usually a single karyosome is seen to occupy the central portion of the body. The cytoplasm is homogeneous and contains refractive granules of variable number scattered both in the anterior and posterior regions of the body. Ordinarily there is no recognizable move- ments of the body except at the time prior to the emergence from the host cell in which the parasite is lodged. Around the body there is a thin but distinct membrane. When stained, the oblong nucleus assumes two kinds in appearance: one with eccentrically located karyosome and the other with chromatin granules scattered evenly on the linin network. The cytoplasm is highly reticulated and is always denser at the rounded end than at the other end. The nucleus of the host cell seems to degenerate by breaking down into a number of smaller irregular masses, and becomes faintly stained, which condition indicates that the infection probably causes some changes in the chemical nature of the nucleus of the host cell. The host cell containing the haemogregarine becomes stretched and exhibits variable shape and size. The number of parasites present in one host cell is usually one, but frequently two are present, in which case the host cell becomes greatly enlarged and deformed. The intracorpuscular forms while under observation start to turn around in the host cell, and finally breaks through the host cells. W^hether this is due to the pressure caused by the cover-glass and by immersion objective or natural phenomenon cannot be determined. The extracorpuscular stage is gregarine-like in its appearance and movements. The forms found in Rana pipiens and R. clamitans differ 66 R. KUDO somewhat. The former is shorter and thicker and its anterior end is less truncate with a nucleus situated near the anterior extremity, while the latter is longer and thinner and its anterior end is more truncate with a centrally located nucleus. I have not noticed the difference in living condition, as their length and shape changed from time to time due to the movements. The difference noted in the stained preparations may indi- cate different circumstances in preparing them, although practically same methods were used in both cases. P'igs. 14 to 21. Ilaemogrcgar'nni sp. Kig. 14, a normal red blood corpuscle. Fig. 15, a triniicleate erythrocyte containing an intracellular stage (the structure of one of the nuclei is omitted). Figs. 16 to 18, three infected erythrocytes. Fig. 19, an erythrocyte containing two parasites. Fig. 20, the parasite is just leaving the host cell. Fig. 21, an extracellular giant form still covered with an envelope. Scluiudinn: Delafield. x 2100. The free haemogregarines may be seen usually soon after the prepara- tion was made, Jjut they increase in number in a few minutes. I have often noticed the fact that when a fresh preparation of a small portion of the lung of an infected frog was made, the number of e.xtracorpuscular forms ON THE PROTOZOA IN FROGS 67 increased from 1 to 2 to from 12 to 20 in each field (compensation ocular 4 and apochromatic objective 8 mm.) in five minutes while under observation. The body is rounded or truncate at the anterior extremity and tapers to an attenuated posterior end. As was noted by many authors in several species of haemogregarines, the posterior end of the animal is seen con- nected with a thread-like structure sometimes measuring twice as long as the body. It seems to me that it is a portion of the cytoplasm of the host cell which was in direct contact with the parasite before the latter left it, Figs. 22 to 29. ifaemogregarina sp. Figs. 22 to 24, e.xtracellular stages from the lung capillaries of Rana pipiens. Fig. 25, an extracellular form from Rana clamitans. Figs. 26 and 27, erythrocytes of Rana pipiens with small forms. Figs. 28 and 29, erythrocytes with an individual of Hacmogregarina sp. and a smaller form. Fig. 24, Giemsa; Fig. 27, Heidenhain; the rest Delafield. x 2100. and which became left behind as the animal moved forward. This view also seems to be reasonable if one considers the fact that the thread- like connection is most conspicuous soon after the parasite leaves the host cell and disappears in a few minutes. The structure of the body in 68 R. KUDO stained state is similar to that of the intracellular stage. The nucleus assumes sometimes ring form. The average dimensions of forms found in Rana pipiens: length 23.8 /z, largest breadth 3.6 /x: and of those found in Rana damitans: length 27.6ju, largest breadth 2.4/i. Development. — Ordinarily the dimensions of the parasites of both phases described above are somewhat uniform. No stages of division were noted either in the host cell or in free state. Smaller intracellular stages such as shown in Figs. 26 to 29, are often observed. They seem to occur always in the host cells. Oval with a flat or concave and a convex side, they show at each end of the body one to three vacuole-like structures in both fresh and stained conditions. These forms are always present with- in the same host animal with the large forms described above. But no intermediate stages between them have been seen, although a few forms such as shown in Figs. 28 and 29 are noticed. Without infection experi- ment, I cannot say whether they are only different stages in the develop- ment of one and the same parasite or entirely different forms. Stebbins (1904) considered the smaller form as a distinct species and named it Haemogregarina cateshianae. The development of haemogregarines of frogs has not yet been worked out, although Haemogregarina stepanowi found an earnest worker in Reichenow (1910) who described interesting observations. IV Trypanosoma rotatorium (Mayer) Synonyms. — Paramoecium loricalum Mayer 1843, Paramoccium cos- tatum Mayer 1843, Amoeba rotatoria Mayer 1843, Trypanosoma sanguinis Gruby 1843, Monas rotatoria Lieberkiihn 1870, Undulina ranarum Lan- kester 1871, Paramoecioides costatus Grassi 1882. Habitat. — In the blood of Rana esculenta, R. temporaria, R. damitans, R. pipiens, R. castebiana, R. galamensis, ^. oxyrhynchus, R. mascarensis, Rappia marmorata, Bufo vulgaris, Bufo- regular ia, Letodadylus ocellatus, Hyla viridis and H. arborea. A number of host frogs whose specific names were not determined by the original authors are excluded. The trypanosomes are more numerous in the blood vessels of organs such as liver and especially kidney than in the peripheral or heart blood. Historical. — Since Gluge (1842) found the organism, several workers no- ted and studied the flagellate, the chronological review of which is found in Laveran and Mesnil (translated by Nabarro, 1907). Doflein (1910), Lebedefif (1910) and Machado (1911) are more recent conributors to our knowledge concerning this blood parasite of frogs. Distribution. — Europe, Africa, Asia, South and North America. Methods of observations. — The blood should be examined as soon as it is taken from the frog. With a capillary pipette, draw the blood from the frog heart. If it is taken aseptically, the Ijlood can be kc]U in sterile ON THE PROTOZOA IN FROGS 69 condition in test tube with physiological solution and the trypanosomes will live for several days. If one cannot observe the preparation soon after the blood was taken, make temporary hanging drop preparations which may be examined in two to six hours. The trypanosomes are the largest ones known up to date, and its presence can be detected under a low power, although one may have to examine several preparations of the frog blood before finding one individual. When alive one can distinct- ly see the undulating membrane and the characteristic wriggling move- ment of the trypanosomes. To make permanent smears, make ordinary blood smears and fix with either sublimate-alcohol-acetic mixture or with absolute alcohol (for 10 to 20 minutes). Staining with Delafield's haematoxylin, Heiden- hain's iron haematoxylin or Giemsa's stain, will bring out morphological details. The nucleus is sometimes hard to stain and prolonged staining is needed to demonstrate its structure. Morphology. — Polymorphic. Earlier observers held that the difference in size and form among different individuals showed that of the specific characters which view has however been abandoned by modern investiga- Figs. 30 to 33. Trypanosoma rotaforium. Fig. 32, Delafield; rest Giemsa. x 1500. tors. When the blood is examined soon after its removal from the frog heart, one will see broad forms as well as slender ones mingled with inter- mediate forms. After some time, some individuals become rounded. The majority have more or less attenuated extremities. The form of the 70 R. KUDO body changes constantly with the striking movements of the undulating membrane. The flagellum which runs along the outer margin of the undulating membrane is very frequently seen to extend beyond the anterior end of the body. Its length varies, and may not be seen at all. The blepharoplast and nucleus can hardly be seen in actively motile individuals. The cytoplasm is granulated, may contain rounded spaces especially near the posterior margin, and shows in many individuals longitudinal striae. When stained, the small oval or oblong blepharoplast is seen located some distance from the posterior tip of the body. The flagellum seems to take its origin a short distance from the blepharoplast. The nucleus is located near the blepharoplast and on the same side of the body where the latter is situated. It is rounded and shows its structure poorly with any stain. It contains chromatin granules collected along the periphery. A small karysome may sometimes be seen in the central region. The cyto- plasm shows numerous small vacuoles in the posterior half of the body. Dimensions vary considerably. Length, 44 to 70^i and breadth 10 to 35 ix. Development. — Although artificial cultivation in vitro of Trypanosoma rotatorium has successfully been carried out by Bouet, Doflein, Lebedeff and Machado, we know practically nothing concerning its development in the frog body. Some authors such as Laveran and Mesnil, Doflein, etc., have not seen trypanosomes undergoing division in the blood of host frog. My own examination of numerous preparations also leads me to agree with them on this point. On the other hand, Franca and Athias (1906), Dutton, Todd and Tobey (1907) and Machado (1911) observed stages in division in the smears of frog blood or in preparations of fresh blood of infected frogs "kept aseptically at 72° to 89° F. for two or three days" (Dutton, Todd and Tobey). According to the observations of the last named three investigators, the trypanosome after losing its flagellum, became rounded and underwent active division, producing numerous small rounded organisms — "forty-one cells were counted, though they were probably more." Each body became ovoid, then pear-shaped, and from the more rounded end a flagellum was produced. These young forms became active and free from the outer covering of the original trypanosome. They divided rapidly by splitting longitudinally increasing in number. They were herpetomonas- like and remained in this condition until the preparations were discarded. The authors studied further stages in stained smears, and stated that the herpetomonas-like forms developed into inopinatum-Yike forms which were also "found in fresh blood, in contradiction to the forms just described, which were found in kept blood alone." Machado describes stages in division of the trypanosomes in frog. From his statement, it is not clear whether he found these stages in the ON THE PROTOZOA IN FROGS 71 blood or kidney. Figs. 14 to 19 and 36 to 40 given by Machado are rather isolated from others and hard to be reasonably connected with the other stages which he figured. I have not had chance of examining Leptodactylus ocellatus myself, but comparison of the above mentioned Machado's figures with the vegetative stages of a Myxosporidian, Leptolhe- ca ohhnacheri which is described very briefly in this paper and in details in the other paper (Kudo, 1922) and which is not uncommon parasite of the kidneys of Rana clamitans, R. pipiens and Bujo lentiginosus of the United States, leads me to think that the frogs studied by Machado were probably infected by the Myxosporidian or an allied form besides the trypanosomes. Machado states that trypanosomes were abundantly seen in the kidney of the host which fact I also noted. He seems to have mixed the stages of development of a Myxosporidian with those of the trypanosomes. Judging from the trypanosomes of fishes and reptiles, and Trypan- osoma inopinatum, another member of the genus parasitic in Rana escidenta of Algeria, the present species seems to undergo changes in the body of blood sucking invertebrates. Fuller accounts of the life history of the trypanosome awaits future investigations. V Trypanosoma parvum nov. spec. Habitat. — In the blood of Rana clamitans. Fourteen specimens were examined between July and September, 1920. In one of them a fairly heavy infection of a trypanosome was noticed. Five to eight active indi- viduals were recognized in every field (compensation ocular 4 and apo- chromatic objective 8 mm.). The frog also harbored Trypanosoma rota- torium in small number (one individual in every two other field under the same combination as noted above), but no haemogregarine was found. I have not seen it since that time, although I have examined about four dozens of Rana pipens which were purchased from a Chicago biological supply store. Methods of observations. — Same as Trypanosoma rotatorium. For demonstration the unusually long flagellum, Fontana's staining was used with satisfactory result. Morphology. — When alive, the movements can be distinguished into two types: travelling and wriggling movements, of which the first is prominent. The active wriggling movements remind one of those of Try- panosoma lewisi. The undulating membrane is fairly well developed. The nucleus and belpharoplast are faintly visible, while the relatively long flagellum can distinctly be seen with an oil immersion objective. The cytoplasm contains frequently small rounded clear spaces, and is more or less vacuolated at the posterior portion. When stained, one finds in them structures typical to a trypanosome. The body is spindle-shaped usually being curved in an arch or S. The •72 E. KUDO posterior end is ordinarily attenuated and ends in a blunt point, while the anterior extremity is more sharply pointed. The cytoplasm is usually dense from the anterior part to the middle region of the body, while a clear area is frequently seen ju^t posterior to the nucleus, either close to or Figs. 34 and 35. Trypanosoma parvum nov. spec. Fig. 34, Giemsa; Fig. 35, Delafield. X 3300. somewhat separated from it. The posterior portion is more or less vacuolated as was noted in living specimens. The blepharoplast is located very close to the posterior tip of the body. It is relatively large, and rounded or oblong in shape. The flagellum that borders the outer margin of the undulating membrane does not seem to take its origin directly in the blepharoplast, but arises from a point inconspicuously marked at some distance from the latter. The free portion of the flagellum reaches \S n in length, though its length varies most widely. The nucleus is rather large, and is located between the middle and anterior third of the body. It is spherical or oval. In Giemsa stained smears, the peripheral portion stains very deeply, while the central portion is occupied by a few linin threads. A karyosome may sometimes be seen eccentrically located. Dividing forms were not seen. The trypanosomes are strikingly uniform in size, showing little variation in size and general shape, except the length of the flagellum. Measurements of two hundred specimens in smears fixed with sublimate-alcohol-acetic mixture and stained with Giemsa's solution are as follows: length of body, exclusive the flagellum, 11 to 14 n, largest breadth including the undulating membrane, 1.2 to 1.9ju, length of free portion of the flagellum 5 to 15 /i. ON THE PROTOZOA IN FROGS 73 Of all the trypanosomes of amphibians known up to date Trypanosoma inopinatum Sergent et Sergent, 1904, resembles closely to the form just stated. These two forms resemble each other in the dimensions and general resemblance to Trypanosoma lewisi. There are however some differences in the location of blepharoplast, the structure of cytoplasm and the general form of the stained individuals which shows the activity of the two forms is not same. The blepharoplast is located more closely to the posterior tip in this form than in Algerian form. The breadth of the American form is 1.2 to 1.9 n, while that of Trypanosoma inopinatum is 3 II. The cytoplasm of the present form is vacuolated at the posterior por- tion of the body, while the Algerian form, according to Sergent and Ser- gent's figures, is uniformly granulated. Furthermore the activity of the two forms appears to be quite different. In the forms I have studied the body shows an arch or S shape in stained smears, while Sergent and Sergent figure more or less straight form, thus indicating probable differ- ence in their activity when alive. Consequently these two forms should better be separated from each other by different specific names, until I am able to compare the preparations of them. Since the cultivation of Trypanosoma rotatorium in vitro has been attempted by Lewis and Williams (1905), the fact that the trypapnosomes undergo division in the culture media resulting in the formation of small spindle-shaped bodies resembling in appearance to Herpetomonas or Crithidia, became known. But in no case, a structure typical to a try- panosome was noted among these small forms. At our present state of knowledge concerning trypanosomes, it is proper for us to consider the extremely small trypanosome described above as independent from Try- panosoma rotatorium. As it is morphologically distinguishable from a closely allied form, Trypanosoma inopinatum, I propose to name it provi- sionally Trypanosoma parvum nov. spec. Parasitic flagellates in the intestine Number of parasitic flagellates have been described in the intestine of frogs. The reader is referred to Dobell (1909) and Swezy (1915, 1915a) concerning them, VI Opalina sp. The Opalinas described here seem to be identical with Opalina ranarum Purkinje et Valentin 1835. Habitat. — In the rectum of Rana clamitans and R. pipiens. Historical. — A complete chronological review of works on Opalinas will be found in Metcalf (1909). Methods of observations. — The rectum of frog is placed in a small watch glass and opened in physiological salt solution under dissecting microscope. When the preparation is made, the Protozoon will be seen actively moving. In order to retard the active movements, a drop of 74 R. KUDO two of cherry gum solution may be added. The ciliary movements and the structure of the body can easily be studied. For permanent prepara- tions, follow the methods stated for Entamoeba ranarum. Morphology. — The body is broadly oval, with blunt anterior and more rounded posterior extremities. One side is convex, while the opposite side exhibits a shallow depression at the middle part. The body is highly flattened. Parallel rows of cilia run obliquely. The body is covered with cilia of uniform length. The protoplasm is sharply differentiated into Fig. 36. Opalina sp. Delaficld. .\ portion of the body is shown in detail. .\ 400, ectoplasm and endoplasm. The ectoplasm is hyaline near the pellicle, but is alveolated near the endoplasm. The latter is granulated in living individuals, l^ut when stained with Delafield's haematoxylin, it shows a vacuolation. The endoplasm contains a large number of nuclei of uniform size. Cytostome or cytpygc is not observed. Dimensions: length 130 ON THE PROTOZOA IN FROGS 75 to 200 /i, breadth 50 to 120 fi. Occasionally large form reaches 500 /x in length. Development. — The Protozoon divides in the intestine of the frog, stages of which are commonly seen in the rectum in the summer. I have not studied a new infection of a host frog. According to Neresheimer (1907), Opalina rafiartim divides successively in the rectum of host frog in the spring, and produces numerous small individuals, each containing a few nuclei. They encyst by producing a hyaline and resistant membrane around them. The cysts come out of the host body with fecal matters and remain on the bottom of the water. When the young tadpoles swallow the cysts, the contents of the latter leave the membrane in the rectum of the new host. The free young opalinas become differentiated into gametes by division and after fusion form zygotes. The zygotes grow into adult ones as the tadpoles metamorphose themselves into adult frogs. Summary 1) The main object of the present paper is to furnish a brief account of Protozoa parasitic in common North American frogs for general students in Zoology. 2) The occurrence of Entamoeba ranarum in Rana clamitans is stated. 3) A Myxosporidian, Lcptotheca olilmachcri is studied in the kidneys of Rana clamitans and R. pipiens. 4) Trypanosoma rotatormm of Rana clamitans and R. pipiens is studied. 5) Haemogregarina sp. in Rana clamitans and R. pipiens is studied. 6) A new trypanosome, Trypanosoma porvum is described from Rana clamitans. 7) Opalina sp. from Rana clamitans and R. pipiens is studied. 8) Methods of observation and brief review of previous works for each of these forms are given. Bibliography The papers marked with an asterisk contain the summary of and reference to the works of previous investigators on the subject. ^ Enlamoeha ranarum DOBELL, C. C. *1909 Researches on the intestinal Protozoa of frogs and toads. Quart. Jour. Micros. Sc, 5.1:201-276, 4 pi. and 1 textfig. 1918 Are Entamoeba htslolytica and Entamoeba ranarum the same species? An experi- mental study. Parasit., 10:294-310. „ Leplothcca ohlmacherl Kudo, R. ^ *1920 Studies on Myxosporidia. A Synopsis of Genera and Species of Myxosporidia. 111. Biol. Monogr., 5:243-503, 25 pi. and 2 textfig. 1921 On the nature of structures characteristic of Cnidosporidian spores. Trans. Micro. Soc, 40:60-74. 1922 On the morphology and life history of a Myxosporidian, Lcptotheca ohlmacheri, parasitic in Rana clamitans and Rana pipiens. Parasitology, 14, no. 2. 76 R. KUDO Ha emogrega rhics DuTTOx, J. E., J. L. Todd and E. X. Tobey. 1907 Concerning certain Protozoa observed in Africa. II. Ann. Trop. Med. Paras., 1:287-370, 13 pi. and 34 textfig. Franca, C. 1917 Sur la classification des hemosporidies. Journ. Sc. Mate. Fis. Nat. Acad. Sci. Lisboa, 3 ser. 41 pp. HlXTZE, R. 1902 Lebensweise und Entwicklung von Lankeslcrella minima (Chaussat). Zool. Jahrb. Abt. Anat., 15:693-730, 1 pi. Stebbixs, Jr., J. H. 1904 Upon the occurrence of Haemosporidia in the blood of Rana catesbiana, with an account of their probable life history. Trans. Amer. Micr. Soc, 25:55-61, 2 pi. 1905 On the occurrence of a large sized parasite of the Karysolysus order in the blood of Rana cJamata. Centr. Bakter. I Abt. (Orig.) 38:315-318, 2 pi. Billet .\. Trypanosomcs 1904 Sur le Trypanosoma inopinatum de la grenouille verte d'Algerie et sa relation possible avec les Drepanidium. C. R. soc. bioL, 57:161-164, 16 textfig. Brumpt, E. 1906 Role pathogene et mode de transmission du Trypanosoma inopinatum Ed. et Et. Sergent. Mode d'inoculation d'autres trj^anosomes. C. R. soc. biol., 61:167-169. DOFLEIX, F. 1910 Studien zur Xaturgeschichte der Protozoen. VI. Experimentelle Studien iiber die Trypanosomen des Frosches. Arch. Protist., 19:207-231, 3 pi. and 1 textfig. DuTTOx, J. E., J. L. Todd and E. N. Tobey (see haemogregarines). Laverax, a. and E. ;Mesxil (translated and revised by Nabarro). *1907 Tr}-panosomes and trj'panosomiases. Chicago. 538 pp., 1 pi. and 8 textfig. Lebedeff, a. 1910 Ueber Trypanosoma rotatorium Gruby. Festschr. 60sten Geburts. Richard Hertwigs, 1:397-436, 2 pi., 9 textfig. ]Machado, a. 1911 Zytologische Untersuchungen iiber Trypanosoma rolalorium Gruby. Mem. Inst. Oswaldo Cruz, 3:108-135, 2 pi. Sergext, Ed. et Et. 1904 Sur un trypanosome nouveau, parasite de la grenouille verte. C. R. soc. biol., 56:123-124, 1 textfig. Dobell, C. C. Intestinal flagellates *1909 (sec Entamoeba ranarum). SWEZY, O. 1915 Binary and multiple fission in Hexamitus. Uni. Calif. Publi. in Zoology, 16: 71-88, 3 pi. 1915a On a new trichomonad flagellate, Trichomitus parvus, from the intestine of .Amphi- bians. Uni. Calif. Publi. in ZoologA-, 16:89-94, 1 pi. Metcalf, :M. M. Opalinac *1909 Opalina. Its anatomy and reproduction, with a description of infection experi- ments and a chronological review of the literature. Arch. Protist., 13. 181 pp., 15 pi. and 15 textfig. Nereshelmer, E. 1907 Die Fortpflanzung der Opalinen. Arch. Protist., Suppl. 1:1-42, 3 pi. and 2 textfig. A NEW SUCTORIAN FROM WOODS HOLE By Francis Metcalf Root, Ph.D. Department of Medical Zoology, School of Hygiene and Public Health, The Johns Hopkins University The aberrant group of the Infusoria known as the Suctoria have been but little studied in the United States. In Europe, however, the mono- graphic work of Sand and, more recently, of Collin have made them well known. In 1914 I published some notes on the reproduction and food reactions of a fresh- water species of the genus Podophrya which appeared in hay- infusions at Baltimore. Through this work I became interested in this peculiar group of the protozoa and during the summers of 1916 and 1917, while at Woods Hole, Mass., I collected and studied five species of Suctoria which attached themselves to the stalks of the common hydroids, Obelia commissuralis and Obelia geniculata. The four previously described species found were as follows: Ephelota coronata (Wright). Acineta tuberosa Ehrenberg. Paracineta livadiana (Mereschowsky) — This is probably the species referred to by Calkins (1901) as "Acineta divisa Fraipont." Ophryodendron abe itinum Claparede et Lachmann. Besides these four species another was present which was obviously new and extremely remarkable, showing decided resemblances to Acinetopsis rara Robin. The genus Acinetopsis was established by Robin (1879) to contain a sin- gle species, Acinetopsis rara, characterized by the presence of a stalked theca with free apical margin (a "coque" in the terminology of Collin [1912 page 117] and by the presence of a single extensile, flexible tentacle in the center of the apical surface of the body. This species has never been reported by any other observer. Martin (1909) attempted to identify Robin's Acinetopsis rara with one stage in the life history of his new species, Tachyblaston ephelotensis. This identification has not been accepted by Collin in his monograph of the Suctoria, and will hardly be agreed to by anyone who will take the trouble to compare Martin's figures with the original figure of Robin, as reprinted in the Journal of the Royal Microscopical Society (1880). More recently Collin (1909, 1912) has described a new species under the name of Acinetopsis campanuliformis. This species is described as having a bell-shaped "loge" or closed theca 77 78 FRANCIS METCALF ROOT, PH.D. and bears six flexible tentacles. Collin seems to have overlooked the statement of Robin that Acinetopsis rara has a theca with free margin, and has classified Acinetopsis as a genus with closed theca. If this differ- ence be really of generic importance, Collin's Acinetopsis campanuliformis must be placed in a new genus. The species which I found at Woods Hole so closely resembles Acine- topsis rara at one stage of its life-history that I am not willing to erect a new genus for it, in spite of the fact that in this new species I find that the flexible extensile organ proves to be a seizing organ, analogous to the pointed tentacles of Ephelota and without any suctorial function. To this new species I have given the name of "tentaculata," because in its adult condition it is provided with true sucking tentacles as well as with the elongated "probosces" which I consider characteristic of the genus. Description of Acinetopsis tentaculata n. sp. Protoplasmic body enclosed in a flattened, cup-shaped theca with a free apical margin, borne on a slender stalk a little longer than the theca itself. (See Figure 1). Body irregularly flattened-ovoid in shape, bearing on the apical face one or two probosces. Each proboscis is a very mobile organ, which can be bent in any direction, extended until it is a mere thread twice the length of all the rest of the organism, or retracted until it is less than half the length of the protoplasmic body alone. Structurally, the proboscis consists of a homogeneous central strand about whose outer surface is wound spirally a ribbon of protoplasm evidently contractile in nature. Each proboscis is tipped with a large, highly refractive globule of adhesive and viscous character. That the probosces are firmly anchored to the body is self-evident from the observations given later regarding their use in feeding. As is shown in Figure 2, this attachment is well below the surface of the body, but I have not been able to make out the details. Besides these probosces the apical face of the body also bears about twenty or thirty short stout tentacles of the familiar capitate type, these being distributed in two groups, one on either side of the insertion of the probosces. The macronucleus has irregular outlines, but is in general roughly ovoid. One or more small spherical micronuclei are present. A single contractile vacuole is located near the base of the body. The measurements of a typical mature specimen were as follows: Protoplasmic body — 138 microns long, 100 microns wide, 73 microns thick. Theca — 187 microns long, 105 microns wide. Stalk — 287 microns long. Proboscis when fully extended — about 500 microns long. A NEW SUCTORIAN FROM WOODS HOLE 79 Feeding Habits As far as I have been able to observe, Acinetopsis tentaculata feeds only on Ephelota coronata, but it attacks this common species with voracity. The probosces are extended and moved about until the globule at the tip of one of them comes in contact with the body of an Ephelota. It seems to secure a firm attachment at once and in a few cases where the Ephelota was very firmly attached to its stalk I have seen the globule elongate and finally pull in two rather than release its victim. As soon as the attachment is secured the proboscis contracts strongly, drawing the body of the Ephelota within reach of the short sucking tentacles of its captor. Sometimes the stem of the Ephelota is long enough so that this can be accomplished by merely bending it. More often the attachment between the body of the Ephelota and its stem must be broken by a rapidly repeated series of jerks due to sudden contractions of the proboscis. When the Ephelota finally comes within reach it is firmly seized by the sucking tentacles and its internal protoplasm sucked out at leisure. One of the difficulties in the study of this species was the long search which was always necessary to find an individual whose body was not almost entirely con- cealed by several Ephelotas in process of being sucked dry. Reproduction The actual escape of the embryo was not observed, but Figure 3 shows plainly that reproduction occurs by the same process of simple (i. e. not mul- tiple) internal budding which is characteristic of all the genera of the family Acinetidae. Nor have I observed the entire series of stages in the attachment and growth of the free-swimming ciliated embryo. From the young individuals I have found in nature, it seems probable that after attachment and the formation of a stalk and theca, a single proboscis is first formed (Figure 4). This condition is structurally a perfect duplicate of Robin's Acinetopsis rara. Slightly larger individuals still show only a single proboscis but have also formed a number of true tentacles (Figure 5). And finally, in mature individuals, we find the two probosces characteristic of the species. There is a great temptation here to suggest that these growth stages may reflect something of the course of evolution in this genus. It seems quite probable, for example, that the second proboscis, the last organ acquired by the individual, is a recent evolutionary acquisition of the species. It would probably be going too far to intimate that the stage with a single proboscis and no tentacles harks back to some ancestor in which the proboscis was a true sucking tentacle and no other organs for capturing prey were necessary. However, this must be the condition which obtained in Robin's Acinetopsis rara, unless he overlooked the inconspicuous tentacles or was dealing only with a growth stage. 80 francis metcalf root, ph.d. Summary Acinetopsis tentaculata n. sp. is described and figured. It is charac- terized by having a stalked "coque" or theca with free apical margin, and by the presence of one or two extensile seizing organs or probosces as well as suctorial tentacles. It feeds on Ephelota, seizing them and drawing them within reach of its tentacles by means of the probosces. It repro- duces by internal budding, forming free-swimming ciliated embryos which settle down and gradually grow into the adult form. Literature Cited Calkins, G. N. 1901 Marine Protozoa from Woods Hole. U. S. Fish Comm. Bull. Vol. 21, pp. 413-468. Collin, B. 1909 Diagnoses preliminaires d'Acinetiens nouveaux ou mal connus. C. R. Acad. sc. Paris. May 24, 1909. 1912 Etude monographique sur les Acinetiens. II. Morphologie, Physiologic, Syste- matique. Arch. Zool. Exp. et Gen. Vol. 51, pp. 1-457. Martin, C. H. 1909 Some observations on Acinetaria. II. The life cycle of Tachyblaston epheloten- sis. Quart. Journ. Micr. Sci. Vol. 53. Robin, C. 1879 Memoire sur la structure et la reproduction de quelques Infusoires. Journ. Anat. et Physiologie. Vol. 15. (Reviewed with illustrations in Journ. Roy. Micr. Soc. 1880.) Root, F. M. 1914 Reproduction and reactions to food in the Suctorian, Podophrya coUini n. sp. Arch, fiir Protistenkunde. Vol. 35, pp. 164-196. Description of Plate All figures are camera drawings from specimens killed in Dubosque's alcoholic modifica- tion of Bouin's Fluid and stained with picric acid haematoxylin. Figure 1. Acinetopsis tentaculata. Side view of entire mature specimen with one proboscis partly extended and the other nearly retracted, x 185. Figure 2. Acinetopsis tentaculata. Front view of body and theca of mature specimen. Note deep attachment of probosces and arrangement of tentacles in two groups, x 350. Figure 3. Acinetopsis tentaculata. Front view of an individual in which the macronu- cleus is just dividing to form the macronucleus of an internal bud. x 350. Figure 4. Acinetopsis tentaculata. Young form with a single proboscis and no ten- tacles. X 350. Figure 5. Acinetopsis tentaculata. Young form with a single proboscis and a few tentacles, x 350. A NEW SUCTORIAN FROM WOODS HOLE 81 DEPARTMENT OF SUMMARIES DEVOTED TO DIGESTS OF PROGRESS IN BIOLOGY TEN YEARS OF HEREDITY^ By A. Franklin Shull University of Michigan Though no one is likely to be misled by my subject into supposing that the laws of heredity have been in operation only a decade, it may not be universally appreciated that heredity is one of the oldest of biological phenomena. It is at least as old as, probably older than, organic evolution of which we have long been accustomed to speak in terms of millions of years. For, when the first living thing, if ever there was such a being, gave rise to a second, by reproduction, this second Hving thing was either like its parent, or different from it. If like its parent, heredity had begun. If different from its parent, it was almost certainly different in only one or a few respects, but Hke the parent in the rest, in which case both heredity and evolution were in operation. The stipulated ten years to which my title refers are not, however, an arbitrary limit set for the purpose of relieving me of the necessity of covering the whole of a very large subject. They are a period which, in the development of knowledge of heredity, is naturally marked off from the numerous decades that precede. Those of you who possess a little knowledge of heredity, to whom the name of Gregor Mendel has a fasci- natingly familiar sound, and in whose memory lingers the date of 1900 in which year the famous Austrian monk's long hidden experiments were again brought to light, may wonder why I should wish to describe the developments of but the latter half of the period since that rediscovery. It is true that the great interest aroused by the verification of Mendel's Law, with the multiplication of experimental work which was induced by it, was a necessary precursor of the events with which I propose to deal. But about 1910 there began a chain of discoveries, which have followed one another in unbroken series to the present time, and which have led to a conception of the operations of heredity of a degree of complexity, and withal of harmony, which even the most sanguine twenty years ago would not have ventured to predict. ' This lecture, delivered before the Graduate Club of the University of Michigan, was designed to present to persons without biological training, not a r^sum^ of all important work in heredity in the period referred to, but the point of farthest advance and the principal work leading to it. 82 department of summaries 83 Former Lack of Analysis Heredity had long been discussed in terms of averages. In popular discourse it was always so, children were replicas of their mothers, or were chips out of the old block, or the son of a Cholmondeley was the image of a Jones. The ensemble of characters was considered. Even those who were professionally engaged in the study of heredity lumped together many things now known to be partially or wholly distinct from one another, regarding them as a single trait. Stature, obviously made up of many elements, was treated as a single characteristic. Intelligence, likewise compound, was studied as if simple. There was not wanting, it is true, even among the laity, an analytic tendency. A youth would have his father's mouth, his mother's eyes, his grandfather's complexion. But it was not until the emergence of Men- del's work in 1900, and the multiplication of investigations consequent upon that event, that it was realized to what extent inherited traits may be separated from one another as distinct and independent units. Eyes were inherited independently of hair, hair color independently of hair form, color independently of distribution of color, whether uniform or in patches. Unit characters became distinctly vogue. Anyone who could utter the magic expression "unit characters" and speak the name of Mendel with his first name and title, had thereby established his right to be regarded as a thoroughly modern geneticist. Difficulties of the New Conception All this development raised in the biological mind certain difficulties. When it was inquired how all these unit characters were manipulated independently of one another, there were obstacles — that is, when the offered explanation passed a certain point. To speak intelligibly of these difficulties it will be necessary to refer briefly to a few elementary facts of biology. All organisms are composed of units of structure called cells. These cells regularly contain, as part of their structure, a rounded body, the nucleus, which stains deeply in most dyes and which is therefore conspicu- ous in most common microscopic preparations. The size of an organism is increased usually by the multiplication of cells, which is accomplished by the division of the cells already present. In the process of division, the cells develop a complicated figure in which the highly staining material of the nucleus is resolved into a number of distinct bodies called chromo- somes. As the division is completed, the chromosomes lose their distinct form, producing a nucleus in which separate bodies are no longer visible; but at the next division the chromosomes appear again, in the same number as in the previous division. This number is in general constant in all cells of the same individual, and, barring some differences between the 34 A. FRANKLIN SHULL sexes, is constant for all members of the same species. Moreover, in animals in which the chromosomes are not all of the same size or shape, each dividing cell reveals the same number of chromosomes of each shape or size. Since all organisms are composed of cells, the phenomena of heredity must in some way be traceable to cells. The constancy of occurrence of the nucleus, and of a given number of chromosomes in the nucleus, early gave rise to a suspicion which later, on a foundation of fact, ripened into a conviction, that in these structures is the mechanism through which heredity is governed. If it were assumed that the factors of heredity were contained in the chromosomes, many things would be explained. Refer- ence will be made now to only one of these things. One of the new features of discussion of heredity was the attention devoted to unit characters. How were these characters operated as units, independently of one another? Chromosomes provided the answer. It must be understood that in all the higher animals and plants, no parent contributes all of its chromosomes to any one offspring, hut only half of these chromosomes. In the development of the germ cells a peculiar cell division called the reduction division takes place in which the chromosomes separate into two groups, one group being enclosed in the one daughter cell, the other group in the other cell. Chromosomes and Recombination of Characters In the composition of these groups of chromosomes, there is a wide range of different possibilities. In some cases, the chromosomes may be Figure 1. Diagram of a cell in which the chromosomes a.rc capable of arrangement in pairs, the two chromosomes of each pair being precisely alike in all respects. Such a cell, in maturation, divides into two cells, each with half the number of chromosomes, and each exactly like the other in all hereditary factors. The sliapes of the chromosomes are not actual, but only a diagrammatic representation of the likenesses and differences in their hereditary composition. DEPARTMENT OF SUMMARIES 85 capable of assortment in pairs, as in figure 1 ; that is, there are two chromo- somes in each cell that are exactly alike, two other chromosomes that are exactly alike but different from the first pair, and so on. In such a case, by separating the members of each pair, two groups of chromosomes may be made up which are identical. In such a case, therefore, tivo germ cells with exactly the same hereditary possibilities are produced, and the parent may contribute precisely the same hereditary traits to every one of its progeny. Moreover, it can transmit to each of its offspring every hereditary trait of which it is possessed. In other individuals, on the contrary, every chromosome of a cell may differ in one or more respects from every other chromosome, as in figure 2, Figure 2. Diagram of a cell in which the chromosomes may be arranged in pairs, but the chromosomes of one pair are not exactly alike. The chromosomes of a pair may be alike in most respects, but different in one or more features. Such a cell, in maturation, divides into two cells which are alike in the main but differ in certain hereditary factors. The precise hereditary composition of each cell therefore depends on how the chromosomes are distributed to the two cells. In such a case, in the reduction division at which only half the chromo- somes are conveyed to each daughter cell, it is not possible to produce two cells that are identical. Each chromosome in each of these two cells is different from every chromosome in the other cell. Moreover, the paiy^nt is here contributing, with respect to certain characteristics, only half of its hereditary potentialities to any one of its offspring. Between these extremes, in which, on the one hand, the parent hands on all its hereditary traits to all of its offspring, and, on the other hand, transmits only half of its possibilities with respect to certain features to any one offspring, there are all intermediate grades. The result depends 86 A. FRANKLIN SHULL on how the chromosomes are separated into two groups. In that cell division in which each daughter cell receives only half the total number of chromosomes, it appears to be a matter of chance, subject to certain restrictions, how the half number shall be made up. If I have not made this procedure clear, the following analogy will be useful. If it is pro- posed to divide by chance a group of buttons, or poker chips, if that be a more familiar figure, of a variety of colors, into two groups of six each, it is obvious that the groups of six may be very unlike; also, that if the same dozen buttons be divided into two groups again, the second division may b e very unlike the first. If these buttons represent chromosomes, and their colors stand for hereditary traits, it is clear that these traits may be distributed in very difTerent ways to different offspring. The chromosomes, then, because they act to some extent indepen- dently of one another, offer an explanation of the independence of unit characters — provided only that the things which produce these characters are in the chromosomes. There are other reasons, equally good, perhaps better, for believing that the hereditary factors, as they are called, are in the chromosomes, but this additional evidence may pass. Individuality of the Chromosomes All this conception of the operations of heredity, in relation to the chromosomes, was arrived at before the ten year period of which I am eventually to speak. But certain difficulties are inherent in the concep- tion. The number of chromosomes in the cells of an animal is strictly limited. In man, one author fixes the number at 48, another at 24. In other animals there is better agreement, and the number is as low as four, or even two. In man, even the largest number suggested, 48, must be much smaller than the number of traits which he inherits. If this be true, the representatives of several traits must reside in the same chromosome. The difficulty involved in this situation was that the chromosomes were believed to he individuals. That is, the chromosomes which become dis- tinguishable at one cell division were held to be the same identical chrom- osomes, part for part, as were observable at the preceding cell division; and chromosomes occurring in one individual were believed to be identical with those of its parents. There were many facts concerning the shapes of the chromosomes, and their behavior at various times, which lent support to the view that they are persistent individual objects. W.Te this regularly true, two hereditary traits represented by something in the same chromosome would necessarily behave as a single characteristic. They could not be independently assorted, when the chromosomes were separated into two groups in the reduction division in the production of germ cells, but would go together. Traits represented in different chromo- somes would be independently assorted, but those in a single chromosome would act as a unit. department of summaries 87 Requirements of Proof of Linkage Whether this condition actually existed in any animal or plant was for a long time not known. To determine whether inherited traits were ever bound together in groups required an animal in which differences in a con- siderable number of characteristics existed in different individuals. It required also a careful study of such an animal or plant to determine whether the traits were wholly independent, or were grouped. Early in the revival of Mendelism, an association of certain hereditary traits with sex was demonstrated, but indications of an association of hereditary traits with one another were long delayed. The number of traits whose inheritance was understood, in any one species, was too small. Then came the year 1910. In that year a fly was born — or hatched. It belonged to the small brownish gray species which is seen every summer day hovering about fruit stands or garbage pails. This species had been bred for years in a number of laboratories, notably those of Columbia University by Professor T. H. Morgan and his students. Then one afternoon, in a bottle, appeared the fly of which I speak, which differed from all others in the bottle, and from all of its ancestors for many genera- tions, in having white eyes. Flies of this species regularly have red eyes. Since 1910, other eye colors have appeared, vermilion, cherry, eosin, buff, tinged, blood and purple being the names applied to some of them. Other flies were produced which had unusual wings — short, blunt, crumpled, or missing, curled up, curved down like an inverted bowl of a spoon, or spread at an angle. Other parts of the body likewise presented variations. The spines became forked, or reduced in number. Extra legs were produced. The color of the body became yellow or black in certain individuals. Physiological changes not producing any observable structural diff'erences have also been detected. All told, over two hundred such modifications have been discovered in this one species of fly since 1910. Most of these characteristics were found to be definitely inherited. Fortunately, many of these altered flies were quite healthy, were easily reared, and have been carefully studied. The fruitfly was obviously the organism by which the individuality of the chromosomes and their relation to heredity could be tested. Early Demonstrations of Linkage This test came gradually. It was found that these characteristics were not wholly independent of one another. Thus, white eye and yellow body-color were very closely associated with one another. When a white-eyed and yellow-bodied fly was crossed with a normal red-eyed and gray-bodied fly, their offspring in certain subsequent generations, in certain cases should have shown all combinations of the two eye colors and the two body colors with equal frequency. That is, if the four traits were inherited independently of one another, white eye and gray body 88 A. FRANKLIN SHULL ^ should have been combined in one individual as often as white eye and yellow body. But they were not; white eyes and gray bodies were found together in only about one-fiftieth as many cases as would have been expected. White eye was nearly always associated with yellow body in these crosses. It was discovered, also, that white eye color was associated in the same way, though less closely, with sable body color, with club shaped wings, and a number of other characteristics. Moreover, if white eye color was thus bound up with a certain characteristic, yellow body color was also associated with the same characteristic. And all characteristics that were thus associated with white eye and yellow body were found to be linked — that is the word Morgan uses — with one another. All these traits behave, to some extent, as a unit. They are not absolutely bound together, but they hang together more frequently than the chance assortment of chromosomes, or colored buttons, or poker chips, ivould lead one to expect. Independent Linkage Groups Approximately forty of the more than two hundred new character- istics that have arisen in this fly in the past ten years may safely be said to belong to the group that is linked with white eye color and yellow body. Long before all of these had been discovered — indeed, when only a few of them were known — certain other new traits had come into existence which were definitely not linked with white eye. One of these was a short crumpled wing which has been called vestigial. In crosses which involve ves- tigial wing and white eye at the same time, the occurrence of vestigial wing in the individuals of subsequent generations bears no relation to the occur- rence of white eyes in the same individuals. The chances are even, in such crosses, that a fly with a vestigial wing will have white eyes in as large a proportion of cases as will a fly with normal wings. Likewise, there is no relation between vestigial wings and yellow body color. Nor is there any association between vestigial wings and any other characteristic in the entire group that is linked with white eyes and yellow body. Clearly, vestigial wing is not a member of that group. Another character that is independent of white eye color is black body color. In crosses which should test any such su]iposed relation, the distribution of black color of the body among the individuals is wholly unrelated to the white color of the eye. Black body occurs with equal relative frequency in, individuals with red eyes and white eyes. Black body color is also independent of any other characters of the group linked with white eye color. But black body color is associated with vestigial wing. If a cross is made involving an individual with both vestigial wing and black body, then in generations jiroducod by a]ipro]-)riatc crosses among the descendants, black body and vestigial wing will occur together DEPARTMENT OF SUMMARIES 89 much oftener than apart. That is, flies having both black body and vestigial wing will be relatively much more numerous than flies havin^ black body and normal long wing; and much more numerous than flies with vestigial wing and normal gray body. Vestigial wing and black body color are clearly linked with one another. With these two traits are also linked a number of others that concern the wings, the body, the eyes, etc. All characteristics linked with black body or with vestigial wing are, when tested in appropriate crosses, found to be linked with one another. They form a distinct second group, every member of which is linked to some extent with every other member. This group contains about as many characters as does the first group linked with white eyes. It must be made entirely clear that, while all the traits of this second group are linked with one another, none of them is in any way linked with any character of the first group to which white eye and yellow body belong. There is still a third group of characters, and a fourth group quite small in numbers, which are made up, as are the first two, of characters that tend to hang together, once they start together. All members of the third group hang together more than mere chance would permit; and all members of the fourth group are in like manner associated with one another more frequently than can be attributed to accident. But no trait of the third group is in any way bound with any trait of the first, second or fourth groups. And no trait of the fourth group is linked to any extent with any member of any of the first three groups. Three of these groups are rather large, that is, include numerous characters, one is quite small. Chromosomes and the Linkage Groups You will have guessed long since that the reason assigned for the linkage of these various traits is that the hereditary factors responsible for them are located in the same chromosomes. All characteristics of the first group are produced by something in the same chromosome. All characteristics of the second group are likewise represented by something in one chromosome. But that chromosome is a different one from the chromosome that produces the characteristics of the first group. Each of these groups owes its existence as a group to one chromosome, which is a different chromosome for each group. In this connection you will care to know something about the chromo- somes of this fly. Fortunately they are well known. Each cell has eight of them (figure 3), but when, in the formation of germ cells, the reduction division divides these into two groups, there are only four in each germ cell. Three of these chromosomes are large, and one quite small, and three of the linkage groups of characters are large, and one small. Assuming that the hereditary factors for one group are all in one chromosome, and that that is the cause of their linkage with one another, 90 A. FRANKLIN SHULL Figure 3. The chromosomes of the female fruitfly Drosophila melanogaskr. In the body cells and immature germ cells there are eight chromosomes, two each of four kinds. In maturation, at the reduction division, the number is reduced to four, one of each of the four kinds. Three of the chromosomes are large, and one is small. what becomes of the idea of individuality of the chromosomes? It must be modified, of course. As previously pointed out, the characteristics of each group are not absolutely bound together, they merely occur together more frequently than chance would permit in the case of independent characteristics. That is, once they are transmitted from parent to off- spring in conjunction with one another, they separate from one another thereafter less frequently than would be expected. But if their factors are in the same chromosome, how can they separate at all? Breaking the Linkage Grouts To make the proposed answer to this question clear it must be stated that when this separation does occur, there is a fairly even exchange. That is, when white eye is separated from yellow body with which it had been associated, some other eye color takes its place and is thereafter as closely linked with yellow body color as was the white eye color before. This is always the case. Whenever a trait is removed from assoeiatiou with another trait, its place is taken by a trait related to the same part of the body. Eye color is exchanged for eye color; one form of wing is replaced by an- other form of wing. This exchange is made possible, presumably, because of the approximate duplication of the chromosomes in each cell. It has already been pointed out that the chromosomes of a cell may be such that they are exactly alike, two by two (figure 1). But even where the chromosomes arc all DEPARTMENT OF SUMMARIES 91 diflferent from one another (figure 2), nevertheless they can be arranged in pairs of twins such that the members of one pair differ from one another in only one or a few features, but are alike in a host of others. One of them may, for example, include a representative of vestigial wing, the other of the normal long wing, but be alike in everything else. Or they may differ with respect to color of body and color of eye, and be alike in all other respects. The two chromosomes have to do with the same parts of the body, and no other chromosomes of the cell are concerned with those traits in the same way. The chromosomes are truly capable of arrangement in pairs of twins. Mechanism of Crossing-over This arrangement in pairs is not purely a figurative one, it is at certain times an actual bodily one. At a certain time in the formation of the germ cells, these chromosomes come together side by side. What they look like in this operation, is known in relatively few forms. In one of these the chromosomes are long slender threads, and the two twins twist about one another in loose spirals (figure 4). This is not an isolated case. It is iSS^P^^ Figure 4. Some of the chromosomes of Batracoseps twisting about one another in spiral form prior to the reduction division. The chromosomes thus twisting together contain factors for the same hereditary characters. Whether they untwist in the reduction division, or separate in some other fashion, is not known from observation. {M odified from Janssens.) not impossible that, in many or most animals, the twin chromosomes twist about one another at this stage of development. Later they separate from one another in some fashion at what we have called the reduction division. How this separation takes place is not known from direct observation, but several possibilities exist. The chromosomes may unwrap completely and be the same chromosomes as before they twisted. Or they may adhere at points, and the two sides of the spiral in that region exchange places. This is a very important conception, put forward by Professor Morgan and his students, but before it can be developed certain other considerations must be presented. 92 A. FRANKLIN SHULL The hereditary factors contained in a chromosome are, Professor Morgan believes, arranged in linear order, like beads on a string (figure 5). Every cell in an animal's body has a chromosome in which these "beads" are the same as those of one chromosome in each of the other cells of the Vellou/ Bodt/ Nhite £ye I Graif Body Red E(/e. No/^mall^inq yNorwall^inq Venmili on £-ye Mmia/ure k/ino Red Eye I Mmiafurc Rudirner\- Forked Br/sf/es Comp/e^eEye Rijdimen- fory U/ina Forked Bristles Bar Eye Figure 5. Diagram of a pair of chromosomes of the female fruilfly Drosophila melano- gasler. The hereditary factors are arranged in a single row in each chromosome. Both chromosomes have hereditary factors for the same characters. The factors for one character are placed at the same level in both chromosomes, so that when the chromosomes meet in a pair the two homologous genes are side by side. Not all the known factors are represented. {From Principles of Animal Biology, by Shiill, La Rue and Ruthven. McGraw-Hill Book Co.) body. In the same cell with it is another chromosome in which the hereditary factors are precisely the same, or at least they concern the same parts of the body. The hereditary factors in these two chromosomes are held to be arranged in the same order, and to lie at the same level. So that, if the chromosomes are placed side by side, or twisted about one another, the two hereditary factors Jor the same part of the body are side by side. If two such chromosomes twist about one another, as has been de- scribed, and then in separating are not unwrapped carefully, they may exchange hereditary factors. If in one of these chromosomes were a factor for white eye and one for sable body color (figure 6), some distance apart so that the breaking point occurred between them, the linkage that formerly existed between these two characteristics would be broken. Where they DEPARTMENT OF SUMMARIES S 93 Figure 6. Crossing-over between two chromosomes containing factors for eye color and body color. One chromosome has factors for white eye (w) and sable body {s); the other has factors for red eye (W) and gray body (S). If these chromosomes adhere and break at some point between the pairs of factors, after the reduction division one chromosome con- tains factors for white eye (w) and gray body (S), the other has factors for red eye (W) and sable body (s). had formerly necessarily passed to the same individual, they would now necessarily pass to different individuals. There is also much to prove that the chromosomes may break twice, or at three places instead of only one. If one of the two chromosomes that twist about one another (figure 7) has MB Figure 7. Double crossing-over in a pair of chromosomes. The chromosomes adhere and break at two points in their length, so that after the reduction division each chromosome is made up of three fragments, two from one of the original chromosomes, one from the other. In the original chromosomes the factors w, m and B were linked, as were also W, M and h. After the crossing-over, w, M and B form one linked group, IF, m and b the other. See text for explanation of the s>'mbols. in it factors for white eye {w), miniature wing {m), and bar eye {B), the other the contrasted normal characteristics which are red eye {W), 94 A. FRANKLIN SHULL long wing (-M), and round eye (b), and these chromosomes break at two points as they separate from one another, two of the three linked characters may be still linked together, but the third separated from them. The separation of a here4itary factor from another with which it was linked is called crossing-over. Whether a pair of twisted chromosomes shall break at one point or another is, with certain restrictions, held to be a matter of chance. If they break between the factors for black body and vestigial wing, those factors will be released from linkage with one another; that is, crossing-over between these two characteristics occurs. These chromosomes may break at any number of places not between the factors for black body and vestigial wing, and the two traits will remain linked as before. Mapping Chromosomes Inasmuch as breakage presumably occurs at different places in hap- hazard fashion, crossing-over between two traits is likely to occur often if their factors are far apart on the chromosome. Conversely, if the factors are very near together they are seldom separated. It is very easy, by making appro- priate crosses, to pick out immediately those individuals in which certain characteristics, usually associated, have been separated, and hence in whose chromosomes breaking has occurred in a given region. By counting these individuals one may ascertain whether crossing-over between vermil- ion eye and club wing, for example, is frequent or rare; and can judge, therefore, whether the factors for these characteristics are far apart or near one another. By means of such experiments, it has been shown that white eye and yellow body seldom separate; they do so in only one out of a hundred chances, whereas they should cross over fifty times out of a hundred if they were independent of one another. Black body and vestigial wing, on the contrary, separate much more frequently, that is. about seventeen times out of a possible hundred. White eye and yellow body must therefore be very close together, black body and vestigial wing must be rather far apart. On the basis of such computations entire chromosome maps have been prepared. Such maps have been in existence for years, having been gradually developed, and altered as new evidence is procured. An abridged map of one of the chromosomes of the fruitfly is given in figure 8. As new characters appear in this species, experiments are performed to determine in which chromosome their factors are, by determining with which other characters they are linked. And when that chromosome has been discovered, the place in it occupied by the new factor is next to be found. In locating the new factor, it may be necessary to alter the supposed place of certain other factors. That has happened lime and again, for at first the location can be only tentative. DEPARTMENT OF SUMMARIES 95 0 Wiitt eye No croi-ii/om Vermilioneyc tiiiya ^obl« bod 6oir CyG VcllOvv body BfJd vcr, Cut ^,n.^ Ty^3y.?) will give the number for one side and doubling this we get a total of 1944." The apparent asymmetry which Stafford found in the anterior branches is not substantiated by my studies. Still more improbable is Stafford's calculation of the number of ultimate capillaries based on the study of "a few branches followed out to the end organs." Moreover, the irregu- larities which he notes in other parts of the system can only be interpreted as inadequate analysis. This is particularly true in the light of my studies which indicate complete symmetry and regularity in the system of the larva as well as in that of the adult. While Stafford predicates four main branches, each of which proceeds to trifurcate successively five times, I have found only three such main stems and each of these in turn branches trichotomously only four times. Each of these branches has been traced to its distal termini, with the result that complete regularity is found to obtain (Fig. 1). In other words, the system for each side of the body actually consists of 3 stems, each branching four times, with 81 terminal units, giving a total of 243 capillaries and flame-cells for each side of the body. This, as against Stafford's mental calculation of 972 units which is theoretical and obviously based on inadequate analysis. The system may be expressed as (3X3X3X3)-f(3X3X3X3X) + (3X3X3X3) or (3)*+(3)''+(3)^ and reduces to the formula a"+i(3" + 7". These data are supported further by the elemental excretory system which is found in the larva (figs. 5, 6), Here three primitive flame-cells and capillaries are seen which are the basal units of the system, (a-\-0-\-y). These by successive trichotomies give rise to the adult system. NOTES ON THE EXCRETORY SYSTEM 115 It seems desirable, in passing, to note that the larva (fig. 7) has paired cluster of cephalic glands (eg), emptying through a cord of cephalic ducts (cgd) just anterior to the oral sucker. This is analagous to the salivary glands described for the redia of Cercaria equitator (Ssinitzin 1911: 52, fig. 50) and C. flabellijormis (Faust 1918: 34, fig. 4v3) and might lend sup- port to the view that the aspidobothrid is a redia with germinal epithelium highly differentiated. Summary 1. Record is made of the presence in several centers in China of the cosmopolitan worm, Aspidogaster conchicola. In addition to the usual hosts, Amyda sinensis has been found to harbor it. 2. The excretory system of the worm is regular and bilaterally symme- trical. It has three main stems on each side of the body, each stem having a 4-fold trichotomy. 3. This is expressed as (3X3X3X3) + (3X3X3X3) + (3X3X3X3) or (3)^+ (3)^+ (3)^ and may be reduced to the formula a"+i8"+7". 4. The fundamental a-\-^-\-y pattern obtains in the larva where each element is represented by a single flame-cell. 5. The presence of cephalic glands in the larva may support the view that the worm is homologous to a highly differentiated redia. Literature Cited Faust E. C. 1918. Life History Studies on Montana Trematodes. 111. Biol. Monogr., 4:1-21, 9 pi. Huxley, T. 1856. Lectures on General Natural History. Med. Times and Gaz. London, 13:131-134, figs. 1-7. Ssinitizin, D. Th. 1911. La generation parthenogenetique des Trematodes et sa descendence dans les mollusque de la Mer Noire. Mem. Acad. Sci. St. Petersbourg, (8) 30:1-127, 6 pi. Russian. Stafford, J. 1896. Anatomical Structure of Aspidogaster conchicola. Zool. Jahrb., Anat., 9:476-542, 4 pi. VoELTZKOW, A. 1888. Aspidogaster limacoides. Arb. Zool-Zoot. Inst. Wiirtzb. Wiesb. 8:290-292. Key to Figures o, anterior fundamental of excretory system. 8, median fundament of excretory system. 7, posterior fundament of excretory system. b, bi, bii, bladder. eg, cephalid gland. cgd, cephalic gland duct. ct, primary collecting tube. ep, epi, epii, excretor>' pore. rt, reflexed (secondary') collecting tubule. 116 ERNEST C. FAUST Fig. 1 PLATE Xlir Description of Figures Fig. 1 . Lateral view of adult A spidogaslcr conchicola, showing complete excretory system for right side of body, X 75. NOTES ON THE EXCRETORY SYSTEM 117 Figs. 2 to 4. Stages in the escape of the larva from the egg shell, X 360. Figs. 5, 6. Lateral and ventral views of the free larva, showing fundaments of the ex- cretory system. The two sides are seen to develop separately, even to the e.xcretory pores. X J(JO. Fig. 7. View of larva showing cephalic glands and ducts, X 360. A NEW CESTODE FROM LIPARIS LIPARIS* By Edwin Linton University of Missotiri The name Spathebothrium simplex gen. et sp. nov. is proposed for a cestode collected at Woods Hole, Mass., by the late A'^inal N. Edwards from the sea snail, Liparis liparis. Mr. Edwards's record is as follows: March 25, 1904, 2 fish examined, stomachs filled with small sand fleas, 2 tape worms in intestine of one. April 14, 1904, 15 fish examined, stomachs filled with sand fleas; full of spawn, nearly ripe; 7 tape worms from 4 fish. January 14, 1905, 2 fish examined, one tape worm in each. The cestodes in these three lots belong to the same species. Their lengths, in alcohol, were 12, 14, 15, 16, 18, 18, 20, 20, 21, 26 and ic> milli- meters respectively. The maximum breadth was about 2.25 millimeters; ova 0.036 by 0.021 mm. in the two principal diameters. The strobile is flattened, nearly linear, bluntly and smoothly rounded at the two extremities. About" the only difference noted between the anterior and posterior ends, as seen in whole mounts, is that genitalia are wanting for a short distance at the anterior end while the vitellaria continue to the extreme tip of the posterior end. In a specimen 11 mm in length the first cirrus is 0. 44 mm. from the anterior end. The scolex therefore is represented by the short portion which precedes the genitalia and is probably transparent in life. In this specimen the vitellaria began a little posterior to the level of the cirrus. The strobile is not divided into distinct proglottides, the only indication of strobilation being the successive sets of genital apertures, and, in cleared and mounted specimens, the ovaries which are conspicuous, lobed, and lie between the genital apertures at what would be the posterior end of a proglottis, if proglottides were present. The reproductive apertures are situated along the median line and are not restricted to one of the flat surfaces of the strobile. For example, in a specimen which had 20 s^ets of reproductive organs twelve of these o])ened on one of the flat surfaces of the strobile and eight on the other. In another specimen sixteen were counted on one side and nine on the other. In a specimen 16 mm. in length the distance between adjacent sets of reproduc- tive pores was about 0.67 mm., the first set lying about the same distance from the anterior end. The apertures of the cirrus, vagina and uterus are *Contribution from the U. S. Biological Station, Wtxwls Hole, Mass., and the Zoological Laborator>- of the University of Missouri. 118 A NEW CESTODE FROM LIPARIS 119 4 near together, that of the cirrus being a little anterior to those of the uterus and vagina, which are very near together and about at the same level. The vitellaria continue without interruption from near the anterior end to the posterior end, so that the strobile superficially resembles an elongated trematode. The testes lie for the most part in front of the ovary and are medially placed with respect to the vitellaria. In transverse sec- tions through regions where the uterus is filled with ova the testes are lateral and near the vitellaria (Fig. 6). The cirrus pouch is short but with relatively thick muscular walls. The vagina has a strong muscular sphinc- ter near its external opening (Figures 2, 4, 5). In ripe strobiles each set of reproductive apertures is preceded by a mass of ova. The musculature, so far as it is shown in sections, is poorly developed. A few longitudinal fibers were noted in the subcuticula, but no trace of a layer of longitudinal fibers between the subcuticula and central parenchyma was seen, nor was there any indication of a circular layer. The cuticle (Fig. 3) consists of two layers, an outer made up of short rod-like structures, and an inner structureless layer. The outer layer constitutes about two- thirds of the thickness of the cuticle but it may be more or less abraded. The subcuticula in my sections appears as a loose mesh of fine fibers with scattering cells. The thickness of the cuticle in the section from which figure 3 was sketched was 0:01 mm., of the subcuticula 0:07, and of the smaller diameter of the section, representing the thickness of the strobile, 0.5. Sections of the anterior end of a strobile show numerous anastomosing vessels of the excretory system. These vessels were difficult to interpret in transverse sections in regions of the strobile where the reproductive organs had appeared. Nowhere were they satisfactorily seen to be definitely established as dorsal and ventral lateral vessel. In cases where two prin- cipal lateral vessels could be distinguished they lay in about the same hori- zontal plane with reference to the axis of the strobile. From a study of a series of sagittal sections the lateral vessels were interpreted to be two, with thin walls, somewhat tortuous, and giving off transverse branches. This cestode is peculiar in the absence of bothria, and in certain char- acteristics of the genital pores. The three genital apertures, cirrus, uterus, and vagina, are, as a rule, near together on the median line, and irregularly alternate with respect to the so-called dorsal and ventral surfaces of the strobile. This feature stands in the way of referring it to the Pseudo- phyllidae, which group is characterized by having the opening of the uterus always on one of the faces, although the openings of the cirrus and vagina may stand on opposite faces, or on a lateral margin. // is thus seen that the species with which we are dealing is unique in that it is not possible to speak of a dorsal and ventral surface of the strobile. For it will be observed that, not only are the reproductive apertures irregularly alter- nate on the flat surfaces of the strobile, but the reproductive organs themselves are also irregularly alternate with respect to those surfaces (Fig. 2). 120 EDWIN LINTON While examining a large number of transverse sections a single excep- tional disposition of the reproductive apertures was noted. Figures 7 and 8 are sketches of this exceptional condition. Here the aperture of the cirrus is seen to be on one of the flat surfaces of the strobile while the openings of the vagina and uterus are on the opposite side. Since the apertures of the uterus and vagina do not lie in the same horizontal plane it was necessary to make two sketches. In the series of sections in which this anomalous condition was noted two sections intervened between the sections shown in figures 7 and 8. An interesting feature with respect to the relative position of ovary and vaginal aperture is shown in figure 2. In the upper part of the figure the ovary is seen to be on the opposite side of the strobile from the vaginal aperture, in the lower part of the figure it is on the same side. In the former case the vagina crosses from one side of the strobile to the other, in the latter it turns abruptly posteriad near the aperture. Synopsis of genus Spathehothrium No distinct scolex; strobile taenaeiform, bluntly rounded at the extremi- ties, proglottides not distinct, reproductive apertures on median line and irregularly alternate. Explanation of Plate c. cirrus sr. seminal receptacle cp. cirrus pouch /. testes cu. cuticle u. uterus m. sphincter muscle of vagina v. vagina 0. ovary vd. vas deferens sc. subcuticula vg. vitelline glands. sg. shell gland Fig. 1. Sketch of specimen mounted in balsam, somewhat diagrammatic. In this specimen there were 12 sets of reproductive apertures on one side and 8 on the other. Length 16 mm. Fig. 2. Sagittal section near median line showing reproductive apertures on opposite sides of the strobile. The succeeding section to this in the series shows the uterus in about the same relative position as that of the vagina in the lower left of the sketch. Thickness of strobile at this point 0.30 mm. Fig. 3. Cuticle and subcuticula highly magnified. Thickness of cuticle 0.01 mm. Fig. 4. Reproductive apertures as seen in horizontal section. Sketch made from section showing first appearance of the uterus. The vagina had appeared first in the preceding section, and the cirrus in the si.xth preceding section. Diameter of cirrus bulb 0.24 mm. Fig. 5. External apertures of vagina and uterus, transverse section. Long diameter of ovum 0.035 mm. Fig. 6. Transverse section showing uterus with ova, etc. Breadth of strobile at this point. 1.5 mm. Fig. 7. Transverse section showing e.xccptional arrangement of genital pores, the cirrus opening on one side and the vagina on the other. Longer diameter of section 0.98 mm. Fig. 8. From same series of sections as Fig. 7, two sections intervening between 7 and 8. The cirrus bulb still shows and the vagina is replaced by the uterus. A NEW CESTODE FROM LIPARIS 121 PLATE XV A LIST OF THE NEW GREGARINES DESCRIBED FROM 1911 TO 1920* By MiNNTE Watson Kamm Although the gregarines are among the oldest known of the Protozoa (Redi, 1684), they still remained a practically unknown group for a hundred and fifty years, researches on more recently described groups far out- numbering those on these parasites. This may have been due to the fact that gregarines are of little or no economic importance. The hosts as a rule are not animals of such import that the elimination of their parasites is desirable and, moreover, the parasites themselves are generally harmless, living commensal rather than actual parasitic lives within their hosts. Because this is practically a new field, much of the work on the group has been up to the present chiefly systematic; it is often easier to find an entirely new species than to obtain a species already known. Considerable has been done on Life Histories, Effect of the Parasite Upon the Host, and Chromosome Behavior in the Complete Life-Cycle during the last decade and much more is to be expected in these fields. Labbe described the gregarines known up to the year 1899^ and reclass- ified many of the wrongly designated and aberrent species. His paper was probably the incentive for much of the subsequent work on the group. The new forms described from the time of Labbe's Summary up to the year 1911 were listed by Sokolow^ and those described in this decade compared favorably with the complete summary of Labbe. Because the number of new species has been rapidly increasing subse- quent to Sokolow's List, the writer has prepared a list of the species de- scribed in the literature ivpm 1911 to the beginning of the year 1920. During this decade were named many new genera and the genus Gregarina re- ceived many new species. Perhaps the most important researches of the decade were those in the Suborder Schizogregarinae which includes many aberrent and apparently unrelated species- and consequently the classification is considerably confused. The classification which follows is that of Minchin, at present the best known. * Contribution from the Zoological Laboratory of the University of Illinois, No. 204. 'Das Tierreich, Pt. 5, Sporozoa. 'Zool. Anz. 38:277-95; 304-14. 122 NEW GREGARINES DESCRIBED FROM 1911-1920 123 Class Sporozoa Leuckart Subclass 1. Telosporidia Schaudinn 1900. Sporulation at end of vegetative period. Order 1. Gregarinoidea Minchin 1912. Trophozoite parasitic in epithelial cells. Sporont free in a cavity. Spore forms a single zygote. Suborder 1. Eugregarinae Leger 1900. Reproduction by sporogony only. Tribe 1. Cephalina Delage and Herouard 1896. With epimerite in trophozoite stage. Septate in all but one family. Generally parasitic in digestive tract of insects. Tribe 2. Acephalina Delage and Herouard 1896. Without epimerite, non-septate. Generally coelomic. Suborder 2. Schizogregarinae Leger 1900. Reproduction by both sporogony and schizogony. Tribe 1. Monospora Leger and Duboscq 1908. Single spore in sporogonic cycle. Tribe 2. Polyspora Leger and Duboscq 1908. Many spores in sporogonic cycle. An Annotated List of Species in the Tribe Cephalina of the Suborder Eugregarinae' Family LECUNIDAE Kamm (1922). Epimerite simple, symmetrical, gregarines non-septate, spores ovoidal, thickened at one pole. Digestive tract of marine annelids. Genus Lecudina Mingazzini 1891 Characters of the family Lecudina sp. Faria, Cunha, and Fonseca (1918) Mem. Inst. Osw. Cruz, 10:17-19. Body spindle-shaped, nucleus spherical. Host: Polydora social-is (Polych.) Taken at Rio de Janeiro, Brazil. Family POLYRHABDINIDAE Kamm 1922. Septate gregarines inhabiting the digestive tract of marine annelids. Epimerites varied. Genus Polyrhabdina Mingazzini 1891 (See Caullery and Mesnil 1914 C. R. Soc. Biol, 77:516-20.) Dicystid, sporonts flattened, ovoidal, epimerite a corona of hooks. Intestine of polychaetes of the family Spionidae. Polyrhabdina spionis (Kolliker) (New name for Gregarina spionis Koll.) ^ All parasites described are intestinal forms unless otherwise stated. 124 MINNIE W. KAMM Type species. Caullery and Mesnil (1914) C. R. Soc. Biol., 77. 516-20. Host: Scololepsis fnliginosa. (Polych.) Polyrhabdina polydorae (Leger) (New name for Doliocystis p. Leger.) Caullery and Mesnil (1914), C. R. Soc, Biol., 77:516-20. Host: Polydora ciliata. (Polych.) Polyrhabdina brasili Caullery and Mesnil (1914) C. R. Soc. Biol., 77:516-20. Spor. ovoidal, 1. 200;u. Epim. like type, spines shorter. Host: Spio martinensis. (Polych.) Polyrhabdina pygospionis Caullery and Mesnil (1914) C. R. Soc. Biol., 77:516-20. Host: Pygospionis seticornis. (Polych.) Family CEPHALOIDOPHORIDAE Kamm 1922 (this paper) Characters of the type genus Genus Cephaloidophora Mawrodiadi 1908 { = Frenzelina Leger and Duboscq 1907, preocc. See Arch. zool. exper., 46:lix-lxx.) Sporonts biassociative, no epimerite, cyst dehiscence by simple rupture, spores ovoidal with equatorial line. Development intra- cellular. Parasites of Crustacea. Cephaloidophora maculata Leger and Duboscq (1911) Arch. zool. exper., 46:lix-lxx. Spor. ovoidal, max. 1. 80/i. Nucl. spher. cysts spher. 100/z, spores spher. 4/1. Host: Gammarus marinus. (Crust.) Taken at Roscoff, France. Cephaloidophora talitri Mercier (1912) C. R. Soc. Biol., 72:38-9. Spor. ovoidal, average 1. 40//, nucl. spher. Host: Talitrus saltator. (Crust.) Taken at Roscoff, France. Cephaloidophora ( = Frenzelina) delphinia^ Watson (1916) Jour. Parasit., 2:129-35. Spor. ovoidal, largest spor. 115juX64ai. LP:TL::1:4; WP:WD::1:1.5.* Nucl. spher. Cysts spher. SOfx. Host: Talorchestia longicornis. (Crust.) Taken at Cold Spring Harbor, L. I. Cephaloidophora {^Frenzelina) olivia Watson (1916) Jour. Parasit., 2:129-35. Spor. ellipsoidal, largest 118^X36^. LP:TL::1:5; WP:WD::1 :1.3. ♦ The ratios of length of protomcrite to total Icnj^lh of sporont and width of protomerite to width of deutomerite are given for average individuals. These ratios will be abbreviated as above subsequently. Many recurring words will also be abbreviated from now on. NEW GREGARINES DESCRIBED FROM 1911-1920 125 Cysts spher., 60/i. Host: Libinia dubia. (Crust.) Taken at Cold Spring Harbor, L. I. CephaloidopJwra ( = Frenzelina) nigrofusca Watson (1916) Jour. Parasit., 2:129-35. Spor. ovoidal, largest 125/iX75m. LP:TL::1:4; WP:WD::1:1.5. Nucl. spher. Hosts: Uca pugnax, U. pugilator. (Crust.) Taken at Cold Spring Har- bor, L. I. Cephaloidophora ( = Frenzelina) ampelisca Nowlin and Smith (1917) Jour Parasit., 4:83-88. Spor. 62/xX15m. Chromidial body in protomerite. Host: Ampelisca s pint pes. (Crust.) Taken at Woods Hole, Mass. Family STENOPHORIDAE Leger and Duboscq 1904. Spor. solitary, Intracellular development. Dehiscence by simple rupture, spores ovoidal with equatorial line. Epimente absent or rudimentary. Parasites of Diplo- poda. Genus Stenophora Labbe 1899 With the characters of the family Stenophora elongata Ellis (1912) Zool. Anz., 39:685-6. Spor. elongate-cylindr., max. 1. 390/x. LP:TL::1:20; WP:WD::1:1 to 1:1.6. Prot. pentagonal. Host: Orthomorpha coarctata. (Dipl.) Taken at Quirigua, Guatemala. Stenophora cockerellae Ellis (1912) Zool. Anz., 39:681-5. Spor. elongate-cylindr., max. 1. 850/1. LP:TL::1:15; WP:WD::1:1.7. Host: Parajulus sp. (Dipl.) Taken at Quirigua, Guatemala. Stenophora robusta Ellis (1912) Zool. Anz., 40:8-11. Spor. short, avg. 1, 140-180//, w. 67^. LP:TL::1:8; WP:WD::1:2.5. Hosts: Parajulus venustus; Orthomorpha gracilis; O. sp. (Dilp.) Taken at Boulder, Col. Stenophora impressa Watson (1915) Jour. Parasit., 2:29; (1916) 111. Biol. Monogr., 2:280. Spor. ellipsoidal, largest 375/iX48/i. LP:TL::1:12; WP:WD::1:2.3. Cysts spher. 160/t. Host: Parajulus impressus. (Dipl.) Taken at Urbana, 111. Stenophora diplocorpa Watson (1915) Jour. Parasit., 2:29; (1916) 111. Biol. Monogr., 2:284. 126 MINNIE W. KAMM Spor. elongate-cylindr., constricted in mid-deut. LP:TL::1:20; WP:WD:: 1:2. Host: Eiiryurus crythropygus. (Dipl.) Taken at Urbana, 111. Stenophora lactarla Watson (1915) Jour. Parasit., 2:29; (1916) 111. Biol. Monogr., 2:282. Spor. elongate-ellips , largest 480juX39yu LP:TL::1:12; WP:WD::1 :1 2. Cysts spher. 170^1. Host: Calliptis lactarius (Dipl.) Taken at Urbana, 111. Stenophora caudata { = Spirosoma caud.) Ishii (1915) Ann. zool. japon., 9:7-9. Spor. tadpole-like in shape, posterior half reduced to cylindrical 'tail' knobbed at end and spirally striated. Max. 1. 400;/, max. w. 100^. LP:TL::1:12. Prot. papillate at apex. Host: Fontaneria coarctata Pocock. (Dipl.) Taken in Gifu, Japan. (The new genus Spirosoma Ishii is named from the spiral deutomerite, none of the generic characters — epimerite, cystdehiscence, spores — being found. From the few positive characters — shape of sporont, protomerite, and diplopod host — it appears to belong to Stenophora. The author's specimens were described from alcoholic specimens only.) Stenophora cunhai Pinto (1918) Brazil-Medico; (1919) Contribuifao ao estudo das Gregarinas, Rio de Janeiro, 116 pp., 6 pi. Spor. elongate-cylindr., prot. sub-spher., largest spor. 250/iX40/i. LP:TL:: 1:5; WP:WD::1:1. Nucl. spher., in post, part of deut. Host: Rhinocricus pugio. (Dipl.) Taken at Rio de Janeiro, Brazil. Stenophora lutzi Pinto (1918) Brazil-Medico; (1919) Contribuifao ao estudo das Gregarinas, Rio de Janeiro, 116 pp., 6 pi. Spor. elongate-cylindr. prot. cylindr. with constriction below middle. Largest spor. 210;uX15a(. LP:TL::1 :7.5; WP:WD::1:1.2. Nucl. small, spher. Host: Rhinocricus sp. (Dipl.) Taken at Rio de Janeiro, Brazil Stenophora cruzi Pinto (1918) Brazil-Medico; (1919) Contribuijao ao estudo das Gre- garinas, Rio de Janeiro, 116 pp., 6 pi. Spor. elongate-cylindr. conical posteriorly, largest ^spor. [400^1 X 30m. LP:TL::1:13; WP:WD::1:2. Prot. a truncate cone. Nucl. unknown. Host: Rhinocricus sp (Dii)l.) Taken at Rio dc Janeiro, Brazil. NEW GREGARINES DESCRIBED FROM 1911-1920 127 Stephora viannai Pinto (1918) Brazil-Medico; (1919) Contribuiyao ao estudo das Gre- garinas, Rio de Janiero, 116 pp., 6 pi. Spor. stout-cylindr., bluntly conical posteriorly, largest spor. 1000//X150^(. LP:TL::1:16; WP:WD::1:2. Nucl. elongate-cylindr. Host: Rhinocricus sp. (Dipl.) Taken at Rio de Janeiro, Brazil. Stenophora umbilicata Pinto (1918) Brazil-Medico; (1919) Contribui(jao ao estudo das Gregarinas, Rio de Janeiro, 116 pp., 6 pi. Spor. stout-bodied, ovoidal, prot. small, broad. Hat. 320yuX 130//. LP:TL:: 1:6; WP:WD::1:3.7. Nucl. spher. Host: Rhinocricus sp. (Dipl.) Taken at Rio de Janeiro, Brazil. Stenophora tenuicolUs Pinto (1918) Brazil-Medico; (1919) Contribui^ao ao estudo das Gregarinas, Rio de Janeiro, 116 pp., 6pl. Spor. elongated globe-shaped in ant. fourth of deul. constricted to a 'wasp- waist' and widening gradually toward post, end, end broadly-rounded, prot. elongate-conical. Nucl. small, spher. Sporont 400;uX50/x. Host: Rhinocricus sp. (Dipl.) Taken at Manguinhos, Rio de Janeiro, Brazil. Genus Fonsecaia Pinto (1918) Brazil-Medico; (1919) Contribuijao ao estudo das Gregarinas, Rio de Janeiro, 116 pp., 6 pi. Like Stenophora except spores elongate-ellipsoidal, no endospore. Epimerite simple, without protoplasm. (The differentiation of this genus from Stenophora is not convincing.) Fonsecaia polymorpha. Type species Pinto (1918) Brazil-Medico; (1919) Contribuigao ao estudo das Gregarinas, Rio de Janeiro, 116 pp., 6 pi. Spor. 170/iX80/i. Broadly ovoidal, prot. small, conical. LP:TL::1:11.3; WP:WD::1:4.4. Nucl. spher. Spores ovoidal 18^^X8//. Host: Orthomorpha gracilis. (Dipl.) Taken at Rio de Janeiro, Brazil. Family GREGARINIDAE Labbe 1899 Epimerite symmetrical. Sporonts associative or solitary. Cysts with or without spore-ducts. Genus Gregarina Dufour 1828 Biassociative in sporont stage. Epimerite globular or cylindrical. Spores regular. Cysts with spore-ducts. Gregarina ctenocephalus { = G. ctenocephalus canis) Ross (1909) Ann. Trop. Med. Par., 2:359-63. Spor. spherical, no dimensions given. Epimerite pyriform, spores barrel- shaped. 128 MINNIE W. KAMM Host: Ctenoccphalus serraticeps (Acarinidae.) Taken at Port Said, Egypt. Omitted from Sokolow's List.) Gregarina creda Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:115-6. Spor. elongate-cylindrical, largest spor. 730yuX60/x. LP:TL::1:5; WP:WD ::1:1. Nucl. spher., cysts spher., SOOfx, spores typical, 6.4/iX3.2/i. Host: Broscus cephalotes. (Col.) Taken in East Prussia. Gregarina ovoid ea Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:117. Spor. obese, max. 1. 200ju. LP:TL::1:5; WP:WD::1:1.8. Nucl. spher. Cyst spher. ISO/i. Host: Crypticus quisquilins. (Col.) Taken in East Prussia. Gregarina polyaulia Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:118-9. Spor. cylindr., largest spor. 470^X250^. LP:TL::1:6; WP:WD::1:1.8. Cysts spher., 450/i, spores typical, 8.2^tX3.8;u. Hosts: Harpalus aeneus and H. ruficornis. (Col.) Taken in East Prussia. Gregarina rostrata Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:120-1. Spor: elongate-ovoidal, largest spor. 200/i long. LP:TL::1:7; WP:WD:: 1:2. Nucl. spher., epimerite elongate-cylindrical. Cysts spher., 205/x, spores ovoidal, 5.6juX3.2/i. Host: Lagria hirta. (Col.) Taken in East Prussia. Gregarina (Gigaductus) exiguus Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:121-2. Spor. obese, max. length 75m. LP:TL::1:4; WP:WD::1:2. Cysts spher., 35m, one long spore-duct. Spores cylindr., 11.3mX4.8m. Host: Pterostichus niger. (Col.) Taken in East Prussia. The genus Gigaductus has been dropped. See Watson (1916) 111. Biol. Monogr., 2: 317, 389. Gregarina guatemalensis Ellis (1912) Zool. Anz., 39:687-8. Spor. somewhat rectangular, max. 1. 276/x. LP:TL::1:3; WP:WD::1:1.5 Nucl. spher. Host: Ninus inter stitialis. (Col.) Taken at Quirigua, Guatemala. Gregarina consohrina Ellis (1913) Trans. Amer. Micr. Soc, 32:267. Spor. obese, average sporont 600m 1., 450m, w. LP:TL::1:5; WP:WD:: 1:1.5. Cysts spher., 300m. Sporeducts up to 1200m in 1. Spores i-lnX^n. Host: Ceuthophilus valgus. (Orth.) Taken near Boulder, Colo. NEW GREGARINES DESCRIBED FROM 1911-1920 129 Gregarina grisea Ellis (1913) Zool. Anz., 42:200. Spor. ellipsoidal, max. 1. 540/z. LP:TL::1:4; WP:WD::1:1.1. Nucl. spher. Host: Tcnehrio castaneus. (Col.) Taken at New Orleans, La. Gregarina longiducta Ellis (1913) Zool. Anz., 43:78-82. Spor. obese, associations avg. 800-900^1 in 1. LP:TL::1:3; WP:WD::1:1. Cysts spher., 560/x. Spores 3aiX6.5^(. Hosts: Ceuthopilus latcns,C. maculatus. (Orth.) Taken at Douglas Lake, Mich. Gregarina typographi Fuchs (1915) Zool. Jahrb., Syst., 38:109-222. Spor. stout-bodied, bluntly ovoidal. No measurements given. LP:TL:: about 1:3; WP:WD::1:1. Nucl. small, spher. Cysts spher., one large spore-duct. Spores 34X22jU. Host: Ips typographus. (Col.) Taken in Southern Germany. Gregarina { = Clepsidrina) hylohii Fuchs (1915) Zool. Jahrb., Syst., 38:109-222. Spor. long-ellipsoidal, largest spor. 847/iX304/z. Nucl. elongate-ellip- soidal, one elongate karyosome. Cysts ovoidal 420 X370/x, without hyalin envelope, spore-ducts numerous, spores rectangular with spine at each corner, 6X4^. Host: Hylobius ahieies. (Col.) Taken in Southern Germany. Gregarina niinnta Ishii (1914) Ann. Zool. japon, 8:436-8; Watson (1916) 111. Biol. Monogr. 2:343, 392, 409. Spor. elongate-cylindr., assn. 1. 118/x. LP:TL::1:9; WP:WD::1:1.7. Nucl. spher., cysts spher., 48/^. Host: Tribolium ferrugineum. (Col.) Taken in Prov. of Izu, Japan. Gregarina globosa Watson (1915) Jour. Parasit. 2:31; (1916) 111. Biol. Monogr., 2:401. Spor. subglobose, 260^X180^. LP:TL::1:8.6; WP:WD::1:2.4. Nucl. spher. Host: Coptotomus interrogatus. (Col.) Taken at Urbana, 111. Gregarina monarchia Watson (1915) Jour. Parasit., 2:31; (1916) 111. Biol. Monogr., 2:400. Spor. elongate-cylindr., largest spor. 570/xXl30ju. LP:TL::1:7; WP:WD:: 1:1.3. Host: Pterostichus stygicus. (Col.) Taken at Urbana, 111. 130 MINNIE W. KAMM Cregarina barbarara Watson (1915) Jour. Parasit., 2:31; (1916) 111. Biol. Monogr., 2:394. Spor. ovoidal, largest spor. 145^X90^. LP:TL::1:6; WP:WD::1:2. Nucl. small, spher. Host: Coccinella sp. (Col.) Taken at Oyster Bay, L. I. Gregarina katherina Watson (1915) Jour. Parasit., 2:31; (1916) 111. Biol. Monogr., 2:392. Spor. ellipsoidal, largest spor. 78/iX35/x. LP:TL::1:7; WP:WD::1:1.7. Nucl. spher. Host: Coccinella novemnotata. (Col.) Taken at Oyster Bay, L. I. Gregarina intestinalis Watson (1915) Jour. Parasit., 2:32; (1916) 111 Biol. Monogr., 2:399. Spor. broadly ellipsoidal, largest spor. 160/xX80/.t. LP:TL::1:5; WP:WD:: 1:2. Host: Pterostichus stygicus. (Col.) Taken at Urbana, 111. Gregarina gracilis Watson (1915) Jour. Parasit., 2:32; (1916) 111. Biol. Monogr., 2:398. Spor. elongate-ellipsoidal, largest spor. 190aiX80/i. LP:TL::1:8; WP:WD:: 1:2. Nucl. spher. cysts spher. 90/i. Host: Larva of Elateridae. (Col.) Taken at Urbana, III. Gregarina tenebrionella Watson (1915) Jour. Parasit., 2:32; (1916) 111. Biol. Monogr., 2:397. Spor. sub-globose, largest spor. 70/iX42M. LP:TL::1:4; WP:WD::1:1.7. Nucl. spher. Host: Larva of Tenebrionidae. (Col.) Taken at Urbana, 111. Gregarina fragilis Watson (1915) Jour. Parasit., 2:32; (1916) 111. Biol. Monogr., 2:395. Spor. ellipsoidal, largest spor. 110^X60^:. LP:TL::1:5; WP:WD::1:2. Nucl. spher. Host: Coccinella sp. (Col.) Taken at Urbana, 111. Gregarina nigra Watson (1915) Jour. Parasit., 2:33; (1916) 111. Biol. Monogr., 2:326. Spor. cylindrical, largest spor. 530aiX180m. LP:TL::1:4; WP:WD:: 1:1.4. Nucl. spher. Hosts: M elanoplus femur-rubrum , M. diferentialis, Encoptolophus sordidus. (Orth.) Taken at Urbana, 111. Gregarina galliveri Watson (1915) Jour. Parasit., 2:33; (1916) 111. Biol. Monogr., 2:321. NEW GREGARINES DESCRIBED FROM 1911-1920 131 Spor. 300aiX180//. LP:TL::1:5; WP:WD::1:1. Prot. flat, broad, deut. widest in post. half. Nucl. spher. Cysts spher., 350//. Host: Gryllus abbreviatus. (Orth.) Taken at Oyster Bay, L. I. Gregarina stygia Watson (1915) Jour. Parasit., 2:33; (1916) 111. Biol. Monogr., 2:324. Spor. obese, largest 180/xXlOO/x. LP:TL::1:6; WP:WD::1:1.6. Nucl. spher., cysts spher. 150/z. Host: Ceuthophilus stygius. (Orth.) Taken at Cold Spring Harbor, L. I. Gregarina illinensis Watson (1915) Jour. Parasit., 2:34; (1916) 111. Biol. Monogr., 2:318. Spor. elongate-cylindr., largest spor. 550yuXl80yu. LP:TL::1:5; WP:WD:: 1:1.5. Nucl. small, spher. Host: Ischnoptera pennsylvanica. (Orth.) Taken at Urbana, 111. Gregarina platyni Watson (1916) 111. Biol. Monogr., 2:402. Spor. elongate-cylindr., max. 1. 610ai. LP:TL::1:4; WP:WD::1:1. Prot. constricted in middle. Nucl. spher. Host: Platynus ruficollis. (Col.) Taken at Oyster Bay, L. I. Gregarina udeopsyllae Watson (1916) 111. Biol. Monogr., 2:327. Spor. obese, largest 310/xX200/i. LP:TL::1:5; WP:WD::1:1.5. Host: Udeopsylla nigra. (Orth.) Taken at Urbana, 111. Gregarina neglecia Watson (1916) Jour. Parasit., 3:65-75. Spor. ovoidal, largest spor. 500yuX230ju. LP:TL::1:6; WP:WD::1:1.5. Cysts spher., 300/z. Host: Ceuthophilus neglectus (Orth.) Taken at Oyster Bay, L. I. Gregarina platydenia Kamm (1918) Jour. Parasit., 4:159-63. Spor. cylindr, slender, largest spor. 1210^X150^. LP:TL::1:12; WP:WD:: 1:1.5. Nucl. spher. Epim. a simple cone. Host: Platydenia excavatum. (Col.) Taken at Urbana, 111. Gregarina diabrotica Kamm (1918) Jour. Parasit., 4:159-63. Spor. elongate-cylindr. largest spor. 270/xXl05/z. LP:TL::1:3.5; WP:WD:: 1:1.6. Nucl. spher. Epim. a sessile knob. Host: Diabrotica vittata. (Col.) Taken at Urbana, 111. 132 MINNIE W. KAMM Gregarina watsoni Pinto (1918) Brazil-Medico; (1919) Contribuifao ao estudo das Gre- garinas, Rio de Janeiro, 116, pp.; 6 pi. Spor. elongate-ovoidal, largest spor. 350mX152m. LP:TL::1:7; WP:WD:: 1:1.5. Nucl. spher. Epim. globular. Host: Omoplata nor?nalis. (Col.) Taken at Nictheroy, Brazil. Gregarina chagasi Pinto (1918) Brazil-Medico; (1919) Contribuifao ao estudo das Gre- garinas, Rio de Janeiro, 116 pp., 6 pi. Spor. sub-globular to cylindrical. Largest spor. ISO^iXSO^u. LP:TL:: 1:3.6; WP:WD::1:1.5. Nucl. spher. Cysts ovoidal. Host: Conocephalus f rater. (Orth.) Taken at Manguinhos, Brazil. Gregarina aragaoi Pinto (1918) Brazil-Medico; (1919) Contribuifao ao estudo das Gre- garinas, Rio de Janeiro, 116 pp., 6 pi. Spor. elongate-ovoidal, max. 1. 170/z, max. w. 70/i. LP:TL::1:5.7; WP: WD::1:1.7. Nucl. spher. Epim. a small papilla. Cysts subspherical. Host: Systena sp. (Col.) Taken at Manguinhos, Brazil. Gregarina sp. Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:146. Host: Sviinthurns fuscus. (Thysan.) Taken in East Prussia. Gregarina sp. Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:148. ' Host: Oribata geniculata. (Arachn.) Taken in East Prussia. Genus Hirmocystis Labbe 1899 Associations of two to twelve or more. Epimerite a small papilla. Cysts dehisce by simple rupture. Spores ovoidal. Hirmocystis harpali Watson (1916) 111. Biol. Monogr., 2:378. Spor. elongate, largest 500^X80/1. LP:TL::1:7; WP:WD::1:1.2. Max- imum of four in a chain. Nucl. spher. Epim. large and spherical. Host: Harpalus pennsylvanicus erythropus. (Col.) Taken at Urbana, 111. Genus Uradiophora Mercicr 1912 Arch. zool. exper., (5) 10:198. Sporonts associative, cysts without spore-ducts. Spores spherical or sub-spherical with equatorial line, extruded in chains. Epimerite an elongated papilla. Deut. with small appendix. Uradiophora cuenoti { = Cephaloidophora cucnoti) Type species. Mercier (1911) C. R. Soc. Biol., 71:51-3; (1912) Arch. zool. exper., (5) 10:177-202. NEW GREGARINES DESCRIBED FROM 1911-1920 133 Spor. associated in chains of from 2 to 4 individuals, very elongate, max. 1. spor. 700fx. Nucl. spher. Epim. an elongated papilla. Deut. with small atrophied appendix. Cysts ovoidal, 44/i in 1., spores 4/i. Host: Atyaephyra Desmaresti. (Crust.) Taken at Nancy, France. Genus Pyxinoides Tregouboff 1912 Arch. zool. exper., (5) 10:liii-lxi. Sporonts in twos, development extracellular, epimerite a slightly stalked globular papilla with 16 longi- tudinal furrows, with small conical papilla at apex. Pyxinoides balani. Type species Tregouboff (1912) Arch. zool. exper., (5) 10:liii-lxi. Max. I. primite 130;u, satellite 60^. Nucl. spher. Hosts: Balaims amphitrite, B. eburneus. (Crust.) Taken at Cette, France. Genus Leidyana Watson 1915 Jour. Parasit., 2:35. Sporonts solitary, epimerite a small sessile knob, dehiscence by spore-ducts, spores in chains, dolioform. Leidyana { = Stenophora) erralica. Type species. Crawley (1903) Proc. Acad. Nat. Sci., Phila., 55:45. Watson (1916) III. Biol. Monogr., 2:328-30. Leidyana tinei Keilin (1918) Parasit., 10:406-10. Spor. long-ellipsoidal, max. I. 300At, w. 85^. LP:TL::1:1.5; WP:WD::1: 1.7. Cysts spher. llO/x, spores barrel-sh., 7/x long. Host: Endrosis fenestrella. (Lepid.) Taken at Cambridge, Eng. Genus Protomagalhdensia Pinto 1918, Brazil-Medico Spores barrel-shaped with spine at each corner, sporonts attenuated, several individuals in an association, often attached laterally. Myonemes prominent. Protomagalhaensia { = Gregarina) serpentula. Type species. Magalhaes (1900) Arch, parasit., 3:34-69; Pinto (1918) Brazil-Medico. Family DIDYMOPHYIDAE Leger 1892 Associations of two or three individuals. None-septate in satellites. Genus Didymophyes Stein 1848 Epimerite a small pointed papilla. Cyst dehiscence by simple rupture. Spores ellipsoidal. Didymophyes { = Gregarina) minuta Ishii (1914) Ann. Zool. japon., 8:435-41. Watson (1916) 111. Biol. Monogr., 2:343. Sporonts elongate, 188/^X26/^. Ratio LP:TL::1:23; WP:WD::1:1.5. Nucleus spherical. Cyst and spores unknown. Host: Tribolium ferrugineum. (Col.) Taken in Prov. of Izu, Japan. 134 MINNIE W. KAMM Family ACTINOCEPHALIDAE Leger 1892 Sporonts solitary, epimerites varied, simple rupture of cysts. Genus Actinocephalus Stein 1848 Epimerite with many upwardly-directed spines, spores biconical. Actinocephalus permagnus (?.4. sp. Pfeiflfer 189vS; A. steUiformis Wasielew- ski 1896) Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52: Spor. elongate, max. 1. 1.3 mm. LP:TL::1:17; WP:WD::1.1 :1. Nucl. ellipsoidal, cysts nearly spher., 750^t. Spores diamond-shaped, 7.6./iX5)U. Host: Procrustes coriaceus. (Col.) Taken in East Prussia. A ctinoccphalus parvus Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:?. Spor. ovoidal, largest 140^X75/^. LP:TL::1:5; WP:WD::1:1.3. Nucl. ovoidal. Epim. a corona of digitiforn processes upon a short neck. Hosts: CeraiophyUiis fringiUae, C. galUnae larv. (Dipt.) Taken in East Prussia. A ctinoccphalus echinatus Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:? Spor. cylindro-conical, largest 400m in 1. LP:TL::1:5; WP:WD::1.1 :1. Cysts spher., 330^, spores biconical, 8^X4.8^. Hosts: Pterostichus niger, P. vulgaris. (Col.) Taken in East Prussia. Actinocephalus zophus { = Stephanophora zopJia Ellis (1913) Zool. Anz., 42:200-2). Ellis (1913) Trans. Amer. Micr. Soc, 32:278. Spor. elongate-cylindr., max. 1. 1600^. LP:TL::1:12; WP:WD::1:1.7. Epim. persistent, stout-necked, constricted at base, and terminating in corona of 9 or more small digitiform processes. Hosts: Xyctotheres barbarata {X. barbata), Alobates pennsylvanicus. (Col.) Taken at New Orleans, La. Actinocephalus brachydactylus Ellis (1913) Trans. Amer. Micr. Soc, 32:279. Spor. elongate-ovoidal, 1. 500^. LP:TL::1:4; WP:WD::1:1. Host: Nymphs of Aeschna sp. (Neur.) Taken at Douglas Lake, Mich. Actinocephalus crassus (^ = Stephana phora crassa Ellis (1912) Zool. Anz., 39:688-9). Ellis (1913) Trans. Amer. Micr. Soc, U:!!?^. Avg. spor. 50m-60m in 1. LP:TL::1:3.5; WP:WD::1 :1.5. Nucl. spher. Host: Leptochirus edax. (Col.) Taken at Quirigua, Guatemala. Actinocephalus gimbeli { = Stenophora gimbeli Ellis (1913) Zool. Anz., 41:464.) Watson (1916) III. Biol. Monogr., 2:353. NEW GREGARINES DESCRIBED FROM 1911-1920 135 Spor. obese, 1. 500/x. LP:TL::1:5; WP:WD::1:1.2. Host: Harpalus pennsylvanicus. (Col.) Taken at Vincennes, Ind. Genus Pyxinia Hammerschmidt 1838 Epimerite a flat crenulate crateriform disc with central style. Spores l)iconical. Pyxinia bulhifera Watson (1916) Jour. Parasit., 3:65-75. Spor. long, slender, longest spor. 850/iXl60/i. LP:TL::1:5. WP:WD:: 1:1.3. Epim. typical, 60m— lOO/i 1. Nucl. spher. Host: Dermestes lardarius. (Col.) Taken at Oyster Bay, L. I. Genus Amphorocephalus Ellis 1913 Zool. Anz., 41:462. Epim. dilated in middle, terminating in a concave disc peripherally fluted at ant. end. Prot. constricted across middle. Spores not known. Amphorocephalus amphorellus. Type species. Ellis (1913) Zool. Anz., 41:463-4; Trans. Amer. Micr. Soc, 32:276-7. Spor. elongate, 1. 500m -970m. LP:TL::1:17; WP:WD::1:2. Host: Scolopendra heros. (Chil.) Taken at Boulder, Col. Genus Boihriopsis Schneider 1875 Epimerite with long slender filaments. Prot. very large. Spores biconical. Boihriopsis ( = Legeria) terpsichorella Ellis (1913) Trans. Amer. Micr. Soc, 32:276; Watson (1916) 111. Biol. Monogr., 2:356. Prot. of spor. equal to or longer than deut. Avg. 1. 720m, w. 145m. LP:TL:: 1.5:1; WP:WD::1.3:1. Host: Hydrophilus sp. (Col.) Taken at Douglas Lake, Mich. Boihriopsis claviformis Pinto (1918) Brazil Medico; (1919) Contribuijao ao estudo das Gre- garinas, Rio de Janeiro, 116 pp., 6 pi. Spor. elongate-triangular, widest at ant. end, bluntly acuminate. LP:TL:: 1:7; WP:WD::1.4 : 1. Host: Aeschnida larva. (Odon.) Taken at Manguinhos, Brazil. Boihriopsis osivaldocruzi Hasselmann (1918) Brazil-Medico, Nov. 2, 1918. Genus Siylocysiis Leger 1899 Epimerite a sharp recurved cone. Spores biconical. Siylocysiis ensiferiis { = Siylocephalus en. Ellis 1912 Zool. Anz., 39:686) Eliis (1913) Trans. Amer. Micr. Soc, 32:274. Avg. 1. spor. 40-65m. LP:TL::1:2.5; WP:WD::1:1.2. Host: Lepiochirus edax. (Col.) Taken at Quirigua, Guatemala. 136 MINNIE W. KAMM Genus Steinina Leger and Duboscq 1904 Epimerite a short digitiform process changing into a flat button. Spores biconical. Steinina rotundata Ashworth and Rettie (1912) Proc. Roy. Soc. Lond., B 86:31. Spor. 180/i long, 80/i wide. Epim. sometimes a blunt cone with central st3^1e, again a saucer-shaped disc with crenulate periphery. Spor. ovoidal, nucl. spher. Cysts spher. 185/x, dehiscing in int. of host, spores ovoidal, 12aiX7/x. Hosts: Ceratophylkis styx, C. farreui, C. galUnae. (Dipt.) Taken near Edinburgh, Scotland. Steinina obconica Ishii (1914) Ann. zool. Japon., 8:439. Spor. ovoidal, largest 148^X80^. LP:TL::1:5; WP:WD::1:1. Epim. a minuted style. Prot. compressed ant. -post. Nuck spher. Cysts ovoidal. Host: Tribolium ferrugineum. (Col.) Taken in Prov. of Izu, Japan. Steinina rotunda Watson (1915) Jour. Parasit., 2:32; (1916) 111. Biol. Monogr., 2:364. Spor. globose, largest 250/^X130^. LP:TL::1:2.3; WP:WD::1:1.1. Epim. spher. Host: Amara angustata. (Col.) Taken at St. Joseph, 111. Steinina harpali Watson (1916) 111. Biol. Monogr., 2:365. Coelomic. Spor. small, obese, largest spor. 200/iXlOOM. LP:TL::1:4; WP:WD::1:1 .3. Epim. a short cone changing into a sphere then cup- shaped. Nucl. small, spher. Cysts spher. 12/i. Host: Harpalus pennsylvanicus longior. (Col.) Taken at Urbana, HI. Family ACANTHOSPORIDAE Leger 1892 Spor. solitary, epim. varied. Dehiscence by simple rupture, spores with equatorial and polar spines. Genus Corycella Leger 1892 Epim. globular with 8 large recurved hooks, spores biconical, 4 spines at each pole. Corycella orthomorpha Hasselmann (1918) Brazil-Medico, Oct. 5, 1918. Genus Prismatospora Ellis 1914 Trans. Amer. Micr. Soc, 33:215. Spores hexagonal, truncate at ends with one row of long spines at each pole. Epim. subglobose with lateral recurved hooks. NEW GREGARINES DESCRIBED FROM 1911-1920 137 Prismatospora evansi. Type species Ellis (1914) Trans. Amer. Mic. Soc, 33:215. Spor. 400/x in avg. 1., broadly conical, LP:TL::1:3; WP:WD::1:1; Prot. broad, blunt, deut. tapering. Nucl. small, spher. Cysts subspher., 370yu, dehiscence by simple rupture, spores ll/iX5.8/z Hosts: Nymphs of Tramea lacerata and Sympetrum rubicunduliim. (Neur.) Taken at Douglas Lake, Mich. Genus Cometoides Labbe 1899 Epim. a sphere with long slender filaments. Spores biconical with one polar and two equatorial rows of spines. Cometoides sp. Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:138. Spor. cylindro-conical. Max. 1. 360/x. LP:TL::1:5; WP:WD::1:1. Nucl. ellipsoidal. Epim. a flattened papilla with long filaments. Cysts spher. 160^. .Host: Carabus sp. larva. (Col.) Taken in East Prussia. Family STYLOCEPHALIDAE Ellis New name for Stylorhynchidae Schneider 1886 preocc. Ellis (1912) Zool. Anz., 39:25. Spor. solitary, epim. varied, nucl. ovoidal, dehiscence by pseudocyst, spores irregular, in chains. Genus Stylocephalus Ellis New name for Stylorhynchus Stein 1848 preocc. Ellis (1912) Zool. Anz., 39:25. Epim. a papilla at end of a long slender neck. Cysts papillate, spores hat-shaped. Stylocephalus giganteus Ellis (1912) Zool. Anz., 39:25-7. Spor. elongate, 1200-1800/1 in 1. LP:TL::1:15; WP:WD::1:1. Cysts spher., 450/x. Spores 7 X 11m- Hosts: Eleodes sp.; Asida opaca; Asida sp.; Eusattus sp. (Col.) Taken at Boulder and Denver, Col. Genus Bulbocephalus Watson 1916 Jour Parasit., 3:66. Epim. a dilated papilla in middle of rather long slender neck. Nucl. ellipsoidal. Bulbocephalus wardi. Type species Watson (1916) Jour. Parasit., 3:66. Spor. stout, widest at shoulder, largest spor. 290/zX45/i. LP:TL::1:5; WP:WD::1:1. Epim. as above. Nucl. placed diagonally. Cysts and spores unknown. Host: Clerid larva. (Col.) Taken at Oyster Bay, L. I. 138 MINNIE W. KAMM Bulbocephalus elongatus Watson (1916) Jour. Parasit., 3:66. Spor. very long and slender, max. 1. 600/z, w. 50/i. LP:TL::1:11; WP:WD:: 1:1. Epim. as above. Nucl. diagonally placed. Host: Cucujus larva. (Col.) Taken at Oyster Bay, L. I. Family DACTYLOPHORIDAE Leger 1892 Epimerite complex, sporonts solitary, cysts dehisce by lateral pseudo- cyst or simple rupture, spores elongate. Genus Nina Grebnecki 1873 Protomerite two long lobes fused at one end, peripherally set with teeth and long slender filaments. Spores in chains. Nina indicia Merton (1911) Abh. Seneck. nat. Ges. Frankf., 34:119-26. Spor. elongate, max. 1. ISOO/x. LP:TL::1:20; WP:WD::4:1. Prot. low, very broad, two long narrow parallel plates attached laterally, free at one end, each plate armed with a ridge of short sharp teeth. Nucl. spher. Host: Scolopendra subspinipcs. (Chil.) Taken at Heidelberg, Germ. Nina ( = Pterocephalns) leitdodacimhai Hasselmann (1918) Brazil-Medico, Sept. 21, 1918. Genus Echinomera Labbe 1899 Epimerite an eccentric cone with short digitiform processes. Dehiscence by simple rupture. Spores cyllindrical, in chains. Echinomera ( = Gregarina) magalhaesi^ Pinto (1918) Brazil-Medico; (1919) Contribuifao ao estudo das Gre- garinas, Rio de Janeiro, 116 pp., 6 pi. Spor. elongate-conoidal, widest at shoulder. Largest spor. 300/zX70/i. LP:TL::1:4.3; WP:WD::1:1 . 1. Epim. a polymorphic eccentric cone. Nucl. ellipsoidal. Host: Scolopendra sp. (Chil.) Taken at Rio de Janeiro, Brazil. Genus Seticephaliis Kamm 1922 (this paper) A dense tuft of short, upwardly-directed brush-like bristles super- imposed upon the broad, flat-topped protomerite, persistent. A chromi- dial body in protomerite. Parasitic in Chilopoda. Seticephalus { = Gregarina) elegans. Type species. Pinto (1918) Brazil-Medico; (1919) Contribuiffio ao estudo das Gre- garinas, Rio de Janeiro, 116 pj)., 6 pi. Spor elongate-conoidal, acuminate, largest spor. 75//X32"'. LP:TL:: 1:7.5; WP:WD::1 :1 .2. Prot. broad, flat, nucl, small ellipsoidal. Epim. * This species was placed by the author in the Genus Gregarina. NEW GREGARINES DESCRIBED FROM 1911-1920 139 short bristle-like filaments across whole ant. end of prot. Cyst and spores unknown. Host: Scolopendra sp. (Chil.) Taken at Rio de Janeiro, Brazil. (This species was placed by the author in the genus Gregarina but it is unlike any member of that genus or any hitherto described genus and is therefore made the type species of a new genus.) Genera of Uncertain Position Genus Agrippina Strickland 1912 Parasit., 5:108 Sporonts solitary, epim. a circular disc armed with peripheral digitiform processes, with short neck. Spores long-ovoidal. Agrippina bona. Type species Strickland (1912) Parasit., 5:108. Spor. elongate, conical, avg. 1. 175/i. Nucl. ellipsoidal. Epim. as stated. Cysts spher. dehiscing by simple rupture. Spores 6.6/xX7)U- Host: Ceratophyllus fasciatns. (Dipt.) Taken at Cambridge, England. Genus Metamera Duke 1910 Q. J. Mic. Sci., 55:261-86. Epimerite subconical, apex eccentric, with corona of numerous branched digitiform appendages. Cysts dehisce by simple rupture. Sporonts solitary. Metamera schubergi. Type species Duke (1910) Q. J. Mic. Sci., 55:261-86. Spor. 150/1 X45;u. Deut. with one to three septa posterior to nucleus. Cysts spher., spores ovoidal, 9/^X7 /jl. Hosts: Glossophonia complanata, Hemiclepsis marginata. (Annelida.) Taken at Heidelberg, Germ, and Cambridge, Eng. (This species was left out of Sokolow's Synopsis (1911).) Genus Ganymedes Huxley 1910 Q. J. Mic. Sci., 55:155-75. Non-septate, with motile extensile fixation- organ, cupped posterior end for association, nucleus large, spherical. Inhabit intestine and liver of host. Ganymedes anasidis Huxley (1910) Q. J. Mic. Sci., 55:155-75. Characters of the genus. Avg. 1. 250-300/1, w. 17-2b/t. Spor. elongate- cylindrical. Host: Anaspides tasmaniae. (Crust.) Taken in Tasmania. (This species was omitted from Sokolow's List — 1911.) Species of Uncertain Position Gregarina crassa Ishii (1915) Ann. zool. japon., 8:438-9. Spor. ovoidal, max. 1. 242/1, w. 64/i. Nucl. spher. LP:TL::1:19; WP:WD:: 1 :4. 140 MINNIE W. KAMM Host: Triholium jerrugmeum. (Col.) Taken in Prov. of Izu, Japan. Prot. lacking in satellite. See Watson (1916) 111. Biol. Monogr., 2:409. Gregarina coptotomi Watson (1916) Jour. Parasit., 2:406. Spor. solitary, epim. and cysts unknown. Spor. 210/i 1. LP:TL::1:7; WP:WD::1:2.3. Nucl. ellipsoidal. Host: Co ptotomus interrogatus. (Col.) Taken at Urbana, 111. Gregarina brasUiensis Pinto (1918) Brazil-Medico; (1919). Contribuicao ao estudo dos Gregarinos Rio de Janeiro, 116 pp., 6 pi. Spor. not associative, pyriform, acutely acuminate, largest spor. 92/xX35/i. LP:TL::1:2.4; WP:WD::1:1.1. Prot. ovo-cylindrical, nucl. ovoidal. Host: Scolopendra sp. (Chil.) Taken at Rio de Janeiro, Brazil. Gregarina legeri Pinto (1918) Brazil-Medico; (1919). Contribuicao ao estudo dos Gregarinos Rio de Janeiro, 116 pp., 6 pi. Spor. not associative, rectangular with bulbous post, extremity, largest spor. 290/zX80/i (at post, end of deut.) LP:TL::1:4.8; WP:WD::1:1. Prot. square, nucl. ellipsoidal, in dilated post, portion of deut. Host: Stylopyga americana. (Orth.) Taken at Rio de Janeiro, Brazil. Taeniocystis legeri Cognetti de Martiis (1911) Arch. Protistenk., 23:247. Spor. segmented in both prot. and deut., max. 1. 1600/x. Up to 19 segments. Nucl. ovoidal. Epim., cysts, and spores unknown. Host: Kynotus PittarelUi (Oligoch.) Taken at Moramanga, Madagascar. This species is placed among the 'Uncertain Species' because the 'protomerite' is divided into three segments and the parasite is coelomic. Miscellaneous Unnamed gregarines: Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:46-7. From the fol- lowing hosts: Heledona agricola (Col.), Polyporus sulphureiis (Plathy.), Tritoma quadripitstulata (Col.), Cychrus rostratiis (Col.), Scolopcndrclla sp. (Chil.). Unnamed Cometoides-like form: Wellmer (1911) Schr. Physik. Ges. Konigsbg., 52:46-7. Host: Ilydro- philus aterrimus. (Col.). Unnamed gregarines of several species: Pantel (1913) La Cellule, 29 (1): 142-4. Host: Forficula auricularia. (Orth.) NEW GREGARINES DESCRIBED FROM 1911-1920 141 Unnamed gregarine: Buddington (1910) Science, 31:470. Host: Balanus ebiirneus. (Crust.) Unnamed gregarines similar to Leidyana tinei: Keilin (1918) Parasit., 10:406. Hosts: Oecophora pseudospretella, Tinea pallescentella. (Lepidopt.) An Annotated List of Species in the Tribe Acephalina of the Suborder Eugregarinae Genus Monocystis Stein 1848 Non-septate, irregular, motile sporonts, cysts with incomplete sporu- lation, spores navicular, octozoic. (All herein described are coelomic or inhabit seminal vesicles unless stated otherwise.) Monocystis pareudrili Cognetti de Martiis (1911) Arch. Protistenk., 23:216-40. Spor. subspherical, max. diam. 60;u. Spores ovoidal, 10X5/z. Host: Pareiidrilus pallidus. (Polych.) Taken in 'Equatorial Africa.' Monocystis thamnodrili { = M. sp. Cognetti 1906) Cognetti de Martiis (1911) Mem. R. Accad. Sci. Torino, 46: (2) 147-262. Host: Rhinpdrilus {=21iamnodrilus) incertus. (Polych.) Taken in Ecuador. Monocystis rostrata Muslow (1911) Arch. Protistenk., 22:20-55. Sem. ves. Spor. spindle-shaped, cysts spher., spores spindle-shaped. Host: Lumhriciis lerrestris. (Oligoch.) Taken in Munich. Monocystis catenata ( = partim. M. herculea Hesse 1909) Muslow (1911) Arch. Protistenk., 22:51. Spor. spher. 425/i, in chains. Cysts nearly spher. SOO^u. Spores 14X6;u. Host: Lumbricus terrestris. (Oligoch.) Monocystis minima Konsulofif (1916) Arch. Protistenk., 36:353-61. Spor. ovoidal, 42/^, spores ellipsoidal. In long. Intestinal par. Hosts: Euchlanis dilatata. (Rotif.); Salpina mucronata Ehrbg. (Rotif.) Taken at Sofia. Monocystis perforans Pinto (1918) Brazil-Medico; (1919) Contribuijao ao estudo das Gre- garines, Rio de Janeiro, 113 pp., 6 pi. Sem. ves. Spor. ovoidal to cylindr. in chains. 1200/iX800At. Nucl. ellip- soidal, cysts spher. spores 24X7.5;u. Host: Glossoscolex wiengreeni. (Ann.) Taken at Rio de Janeiro, Brazil. 142 MINNIE W. KAMM Monocystis niichaelseni Hesse (1916) Tolosani Monit. Zool. ital , 27:217-22. Monocystis sp. Wellmer (1911) Schr. Physik. Ges. Konigsb., 52:147. Coleomic. Host: Helophorus aquaticus. (Col.) Taken in East Prussia. Genus Lithocystis Giard 1876 Emend. Pixell-Goodrich (1915) Q. J. Mic. Sci., 61:81-104. Spor. elongate, very motile. Spores in rosettes, long ovoidal, truncate. Epispore a funnel at one end through which 8 sporozoites escape, other end a tubular tail. Lithocystis foliacea Pixell-Goodrich (1915) Q. J. Mic. Sci., 61:81-104. Coelomic. Max. 1. sporont 1.3 mm. Cysts spher. 600yu. Spores long- ovoidal, 24X9/X, tail three times as long as spore, with leaf-like expansion, funnel at other end. Host: Echinocardium cordatum. (Echinodermata.) Taken at Naples and Plymouth, Eng. Lithocystis microspora Pixell-Goodrich (1915) Q. J. Mic. Sci., 61:81-104. Coelomic. Max. 1. 1mm. Cysts spher. 300^t. Spores 13X7/x, with tail two or three times as long, narrow, tapering. Host: Spatangus purpureus. (Echinod.) Taken off Plymouth. Genus Urospora Schneider 1875 Emend. Pixell-Goodrich (1915) Q. J. Mic. Sci., 61:81-104. Tropho- zoites coarsely granular, elongate. Female gamete with long flagellum, male non-motile. Cysts spher. 8 sporozoites which escape from one end of funnel-shaped epispore other end a filamentous tail. Urospora synaptae { = Syncystis syn. Cuenot (1891) Rev. Biol. nord. Fr., 3:295.) Cuenot (1912) Bull. sta. biol. Arcachon, Bord., 14:85. One form spor. rotund, 300;u max. diam, other vermiform SOO/jl long, very motile. Cysts spher. 150/x, spores ovoidal, 20/x, one end cupped other a long filament. Coelomic and intestinal. Host: Synapta gaUicnnci. (Echinod.) Taken at Arcachon and Roscoff, Fr. Urospora travisiae Urospora ovalis Mawrodiadi (1914) Varsava Univ. izv., No. 8, 1-164. NEW GREGARINES DESCRIBED FROM 1911-1920 143 Urosporz neapolitana Pixell-Goodrich (1915) Q. J. Mic. Sci., 61:81-104. See also Q. J. Mic. Sci., 60:159-74. Spor. 200-300^ 1., 40^ w. Cysts spher. 100-200^. Spores 12X7^, ovoidal, cupped at one end, tail twenty times as long as spore and tightly coiled at other end. Host: Echinocardiuni cordatum. (Echinod.) Taken at Naples. Urospora echinocardii Pixell-Goodrich (1915) Q. J. Mic. Sci., 61:81-104. Troph. and cysts same as U. neapolitana. Spores \9n long, tails 6 or 7 times as long as spore, not tightly coiled. Hosts: Echinocardium sp. and Spatangus sp. (Echinod.) Taken at Ply- mouth, Eng. Genus Gonospora Schneider 1875. Emend Pixell-Goodrich (1916) Q. J. Mic. Sci., 61:205-16. Polymorphic, nematoid, pyriform or ovoidal. Cysts spher., spores with funnel at one end refringent endo-spore which gives oflf processes supporting thick trans- parent ectospore and funnel at other. Gonospora mercieri { = Lithocystis miilleri Giard 1886.) Cuenot (1912) Bull. sta. biol. Arcachon, Bord., 14:88-90. Spor. spher., max. diam. 160/x. Cysts 180/i in diam., spores 23/z long, ovoidal, no caudal filament. Intestinal par. Kost: Synapta digiiaia. (Echinod.) Taken at Arcachon, France. Gonospora glycerae Pixell-Goodrich (1916) Q. J. Mic. Sci., 61:205-16. Coelom par., generally surrounded by host epithelium. Spor. 1 to 5 mm. long, widest near ant. end and tapering to blunt point. Nucl. spher. cysts spher. spores 10X8^t. Refringent endospore with many supporting proc- esses. Associations of spor. by 'ball-and-socket' dovetailing of ant. ends. Host: Glycera siphonostoma. (Polych.) Taken at Naples. Gonospora testiculi ( = Cysiobia test.) Tregouboff (1916) C. R. Soc. Biol, 79:652-5; (1918) Arch. zool. exper., 57:471-509. L. 250//, elongate-ovoidal rounded at ant. end, pointed at post. end. Cysts 60-IOOfjL in diam. Spores 8 to 10/x in 1. Testicle par. Host: Cerithium vulgatum. (Moll.) Taken at Villefranche-sur-Mer, France. Gonospora intestinalis { = Cystobia int.) Sokoloff (1914) Arch. Protistenk., 32:221-8.; Tregouboff, (1918) C. R. Soc. Biol., 79:652-55, Pixell-Goodrich (1916) Q. J. Mic. Sci., 61-205-16. 144 MINNIE W. KAMM Intestinal par. Spor. elongate, max. 1. 300/1. Cysts nearly sphcr. SOO^t. Spores ovoidal, IO/jl 1. Host: Glycera siphonstoma. (Polych.) Taken at Naples. Genus Rhynchocystis Hesse 1909 Spor. ovoidal to cylindr. ant. end conical. Spores biconical with like poles. Rhynchyocystis hessei Cognetti de Martiis (1911) Mem. R. Accad. Sci. Torino, 46:207-16. ^ax. 1. spor. 116/i, w. 88/x. Coelomic par. Spores 13X2.5/i. Host: Pareudrilus palUdus. (Polych.) Taken in 'Equatorial Africa.' Rhynchocystis geoplanae Fuhrmann (1916) Centrallbl. Bakt. Parasit., 77:482-5. Parenchymatous and intestinal par. Largest spor. 280X80/x. Nucl. large, spher. Cysts spher. 180/^. 'Pseudoepimerite' a rosette. Hosts: Geoplana backi, G. amagensis. (Furh.) Taken in Columbia, S. A. Genus Diplocystis Kiinstler 1887 Coelomic, associating early to form spherical masses. Spores spherical or oblong. Eight sporozoites. Diplocystis phryganeae Berg-von-Emme (1913) Arch. Protistenk., 28:43-51. Spor. subspher. nucl. spher. Host: Pkryganea grandis (Neur.) Taken at Petrograd. Genus Lankesteria Mingazzini 1891 Trophozoites spatulate, cysts spher., spores ovoidal. Lankesteria sp. Swarczewsky (1910) Festschr. Geburtst. R. Hertwig 1: 635-74; (1911) Arch. Protistenk., 22:236. Intestinal par., encysted in parenchyme. Cysts spher. lOO/i. Cyst walls dissolve and spores are carried to organs, set at liberty at death of host. Hosts: Planaria sp. and Sorocoelis sp. (Plath.) Lankesteria culicis Stevenson and Wenyon Jour. Trop. Med. and Hyg., 18:196; Macfie (1917) Report of the Accra Lab. for 1916, London, pp. 67-75. Host: Stegomyia fasciata larv. (Dipt.) Taken at Accra, Gold Coast, Africa. . Genus Ancora Labbe 1899 Anchor-shaped spor. two long lateral backwardly-directed prolonga- tions from ant. end, body tapering to sharp point. Ancora lutzi {?A. sagittata Lcuckart Arch. Naturg., 26 (2):263) Hasselmann (1918) Brazil-Medico, Aug. 10, 1918. Host: Capitella capitata Fabr. (Ann.) Taken at Manguinhos, Rio de Janeiro, Brazil. new gregarines described from 1911-1920 145 Uncertain Genus in the Acephalinae Genus RhytidocysHs Henneguy 1908 Trophozoite stage intracellular, encystment solitary, two sporozoites in spore. Rhytidocystis henneguyi deBeauchamp (1912) C. R. Acad. Sci. Par., 154:1384; (1913) Arch. Protistenk., 31:138. Spor. ellipsoidal, encystment solitary. Nucl. spher. Spores 12X7yu, ovoidal, symmetrical. Epithelium and lumen of intest. Host: Ophelia negleda. (Polych.) Taken off Roscoff, France. Uncertain Species in the Acephalinae Unnamed sp. Guenther (1914) Zool. Anz., 44:264-7. Host: Ficalbia dofleini. larv. (Dipt.) Taken on Island of Ceylon. Habi- tat: Tracheae and coelom. Unnamed sp. Pixell-Goodrich (1916) Q. J. Micr. Sci., 61:205-16. Max. 1. 1.6 mm., w. 1 mm. Coelomic and attached to body or intest. walls. Nucl. large, ovoidal. Host: Glycera siphonostoma. (Echinod.) Taken at Naples. Two other unnamed sp. found by same author, and in same host as last. An Annotated List of the Species in the Suborder Schizogregarinae Tribe 1. MONOSPORA Leger and Duboscq 1908 Family 1. OPHRYOCYSTIDAE Leger and Duboscq 1908 Tribe 2. POLYSPORA Leger and Duboscq 1908 Family 2. SCHIZOCYSTIDAE Leger and Duboscq 1908 Genus Schizocystis Leger 1900 Schizonts extracellular, vermiform, multinucleate. Gametes ovoidal, pointed at one end. Cysts subspher. or ovoidal, spores octozoic, biconical. Schizocystis spinigeri Machado (1913) Mem. Inst. Oswaldo Cruz, Rio de Jan., 5:5-13. Spor. slender, striated longitudinally, cysts ovoidal, spores ovoidal, pointed. Both sporogony and schizogony noted. Host: Spiniger sp. (Hemipt.) Taken near Manguinhos, Rio de Janeiro, Brazil. Family 3. SELENIDIIDAE Brazil 1907 Schizonts intracellular, multinucleate, at close of development. Game- tocystes mobile, longitudinal myonemes. Parasitic in Polychaetes. 146 MINNIE W. KAMM Genus Sclenidium Giard 1884 Schizogony in the intracellular stage. Selenidium cruzi Faria, Cunha and Fonseca (1917) Brazil-Medico, 31:243; (1918) Mem. Osw. Cruz, 10:17. Largest trophoz. 160mX25;u, vermiform, slightly flattened, ant. end blunt with small pointed epimerite. Nucl. ellipsoidal. Host: Polydora socialis. (Polych.) Taken at Rio de Janeiro, Brazil. Selenidium mechnikovi Leger and Duboscq (1917) Ann. Inst. Past., 31:69. Intestinal par. Intra- and extra-cellular, schizozoites pyriform, 5/i. Sporonts cucumber-shaped, 30-34^t, longit. striated, nucl. sub-spher. Host: Glossobalanus minutus. (Enteropneusta.) Taken at Sainte-Jean- de-Luz, France. Family 4. MEROGREGARINIDAE Porter 1908 Family 5. SPIROCYSTIDAE Leger and Duboscq 1915 Arch. Protistenk., 35:199-211. Mono-sporic and monozoic, schizogony and sporogony in same host. Genus Spirocystis Leger and Duboscq 1911 Bull. Soc. zool. Fr., June, 1911. Spirocystis nidula. Type species Leger and Duboscq (1911) Bull. Soc. zool. Fr., June 1911; C. R. Soc. Biol., 76:296; Arch. Protistenk., 35:199. Sporocyst ovoidal, 35n long, rejected with excrement. Releases in its next host through a micropyle a single folded sporozoite 40^i long. Sporozoite gives rise to helix-shaped schizont found in somatic or visceralperitoneum. This becomes multinucleate of max. 1. 35ju and gives rise to macro- and micro-gametes, the copulation of which produces the spore, found in the chloragogue cells. Host: Lumbricus variegatus. (Oligoch.) Taken near Grenoble, France. Tribe 3. OCTOSPOREA Keilin (1914) C. R. Soc. Biol., 76:768 With eight spores in the sporogonic cycle. Family 6. CAULLERYELLIDAE Keilin (1914) C. R. Soc. Biol., 76:768. Genus Catdleryella Keilin (1914) C. R. Soc. Biol., 76:768 Intestinal par. Schizogony extracellular, veg. nucleus gives rise to 16 merozoites. Each of the two sporonts in a cyst produces 8 gametes. These 16 conjugate by twos to form 8 spores which produce 8 sporozoites. Caulleryclla aphiochaetae. Type species Keilin (1914) C. R. Soc. Biol., 76:768. Veg. stage 22^ long, ovoidal, pointed at end, embedded in epithelium. NEW GREGARINES DESCRIBED FROM 1911-1920 147 Nucl. divides four times, giving rise to 16 merozoites liberated and affix themselves to epithelium. Sporulation by twos with 16 gametes produced. Cysts and gametes spherical. Host: Aphiochaeta rnfipes larv. (Dipt.) Taken at Paris. Caulleryella anophelis Hesse (19?) C. R. Acad. Sci. Par., 166:569. Spor. 35X30^, syzygy in twos, cysts spher. 24/x to 3>2ix. Spores sub-spher. 12.5X11m- Dehiscence of cyst in host intest. Host: Anopheles bifurcatus. larv. (Dipt.) Taken in the Dauphine, France. Family of Uncertain Position Family 7. POROSPORIDAE Leger and Duboscq 1908 1915 C. R. Soc. Biol., 75:95-8. Many sporozoites from a sporoblast. No sporocyst. Genus Porospora Schneider 1875 Epim. minute, button-like, spor. septate, usually solitary. Porospora legeri deBeauchamp (1910) C. R. Acad. Sci. Par., 151:997-9. Spor. associative prot. of primite depressed at apex, satellite longer with no prot. 750/iX75iU. Cysts spher. from two sporonts. Intestinal par. Host: Eriphia spinijrons. (Crust.) Taken at Saint- Jean-de-Luz, Fr. (This species was omitted from Sokolow's List-1911.) Porospora portunidarum Leger and Duboscq 1911 ( = Aggregata p. Frenzel) (1911) Arch. zool. exper., (5) 6:lix-lxx; (1913) C. R. Acad. Sci. Par., 156:1932; (1913) C. R. Soc. Biol., 75:95. Porospora pisae Leger and Duboscq (1911) Ann. Univ. Grenoble, 23:403; Tregouboff (1916) Arch. zool. exper., 55:xxxv-xlvii. 1 mm. long, eel-shaped. Encystment from one or two spor. Host: Pisa gibosii. (Crust.) Taken at Cette and Villefranche-sur-AIer, Fr. Porospora maraisi Leger and Duboscq (1912) Ann. Univ. Grenoble, 23:399. Host: Portunns depuraior. (Crust.) Porospora nephropsis Leger and Duboscq (1915) C. R. Soc. Biol, 75:368-71. Spor. elongate, ellipsoidal, blunt at ends, max. 1. 240;Lf, max. w. 44/i. Nucl. spher. Solitary vermiform enigmatic individuals 1300X36 also present. Cysts 160/i in diam. Schizogonic spores 5 in diam. Host: Nephrops norvegicus. (Crust.) 148 MINNIE W. KAMM The classification of this family is uncertain because the sporonts are apparently typical cephaline Eugregarinae yet a schizogonic cycle exists. Minchin (1912) says 'The classification of the future will probably be one which divides all gregarines into Cephalina and Acephalina and distributes the schizog^egarines amongst these two divisions.' Genus of Uncertain Position Genus Selysina Duboscq 1917 C. R. Acad. Sci., 164:? Selysina perforans. Type species Duboscq (1917) C. R. Acad. Sci., 164:? (1918) Arch. zool. exper., ?:l-53. Host: Stolonica socialis. (Ascid.) Taken off Roscoff, France. Unnamed gregarine Strickland (1913) Jour. Morphol., 24:84. Schizogonous. Pathogenic effect upon host. Inhabits various tissues. Cysts spher. 250^t. Host: Simulium bracteatum larv. (Dipt.) Taken near Boston, Mass. A species described as Microtaeniclla clymenellae n.g., n. sp. from Clymenella torqtiata (Ann.) by Calkins (1915) Biol. Bull., 29:46 is regarded by the author as a colonial gregarine resembling the scolex and proglottids of the cestodes, each segment being nucleated. This polynucleate condition makes its inclusion in this group doubtful. Poche (Arch. Protistenk., 37:6) considers it identical with the genus Haplozoon. List of Hosts With Their Gregarine Parasites Host Parasite Platyhelminthes Geo plana hacki Rhynchocystis geoplanae Fuhrman G. amagensis Rhynchocyslis geoplanae Planaria sp. Lankcstcria sp. Swarczewsky Polyporus sulphureus Gregarine form, Wellmer Sorocoelis sp. Lankestcria sp. Swarczewsky Annelida: Polychaeta Capitella capHala Aticora lutzi Hasselmann Clymenella torquata Microtaeniella clymenellae Calkins Glycera siphotrostoma Gonospora glyccrae Pi.xell-Goodrich Glycera siphonosloma Gonospora inlcstinalis Pixcll-Goodrich Glycera siphonosloma Three unnamed parasites Pixell-Goodrich Ophelia neglecla Rhytidocyslis henneguyi dcBeauchamp Parendrilus pallidus Monocyslis pareiidrili Cognetti de Martiis Pareiidrilus pallidus Rhynchocyslis hessci Cognelti de Martiis Polydora ciliala Polyrhabdina polydorae Caullerj'^ and Mesnil Polydora socialis DoUocyslis sp. Faria, Cunha and Fonseca Polydora socialis Selcnidium cruzi Faria, Cunha and Fonseca Pygospionis scticornis Polyrhabdina pygospionis Caullcr>' and Mesnil i NEW GREGARINES DESCRIBED FROM 1911-1920 149 Host Rhinodrilus inccrlus Scolclepsis fuliginosa Spio marlinensis ANNELroA: Oligochaeta Kynotus Pittarcllii Lumbricus terrestris Lnmhricus terrestris Lumbricus variegatus Glossoscolex mengreeni Annelida: Hirudinea Glossophonia complanata Hemiclepsis marginala ROTIFERA Euchlanis dilatata Salpina mucronata ECHINODERMATA Eckinocardium cordatum Echinocardium cordatum Echinocardium sp. Spatangus sp. Synapla pitrpurcus Synapta galliennei Synapta digitata MOLLUSCA Cerithium vulgalum Crustacea Ampelisca spinipes Anaspides tasmaniae Atyaephyra Dcsmaresti Balanus amphilrite Balanus eburneus Balanus eburneus Eriphia spinifrons Gammarus marimis Libinia dubia Nephrops norvegicus Portumis depurator Pisa gibosii Talitrus saltator Talorchestia longicornis Uca pugnax Uca pugilator Chilopoda Scolopendra heros Scolopendra subspinipes Scolopeiidra sp. Scolopendra sp. Scolopendra sp. Scolopendrella sp. Parasite Monocystis Ihamnodrili Cogn. dc Marliis Polyrhabdina spiofiis Caullery and Mesnil Polyrhabdina brasili Caullery and Mcsnil Taeniocystis legcri Cogn. de Martiis Monocystis roslrata AIuslow Monocystis catenala Muslow Spirocystis nidula Leger and Duboscq Monocystis perforans Pinto Metamera schubergi Duke Metamera schubergi Duke Monocystis minima Konsuloff Monocystis minima Konsulofif Lithocystis foliacea Pixell-Goodrich Urospora neapolitana Pixell-Goodrich Urospora echinocardii Pixell-Goodrich Urospora echinocardii Pixell-Goodrich Lithocystis microspora Pixell-Goodrich Urospora synaptae Cu6not Gonospora mercieri Cuenot Gonospora tesliculi Tregouboff Ceplmloidoplwra ampelisca Kamm Ganymedes anaspides Huxley Uradiophora cuenoti Mercier Pyxinoides balani Tregouboff Pyxinoides balani Tregouboff Unnamed parasite, Buddington Porospora legeri deBeauchamp Cephaloidophora macidata Leger and Duboscq Cephaloidophora olivia Kamm Porospora nephropsis Leger and Duboscq Porospora maraisi Leger and Duboscq Porospora pisae Leger and Duboscq Cephaloidophora talilri Mercier Cephaloidophora delphinia Kamm Cephaloidophora nigrofusca Kamm I Amphorocephalus amphorellus Ellis Nina indicia Merton Echinomera magalhaesil Kamm Seticephalus elegans Kamm Gregarina brasilietisis Pinto Gregarine form, WeUmer 150 MINNIE W. KAMM Host DiPLOPODA Callipits laclarius Euryiirus crythropygiis Fontaneria coarctata Orthomorpha coarctata Orthomorpha gracilis Orthomorpha sp. Orthomorpha sp. Parajulns impressus Parajulus venustus Parajulns sp. Rhinocricus pugio Rhinocricus sp. Rhinocricus sp. Rhinocricus sp. Rhinocricus sp. Rhinocricus sp. Thysanura Sminthurus jiiscus Orthoptera Ceuthophilus latcns Ceuthophilus maculatus Ceuthophilus ncglcctus Ceuthophilus stygius Ceuthophilus valgus Conocephalus f rater Encoptolophus sordidus Forficularia auricular ia Gryllus ahbrcviatus Ischnoptera pennsylvauicus Mclanoplus differential is M elano plus femur-ruhr urn Udeopsyllae nigra Hemiptera Spiniger sp. Neuroptera Aeschnidae Iv. Acschna sp. Phryganea grandis Sympelrum rubicundulum Tramea lacerata DiPTERA Anopheles bijurcalus Iv. , Aphiochacta rufipes Iv. Ceratophyll us fasciatus CcratophylUis farreni Ccratophyllus Jringillac Iv. Ccralophyllus gallinae Iv. Ceralophyllus gallinae ad. Ccratophyllus slyx Stenophora Stenophora Stenophora Stenophora Stenophora Stenophora Fonsecaia j. Stenophora Stenophora Stenophora Stenophora Stenophora Stenophora Stenophora Stenophora Stenophora Parasite lactaria Watson diplocorpa Watson Cauda ta Watson clongata Ellis robusta Ellis robusta Ellis wlymorpha Pinto imprcssa Watson robusta Ellis cockcrellae Ellis cunhai Pinto lutzi Pinto cruzi Pinto viannai Pinto umbilicata Pinto tenuicoUis Pinto Gregarine form, Wellmer Grcgarina Grcgarina Grcgarina Grcgarina Grcgarina Grcgarina Grcgarina Gregarine Grcgarina Grcgarina Grcgarina Grcgarina Grcgarina longiducta Ellis longiducta Ellis neglecta Watson stygia Watson consobrina Ellis chagasi Pinto nigra Watson form, Pantel galliveri Watson illinensis Watson nigra Watson nigra Watson udeopsyllae Watson Schizocystis spiniger Machado Bolhriopsis claviformis Pinto Aclinocephalus brachydactylus Ellis Diplocyslis phrygancac Berg-von-Emme Prismatospora cvansi Ellis Prismatospora cvansi Ellis Caullcryclla anophelis Hesse Caulleryclla aphiochaelae Keilin A grip pi na bona Strickland Steinina rotundata Ashworth and Rettie Aclinocephalus parvus Wellmer Aclinocephalus parvus Wellmer Stein itui rotundata Ashworth and Reitie Steinina rotundata Ashworth and Rettie NEW GREGARIXES DESCRIBED FROM 1911-1920 151 Host Ficalbia dofleini Iv. Simidiiitn hractealum Iv. Stegomyia fasciaca Iv. COLEOPTERA Alobaks poinsyliHuticus A viara anguslota Asida opaca Asida sp. Broscus ccphaloks Carabus sp. Clerid Iv, Coccinella sp. Coccinella sp. Coccinella novcnniolala Coptotomus inkrrogatus Coploiomus intcrrogalus Crypticiis quisquilius Ciicujus Iv. Cyclirus roslralus Dermesles lardariiis Diabrotica vitkila Elakridae Iv. Ekodes sp. Eusattus sp. Harpalus aeneus Har pains pennsylvankus Harpalus pennsylvanicus erythropiis Harpalus pennsylvankus longior Harpalus ruficornis Heledona agricola Helophorus aquaticus Hydrophilus akrrimus Iv. Hydrophilus sp. Hylobius abktis Ips typographus Lagria hirla Lepkchirus edax Leptochirus edax Ninus inkrstitialis N ycktheres barbaraia Omoplaia normalis Platydema excavatum Platynusruficollis Procrusks coriaceus Pkrostkhus niger PkrosHchus niger Syskna sp. PkrosHchus slygkus PkrosHchus stygicus Pkrostkhus vulgaris Par.\site Unnamed par. Guenther Unnamed par. Strickland Lankeskria cidicis Stevenson and Wenj'on Actinocephalus zophus Ellis Skinina rotunda Watson Stylocephalus giganteus Ellis Stylocephulus giganteus Ellis Gregarina erecta Wellmer Cometoides sp. Wellmer Bulbocephalus wardi Watson Gregarina Jragilis Watson Gregarina katherina Watson Gregarina katherina Watson Gregarina globosa Watson Gregarina coptolomi Watson Gregarina ovoidea Wellmer Bulbocephalus elongatus Watson Gregarine form, Wellmer Pyxinia bulbifera Watson Gregarina diabrotica Kamm Gregarina gracilis Watson Stylocephalus giganteus Ellis Stylocephalus giganteus Ellis Gregarina polyaulia Wellmer Actinocephalus gimbeli Watson Hirmocustis harpali Watson Steinina harpali Watson Gregarina polyaulia Wellmer Gregarine form, Wellmer Monocystis sp. Wellmer Cometoides-like form, Wellmer Bothriopsis tcrpsichorella Ellis Gregarina hylobii Kamm Gregarina typographi Fuchs Gregarina rostrata Wellmer Actinocephalus crassus Ellis Stylocystis ensiferus Ellis Gregarina guatemalensis Ellis Actinocephalus zophus Ellis Gregarina watsoni Pinto Gregarina platydema Kamm Gregarina platyni Watson Actinocephalus permagnus Wellmer Gregarina exiguus Kamm Actinocephalus echinatus W^ellmer Gregarina aragaoi Pinto Gregarina monarchia Watson Gregarina intestinalis Watson Actinocephalus echinatus Wellmer 152 MINNIE W. KAMM Host Tenehrio castaneus Tenebrionidae Iv. Tribolium ferrtigineum Tribolium ferrugineum Tribolium ferrugitieum Tribolium ferrugineum Tritoma quadripustulata Lepidoptera Endrosis fenestrella Iv. Oecophora pseudospretella Stain Tinea pallescentella Stain Arachnida Ctenocephalus scrraticeps Oribata geniculala TUNICATA Stolonica socialis Enteropneusta Glossobalanus minutus Parasite Gregarina grisea Ellis Grcgarina tenebrionella Watson Gregarina minuta Ishii Gregarina crassa Watson Disymophyes minuta Kamm Steinina obconica Jshii Gregarine form, Wellmer Leidyana tinei Keilin Unnamed greg. Unnamed greg. Grcgarina ctenocaphalus Ross Gregarina sp. Wellmer Selysina perforans Duboscq Selenidium metchnikotd Leger and Duboscq; DEPARTMENT OF METHODS, REVIEWS, ABSTRACTS, AND BRIEFER ARTICLES ABNORMAL EARTHWORM SPECIMENS, HELODRILUS SUBRU- BICUNDUS AND H. TENUIS* By Frank Smith University of Illinois Comparatively little attention has thus far been given to abnormalities in the relations of the reproductive organs of earthworms. Variations from the normal positions and number are sometimes found, and asymmetri- cally placed gonads and openings of efferent ducts are not infrequently encountered. Since further investigation of such abnormalities may lead to at least a partial understanding of their relation to disturbances in the normal developmental activities of the animals concerned, it seems advisable to record the more important details in the structure of specimens repre- sentative of some of the more common types of such abnormalities, if indeed it be found that there are such types. A specimen of Hclodrilus siibriibicimdus (Eisen) recently collected at Urbana, Illinois, in the banks of a stream heavily contaminated with sewage was found to have the spermiducal pores on somite 14 instead of in the usual position on the fifteenth somite. Sagittal sections of the left half of the anterior part were made and unexpected irregularities were found. Spermaries and spermiducal funnels are present in the usual positions in 10 and 11. An ovary and oviducal funnel are present in the usual positions in 13; but an additional one of each, equally well developed, have similar positions in the twelfth somite which normally has no gonads. An oviducal pore is present in the usual position on 14, and in addition there is a super- numerary one on 13, related to the oviducal funnel of 12. The spermiducal pores on 14 are slightly laterad of the oviducal pores of the same somite. Sperm sacs in 9, 11, 12, and an ovisac in 14 have the usual location and relations. The calciferous gland, crop, and gizzard also have the usual location and relations; but the most posterior heart is in 10 instead of in the usual position in 11; and the lateral longitudinal vessel branches off from the dorsal vessel in 11 instead of in the usual place in 12. The ventral setae of 9 are modified to genital setae which are of about twice the length of ordinary setae and relatively more slender. *Contribution from the Zoological Laboratory of the University of Illinois, No. 205. 153 154 FRANK SMITH The presence of extra gonads in 12 is not infrequently met with, but the presence of spermiducal and oviducal pores on the same somite (14) is decidedly unusual, in the experience of the writer, but has been found also in another specimen, described below. A specimen of Helodrilus tenuis (Eisen) collected near Urbana, Illinois in a fallen and decaying tree, attracted attention because the spermiducal pores were asymmetrically placed, the one on the right side being normally situated on 15, while that of the other side opened on the somite next ante- rior. Sections were made and the asymmetrical relations were found to extend to internal organs. Reproductive organs of the right side were found in normal positions and relations, as follows: spermaries and spermi- ducal funnels in 10 and 11; an ovary and oviducal funnel in 13; oviducal pore on 14; and the spermiducal pore on 15. In the left half of the worm, there are spermaries and spermiducal funnels in 9, 10, 11; ovaries and oviducal funnels in 12 and 13; oviducal pores on 13 and 14; and a spermidu- cal pore on 14, laterad of the oviducal pore of that somite. The extra gonads and associated funnels are as large and well developed as the normal ones. Paired sperm sacs in 11 and 12 are in the locations normal for this species. No irregularities in the location of hearts and lateral longitudinal vessels have been noticed; and the alimentary tract has normal relations, except that the anterior evagination of the calciferous gland in the left half of the worm is found anterior to the septum 9/10, and the one in the right half is anterior to 10/11 which is the more normal position. Asymmetry in the number and position of various organs in the right and left halves of specimens is of fairly frequent occurrence and often involves circulatory and alimentary systems as well as the reproductive organs. It will be noticed that the presence of both spermiducal and ovi- ducal spores on 14 is associated, in the two specimens described above, with the presence of ovaries and oviducal funnels in both 12 and 13; but such association may be a mere coincidence rather than an actual correlation. SUBSTITUTES FOR ABSOLUTE ETHYL ALCOHOL By Lawrence E. Griffin Reed College During the past year the writer has had his interest attracted to the question of whether other alcohols can be successfully substituted for anhydrous ("absolute'') ethyl alcohol in histological work. For a consider- able part of the year the air of Portland, Oregon, is nearly saturated with water vapor, and the tendency of absolute ethyl alcohol to absorb water constitutes one of the difficulties in its use. More potent reasons for seeking substitutes were, however, the high cost of the absolute ethyl alcohol and annoyances resulting from tax and prohibition laws. Even when alcohol can be secured by institutions free of tax there are regulations to be ob- served which entail a certain amount of delay in securing it, as well as much supervision of its use. So it would be advantageous if other alcohols can be used which are not subject to the regulations of the Bureau of Internal Revenue, and which are not sought as beverages. Fortunately, there are at least three such alcohols which have proved to possess merit. METHYL ALCOHOL. The ordinary commercial quality of methyl alcohol contains about 95% of alcohol, the remainder consisting mostly of acetone, with traces of a large variety of other impurities. Purified methyl alcohol, anhydrous, and nearly free from acetone and other impurities, is put on the market under various trade names. The brand which we have tested is known as Diamond Methyl alcohol. Being practically free from acetone it not only lacks the strong disagreeable smell of ordinary wood alcohol but, in fact, has a pleasant odor much like that of refined grain alcohol. This alcohol dehydrates sections and tissues as well as the absolute ethyl alcohol, and is a little more reliable because it will dehydrate more sections than an equal amount of absolute grain alcohol. We have found it to be a good solvent, and have used it with success in the compounding of a number of reagents. As regards cost, it was not only far cheaper than anhy- drous ethyl alcohol, but was considerably cheaper than the tax-free 95% grain alcohol which we bought a short time before we secured the methyl alcohol. We are now using this alcohol as our standard reagent in dehydra- tion. In the course of eight months our stock, kept in a large glass stop- pered bottle, has not absorbed enough water vapor to be noticeable. We purposely made no particular effort to seal the stock bottles tightly from the atmosphere. Anhydrous ethyl alcohol kept under the same conditions would have been useless for the dehydration of tissues. 155 156 LAWRENCE E. GRIFFIN BUTYL ALCOHOL. Professor George W. Martin has called attention (Science, April 21, 1922), to the use of butyl alcohol in dehydration and infiltration with parafin. This alcohol has been under test in our laboratory as a dehydrating reagent for several months and has given excellent results. We have used it, however, only in the last stage of dehydration, passing slides from 90% methyl or 95% ethyl alcohols to the butyl alcohol. It appears to us to be superior to either ethyl or methyl alcohols for dehydra- tion, but its use is slightly disagreeable on account of the pungent, char- acteristic odor. Inhalation of its fumes causes a slight, temporary irritation of the throat. In reply to our inquiry as to whether this property of butyl alcohol might be removed, the Commerical Solvents Corporation, which made our sample, replied: "The irritation of the throat, caused by the use of Butanol, is quite characteristic of this compound and is a property which it would be difficult to obliterate. However, we do not believe it has any harmful effect, as some of the men who work on the distillation end of the process have been subjected to this for years and have experienced no ill effects. They are much less susceptible to the irritation after having worked with this com- pound for some time." Butyl alcohol is, at any rate, a valuable reagent for dehydration, our observation being that the sections dehydrated with it are slightly more brilliant than those cleared with the previously mentioned alcohols. As Professor Martin also states, butyl alcohol is a solvent of paraffin, and can be used for infiltration of tissues likely to shrink or harden in the usual infiltrants. We believe, however, that for this purpose it is surpassed by Terpeneol. TERPENEOL. The use of terpeneol (terpineol) in place of absolute ethyl alcohol was suggested a number of years ago, but it is only lately that I have been able to test it thoroughly. Terpeneol is a pleasant smelling, aromatic liquid, of about the consistency of thin cedar oil. It is tolerant of large amounts of water in dehydration, and also dissolves paraffin and resins. On account of its consistency we have found it advisable to use first a mixture of terpeneol and methyl alcohol before placing tissues into pure terpeneol. Terpeneol may be used as a dehydrating agent for sections, but does not have any advantages over methyl or butyl alcohol when used in that way. As it dissolves parafin readily it is more useful as a dehydrant and infiltrant of tissues to be embedded. Terpeneol dissolves parafin better than butyl alcohol. Our experience has been that tissues which had been dehydrated and infiltrated with terpeneol were less shrunk and hardened than when embedded by the ordinary methods. Terpeneol is of rather high refractive index, so that it serves as a clearing agent also. Sections may be transferred directly from terpeneol to Xylol-damar. As the terpeneol SUBSTITUTES FOR ETHYL ALCOHOL 157 does not make tissues so brittle as does Xylol it can be used advantageously in the preparation of whole mounts. We have also found that damar dissolved in terpeneol makes a mounting medium which, on account of the refractive index being lower than that of xylol-damar or xylol-balsam, shows some details of cell structure which are obscured in these commonly used mounting media. /yt I I TRANSACTIONS OF THE American Microscopical Society Organized 187S Incorporated 1891 PUBLISHED QUARTERLY BY THE SOCIETY EDITED BY THE SECRETARY PAUL S. WELCH ANN ARBOR, MICHIGAN VOLUME XLI Number Four Entered as Second-class Matter August 1.?, 1918, at the Post-office at Menasha. Wisconsin, under Act of March 3, 1879. Acceptance for mailing at the special rate of postage provided for in Section 1 103, of the Act of October 3, 1917. authorized Oct. 21, 1918 iSltr dollrgiate Prraa Geopoe Banta Publishing Compan-v Menasha. Wisconsin 1922 TABLE OF CONTENTS For Volume XLI, Number 4, October, 1922 The Anatomy of Some Sexually Mature Specimens of Dero limosa Leidy, with one plate, by R. L. Mayhew 159 Studies on American Naid Oligochaetes, by H. E. Hayden, Jr 167 Excessive Sexual Development in Hydra oligactis with Spermary on Tentacle, with one figure, by A. W. Schmidt 172 Some Suggestions for Teaching Mycology, with two figures, by F. D. Heald 175 List of Members 179 Index to Volume XLI 189 TRANSACTIONS OF American Microscopical Society (Published in Quarterly Instalments) Vol. XLI OCTOBER, 1922 No. 4 THE ANATOMY OF SOME SEXUALLY MATURE SPECIMENS OF DERO LIMOSA LEIDY.^ By Roy L. Mayhew University of Illinois Introduction Ecology. The material on which this paper is based was collected during the summer of 1921 at the Biological Station of the University of Michigan, on Douglas Lake, Michigan. The particular locality was a small bog draining into Burt Lake, about four and one half miles from the Station. A thick mat of algae taken from a plank at the surface of the water proved to be especially rich in the following genera of Naididae: Nais, Chaetogaster, Pristina, and Dero. The families Lumbriculidae and Enchytraeidae were represented by a number of specimens. Plankton taken from the water near by contained specimens of the genera Slavina, Stylaria, Nais, Dero, and Pristina. During the examination of the algae it was frequently noticed that a number of worms of the same species would be found close together. Notes made at the time showed that four sexually mature specimens of Dero limosa were thus found in close proximity. At other times Pristina would be represented in great abundance. Five specimens belonging to the genus Nais were taken from an area about two inches square and were the only specimens of the genus found in the algae. Only one or two specimens belonging to the genus Nais were taken from the plankton. So commonly was the grouping of worms of the same kind observed, that when one of a particularly desirable type was located, the algae of the immediate vicinity was examined with special care. Technique. Specimens of Dero were found to be the most difficult of any of the worms encountered, to kill and fix for histological study. Anesthesis with chloretone often resulted in the death and maceration of 1 Contribution from the Zoological Laboratory of the University of Illinois, No. 212 and from the University of Michigan Biological Station. 159 160 ROY L. MAYHEW the posterior part of the worm before the anterior ceased to move or came sufficiently under the influence of the drug to prevent bending and distor- tion when subjected to the killing fluid. A concentrated aqueous solution of corrosive sublimate was used as a fixing fluid. The specimens sectioned were cut transversely, 6 ^ in thickness, and stained with Delafield's haema- toxlin and eosin. Mounts, in toto, were stained in borax carmine and cleared in oil of wintergreen. Identification. Seven sexually mature and numerous immature speci- mens of Dero limosa Leidy were taken from the algae referred to above during the first two weeks of August, 1921. The determination of the species proved a little difficult since living specimens not anesthetized were constantly in motion, and, when anesthetized or kijled for preserva- tion, were usually found to have completely closed the branchial pavilion. The gill arrangement seems, however, identical with that of the above named species as described by Michaelsen ('00, '03, & '09) and others. Examination of sections of the closed pavilion reveals the fact that the lateral portions of the lip, which are carried medially in the process of closing, may easily be mistaken for a third pair of gills on the ventral portion of the structure, since they are intimately associated with the true gills in position and have similar cell arrangement (fig. 16). Since the only descriptions found of sexually mature specimens be- longing to the genus are those given by Michaelsen ('00) for D. perrieri and D. imiltibranchiata, and by Walton ('06) in his general description of the genus, it seems desirable to give a rather extended account of the new material mentioned above. The descriptions of the sex organs as given by Michaelsen are brief and are here quoted for comparison. D. perrieri, "1 Paar Samentrichter im 5. Segm.; Samenleiter allmahlich in die driisenlosen Atrien iibergehend. 1 unpaariger Eiersack. Samentas- chen im 5. Segm., mit scharf abgesetztem, ziemlich kurzem, tonnenformi- gem Ausfiihrungsgang." D. multibranchiata, "Unpaariger Samensack vom 5. Segm. nach hinten gehend. Ovarien (Eiersacke?) im 7. Segm." Length and Number of Somites The length of preserved sexually mature specimens varied from 5 to 10 mm. and the total number of segments from 40 to 55. Immature specimens showed an equal range of variation in length and contained from 40 to 62 somites; those with one or two budding zones containing from 50 to 59. Setae In respect to the setae, the sexually mature specimens differ from the immature only in the entire absence of ventral bundles in the 6th somite \ ANATOMY OF DERO LIMOSA LEIDY 161 in contrast with the occurence of special or genital setae in Nais obtusa and a number of other species described by Piguet ('06 & '09). The dorsal bundles, which begin on the 6th somite in both mature and immature specimens, have been found to contain one long capilliform and one short, slightly cleft, biuncinate seta in each of the bundles examined (fig. 13). The long capilliform setae are about equal in length to the diameter of the body, and are without serrations. The biuncinate setae extend but little beyond the surface of the body, and are more or less curved and somewhat tapered distally. Their distal portions are much stouter than figured by Michaelsen ('09) and by Bousfield ('87), and the tips 6f the teeth are not sharp pointed but slightly rounded. One biunci- nate seta of a sexually mature specimen was found to be somewhat smaller in diameter and to have a slight nodulus. Piguet ('09) says that mature specimens of the genus A^ais lose the setae of the dorsal bundles in the clitellar somites, but such is not the case in D. limosa. The ventral bundles (fig. 9-12) contain from 2 to 5, in the majority of instances 3 or 4, biuncinate setae. The distal tooth, or the one farthest from the nodulus, is about equal in length to the proximal, or but slightly longer. The teeth are usually sharp pointed, though occasionally the proximal one is thickened and round pointed. They often appear to have round tips due to their being turned so that an exact lateral view is not obtained. Each of the ventral setae is provided with a well defined nodu- lus. The ventral setae of somites 2-5 are longer than those from the re- mainder of the body, in both mature and immature specimens. Setae from each of the somites 2-5 were measured and averaged for each speci- men with the result that the averages varied from 120/i to 160/x. Averages obtained in like manner from a number of somites posterior to 6 varied from 80 to 100;u. The extreme difference between the averages so obtained for any specimen was 65 /x, the measurements being 95^1 to 160/u. Definite ratio relations have been noted between the portions of the setae proximal and distal to the noduli in particular parts of the worm. The portions of the setae distal to the noduli in somites 2-5 are usually about equal in length to the proximal parts but may be as much as 1.5 times as long, while the distal portions of setae in somites posterior to 6 are only .6 to .8 as long as the proximal. The. Reproductive Organs Clitellum. This organ extends from the anterior part of the 5th somite almost to the setae of the 8th (fig. 1, cL). On the ventral side it begins just back of the openings of the spermathecae, but dorsally farther forward. In life the clitellum is yellowish or cream colored, and, when sectioned, is found to be made up of a single layer of large columnar cells 162 ROY L. MAYHEW containing large globules of a substance, which is probably the secretion that later forms the cocoon, and relatively small nuclei irregularly dis- tributed in the cells. Spermathecae. These are conspicuous paired organs almost filling the ventral two thirds of the 5th somite (fig. 1-3, spm.) The upper part is a relatively thick walled sac with deeply staining, irregularly arranged nuclei, and well filled with a compact mass of sperm cells indicating that copulation has taken place. The duct arises a little laterad of the most ventral portion of the sac and extends directly to the body wall where it turns directly posteriad, a very short distance, and opens on the surface through the pore slightly anteriad and laterad of the ventral seta bundle of the same side of the somite (fig. 2 & 3). In one specimen the duct passes almost directly to the pore. The walls of the duct are thickly set with nuclei of about the same size as those of the sac. Dorsal to the spermathecae there is a quantity of sperm cells in the body cavity. Sperm ducts. A pair of sperm ducts lie in the 6th somite and have their funnels in the posterior part of the 5th. Each of the pair consists of four portions, (1) the spermiducal funnel, (2) a duct joining the funnel to the atrium, (3) the atrium, and (4) the duct connecting the atrium with the hypodermal invagination. The spermiducal funnels are found in the ventral posterior part of the 5th somite anterior to septum 5/6. They are shaped as if the distal end of the duct had been split on the dorsal side and the lateral portions flattened out in the process of formation. The part of the duct between the funnel and the atrium tapers posteriorly, bends abruptly upon itself (figs. 1 & 7), and extends anteriorly to its point of union with the atrium, on the median anterior ventral surface of the latter. The atrium is a cylindrical sac occupying much of the anterior ventral portion of the corresponding half of the 6th somite. The walls are relatively thick and are made up of a single layer of large irregular cells containing large vacuoles. The duct leading from its posterior end is short and opens into a conspicuous invagination of the hypodermis on the ventral wall of the body. In one specimen one atrium was displaced so that it lay dorsad of the nerve cord in the median line of the body. Spcrmaries. Nothing was found which could be identified as such. This fact is probably due to the advanced stage of sexual maturity of the specimens, as the sperm sac and spermathecae were filled with sperm cells. When developed, they should be present on the posterior side of septum 4/5, since sperm cells were found above the spermathecae, and the sperm sac extended posteriad from the 5th somite. Sperm sac. The sperm sac is a posterior evagination of septum 5/6, and extends a short distance posteriad of the setae of the 7th somite in one specimen. ANATOMY OF DERO LIMOSA LEIDY 163 Oviducal pores and funnels. The pores are paired and are in the posterior part of the 6th somite about midway between a line joining the ventral seta bundles and one joining the dorsal seta bundles of the 6th and 7th somites (figs. 1 & 14). No distinct funnels could be identified comparable with those figured and described by Piguet ('09) for Nais obiusa, although there appeared to be several cells on each side which might properly be interpreted as belonging to a funnel since they are sharply dififerentiated from the clitellum (fig. 15). The cells of the clitellum are so graded in length as to form a distinct depression with the funnel cells at the base. The paired funnels are in the same relative position as those of A^ais obiusa. The lumen of the pore could not be located, probably because of the thickness of the transverse sections, and, for the same reason, the opening in the muscular layers was not observed. The muscular layers were found separated from the pore cells in that region. The funnel and pore would no doubt be much more conspicuous at the time of emission of the eggs. 5 6 7 ^'- R Fig. 1. Diagram showing the general plan of arrangement of the sex organs in somites 5 to 8. Reconstructed from camera lucida outlines of serial transverse sections. Lettering as for other figures. The numbers indicate the position of the setae bundles. 120X. Ovisac, (fig. 1). The ovisac is formed by septum 6/7 and extends posteriad, in one specimen through the 12th somite, in another, just be- yond the seta bundles of the 9th. Its extent is no doubt dependent upon the quantity of ova present. It almost fills the body cavity for the major part of its length, and is distended with a granular appearing material which stains pink with eosrn, but contains no ova. The sperm sac lies within its anterior portion. Ovaries. No ovaries could be found. They should be located on the posterior side of septum 5/6 since the ovisac extends posteriad from 6 and the oviducal pore is in the posterior part of this somite (fig. 1). Their absence is no doubt due to the advanced stage of development of the speci- mens. The absence of ova and the presence of abundant sperm cells suggests the possibility that an interval of time elapses between the 164 ROY L. MAYHEW functioning of the spermaries and ovaries. However this does not seem probable because of the very extensive development of the ovisac. It seems more probable that ova have occupied the latter and have been discharged. Piguet ('06) refers to the appearance of gonads as follows: "Michael- sen suppose que, chez les Na'ididees, les gonades disparaissent entierement avant le developpement des autres organes genitaux; cela est sans doute vrai en general, mais il pourrait y avoir la une exception. Frank Smith (1896, PI. 35, fig. 4, t.) figure un reste de testicule chez Pristina Leidyi." In several mature specimens of Paranais uncinata Piguet has observed vestiges of ovaries. It seems, therefore, that there are individual excep- tions, but the few observations that have been possible upon Dero limosa indicate that gonads are developed only during the period of production of the germ cells. LITERATURE CITED BousFiELD, Edward C, 1887. The Natural History of the Genus Dero. Jour. Linn. Soc. Zool. London, 20:91-106. 3 pi. MiCHAELSEN, W., 1900. Oligochaeta. Das Tierreich. 10. Lief. Berlin. 1903. Oligochaeten (Hamburgische Elb-Unlersuchung 4). Jahrbuch der Hamburgis- chen Anstalten, 19:169-209. 1 pi. 1909. Oligochaeta. Siisswasserfauna Deutschlands. Heft 13. Piguet, Emile, 1906. Observations sur les Naididees et revision systematique de quelques especes de cette famille. Revue Suisse de Zoologie, 14:185-315. 4 pi. 1909. Nouvelles observations sur les Naididees. Revue Suisse Zoologie, 17:171-216. Ipl. Walton, L. B., 1906. Naididae of Cedar Point, Ohio. Am. Nat., 40:683-706. 12 fig. ABBREVIATIONS at. Atrium nc. Nerve cord at. 1 Posterior end of atrium ov. du. Oviducal funnel and pore b. 1. Borders of dorsa: lip ov. s. Ovisac cl. Clitellum, in fig. 1 the e.xtent of the sp. Sperm cells organ is indicated spm. Spermathecae du. s. Duct of spermatheca sp. du. Sperm duct gil. 1 True gills sp. du. 1 Sperm duct opening into atrium gil. 2 Secondary gills sp. du. 2 Sperm duct in transverse section at hyp. Hypodermis its bend int. Intestine sp.f. Spermiducal funnel mus. Muscular layers sp.s. ^ Sperm sac DESCRIPTION OF FIGURES Fig. 2. Transverse section in the 5th somite showing spermathecae. 120X. Fig. 3. Portion of left spermatheca, its duct and adjacent body wall in transverse section. 270X. Fig. 4. Transverse section of si)crmi(lucal funnel with sperm cells. 270X. Fig. 5. Transverse section of spermiducal funnel posteriad of section represented in fig. 4. 270X. ANATOMY OF DERO LIMOSA LEIDY 165 Fig. 6. Transverse section of sperm duct and anterior end of| atrium at the point of entrance of the duct. 270X. Fig. 7. Transverse section of the left ventral portion of a specimen at the point where the sperm duct bends anteriad. 270X. Fig. 8. Transverse section of the left ventral portion of the same specimen, as represented in the preceding figures, at the point where the hypodermal invagination receives the sperm duct. 270X. Left ventral setae bundle of the 7th somite. 270X. Left ventral setae bundle of the 30th somite. 270X. Left ventral seta of the 2nd somite. 270X. Left ventral seta of the 3rd somite. 270X. Right dorsal setae bundle on the posterior half of a specimen. 340X. Diagram showing the position of the oviducal funnels. The position of the setae of the 7th somite is shown, as obtained by superimposing the sections containing them (21 sections posteriad) upon the outline of the sections containing the funnels, by means of a camera lucida. 120X. Fig. 15. Diagram showing the differentiation of the oviducal funnel from the clitellum 270X. Fig. 16. Transverse section of the closed branchial pavilion. 150X. Fig. 9. Fig. 10. Fig. 11. Fig. 12. Fig. 13. Fig. 14. 166 ROY L. MAYHEW spm PLATE XVI STUDIES ON AMERICAN NAID OLIGOCHAETES 1. Preliminary Note on Naids of Douglas Lake, Michigan* By H. E. Hayden, Jr. University of Richmond During July and August, 1921, I was engaged in a study of the Naididae of the region around the University of Michigan Biological Station on Douglas Lake in the upper end of the southern peninsula. This locality afiForded a wealth of material for such a study, including a number of sexually mature forms. A full discussion of the systematic aspects of this work is in course of preparation, to be followed by papers dealing with the morphology and histology of the various naids, especially of the mature individuals. As a preliminary report on this work, I wish to place on record here, for the benefit of other students of this family of the Oligochaeta, a list of the species noted, together with brief diagnoses of two new species and notes on two new varieties. The following established species were represented by forms which did not differ appreciably from the published descriptions: » Aulophorus furcatus (Oken) Chaetogaster diaphanus (Gruithuisen) Chaetogaster langi Bretscher Chaetogaster limnaei K. von Baer Dero liniosa Leidy Dero perrieri Bousfield Nais communis Piguet Nais pseudoobtusa Piguet Nais simplex Piguet Nais variabilis Piguet Pristina longiseta Ehrenberg Slavina appendiculata (d'Udekem) Stylaria fossularis Leidy Stylaria lacustris (L.) Vejdovskyella comata (Vejdovsky) 'Contribution from the University of Michigan Biological Station, and contributions from the Biological Laboratory of the University of Richmond, No. 1. 167 168 H. E. HAYDEN, JR. With the exception of Stylaria fossularis and Vejdovskyella comata, all these species were found in consideFable numbers. Of these two species only one individual of each was found. In each case, however, the species is unique and its characteristics sufficiently pronounced to prevent any error in the identification. The following species was represented by forms which showed only one marked difference from the type. Pristina acquiseta Bourne The individuals of this species agreed closely with the description of Naidium tentaculatum given by Piguet (1906), which species was later united by the same writer (1909) with Pristina aeqmseta Bourne (1891). In the forms described by Piguet and Bourne, however, there are pecu- liarly enlarged setae in the ventral bundles of segment 4; while in the forms that came under my observation these setae were, with one excep- tion, on segment 5. Whether this amounts to a varietal difference or whether the position of these setae is a matter of no importance, remains to be seen. The following is, in my opinion, entitled to rank as a variety: Chaetogaster diaphanns, var. cy clops, var. no v. This is in most respects similar to the type form of the species, but differs from it in the presence of a very definite median pigmented body intimately associated with the brain and strikingly like an eyespot. The following species have not hitherto been described: Dero polycardia sp. nov. Worms quite large, 7-10 mm. in length, about 300 microns in diameter. Color reddish. Swimming actively. Ventral setae of segments 2-5, four to six in number, about 135 microns long, nodulus proximal, distal tooth longer than proximal and with a slight swelling at base. Ventral setae of other segments, four to six in number, about 95 microns long, nodulus a trifle distal, teeth about equal, distal tooth half as thick as proximal, and with a slight swelling at base. Dorsal setae beginning on segment 6, with one or two capilliform setae, somewhat longer than the diameter of the body, and one or two needle-like setae, about 87.5 microns long, slender, bifid, nodulus distal, di!?tal tooth longer than proximal, proximal part of the seta almost straight, distal part strongly curved. Contractile trans- verse vessels ("hearts") up to eight pair, in segments 6-13 inclusive, though one or more of the last few pair may be lacking. Blood quite red. Intestinal dilation in segments 9 and 10. First nephridia in segment 7. Respiratory bursa with dorsal lip, consisting of a median portion and two lateral ciliated processes. Gills, two pair, of the pyramidal type. Budding takes place between segments 25 and 36. Sexually mature forms STUDIES ON AMERICAN NAID OLIGOCHAETES 169 not yet observed. Habitat, in felted masses of blue-green algae attached to slightly submerged logs in a marshy pond near Burt Lake, Michigan. Haemonais ciliata sp. nov. Worms large, as much as 16 mm. in the case of double chains, but able to contract to about one third of their length. Diameter, about 500 microns. Very active and, because of their rapid contractions and ex- pansions, rather leech-like in their movements. Color, light reddish. Number of segments up to 55 in individual worms, and up to 100 in double chains. Prostomium rather acuminate; when expanded, slightly longer than broad at the base. Eyes absent. Prostomium covered with fine, straight tactile processes; a zone of similar processes around each segment. Remainder of body surface bears frequent smaller processes which are sharply reflexed and terminate in a bulbous swelling. As far back as the first segment bearing dorsal setae, body surface ciliated. Setae about middle of segment. Ventral setae usually three in number, about 90 microns long, sigmoid, nodulus about middle, teeth equal in length, distal tooth half as thick as proximal, and with a slight swelling at base. In all the individuals observed, nine in number, the first four or five segments were very short, and the setae of these segments, while having the same form as those following, v/ere relatively smaller. Dorsal setae beginning on any segment from 14 to 22 inclusive: with one capilliform seta, about 160 microns long, slightly sigmoid, distal half more curved than proximal; and one biuncinate seta, about 110 microns long, slightly sigmoid, nodulus barely distal, teeth long, distal tooth longer and a trifle thinner and with a very slight swelling at base. Pharynx short, pigmented at both ends. Remainder of canal not highly specialized. Contractile transverse vessels ("hearts") in most of segments 4-20 inclusive. Circulatory system more like the usual naid type than that of H. waldvogeli Bretscher (1900). Budding takes place after segment 40. Mature forms not yet observed. Habitat, in felted masses of blue-green algae attached to slightly sub- merged logs, and in water-macerated wood, from a marshy pool near Burt Lake, Michigan. In Bretscher's (1900) description of H. waldvogeli, no mention is made of the presence of cilia on the body surface, and as this is such a noticeable feature of H. ciliata, I have ventured to indicate this fact in the specific name. The attention of systematic zoologists is called to the existence in this country of representatives of two genera not given in the key to the Naididae on pages 638-640 of Ward and Whipple's "Fresh- Water Biology": namely, Haemonais and Vejdovskyella. The latter will be found in Michaelsen (1909), as well as, under the older name Bohemilla, in Michael- sen (1900). Haemonais, hitherto known only through the single species, H. waldvogeli, is described in Bretscher (1900). These two genera may be 170 H. E. HAYDEN, JR. added to the key in Ward and Whipple by altering the text of page 639 as follows: 10 (11) Setae of dorsal bundles all uncinate Paranais Czerniavsky 18S0. 11 (10) Dorsal setae nearly straight, slightly toothed or simple- pointed OpJiidonais Gervais 1838. 12 (9) Capilliform setae present in dorsal bundles 13. 13 (13J/2, 21) First anterior dorsal setae on XII to XXII Haemonais Bretscher 1900. 133^ (13, 21) First anterior dorsal setae on V or VI 14. 14 (18) Posterior end not modified into a gill-bearing respiratory organ 15. 15 (I53/2) Capilliform setae of dorsal bundle with a series of very prominent teeth; first anterior dorsal setae on V. VcjdovskyeUa Michaelsen 1903 1534 (16, 17) Capilliform setae without teeth; one or more capilliform setae of VI much longer than those of other somites and equal to three or four times the diameter of the body Slavina Vejdovsky 1883. 16 (153/^, 17) Prostomium elongated to form a proboscis; dorsal setae of VI similar in length to those of other somites Siylaria Lamarck 1816. 17 (153/2. 16) Without proboscis; dorsal setae of VI similar in length to those of other somites Nais Miiller 1774. 18 (14) Posterior end modified into a gill-bearing respiratory organ, the branchial area 19. 19 (20) Ventral margin of the branchial area with a pair of long processes Aulophorus Schmarda 1861. Two ecological notes may be made very briefly here. The observation of Mrazek (1917) as to the ingestion of trematode larvae by Chaciogaster limnaei is similar in all respects to observations made in the course of my study of this form in Michigan. Chactogaslcr is in general carnivorous, especially Ch. diaphanns. This latter species, particularly the cyclops variety, is actively predaceous and even cannibalistic, and those who are just beginning the study of these worms are warned to keep their chaeto- gasters away from vessels containing other genera, as they will depopulate a culture of naids in a very little time. BIBLIOGRAPHY Bourne, A. G. 1891. Notes on the Naidiform Oligochacta, etc., Quart. Jour. Micro. Sci. (n. s.), 32:335-356. STUDIES ON AMERICAN NAID OLIGOCHAETES 171 Bretscher, K. 1900. Mittheilungen iiber die Oligochaetenfauna der Schweiz. Rev. Suisse de Zool., 8:1-44. MiCHAELSEN, W. 1900. Oligochaeta, Das Tierreich, 10. Berlin. 1909. Oligochaeta, Die Sussvvasserfauna Deutschlands, 13. Jena. Mrazek, a. 1917. The feeding habits of Chaetogaster limnaei. Sbornik Zoologicky, 1 :22-23. Prague. PiGUET, E. 1906. Revision des Naididae. Rev. Suisse de Zool., 14:185-315. 1909. Nouvelles observations sur les Naididees. Rev. Suisse de Zool., 17:171-218. Ward, H. B. and Whipple, G. C. 1918. Fresh-Water Biology. New York. EXCESSIVE SEXUAL DEVELOPMENT IN HYDRA OLIGACTIS WITH SPERMARY ON TENTACLE By Arthur W. Schmidt Department of Zoology, University oj Nebraska^ This specimen of Hydra oligactis was found in an aquarium with other normal individuals of the same species in February of 1918. The culture was abundantly supplied with food, such as Daphnia, Cyclops, and various other Crustaceans. The specimen was killed with a solution of corrosive sublimate and acetic acid, stained in a special preparation of borax car- mine, and mounted in Canada balsam. It measures 11 mm. in length, the body 83^ mm. and the tentacles 2}/^ mm. as it is miounted on the slide. Its extreme length when living was 13 mm. Fifteen spermaries and two ovaries occur on the body and one spermary on one of the tentacles of the specimen (See figure). The spermary on the tentacle appears to be perfectly normal in all respects except location. The question now arises as to the cause of its occurrence on the tentacle. Parke cites the following instances in the establishment of normal tentacles from abnormal ones. "A nine-tentacled Hydra fusca with one branching tentacle was isolated on February 27th. On February 28th one branch had revolved about 45° till it was in line with the longitudinal axis of the tentacle, while the other one appeared somewhat shorter than it did at first. On March 1st the small branch was almost entirely resorbed. It was much nearer the end of the tentacle than before and appeared as a small outgrowth from the tentacle. This apparent shifting of the small branch, by a migration of the short branch, may have taken place in three ways: by a shortening of the long branch, by a migration of the short branch towards the end of the tentacle, or by a fusion of the two branches along the median line. The first or last explanation seems_ the most plausible since similar instances were seen in which there could be no doubt that these were the processes involved. On March 3rd the small branch was entirely resorbed, leaving the Hydra with nine normal tentacles. Two other instances of regulation of forked tentacles in the same manner were observed." He states further that his observations show that branching tentacles may arise by the fusion of two tentacles and may regulate them- selves by a complete fusion along their median sides so as to form a single tentacle. * Studies from the Zoological Laboratory, The University of Nebraska, No. 130. 172 SEXUAL DEVELOPMENT IN HYDRA OLIGACTIS 173 In further observations he found that by regulative processes two distinct tentacles could be reformed out of two fused tentacles. He cites several instances in which two of the tentacles were fused near their ends and not near their bases along the median line. "This fusion," he states, "may have been caused by an injury to one of the tentacles, the other tentacle having become attached to it. This seems probable from the fact that alternate tentacles as well as adjacent tentacles were found fused in this manner, One eight-tentacled PI\dra was found Hydra oligac is: A, showing 18 gonads; s, spermary; o, ovary; d, basal disc. B, showing position of spermary on tentacle. in which two alternate tentacles had stuck together at a point about three fourths of the distance from the base to the tips of the two tentacles at the point of fusion. There was no connection between the cavities of the two tentacles at the point of fusion. The next day after the Hydra had been isolated, one of the tentacles had constricted off from the other tentacle just below the point of fusion of the two, leaving the tip of its tentacle attached to the other branch. The cavities of the two branches 174 ARTHUR W.'^SCHMIDT were in direct communication. The process of regulation that now took place was exactly as in the Hydra described above. This is a good ex- ample of how branching tentacles may originate." Both tentacles became normal. He summarizes his conclusions with the statement: "It appears that three regulative processes may take place in the establishment of normal tentacles; viz. (1) fusion, (2) resorption, (3) constriction." The theories of fusion and constriction suggest the idea that by a contact of the tentacle with the spermary in its original position on the body of the Hydra the two were fused; and then by a process of constric- tion the spermary was severed from the body, leaving it in its present position on the tentacle. Parke's reference to the migration of a short branch of a tentacle, mentioned above, also suggests the idea that the spermary migrated from an original position on the body to its present position on the tentacle. Probably the most plausible suggestion, however, is that the position of the spermary is due to an unusual local stimulation of a group of interstitial cells in the ectoderm of the tentacle, causing ab- normal development in that location. Due, however, to the absence of facts regarding previous conditions in the aquarium, it is impossible to throw any light upon the real cause of this abnormal condition, except by way of suppositions from previous investigations. Whitney, in his observations on Hydra viridissima, found that when they are subjected to a low temperature and starvation they develop testes and eggs. Hertwig, however, found that if Hydra oligactis is kept at a temperature^ of 8°-10° C. it will develop testes irrespective of the food con- ditions. At the time of the discovery of this specimen of Hydra oligactis that was about the temperature of the room in which the aquarium that contained this specimen was kept. Extensive experimental work on sexually reproducing Hydras would undoubtedly reveal the generality or abnormality of this occurrence. The writer desires to express his thanks to Drs. Robt. H. Wolcott and David D. Whitney for access to materials and laboratory facilities, also for their suggestions and generous interest. BIBLIOGRAPHY Hertwig, Richard. 1906 tjber Knospung und Geschlechtsentwickelung von H\c]ra fusca. Biol. Centralbl., Bd. XXVI, No. 16, pp. 489-507. Parke, H. H. 1900 Variation and Regulation of Abnormalities in Hydra. .Arch. f. Entw.- Mech., Bd. X, Heft 4, pp. 692-710. Peebles, Florence. 1900 Experiments in Regeneration and Grafting of Hydrozoa. Arch. f. Entw.-Mech., Bd. X, Heft 2 & ?>, pp. 435-487. Rand, Herbert W. 1899 Regeneration and Regulation in Hydra viridis. Arch. f. Entw.- Mech., Bd. VHl, Heft 1, pp. 1-34. Whitney, David Day. 1907 The Influence of External Factors in Causing the Development of Sexual Organs in Hydra viridis. Arch. f. Entw.-Mech., Bd XXIV, Heft 3, pp. 524-537. SOME SUGGESTIONS FOR TEACHING MYCOLOGY By F. D. Heald The study of any group of plants as to its taxonomy may proceed along two widely divergent lines: First, the student may be taught to use artificial keys and determine species, which are put in their respective species pigeon-holes and properly labeled, the prime object being to deter- mine the binomial, but little concern being given to the relationship of the various forms studied; or Second, the student may be taught to construct diagrammatic keys to the various groups, which will express natural rela- tionship. These natural keys, which give a graphic representation of relationship, are clearer than the obscurely worded artificial keys crowded full of technical terms. The writer has followed the latter method with marked success in presenting the taxonomy of seed plants with successive classes through a period of years and more recently has used the same plan with classes in mycology. The method has a number of features to recommend it, some of which are: (1) The creation of a greater interest on the part of the student in his work; (2) The development of the stu- dents' ability to reason and weigh evidences; (3) The cultivation of the scientific imagination; (4) A better understanding of evolution and what it means; and (5) The possibility of emphasizing natural descent of the various groups and bringing out the fact that classification is in reality but a means to an end,^ — an expression of relationships. The work in mycology in our laboratory is offered to students who have had general elementary botany and also to those who have had in addition a semester in general pathology, in both of which they gain some familiarity with fungi. The minimum time which suffices for anything like a satisfactory presentation of the subject is six hours of laboratory work throughout the year. The general method of procedure may be briefly presented. Very early in the beginning of the work the class is given a skeleton outline of the great groups, somewhat as shown in the accompanying diagram (Fig. 1), except that the diagrams are omitted and the student is required to select diagrams and make any adjustments that may seem necessary to present concepts of the great groups by the visual channel, or to bring out more clearly the natural relationships. Various mycological works, such as Engler & Prantl, Rabenhorst's Kryptogamen Flora, special monographs, etc. must be available for reference. Suggestions are 175 176 F. D. HEALD given to the student, but they are encouraged to use their own originality and independent thought as well as to consult authorities. They are also ,.-~^ 16.HYMENJ0MYCETALES *" ^^ /'' 14.EX0BAS1D1ALES ' 15.GASTER0MYCETALES ^*'- ALLIES 13. AURICU L ARIALES ^^''ALLl E S 12.USTILAGINALES MYCELIUM SEPTATE I I ll.UREOINALES 10. TUBERALES •'^ALLIES 9. pezizalesAllies 7.sphae:riales»*''allies 6. PERISPORIALES /ALLIES MONILIkLCS 8.FUNGI IMPERFECTI 5. EXOASCALES ^'^'ALLIES MYCELIUM NON-SEPTATE yio % lOWNV MILOCWS' |(? tiOo#e. I.SCHIZOMYCETES BlCrcHiA /5>V .4. OOMYCETES. TT 7 ^vnnMvr-irTrc 3. ZYGOMYCETES -^.PRIMITIVE FORMS --' i,, MYXOMYCETES si-'xi Ht^n Fig. 1. Chart ShowinR the (Jrcat Group of Fungi. SOME SUGGESTIONS FOR TEACHING MYCOLOGY 177 given the understanding that schemes of natural relationship can be nothing more than the expression of individual opinion, which should be arrived at by weighing all the evidence that can be brought to bear in any specific case. No two students will choose the same illustrations and the more intimate details of relationship as expressed in the charts are certain to show variations. These variations afTord excellent material for class discussions which can frequently be held with much profit. The logical order for the study of the great groups would be to begin at the bottom of the family tree with the most primitive forms and proceed to the more complex and higher forms later. In actual practice, however, it seems better to sacrifice logic and begin with some group which more readily lends itself to the method in question. The Erysiphaceae, or powdery mildews of the order Perisporiales, is a family well suited to introduce the plan of study: (1) Because species determination is relatively easy; (2) Because representatives of all the genera can very readily be obtained in most environments. Our plan would call for a careful and detailed study of some type of each genus, accompanied by drawings. Following this the student is asked to construct a diagrammatic key to the genera of powdery mildews, which will express relationship and afford generic concepts, mainly through the visual channel. Before this key is made, a general class discussion is held and the more important characters which may indicate relationship are briefly reviewed, with emphasis on those which are primitive and those which are more advanced. These keys are then presented for comparison and discussion (Fig. 2). After the completion of the keys, the class is asked to determine the species of all the powdery mildews which they have collected on some of their special field trips. T^OtXBPMACRA Fig. 2. Chart Showing the Genera of Powdery Mildews Essentially the same plan is followed with all the great groups or alliances, or with representative families of these groups. It will be at once evident that all groups can not be treated as fully as the Erysipha- 178 F. D. HEALD ceae, which we have used for our introduction to the method. For example, in the study of the Sphaeropsidales of the Imperfect Fungi, attention is given to the genera furnishing parasites and only these are included in the graphic key which the students are required to construct. In other cases, as in the Sphaeriales and allies with numerous families, the graphic keys may be limited to a representation of the families. As previously stated, the minimum time for a course in mycology according to the plan outlined is six hours of laboratory work per v/eek throughout one school year. With this minimum time there must of necessity be many omissions and consequently much of the success of the course depends on the judgment of the instructor in making wise selec- tions. There is no doubt that the same plan could be followed with much profit throughout an additional year of work. This brief note has been prepared at the suggestion of several of my former students who have been stimulated to further mycological study by the use of the method outlined. It is hoped that it may offer some suggestions to some of our younger mycologists who have received their instruction by the pigeon-hole method. Department of Plant Pathology, Washington Stale College, Pullman, Washington. LIST OF MEMBERS Honorary Members Crisp, Frank, LL.B., B.A., F.R.M.S 5 Landsdowne Road, Netting Hill, London, Eng. Pflaum, Magnus 2334 S. 21st St., Philadelphia, Pa. Life Members Brown, J. Stanford, Ph.B., A.M P.O. Box 38, Far View, Black Hall, Conn. Capp, Seth Bunker P.O. Box 2054, Philadelphia, Pa. Duncanson, Prof. Henry B., A.M R.F.D. 3, Box 212, Seattle, Wash. Elliott, Prof. Arthur H 52 E. 41st. St., New York City. Hately, John C Chicago Beach Hotel, Chicago, 111. Members The figures denote the year of the member's election, except '78 which marks an original member. The TRANSACTIONS are not sent to members in arrears, and two years' arrearage forfeits membership. (See Article IV of By-Laws.) Members .\dmitted Since the Last Published List BucHHOLz, J. T. Linton, Edwin Challis, Frank E. McCauley, David V. Cheavin, William S. Noland, Lowell E. Faust, E. C. Plunkett, Orda A. Hartman, Ernest Ryan, Ruth Harris, D. F. Titus, C. P. Jewell, Mina E. Young, Paul A. Kamm, Minnie Watson Zimmerman, Naomi B. List of Members Ackert, James Edward, Ph.D., '11 Kas. State Agr. Col., Manhattan, Kans. Adams, Frederick, C. E., '19 Apartado 560, Mexico, D.F., Mexico. Allen, Harrison Sanborn, M.A., '15 442 Farmington Ave., Waterbury, Conn. Allen, Wm. Ray, M.A., '15 Dept. Zoology, Univ. of Kentucky, Lexington, Ky. Allen, Wynfred E., A.M., '04 Scripps Inst., La Jolla, Calif. Anderson, Emma N., '16 Station A, Lincoln, Nebraska. Andras, J. C, B.A., '12 540 S. Main St., Manchester, 111. Arnold, L. P., O.D., '20 Vulcans Temple, Carlisle, Ark. Arnold, Wm. T., '17 21 Park Rd., Wyomissing, Pa. Ashley, Frank M., M.E., '20 Tribune Building, New York City, N. Y. Atchison, Mrs. W. S., A.M., '16 263 Walnut Ave., Elgin, 111. Atherton, Prof. L. G., A.B., M.S., '12 State Normal School, Madison, S. D. Atwood, H. F., '78 16 Seneca Parkway, Rochester, N. Y. Baldwin, Herbert B., '13 927 Broad St., Newark, N. J. Barker, Fr.anklin D., PH.D. '03 Univ. of Nebraska, Lincoln, Nebr. Barre, H. W., B.Sc., M.A., ^2 Clemson College, S. C. Bausch, Edward, '78 179 N. St. Paul St., Rochester, N. Y. 179 180 LIST OF MEMBERS Bausch, William, '88 St. Paul St., Rochester, N. Y. Bean, A.M., M.A., '15 1501 Palm Ave., Fresno, Calif. Beck, William A., M.Sc, '16 Univ. of Dayton, Dajton, Ohio. BiCKNELL, Anna (Miss) B.S., '21 1713 Lamont St., X.W., Washington, D. C. BiERBAUM, C. H., '21 Mutual Life Bldg., BufTalo, N. Y. BiRGE, Prof. E. A., Sc.D., LL.D., '99 772 Langdon St., Madison, Wis. Black, J. H. M.D., '12 530 Wilson Bldg., Dallas, Texas. Bo\T)EN, Alan Arthi^r, '21 1421 Oakridge Ave., Madison, Wis. Boyer, C. S., A.m., '92 6140 Columbia Ave., Philadelphia, Pa. Brown, Alice L., '19 Kans. St. Ag. Col., Manhattan, Kans. Brunn, Ch.arles a., LL.B., '16 314 Reliance Bldg., Kansas City, Mo. Bryant, Prof. Earl R., A.M., '10 Muskingum College, New Concord, O. BucHHOLz, Prof. John T., Ph.D., '22 Dept. of Botany, Univ. of Arkansas, Fayetteville, Ark. Bltfalo Society of Natural Sciences Library Building, BufTalo, N. Y. Bull, James Edgar, Esq., '92 141 Broadway, New York City. Bullitt, Prof. J. B., M.A., M.D., '12 Chapel Hill, N. C. BuswELL, A. M., M.A., '16 Univ. of Illinois, Urbana, III. Caballero, Prof. Gust.w A., '16 Fordham Univ., New York City. Carlson, C. O., A.B., '13 Doane College, Crete, Nebr. C.A.RTER, Prof. Charles, 'U Parsons College, Fairfield, la. Carter, John E., '86 5356 Knox St., Germantown, Philadelphia, Pa. Challis, Frank E., '22 252 Central Ave., Aurora, 111. CHEA^^N, William Squier, F.R.M.S., F.E.S. (Lond.), F.C.S. (Lond.), '22 ^liddlesex Hosp. ^led. School, London, W. England. Chester, Waylant) Morgan, M.A., '15 Colgate University, Hamilton, N. Y. Chickering, A. M., A.M., '16 Albion, Michigan. Clark, George Edw., M.D., '96. . Sheppard & Enoch Pratt Hospital, Towson, Maryland. Cl.^rk, Howard W., A.M., '12 Fairport, Iowa. Cleveland, L. R., B.S., '21 310 W. Monument St., Baltimore, Md. Cobb, N. A., Ph.D., '14 Falls Church, Va. Coghill, Prof. George E., Ph.D., '11 R.F.D. 9, Lawrence, Kans. Colton, Harold S., Ph.D., '11 Zoological Lab., Univ. of Pa., Philadelphia, Pa. Cone, Albert, '12 . .Associate Editor, Lumber World Review, 608 So. D arborn St., Chicago, lU. Conger, Allen C, M.A., '15 527 Forest St., East Lansing, Mich. Conlon, James J., Ph.D., '14 717 Hyde St., San Francisco, Cal. Cornell Univ. Library (Prof. S. H. Gage) Ithaca, N. Y. CoRT, W. W., Ph.D., '11 . . J. H. U. School of Hygiene, 310 W. Monument St., Baltimore, Md. Covey, George W., '11 2017 South 26th St., Lincoln, Nebr. Danheim, Miss Bertha L., B.S., '21 Blue Rapids, Kansas. Darbaker, Leasxjre Kline, Ph.D., M.D., '11 7025 Hamilton Ave., Homewood Sta., Pittsburgh, Pa. Davis, Prof. H. S., Ph.D., '12 Bureau of Fisheries, Washington, D. C. Dayton, Miss Edna B., M:D., '21 1512 N. Gratz St., Philadelphia, Pa. Deere, Emil Olaf, A.M., S.M., '13 Bethany College, Lindsborg, Kans. Depew, Ganson, '21 167 Summer St., BufTalo, N. Y. De puy, Percy Leroy, B. S., '19 Federal Bldg., El Reno, Okla. Diago, Dr. Joaquin, '21 Aguila 72, Havana, Cuba. Disbrow, William S., M.D., Ph.G., '01 151 Orchard St., Newark, N. J. Dodge, Carroll W., Ph.D., '14. .Dept. Cr>'ptogamic Botany, Harvard Univ., Cambridge, Mass. LIST OF MEMBERS 181 DoLBEY, Edward P., '06 3613 Woodland Ave., Philadelphia, Pa. DouBLEDAY, ARTHUR W., M.D., '16 5 Marlborough St., Boston, Mass. Drescher, W. E., '87 Care Bausch & Lomb Opt. Co., Rochester, N. Y. Duncan, Prof. F. N., Ph.D., '16 So. Methodist Univ., Dallas, Tex. Edmon'dson, Charles H., Ph.D., '15 College of Hawaii, Honolulu. Eggleston, H. R., M.A., '13 Marietta College, Marietta, Ohio. Eigenmann, Prof. C. H., '95 630 Atwater Ave., Bloomington, Ind. Elliott, Frank R., M.A., '15 324 Kinsey St., Richmond, Ind. Ellis, Prof. M. M., Ph.D., '12 Dept. Physiol., University of Mo., Columbia, Mo. Elmore, Prof. C. J., '19 706 West St., Emporia, Kan. Elrod, Prof. Morton J., M.A., M.S., '98 University of Montana, Missoula, Mont. Enburg, J. M., '20 5207 Baltimore St., Philadelphia, Pa. Essenberg, Mrs. Christine, M.S., '16 Scripps Institute, La Jolla, Cal. Esterly, Calvin O., '15 Occidental College, Los Angeles, Cal. Eyre, John W. H., M.D., M.S., F.R.M.S., '99. .Guy's Hospital, London, S. E., England. Fattig, Prof. P. W., B.S., M.S., '12 207 Pine St., Farmville, Va. Faust, Ernest C, Ph.D., '22 Peking Union Medical College, Peking, China. Fellows, Chas. S., F.R.M.S., '83 107 Cham, of Comm., Minneapolis, Minn. Fellows, Harriette L., '21 220 S. Prairie Ave., Siou.x Falls, South Dakota. Fernandez, Fr. M.anuel, B.S., '16 San Juan de Latran College, Manilla, P. I. Findlay, Merlin C, A.M., '15 Park College, Parkville, Mo. FooTE, J. S., M.D., '01 Creighton Dental College, Omaha, Nebraska. Furniss, H. W., M.D., Ph.D., '05 56 Brazos St., West Hartford, Conn. Gabriele, H. J., '16 2659 California St., San Francisco, Cal. Gage, Prof. Simon H., B.S., '82 Stimson Hall, Ithaca, N. Y. Galloway, Prof. T. W., A.M., Ph.D., '01 Penn Terminal Bldg., 370 Seventh Ave., New York, N. Y. Gilbert, E. M., Ph.D., '19 Biology Building, U. of Wis., Maidson, Wis. Goldsmith, G. W., B.A., '13 123 E. Washington, Colorado Springs, Colorado. Gowen, Francis H., '14 R. D. 1, Box 14, Exeter, N.H. Graff, John H., '19 Research Dept., Brown Company, Berlin, N. H. Graham, Charles W., M.E., '11 1033 Mills Bldg., San Francisco, Cal. Graham, John Young, Ph.D., '14 University, Alabama. Gravelle, p. O., '19 114 Prospect St., South Orange, N. J. Griffin, Lawrence, E., '13 Reed College, Portland, Ore. Gross, F. O., M.D., '19 1816 Erie Ave., Philadelphia, Pa. GuBERLET, John E., Ph.D., '11 A. & M. College, Stillwater, Okla. GuNNS, Cecil Aguila, '21. .Dept. Zoology, Kansas State Agr. College, Manhattan, Kansas. GuYER, Michael F., Ph.D., '11 University of Wisconsin, Madison, Wisconsin. Hagelstein, Robert, '16 165 Cleveland Ave., Mineola, Nassau Co., N. Y. Hague, Florence, A.M., Ph.D., '16 . .Dept. Zoology, Oregon Agr. Coll., Corvallis, Oregon. Hall, F. Gregory, B.A., '17 Biology Building, Univ. of Wis., Madison, Wis. Hallinen, J. E., B.S., '21 Cooperton, Okla. Hance, Robert T., B.A., '13 Zool. Lab., N. Dakota Agr., College, North Dakota. Hankinson, T. L., B.S., '03 St. Normal School, Ypsilanti, Michigan. Hansen, James, '15 St. Johns Univ., Collegeville, Minn. Hardy, Eugene H 1230 S. Keystone Ave., Indianapolis, Ind. Harman, Mary T., Ph.D., '13 Kansas State Agr. College, Manhattan, Kansas. Hartman, Ernest, B.S., '22 Kans. St. Agr. College, Manhattan, Kansas. Harris, David Eraser, M.D., D.Sc, F.R.S.S., '22 Dalhousie University, Halifax, N. S. Hayden, H. E., A.m., '21 Department of Biology, Univ. of Richmond, Va. 182 LIST OF MEMBERS Hayes, W. P., M.S., '19 319 N. 18th St., Manhattan, Kans. Heald, F. D., Ph.D., '06 Wash. State College, Pullman, Wash. Heath, Roy Franklin, M.Sc, '18 P.O. Box 270, Billings, Montana. Henderson, William, '11 Mellon Inst., Univ. of Pittsburgh, Pittsburgh, Pa. Herrick, Chester A., B.S., '21 Kansas State Agri. College, Manhattan, Kansas. Hickman, J. R., A.B., '19 Bristol, West Virginia. Hilton, William A., Ph.D., '15 Claremont, Cal. Hisaw, F. L., M.S., '19 Kans. State Agr. College, Manhattan, Kans. Holy Cross College, Professor of Biology Worcester, Mass. Hopkinson, D., M.D., '20 1008 Third St., Milwaukee, Wis. Hoskins, Wm., '79 49 6th St., LaGiange, 111. Hottes, C. F., Ph.D., '20 Nat. Hist. Bldg., Univ. of 111., Urbana, 111. Hubert, H. E., B.S., '20 3615 Melpomene St., New Orleans, La. Hltdson, D. v., B.S., '20 Johns Hopkins Medical School, Baltimore, Md. Ives, Frederic E., '02 1327 Spruce St., Philadelphia, Pa. Jackson, F. S., M.D., '19 Mc Gill University, Montreal, Canada. Jacot, A. P., A.B., '19 Biology Dept., Shantung Univ., Tsinanfie, Shantung, China. Jeffs, Prof. R. E., '11 624 N. Johnson St., Iowa City, Iowa. Jewell, Mina E., Ph.D., '22.. Dept. Zoolog>', Kans. St. Agr. College, Manhattan, Kansas. Jordan, Prof. H. E., '12 34 University Place, Charlottesville, Va. Juday, Chancey, '00 Biology Bldg., U. of Wis., Madison, Wis. JuDD, H. D,, Opt.D., '19 460 W. Philadelphia Ave., Detroit, Michigan. Kamal, Mohammed, B.S., '21.. . .Box 223, Kansas State Agr., College, Manhattan, Kansas. Kamm, Minnie Watson, Ph.D., '22. . .263 Windermere Rd., Walkerville, Ontario, Canada. Kincaid, Trevor, A.M., '12 University of Washington, Seattle, Wash. KiRSCH, Prof. Alexander M., M.G., '16 Notre Dame (Univ.), Ind. Knight, F. P. H., '11 1015 Blondeau St., Keokuk, Iowa. Kofoid, Charles A., Ph.D., '99. . . .University of California, 2616 Etna St., Berkeley, Cal. Kostir, W. M., M.A., Ph.D., '20 Dept. Zoology, Ohio State Univ., Columbus, Ohio. KoTZ, A. L., M.D., '91 302 High St., Easton, Pa. Krecker, Frederic H., Ph.D., '15 Ohio State University, Columbus, Ohio. Kudo, R., Ph.D., '20 Dept. Zoology, Univ. of 111., Urbana, 111. Lambert, C. A., '12 Bank of New South Wales, Warwick, Queensland, Australia. LaRue, George R., Ph.D., '11 University of Michigan, Ann Arbor, Michigan. Latham, Miss V. A., M.D., D.D.S., F.R.M.S., '88 1644 Morse Ave., Rogers Park, Chicago, 111. Latimer, Homer B., Ph.D., 'U 1226 So. 26th St., Lincoln. Nebr. Lewis, Ivey Foreman, Ph.D., '18 University, Va. Lewis, Mrs. Katherine B., '89 656 Seventh St., Buffalo, N. Y. Linton, Edwin, A.B., A.M. (Wash. Jeff) Ph.D. (Yale) '22 1 104 Milledge Road, Augusta, Ga. Litterer, Wm., A.M., M.D., '06 Nashville, Tenn. Lofton, Robert Elwood, A.B., '21 Bureau of Standards, Washington, D.C. Lome, Adolph, '92 289 Westminster Road, Rochester, N. Y. Longfiliow, Robert Caples, M.S., M.D., '11 1611 22nd St., Toledo, Ohio. LowDEN, Hugh B., '16 1312 York St., Denver, Colo. Lowrey, Eleanor C, '19 1826 D. St., Lincoln, Nebr. Lyon, Howard N , M.D., '84 828 N. Wheaton Ave., Whcaton, 111. MacGillivray, Alexander D., '12 603 W. Michigan .\ venue, Urbana, III. MacKay, Alexander H., B.A., B.Sc, L.L.D., F.R.S. Canada, '21 61 Queen Street, Dartmouth, Nova Scotia, Canada LIST OF MEMBERS 183 Magath, T. B., M.S., Ph.D., M.D., '13 Mayo Clinic, Rochester, Minn. Manchee, E. D., '19 200 Glen Cairn Ave., Toronto, Can. Mannhardt, L. a., Ph.B., '21 ... .N. Y. University, Washington Square, New York, N. Y. Mark, George Henry, M.E., '11 94 Silver St., Waterville, Maine. Marshall, Collins, M.D., '96 2507 Penn Ave., Washington, D. C. Marshall, Ruth, Ph.D., '07 Rockford College, Rockford, 111. Marshall, W. S., Ph.D., '12 139 E. Gilman St., Madison, Wis. Martland, Harrison S., A.B., M.D., '14 1138 Broad St., Newark, N. J. Mather, E., M.D., Ph.D., '02 228 Gratiot Ave., Mt. Clemens, Mich. May, Henry Gustav, Ph.D., '15 Agr. Exp. Sta., Rhode Island State College, Kingston, R. I. Maywald, Frederick J., '02 222 Grand Ave., Nutley, N. J. McCauley, David V., S.J., M.A., '22 Fordham University, New York, N. Y. McCoLLOCH, J. W., B.S.j '19 Kans. Agr. Exp. Sta., Manhattan, Kans. McGreery, Geo. L., '13 110 Nevada St., Carson City, Nev. McCuLLOCH, Irene, Ph.D., '20 Dept. Biology, Sophie Newcomb Memorial College, Tulane Univ., New Orleans, La. McEwAN, A., '15 Fifth Ave., Guarantee Building, 522 Fifth Ave., New York, N. Y. McKay, Joseph, '84 259 Eighth St., Troy, N. Y. McKeever, Fred L., F.R.M.S., '06 P.O. Box 210, Penticton, B. C. McLaughlin, Alvah R., M.A., '15 108 S. 6th St., Columbia, Mo. McWilliams, John, '14 Lock Box 91, Greenwich, Conn. Mercer, A. Clifford, M.D., F.R.M.S., '82 324 Montgomery St., Syracuse, N. Y. Mercer, W. F., Ph.D., '99 200 E. State St., Athens, Ohio. Metcalf, Prof. Zeno P., B.A., '12 St. College Station, Raleigh, N. C. Miller, Charles H., '11 Med. School, Johns Hopkins U., Baltimore, Md. Miller, John A., Ph.D., F.R.M.S., '89 44 Lewis Block, Buffalo, N. Y. MocKETT, J. H., Sr., '01 2302 Sumner St., Lincoln, Nebr. Moeller, H., M.D., '07 341 West 57th St., New York, N. Y. Moody, Robert P., M.D., '07 Hearst Anat. Lab., U. of Cal., Berkeley, Cal. Morgan, Anna Haven, Ph.D., '16 Mt. Holyoke Coll., So. Hadley, Mass. MuTTKOWSKi, R. a., Ph.D., '19 Univ. of Idaho, Moscow, Idaho. Myers, Frank J., '13 15 S. Cornwall Place, Ventnor City, N. J. NoLAND, Lowell E., M.A., '23 Biology Building, Univ. of Wis., Madison, Wis. NoRRis, Prof. Harry Waldo, '11 Grinnell, Iowa. Norton, Charles E., M.D., '11 118 Lisbon St., Lewiston, Me. OsBORN, Prof. Herbert, M.S., '05 Ohio State University, Columbus, Ohio. Ott, Harvey N., A.M., '03 Spencer Lens Co., Buffalo, N. Y. Patrick, Frank, Ph.D., '91 822 West 58th Street, Kansas City, Mo. Payne, Miss Nellie M., B.S., '21 Division of Entomology, University Farm, St. Paul, Minn. Pease, Fred N., '8/ P.O. Box 503, Altoona, Pa. Pennock, Edward, '79 3609 Woodland Ave., Philadelphia, Pa. Peterson, Niels Fr3:derick, '11 Plainview, Nebr. Pickett, F. L., Ph.D., '20 Dept. Botany, State College of Washington, Pullman, Wash. Piatt, H. S., Ph.D., '19 561 W. 141st St., New York, N. Y. Pitt, Edward, '11 Brandhock, Gerrard's Cross, Bucks, England. Plough, Harold H., A.M., Ph.D., '16 Dept. Biology, Amherst Coll., Amherst, Mass. Plunkett, Orda a., am., '22 Dept. Botany, Univ. of Illinois, Urbana, 111. POHL, John C, Jr., '17 204 N. 10th St., Easton, Pa. Pool, Raymond J., Ph.D:, '15 Station A, Lincoln, Nebr. 184 LIST OF MEMBERS Pound, Roscoe, A.M., Ph.D., '98 Harvard Law School, Cambridge, Mass. Powers, E. B., A.B., Ph.D., '12 Univ. of Tenn., College of Medicine, 718 Union Ave., Memphis, Tenn. Praeger, Wm. E., M.S., '14 421 Douglas Ave., Kalamazoo, Mich. Procter, William, Ph.D., '19 Suite 423, 30 East 42nd St., New York, N. Y. PuRDY, William C, M.Sc, '16 1311 Burdette Ave., Cincinnati, Ohio. PuTZ, Alfred, '21 5117 Locust St., Philadelphia, Pa. Qulllian, Marvin C, A.M., '13 .• Wesleyan Col., Macon, Ga. Rankin, Walter M., '13 Princeton University, Princeton, N. J. Ransom, Brayton H., '99 U. S. Bureau of Animal Industry, Washington, D. C. Reese, Prof. Albert M., Ph.D., (Hop.) '05 W. Va. Univ. ,Morgantown, W. Va. Richards, Aute, Ph.D., '12 Dept. Zoology, Univ. of Oklahoma, Norman, Oklahoma. Riley, C. F. Curtis, M.S., '15 Univ. of Manitoba, Winnipeg, Can. Roberts, E. Willis, '11 65 Rose St., Battle Creek, Mich. Roberts, J. M., '11 460 E. Ohio St., Chicago, 111. Robinson, J. E., M.D., '15 Box 405, Temple, Texas. Roe, G. C, A.B., '17 113 R. Street, N.W., Washington, D. C. Rogers, Walter E., '11 Lawrence College, Appleton, Wis. 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Unr'ersity of Iowa Library Iowa City, Iowa. University of Kansas Library Lawrence, Kans. University of Michigan Library Ann Arbor, Mich. University of Minnesota Library .Minneapolis, Minn. University of Missouri Library Columbia, Mo. University of Montana Library Missoula, Mont. University of Nebraska Library Lincoln, Nebr. University of Oklahoma Library Norman, Okla. University of Oregon Library Eugene, Oregon. University of Pennsylvania Library Philadelphia, Pa. University of Southern California Library Los Angeles, Cal. University of Texas Library Austin, Texas. University of Toronto Library Toronto, Canada. University of Utah Library Salt Lake City, Utah. University of Virginia Library Charlottesville, Virginia. University of Washington Library Seattle, Wash. University of Wisconsin Library Madison, Wis. University of Wyoming Library Laramie, Wyo. Vassar College Library Poughkeepsie, N. Y. Washington and Lee Biological Dept. Library Lexington, Va. Washington State College Library Pullman, Wash. Western College for Women Library Oxford, Ohio. Yale College Library New Haven, Conn. I i INDEX TO VOLUME XLI Abnormal Earthworm Specimens, Helod- rilus subrubicundus and H. tenuis, 153. Absolute Ethyl Alcohol, Substitutes for, 155. Alcohol, Substitutes for Absolute Ethyl, 155. American Microscopical Society, Proceed- ings of, 57. Anatomy of Some Sexually Mature Speci- mens of Dero limosa Leidy, 159. Annual Report of the Treasurer, 110. Aquatic Lepidoptera, Respiratory- Mechan- ism in, 29. Aspidogaster conchicola, Notes on the Excretory System in, 113. B Barber Pipette, A Modified, 55. C Cestode from Liparis liparis, A New, 118. Cleaning Slides and Covers for Dark-field Work, 56. Covers for Dark-field Work, Cleaning Slides, 56. Cunningham, Bert. ]\Iodified Barber Pi- pette, 55. Custodian's Report for the Year 1921, HI. D Dark-field Work, Cleaning Slides and Covers for, 56. Dero limosa Leidy, The Anatomy of Some Se.xually Mature Specimens of, 159. Dichromatic Illumination for the IMicro- scope, 51. Douglas Lake, Michigan, Preliminary' Note on Naids of, 167. Earthworm Specimens, Helodrilus subrubi- cundus and H. tenuis. Abnormal, 153. Ethyl Alcohol, Substitutes for .\bsolute, 155. Excessive Sexual Development in Hydra oligactis with spermar>- on Tentacle, 172. Excretory System in Aspidogaster conchi- cola, Notes on, 113. Faust, E. C, Notes on the Excretory System in Aspidogaster conchicola, 113. Frogs, On the Protozoa Parasitic in, 59. Gage, S. H., Cleaning Slides and Covers for Dark-field Work, 56. Gregarines described from 1911 to 1920, A List of the New, 122. Griffin, L. E., Substitutes for Absolute Ethyl .-Vlcohol, 155. H Hausman, L. A., Dichromatic Illumination for the Microscope, 51. Hayden, H. E., Preliminary Note on Naids of Douglas Lake, Michigan, 167. Heald, F. D., Some Suggestions for Teaching Mycolog>s 175. Helodrilus subrubicundus and H. tenuis. Abnormal Earthworm Specimens, 153. Helodrilus tenuis, .Abnormal Earthworm Specimens, Helodrilus subrubicundus and, 153. Heredity, Ten Years of, 82. Hydra oligactis with Spermary on Tentacle, Excessive Sexual Development in, 172. I Illumination for the Microscope, Dichro- matic, 51. Index, 189. K Kamm, Minnie Watson, List of the New Gregarines described from 1911 to 1920, 122. Killing, Staining and Mounting Parasitic Nematodes, 103. Kudo, R., On the Protozoa Parasitic in Frogs, 59. L Lepidoptera, Respiratory Mechanism in Certain Aquatic, 29. Linton, Edwin, A New Cestode from Liparis liparis, 118. 189 190 INDEX TO VOLUME XLI Liparis liparis, A New Cestode from, 118. List of members and subscribers, 179. List of the New Gregarihes described from 1911 to 1920, .\, 122. IM Mature Specimens of Dero limosa Leidy, The Anatomy of Some Sexually, 159. May, H. G., Killing, Staining and Mounting Parasitic Nematodes, 103. Mayhew, R. L., The Anatomy of Some Sexu- ally Mature Specimens of Dero limosa Leidy, 159. Members, List of, 179. Michigan, Preliminary Note on Naids of Douglas Lake, 167. Microscope, Dichromatic Illumination, 51. Micro-slip, A New, 101. Mounting Parasitic Nematodes, Killing, Staining and, 103. Mycology, Some Suggestions for Teaching, 175. Myers, F. L., A New Micro-slip, 101. N Naids of Douglas Lake, Michigan, Prelimi- nary Note on, 167. Nematodes, Killing, Staining and Mounting Parasitic, 103. New Cestode from Liparis liparis. A, 118. New Gregarines described from 1911 to 1920, 1920, A, List, 122. New Locality for Spongilla wagneri Potts, A, 106. Notes on the Excretory System in Aspido- gaster conchicola, 113. P Parasitic in Frogs, On the Protozoa, 59. Pipette, A Modified Barber, 55. Preliminary Note on Naids of Douglas Lake, Michigan, 167. Proceedings of the .\mcrican IMicroscopical Society, 57. Protozoa Parasitic in Frogs, On the, 59. R Report for the year 1921, Custodian's, 111. Report of the Treasurer, Annual, 110. Respirator>' Mechanism in Certain .\f|ualic Lepidoptera, 29. Roberts, E. W., Some Interesting Studies on Spider Anatomy, 107. Root, F. M., A New Suctorian from Woods Hole, 77. S Schmidt, A. W., Excessive Sexual Develop- ment in Hydra oligactis with Spermary on Tentacle, 172. Sexual Development in Hydra oligactis with Spermary on Tentacle, Excessive, 172. Sexually Mature Specimens of Dero limosa Leidy, The Anatomy of Some, 159. Shull, A. Franklin, Ten Years of Heredity, 82. Slides and Covers for Dark-field Work, Cleaning, 56. Specimens of Dero limosa Leidy, The Anat- omy of Some Se.xually Mature, 159. Smith, Frank, Abnormal Earthworm Speci- mens, Helodrilus subrubicundus and H. tenuis, 153. Smith, Frank, a New Locality for Spongilla wagneri Potts, 106. Specimens, Helodrilus subrubicundus and H. tenuis. Abnormal Earthworm, 153. Spermary on Tentacle, Excessive Sexual Development in Hydra oligactis with, 172. Spider Anatomy, Some interesting Studies on, 107. Spongilla wagneri Potts, A New Locality for, 106. Staining and Mounting Parasitic Nematodes, KUlmg, 103. Studies on Spider Anatomy, Some Interest- ing, 107. Subscribers, List of, 185. Substitutes for Absolute Ethyl Alcohol, 155. Suctorian from Woods Hole, A New, 77. Suggestions for Teaching Mycology, 175. Teaching Mycology, Some Suggestions for, 175. Tentacle, Excessive Sexual Development in Hydra oligactis with Spermary on, 172. Treasurer, .Annual Report of the, 110. W Wagneri Potts, a New Locality for Spongilla, 106. Welch, P.S., Respiratory Mechanism in Certain .Aquatic Lepidoptera, 29. Woods Hole, .A New Suctorian from, 77. A, TRANSACTIONS OF THE American Miscroscopical Society Organized 1878 Incorporated 1891 PUBLISHED QUARTERLY BY THE SOCIETY EDITED BY THE SECRETARY PAUL S. WELCH ANN ARBOR, MICHIGAN VOLUME XLII Number One Entered as Second-class Matter August 13, 1918, at the Post-office at Menasha, Wisconsin, under Act of March 3, 1879. Acceptance for mailing at the special rate of postage provided for in Section 1103, of the Act of October 3, 1917, authorized Oct. 21, 1918 (ilje (Tnllrginte 'J^rtsB }L'CRGE BANTA I'UBLISHING COMPANY MENASHA, WIS. 1923 TABLE OF CONTENTS For Volume XLII, Number 1 , January, 1923 Studies on Sparganophilus eiseni Smith, with four plates, by Florence S. Hague 1 A Systematic Presentation of New Genera of Fungi, by O. A. Plunkett, P. A. Young, and Ruth W. Ryan 43 Department of Methods, Reviews, Abstracts, and Briefer Articles Hemistomum Confusum, a Homonym, by John E. Guberlet 68 The Use of Sodiumi Silicate as a [Mounting Medium, by Charles W. Crcaser and William J. Clench .' 69 New Pocket Dissecting Microscope 71 The Physiology of Reproduction, a review b)- T. W. Galloway 72 Mar>' AUard Booth, by Bessie Perrault Titus 73 Proceedings of the American IMicroscopical Society 76 American TRANSACTIONS OF Microscopical Society (Published in Quarterly Instalments) Vol. XLII JANUARY, 1923 No. 1 1 STUDIES ON SPARGANOPUILUS EISENI SMITH* By Florence S. Hague TABLE OF CONTENTS I. Introduction 1 II. Specific Characters 3 III. Material and its Preparation 3 IV. Development 4 1 . Observ^ations on the Cocoons and Living Embryos 4 2. Embrj'olog}^ 6 V. ]Morpholog3' 7 L External Characters 7 2. Pharyngeal Glands 8 3. Digestive System 8 4. Vascular System 9 5. Excretory System 9 . (a) Structure of a Nephridium 9 (b) History of the Anterior Nephridia 12 Table 1 13 Table II 15 Table III 16 6. Reproductive Sj'stem 17 (a) Time of Development 17 (b) Genital Funnels and Ducts 18 (c) Sperm Sacs 20 (d) Spermathecae 21 (e) Accessory Reproductive Glands 21 Table IV 24 VI. Systematic Relations 32 VII. Summary 34 VIII. Literature Cited 34 I. Introduction The genus Sparganophilus was established by Benham (1893) when he described the first species, 5. tamesis. He found the specimens of this species in one restricted area along the Thames River. Since they had not been reported from other places in England, and since he was unable to *Contributions from the Zoological Laboratory of the University of Ilhnois, No. 215. 1 I FLORENCE S. HAGUE find them in other places along the River, he concluded that they had been introduced there from some other part of the world. He thought it possible that they had been brought with timber or plants from North America, for America was considered the home of the most closely related forms. In 1895, H. F. Moore reported the presence of this same species in the banks of streams in the vicinity of Philadelphia. However, some of the Philadelphia worms were examined by Michaelsen (1917) and, al- though they were not adequately preserved for dissection, he found from external characters that they were not S. tamesis. In response to a request for material Doctor J. P. Moore, also of Philadelphia, wrote that he was unable to find any of the worms in the spring of 1920, in places where they had previously been found. The next species reported was 5. eiseni. The specimens were found in the Illinois River at Havana, Illinois, by Professor Frank Smith, and described by him in 1895. .5'. eiseni has since been reported from Ohio, Michigan, Indiana, and Florida. Eisen in 1896 described 5'. henhami from Mexico; S. guatemalensis and S. carneus from Guatemala and Iowa, respectively; 5. smithi and 5. sonomae both from California. The genera which, according to Michaelsen (1917), are most closely related to Spar- ganophilus are found in Central and South America. This paper deals with certain organs and with variations in specimens of S. eiseni from Florida, Iowa, Illinois, and Michigan, from which states the genus has previously been reported, and also with specimens from Louisiana and Wisconsin. Although certain points in embryology and in the anatomy of several systems are given some attention, the sub- sequent discussion is concerned chiefly with the excretory and repro- ductive systems. The structure of a nephridium of S. eiseni is compared with that of the nephridia of other genera which have been studied. Although the development and subsequent loss of the nephridia of the anterior somites has been known for some time among limicoline Oligo- chaeta, a similar process has been reported in an earthworm only once. The detailed developmental history of the nephridia of the anterior somites of an earthworm is here presented for the first time. It has been found that they develop and then disintegrate while the worms are still quite young. The disintegration of the nephridia in the somites which contain the genital ducts throws additional light on the relation of genital ducts and nephridia. While accessory reproductive glands have been reported in numerous species of earthworms and their structure described in some of these species, the published accounts have been based on com- paratively few specimens, and have not dealt with their development. The data in the present paper have been taken from over 150 specimens which were difTerent in age and which were collected at various seasons of the year. STUDIES ON SPARGANOPHILUS EISENI SMITH 6 The writer is indebted to Doctor C. P. Alexander of the State Labora- tory of Natural History for the material collected at Havana, Illinois in 1920. Material collected in earlier years at Havana, and at Douglas Lake, Michigan, as well as that from various other places, is in the collection of Professor Frank Smith. The writer wishes to express her appreciation for the use of this material and for other material which he has recently collected. She is further indebted to Professor Smith for his suggestions and interest during the progress of this study. II. Specific Characters Sparganophilus eiseni is a rather slender earthworm, which varies in length from 80 mm. to 200 mm. Setae c and d are in the dorsal half of the worm. The clitellum extends approximately from somites 15-25; the tubercula pubertatis, from 17-22. The spermiducal pores are on somite 19, and the oviducal pores on 14, but both are inconspicuous; the sperma- thecal pores are just ventrad of the seta line c, in the intersegmental grooves 6/7, 7/8 and 8/9. There is no trace of gizzard or of calciferous glands in the digestive tract. Moniliform hearts are present in somites 7-11. The spermathecae are in somites 7, 8 and 9; the ovaries, in 13; the ovisacs, in 14; the spermaries and spermiducal funnels, in 10 and 11. The sperm ducts pass through the longitudinal muscular layer of the body wall in somites 11 and 12, and, from there to the pores, lie in the circular muscular layer, or between it and the epidermis. The lobed sperm sacs are paired and are in somites 11 and 12. The first typical nephridia are in somite 13 or 15. Accessory reproductive glands may be present in some one or more of somites 3 to 10, and are regularly present in 23-26 or adja- cent somites. Both worms and cocoons are figured by Smith (1915). III. Material and Its Preparation Material for this study was collected between July 1919 and February 1921, in the region of Douglas Lake, Michigan, and at Homer Park and Havana, Illinois. The collecting in the former locality was done while the writer was in attendance at the University of Michigan Biological Station, which is on the shore of Douglas Lake. Although search was made for Sparganophilus at many points along the shores of Douglas Lake and of its tributaries, specimens were found only in six places. These places are near, and perhaps have been a part of the shore line at some past time, or are now connected with the lake shore. They are supplied with decaying organic matter but are separated from each other by stretches of shore, parts of which are bare sand. Two mature worms were collected in one spot on the northwest shore of the Lake, but since they were the only ones in the vicinity, they may have been carried there from some other place. Large numbers of worms were found in Diogenes Pond, at Hook Point, in 4 FLORENCE S. HAGUE Sedge Pond, in the banks of Bessey Creek, and in the banks of Maple River. Maple River flows out from Douglas Lake, and, after a long and tortuous course, empties into Burt Lake which at the nearest point is less than two miles from Douglas Lake. One specimen was brought in with some plants collected by Doctor F. C. Gates at the mouth of this River (Burt Lake). At the entrance of Carp Creek into Burt Lake, specimens of Sparganophilus were abundant. Two unidentified lumbricids' were also found here. This was the only place at which the writer collected another species of earthworm with Sparganophilus eiseni. At Homer Park, Illi- nois, specimens of S. eiseni have been collected in May, June, August, September, October, December and February. They live along the bank and in the bed of a small stream, Salt Fork, sometimes among gravel and small stones, sometimes in soft, mucky soil. In all these places in which worms were collected, decaying organic matter was present. They were never more than 12 to 18 inches from the edge of the water, and as the water gradually receded in Sedge Pond, the fresh castings were always close to its edge. The worms were usually found at a depth of one to four inches. Frequently both worms and cocoons were found among the roots of grasses growing in or near the water. The first step in preparing the worms for study was to clean out the digestive tract. This was accomplished by keeping the worms in a vessel with wet filter paper or cloth for 36 to 48 hours. They were then anes- thetized with chloretone solution, straightened out between sticks and killed. A solution of alcohol and formalin; a saturated solution of corro- sive sublimate; and a solution of corrosive sublimate and acetic acid were used as killing fluids. The latter was used exclusively for the smaller embryos. Sections were stained with Ehrlich's haematoxylin, and a few were counterstained with eosin or orange G. IV. Development (1) Observations on the Cocoons and Living Embryos The cocoons of S. eiseni are elongated, but not all of them are as slender and long as those figured by Smith (1915, figs. 8 and 9). The formation of cocoons evidently occurs chiefly during July and August, and to a limited extent during the last half of June and the first two weeks of September. When first formed the cocoons are soft, colorless and transparent or semi-transparent. They are enclosed in a slime tube which may be two or three times as long as the cocoon. The cocoons gradually assume a straw color, become less flexible, and lose the slime tube, probably within six hours. They usually remain suflficicntly transparent so that the eggs within them are visible. The cocoons which were formed after the worms had been in the laboratory (in dishes with wet filter paper) for three or STUDIES ON SPARGANOPHILUS EISENI SMITH 5 four days were misshapen and opaque in comparison with those which were formed during the first day or two in the laboratory, or those which were found in their natural environment. Although it is not uncommon to find eight or ten eggs in a cocoon, the number of fairly well developed embryos is usually one to four. Seven such embryos have been found in one cocoon, and five or six in each of several cocoons. From a large number of cocoons, perhaps 25% of those which were brought into the laboratory and were apparently normal, worms did not emerge. Of those cocoons from which worms failed to hatch, some remained perfectly transparent, and some became more or less opaque. Many of the latter, when opened, contained disintegrated embryos. In some of both transparent and opaque cocoons, but chiefly the latter, ciliates were found. Some of these cocoons were not tightly sealed, for a slight pressure caused them to emit their contents. Ciliates from the surrounding water might have invaded such cocoons. However, other cocoons were so tightly sealed that they had to be cut open, and the ciliates were seen in the albumen as it was forced out of the cocoon. In a few instances rhabdocoels, rotifers and nematodes were apparently in the cocoons. Beddard (1892) reported finding nematodes in the cocoons of Octockaetus (originally Acanthodrilus) multiporus. From two cocoons which were brought into the laboratory when the eggs were in early cleavage or the blastula stage, worms hatched out in 24 and 26 days, respectively. Other cocoons were kept for 2>3 days before the worms emerged. Empty cocoons are open only at one end. Some of the worms, at hatching, are twice the size of others. Such a difi^erence in size is found frequently, when there are two to six embryos in one cocoon. Again one cocoon may contain eggs in cleavage stages and also well formed gastrulae. It scarcely seems that the difference in the time of fertilization would be great enough to cause such a difference in size. Possibly there are periods of cessation of development, or variations in the rate of metab- olism. Four bifid embryos have been taken from cocoons. Each has a single anterior part and two posterior ends. Two of these embryos were the complete contents of one cocoon and were removed before they had attained the degree of development which usually occurs within the cocoon. The third was noticed in a cocoon which had been in the laboratory 34 days. It was moving actively, but, although the opening at the end of the cocoon was enlarged, it did not escape. After eight more days the posterior ends began to disintegrate and it was removed and fixed. A study of these embryos shows a type of dorsal union. A single anterior digestive tract bifurcates into the two digestive tracts of the posterior ends. On opposite sides of the single digestive tract and continuing directly posteriad into each branch, is a nerve cord with four pairs of setae in the 6 FLORENCE S. HAGUE normal relative position. The anterior portions contain from 7 to 20 or 25 somites, and equal from one-seventh to three-fifths of the total lengths. One bifurcated worm which had probably been out of the cocoon only a few days was found in debris which had been brought into the laboratory. An unsuccessful attempt to keep it alive resulted in the disintegration and loss of the bifurcated portion. Figure 1 shows the manner of bifurcation, which was posterior and which extended through about one-fifth of the total length. The branch was somewhat shorter than the end which was in line with the main axis, but, like it, had an anus, a growing region, a ventral vessel and a pulsating dorsal vessel. (2) Embryology The study of the embryology of Sparganophilus eiseni is incomplete, but because of the difiference of opinion in regard to several points in the embryology of earthworms, it seems best to mention the more important facts noted. Wilson described the following parts in the germ bands of Lumbricus: a thin outer layer of ectoderm; a middle layer formed from four pairs of large cells which are known as teloblasts and which are ecto- dermal in origin; and an inner layer of mesoderm formed from large cells which are known as mesoblasts. Two large mesoblasts with the meso- dermal bands extending forward from them are present in embryos of Sparganophilus eiseni. The teloblasts are somewhat anterior to the mesoblasts and are less readily identified. In sections of 0.8 mm. and 1 mm. embryos they form a part of the surface ectoderm, but in 2 mm. embryos they are more or less sunken beneath the ectoderm. Continuous with and anterior to each teloblast is a row of cells to which it has given rise. Because of the structures which develop from these rows the median or first pair of rows are known as the neural rows, and the second as the nephridial. Staff attributed the formation of the circu- lar muscular layer of the body wall to the third and fourth rows. Earlier investigators (Wilson, Bourne 1894a) had not reached the same conclusion. In longitudinal sections of the 0.8 mm. embryo of 5. eiseni, the neural rows are covered by a thin cellular layer and can be traced to the sides of the mouth, but the second and third rows are at the surface and can be traced forward through only a comparatively few sections. Even in sections of a 2 mm. embryo the third and fourth rows are lost at times. In whole mounts of 2 mm. to 3 mm. embryos the four pairs of rows show definitely. The first pair are closely approximated in the midventral line; the second row shows the heavy masses or coils of the early nephridia; the third row is a straight line in which no structural differentiation shows; and the fourth row is a series of oval bodies, one in each segment. The development was not followed further, but the difference in the appearance of the third STUDIES ON SPARGANOPHILUS EISENI SMITH 7 and fourth rows would scarcely indicate that they form the same adult structure, namely, circular muscles. V. Morphology (1) External Characters There is a wide variation in the size of the mature specimens of S. eiseni collected. The largest are over 200 mm. in length and 2.6 mm. in diameter, and the smallest are only 80 mm. to 100 mm. in length and about 1 mm. in diameter. The former are from certain places in the vicinity of Douglas Lake, Michigan, and the latter from Havana, Illinois. In these places there are also worms of intermediate size. No regular difference other than size has been noted among these worms. The clitellum begins dorsally on somite 15 but may not include all of that somite. The posterior end of the clitellum is somewhat variable. There is usually a gradual decrease in thickness, beginning on 24 and sometimes extending onto 26. Ventrally the clitellum is thin, and scarcely distinguishable except in sections. This ventral part of the clitellum extends from 14-26 or 27, and is usually somewhat thicker in the "region of 22 and 26. On worms, which were killed in the manner described, the clitellar region is definitely enlarged; but in some specimens which were apparently put directly into formalin or alcohol, it is no greater in diameter than the somites adjacent to it. It is, however, distinct because of a slightly darker color and because of the absence of intersegmental grooves. The tubercula pubertatis seem to be typically on somites 17-22, but may extend onto 16 and 23. A specimen from Wisconsin has the tubercula pubertatis on 18-22, and two from Homer Park, Illinois, in which the clitella and tubercula pubertatis are developing, have the latter on somites 18-22. Ventrally the clitellar somites are flattened and the tubercula puber- tatis form longitudinal ridges along the lateral edges of part of the flattened area. The flattened area usually extends posteriad onto 26 or 27, which extent is one or two somites posteriad of the dorsal clitellar thickening. Sometimes a pair of narrow ridges extend posteriad from the tubercula pubertatis, along the lateral edge of this flattened area and border its rounded posterior end. The posterior end is always rounded and the whole flattened area definitely outlined, when the clitellum is well devel- oped, even if the ridges are not present. Narrow ridges also extend anteriad from the tubercula pubertatis in some specimens. The tubercula pubertatis do infringe on the width of the flattened area between them, but in neither the complete ventral flattening, nor the anterior part of it, has the writer noted in any specimen the hour-glass shape which was mentioned by Eisen (1896) as occurring in S. eiseni. 8 FLORENCE S. HAGUE (2) Pharyngeal glands The term, pharyngeal glands, is used in this discussion for those masses of deeply staining cells, which are associated with the muscles extending from the pharynx to the body wall of 5. eiseni. In somites 5, 6 and 7 the cells are aggregated in masses which are attached to the large pharyn- geal muscles extending through those somites. The septum 3/4 is incom- plete and the cells of this region are scattered singly and in small groups between the muscles, near their attachment to the pharynx. Eisen (1896) called the latter salivary or suprapharyngeal glands, and the former septal or intestinal glands. He described ducts, which open into the pharynx, from both kinds of glands. The writer has not found such ducts in S. eiseni. Stephenson (1917) was unable to find any ducts from similar glands in several species of Pheretima and of Helodrilus. He concluded, f ro;n his studies, that both pharyngeal and septal glands were of peritoneal origin, and were not related to the pharynx in the manner originally supposed. The name, chromophil cells, was suggested by him for these structures. It seems to the writer that it would be best to retain the name, pharyngeal, until the origin of these cells from some source other than the pharyngeal wall is definitely established. The term gland is used for convenience. Eisen stated that the glands of somite 6 were as large as those of 5 in some species, but smaller in others. Table IV (p. 24) shows that the relative size of the glands of 6 is variable, and that glands are sometimes present in somites 7 and 8. Except in the immature specimens, the glands of 6 are usually somewhat smaller than those of 5, in worms which were collected in Michigan. In some of the specimens collected at Homer Park, Illinois, the glands in 6 are as large as those in 5, and in others, they are smaller. The writer could find no fixed relation between the presence of the anterior accessory reproductive glands and the size of the pharyngeal glands of 6. It has been suggested that such pharyngeal glands are homologues of the nephridia in certain worms in which the latter are absent in the anterior somites. The subsequent discussion of the development and disintegration of the anterior nephridia (p. 12) and the theory (Hesse) of the origin of the pharyngeal glands from (he pharyngeal wall would both lend to contradict the suggestion. (3) Digestive System The digestive tract has neither gizzard, esophageal glands, nor typhlo- sole. A dorsal sac opens into the pharynx and the inner surface of the esophagus usually has numerous irregular, but cliiclly transverse folds in somites 4 and 5. In the succeeding somites it is folded longitudinally. In 9 or 10 and one or more succeeding somites the diameter of the digestive STUDIES ON SPARGANOPHILUS EISENI SMITH 9 canal is frequently greater, and the thickness of the walls is less than in the somites immediately anteriad or posteriad. These facts indicate that the enlargement is a temporary expansion. Since the perienteric blood sinus begins in somite 9, the intestine may be said to begin in 9, although no other structural difference has been found by which to distinguish esophagus and intestine. (4) Vascular System The main vessels of the vascular system of S. tamesis and S. eiseni are similar. They have been described by Benham (1893) and Smith (1895), respectively. Eisen (1896) added the description of the blood glands, but he was in error in stating that the hearts are in somites 8 to 11 in 6*. eiseni. In all specimens studied, including one identified by Eisen as .S". henhami, the hearts are in somites 7-11. The hearts decrease in size from posterior to anterior, but all five pairs can be seen pulsating in living worms. The hearts do not contract simultaneously, but quickly and in rapid succession beginning with the most posterior one. It is evident that the condition of the hearts at the time when the worms were killed (whether they had just contracted, or were fully expanded) would make a difference in their size. Among the worms studied, the hearts are all moniliform, but in some they are more contorted and of relatively greater diameter. (5) Excretory System (a) Structure of a Nephridium The excretory organs of S. eiseni are paired meganephridia which open to the exterior through pores placed anteriad of the ventral setae. These nephridia are large, compact organs, consisting of a lobed mass of coelomic epithelium in which the nephric tubule is embedded. Similar nephridia have been described in other species of Sparganophilus and in other genera. They present an appearance quite different from that of the nephridia of Lumbricus (Benham 1891), of M aoridrihis rosae (Camer- on) or of the widely distributed Helodrilus caliginosus trapezoides. In the latter a nephridium does not appear as a mass of tissue but as a series of loops or a group of convoluted tubules. The tubule, however, is the essential part in both types of nephridia. The nephridia of Sparganophilus eiseni are so compact that tracing the complete course of the tubule in the entire nephridium is impracti- cable, if not impossible. Tracing the tubule in sectioned nephridia is equally difhcult. Of several nephridia which were dissected out, stained in Delafield's haematoxylin and cleared in glycerine, one was sufficiently spread out to trace the greater part of the course of the tubule. The nephridium was then sectioned, approximately in the plane outlined. 10 FLORENCE S. HAGUE The single line in figure 2 represents the general course of the tubule as it was worked out from the study of the whole mount and the sections. The septum, nephrostome and duct connecting the latter with the nephrid- ial mass were torn off, and are consequently shown by broken lines. The break (br) was caused by displacing the lobe from its position over the body of the nephridium. Aside from these interruptions the diagram shows an unbranched and continuous tubule from the nephrostome to the nephridiopore. Most of it is in two long and somewhat convoluted loops. Each nephridium, as is generally true, consists of a pre-septal and a post-septal part. The former includes a nephrostome or funnel and a short duct. The funnel is really a broad tube with two extensions, a large lip and, opposite it, a very small lip. In the large lip the large marginal cells and, outside of them, a row of extra-marginal cells are distinctly visible from the front or inner side of the lip. When this lip is sectioned, there is a noticeable thickening of the edge produced by one or two addi- tional rows of extra-marginal cells, which curve over the edge onto the outside of the lip. The smaller lip is merely a slight extension of the cells of the tube and presents no special structures. Flattened coelomic epi- thelium covers the outside of the entire funnel and continues over the duct. The broad tube which forms the body of the funnel gradually narrows into the duct which connects the nephrostome with the nephridial mass. The cell walls are not visible, but judging by the number and position of the nuclei, the tubular portion of the funnel is made up of two series of cells. Just anterior to the septum the number of nuclei indicates that a single row of perforated cells forms the duct. There is probably a gradual change from the intercellular to the intracellular condition, as the funnel narrows into the duct. There is nothing to indicate that the post-septal part of the nephridium is not intracellular throughout its length. It may be distinguished, accord- ing to its structure, into three parts which are indicated in figure 2. They are the narrow tube (nar) which forms the first loop; the middle tube (mid) which extends to the apex of the second loop; and the wide tube (wi) which extends from the apex of the second loop to the nephridiopore (ne). The narrow tube is slender and thin-wallcd. Parts of it are ciliated. The middle tube has a thicker wall and is ciliated throughout its length. The first part of this tube is coarsely granular. Somewhat beyond the middle the coarse granules gradually disappear and scattered groups of fine granules appear. The fine granules increase in number and become aggregated so that in the last of the middle tube they form a layer near the middle of the wall of the tube (fig. 9). The granules are arranged rather regularly in closely packed radial rows, with occasional strings of granules extending out into the peripheral portion of the wall. In some ncphridia STUDIES ON SPARGANOPHILUS EISENI SMITH 11 these strings are sufficiently numerous and branched to form a network. This layer of granules stains differently from other parts of the nephridium and is distinct in all nephridia. A constriction separates the middle and wide tubes (fig. 9). The wide tube is without cilia in all parts but the structure of the wall is not the same throughout. A short portion, the ampullar region, at the begin- ning of the wide tube is made up of rather large deeply stained and defi- nitely outlined cells. Both cross and longitudinal sections of this portion show very distinctly the intracellular nature of the nephric tubule. The first third or half of this region is an enlargement, the ampulla (am), which contains deeply staining masses in its lumen. The sectioned walls show a darker inner and a lighter peripheral cytoplasm. In the peripheral cytoplasm there is a network of granules which is similar to that in the peripheral cytoplasm of the middle tube, and which seems to condense into the darker inner cytoplasm. The inner cytoplasm differs from the layer in the middle tube in that it lacks the radial striations and stains less intensely. The distinction between inner and peripheral cytoplasm gradually disappears in the large cells beyond the ampulla. These large cells abruptly give place to a thin-walled tube in which the cell boundaries are indistinguishable. The last part of the wide tube (fig. 5) begins with cells similar to those of the ampullar region, but in its distal part the wall becomes thinner and the cell boundaries gradually disappear. Figure 7 represents the distal part of the wide tube, including that which passes through the body wall to the epidermal^ invagination, in a nephridium which had been dissected out and sectioned. The number of nuclei in this and adjacent sections of the tube indicates that it is intracellular. Figure 8 shows a section of the complete epidermal invagination of the same nephridium. Since adjacent sections show no cellular structure other than that which is shown in these two figures, it is evident that the wide tube opens directly into the nephridiopore, and that there is no muscular duct. Serial sections of nephridia in situ substantiate this conclusion. The shape of the nephridium and position of the different parts of the tubule within it are variable. However, the two long loops are regu- larly approximately parallel, and for the greater part of their length, are embedded in large lightly staining cells (fig. 6). These large cells are usually found about the periphery rather than in the central part of the nephridium, and they have not been noted except in relation to the nephric tubule. These facts suggest that the large cells may have a direct relation to the process of excretion. ^ The outer cellular layer covering the earthworm has been called epidermis, and also hypodermis. Neither name is satisfactory, but because of its moie general usage the former is used here. 12 FLORENCE S. HAGUE The smaller, evenly granular cells, which form the nephridial mass, are probably epithelial. The septa are covered by delicately stained projections, some large, some small and closely crowded; some with nuclei, others without nuclei. These projections are parts of vesicular epithelial cells. Similar projections, without nuclei, cover the surface of the nephrid- ium. These projections are frequently found to be parts of the evenly granular cells of the nephridial mass. Evidently, then, these smaller cells in the nephridial mass are epithelial cells, but only those at the surface are vesicular. The structure of the nephridial tubule of Lumbricus has been de- scribed by Gegenbauer, Benham (1891) and Maziarski (1905). The last paper is available only in the form of a review by Meisenheimer. K. C. Schneider studied the nephridium of Eisenia rosea, one of the Lumbricidae, and Cameron, the nephridium of Maoridrihis rosae, one of the Megas- colecidae. The nephridium of each of these has three parts similar to those of SparganophUus eiseni, and in addition a muscular duct which is located between the wide tube and the pore. Intermittent ciliation of the narrow tube, complete ciliation of the middle tube and lack of ciliation in the wide tube are characters common to all. Histologically there are differences in the structure of corresponding parts of the tubule. None, except S. eiseni, have the more deeply staining layer in the wall of the middle tube. The figure by Maziarski (Meisenheimer) shows some vari- ation in the wide tube, but it is not the same as that found in S. eiseni. The nephridia of Moniligastcr grandis (Bourne 1894b), and of Perieodrilus ricardi and P. montanus (Benham and Cameron) are distinctly different in structure from those of the above mentioned earthworms. From the foregoing discussion it appears that although there is a general agreement between the nephric tubule of SparganophUus eiseni and that of Lumbricus, from the nephrostome to the muscular duct, the former has a higher degree of specialization in the middle and wide tubes. (b) History oj the A nterior Nephridia Sparganophilus is one of several genera of earthworms which resemble certain limicoline forms in lacking nephridia in the anterior somites. Smith (1895) and Eisen (1896) both listed somite 13 as the first one contain- ing nephridia in S. eiseni. In other species the first nephridia have been recorded in 12, 13 and 16. However, a study of embryos shows that nephri- dia are present in some of the somites anterior to 13 (Table I). The first distinct ne])hridia are in somite 3 of seven embryos; in somite 4 of five em- bryos; in somite 6 of four embryos and in somite 12 of two young worms. Unfortunately no first nephridia have been found in somites 9, 10 and 11, and only one in each of somites 7 and 8. In addition to the fact that, generally in the larger embryos, the nci)hridia arc absent in the more STUDIES ON SPARGANOPIIILTJS EISKNI SMITH 13 anterior somites, there are signs of disintegration of the anterior nephridia. The first nephridia are called distinct rather than well developed or typi- cal because, while there can be no doubt that they are nephridia, the canal in some is indistinct if visible at all. This must be due to disintegration for the canal develops very early as is shown by its presence in figure 11, which is typical of the nephridia of three successive somites, probably 3, 4 and 5, of a 0.5mm. embryo. Figures 10 and 12, from sections through nephridia of somite 3 of embryo No. 2 and of somite 5 of embryo No. 7, respectively, show very distinct canals; but in figure 13 from a section through one of the nephridia of somite 3 of embryo No. 7, only one indis- tinct section of the canal is recognizable. The canal shows more definitely in this section than in any other sections of the nephridia of this somite. Table I First Nephridia in Embryos and Very Young Worms Nos. 20 and 21 are the same as 99a and 100a of Table IT, respectively. Kiiiiibcr Length Somite Number Length Somite 1 1 mm. 3 12 15 mm. 4 2 1 .5 mm. 3 13 8 mm. 5 3 2 mm. 3 14 5-6 mm. 6 4 3-4 mm. 3 15 6 5 4-5 mm. 3 16 7-8 mm. 6 6 3 17 10 mm. 6 7 7 mm. 3 18 7 8 4 mm. 4 19 8 mm. 8 9 4.5 mm. 4 20 17 mm. worm 12 10 5 mm. 4 21 15 mm. worm 12 11 6 mm. 4 Other first or first and second nephridia show similar conditions and in somites anterior to the first nephridia there are frequently small masses of tissue which are located and stained similarly to nephridia. These are evidently rudiments of nephridia. They are in somite 3 of Nos. 9, 10, 11 and 12, and in one to three somites anterior to the first nephridia of Nos. 13 to 20. In somite 2 of several embryos there are small masses of tissue which may be nephridial rudiments, but no nephridia, as distinct as the smallest of those of somite 3, have been definitely located in somite 2. All these facts indicate that the nephridia develop in the anterior somites and then disintegrate in an antero-posterior order. If the number of somites rather than the length of the embryo had been used as a measure of relative development, there might be less variation among those worms which have the first nephridia in 3, 4 or 6. Part of the variation in size is doubtless due to the degree of contraction or relaxation of the embryos. However, such 14 FLORENCE S. HAGUE a marked divergence as is shown by Nos. 7, 12 and 19 seems to indicate variations in the rate of disintegration of the nephridia. Possibly such variations are correlated with differences in the rate of metabolism, which differences, as was suggested above, may be related to the distinct differ- ences in size frequently found among embryos in the same cocoon. The development of the nephridia takes place in an antero-posterior direction. Each nephridium is consequently visibly farther advanced than those from eight to ten somites posteriad of it. But, since the rate of devel- opment is more rapid than that of disintegration, the nephridia of somites 10-12 reach a more advanced stage of development than do those of somites 3-6. In No. 14 the nephridia of somite 7 have more convolutions and a canal of proportionately greater diameter than do the nephridia of somite 3. In somites 7 and 8 of No. 18, large cells, similar to those in which part of the nephric tubule of the adult is embedded, can be recognized. In No. 20 the epithelial masses have developed about the nephridia of 12. The development of the nephrostome has not been followed, but there are funnels in 9 and some of the succeeding somites of No. 19, and in several somites of No. 18. The nephridiopores are readily recognizable in somite 8 of No. 19 and in somites 7-10 of No. 18, but not in other worms in which the nephridia extend as far forward as somites 7 and 8. In specimens in which typical nephridia are present in 12 the pores are distinct in that somite. A study of mature and immature worms discloses a variation in the position of the first typical nephridia. Table II shows the condition of the nephridia in those worms in which they were studied. Table III is a summary of Table II. A typical nephridium is one, such as has been de- scribed, with its tubule embedded in a mass of cells, and having a definite nephrostome and nephridiopore. The reduced nephridia vary from a small mass of epithehal cells without a tubule to a mass about the size of a normal nephridium, but with reduced tubule and without a nephrostome or a nephridiopore. The typical and reduced nephridia, especially of 13, grade into each other so that it is sometimes difficult to distinguish them. This is because the disintegration of the nephrostomes is variable, and the nephridiopores may persist and retain an indistinct connection with a nephridium in which the tubule is reduced. Table III indicates that reduced and typical nephridia are present in increasing numbers in somites 11, 12, 13 and 15, and that all nephridia in 15 are typical. All nephridia in somites posteriad of 15 are also typical. The increasing number of nephridia in somites 11, 12, 13 and 15 is doubtless due to the antero-posterior order of disintegration. The dif- ferent conditions of the nephridia of 12, of 13 or of 14 in different individ- uals of S. eiseni are probably due to individual variations. It is possible that the nephridia of these somites are reduced increasingly wilh age or successive breeding seasons; Init, since no method has been found for STUDIES ON SPARGANOPHILUS EISENI SMITH 15 distinguishing worms of different ages, there is no evidence for such a progressive reduction. On the other hand, individual variations have been noted in the nephridial condition of embryos. Specimen No. 101a Table II Nephrtdia of Young and Adult Worms I indicates a typical organ: s, a reduced organ: o and a blank, absence of the organ. Two symbols are used when the nephridia of the two sides are dissimilar. Identical specimens have the same number as in Table IV. No. of Size of speci- worm or men clitellum 15 I 22 l 31 i 31a I 32, 0 34 t 35 i 36 t 37 t 38 t 40 0 41 s 42 s 43 0 44 0 45 s 46 s 52 s 53 0 54 0 55 s 57 s 58 0 59 0 60 0 83 s 84 i 85 t 99 45 mm. 99a 17 mm. 100a 15 mm. Nephridia in somites 11 12 so s 13 14 15 No. of speci- men Size of worm or clitellum Nephridia in somites 12 13 14 100 35 mm. t 101 35 mm. so 101a 17 mm. to 102 immature s 103 0 to 0 104 s Is 0 105 s 0 106 0 107 s 108 so 109 so 110 to 0 111 0 112 0 113 0 114 0 s s 121 0 s s 122 0 so s 123 s s 128 0 129 so 0 138 0 139 s 140 s 0 141 s s 142 so 0 143 0 0 144 0 0 145 t 0 152 s s 155 t 0 shows a marked difference from others of its size. Adult specimens from different localities show greater or lesser tendencies toward the dis- appearance of nephridia in certain somites. 16 FLORENCE S. HAGUE Other worms in which the most anterior nephridia are in some one of somites 10 to 16 are Sparganophilus tamesis, Criodrilus lacuum, AUuroides pordageijGlflssoscolex giganteus (originally Titanus hrasiliensis), Haplotaxis heterogyne and several species of Pontodrilus. The embryonic condi- tion of the anterior nephridia of these is not known. Vejdovsky (1888- 1892) described the development and subsequent disintegration, during embryonic life, of the nephridia of 1-6 of Rhynchelmis, and mentioned a similar process in Chaetogaster, Aeolosoma and Nais. Bourne (1894a) found that the nephridia of Diporochaeta (originally Perichaeta) pellucida attained a well developed condition in somites 2-6 and then degenerated, while those of 7-11 became complex. All of the worms mentioned, which lack nephridia in ten or more anterior somites, live in or at the edge of the water. Bage and Stephenson (1915) found that, in certain Megascolecidae, micronephridia are present in all somites of the body, and that in the pos- terior somites, sometimes as far forward as 12, there are also meganephridia. Table III Summary of Table II All with nephridia Is or to are counted with /. All with nephridia so are counted with s. 0 Nephridia s t Somite 11 Mature 55 5 37 1 2 0 ly 0 0 0 0 2 18 3 8 0 36 0 0 0 0 Immature 0 Somite 1 2 INIature 0 Immature 3 Somite 13 Mature 45 Immature 7 Somite 14 Mat ure d Immature 7 Somite 15 Alature 55 Immature • 7 Still other species of earthworms have a thick ccelomic covering on the posterior but not on the anterior nephridia, or the muscular duct may be more highly developed in one part of the body than in another. In a few species the nephridia arc larger in a few anterior somites than in the poste- rior ones. Such modifications are similar to the absence of nephridia in the anterior somites. 1 STUDIES ON SPARGANOPHILUS EISENI SMITH 17 It has been thought that the absence of anterior nephridia in earth- worms is correlated in some way with the reproductive system. However, disintegration of the nephridia of Sparganophilus eiseni begins before there is any trace of the reproductive organs. The gonads, which are the first parts of the reproductive system to develop (p. 17), are not recognizable until the nephridia of somite 8 are beginning to disintegrate (No. 19). Furthermore, if the nephridia disintegrated as the reproductive organs developed, they would disappear not in an antero-posterior sequence, but first in somites 10, 11, 13; then in 11, 12, 14; lastly in 7, 8, 9; and probably not at all in the somites anteriad of 7, since there are no reproductive organs in those somites. The probable influence of the genital ducts on the nephridia of 11, 12 and 14 will receive attention in the discussion of the genital ducts. The time and order of the disintegration of the nephridia of 3-10 indicate that this process is not directly related to the development of the reproductive organs. The adaptation to the aquatic habitat or some other factor may have produced a physiological condition which is different from that in most earthworms and which has resulted in the loss of the anterior nephridia. (6) Reproductive System (a) Time of Development The development of the reproductive organs begins about the time that the worms emerge from the cocoons, and is not closely related to the size of the worms. The gonads appear first; then the spermiducal funnels, sperm ducts and sperm sacs develop in quick succession. The oviducal funnels, oviducts and ovisacs develop later, only a short time before the accessory glands and spermathecae which develop as the worm approaches adult size. Finally, with the approach of the breeding season, the clitellum appears. Specimen No. 19 of Table I is the youngest specimen in which the gonads can be identified, and in that only on one side. Nos. 20 and 21 are the same as Nos. 99a and 100a of Table II, respectively. These two and the other immature worms, Nos. 99 to 102, inclusive, (Table II) have distinct spermaries and ovaries. Nos. 99, 101a, 101 and 102 have anlagen of the spermiducal funnels, and the latter three, of the sperm sacs and sperm ducts also. Tiny anlagen probably of the oviducal funnels, are present in Nos. 101 and 102, and definite anlagen of these funnels are present in a 55mm. worm which is not included in the tables. In Nos. 121 and 122, which have reached adult size but probably not sexual maturity, the oviducal funnels are still small and the anlagen of the oviducts are recognizable. Accessory reproductive glands and spermathecae are not developing in any of these specimens except Nos. 121 and 122. 18 FLORENCE S. HAGUE Bergh (1886) found that the spermaries and ovaries were the only parts of the reproductive system of Lumbricus, which developed during embryonic life. In Octochaetus (originally Acanthodrilus) nmltiporus, Beddard (1892) found, in addition to the gonads, prominent genital funnels in somites 10-13 of embryos that were not yet ready to em.erge from the cocoon. (b) Genital Funnels and Ducts The anlagen of the genital funnels first appear as deeply staining areas on the anterior faces of septa 10/11, 11/12 and 13/14, just laterad of the nerve cord and near the point at which the nephridial tube penetrates the septum. This area thickens into a mass with deep indentations, from which a deeply staining strand of tissue can be traced through the septum, and toward or into the body wall of the following somite at the point where the nephridium (if present) penetrates to the exterior. This strand is the anlage of the genital duct. It enlarges and as sexual maturity approaches, acquires a lumen. The oviduct opens directly to the exterior in 14, but the sperm ducts pass into the body wall in 11 and 12 and then turn posteriad; those of each side unite and extend, in the body wall, to the spermi- ducal pores on somite 19. An early stage in the development of the longi- tudinal ducts has been found in a worm in which the genital funnels are not of maximum size, and the spermathecae and accessory glands are very small. In longitudinal sections the duct appears as two narrow parallel bands in the longitudinal muscular layer. In each band are many nuclei, while numerous similar nuclei are located close to the duct and scattered through the longitudinal muscular layer. Since the normal position of the longitudinal part of the sperm duct in the adult is between the epidermis and the circular muscular layer or in the latter, there must be a shifting of this duct as it develops. No difference has been noted in the development of the spermiducal and oviducal funnels or in the ducts from them to the body wall. The mature spermiducal funnel is much larger than the oviducal funnel, but sections show that the difference is one of size rather than structure. Be- tween periods of breeding activity the funnels revert to small solid masses and the ducts to soHd strands of tissue. Although the nephridia degenerate more or less completely before the genital funnels develop, there is a limited amount of evidence in regard to the morphological relations of excretory and reproductive systems. The genital funnel anlagen first appear as parts of the epithelium of the septa, while the nephridial funnels, at all stages seen, arc separated from the septa by the short prc-septal portions of the ducts. In Nos. 101 and 102, the nephridial funnels are still typical in somite 13, and there are also small deeply stained areas which are pr()bal)ly the anlagen of the oviducal fun- STUDIES ON SPARGANOPHILUS EISENI SMITH 19 nels. On one side of somite 13 of No. 122 there is a small mass of tissue, which has the shape of a nephridial funnel, and which is in line with the nephridial funnels of 12 and 14. It is probably the remnant of the nephrid- ial funnel, and laterad of it is the very definite anlage of the oviducal funnel. From the latter, the anlage of the oviduct can be traced through the septum and toward the body wall. These facts indicate that the geni- tal funnels are not derived from the nephridial funnels. The development of the genital ducts from the genital funnels has been described above. The oviduct passes into the body wall close beside the nephridium and opens to the exterior through an epidermal invagination, which, if one may judge by its position, is the nephridiopore. The sperm ducts likewise pass into the body wall close beside the nephridia, but they open to the exterior on somite 19, and entirely independent of the nephridia. Vejdovsky (1884) described the anlagen of the genital funnels as thickenings of the septa in Naididae, Enchytraeidae, Tubificidae and Lumbriculidae. Bergh (1886) found the same type of development in Lumbricidae. In Tubifex riviilorum, Gatenby found a difference in the development of spermiducal and oviducal funnels, but both developed from coelomic epithelium. All these investigators found that the genital ducts developed as outgrowths from the genital funnels. Stole (Beddard 1892) reported that in Aeolosoma the spermatozoa passed out by way of the nephridia, especially those of somite 6, and that during the sexual period the nephridia of certain somites disappeared wholly or in part. In Marionina sp. (originally Enchytraeoides marioni) Roule found that the nephridia in 1-8, and in 12 were lacking in early stages of embryos; that the sperm ducts subsequently developed in 12, and that with the attainment of sexual maturity the remaining nephridia of 9-14 disappeared more or less completely. He regarded the sperm ducts as nephridia whose development had been delayed. It seems to the writer more probably that the nephridia of 1-8 and of 12 of Marionina had developed and disintegrated in earlier stages, and that the sperm duct probably originated as Vejdovsky described it in Enchytraeidae. Beddard (1892) concluded that the genital funnels developed from the nephridial funnels, and the genital ducts, from parts of the nephridial ducts in Octochaetus multiporiis. The conditions in this species are similar to those in Sparganophilus eiseni, except that in the latter there is no apparent continuity between the genital and nephridial ducts. Benham (1904) found, in both mature and immature specimens of Haplotaxis heterogyne, that the most anterior nephridia were in somite 10 and were somewhat degenerate, and that there were no nephridia in somites 11-13. Because of the marked structural similarity between sperm ducts (in 11 and 12) and nephridia, he concluded that the sperm ducts were nephridia onto which genital funnels had been grafted. Since the oviducts had no resem- 20 FLORENCE S. HAGUE blancc to the nephridia, they were considered different in origin. No explanation of the absence of nephridia in 13 was offered. Reduction of the nephridia in the genital somites alone was reported in Moniligaster (Benham 1886) and in Gordiodrilus (Beddard 1894). In Libyodrilus violaceous Beddard (1891) found nephridia in all somites posterior to 3 in a young specimen; but in the adult all that remained of the nephridia of 13-17 was the integumental network. Beddard attributed the loss of nephridia to the presence of very large spermathecae which extended through these somites. It has been concluded (p. 17) that the disintegration of the nephridia of somites 3-10 of Sparganophilus etseni is not directly related to the devel- opment of the reproductive organs; and (p. 19) that the genital funnels and ducts develop independently of the nephridia, although the same pore may serve first for a nephridium and subsequently for an oviduct. With the exception of definitely immature specimens, none have typical nephridia in 11, 12 and 14, in which somites the genital ducts are located. The nephridia of 11 are merely rudiments or are lacking in those specimens in which the anlagen of the spermiducal funnels are found. Of the imma- ture specimens, Nos. 99a, 100a, and 100 have no spermiducal funnels but do have typical nephridia in 12; Nos. 99, 101, 101a and 102 have the anlagen of spermiducal funnels but have reduced or no nephridia in 12. Nos. 101 and 102 have tiny anlagen of the oviducal funnels and typical nephridia in 14; Nos. 121 and 122 have distinct anlagen of the oviducal funnels and ducts, but the nephridia are reduced. Finally, 45 mature worms have typical nephridia in 13 but reduced or no nephridia in 14 (Table III). Because of these facts, it seems that the disintegration of anterior nephridia which is produced in the embryo by the physiological condition, is augmented in 11, 12 and 14 by the development of the geni- tal funnels and ducts of the respective somites. Since the nephridia are not transformed into genital organs, probably this effect is produced by introducing some different physiological condition, possibly absorption of the food supply or a hormone secretion. (c) Sperm Sacs The sperm sacs extend into somites 11 and 12 from septa 10/11 and 11/12. During the breeding season, the sacs of 12 are often so large that they push the septum 12/13 back against septum 13/14. Small out- growths which somewhat resemble the sperm sacs are frequently found on septum 12/13. The sperm sacs have been described as lobulate. Eisen (1896) distinguished between a minutely lobulate condition in .S". eiseni and a less minutely lobulate condition in S. bctihami. There is a great variation in the size of the k)bules in S. eiseni at different seasons. When free from spermatozoa the lobules are quite small, but at the height of STUDIES ON SPARGANOPHILUS EISENI SMITH 21 the breeding season, they may be so distended and the whole sperm sac so compact that the lobulation is not readily recognizable. Figures 99 and 119D (Eisen 1896) of S. benhami represent conditions frequently found m S. eiseni. (d) Spermathecae Between periods of breeding activity a spermatheca is represented by a small mass of undifferentiated cells. The mass is without a definite lumen, and is placed at the inner end of a more deeply staining strand of tissue which extends through the body wall. The strand is the rudiment of the spermathecal duct. The spermathecae of the sexually mature worm are variable in shape. Often they are cylindrical with a spherical enlargement at the free end; but the enlargement may be elongated at the expense of the cylindrical portion. Sometimes the contour is smooth, again it is irregular as if the spermatheca had been crowded into a space shorter than the spermatheca itself. Eisen (1896) figured spermathecae of S. benhami (figs. 118a and 118b), of S. carneus (figs. 140a and 140b), and of S. guatemalensis (figs. 141a, 141b and 141c). A specimen, identified by Eisen as .S. benhami, is in the collection of Professor Smith. It has spermathecae which are slightly irregular in outline, but not so irregular as those figured by Eisen. Some specimens of S. eiseni, which have the anterior accessory glands, have spermathecae of the same shape as those of the above mentioned specimen of S. benhami. Figures 140a, 140b and 141b (Eisen) are quite typical of the shape of the spermathecae of S. eiseni. A sac, similar to the ones which he mentions and figures at the end of the spermathecae of S. guatemalensis, has been noted in some specimens of S. eiseni. (e) Accessory Reproductive Glands These are glands which are evidently related to the reproductive activities but are not connected with the sperm ducts or other reproductive organs. There are two kinds, of which one is found in some of somites 3 to 10, and the other in some of somites 15 to 17 and 22 to 27. For the sake of brevity, the first mentioned glands will be called the anterior glands and the last mentioned, the posterior glands, in the following discussion. The posterior glands were called prostates in the original description of the species by Smith (1895). Eisen (1896) followed the nomenclature of Beddard (1895) and applied the term spermiducal to these same glands. Michaelsen preferred to limit the term prostates to the glands which were directly associated with sperm ducts and described the glands of S. eiseni as prostate-like. The presence of the anterior glands was first noted by Eisen (1896). He called them parietal glands. This name is in no way suggestive of the function, neither are the posterior glands true prostates. 22 FLORENCE S. HAGUE Consequently, since the anterior and posterior glands are similar in origin and structure, and are related to the reproductive activities, which facts will be shown subsequently, the writer has chosen to use the term, acces- sory reproductive glands for both kinds, and to distinguish them from each other by the adjectives, anterior and posterior. The term prostates will be used for those glands which are connected with the sperm duct. Each anterior gland (fig. 4) is a spherical or somewhat elongated mass from which a duct passes through the body wall close beside seta a and opens into the follicle of that seta. The glandular part (fig. 16) consists of a tubular lumen surrounded by epithelial, muscular and glandular layers; the duct lacks the glandular layer. The epithelial layer is a con- tinuation of the body epidermis (fig. 18); the muscular, of the circular muscular layer of the body wall. The proximal ends of the glandular cells are attenuated and can be traced into the muscular layer. They probably extend into the epithelial layer. A posterior gland (fig. 3) consists of a tubular and somewhat convoluted glandular part (fig. 14), and a duct (fig. 15) which opens to the exterior close beside and usually through the follicle of seta b. The duct consists of an epithelial layer which is continuous with the epidermis, and a muscular layer. The peritoneum covering the duct is prominent. The glandular part consists of epithelial cells with attenuated processes which extend toward or to the lumen. Because of the regularity of cell walls and of the position of the nuclei about the lumen there appears to be an epithe- lial lining surrounded by a glandular layer. The earliest stage in the development of an anterior gland was found in specimen No. 68, which was collected in May and was without a trace of a clitellum. In this gland (fig. 18) the epidermal invagination is distinct and cells from the circular muscular layer can be seen extending part way around it. Figure 19 is a section through the deepest part of the invagina- tion, which is as deep as the thickness of the body wall. In later stages mitotic figures are present in the epithehum. The epithehal nuclei are very numerous and are arranged in two or three irregular series. The muscular layer appears as a thin strand of cells which are not typical muscle cells but which are connected with the circular muscular layer. At this stage the glandular layer first appears. It consists of numerous nuclei and a comparatively small amount of cytoplasm, but is without cell walls. The nuclei are stained like those of the epithelium, are found close to the muscular layer and occasionally are seen in sections directly over the thin muscular strands. Because of these conditions and the attenuated bases of the glandular cells in the fully developed gland, it seems that the glandular cells are epithelial in origin. Nothing has been found which would indicate their development from the body wall or from the coelomic epithelium. In later stages the epithelial cells become definitely columnar STUDIES ON SPARGANOPHILUS EISENI SMITH 23 and arranged in a single layer. The muscular and glandular layers gradu- ally increase in thickness and cell walls appear in the latter. In the devel- opment of a posterior gland, which is similar to that of an anterior one, the continuity of the epithelial and glandular cells shows as soon as cell walls appear. Variations in size and position have been found in both kinds of glands. Table IV gives the condition and location of these glands, in so far as they were ascertained, in the various worms studied. Worms numbered from one to 36 are in the collection of Professor Smith and the remainder have been collected during the course of this study. The specimens from a given locality are listed together in each of the two series. This table shows that, so far as is known, the anterior and posterior glands in each worm are in similar stages of development. Of the worms collected at Homer Park in June and July, all have typical glands; in October, Decem- ber and February, all have small glands. Furthermore, a distinct clitellum is, without exception, accompanied by typical glands; an imperfectly developed clitellum is most frequently, and an absence of clitellum, in mature worms, is always accompanied by small glands. There is then a decrease in the size of the glands between breeding seasons. This decrease in size is not merely a shrinkage but an actual disintegration and loss of certain layers. Muscular and glandular layers degenerate into a mass (fig. 17, deg), in which cell outlines and nuclei are indistinct or wanting, and at the surface of which are numerous bloodvessels. This mass dis- appears leaving only the epithelial layer, which apparently does not degen- erate, for mitosis occurs in its cells both before and after the loss of the other layers. Mitotic figures have been found in the epithelium of worms collected in December, February and May. The subsecjuent development is similar to that already described. In the posterior glands there is a similar process of degeneration and regeneration. The original development of these glands is probably not closely re- lated to the breeding season, for in Nos. 94 and 95, which were collected in September, the glands were apparently developing. They were also developing in No. 68 which was collected in May. The posterior glands were found so regularly in three or four of somites 23-26 in the worms studied first, that, later, only somites 1-15 of most of the worms were sectioned. Sometimes a part of somites 23-26 was sec- tioned in order to ascertain the condition of the glands. Of all the worms in which the location of the posterior glands has been investigated, only specimens from Havana, Illinois have the glands in somites other than 23-26. Several of these worms have glands in three or four of somites 22- 27, and one or more of 15, 16 and 17. Aside from the seasonal changes, there are in the anterior glands vari- ations in size due chiefly to the thickness of the glandular layer; and vari- 24 FLORENCE S. HAGUE Taule IV Accessory Refroductive and Pharyngeal Glands Some of the specimens were dissected: from one to 27 somites of the others were sectioned. A blank means that the condition of the organ is unknown, t indicates a tj'pical organ: 5, a small organ : 5^, a very small organ : 0, absence of the organ. The numbers which follow these letters indicate the somites. No. of Pla-ce or dak Clitel- Anterior Posterior Pharyngeal specimen of collection liim glands glands glands 1 ? 2t,A 2 > 0 3 ? 0 4 ? 0 5 ? 0 6 Urbana, 111. 1/, 8 7 ii « 0 8 ? 2t,6 9 ? I5, 4 10 Havana, 111. (2/, 3 \U,4 11 K U 2^,6 12 1895 U,7 t, 16 13 1895 t, 16, 25-27 14 111. 2 /, 3, 4 t 15 u « 0 t 16 u « 2/, 4 t, 23-26 17 a u 2 /, 3, 4 18 u u 2 /, 3, 4 19 u (' .( 2 5,3 20 u u 1 ^2/, 3 1U,4 21 u u 1,3,4 22 [Diogenes t K, 3 t 23 [ Pond I U,3 24 Douglas Lake 2 5,3 • 25 " 2/, 3 26 u u n,4 27 u « u, 3 28 (1 II 2/, 3 29 II II 1 /, 3 30 II II 2/, 3 31 Florida 2/, 4 t 32 II 2/, 4 i :ii 11 1 /, 8 I, 23-25 34 Louisiana (' /, f 23-25 \ 23-26 35 Wisconsin 0 I, 23-26 36 Iowa 2/, 6 /, 24-26 STUDIES ON SrARGANOPlIILUS EISENI SMITH m Table IV, continued 25 'No. of Dak of Clikl- A nlcrior Poskrior Pharyngeal specimen collection lum glands glands glands Homer Park, 111. 37 July 1918 t 2/, 4 t, 23-26 t,6 38 it u I n, 4 /, 23-26 /, 6 39 \u^. 1919 I 0 5, 23-26 40 u u 0 1 5,4 5, 23-26 41 ii a s 2/, 4 / 5,6 42 u u s 2^,4 / 1,6 43 U 11 0 1 5,4 5 5, 6 44 Oct. 1919 0 l5, 4 5 1,6 45 (I It s 2 5,4 5, 23-26 s, 6:55,7 46 « it s l5, 4 s 5, 6 47 July 1918 2t,4. 48 ti it 0 49 11 u 2 1,4 50 It ii 2t,A 51 ti » 2 1,4 52 IMay 1920 s 0 I, 23-26 ss, 6 53 (( « 0 2 s, 4 5, 23-26 t,6 54 it K 0 2 s, 4 5 s, 6 55 K (1 s Is, 4 s, 23-26 1,6 56 l( (I s 2 1,4 t,6 57 Ii II t 2 1,4 5,6 58 II II 0 2 5,4 5 5,6 59 II II 0 2 5,4 5 5,6 60 II II 0 l5, 4 5,6 61 II II 0 2 5,4 62 U II 0 l5, 4 63 11 II 0 2 5,4 64 May or June I 1 /, 4 65 June 1920 I 2/, 4 66 11 II I 2/, 4 67 May 1920 s 2 5,4 68 11 11 0 2 5,4 69 Oct. 1920 0 1 5,4 70 11 « 0 2 5,4 71 Dec. 1920 0 1 5,4 5, 23-25 72 II (1 0 1 5,4 73 II 11 0 2 5,4 74 II 11 0 0 5, 23-26 75 II 11 0 Is, 4 76 11 II 0 2 s, 4 77 11 11 0 0 78 Feb. 1921 0 Is, 4 79 II 11 0 1 5,4 80 11 11 0 l5, 4 81 11 « 0 0 82 11 11 0 0 5, 23-26 26 FLORENCE S. HAGUE Table IV, continued No. of Date of Clitel- A nkrior Posterior Pharyngeal specimen collection lum glands glands glands Havana, Illinois. 83 Sept. 192« s 0 s, 15-17, 23-26 ss, 6 84 (1 u t 2t,6 s, 6 85 U il t 1 i,6 s, 6 86 t 1/, 7 t, f 15-17, 24-27 1 16, 24-26 87 a u t 2/, 6 88 " " f 2 /, 6 /, 23-26 89 " " i 2/, 6 t, 23-25 90 u u I 0 t, i 23-26 [24-26 91 u u I 1 /, 6 92 u u t 2/, 6 93 u u i 1 /, 6 t, 23-26 94 " " 0 2^,7 5,15-17,25-27 95 u u 0 0 s, 23-26 96 u u i It, 4 t, 22-25 97 " " s 0 s, 23-26 98 I 1 /, 4 Vicinity of Douglas Lake, Michigan Sedge Pond. 99 July 1919 immature 0 0 t, 6:s, 7 100 " " 11 0 0 f, 6,7:5, 8 101 " " 11 0 /, 6:ss, 7 102 u u 11 0 t, 6s, 7 103 AvK 1919 0 0 s, 23-26 104 " " s 0 s, 23-26 105 s 0 5, f 23-25 [23-26 106 " " 0 / s,6 107 11 It 0 /, 23-26 s,6 108 (1 K 0 t 5,6 109 July 1919 0 5,6 110 11 11 0 5, 6 111 11 (1 0 5,6 112 11 11 0 I 5,6 113 11 u 0 I 5,6 114 a 11 0 0 s 5.6 115 11 .1 0 .t,6 116 11 11 0 S.6 117 11 11 0 s, 6 118 11 11 0 5, 6 119 11 11 0 5,6 120 11 11 0 s, 6 121 11 11 0 0 s 1,6 122 11 ii ° 0 s, 23-26 1,6 I STUDIES ON SPARGANOPHILUS EISENI SMITH 27 Table IV, concluded iVo. of Dale of Cliiel- A nkrior Posterior Pharyngeal specimen Collection him glands glands glands Bessey Creek. 123 July 1919 t 0 s, 23-26 s,6 124 July 1920 t 0 t, 23-26 125 u u t 0 126 Aug. 1920 1 0 127 Northwest Shore of Lake. t 0 128 Aug. 1920 i 0 s, 6 129 u u Maple River. t 0 /, 23-26 ss, 6 130 July 1919 i 0 s, 6 131 u u t 0 ^,6 132 Aug. 1920 t 0 133 a 11 t 0 134 u « t 0 /, 23-26 135 U (1 Hook Point. t 0 136 Aug. 1920 t 0 /, 23-26 137 (( a t 2t,3 138 u u t 0 ^,6 139 Diogenes Pond. t 2 1,3 /, 23-26 .,6 140 June 1920 t 0 /, 23-26 s, 6 141 11 u t It, 3 ^,6 142 July 1920 I i 1,3,4 ^,6 143 11 11 t 0 s, S:o, 6 144 11 11 t 2 1,3 ss, 6 145 a 11 t 2t,3 5,6 146 June or July 1920 t 0 147 11 11 11 i 1 s,3 148 11 U 11 t 1 1,3 149 11 11 11 t 0 150 11 11 11 Burt Lake, Carp Creek. t 1^,3 151 Aug. 1920 t 2 1, 10 t, / 24-26 [25-26 s,6 152 11 11 1 0 ss, 6 153 11 11 t 0 154 11 It Burt Lake, Maple River. t 0 t, 23-26 155 Aug. 1920 t 0 t, 23-26 .,6 28 FLORENCE S. HAGUE ations in shape, due to the configuration of the coelome and possibly to the contraction of the muscles of the gland. In several worms there is a slight, and in No. 151a very definite epidermal thickening surrounding the gland pore. Typically, the anterior glands open into the follicles of setae a of the somites in which the glands are located. These setae are not ornamented or otherwise different from the usual type, but they are lacking in two worms. No. 36 lacks setae a and has glands which are larger than usual and which have a heavier muscular layer. No. 151 lacks setae a and b. Since No. 36 is the only mature worm available from its locality, and No. 151 is the only one from its locality which was studied and which has glands, it is not known whether their conditions are exceptional or usual for their respective localities. Variations in number and position of the anterior glands are numerous. These glands occur most frequently in somites 3, 4 and 6. They are in somite 7 of three worms; in somite 8 of two worms, and in somite 10 of one worm. Most of the specimens which have the glands in 6 are from Havana, Illinois (1920); most of those which have them in 4, from Homer Park, Illinois and the earlier collections at Havana; most of those which have them in 3, from Michigan and the earlier collections at Havana. There may be a pair of glands symmetrically placed in the somite, or there may be a gland on one side only. Each of about half of the worms from the earlier collections at Havana has three or four glands in somites 3 and 4. None of the recently collected worms have more than two glands, although one of them (No. 142) has its two glands in two successive somites. A summary of the condition of the anterior glands in the worms col- lected in different localities shows that the glands are not always present. Of a total of 46 worms from Homer Park, 39 have anterior glands in somite 4, and seven have no anterior glands; of 16 worms from Havana (1920) eight have glands in 6; two, in 4; two, in 7; and four lack glands. Among the Michigan specimens, the 22 from Sedge Pond have no anterior glands; the same is true of five from Bessy Creek, two from the northwest shore of the Lake, and six from Maple River. Of four worms from Hook Point, two have glands in somite 3, and two lack glands; of eleven from Diogenes Pond, seven have glands in 3, and four lack glands. Three worms from Carp Creek lack glands and one has glands in 10. Of eleven worms from the earlier collection at Havana, seven have glands in somites 3 and 4; one, in 4; one, in 6; one, in 7; and one lacks glands. Of nine worms pre- viously collected at Douglas Lake, one has an anterior gland in 4, and all the others have glands in 3. In all these groups mentioned, only certain ones from the Douglas Lake region have a uniform condition in respect to the anterior glands. These are the Sedge Pond, Bessey Creek, and Maple Ri\er groups, in all of which such glands arc lacking. It is possible that a stud)- of more speci- STUDIES ON SPARGANOPHILUS EISENI SMITH 29 mens from Bessey Creek and Maple River would result in finding some worms with glands. Of the groups of four or more worms which were col- lected in 1919 and 1920, and in some of which glands are present, the per- centage having glands varies from 25 to 85. The significance of these percentages lies in the fact that they show a wide variation. The position of the glands is least variable in the worms from .Homer Park. All of these have the glands in somite 4. The worms with the greatest variation in the position of the glands are those from Havana. Those of the 1920 col- lection have glands in somites 4, 6 and 7, with most of them in 6; those of the earlier collections have glands in somites 3, 4, 6 and 7, with most of them in 3 and 4. The fact that glands, probably similar in function and differing only slightly in structure, are found ventrally in different worms in several of somites 3, 4, 6, 7, 8, 10, 15, 16, 17, 22, 23, 24, 25, 26 and 27 suggests that these glands are remnants of a once continuous series of ventral glands. Eisen (1896) wrote: "Of the same nature (as the spermiducal glands) I consider the forward parietal glands in somite 3 of S. eiseni, and it seems not unlikely that originally this genus possessed many more pairs of sper- miducal glands, perhaps one in every somite." In order to account for the two kinds of accessory glands, it would have to be assumed that there had been two series of glands, or that one series had become differentiated into two kinds of glands. The facts that all of the earlier (1913) collection of worms from Douglas Lake, probably Diogenes Pond, have anterior glands, but that over 2)Z 1/3% of those of the 1920 collection from Diogenes Pond, which were studied, lack these glands; and that over half of the earlier (1895) collection of worms from Havana had three or four glands, while none of those from the recent collections have more than two glands, suggest the possibility that these changes may be continuing even now. Because of the variable condition of these glands, it scarcely seems that their presence or absence can be regarded as a specific character. The first mention in the literature of anything that seems comparable to the accessory glands described above, is the description (Perrier 1874) of 40 pairs of pyriform bodies located in the posterior somites of Ponto- scolex (originally Urochaeta) corethrurns. Dissection and sections of a few somites of a specimen of P. corethrurus, which is in the collection of Pro- fessor Smith, have established the fact that each pyriform body is part of a nephridium. In the glossoscolecid genera, Andiorrhinus, Criodrilus, Kynotus, Micro- chaeta, Rhinodrilus and Tritogenia, glands have been described which are associated with the ventral setae. In Criodrilus these are conspicuous glandular areas about each pair of ventral setae on the clitellum. Some of the setae are modified as genital setae. In the genus Microchaeta, the glands are located in some one or all of somites 9 to 35. There may be a 30 FLORENCE S. HAGUE single gland or there may be several glands opening into a setal follicle. Some of the setae are modified as genital setae, and sometimes external papillae mark the location of the glands. The glands of M. papillata (Benham 1892) and of a specimen of M. algoensis in the collection of Pro- fessor Smith, are structurally like those of Sparganophilus eiseni, except that those of Microchaeta algoensis have both circular and longitudinal bands in the muscular layer. Such structures are not unknown among Lumbricidae. The so-called papillae, on which setae ah of 9, 10 and 11 of Helodrilus caliginosus trape- zoides are located, are thickenings of the epidermis into which irregular projections of the cavity of the setal follicle extend. There are glandular structures accompanying the copulatory setae of Lumhricus terrestris (Hering), and the ventral setae of 13-16 of Bimastns palustris (H. F. Moore). Glands are associated with ventral setae, which in some are modified as genital setae, in the megascolecid genera, Acanthodrilus, Octochaetus, Dichogaster, Diplocardia and Pheretima. Diplocardia (Eisen 1900) has glandular areas consisting of cells which lie between the epidermal and muscular layers and which have long ducts opening into the setal follicles. In other genera the relation of the glands near the genital and spermathecal orifices to the ventral setae is not known, but from brief accounts of their structure it seems probable that they are similar to the accessory repro- ductive glands described. The accessory glands described by Sweet are not connected with the setae but with the sperm duct. In one of the Moniligastridae, Syngenodrilus lamuensis (Smith and Green), "prostates" have been described in somites 11-13. They are not connected with the sperm ducts which open on the anterior margin of 13. Each gland opens to the exterior slightly laterad of seta b, and has a tubu- lar lumen surrounded by epithelial, glandular and muscular layers. Bed- dard (1887) described a similar position for the muscular layer in the prostates of Eudrilus eugeniae (originally sylvicola), but in all other glands of the accessory type of which descriptions have been found, the muscular layer is between the epithelial and glandular layers. The glandular structures which have just been mentioned are so vari- able and the data on their development and structure are so meager that it is difficult to interpret their relations to each other. Lankester sug- gested that the capsulogenous glands of Lumbricus were excessive develop- ments of the setigerous glands. Beddard (1895) concluded that the accessory, or copulatory glands as he called them, had developed as a result of the invagination of a glandular area, and that the accessory and prostate glands were serially homologous. It was suggested, also by Beddard, that the glands may have developed differently in difTerent families of earthworms. Stephenson and Haru concluded that the prostate glands of Pheretima hawayana were mesodermal in origin, whereas it had STUDIES ON SPARGANOPHILUS EISENI SMITH 31 previously been assumed that all prostates and accessory glands were ectodermal. It has been shown, (p. 21) that the glands of Sparganophilus eiseni are ectodermal in origin. The accessory glands have been thought by different investigators to produce slime, egg capsule (cocoon), albumen, a poisonous secretion, or an irritating secretion. Rosa thought that the glands of Microchaeta benhami had assumed a prostate function. Eisen (1896) held a similar view in regard to the glands of Sparganophilus eiseni. Benham (1892) stated that the copulating individuals of Lumbricus were held together by a sucking action of the clitellum and not by a slime tube. He, accgrdingly, attributed a sucking action to the accessory glands about the setae of Microchaeta papillata. Buchanan suggested that the secretion of the accessory glands which are near the spermiducal and oviducal pores of Notoscolex (origi- nally Cryptodrilus) saccarius might aid in the passage of the body of the worm through the cocoon without friction. Since these last mentioned glands are not related to the setae, they may not belong to the same cate- gory as those under discussion, and yet they seem to be similar to those of Pheretima which Beddard (1895) considered capsulogenous. No evidence for a definite conclusion in regard to the function of these glands has been obtained from the present study. The writer did not see any living specimens of Sparganophilus etseni in copulo, but did study sections of a pair of worms tn copulo which had been collected by Professor Smith at Havana, Illinois in 1895. As is generally the case in copulating earthworms, the anterior ends are turned in opposite directions and are closely approximated along their ventral surfaces. The ventral side of somites 18-27 is concave and somites 1-9 of the opposite worm lie in this concavity. These parts of the two worms are held closely together by a slime tube which is constricted at both ends. There is such a slime tube about one anterior end of each of two copulating pairs, but the other an- terior ends have evidently pulled away from the opposed somites. It is probable that the slime tube was continuous from somites 1-27 of both worms. The anterior end of the worm is pushed into the concavity of somites 18-27 so far that setae cd of somites 1-9 are just outside the ventro-lateral edges of somites 18-27. Setae cd of somites 26 and 27 are on the convex surface of those somites but close to their ventro-lateral edges. The closest contact of the worms is in and immediately below the seta line, c, of somites 1-9. In sagittal sections the epidermal cells of the two worms are so intimately associated in places that the dividing line between them is not readily recognizable. Medially there is an irregular slit-like space between the worms. The slime tube apparently is not formed as a single tube enclosing the worms, but by the fusion of two tubes, one around each worm. A thicken- ing in the slime tube, just laterad of the line of contact of the worms, is 32 FLORENCE S. HAGUE interpreted as the line of fusion of the two tubes. While the slime, which forms the tube enclosing the worms, is thin and deeply stained, that which is between the worms is thicker and only faintly stained. Dorsally the slime tube does not extend deep into all the narrow crevices of the inter- segmental grooves, and does form a smooth, continuous outer surface, which obliterates the outlines of segments. Ventrally the slime covering extends into the various grooves, although not always closely, and forms a small mass filling the anterior end of the slit-like space between the worms. The cuticula is recognizable in different places, both dorsally and ventrally, between the slime and the epidermis. The accessory reproductive glands of somites 7, 25, 26 and 27 open into the irregular slit-like space between the worms. The pores of the accessory glands are not in contact with the body of the opposed worm, therefore, these glands cannot be sucking organs as was suggested for similar structures in Microchaeta papillata. The spermathecal pores open very close to, but slightly laterad of the line of closest contact. The spermiducal pore could not be found. The intersegmental grooves 19/20 and 8/9 are opposite each other. Since the accessory glands open into the median space, from which the spermathecal pores are apparently separated, it hardly seems that the secretion of the former can facilitate the transfer of spermatozoa, if this transfer is accomplished as it is in Lumbricus (Andrews). Different investigators have thought that these glands helped in the for- mation of the slime tube which is present on worms in copulo and at the time of cocoon formation. Since the posterior glands are in somites at the posterior end of the clitellum, and the anterior glands in somites opposite the posterior end of the clitellum of worms in copulo, and since these glands open on the ventral side where the clitellum is thin, it is possible that they do produce a part of the slime tube, or the slime which plugs up the opening at the anterior end of the worm. The glands of 15, 16 and 17, when present, are between the clitella of the copulating worms and might help in the completion of the slime tube. Or, the secretion of the accessory glands might help to fasten the slime tubes together. This study of the accessory reproductive glands shows that the pres- ence and position, especially of the anterior glands, is variable; and that the function of both anterior and posterior glands is related to the repro- ductive activities but is not definitely known. VI. Systematic Relations Eisen (1896) first reported the presence of the anterior accessory reproductive glands in SparganophUus eiseni. Since he did not find such glands in other specimens of Sparganophilus from Central and North America, he concluded that these glands distinguished 5. eiseni from all other species. Because of the presence of such glands and of some minor STUDIES ON SPARGANOPHILUS EISENI SMITH 33 diflferences, he separated S. eiseni from S. benhami and the less distinct species, S. giiatemalensis and S. carneus. He also stated that he had in- sufficient well-preserved material of the two latter species, and that both might prove to be varieties of S. benhami. The most important difference between S. benhami and S. eiseni, as defined by Eisen, was the presence in the latter of the anterior accessory reproductive (parietal) glands. In the present discussion of these glands it has been concluded (p. 29) that they are too variable in presence and absence to be properly regarded as a basis for separating species. These species were also said to differ in size; in position of the tubercula puber- tatis, and of the anterior nephridia; in the shape of the spermathecae; in the lobulation of the sperm sacs and in the relative sizes of the pharyngeal (septal) glands. Again facts have been presented to show that there is quite a variation in each of these characters among the specimens of S. eiseni studied, and that there are conditions among some of these specimens which are similar to the conditions described in S. benhami. A specimen, identified by Eisen as S. benhami, is in the collection of Professor Smith. It does not have the clitellum limited ventrally to somites 17-26, as de- scribed for the species, but on 15-27, with a thicker portion on 22-27. It also has the spermiducal pores on somite 19, not on 20. It has no dorsal pores. Sections reveal nothing which is different from conditions found in S. eiseni. Therefore it seems that S. benhami should be united with S. eiseni. Since the presence of anterior glands is insufficient to distinguish species, the only significant difference between S. guatemalensis and ^S. eiseni is the fact that the clitellum in the former is on somites 16-26. In a specimen of S. eiseni collected in October, the clitellum appears to begin on somite 16, probably because the clitellum was degenerating. Since the material, on which the description of S. guatemalensis was based, was not in good condition; since the difference is small, and especially since, in other points, it agrees with the conditions found in S. eiseni, there seems to be insufficient basis for making this a separate species. The differences between S. carneus and S. eiseni are also such as may be accounted for in the variations of the latter. Eisen suggested that S. carneus might be a northern form of S. benhami, and mentioned that the former resembled S. eiseni in the shape of the spermathecae. In the original description of the genus Sparganophilus, Benham placed it in the family Rhinodrilidae, which he had defined (1890), but which has since been made a part of the family Glossoscolecidae. Michaelsen (1917) united the Glossoscolecidae and the Lumbricidae into the Lumbricidae s. I., and subdivided the former Glossoscolecidae into five subfamilies: Glossoscolecinae, . Sparganophilinae, Microchaetinae, Criodrilinae and Hormogastrinae. These were given equal rank with the Lumbricinae, formerly Lumbricidae, in the new family Lumbricidae s. I. This was done 34 FLORENCE S. HAGUE because additional studies had made a separation of the Glossoscolecidae and Lumbricidae, as two families, seem to him impracticable. According to this classification, Sparganophilus in the only genus of the subfamily Sparganophilinae. Whereas, it was formerly considered the ancestral form of the Glossoscolecidae, Michaelsen now considers it a degenerate descendant of the Glossoscolecinae. In a more recent paper (1921), Michaelsen has created a Familienreihe Lumbricina, in which he has placed the families Glossoscolecidae, Sparganophilidae, Microchaetidae, Hormo- gastridae, Criodrilidae and Lumbricidae. VII. Summary 1. The embryology, as far as it was followed, presents no marked differences from that of other earthworms. 2. Aside from the somewhat greater histological differentiation, the nephridium of Sparganophilus eiseni is similar to that part of the nephrid- ium of Lumbricus, which extends from the nephrostome to the muscular duct. There is no muscular duct in the nephridium of Sparganophilus eiseni. 3. The nephridia begin to develop in somite 3 and the somites poste- rior thereto in embryos of 5. eiseni. Disintegration soon sets in at the anterior end, and causes the complete loss of the nephridia of somites 3-11 and the loss or degeneration of the nephridia of 12 and 14, and sometimes of 13. 4. The genital ducts and funnels, although closely associated with the nephridia, develop independently of them. 5. The accessory reproductive glands, of the various specimens ex- amined, are found in three or more of 15 different somites, and probably are remnants of a once continuous series of glands. The anterior accessory reproductive glands are too variable to be of value in distinguishing spe- cies. 6. Sparganophilus eiseni is a variable species in several respects. Since each of the combinations of structural characters which are included in the descriptions of the various species: S. hcnhami, S. guatemalcnsis and S. carneus are found represented among various individuals of S. eiseni, it seems necessary to unite them into the one species, .S". eiseni. VIII. Literature Cited Andrews, E. A. 1895 Conjugation of the Brandling. Amer. Nat., 29:1021-1027, 1121-1127. Bage, F. 1910 Contributions to our Knowledge of .Vuslraliau Earthwonns. The Nephridia. Proc. Roy. Soc. Victoria, 22:224-243. STUDIES ON SPARGANOPHILUS EISENI SMITH 35 Beddard, F. E. 1885 On the Specific Characters and Structure of certain New Zealand Earthworms. Proc. Zool. Soc. London, 1885: 810-832. 1886 Note on the Structure of a large Species of Earthworm from New Caledonia. Proc. Zool. Soc. London, 1886:168-175. 1887 Contributions to the -Vnatomy of Earthworms. Nos. I, II, III. Proc. Zool. Soc. London, 1887:372-392. 1889 On Certain Points in the Structure of Urochaeta, E. P., and Dichogaster, nov. gen., with further Remarks on the Nephridia of Earthworms. Quart. Journ. Micros. Sci., 29:235-282. 1890 Contributions to the Anatomy of Earthworms, with Descriptions of Some New Species. Quart. Journ. Micros. Sci., 30:421-479. 1891 On the Structure of an Earthworm allied to Xemertodrilus, Mich., with Observa- tions on the Post-embr\-onic Development of Certain Organs. Quart. Journ. Micros. Sci., 32:539-586. 1892 Researches into the Embrj'ology of the Oligochaeta. No. 1. 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Micros. Sci., 34:155-179. 1904 On a New Species of the Genus Haplotaxis: with some Remarks on the Genital Ducts in the Oligochaeta. Quart. Journ. Micros. Sci., 48:299-322. Benham, W. B. and Cameron, G. 1912 The Nephridia of Perieodrilus ricardi and of P. montanus. Trans. N. Zealand Inst., 45:191-198. Bergh, R. S. 1886- Untersuchung liber den Bau und die Entwickelung der Geschlectsorgane der Regenwiirmer. Zeit. wiss. Zool., 44:303-332. 1899 Nochmals iiber die Entwicklung der Segmentalorgane. Zeit. wiss. Zool., 66:435- 449. Bourne, A. G. 1894a On Certain Points in the Development and Anatomy of some Earthworms. Quart. Journ. Micros. Sci., 36:11-34. 1894b On JMoniligaster grandis, A. G. B. from the Nilgiris, S. India: together with Descriptions of other Species of the Genus Moniligaster. Quart. Journ. Micros. Sci., 36:307-384. Buchanan, G. 1911 Note on the Accessory Glands of Cryptodrilus saccarius (Fletcher). Proc. Roy. .Soc. Victoria, 22:221-223. 36 FLORENCE S. HAGUE Cameron, Gladys M. 1912 The Minute Structure of the Nephridium of the Earthworm Maoridrilus rosae Bcddc-rd. Trans. N. Zealand Inst., 45:172-190. Cole, F. J. 1893 Notes on the Clitellum of the Earthworm. Zool. Anz., 16: 440-446. Collin, Ant. 1888 Criodrihis lacuum Hoffrn. Zeit. wiss. Zool., 46:471-497. ElSEN, G. 1895 Pacific Coast Oligochaeta. I. Mem. California Acad. Sci., 2:63-122. 1896 Pacific Coast Oligochaeta. II. Mem. California Acad. Sci., 2:123-198. 1900 Researches in American Oligochaeta, with Especial Reference to those of the Pacific Coast and Adjacent Islands. Proc. California Acad. Sci., (3) 2:85-274. Foot, K. and Strobell, E. C. 1902 Further Notes on the Cocoons of AUolobophora foetida. Biol. Bull., 3:206-213. Gatentby, J. B. 1916 The Development of the Sperm Duct, Oviduct and Spermatheca in Tubifex rivulorum. Quart. Journ. Micros. Sci., 61:317-336. Gegenbauer, C. 1853 Ueber die sogenannten Respirationsorgane des Regenwurms. Zeit. wiss. Zool., 4:221-232. GOEHLICH, G. 1890 Uber die Genital- und Segmental-Organe von Lumbricus terrestris. Zool. Beitrage, 2:133-167. Breslau. Goodrich, E. S. 1895 On the Coelom, Genital Ducts and Nephridia. Quart. Journ. Micros. Sci., 37:477-510. Heimberger, H. V. 1915 Notes on Indiana Earthworms. Proc. Indiana .\cad. Sci., 1914:281-285. Hering, E. 1857 Zur Anatomic und Physiologie der Generationsorgane des Regenwurms. Zeit. wiss. Zool., 8:400-424. Hesse, R. 1894 tjber die Septaldriisen der Oligochaetcn. Zool. Anz., 17:317-321. HORST, R. 1888 Descriptions of Earthworms. Notes Leyden Mus., 10:123-128. 1895 Descriptions of Earthworms. Notes Leyden Mus., 17:21-27. Lankester, E. R. 1864 The Anatomy of the Earthworm. Quart. Journ. Micros. Sci., 4:258-268; 5:7-18, 99-114. Lehmann, O. 1887 Beitrage zur Frage von der Homolgie der Scgmentalorgane und Ausfuhrgange der Geschlectsproducte bci den Oligochaeten. Jena. Zeit. Naturw., 21:322-354. Meisenheimer, J. 1910 Die Excretionsorgane der wirbellosen Ticrc. Ergebn. und Fortsch. Zool., 2:275- 366. MiCHAELSEN, W. 1891a Tcrricolcn der Berliner Zoologischen Sammlung. I. Afrika. Arch. Naturg., 57, 1:205-228. 1891b Bcschreibung der von Ilcrrn Dr. Fr. Stuhlmann auf Sansibar und dem gogcniiber- liegcnden Festlande gesammelten Terricolen. Jahrb. Hamburg Wiss. .Anst., 9:1-72. STUDIES ON SPARGANOPHILUS EISENI SMITH 37 1892 Terricolen der Berliner Zoologischen Sammlung. II. Arch. Naturg., 58, 1 :209-261. 1894 Die Regenwurm-Fauna von Florida und Georgia. Zool. Jahrb., Syst., 8:177-194. 1895 Zur Kenntnis der Oligochaeten. Abh. nat. Ver., Hamburg, 13:3-37. 1899a Terricolen von verschiedenen Gebieten der Erde. Mitt. Nat. Mus. Hamburg, 16:1-122. 1899b Revision der Kinberg'schen Oligochactcn-Typen. Ofv. Vet. Akad. Forh., 56: 413-448. 1900 Oligochaeta. Das Tierreich, 10 Lief. XXIX+575 pp. Berlin. 1907 Oligochaeten von Natal und dcm Zulu-land. Arkiv. Zool., 4. No. 4, 12 pp. 1910 Oligochaeten von verschieden Gebieten. Mitt. Nat. Mus. Hamburg, 27:47-169. 1913a The Oligochaeta of Natal and Zululand. Ann. Natal. Mus., 2:397-^58. 1913b Die Oligochaeten des Kaplandes. Zool. Jahrb., Syst., 34:473-556. 1917 Die Lumbriciden mit besonderer Beriicksichtigung der bisher als Familie Glos- soscolecidae zusammengefassten Unterfamilien. Zool. Jahrb., Syst., 41 :l-398. 1921 Zur Stammesgeschichte und Sj^stematik der Oligochaeten, insbesondere der Lumbriculiden. Arch. Naturg., 86:129-142. Moore, H. F. 1895 On the Structure of Bimastos palustris, a new Oligochaeta. Journ. Morph., 10:473-496. Moore, J. P. 1906 Hirudinea and Ohgochaeta collected in the Great Lakes Region. Bull. Bur. Fisheries,. 25:153-171. Orley, L. 1887 Morphological and Biological Observations on Criodrilus lacuum Hoffmeister. Quart. Journ. Micros. Sci., 27:551-560. Perkier, E. 1874 Etudes sur I'organisation des Lombriciens terrestres. Arch. zool. exper., 3:331- 350. 1881 Etudes sur I'organisation des Lombriciens terrestres. Arch. zool. exper., 9:177- 248. 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Stephenson, J. 1915 On some Indian Oligochaeta, mainly from Southern India and Ceylon. Mem. Indian Mus., 6:35-108. 38 FLORENCE S. HAGUE 1917 On the So-called Pharyngeal Gland-cells of Earthworms. Quart. Journ. Micros. Sci., 62:253-286. Stephenson, J. and Haru, Ram. 1919 The prostate glands of the Earthworms of the Family Megascolecidae. Trans. Roy. Soc. Edinburgh, 52, 2:436-453. Sweet, Georgina. 1900 On the Structure of the Spermiducal Glands and Associated parts in Australian Earthworms. Journ. Linnean Soc, 28:109-139. Vejdovsky, F. 1884 System und Morphologie der Oligochaeten. 166 pp. Prag. 1888-1892 Entwicklungsgeschichtliche Untersuchungen. IV+401-f 24 pp. Prag. 1892 Zur Entwicklungsgeschichte des Nephridialapparates von INIegascolides australis. Arch. Mikros. Anat., 40:552-562. Wilson, E. B. 1889 The Embrj-olog}- of the Earthworm. Journ. Morph., 3:387-462. EXPLANATION OF PLATES Abbrev iations a ace. anterior accessor}' reproductivi s k. large cell gland Im. longitudinal muscles am. ampulla In. lumen • ant. anterior mi. mitotic division ha. body axis mid. middle tube hi. branch mil. muscular layer hr. break in nephridium n. nucleus of nephridial cell hv. blood vessel na. cut edge of narrow tube bw. body wall nar. narrow tube h'li'U'. width of body wall n c. nerve cord c. cuticula ne. nephridiopore ca. canal ncs. nephrostome ce. nephridial cell n Ic. nucleus of large cell cir. connection with circular muscles P- gland pore cm. circular muscles p ace. posterior accessor}' reproductive coe. coelome gland con. constriction between middle and P O'- peripheral cytoplasm wide tubes pilar. phar}-nx dcg. mass of degenerate muscular and post: posterior glandular tissues rcg. regenerating epithelium dor. dorsal s. septum d sac. dorsal sac se. seta e. coelomic epithelium sef. setal follicle en. nucleus of epithelial cell sc m. setal muscles ep. epidermis th w. thin walled part of wide tube epn. nucleus of epidermal cell Ik u: thick walled part of wide tube gl. glandular cells vcn. ventral gl c. glandular epithelium wa. waste in ampulla gr- granular layer ■ui. wide tube i. beginning of last part of wide tube 28, ; somite numbers i cy. inner cytoplasm 34, S All drawings except figures 1 and 2 were made with the aid of a camera lucida. ''/ dor X45. Plate I Fig. 1. Outline of tlie bifurcation in a young worm. Dorsal view. Fig. 2. Outline of a nephridium and its canal. Fig. 3. A somite with a posterior accessory reproductive gland. Longitudinal section. Fig. 4. Somite 4 with an anterior accessory reproductive gland. Cross section. X 45. epn [0^ cm wi '^W-^. ©pn Wl^ — c ne Plate II l-'ig. 5. Section through the wide tube at i (fig. 2). X 46*). Fig. 6. Part of a section through a ne[)hridium. X 469. Fig. 7. Section through the wide tube at its entrance into,the body wall. X 46A Fig. 8. Section through the ncphridioporc. X 409. 10 13 Plate III Fig. 9. Section through the junction of middle and wide tubes. X 469. Fig. 10. Section through a developing nephridium. X 1173. Fig. 11. Section through a developing nephridium of a 0.5 mm. embryo: an earlier stage than figure 10. X 1173. Fig. 12. Section through a ty-pical young nephridium. X 1173. Fig. 13. Part of a section through a disintegrating nephridium. X 1173. IS Im— ^. cm Plate IV Fig. 14. Posterior accessory reproductive gland ; part of a section througli the glandular portion. X 300. Fig. 15. Posterior accessor}- reproductive gland; section through the transition of duct into glandular portion. X 300. Fig. 16. Anterior accessory reproductive gland; part of a section tlirough the glandular portion. X 300. Fig. 17. Part of a section through a degenerate anterior accessor}- reproductive gland. X514. - ^ ■ y A Figs. 18 and 19. Sections through a developing anterior accessory reproductive glana. Fig. 18. The invagination. X 514. Fig. 19. The inner part. X 514. A SYSTEMATIC PRESENTATION OF NEW GENERA OF FUNGI By O. A. Plunkett, p. a. Young, and Ruth W. Ryan University of Illinois The new families and genera of fungi described since volume 22 of Saccardo's "Sylloge Fungorum" was compiled are here assembled from all available literature and presented in a concise, classified form with the reference accompanying each new name. This paper is necessarily incom- plete because some publications and parts of others were unavailable. Only small parts of Broteria, The Botanical Magazine of Tokyo, and Osterreichische Botanische Zeitschrift were found. As far as is known, there has been no previous compilation of the new genera of fungi described since 1910. Mycologists have been compelled to search through an extensive, scattered literature for the special types in which they were interested. This paper will be of value to mycologists in that it contains, with necessary references, most of the new genera of fungi published since the last volume of the "Sylloge Fungorum" was compiled. The authors wish to thank Dr. F. L. Stevens for numerous helpful suggestions. To him is due the credit for planning the work of assembling all the new families, genera, and species of fungi described since 1910. They also wish to acknowledge the assistance of Messrs H. L. Dixon, J. M. Mendoza, P. J. Byrd, H. C. Abbott, and Miss Ruth Dowell who aided in the search through the literature. Approximately 7,000 new species of fungi were listed on cards and catalogued in taxonomic order. The cards bear the citation, classification, the name of the genus, species, and generally the host of the fungus. Of these about 800 belong in the Sphaerioidaceae, 700 in the Agaricaceae, 300 in the Pucciniaceae, 200 in the Dematiaceae, 200 in the Microthy- riaceae, 200 in the Pleosporaceae, 150 in the Mycosphaerellaceae, and 100 in each of the following families: Dothideaceae, Hypocreaceae, Melan- coniaceae, Moniliaceae, Polyporaceae, Sphaericaeae, Thelephoraceae, Tuberculariaceae, and Valsaceae. This list is too voluminous to print at the present time. Explanation of Tables The name of the new genus is given in the first column. The name in the second column is that of a genus nearly related or similar to the new 43 44 O. A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN genus named in column 1 or sometimes some other significant name. A number has been given to each publication. This number is given in the 3rd column and refers to the bibliography. The number in column 4 refers to the volume, that in column 5 to the page, and the one in column 6 to the date of publication. At the end of the list is a group of new genera the positions of which were not definitely stated by the authors and the afiinities of which were not sufficiently clear to warrant us in assuming their relationships. The classification system used in this paper is mainly that of Engler and Prantl as given in "Die Naturlichen Pflanzenfamilien" except in the Dothideales and Hemisphaeriales in which the keys of Theissen and Sydow are followed as closely as possible. For the most part, the genera are classified where the authors placed them even though the positions of many were subsequently changed. See the "Synoptische Tafeln" of F. Theissen and H. Sydow in Annales Mycologici 15:389-491. 1917. M\^OMYCETES Plasmodiophorales Plasmodiophoraceae Anisomyxa 23 Clathrosorus 9 IMolliardia 7 Ostenfeldiella 9 Sorodiscus 11 Sorolpidium 23 Trematophlyctis 54 PHYCOMYCETES Chytridiales Synchytriaceae Mitochytridium 54 27 202 191 1 Monochytrium Synchytrium 45 10 3, 50 1910 Saprolegniales ' Saprolegniaceae Isoachlya . .Achlya 3 Jaraia 23 Pythiomorpha 68 Rheosporangiuni 82 Leptomilaccae AUomyces Blastocksia 9 25 1023 1911 1 1913 34 467 1920 9 236 1911 28 643 1914 12 9,23 1913 19 1911 34 86 1918 8 231 1921 1 1913 29 391 1909 4 280 1915 58 353 1914 58 361 1914 58 363 1914 A PRESENTATION OF NEW GENERA OF FUNGI 45 Peronosporales Peronosporaceae Bremiella Brcmia 41 6 195 1914 Nozemia 52 13 566 Stigeosporium Phytophthora 9 30 357 1916 MUCORALES Mucoraceae Blaskestca Choanophora 18 Dissophora Mortierella 18 Haplosporangium Mortierella 18 PHYCOMYCETES STERILIA Zoophagus 46 61 368 1911 ASCOMYCETES Protomycetales Hemiascaceae Dipodascus 85 5 1 1921 Taphridium 85 5 1 1921 Saccharomycetales Endogonaceae Protascus 42 3 155 1913 Saccharomycetaceae Aleurodomyces 13 Blastocystis Dermatocystis 28 Coccidiascus 28 Cycadomyces 73 Guillermondia 65 Medusomyces Mycoderma 16 Nectaromyces 22 Psillidomyces 13 Helvellales Helvellaceae Geomorum 89 23 1921 Pezizales Pezizaceae Aleurina Aleuria 41 Aparyphysaria 89 Manilaea 7 Mollisina 16 Pezizellaster Pezizella 7 26 100 1912 71 296 75 117 1913 3 1 1910 23 1912 31 243 1913 11 176 1919 26 96 1912 6 277 1914 25 1921 12 569 1914 37 109 1919 15 349 1917 46 O. A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN Helotiaceae Belonioscyphella Belonium 53 127 590 1918 Calycellina Helotium 53 127 601 1918 Helotiopsis Mollisiella 53 119 623 1910 Lachnobelonium Belonium 16 37 109 1919 Lambertella Belonioscypha 53 127 375 1918 Leptobelonium Belonidium 16 37 108 1919 Microscypha Dasyscypha 7 17 38 1919 Neobulgaria Ombrophila 7 19 44 1921 Psilachnum 16 37 109 1919 Stereolachnea Lachnea 7 15 353 1917 Tanglella 53 127 606 1919 Torrendiella Dasyscypha 54 27 133 1911 Patelliariaceae Pleoscutula Scutula 54 29 434 1913 Rodwaya Woodiella 86 13 425 1917 Siscocera Nesolechia 59 6 48 1917 Cenangiaceae (Including Bulgariaceae) Asterocalyx 53 121 402 1912 Bulgariastrum 47 8 497 1913 Caloriopsis 7 15 254 1917 Caloriella Cenangina .53 127 345 1918 Discomycella Ascosorus. . 53 121 401 1912 Encoeliella 53 119 619 1910 Stegopeziza Dermataceae 53 126 308 1917 Phacidiales Stictidiaceae Eupropolella 7 Hysteropezizella Stegia? 53 Hystciostegiella Sarcotrochila 53 Phacidiella Char, emend 53 Propoliopsis 36 Sarcotrochila Trochila 53 Tryblidiaceae Odontoschizon Helerosphaeria . 7 12 568 1914 Phacidiaceae Leptophacidium 53 Myxophacidiella 53 Myxophacidium '. 53 Phacidiella 67 Hysteriales Hypodermataceae Bifusella Hypoderma 7 Haplophyse 7 Hypodermellina Hypoderma 7 15 311 1917 126 311 1917 126 313 1917 126 304 1917 6 2279 1914 126 310 1917 127 332 1918 126 303 1917 126 301 1917 22 147 1912 15 318 1917 14 267 1916 15 302 1917 13 228 1915 12 194 1914 14 452 1916 23 87 1912 A PRESENTATION OF NEW GENERA OF FUNGI 47 Hysteriaceae Hysterostomina Hysterostomella 7 Parmulina Parmularia 7 Periaster Erikssonia 7 Polhysterium Hysterographium 8 TUBERALES Tuberaceae Hydnotryopsis Geopora 62 6 336 1916 Agyriaceae Ramosidla 7 15 254 1915 ASPERGILLALES Asptrgillaceae Acaulium 64 Aspergillopsis 64 Crollium 64 Rhopalocystis 35 Periosporales Erysiphaceae Chilemyces 11 42 1912 11 202 1912 11 98 1912 6 140 1911 ]•'; Leucoconis 7 Schistodes nov. nom 7 Typhulochaeta 19 Perisporaceae Acanthostoma 15 Chaetostigme Dimeriella 7 Chaetostigmella Phaeodimeriella 7 Cleistosphaeria 7 Diblastospermella Dimeriorum 77 Dichothrix 15 Dimeriopsis Dimerium 60 Eosphaeria 7 Euantennaria 77 Eudimeriolum 8 Guttularia Orbicula 42 Haraea 7 Jaffuela 77 Meliolina 7 Nematothecium 36 ParodielJa Char, emend 7 Parodiopsis Parodiella 55 Perisporiopsis Perisporium 60 Phaeodimeriella 15 Phaeostigme Dimerium 7 Phycopsis Seuratia 27 Pilene 7 27 1910 15 454 1917 15 456 1917 15 456 1917 29 22 1915 29 46 1912 15 199 1917 15 199 1917 14 74 1916 23 579 1918 29 260 1912 10 171 1917 15 362 1917 23 549 1918 23 36 1912 3 9 1913 11 312 1913 25 39 1921 12 553 1914 5 1534 1912 15 132 1917 31 22 1915 10 170 1917 29 45 1912 15 199 1917 154 1480 1912 14 409 1916 48 O. A. PLUNKETT, P. A. YOUNG, AND RUTH \V. RYAN Plenophysa 7 17 142 1919 Pseudoparodia Dimerosporina 7 15 138 1917 Rizalia ; . 7 12 546 1914 Rhizogene Lasiobotrys 7 18 181 1920 Rhizosphaerella Perisporium 32 59 254 1917 Setella 7 14 359 1916 Stigme Dimerina 7 15 199 1917 Stomatogene 7 14 404 1916 Teratonema 7 15 180 1917 Trichospermella 8 23 38 1912 Winteromyces Parodiclla 8 23 37 1912 Englerulaceae 81 66 296 1916 Diathrypton 47 21 137 1922 Euthrypton 70 66 296 1916 Linotexis Parcnglerula 7 15 198 1917 Oothecium 77 23 519 1918 Ophiolexis 70 66 296 1916 PhaeoschifiFnerula 20 12 21 1914 Rhizotexis 7 15 141 1917 Syntexis 70 66 296 1916 Theissenula Schiffnerula 7 12 198 1914 Thrauste 70 66 296 1916 Capnodiaceae Adelopus nov. nom 7 15 482 1917 Aithaloderma 7 11 257 1913 Antenella 7 15 473 1917 Balladynopsis Balladyna 7 15 435 1917 Balladynella 7 15 478 1917 Calyptra 7 15 478 1917 Ceratochaete Sitella 7 15 179 1917 Chaetothyrina 7 11 495 1913 Chrysomyces 7 15 475 1917 Crytopus 7 12 72 1914 Microtyle .77 23 458 1918 Neohoehnelia 7 15 476 1917 Phragmicapnis 7 15 480 1917 Schizocapnodiiim Capnodium 49 6 91 1921 Parodiellaceae Acantharia 7 16 15 1918 Epiphyma 70 306 1916 HjTJophlegma 7 15 135 1917 Parodiellinaccae N. Earn . . . . 4 16 21 1918 HEMISPHAERIALKS I Ifmisphaeriaccac Chaetoplaca 7 15 232 1917 Dictothyriella 20 12 92 1914 Dictyothyrium Dictyopeltis 46 62 277 1912 Epipeltis 1 7 3,26 1913 Eremotheca Eremothecella 7 15 235 1917 15 236 1917 12 88 1914 23 84 1912 23 199 1916 12 87 1914 12 67 1914 9 180 1914 12 85 1914 12 85 1914 39 625 1913 7 30 1913 12 563 1914 32 3 1914 39 636 1914 416 1911 15 146 1917 A PRESENTATION OF NEW GENERA OF FUNGI 49 Eremothecella Phragmothyriella 7 Haplopeltis 20 Hormopeltis o Myiocoprella ^ Plochmopeltis 20 Polyclypeolum Microthyriella 7 Stephanotheca 47 Stomiopeltis 20 Stomiopeltella 20 Trichopeltaceae N. Fam 26 Pycnocarpon j Pycnoderma ^ Trichopeltina 2 c Trichopeltula 26 Trichosphaeriaceae Melanopsomella 7 J7^ J21 1919 Microthyriaceae Actinocymbe 15 ^ .20 Actinomyxa 7 f'^'^^p^^^'^ •■■■■••'•^^■'^^■■^■^■::':::::::::::: 7 n 315 1913 ^TZ^- 7 11 499 1913 Asterolibertia Dimerosporium 4 16 166 1918 Asteromyxa 7 j5 ^^^ ^^^^ Asterostemula Asterinella. ... 7 14 270 1916 ^T'^^'^tu 20 10 101 1912 Auographiella 7 ^5 3^^ ^^^^ ^"""^'^"•; •. 4 16 123 1918 Calothyriella Calothyrium 7 15 371 1917 Calothyrium / 7 10 160 1912 r Au 120 82 1914 ?"^^"^: 7 14 90 1916 Caenothyrium 7 j^ ^j^ ^^j^ Lamposa 77 Chaetothyrium 77 ^^^^°^i",; ■•■:■■:;;::;■;::::::::::::;::::: 4 i6 ^ 1918 ^r^^'ft 4 16 129 1918 ^^yP'^^'^^^ 53 119 403 1910 Clypeolina /26 34 234 1912 ^ . . 1 7 15 419 1917 Locconiopsis . Dictyothyrium Echiclnodes Lembosia. '. ^' .'.'.''.' .' 7 15 422 1917 Echidnodella Morenoella 7 15 422 1917 l-f'f^'^^ 53 119 454 1910 ^"°Pf^ 53 119 420 1910 !!t^"!^"^ 7 14 430 1916 2"^?^"T Halbania 4 16 163 1918 ?^"^^"^^ Microthyrium 27 164 890 1917 ^'^""^ ■ Meliola 7 15 194 1917 25 90 1921 23 522 1918 16 113 1918 46 62 277 1912 50 O. A. PLUNKETT, P. A. YOUNG, AND RUTH \V. RYAN Lembosia Char, emend 7 11 427 1913 Lembosina 7 11 437 1913 Lembosiopsis 7 11 435 1913 Kriegeriella 7 16 39 1918 Maublancia Asterina 4 16 159 1918 Maurodothella 4 16 124 1918 Melanochlamys 39 438 1913 Meliolaster 61 8 123 1919 Micropeltella 7 11 405 1913 Morenoina 7 11 434 1913 Mycolangloisia Lembosia 4 16 157 1918 Niesslella 16 36 468 1918 Parengkrula 53 119 465 1910 Patouillardina 27 164 890 1917 Peltella Myiocopron 7 15 237 1917 Phaeopeltis 83 7 1 1919 Prillieuxina Asternella 4 16 161 1918 Protothyrium 27 164 575 1917 Pycnoderma 7 12 563 1914 Pycnopeltis 7 14 365 1916 Questeria Dimerosporium 4 16 186 1918 Seynesiella Myiocopron 4 16 196 1918 Sirothyriella 53 119 451 1910 Stegothyrium 53 127 382 1918 Symphaeophj-ma Microphaeophyma 8 23 97 1912 Symphaster 7 13 217 1915 Thallochaete 7 11 501 1913 Thyrosoma 7 19 307 1921 Trichasterina Asterina 4 16 172 1918 Wardina Asterina 4 16 165 1918 Yatesula Stephanotheca 7 15 237 1917 Microthyriopsidaceae N. Fam. 4 16 99 1918 Leprieurina 4 16 210 1918 Manginula : 4 16 99 1918 Trichothyriaceae 15 32 3 1914 Trichothyriella 15 32 4 1914 Trichothyriopsis 15 32 4 1914 Polystomellaceae 7 13 158 1915 Armalella Polyrhizon 7 13 235 1915 Asterodothis 7 10 179 1912 Chaetaspis Parmulina 7 15 279 1917 Cyclotheca 7 12 7 1914 DothithjTcUa 7 16 171 1918 Ellisiodothis , 7 12 73 1914 Hysterostomina Ilystcrostomella 7 13 228 1915 Inocyclus Polycyclus 7 13 211 1915 Marchalia Char. Emend 7 13 251 1915 Mclanoplaca Marchalia 7 15 29S 1917 Monorhiza uleopcltis 7 13 318 1915 Monorhizina Monorhiza 7 13 320 1915 Pleiostomella Uleopcltis 7 15 221 1917 u 212 1916 10 456 1912 13 197 1915 15 410 1917 15 221 1917 14 426 1916 15 134 1917 13 261 1911 12 1 1914 A PRESENTATION OF NEW GENERA OF FUNGI 51 Poiycjclina Polycyclus 7 Rhipidocarpon { - Scoleoncma IJothidastromclla 7 Synpeltis 7 Stigmataceae N. Fam 7 Aphysa 7 Isomunkia Coccinopeltis 7 Vizdla 20 Hypocreales Hj'pocreaceae Balansiopsis Balansia 53 Borinquenia 60 Bronectria 77 Chromocrca 41 Chromocreophis 41 Cylindrocarpon Nectria 69 Dextria Caloncctria 60 Dialhypocrea 77 Epinectria 7 Epispora 54 Hyalocrea 7 Hyalosphaera 60 Hypocreophis 77 Leptocrea 7 Linearis troma 53 Mastigocladium 27 Nectriopsis Nectria 7 Neonectria Nectria 7 Orcadia 59 Patellonectria 77 Phy llocrea 7 Podonectria Ophinectria 59 Sterocrea 7 Trailia 59 Uropolystigma Polystigma 54 Dothideales Plectodiscellaceae N. Fam. . .42 Plectodiscella 42 Myriangiaeceae 7 Ascostratum 7 Butleria 7 Symphaeophyma '. 8 Myxomyriangiaceae AT • • / 7 Myxomyriangium < „ Saccardiaceae Byssogene 47 Calopeziza .47 119 936 1910 10 173 1917 23 563 1918 2 58 1910 2 63 1910 3 225 1913 10 174 1917 23 475 1918 15 215 1917 38 84 1922 15 214 1917 10 172 1917 23 480 1918 14 87 1916 119 938 1910 152 326 1911 9 323 1911 15 52 1917 5 151 1914 25 477 1918 16 38 1918 3 146 1920 15 216 1917 5 151 1914 36 36 1920 4 232 1914 4 232 1914 15 438 1917 10 41 1912 12 302 1914 23 97 1912 11 507 1913 15 438 1917 21 144 1922 8 499 1913 52 O. A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN Dothideaceae Char, emend.. . 7 Achorella Sj'stremma 7 Actinodothis 47 Amerodothis 7 Angatia 7 Auerswaldiella 7 Aulacostroma 47 Bagnisiopsis ; 7 Benguetia 7 Botry ostroma 53 Castagnella 54 Catabotrys Amerodothis 7 Catacauma Sphaerodothis 7 Clypeostroma 7 Coccochora Char, emend 53 Coccochorella 53 Coccodothis Phyllachora 7 CoccodotheUa Coccodothis 7 Coccostroma Yoshinagella 7 Cyclodothis 7 Cyclotheca 7 Dictyochora Curreya 7 Dictyodothis Phragmodothis 7 Diplochora 7 '53 Dothidastromella , „ Dothideopsella 53 Dothidina 7 Pvllisiodothis Asterula 7 Ehnerococcum Coccidella 7 Englerodothis Leveillella 7 Halstedia 18 Haplodothis , 53 Haplotheciella 53 Heterodothis 47 Hysterostoma 7 Ixptodothis 7 LeveilHna 7 Leveillella 7 Melanopsamnopsis Dothidella 76 Metachora 7 IVIetameris Achorella 7 Microdothella 47 IMicrocylella 7 Microphrodothis Ophiodothis 77 Ophiodothiella Ophiodothis 53 Palawania 47 Parmulina Parmularia 7 Perischizon Dothidea 7 Phaeodothiopsis Phaeodothis 7 Phragmodothella Achorella 7 13 174 1915 13 340 1915 9 175 1914 13 295 1915 12 566 1914 12 278 1914 9 176 1914 13 290 1915 15 252 1917 120 424 1911 30 358 1914 13 247 1915 12 280 1914 12 272 1914 119 432 1910 119 431 1910 12 271 1914 13 280 1915 12 269 1914 11 266 1913 12 71 1914 12 275 1914 13 346 1915 11 60 1913 119 421 1910 13 229 1915 124 70 1915 13 302 1915 12 74 1914 13 282 1915 13 285 1915 69 253 1920 20 422 1911 128 615 1919 9 170 1914 11 509 1913 12 268 1914 13 286 1915 13 284 1915 34 1917 9 400 1911 13 342 1915 9 169 1914 12 68 1014 23 495 1918 119 940 1910 9 172 1914 12 194 1914 12 265 1914 12 192 1Tnella 7 16 66 1918 Epipolaeum Hypolyma 7 16 7 1918 Phanerococcus Hypoplegma 7 16 9 1918 Physalosporina Physalospora 7 9 288 1911 Plectosphaeria Physalospora 7 14 413 1916 Scleropliella Leptosphaeria 7 16 158 1918 Massariaceae Leptomassaria Phorcys 7 12 474 1914 Myelosperma Pseudomassaria 7 13 38 1915 Trematosphaeria Massaria 53 123 99 1914 Gnomoniaceae Desmotascus Phomatospora 18 68 476 1919 Stegasphaeriaceae N. Fam. . . 7 14 364 1916 Stegasphaeria 7 14 362 1916 Clypeosphaeriaceae Amerostegi 70 66 297 1915 Euacanthe Clj^osphaeria 7 15 272 1917 Linocarpon 7 15 210 1917 Schizostege 7 14 415 1916 56 O. A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN Stigastroma 7 14 81 1916 Teratosphaeria 7 10 39 1912 Trabutiella Trabutia 18 70 401 1920 Valsaceae Allanthoporthe Diaporthe 32 62 289 1920 Clypeoporthe Diaporthe 53 128 584 1919 Discoidaporthe AUantoporthe 32 62 293 1920 Macrodiaporthe Diaporthe 7 17 94 1919 Phaeodiaporthe Diaporthe 7 17 99 1919 Valseutypella Valsa 7 18 72 1920 JMelanconidiaceae Amphicytostroma Crytosporella 7 19 63 1921 Cryptoceuthospora Crytosporella . 7 19 56 1921 Diatrypaceae Apioporthe Diatrype 53 126 381 1917 Ectosphaeria Diatrype 77 25 48 1921 Phaeotrj-pe Diatrype 41 12 201 1920 Melogranimataceae Anisomyces 7 12 270 1914 Causalis 7 16 184 1918 Pseudothis 7 12 274 1914 Phaeobotryon Botryosphaeria 7 13 664 1915 Xj'lariaceae Theissenia 54 30 52 1914 Laboulbeniales Laboulbeniaceae Amphoropsis Platysthethus 10 Aposporella 18 Autophagomyces 48 Cantharosphaeria 18 CochHomyces 8 Coreomycetopsis 18 Crytandromyces 48 Cucujomyces Monicomyces 8 Diandromyces 48 Eudimeromj'ces 48 Endosporella 18 Entomocosma Anformortidea 10 Helodiomyces ^/ 54 Ilytheomyces Ji 48 Laboulbeniella '. 8 Laboulbeniopsis 18 Mimeromyces Sphaleromyces 48 Myriapodophila llcrpomyccs 10 Nyctcroinyces 48 Pselaphidomyces Stichomyces 8 Scaphidiomyces 48 85 312 1917 69 11 1920 48 172 1913 69 3 1920 23 180 1912 69 13 1920 48 173 1913 29 506 1917 55 209 1920 55 215 1920 69 16 1920 85 315 1917 29 557 1913 52 704 1917 23 188 1913 69 17 1920 48 163 1913 85 313 1917 52 653 1917 29 662 1917 48 219 1913 48 210 1913 29 671 1917 48 174 1913 48 218 1913 48 177 1913 69 8 1920 48 168 1913 85 314 1917 25 155 1909 A PRESENTATION OF NEW GENERA OF FUNGI 5/ Scelophoromyces 48 Stephanomyces Monicomyccs 8 Sj-nandromyces 48 S>'naptomyces 48 Tengandromyces 48 Termitaria 18 Tetrandromyces 48 Thaxteriola 10 Trenomyces Dimeromyces 54 BASIDIOMYCETKS USTILAGINALES Ustilaginaceae Anthracocystis Ustilago 63 15 53 1912 Mycocoscoma 63 15 50 1912 Uredinales Melampsoraceae Bottyorhiza '. . . Endophyllum 3 4 47 1917 Crossopsora 7 16 243 1918 Endophylloides Endophyllum 3 4 50 1917 Oliveo 41 9 61 1917 Pucciniaceae Alevomyces Uromyces 5 28 190 1914 Anthomycetella '. 7 14 353 1916 Calidion 7 16 42 1918 Caronotelium 7 19 174 1921 Cephalotelium 7 19 165 1921 Chrysocelis 39 5 542 Clenoderma 4 17 103 1919 Cleptomyces Calliospora 18 65 464 1919 Ctenoderma 7 17 102 1919 Cystopsora 7 8 448 1910 Cystotelium Longia 7 19 165 1921 Desmella 7 16 241 1918 Dichlamys 7 19 105 1919 Graveola 7 19 173 1921 G>Tnnotelium 7 19 170 1921 Haplaravenelia 7 19 165 1921 Haplopyxis 7 17 105 1919 Kunkelia 18 63 504 1917 Linkiella 7 19 173 1921 Longia 7 19 165 1921 Miyagia 7 11 107 1913 Nielsenia 7 19 171 1921 Nothoravenelia Ravenelia 7 8 310 1910 NyssopsoreDa 7 19 169 1921 Ontotelium '. 7 19 174 1921 Oplophora 7 19 170 1921 19 175 1921 19 167 1921 19 171 1921 19 172 1921 19 168 1921 19 168 1921 19 169 1921 4 283 1912 17 106 1919 30 78 1914 7 216 1912 18 178 1920 58 O, A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN Peristemma 7 Phragmotelium 7 Pleomeris Nielsenia 7 Sclerotelium 7 Teloconia 7 Trachizsporella 7 Triactella 7 Tricella Calliospora 41 Trocochodium 7 Triphragmiopsis 54 Uredinales Imperfecti Argom^'ces 43 Xenostele 7 Auriculariales Auriculariaceae Hoehnelomyces Pilacrella 16 37 514 1919 Tremellales Tremellaceae Gloeosoma 7 18 51 1920 Phaeotremella 59 5 376 1911 Dacryomycetales Dacr\-omycetaceae Dacrj'opsella ■ 53 124 49 1915 Agaricales Thelephoraceae Duportella Stereum 47 Jaapia 7 Peniophorina Peniophora .53 Hydnaceae Gloiothele 7 H3-dnodon Hydnum 41 Polj^oraceae Echidnodia 54 Ermeria Daedalca 32 Pseudopolj'porus 41 Xanthoporia 41 Agaricaccac Amanitella \manita 7 Catathelasma 18 Chloroph}-llum 43 Chlorosperma 41 Copelandia 32 Lentodicllum 41 Micropsalliota Agaricus 53 10 87 1915 9 428 1911 126 285 1917 18 44 1920 5 297 1913 34 199 1918 51 318 1912 2 93 1910 8 56 1916 11 337 1913 50 383 1910 9 172 1910 14 9<) 1922 53 51 1913 7 216 1915 123 79 1914 9 172 1910 9 171 1910 11 337 1913 63 214 1913 30 424 1914 30 349 1914 A PRESENTATION OF NEW GENERA OF FUNGI 59 Plicaturella 43 Pol}'ozellus 33 Rhodopaxillus Tricholoma 7 Phallales Phallaceae Protophallus Phallogaster 41 2 25 1910 Clathraceae Pharus Lysurus 83 7 60 1919 Hymenogastrales Hysterangiaceae Jaczerwskia 37 Phaeocryptopus Cryptopus 54 Hymenogastraceae Stephanospora Hydnangium 54 Sclerodermataceae Neosaccardia Scleroderma 75 56 6 1921 Lycoperdales Lycoperdaceae Geasteroides Geasteropsis 41 9 271 1917 Lycoperdellon Lycoperdon 20 11 92 1912 FUNGI IMPERFECTI Sphaeropsidales Sphaerioidaceae Amphiciliella 32 Amphorula Kellermania 43 Amylirosa Ephclidium . 10 Angiopomopsis 53 Bakerophoma 7 Botryella 7 Botryogene 7 Botrj'osphaerostroma Diplodia 32 Calopactis 7 Camarographiuni 16 Caudosporella Harknessia 53 Ceratophoma Sphaeronema 32 Ceratopycnis Hendersonia 53 Chaetocytostroma Fusicoccum 7 Chondropodiella 32 Cladochaete 7 Collonaemella 53 Columnothyrium 16 Cornucopiella 53 Cr>'ptorhynchella Sphaerographium 53 Crytosphaerella ' 53 62 58 1920 60 82 1922 90 178 1920 121 407 1912 14 62 1916 14 94 1916 15 259 1917 62 302 1920 10 82 1910 34 306 1916 123 135 1914 59 276 1917 124 86 1915 17 91 1919 59 261 1917 10 318 1912 124 82 1915 34 306 1916 124 118 1915 124 88 1915 126 360 1917 60 O. A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN Cytonaema Cytospora 53 Cytophoma Cytospora 53 Cytoplacosphaeria 7 Cytosphaera 7 Cytostagonospora Stagonospora 7 Cytotriplospora Cytospora 59 Dasypyrena 8 Dasysticta 8 Dearnessia Stagonospora 32 Diplodothiorella Dothiorella 7 Diploplacosphaeria Thoracella 32 Dothorellina Dothiorella 16 Dothisphaeropsis 53 Ectosticta 8 Endogleoea Sirostomella 66 Fumagospora 4 Gamonaemella Gamospora 49 Haplosporidium Pyrenochaete? 8 Hemidothis 7 Hendersonina 38 Herpotrichopsis 53 Jahniella 7 Lasiostroma 54 Leptophoma 53 Lichenophoma Dendrophoma 32 Linochora 53 Linchorella Linchora 7 Malachodermis 32 Macrophomella Macrophoma 7 Mastigosporella Harknissia 53 Myriellina 53 Myxofusicoccum Fusicoccum 7 Neohendersonia Hendersonia 7 Neoplacosphaeria Placosphaeria 7 Neosphaeropsis Sphaeropsis 7 Phacidiopycnis 67 Phaeocytostroma Cytospora 7 Phellostroma 47 Phyllostictina 7 Placodiplodia 16 Placonaemina 7 Placophomopsis Phomopsis 34 Placothyrium Cytosporina 16 PlectonaemcUa 53 Pleocouturea ^^^ 4 Pleosi)haeropsis ' Sphaeropsis 7 Pleuronaema Sphacronema M Pleurophoma Dendrophoma 53 Plcurophomella Dothiorella 53 Polyopeus Phoma 43 Pseudodiplodia Diplodia 10 Pseudohaplosporeila 10 123 131 1914 123 133 1914 17 79 1919 14 205 1916 14 150 1916 7 47 1921 23 109 1912 23 108 1912 58 25 1916 14 151 1916 62 308 1910 29 70 1911 128 616 1919 23 107 1912 5 207 1915 10 326 1911 6 123 1922 23 106 1912 14 95 1916 6 198 123 115 1914 18 123 1920 27 472 1911 124 73 1915 50 296 1910 119 638 1910 10 43 1912 52 344 1912 14 63 1916 123 135 1914 124 100 1915 10 68 1912 19 190 1921 19 74 1921 19 67 1921 22 147 1912 19 45 1921 9 185 1914 14 185 1916 34 305 1916 19 197 1921 59 315 1921 34 302 1916 124 81 1915 10 326 1911 14 203 1916 59 257 1917 123 117 1914 123 123 1914 58 239 1920 90 183 1920 90 192 1920 A PRESENTATION OF NEW GENERA OF FUNGI 61 Pycnis Sclcrophoma 53 Pycnosporium Cicinnobolus 26 Pyrenochaetina 7 Rhodoseptoria 54 Sarcophoma Sclerophoma 53 Sclerochaetella Plendomus 32 Sclerophomella Phoma 32 Sclerophomina Plioma 32 Scleropycnis 7 Sclerosphaeropsis Sphaeropsis 5 Sclerostagonospora Stagonospora 32 Sclerotheca Camarosporium 57 Scleroth>'rium Sclerophoma 32 Scolecosporella Hendersonia 7 Septoriopsis Septoria 41 Sirophoma Phoma 32 Sirospenna 72 Sirosphaera 47 Sirostomella 53 Sphaeriostromella Phomopsis 16 Sphaeronaemina Sphaeronema 32 Sphaerothyrium Sclerophoma 16 Steganopycnis 7 Stenocarpella 7 Subulariella Sphaerographium 53 Traversoa Sphaeropsis 7 Trotteria 87 \"ermiculariopsis Vermicularia 20 Verrucaster 2 Nectrioidaceae Blennoriopsis Sirothyrella 7 Cyanophomella 32 Cyanochyta 53 Dothiorina 53 Gyrostroma 54 Leptodermella 66 Mycorhyuchella 32 Pycnidiella 53 PIenoz3lhia ' 7 Scleropycnium 58 Sirocyphella 53 Stylonectria (Fam. doubtful) 53 Stylonectriella 53 Leptostromiitaceae Chaetopeltiopsis Chactothyrium 19 Didymochora 32 Diedickea Pycnothyriaceae 7 Discosiella Discosia 36 Discothecium 7 Ichnostroma ■ 47 123 129 1914 51 515 1909 14 94 1916 29 178 1913 125 75 1916 59 251 1920 59 237 1917 59 240 1917 9 278 1911 28 209 1914 59 252 1917 11 314 1917 60 181 1918 19 30 1921 11 4 1919 59 257 1917 54 246 1916 8 502 1913 125 75 1916 34 297 1916 59 275 1917 34 299 1916 14 370 1916 15 258 1917 124 118 1917 11 317 1913 10 54 1917 10 1912 21 383 1913 17 92 1919 60 156 1918 124 92 1915 120 464 1911 30 387 1914 5 212 1914 60 135 1918 124 93 1915 14 215 1916 31 5 1912 119 650 1910 124 52 1915 124 53 1915 27 253 1913 60 172 1918 11 268 1913 5 1546 1912 14 371 1916 9 186 1914 62 O. A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN Khekia 32 Lasiothyrium 47 Leptothyrina Leptothyrium 53 Massalongina Leptostroma 16 Peltaster Asterostomula 7 Phaeolabrella Labrella 8 Pirostomella 7 Pleurothyrium Leptostromella 16 Pycnothyrium 7 Rhabdothyrella 53 Rhabdothyrium 53 Rhizothyrium Septothyrella 54 Sirothyriella 53 Sirothyrium 7 Sphaerothyrium Leptostroma? 16 Thyriostroma 7 Trachythyriolum 77 Pachystromaceae Hypodermina 53 Microdiscula 53 Pachydiscula Scleropycnis 66 Rhabdostromella 53 Tryblidiopycnis 53 Xenostroma 53 Excipulaceae Acleistia 59 Acrosporium 16 Bacterexcipula 32 Chaetodiscula 32 Desmopatella 16 Dinemasporiella. . . , Dinemasporium 32 Excipuella Excipula 53 Exotrichum 7 Falcispora 32 Phaeopolynema Excipula 8 Psalidosperma 7 Pseudolachnea Pseudopatella 7 Ramulariospora 5 Stauronema 7 Stictepatella 32 Melanconiales Melanconiaceae Basilocula 7 Cheiroconium 53 Colletotrichella Labrella 53 Cryptosporiopsis 32 Didmosporina Chalara 53 Discosporium 66 Elaedemia 7 62 284 1920 8 503 1913 124 123 1915 34 319 1916 15 261 1917 23 117 1912 12 308 1914 34 322 1916 11 175 1913 126 290 1917 124 125 1915 30 427 1914 119 451 1910 14 218 1916 34 298 1916 11 176 1913 23 523 1918 125 55 1916 124 142 1915 5 210 1914 124 145 1915 127 562 1918 124 149 1915 5 420 1914 29 385 1911 60 161 1918 50 44 1910 37 159 1919 52 385 1912 124 109 1915 12 571 1914 52 269 1912 23 117 1912 12 571 1914 8 393 1910 28 216 1914 14 217 1916 60 166 1918 12 210 1914 119 664 1910 125 S3 1916 52 360 1912 125 83 1916 5 196 1914 20 62 1922 62 318 1920 19 214 1921 13 136 1915 125 108 1916 10 448 1912 14 362 1916 14 345 1916 17 112 1919 A PRESP.XTATION OF NEW GENERA OF FUNGI 63 Gloesporidiella Gloeosporium 32 Gloeosporidina Gloeosporidiiim 7 Heteroceras 7 Marssoniella Marssonina 53 Myrioconium 7 Stegosphaeria Marsonia 7 Titaespora 7 Titaesporina 7 MOXILIALES Moniliaceae Amblyosporiopsis Amblyosporium 49 Beauveria Spicaria 21 Cristulariella Cristularia 53 Gemmophora 16 Helicodendron 44 Hormactinia 32 Oosporoidea Oospora 41 Pachybasidiella .' 7 Polymorphomyces 71 Ramulispora Ramularia 88 Sporoclenia 12 Triposporina . . . 53 V'erticilliastrum Verticilliopsis 22 Dematiaceae Casaresia 80 Ceratosporella 16 Chalaropsis 55 Cheiropodium Clasterosporium 7 Columnophora Chalara 7 Cystodendron 7 Dendryphiella 7 Dichotomella 7 Didymolrichum 53 Endophragmia 54 Eriomenella 44 Harziella Cladosporium. 84 Hormisciopsis Hormiscium 41 Lacellina 7 ]\Ielanopsamella Conytrichium 16 Microbasidium Haplobasidium 7 Muiaria Macrosporium 18 Muiogone Sporodesmium 18 Phialophora 41 Piricauda Stigmella 7 Pleurothecium Acrothecium 16 Septoidium 6 Sirosporium Macrosporium 32 Stigmopsis Stigmella 7 Toruloidea Torula 41 6 127 1922 59 40 1912 125 124 1916 30 478 1912 25 460 1912 57 336 1915 5 53 1913 13 9 1915 24 248 1914 21 56 1921 7 302 1912 121 410 1912 24 302 1912 20 113 1920 37 154 1919 49 584 1916 13 42 1915 14 349 1916 12 212 1914 12 417 1914 12 312 1914 123 139 1914 36 86 1920 25 447 1918 313 460 1916 6 32 1914 11 418 1913 37 112 1919 12 415 1914 58 244 1914 58 239 1914 7 202 1915 12 218 1914 37 154 1919 7 50 1921 52 278 1912 12 218 1914 5 53 1913 64 O. A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN Slilbaceae Calostilbella 16 Cladographium 44 Coeleographium 24 Coremiella 7 Melanographium Sporocybe 7 Phaeostilbella Stilbella 16 Sporostachys 87 Stilbodendron 7 Synnematium Hirsutella 41 Tuberculariaceae Amphichaete Chaetospermum 53 Anomomyces 16 Cheiromycella 55 Clathrococcum Epicoccum 53 Discofusarium 59 Exosporella 53 Leucodochium 7 Marcosia 7 Periolopsis 9 Petrakia 7 Phanerocorynella 16 Sigmatomyces 7 Sirodochiella 7 Thyrostroma Epicoccum 53 Tuberculariella 66 Verticilliodochium Tubercularia 7 Xiphomyces 7 The following fungi are of UNKNOWN AFFINITY. Graphiolaceae Stylina 7 18 192 1920 Coccoidaceae Coccodiella 19 25 222 191 1 ASCOMYCETES Haplostroma 7 Konenia 19 Melanomyces Pseudoparodia 7 Miyakeamyces 19 Solanclia 40 FUNGI IMPERFECTI Chlamydosporium ii Gloeodes 60 Menezesia 20 Nothospora ' 33 Saprophorum (Hyphomycete) 72 Sirostomella 53 Spirospora 54 37 154 1919 25 439 1918 2 13 1920 10 52 1912 11 558 1913 37 153 1919 10 54 1917 14 260 1916 12 74 1920 123 142 1914 37 153 1919 119 864 1910 120 473 1911 7 164 1920 121 414 1912 15 266 1917 14 96 1916 11 357 1913 11 407 1913 37 157 1919 11 319 1913 19 144 1921 120 472 1911 5 209 1914 12 220 1914 14 374 1916 14 80 1916 27 250 1913 15 196 1917 27 248 1913 3 268 1910 18 1913 13 157 1920 9 1913 20 1913 54 246 1916 125 78 1916 36 96 1920 15 258 1917 73 1910 37 158 1915 58 249 1914 31 1911 58 247 1914 114 1911 54 220 1916 90 184 1920 37 155 1919 18 1912 23 93 1918 178 1911 374 1910 149 239 26 798 1912 30 424 1914 17 142 1919 17 465 1913 52 21 1919 17 39 1919 65 245 1918 A PRESENTATION OF NEW GENERA OF FUNGI 65 StenocarpcUa 7 Trichodiscula 51 No knowfedge as to position Alichora 16 Amphoromorpha 18 Candelospora 50 Canthransiopsis 18 Cryptoacsus 56 Diploospora 34 Ephelidium .10 Fusicladiella Carlia 16 Heptasporium 63 Mitopeltis 77 Neofabraea Mollisiaceae 17 Parendomyces 25 Peltomyces 27 Pericystis 9 Phaeocryptopus Cryptopus 79 Plenophysa 7 Rachisia 30 Rachodiella 55 Xenopeltis 7 Mycelia Sterilia Graillomyces 18 BIBLIOGRAPHY 1. Abhandlungen Zool.-Bot. Gesellschaft Wein. 2. Abhandlungen Naturwissen. ver Bremen. 3. American Journal of Botany. 4. Annales de I'Ecole d'Agriculture de Montpelier. 5. Annalen des K. K. Naturhistorischen Hofmuseums in Wein. 6. Annales des Epiphytes. 7. Annales Mycologici. 8. Annales del Museo Nacional de Historia Natural de Buenos Aires. 9. Annals of Botany. 10. Annales Sociedad Cientifica Argentina. 11. Arkiv. fiir Botanik. 12. Arch, fiir Hydrobiol. und Plank tonk. 13. Archiv. fiir Protistenkunde. 14. Atti Inst. Bot. Univ. Pavia. 15. Beihefte Botanisches Centralblatt. 16. Berichten der Deutschen Botanischen Gesellschaft. 17. Biennial Crop Pest and Hort. Report Oregon Agricultural Experiment Station. 18. Botanical Gazette. 19. Botanical Magazine of Tokyo. 20. Broteria. Botanical Series. 21. Bulletin de la Societe Botanique de France. 22. Bui. de la Soc. de Bot. Geneve. 23. Bui. Intemat. Ac. Sci. Boheme. 24. Bui. du Jardin Botanique de Buitenzorg. 66 O. A. PLUNKETT, P. A. YOUNG, AND RUTH W. RYAN 25. Bul. Soc. France Dermat. et Syphil. 26. Centialblatt fiir Bakteriologie, Parasitenkunde, und Infektionskranheiten. 27. Compte Rendu de I'Academie Sci. de Paris. 28. Compte Rendu de la Socicte Biologique de Paris. 29. Die Wurzelpilze die Orchideen. 1909. 30. Deutsche Essingundustrie. 31. Fungi Chilensis. 32. Hedwigia. 33. Inaug-Dissert Padova. 34. Journal of Botany. 35. Journal of Economic Biology. 36. Leaflets of Philippine Botan}'. 37. Memoire della Reale Accademia delle Scienz di Torino. 38. Memoirs of the Department of Agriculture in India. 39. Mem. Soc. neuchat. Sci. Nat. Vienna. 40. JNIonatshefte fiir Landwirtschaft. 41. Mycologia. 42. Mycologisches Centralblatt. 43. North American Flora. 44. Nuovo Giomale Botanico Italiano. 45. Ohio Naturalist. 46. Osterreichische Botanische Zeitschrift. 47. Philippine Journal of Science. 48. Proceedings of the American .\cadem}- for the Advancement of Science. 49. Proceedings of the Rochester Academy of Science. 50. Proceedings of the Royal Irish Academ}'. 51. Recherches Lichens Dunquerque. 52. Scientific Proceedings of the Royal Dublin Society. 53. Sitzenberichte der Kaiserlichen Akademieder Wissenschaften Wein. ^Mathematisch- NaturwissenschaftUche Klasse. 54. Societe JNIycologique de France, Bulletin de la. 55. Staz. Sper. Agar. Ital. 56. Studi :\Ialitti Olivo. Roma. 1911. 57. Svensk Botanisk Tidskrift. 58. Transactions of the American ilicroscopical Society. 59. Transactions of the British Mycological Society. 60. Transactions of the Illinois State Academy of Science. 61. Transactions of the Royal Society of South Africa. 62. University of California. Publications in Botany. 63. Untersuch. gesamt. Gcbiet. Mykol. 64. Vidensk. Selsk. Skrift. Mat.-Nat. Kl. 65. Wochenschr. fiir Brauerie. 66. Zeitschrift fiir Garungsphysiologie. 67. Zeitschrift fiir Pflanzenkrankheilen. 68. Botanisk Tidsskrift. 69. Phytopaihology. 70. Vcr. Zool.-Bol. Gcsellschaft Wein. 71. Revue Generalc Botanique. 72. Englcr's Botanisches Jahresbericht. 73. Sitzenberichte Kais. Bohn. Ges. Wiss. Math, Classe. 74. BuUetino dell'otro Botanico. di Napoli. A PRESENTATION OF NEW GENERA OF FUNGI 67 75. Atti della Reale Accademia delle Scienze di Torino. 76. Bui. Dep't. Landb. Suriname. 77. Boletin de la Accademia Nac. de Ciencias en Cordoba. 80. Bol. Real. Soc. Esp. Hist. Nat. 82. Journal of Agricultural Research. 83. Ann. Roy. Bot. Gar. Perademja. 84. Atti Reale .\ccad. Lincei. 85. Nova -Acta Reg. Soc. Sci. Upsal. 86. South African Journal of Science. 87. .\tti dell'Acad. Veneto-Trentino-Istriana. 88. Bui. S. Manchuria R. R. Co., .Agr. E.xp. Sta. (Kunchu-ling, Manchuria.) 89. Resultados de la Primera E.xpedicion a Tierra del Fuego (1921) (Univ. Nac. Buenos Aires.) DEPARTMENT OF METHODS, REVIEWS, ABSTRACTS, AND BRIEFER ARTICLES HEMISTOMUM CONFUSUM, A HOMONYM By John E. Guberlet Parasitologist, Oklahoma Agricultural Experiment Station Dr. Maurice C. Hall recently called the attention of the writer to the use of a homonym in connection with the description and naming of a new species of holostome, Hemistomum confiisum Guberlet, 1922 (Jour. Parasit., 9:6-14). The specific name conjusum is a homonym of a species described by Krause, 1914, (Ztschr. f. Wissensch. Zool., Leipz. & Berl., 112:93-238), whose work on holostomes was unknown to me during my investigations. Therefore, a new specific name must be submitted for my species, for which I propose indistincta in place of confusum. Hall and Wigdor, 1918 (Jour. Am. Vet. Med. Assoc, 53:616-626), whose work was overlooked by me, were also apparently unaware of Krause's work but arrived at the same conclusion as Krause relative to the nomenclature of the genus Hemistomum Diesing, 1850. In both works it is shown that the name Hemistomum is not in good standing and that the name Alaria Schrank, 1788, is the correct generic term with alala Goeze, 1782, as the specific name for the type species. Therefore, according to priority, the combination should be Alaria aJata (Goeze, 1782) Krause, 1914. In view of the foregoing and following the suggestion of Hall by com- munication, I wish to substitute the generic name Alaria, in place of Hemistomum, for the holostomes recently described by me (1922), i.e. H. gavium and H. indistincta. Hence the substitution will change the names to Alaria gavia and .4. indistincta. 68 THE USE OF SODIUM SILICATE AS A MOUNTING MEDIUM By Charles W. Creaser and William J. Clench Museum of Zoology, Universily of Michigan In preparing slides for use in two widely different fields the writers have had occasion to use a solution of sodium silicate (water-glass) as a mounting medium. Several investigators have used this substance but there seems to be no literature on the subject. Mr. W. F. Clapp of Cam- bridge, Massachusetts, has used it in mounting the radulae of certain mollusks. Through a knowledge of his use of sodium silicate as a mounting medium, the present development had its inception. Several investigators have inquired about the methods best adapted for working with it, and its availability for their problems. Our experience with this substance is made known here. Sodium silicate is soluble in water, and basic in reaction, therefore, it can only be used with basic stains. Fish scales may be mounted as total objects from water without staining. In mounting radulae of certain fresh water mollusks, basic fuchsin has given very good results. For the stock of sodium silicate, we have used that sold by the ordinary drug store for preservation of eggs. We find it as good as any of the more carefully prepared solutions. Care must be used in the selection of the stock since this substance not uncommonly contains a fine white precipitate in suspension. This kind of material produces a mount with a milky or clouded appearance. Sodium silicate may be kept in the ordinary balsam bottle, but it should not be stored in glass-stoppered bottles. The ordinary water solution of sodium silicate, in its commercial form, may be used as described here. It has, however, certain defects that render the slides unfit for use after approximately three months. We have used it to prepare mounts that are to be studied in a vertical position. By the use of this material, solid, durable mounts may be produced which do not melt or run when heated and in a very short time may be used in a vertical position. It is for this specific purpose that we have found sodium silicate very useful. Objects mounted in glycerine- jelly will not stay in position under the influence of heat and gravity until after they have been prepared for some time. Glycerine jelly and similar media have a tendency to clear, but it is necessary to avoid this effect in fish scales and moUusk radulae. In field work where it is difficult 69 70 CHARLES W. CREASER AND WILLIAM J. CLENCH to make mounts with a medium that must be heated the use of a cold solution of sodium silicate has much in its favor. Optically, these slides are excellent in revealing the characters of fish scales. It has been found that mounts made, of the commercial solution of sodium silicate are not permanent since this medium will crystallize in three or four months. However, this difficulty seems to have been over- come by the use of glycerine in connection with the sodium silicate. Our slides, made according to this revised formula, have not been kept long enough to show what the ultimate outcome will be, but after several months they show no tendency to crystallize. The fish scales are placed in water, cleaned, and allowed to soak for a time. They are then transferred to clean water or to dilute solutions of glycerine or of sodium silicate. Mounting from either water or glycerine into sodium silicate has been found very satisfactory. Care must be used that the object is free from all alcohol as a trace of this will cause a white precipitate to form over the object when it is placed in the sodium silicate. It has been our practice to put the sodium silicate solution on the slide by the use of a solid glass rod or to pour it direct from a small bottle. The medium should be spread over most of the area to be covered by the cover glass. The objects are then transferred from the water or the dilute solutions of water-glass or glycerine before they dry and placed in the desired position on the slide. Objects may be placed on the slide lirst in a small drop of water, the water-glass and the cover glass being put on afterwards. Since the water solution of sodium silicate quickly forms a tough film over the exposed surface much care and speed must be used in placing on the cover glass, in order to prevent air bubbles and to insure the spread- ing of the medium to all parts of the area under the cover glass. The slides are allowed to set. Air bubbles will work their way out if the slide is at a very slight angle during this time. These slides will set in two or three days and can then be used in a vertical position and the objects will not be displaced by heat or gravity. Cleaned radulae are transferred directly from water to a water soluble, basic stain and then returned to water where the excess stain is removed. The" are then mounted directly in a drop of water-glass in the same manner as are the fish scales. Ringing the slides in the ordinary manner will insure greater permanence in these mounts. M Glycerine sodium silicate may be prepared as follows: To a solution of commercial sodium silicate, slightly diluted glycerine is added and the liquids mixed by shaking. The proportions may vary but the best results are obtained by using twelve parts of water-glass to one part of glycerine. If the mixture does not go into a homogeneous solution a little water should be added. This medium does not set as rapidly as the sodium sili- SODIUM SILICATE AS A iMOUNTING MEDIUM 71 cate solution and is therefore easier to handle. Objects may be mounted in it in the manner described above. An excess of medium may be removed with warm water and discarded slides placed in water may be easily cleaned after a few hours. Slides prepared from this medium are excellent for the study of fish scales and mollusk radulae. They seem to be quite permanent. NEW POCKET DISSECTING MICROSCOPE E. G. Campbell of Purdue University has described and illustrated (Science, 192v3, 57:179-180) a simple, efficient instrument for the examina- tion of small objects in the field. Focusing, rotation of the object, and dissection may all be performed simultaneously and with ease as the microscope is held in the hands of the observer. The pocket adjustment of the instrument provides not only for concealment and protection of the working parts but also storage space for dissecting instruments. Change from pocket to working adjustment is simple and quickly accom- plished. Two figures show clearly the details of construction of this instrument. THE PHYSIOLOGY OF REPRODUCTION, by F. H. A. Marshall. Second revised edition; 770 pages, 189 illustrations. Longmans, Green and Co., London and New York, 1922. Price $12.00. Since Dr. Marshall's "Physiology of Reproduction" first appeared it has been the standard and very useful text and reference book in its field. The excellent arrangement of its materials, its brevity of statement, and the clearness and cogency of its summaries have made it one of the most satisfying and dependable reviews of knowledge in any field of comparative biology. The manner of handling the references to original papers is adequate, and gives a historical view without being overwhelming. The new edition loses none of the admirable qualities of the first and brings the survey up to date by recording the progress of the last twelve years. Aside from such detailed revision certain large topics have been rewritten and elaborated. Some of these are: The Biochemistry of th^ sexual organs; Changes in the maternal organism during pregnancy; Fertilization; Internal secretory functions of the reproductive glands; Sex-determination, and the causes of birth. A special discussion is included of Child's theory of rejuvenescence and senescence as these bear upon certain types of reproductive and life cycles not so well explained by the theory of the continuity of germplasm and the segregation of germ cells. Aside from its indispensable use to all teachers and students of biology, zoology, and physiology, the book is of high value to gynecologists, veterina- rians, and animal breeders. Subject and author's indices are excellent; the printing, illustrating, and mechanical appearance are worthy of the matter. T. W. Galloway 72 MARY ALLARD ROOTH September, 8, 1843 September, 15, 1922 Ne^^r has a family motto been typified so truly as in the life of Mary Allard Booth. "What I hope to accomplish I shall accomplish" seems to have been the daily inspiration which aided her, in spite of manv obstacles, to the highest achievement in the labors undertaken. Born at Longmeadow, Massachusetts, on September 8th, 1843 the years of her childhood and young womanhood were a record of ill health and affliction, and during her early education in the local schools, much of tiie work and play of ordinary youth were denied her. In speaking of her family, Miss Booth said, "My mother's people were a literary family, the Bartons of Vermont, of whom Clara Barton, Presi- dent of the Red Cross, was one. From my father I inherited mv love of science. During a visit to the shores of Long Island Miss Booth's attention was first directed to the interesting forms of aquatic life. While seated in her wheel chair she watched a woman near bv who was intent upon gathering sea weeds. The woman was Miss Mary Halliday of Brooklyn, and during the chance acquaintance which the occasion offered. Miss Halliday took pleasure m explaining to the invalid girl the wonderful structure and life history of some of the marine algae. This incident seemed to be just what was needful to awaken the interest which her father had long sought to arouse, and with his help and knowl- edge at her constant disposal she began the work which was to fill her long life with an unfailing enthusiasm and which bv painstaking application placed her at last in the foremost rank of scientific workers. In 1877 she purchased her first compound microscope and step bv step advanced into the wonderland which it disclosed. The delicate manipu- lation necessary in the work, the skill in preparation and mounting of specimens were the result of long and patient endeavor due to love of the work. No object in the field of nature was too minute for careful study VA/hen the wonders of this new world were thus revealed to her. Miss Booth sought some method by which she could make this microcosmos intelli- gible to others and there the camera seemed to fill the required need. With microscope and camera, then, she was able to make of vast impor- tance to humanity the line of study so assiduouslv pursued. When the invasion of bubonic plague seemed imminent in this country Surgeon 73 74 BESSIE PERRAULT TITUS General Blue turned to Miss Booth with a request for photomicrographs of the plague fleas infesting rats in San Francisco, and these photographs were used in a nation-wide lecture campaign for the extermination of the plague. Not only this Government but those of France and Fngland as M AKY A. HocJTII well sought her aid in the tight against such subtle enemies. Many infini- tesimal creatures were sent on the long journey to her laboratory in Spring- field, Massachusetts, there to be photographed and studied scientifically. MARY ALLARD BOOTH 75 All the steps in the painstaking process from the preparation of the object, proper staining to emphasize structural detail, delicate mounting on microscopical slide, photographing, developing the negative and making the prints, were performed by this patient worker, and each picture was a masterpiece of its kind. And in the midst of all this patient endeavor and accomplishment, Miss Booth found time to make for herself an honored place in the lecture field and editorship of several scientific journals. The great esteem in which she was held at home and abroad is evidenced by the honors conferred upon her. Few women have shared with her the distinction of fellowship in the American Association for the Advancement of Science, and she was likewise one of the few women elected a fellow of the Royal Microscopical Society of London. As a higher reward for her humanitarian services the privilege was given her to continue her activity in full strength and vigor of mind up to the very close of her life. Her latest photographs were those of a family of busy little squirrels who made their home in a neighboring tree and who had learned to know and love the good friend who cared for them so faithfully. Indeed, as was a fitting close for such a lover of nature her last act on earth was the carry- ing of the evening meal to these little creatures; and in the great out-of- doors, at the close of a September day, surrounded by these appreciative friends, she passed into the great beyond. Bessie Perrault Titus PROCEEDINGS OF THE AMERICAN MICROSCOPICAL SOCIETY Minutes of the Boston Meeting The 41st annual meeting of the American Microscopical Society was held in affiliation with the American Association for the Advancement of Science at Boston, Mass., December 28, 1922. President N. A. Cobb presided at this meeting. The report of the Treasurer for the year 1922 was read by the Secretary and was referred to an Auditing Committee composed of Professors F. H. Krecker and J. W. Kostir. The report of the Custodian was read by the Secretary and was referred to an auditing committee composed of Messrs. Edw. Pennock and F. E. Ives. The meeting voted to send hearty congratulations to the Custodian on the growth of the Spencer-Tolles Fund. The Secretary presented a report on the affairs of his office, this report covering the previous three years. The following officers were nominated and elected: President, Professor Chancey Juday, University of Wisconsin, Madison, Wis.; First Vice-President, Dr. B. H. Ransom, Bureau of Animal Industry, Washington, D. C; Second Vice-President, Dr. W. W. Cort, Johns Hopkins University, Baltimore, Md.; Secretary, Professor Paul S. Welch, University of Michigan, Ann Arbor, Mich.; Treasurer, Dr. Wm. F. Henderson (for two years), University of Pittsburgh, Pittsburgh, Pa. Professor Geo. R. La Rue, University of Michigan; Professor Z. P. Metcalf, North Carolina State College of Agriculture and Engineering, and Professor E. M. Gilbert, Univer- sity of Wisconsin, were chosen as the elective members of the Executive Committee for 1923. Dr. B. H. Ransom, Bureau of Animal Industry, was chosen as a representative of the Society on the Council of the American Association for the Advancement of Science. Dr. N. A. Cobb, Bureau of Plant Industry, was appointed as a member of the Spencer- Tolles Fund Committee. Adjourned. Paul S. Welch, Secretary. CUSTODIAN'S REPORT FOR THE YEAR 1922 Spencer-Tolles Fund Balance reported for the yeaj 1921 S9104 . 56 Interest on Bonds 250.00 Dividends Penna. R. R. Co 96.75 *Build'g. & Loan Ass'n 144. 16 490.91 9595.47 Less Grant to Wm. P. Hayes 60 00 95,>5 47 Net Increase during the year S4.?0.91 *Estimates, proved correct by letter of B. & L. Ass'n. of Dec. 19-"22. M.P . 76 PROCEEDINGS OF THE AMERICAN MICROSCOPICAL SOCIETY / / Totals Receipts All contributions 802 .03 All sales 1193.38 • All life memberships 300.00 All interest, dividends, profits 7590 . 06 9885.47 Disbursements All Grants 310.00 All life membership dues 40.00 350.00 9535.47 Investments Stock in Keystone State Bldg. & L. Ass'n 2385 .47 Bonds, Rio Grande Junction R'y 5000.00 Stock, 43 shares Penna. R. R. Co 2150.00 9535.47 Life members: (Robert Brown, dec'd.); J. Stanford Brown, Seth Bunker Capp, Harry B. Duncanson, A. H. Elliott (dec'd.) and John Hately (dec'd.). Contributions of $50 and over: John Aspinwall, Iron City Microscopical Society, Magnus Pflaum and Troy Scientific Society. (signed) Magnus Pflaum Custodian Philadelphia, Pa. Dec. 30, 1922 Philadelphia, Feb. 1st. 1923 Having examined the above account, and the securities on hand as shown therein, we find jj^em correct. F. E. Ives Edward Pennock ANNUAL REPORT OF THE TREASURER OF THE AMERICAN MICROSCOPICAL SOCIETY December 24, 1921 to December 13, 1922 Receipts Balance on hand, December 24, 1921 $ 592 . 71 Dues received for Volume 40 or before 45 . 00 Dues received for Volume 41 170 . 10 Dues received for Volume 42 252 . 00 Dues received for Volume 43 2 . 00 Initiation Fees 24 . 00 Subscriptions for Volume 40 or before 3 . 00 Subscriptions for Volume 41 315.02 Subscriptions for V' olume 42 54 . 00 Sales of Transactions, duplicates, back numbers 26.21 Advertising, Volume 39 10 . 00 Advertising, Volume 40 250.00 Authors, for preparation of plates 34 . 30 Grant, from Spencer-Tolles Fund 60 . 00 Sundries , -50 Total S1838.84 78 PROCEEDINGS OF THE AxMERICAN MICROSCOPICAL SOCIETY Expenditures Printing Transactions, Volume 40, No. 4 $ 224.02 Printing Transactions, Volume 41, No. 1 257 . 16 Printing Transactions, Volume 41, No. 2 256. 77 Printing Transactions, Volume 41, No. 3 200.05 Postage and Express for Secretary 21 . 00 Postage and Express for Treasurer 13 .00 Office expenses of Secretary 47 . 82 Ofifice expenses of Treasurer 28 . 65 Secretary, trip to Toronto 39 . 89 Balance on hand 750 . 48 Total $1838.84 December 13, 1922. \V. V. Henderson', Treasurer. Report of the Auditing Committee of the American Microscopical Society The accounts of W. F. Henderson, Treasurer of the American Microscopical Society, for the period beginning December 24, 1921, and ending Dec. 13, 1922, have been examined by the Auditing Committee and have been found to be correct. Respectfully submitted, W. J. KOSTIR Feb. 28, 1923. F. H. Krecker TRANSACTIONS OF THE American Microscopical Society Organized 1878 Incorporated 1891 PUBLISHED QUARTERLY KY THE SOCIKTV EDITED BY THE SECRETARY PAUL S. WELCH ANN ARMOR, MICHIC.AN VOLUME XLIl Number Three Entered as Second-class Matter August 13, 1918, at the Post-office at ^lenasha, Wisconsin, under Act of March 3, 1879. Acceptance for mailing at the special rate of postage provided for in Section 1103, of the Act of October 3, 1917, authorized Oct. 21, 1918 W^i OlolUsiatc Prcaa GEORGE BANTA PUBLISHING COMPANY MENASHA, WISCONSIN 1923 TABLE OF CONTEXTS For Volume XLTI, Number 2, April, 1923 The Distribution of Frog Parasites of the Douglas Lake Region, Michigan, by Harry C. Fortner 79 New Records of North American Enchytraeidae, by Paul S. Welch 91 Primitive Microscopes and Some Early Observations, by William A. Locy 95 An Illuminating Device for Microscopes, by William A. Beck 108 Abnormal Specimens of Helodrilus caliginosus, trapezoides (Duges) and Hclodrilus roseus (Savigny), by Bess R. Green 122 Department of Methods, Reviews, Abstracts and Briefer Articles A Study of the Stability of Staining Solutions, by F. L. Pickett 129 Modern Microscopy, a Handbook for Beginners and Students, a review by George R. La Rue 133 TRANSACTIONS OF American Microscopical Society (Published in Quarterly Instalments) Vol. XLII APRIL, 1923 No. 2 THE DISTRIBUTION OF FROG PARASITES OF THE DOUGLAS LAKE REGION, MICHIGAN^ By Harry C. Fortner University of Vermont Introduction During the summers of 1917 and 1919 the writer made a study of the parasites of frogs and their distribution in the Douglas Lake Region, Cheboygan County, Michigan. It was thought that a study of the distri- bution of frog parasites, including detailed statistical data, might present some interesting as well as valuable information. Three species of frogs were examined within a limited area, thus furnishing data contributing to the geographical distribution of frog parasites. In all, two hundred eight hosts were examined from eleven local stations (See Map Fig. 1.). The collections were made during the months of July and August of the two years, 1917 and 1919. Some differences occurred in the two seasons which are worth noting and these will be stressed in the discussion. The particular habitats from which hosts were taken are described below. Bryant's Bog is a typical bog association with plant life slowly encroach- ing upon the water. As a matter of fact, very few frogs inhabit this place. So few frogs were taken there that a lengthy description of the habitat is not warranted. Carp Creek is a short, rapid, spring-fed trout stream. The majority of specimens taken from this habitat came from the roadside among the grass which grew around two large moss-covered logs. A few were taken from an abandoned road along the stream where mosses and liverworts were abundant. On account of the swift current, breeding could take place here only in the side pools. In 1919, but two specimens were taken in this locality from the roadside. The vegetation of the abandoned road was dense and practically no open places existed as in 1917. Fairy Island furnished but one specimen each season. Here the shore and bottom consisted mostly of gravel. Sedges were the only vegetation ' Contribution from the University of Michigan Biological Station. 79 80 HARRY C. FORTNER along shore, but they afiforded resting places for several species of Cole- optera, Hemiptera, and Hymenoptera, which served as food for the frogs. Lancaster Lake is a small body of water near Douglas Lake, having an area of several acres. A small stream, Bessey Creek, connects Lancaster Lake with Douglas Lake, thus presenting a very easy path of migration for frogs from one lake to the other. The shore was thickly matted with Fig. 1. Collecting Stations sedges. Small bushy willows provided a shaded shelter between the sedges and the edge of the wooded area. It was here that most specimens were taken. The soil between the water and the wooded area consisted mostly of muck. Small depressions were present in which numerous aquatic insects thrived. There was practically no visible change in this habitat since 1917. Maple River, the outlet of Douglas Lake, runs tiirough a comparatively level stretch of country. Scattered along its course are numerous bayous FROG PARASITES OF DOUGLAS LAKE 81 affording excellent breeding places for frogs. Sedges and grasses were very dense and stumps and roots of trees lined the banks. Cat-tail societies were also present. The bottom was muddy or sandy, and at places stones and gravel were prevalent. Deep holes and shallows were common. North Fish Tail Bay (N. F. T. Bay in tables), an arm of Douglas Lake, presents a habitat consisting of a level sandy shore with a thick growth of sedges. The bottom consists of sandy marl and muck. A few potomogetons and some chara are present in the water. Here the frogs have access to many species of insects and some few snails. Trout Creek is a small trout stream rising in bogs to the north and east of Douglas Lake. The vegetation along this stream is varied. Typical roadside plants and trees are present along the banks of the stream. Small areas of water lilies occur in quiet stretches of water. Algae are also present. Grasses overhang the water in many places. At some places the bottom consists of muck; at others gravel forms the main constituent of the bed of the stream. The stream is swift in some few places; at others it is slowly running or even stagnant. Some places are in dense shade and others in bright sunlight. The depth of the water varies from a few inches to a foot and a half. Reese^s Bog is a large Thuja bog on the north shore of Burt Lake. Conditions in this bog had undergone considerable change between the years 1917 and 1919. At one place where many specimens were taken in 1917 moisture was lacking in 1919 and very few frogs were taken. At another place the grass had become very dense and it was too dry for a frog habitat. In still another area certain mosses and grasses had become denser, thus holding back more of the surface drainage which made the habitat too moist for certain kinds of insects. On slightly higher ground conditions were more favorable but frogs were not as abundant as in 1917. The Rana clamitans which were taken here were found near the water and in secluded and well sheltered spots. Rana pipiens wanders a considerable distance from the stream in contrast to Rana clamitans, which is always found near the main body of water. Sedge Point is situated on the north shore of Douglas Lake, not far from the western margin of North Fish Tail Bay. Here several pools are cut off from the lake as a result of wave action. Around these pools there was a zone of water plants. One pool usually containing water was devoid of water in 1919, but the soil was somewhat moist. Here insect life was very abundant. Snails, too, were very numerous, eight species being present; these no doubt figuring largely in the life histories of many of the parasites. South Fish Tail Bay (S. F. T. Bay in tables) is the site of the Biological Station. Here the shore is sandy. There is a very scant growth of plants in the water, and but few sedges along the shore. Very few frogs make this a permanent habitat. 82 HARRY C. rORTNER Sunny Strand is a sedge-covered beach situated at the north-western part of Douglas Lake. The water along this shore is very shallow for fifteen or twenty yards out. On account of the direction of the prevailing winds this is a sheltered shore. No hosts were taken from this place in 1917, but in 1919 four were taken. Discussion Three species of hosts were examined, one hundred seventy-seven specimens of Rana pipiens, twenty-nine Rana clamitans, and two Rana cantabrigensis, making a total of two hundred eight. No Rana catesbiana were seen at any time during the two summers, altho they are recorded for the region. Of the number collected, seven individuals, all of small size, seemed to be entirely free from parasites. A total of ten species, excluding the nematodes, parasitic in frogs were found. Six, however, was the highest number of species found in any individual host. The nematodes are in the hands of an expert and their distribution will have to be reported at a later date. It might be interesting to note that when frogs were fed daily in captivity, then examined, no loss of intestinal and urinary bladder para- sites were noted. Loss occurred, however, where no feeding was done within 12-24 hours, and the parasites were recovered from the bottom of the aquarium. Table No. I shows the number of hosts taken from each habitat in each of the two seasons. Table No. I Number of Hosts Taken from Each Habitat R. pi 1917 pi ens 1919 R. clai 1917 nitans 1919 R. cant a 1917 Wigensis 1919 Bryant's Bog 3 19 1 7 11 1 4 20 1 9 1 1 Carp Creek Fairy Island 2 1 12 28 7 3 22 20 2 4 Lancaster Lake 1 16 Maple River 6 1 N. F.T.Bay Trout Creek 1 2 Reese's Bog Sedge Point 1 1 S. F. T Bay Sunny Strand Total collected 76 101 20 9 2 Grand total 208 FROG PARASITES OF DOUGLAS LAKE 83 Table No. II shows the parasites found and their local distribution. Table No. II Parasites Found and Habitat Distribution Octomitus intestinalis Prowazek Opalina obtrigonoidea Metcalf (An as yet un- published species) Nyctotherus cordiformis Stein Diplodiscus temperatus Stafford Gorgoderina attenuata Stafford Pneumoneces medioplexus Stafford Cephalogonimus americanus Stafford Clinostomum attenuatum Cort Proteocephalidae Pneumoneces similiplexus Stafford M a; ►5 pa n Pi (X, a 3 C/2 The identification of trematodes was confirmed by Dr. W. W. Cort; the Opalina by Dr. M. M. Metcalf; and the other forms by Dr. G. R. LaRue. Octomitus was present in the Rana cantabrigensis from Maple River, and Opalina and a lung nematode were found in the host from Sedge Point. Octomitus and Opalina were present in all localities. Nyctotherus was not secured in frogs from three habitats, probably due to the small number of hosts taken. Specimens of Diplodiscus were not taken from five of the eleven localities. Their absence from collections made at Lancaster Lake and Sedge Point is not to be explained on the basis of small numbers of hosts examined since large numbers of hosts were taken from those localities. Gorgoderina seems to be evenly distributed. The heaviest infection of this parasite noted was fourteen from a single specimen. Specimens of Pneumoneces medioplexus were taken from all localities except North Fish Tail Bay. There is a possibility of its being present there also, as but eight hosts were examined from that region. The heaviest infection with this species in any one frog was from the Maple River habitat, one lung containing thirty-eight, the other thirty-four. Another host from Sedge Point had thirty-six in one lung and thirty-four in the other. Pneumoneces similiplexus was taken only in the one locality, Maple River. Six specimens of Cephalogonimus were taken during the two summers from, three hosts and two localities. Since these two localities 84 HARRY C. rORTNER are not far apart it would be possible for hosts to migrate readily from one of these habitats to the other in a short time, and thus account for its presence in both places. Clinostomuni attenualum was taken in the same year, 1917, from but two hosts, which were very heavily infested. Proteo- cephalidae were obtained from three stations from both Rana pipiens and Rana clamitans. Protozoan parasites alone infested the very small frogs which leads one to believe that the frogs become infested with the metazoan parasites when they feed on animal life. An exception to this fact, however, is that Dr. L. R. Cary found Diplodiscus temperatus in tadpoles. Any one of the protozoans, Opalina, Nyctotherus, and Octomitus, does not seem to be inconvenienced by the presence of the other forms, as nine hosts examined contained hundreds of all three species. One of the striking things about the Protozoa is the fact that Opalinae were found in only one specimen of Rana clamitans of the twenty-nine examined, while the percentage of infestation of Rana pipiens with this organism is 61 and 80 for the two summers respectively. Why the infestation differs in the two species is a question that cannot be answered until more exami- nations have been made paying particular attention to the other inhabitants of the habitat which may serve as food and act as carriers. Dr. R. W. Hegner, in an article published since these notes were written, has pointed out that while the tadpoles of Rana clamitans are infected with Opalinae the adults very rarely are infected. A sufficient number of frogs were not collected at the Sedge Point locality in 1917 to make a fair comparison with those collected during 1919. Collections made at this habitat might shed some light on the adult forms of the cercariae present there. Extensive studies have been and are being made on the cercariae of this habitat. For instance no D. temperatus were found there, and we might conclude if larger numbers of hosts were examined and none found that the cercariae of that form do not exist there. In all the examinations no Acanthocephali were found. This corrobo- rates statements of other investigators who have found a very limited number upon examination of similar hosts in North America. A comparison of the species of the parasites found in Rana pipiens and Rana clamitans is made in Table III. As mentioned before, it is a remarkable fact that Rana clamitans is so lightly infested with Opalina. Diplodiscus, Pneiimoneces medio plexus, Cephalogonimus, and at least three species of nematodes were present in Rana pipiens and not found at all in Rana clamitans. Another striking comparison is the relatively low infestation of Rana pipiens with Pneiimo- neces similiplexus and Proteocephalidae as compared with that of Rana clamitans. FROG PARASITES OF DOUGLAS LAKE 85 Table No. Ill Comparison of Infestation of Two Species of Frogs Expressed in Percentages of Infested Individuals to Entire Number Examined Rana Rana pipicus damitans 1917 Rana Rana pipiens damitans 1919 Octomitus Opalina Nyctotherus Diplodiscus Gorgoderina Pneumoneces medioplexus Pneumoneces similiplexus. Cephalogonimus Clinostomum Proteocephaiidae 30 61 2 30 51 5 1 3 2 0 18 5 15 0 50 0 20 0 0 10 48 80 22 1 38 30 0.9 0 0 1 0 22 0 66 0 11 0 0 0 As is shown in Table IV there is not enough variation in infection between the sexes to justify the consideration of each sex separately. The males and females live under the same conditions and in the same habitat, and, as would be expected, the percentage of infection between the two sexes does not differ to any great extent. Table No. IV Percentage of Infection of Total Number of Frogs (112) Collected During the Year 1919 57 Females 55 Males 112 Males and Females Opalina Nyctotherus Octomitus Bladder flukes. . . Lung flukes Diplodiscus Cephalogonimus . Proteocephaiidae . 70 19 50 36 28 3 1 1 74 25 52 43 27 0 0 0 72 22 51 39.5 27.5 1.5 .5 .5 Comparisons of the percentage of infestation of both sexes of the same locality also give similar results. The comparison of the percentage of infection of the hosts of the two summers in the various collecting places shows but slight variation except where a habitat was affected by drought or other factors. Variation is slight particularly where a large number of hosts were taken and can readily be seen in Table V. 86 HARRY C. FORTNER Table V Comparing Percentage of Infestation of Two Summers in the Various Localities tc o m m -a a d '7a 1— ( a "5. 'e3 rn o 2 u 3 o pa en to C/3 |« o C/3 -a c s c 3 CO Octomitus intestinalis '17 '19 '17 '19 50 50 0 5 0 45 50 5 0 0 100 100 100 100 100 42 33 85 83 0 25 23 65 38 57 11 2 100 14 100 85 0 14 50 50 50 75 25 75 38 20 57 79 9 25 100 80 100 76 100 28 33 100 44 0 0 0 75 Opalina obtrigonoidea 100 Nyctotherus cordiformis •17 '19 0 Diplodiscus temperatus '17 '19 0 30 0 0 0 0 0 3 0 0 14 25 0 33 0 0 0 66 0 0 Gorgoderina attenuata '17 '19 50 65 50 100 0 18 16 42 37 0 28 75 0 47 29 0 42 55 100 50 Pneumoneces medioplexus '17 '19 50 0 0 50 100 0 0 8 0 41 0 14 0 0 4 4 0 42 11 50 50 Pneumoneces similiplexus '17 '19 0 0 0 0 0 0 0 19 2 0 0 0 0 0 0 0 0 0 0 0 Cephalogonimus americanus '17 '19 0 10 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 Clinostomum attenatum '17 '19 '17 '19 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 0 11 0 0 Proteocephalidae 25 An abundance or lack of food appears to be a factor of considerable importance. The more a host eats the greater opportunity it has to become infected. Abundance of food for frogs depends upon abundance of rainfall and vegetation. Among the stomach contents of the hosts examined were noted adults of the following orders of Insects: Hemiptera, Orthoptera, Coleoptera, Lepidoptera, and Hymenoptera. Representatives of the following groups of Coleoptera were recognized: Chrysomelidae, Scoly- tidae, Prioninae, and Cicindelidae. Larval forms of Hemiptera, Coleop- tera, Lepidoptera, Hymenoptera, and Diptera were also found. FROG PARASITES OF DOUGLAS LAKE 87 Conditions of the habitats at Lancaster Lake and Maple River re- mained practically the same from 1917 to 1919, and no great differences were found in the percentage of infection by comparing the two summer's finds. Pneumoneces medioplexus seems to be the exception here. At Carp Creek the frogs were not so abundant in 1919 as in 1917, and the vegetation was very different. With two exceptions, the infestations of Opalinae and Gorgoderinae in frogs from the Carp Creek habitat, were markedly different in the two seasons. Many species found in 1917 were not present in 1919. Table VI gives for each parasitic species the percentage of the entire number of frogs infested. As can be seen, the heaviest infestations were with Octomitus, Opalina, and Gorgoderina. Table No. VI Percentage of Infestation of Entire Number of Frogs (208) Examined, 1917 and 1919 Octomitus intestinalis Opalina obtrigonoidea Nyctotherus cordiformis .... Diplodiscus temperatus Gorgoderina attenuata Pneumoneces medioplexus . . Pneumoneces similiplexus . . . Cephalogonimus americanus. Clinostomum attenuatum. . . Proteocephalidae 112 Females 37 58 15 13 47 13 2 2 0 3 96 Males 44 66 17 10 42 20 2 0 2 0 208 Males and Females 40.5 62 16 11. S 44.5 16.5 2 1 1 1.5 Seasonal differences probably are due to a variety of factors. The amount and time of rainfall certainly affects the presence of parasites in a given locality in any one year. Table VII gives the amount of rainfall and the temperature previous to and during the collecting periods of both years. Table VII Rainfall and Temperature MAY Rainfall 1917 3 . 53 inches 77° F. 29° F. 4.73 inches 86° F. 35° F. 1919 3 . 49 inches Maximum temperature Minimum temperature JUNE Rainfall 83° F. 31° F. 3.78 inches Maximum temperature Minimum temperature Collecting Periods 91° F. 44° F. JULY & AUGUST Rainfall . . ! 3 . 47 inches 1 .25 inches 88 HARRY C. FORTNER The amount of rainfall previous to the collecting seasons totals approxi- mately the same for both years. However, in May 1917 there was an interval of 16 days of dry weather as compared to a shower every two or three days in 1919. During the time collections were being made in 1917, 3.47 inches of rainfall was recorded as compared to 1.25 inches during the collecting period of 1919. Most of the rain that fell during the 1919 collecting season came near the close of the season in one or two heavy showers. The small amount of rainfall during the collecting season of 1919 might account for the low percentage of infection with some forms as compared with the season of 1917. For instance, hosts taken at Reese's Bog during 1919 contained no Diplodiscus, and no Cephalogonimus were found in any hosts examined that year. The percentage in the majority of instances runs lower for 1919 than for 1917. Drought seriously affected the food plants of many insects in some habitats during 1919, and if insects enter into the life history of some parasitic forms, then this might account for the absence of adult parasites. The temperature of this region does not vary enough from season to season to be reckoned as an important factor. Erosion, weathering, and winds affecting the habitats are, no doubt, slight factors but some that cannot be entirely ignored. The writer takes this opportunity to express his appreciation to Dr. George R. LaRue, under whose supervision this work was done, for his encouragement and helpful criticism; and to Dr. W. W. Cort and Dr. M. M. Metcalf for their aid in the identification of certain species. Summary 1. A study was made of the distribution of the parasites of frogs in the Douglas Lake Region, Michigan. 2. Two hundred eight hosts from eleven habitats near Douglas Lake were examined during the months of July and August of the years 1917 and 1919. 3. Three species of hosts were examined, namely, Ratia pipiens, Rana clamitans, and Rana cantabrigensls. 4. Ten species parasitic in frogs (excluding the nematodes) were found. Six species was the highest number found in any individual host. 5. Octomitiis, Opalina, and Gorgoderina were present at all stations. Fourteen individuals of the genus Gorgoderina were taken from one host. Diplodiscus was not taken from all habitats. Pneiimoncccs medioplexus seems to be evenly distributed in habitats studied. As a rule infestations by this species run high, instances of 70 and 72 from one frog being recorded. But very few hosts contained Pneumoncccs simiUplexns, Cephalogonimus americanus, Clinostomum attenuatum, and Proteocephalidae. 6. The light infestation of Rana clamitans with Opalina is remarkable compared to the heavy infestations of Rana pipiens. FROG PARASITES OF DOUGLAS LAKE 89 7. All stages in the life histories of many of the flukes are not known. Many cercariae are present in a given locality. By a study of both adult forms and cercariae of a given station these stages in life histories may be worked out. If an adult does not happen to be present, then the cercariae found there, in all probability, are not of that species. Such information may prove valuable to any one making a study of the cercariae of any given area or locality. Doubtless the cercariae found at Sedge Point are not a stage in the life history of Diplodisciis temperatiis as no adults seemed to be present at that station. 8. No Acanthocephali were found in any hosts examined. 9. No Diplodiscus, Pneumoneces medioplexus, and Cephalogonimus were found in Rana clamitans, and the infestation of this species with Pneumoneces similiplexus and Proteocephalidae is comparatively low. 10. Males and females seem to be infested to about the same degree. 11. The food of the hosts, consisting chiefly of insects, the abundance of which is largely dependent upon rainfall and vegetation, appears to be a factor affecting the presence or absence of certain parasites. This might account for the absence of certain species at the Carp Creek habitat in 1919. 12. The amount and time of rainfall appears to affect the presence of parasites at a given station. The striking examples were that Diplodis- cus was not found to be present at Reese's Bog and that Cephalogonimus was not found at all in the vear 1919. BIBLIOGR.\PHY Adams, C. C. 1913 Guide to the Study of Animal Ecology. New York, 183 pp. 1919 Migration as a Factor in Evolution: Its Ecological Dynamics, .\merican Natura- list, 53:55-78. Gary, L. R. 1909 The Life-History of Diplodiscus temperatus Stafford. Zool. Jahrb., Abt. f. Anat. u. Ont., 28:595-659. CORT, W. W. 1912 North American Frog Bladder Flukes. Trans. Amer. Micr. Soc, 31:151-166. 1913 Notes on the Trematode Genus Clinostomum. Trans. Amer. Micr. See., 32:169- 182. 1914 Larval Trematodes from North .American Fresh Water Snails. Jour. Parasitology, 1 :65-84. 1915 Egg Variation in a Trematode Species. Jour. Parasitology, 2:25-26. 1915 North American Frog Lung Flukes. Trans. Amer. Micr. Soc, 34:203-240. Hegner, Robert W. 1922 The Effects of Changes in Diet on the Incidence, Distribution, and Numbers of Certain Intestinal Protozoa of Frog and Toad Tadpoles. Jour. Parasitology, 9:51-67. Holmes, S. J. 1906 Biology of the Frog. New York. 370 pp. 90 HARRY C. FORTNER Johnston, S. J. 1912 On Some Trematode Parasites of Australian Frogs. Proc. Linn. Soc, N. S. W., 37:285-362. Kent, W. Saville, 1880-1881 A Manual of the Infusoria. 3 vols. London. LaRue, Geo. R. 1914 A Revision of the Cestode Family Proteocephalidae. 111. Biol. Monogr., 1:1-350, 16 pi. Leidy, J. 1851 Contributions to Helminthology. Proc. Acad. Nat. Sc. Phila., 5:205-209. Looss, A. 1894 Die Distomen unserer Fische und Frosche. Biblioth. Zool., No. 16, 226 pp. MiNCHIN, E. A. 1912 An Introduction to the Study of the Protozoa. London. Pratt, H. S. 1903 Descriptions of Four Distomes. Mark Anniv. Vol., pp. 25-38. 1916 Manual of the Common Invertebrate Animals. Chicago. 737 pp. Seeley, L. B. 1906 Two Distomes. Biol. Bull., 10:249-254. Stafford, J. 1905 Trematodes from Canadian Vertebrates. Zool. Anz., 28:681-694. Stiles, Ch. W. and Hassall, A. 1902 Eleven Miscellaneous Papers on Animal Parasites. Bur. An. Industry, Bull. 35. Ward, H. B. 1911 The Distribution and Frequence of Animal Parasites and Parasitic Diseases in N. A. Fresh Water Fish. Trans. Amer. Fish. Soc. pp. 207-241. Ward, Henry B. and Whipple, Geo. C. 1918 Fresh Water Biology. New York, 1111 pp. NEW RECORDS OF NORTH AMERICAN ENCHYTRAEIDAE* By Paul S. Welch Our knowledge of the North American enchytraeid fauna is so meager that at the present time any definite record is of considerable importance. Various collections sent to the writer for identification have yielded specimens representing species previously known only from remote locali- ties and it has, therefore, seemed desirable to make available those records which modify so strikingly present impressions as to the range of the species involved. Marionina forbesae Smith and Welch During the month of August, 1921, Mr. R. L. Mayhew collected sexually mature aquatic enchytraeids from a small pond near the shore of Burt Lake, Michigan. Preparations resulting from a preliminary examination were subsequently transmitted to the writer for identification. Three specimens in the form of serial sections and three as whole mounts, all prepared by Mr. Mayhew, have been the basis of the work, although alcoholic material was also available. Identity. — These enchytraeids exhibit characters which agree exactly with those described for Marionina forbesae Smith and Welch (1913), except that (a) in the Michigan specimens the dorsal blood vessel arises in the posterior part of XIV or the anterior part of XV, instead of in XIII as in the type, and (b) that small scattering unicellular glands occur at the ectal opening and along the surface of the spermathecal duct, such glands being absent in the specimens from Illinois. The writer has not felt justified in regarding these differences as representing more than variations within the species. Mr. Mayhew's records contain no mention of the definitely arranged superficial spots reported in the original description of the species. Since alcohol causes these spots to disappear their absence in the Michigan material may be due to the preservative. Delphy (1919; 1921) holds that the distinction between Marionina and ^^Pachydrilus" is not valid. Smith and Welch (1913) had already noted the close similarity between the two genera. However, pending more extensive study, the writer has followed the older practice. Previous record. — M arionina forbesae was originally described from five sexually mature specimens found in the bottom mud and settlings of the waterworks reservoir at Urbana, Illinois, in October and November, 1895. This constitutes the only previous record. * Contribution from the University of Michigan Biological Station, and from the Zoological Laboratory of the University of Michigan. 91 92 PAUL S. WELCH Habitat. — The specimens on which this identification is based were collected from masses of algae growing upon partly submerged boards in a pond, known as Fontinalis Run, on the northeast shore of Burt Lake, Michigan, about three miles from the University of Michigan Biological Station. This pond is merely an expanded end of a stream surrounded by swamp conditions and opening into Burt Lake through a narrow, shallow passage. A profusion of invertebrate animals and aquatic plants, large quantities of decaying organic matter on the bottom, floating wood bearing masses of algae, very slow current, protection from surface distur- bances and complete absence of artificial influences are outstanding features of this habitat. Fridericia bulbosa (Rosa) \. A collection made at Mound City, Kansas, July 9, 1914, contained sexually mature enchytraeids six of which were studied in detail and found to be typical forms of Fridericia bulbosa. These worms were col- lected from decaying roots of alfalfa, under conditions which indicated an indigenous- species. 2. A collection made at Emporia, Kansas, on June 22, 1921, and sent to the writer by Professor R. C. Smith, Kansas State Agricultural College, contained sexually mature specimens of Fridericia bulbosa. As in the previous case, these worms were found in connection with the dead or dying roots of alfalfa and apparently represent an indigenous species. 3. Sexually mature material of an enchytraeid was found by an in- spector of the Federal Horticultural Board, on March 17, 1917, in the soil around the roots of citrus plants growing in the plant quarantine greenhouse at Washington, D.C. The specimens are clearly Fridericia bulbosa. The original source of these worms is uncertain. Mr. E. R. Sass- cer of the Federal Horticultural Board, who sent the specimens, stated that the plants were originally received from the plant introduction garden of the Office of Foreign Seed and Plant Introduction at Yarrow, Maryland and "in all probability, the soil used comes from that locality." Considering the ease with which these enchytraeids are transported in soil about the roots of plants the original stock may have been imported from a foreign locality. However, the finding of representatives of the same species in central United States under conditions not indicative of foreign importation suggests the possibility that the Maryland material may also be indigenous, Penial bulb. — Specimens from the three above mentioned collections all agree in the structure of the penial bulb. This organ is of the lumbri- cillid type in all respects. The body of the bulb is composed of cells of one kind only, their nucleated portions being near the periphery. A very few nuclei appear irregularly in the central region. Stephenson (1911, NEW RECORDS OF ENCHYTRAEIDAE 93 p. 63; PL II, fig. 17) mentions and figures the penial bulb in specimens from the littoral region of the Clyde and as nearly as can be judged from the very brief description and the small figure there is complete agreement with the American specimens. Chylus cells. — In the Maryland material the chylus cells occur in XIV-XVI, while in specimens from both of the Kansas collections the chylus cells begin in XIII and appear to end in XV. Previous North American Records. — Moore (1895, pp. 343-344) described a new species under the name Fridericia parva from material collected in the vicinity of Philadelphia, Pa. Michaelsen (1900, p. 96) regarded F. parva as a synomym of F. bulbosa and more recent studies indicate the correctness of this view. If, then, the Philadelphia material be regarded as F. bulbosa, it constitutes the first and only North American record in the literature. Fridericia agilis Smith Through the courtesy of the Illinois Natural History Survey the writer had the opportunity to study some enchytraeids collected in the Sangamon River bottoms near Kilburn, Illinois. These worms were found about the roots of winter killed wheat in dark soil having a rather high moisture content and were reported as occurring in considerable abundance in region where the collections were made. About forty speci- mens were collected on April 5, 1912, of which thirty were sexually mature. The color of the living specimens was, in many cases, nearly white with a slight tinge of flesh color. Some individuals were, however, dis- tinctly yellowish throughout their entire length. They were very active and when disturbed showed vigorous writhing movements involving strong side to side motions. Serial sections and dissections showed that the specimens repffesent Fridericia agilis Smith. Aside from certain variations in size they agree with the original description of the species in every respect. The original description gives the variation in length of well-extended living specimens as 25-30 mm. The Kilburn material showed a range of from 20 to 29 mm., with an average of 24 mm. However, these measurements were made on alcoholic material and possibly show a lower range because of a certain amount of contraction during the killing process. The original description gives the number of somites as 57-66, the average being 62, while the Kilburn material shows a range of 52-69, with an average of 57. The diameter of the body in the region of the clitellum is 0.61 mm. -0.75 mm., average 0.68 mm. This is the first time that F. agilis has been taken since the original material was collected by Professor Frank Smith (1895) in the vicinity of Havana, Illinois. . 94 paul s. welch Enchytraeus albidus Henle On June 5, 1914, enchytraeids were found in connection with the roots of house plants at Houghton, Michigan. Professor R. H. Pettit, Michigan Agricultural College, sent to the writer sexually mature specimens which proved to be typical Enchytraeus albidus Henle, the first record of its occurrence in North America west of the Atlantic Coast. Previous North American records have been discussed by the writer (1917, p. 120-121) in an earlier paper. The meager data accompanying the specimens give no information as to their original source. Their occurrence in connection with the roots of house plants makes it uncertain whether they have been transported thither with potted plants or are present in the native soil. LITERATURE CITED Delphy, M. J. 1919 Recherches sur les Oligochetes Limicoles. Bull. Mus. d'Hist. nat., No. 7, 7 pp. 1921 Etudes sur I'Organization et le Developpement des Lombriciens Limicoles Thalas- sophiles. 137 pp. 65 fig. Imprime pour I'Auteur. Paris. MiCHAELSEN, W. 1900 Oligochaeta. Das Tierreich. 10 Lieferung, I-XXIX, 575 pp. 13 fig. Berlin. Moore, J. P. 1895 Notes on American Enchytraeidae. I — New Species of Fridericia from the Vicinity of Philadelphia. Proc. Acad. Nat. Sci. Phil., pp. 341-345. 1 pi. Smith, F. 1895 Notes on Species of North American Oligochaeta. Bull. 111. State Lab. Nat. Hist., 4:285-297. Smith, F. and Welch, P.S. 1913 Some New Illinois Enchytraeidae. Bull. 111. State Lab. Nat. Hist., 9:615-636. 5 pi. Stephenson, J. 1911 On some Littoral Oligochaeta of the Clyde. Trans. Royal Soc. Edinburgh, 48:31-65. 2 pi. Welch, P. S. 1917 The Enchytraeidae (Oligochaeta) of the Woods Hole legion. Trans. Am. Micr. Soc, 36:119-138. PRIMITIVE MICROSCOPES AND SOME EARLY OBSERVATIONS^ By William A. Locy Northwestern University The question "Who first constructed the microscope?" is not one of major importance. The story is somewhat involved. However, the period in which magnifying glasses were brought into general use for the study of nature is quite well established. This was near the close of the sixteenth and in the first part of the seventeenth century. Primitive microscopes and pioneer observations with these instruments are of unusual interest, because they represent the tools employed and the beginnings of a new kind of scientific knowledge. Nothing of this kind comes down to us from antiquity. We should like to believe that Aristotle, the Alexandrines, and Galen had means of increasing their natural vision, but no such evidence exists. The unexpected discovery of so many appliances of antiquity has placed the modern mind in a receptive condition to all sorts of suggestions regarding the equipment of the ancients. A lens-shaped rock crystal, discovered by Layard in the ruins of the palace at Nineveh, has been heralded as a quartz lens of great antiquity. This antique ornament or jewel, dating from 721-705 B.C., is now in the British Museum, and, as Myall, Charles Singer, and others have pointed out, its surface is not ground smooth but is cut into small facets, which disperse the light, so that it cannot act as a lens. Moreover, this piece of quartz is not clear but is clouded by dark bands. "From a number of sites of classical antiquity crystal balls have been recovered and these may or may not have been used as burning-glasses. The point is doubtful, but it is certain that they are not lenses in the usual sense of the word." (Singer.) The fragmentary and usually dubious references to magnifications by ancient writers are not satisfying. The most often quoted statement is from Seneca's Natural Questions (63 A.D.), in which he says: "I may now add that every object much exceeds its natural size when seen through water. Letters however small and dim are comparatively large when seen through a glass globe filled with water." In this connection Seneca is attempting to explain why the rainbow appears so large, and the rest of the text shows that he is merely sustaining his hypothesis that objects seen through water appear enlarged; his mind is not directly concerned with the magnifying properties of transparent curved objects. Passing over the story of the use of lenses by Alhazen in the eleventh, and Roger Bacon in the thirteenth century, we come to the last part of ^ Address of the Retiring Cha!irman of the Section of the History of Science, American Association for the Advancement of Science, Boston, Dec. 27, 1922. 95 96 WILLIAM A. LOCY the sixteenth century where we can trace more directly the manufacture and the use of magnifying lenses. There are various claimants for priority, but it is not clear to whom the credit belongs. There were a number of spectacle makers at that time in the Netherlands, Italy, Germany, etc., and it would seem that combinations of lenses inserted in the ends of tubes were happened upon independently by different parties. In these early days the development of telescopes and of compound microscopes runs a parallel course. The simple microscope, consisting of a single lens, appears to have been used before lenses in combination, but both kinds were often employed by the same observer. After recognizing the English- man, Digges, (in 1571), and the Hollander, Zacharias (called Jensen), about 1590, as prominent among the earliest inventors, we venture to say that to determine who actually was first is a small matter compared with who first made the instrument the common property of science. For this honor, perhaps, Galileo has the best claim. He was, says Charles Singer, the "effective" inventor of the telescope and the compound micro- scope. About 1608 he made his first telescope (soon followed by enlarged and improved forms); and with this combination of lenses he not only made observations on the celestial bodies, but, also, in 1609, published microscopical observations on minute objects. We know, as a matter of fact, that single lenses (and lenses in com- bination) had been used earlier and that the use of magnifying glasses for scientific purposes came about gradually. A considerable number of early works exist of insects, spiders, worms, etc., some of them showing enlargements. For illustration, George Hoefnagel published in 1592 a set of fifty plates of insects engraved on copper. The pictures had been exquisitely drawn by his son, Jacob, at the age of seventeen, and some of them unmistakably indicate the use of magnifying glasses. So far as known the pictures of Hoefnagel are the earliest printed figures of magnified objects. There is reason to believe, however, that the naturalist, Mouffet, had made an earlier use of magnifying lenses. His "Theater of Insects" ("Insectorum sive Animalium Minimorum Theatrum") was prepared in manuscript as early as 1590 but was not published until 1634. Some of the illustrations in this book show magnifications. In the complicated question regarding the invention of microscopes, involving conflicting accounts, Charles Singer offers some deductions as follows: 1. The invention of the microscope probably preceded that of the telescope. 2. The invention of the microscope was the work of Zacharias Jensen, after 1591 and before 1608. It was perhaps formed of two convex lenses. 3. This invention was followed by that of the telescope, about 1608, by Lippershey and Metius. Its military application drew attention to it. 4. The first telescope was of the Galilean type concave eye-piece and convex o1)jective. Galileo, however, made both the telescope PRIMITIVE MICROSCOPES 97 and the microscope the property of science and was the efedive discoverer of both. His instrument was improved by Kepler in 1611. The priority of effective demonstration of the telescope rests with Galileo and of the publication of a mathematical analysis with Kepler. There is plenty of documentary evidence from writings in English, French, German, Dutch, and Italian to establish the fact that the use of the simple microscope was common in the first half of the seventeenth century. By the time of Harvey evidently magnifying glasses were no novelty. In his "De Motu Cordis et Sanguinis" (published 1628), he speaks in a matter of fact way in two places of his use of magnifying glasses. A few years later we have the earliest printed pictures of microscopes, when, in 1637, Descartes published his "Dioptrique" as an appendix to his well-known "Discourse on Method" and supplied two pictures with descriptions of microscopes. Fig. 1 shows Descartes' picture of a simple Fig. 1. Earliest known printed picture of the simple microscope. Descartes, 1637. (After Petri.) lens provided with a means of illuminating the object to be examined. H represents the eye, in front of which, at A, is a plano-convex lens inserted in a blackened frame; behind the lens is a parabolic mirror with a trans- parent central area, through which the object can be viewed; the parallel rays of light from the mirror coming to a focus at the point, E. The object to be examined is attached to an object-holder, G, at the point of greatest illumination. In addition to the foregoing, Descartes published a sketch of a huge clumsy apparatus designated an "ideal microscope." As shown in Fig. 2, 98 WILLIAM A, LOCY this has a sliding tube carrying a combination of lenses; the lens near the eye being plano-concave, and that at the far end of the tube (R) plano- convex. For illuminating the object, there was a concave mirror, similar to that of his simple microscope, and also a plano-convex lens placed in the pathway of light and giving a strong illumination at the point Z. Descartes says that the single lens may be replaced with one having two lenses combined. It is evident from these pictures and descriptions of Descartes that, in 1637, he had represented both the simple and the compound microscope. The large, unwieldy apparatus was later called perhaps in derision, a "megaloscope," but so far as known it remained as a theoretical representation and was never manufactured. Fig. 2. Descartes' representation of an "ideal microscope," 1637. (Petri.) The pictures of Hoefnagel and Mouffet, referred to a moment ago, were merely enlargements of objects visible to the unaided eye, but in the writings of Athanasius Kircher we have the first authenticated notices of microscopically minute living organisms. In his "Ars Magna Lucis et Umbrae," published in 1646, he describes a sphero-hyperbolic lens with which he made his first observations. Later he used an improved com- pound apparatus. Speaking of the different kinds of microscopes known in his time, Kircher says that some use two convex lenses; others use large glass globes filled with water and still others use a new and clever dis- covery of the smallest glass globules not larger than the smallest pearl. With the aid of lenses Kircher saw minute "worms" in all decaying sub- stances, in milk, and in the blood of [)ersons stricken with fever. In 1658, in his "Scrutinium Pestis," Kircher gave a notable anticipation of the germ theory of disease. He described living "corpuscula" as occur- PRIMITIVE MICROSCOPES 99 ring in great numbers in the blood of plague stricken persons and stated that these micro-organisms were the source of contagion. Kircher did not see the organisms that produce bubonic plague — which were discovered a long time afterward — the structures which he saw were probably pus-cells and rouleaux of blood corpuscles, but he did ascribe contagion to living organisms (contagium animatum). More than one hundred years earlier "with remarkable clairvoyance," Fracastorius had attributed diseases to mihute bodies or spores but he did not regard them as living organisms. Kircher's opinion was fortified by his actual observation of minute "vermi- cula" occurring in all putrifying substances and in the blood of the sick; his conclusion had some observational basis and his idea that infection is due to living organisms was a remarkable anticipation which has received merited attention in recent times. In following this idea of infection from living organisms, we note that a hundred years later, in 1762, Plenciz believed that there was a particular organism (seminarium) for each disease with a definite incubation period, but this noteworth example of prevision (together with others of similar import) was forgotten and the matter subject was revived only in the nineteenth century. We now look with interest on the picture of Kircher's early microscopes. Fig. 3 from his "Ars Lucis et Umbrae" shows a short tube with a lens Fig. 3. Kircher's microscope, 1646. (Petri.) at one end and a plain glass at the other. Another picture, Fig. 4, shows ornamentation of the tube. The object to be examined was placed against the flat glass and the lens near the eye was the magnifier. This is the prototype of the simple microscope. Because they were first used for 100 WILLIAM A. LOCY magnifying insects, these instruments came to be known as flea-glasses, and fly-glasses (vitrea pulicaria, vitrea muscaria, etc.). They were small tubes not thicker and longer than the thumb. In the last part of the seventeenth century they had quite a vogue as instruments of diversion, and documentary evidence shows that in 1679 microscopes with spherical lenses (microscopia globularia) were on sale in Paris. Fig. 4. An early "flea-glass" with ornamentation of the tube. Zahm, 1685. In connection with Kircher, we should mention Schott, his colleague and fellow member of the society of Jesus. Kircher being occupied with another work besought his friend, Schott, to finish for him and publish a work on natural magic; this was done, and, in 1657, a year before Kircher's "Scrutinium Pestis" appeared, Schott published a sort of preliminary volume designated "Magia Optica" and giving credit to Kircher. The work was translated and printed in German, in 1671. I have had the use of this German edition through the courtesy of its owner. Dr. A. B. Luckhardt, of Chicago. Fig. 5 is a photograph of the plate of microscopes in Schott's book. The size of these microscopes has been misconceived on account of the full-length human figure represented in connection with them and it has been generally overlooked' that the dimensions of the instruments are mentioned in the text. Schott says of the picture marked 1 in the cut that the microscope is a small tube of wood or bone scarcely longer and thicker than a finger ("das kaum lenger und dicker ist als ein finger Glaich") . At the end near the eye it is provided with a small spherical glass not larger than the smallest pearl. The others also are described as relatively small. The dimensions of picture 4, the largest one represented, is given as having a tube a foot long and thicker than the thumb mounted perpen- dicularly on a small block three feet high. These instruments were not huge "megaloscopes" as represented in Descartes' "ideal miscroscope." PRIMITIVE MICROSCOPES 101 The presumption is that the artist inserted an entire human figure in place of the single eye commonly shown in many similar pictures. In other sources of the nearby period we have an occasional mention of the size of the instruments employed. For illustration, Hooke's com- pound microscope (Fig. 11, about 1660), had a tube six or seven inches long, and a picture supposed to represent the microscope of the Italian, Divini, shows an instrument provided with five lenses, the length of which, by different writers, has been estimated from one foot to sixteen and one-half inches. In connection with the introduction of the microscope as a tool of science there naturally comes the discovery of micro-organisms, both animals and plants, and also the minute structure of tissues, of organic and of mineral substances. Fig. 5. Microscopes from Schott's Magia Optica, 1657. (Petri.) The first to devote a long life to studies with the microscope, and to make a large number of observations — sometimes illustrated with sketches — was the Dutch observer, Antony van Leeuwenhoek of Delft. Through his multitudinous observations, published chiefly in the Transactions of the Royal Society of London and extending over a period of forty years, he made the microscopical world known to a wide circle. We may cluster about the name of Leeuwenhoek the story of early microscopical obser- vations— remembering that there were other men who took part in the development of this kind of knowledge. In particular, Malpighi, the Italian, earlier in the field than Leeuwenhoek, extended his observations to the embryology of animals, to the minute structure of plants, to circu- lation of the blood in the transparent lungs of the frog (1660), etc., and Swammerdam, who used lenses extensively in investigating the structure of insects. 102 WILLIAM A. LOCY Leeuwenhoek made his observations with small microscopes of his own contrivance. Although he made several hundred of these instruments for his own use, he was not, as represented in Dr. Carpenter's article in the ninth edition of the Encyclopaedia Britannica, an optician, nor a manufacturer of lenses for the market. Time does not permit now to demonstrate this point. Twenty-two years before his death, Leeuwenhoek designated twenty-six of his microscopes to go to the Royal Society after his death. His com- munication to the Royal Society was dated Aug. 2, 1701, and since it throws light on the extent to which he prepared his own instruments, it is worth quoting: "I have (says Leeuwenhoek) a small black cabinet, lacker'd and gilded, which has five little drawers in it, wherein are contained thirteen long and square tin boxes, covered with black leather. In each of these boxes are two ground microscopes, in all six and twenty; which I did grind myself, and set in silver; and most of the silver was what I had extracted from minerals, and separated from the gold that was mixed with it; and an account of each glass goes along with them. "This cabinet, with the aforesaid microscopes, (which I shall make use of as long as I live), I have directed my only daughter to send to your Honors, as soon as I am dead, as a mark of my gratitude, and acknowledg- ment of the great honor which I have received from the Royal Society." Baker, in his work 'The Microscope Made Easy' (1742), mentions having had these instruments away from the rooms of the Society for ex- amination. He described them and figured some of them, but soon after they were lost sight of, and, unfortunately, these hierlooms to science have never been recovered. Inasmuch as Baker had these microscopes under observation his testimony as to the shape of the lenses is important. He says: "Several writers represent the glasses Mr. Leeuwenhoek made use of in his Micro- scopes to be little globules, or spheres of glass; which mistake most probably arises from their undertaking to describe what they had never seen; for, at the time I am writing this, the cabinet of Microscopes left by that famous man, at his death, to the Royal Society as a Legacy is standing upon my table; and I can assure the world that every one of the twenty-six Microscopes, contained therein, is a double convex lens, and not a sphere or globule." Leeuwenhoek gave descriptions and some drawings of his microscopes, and those in existence have been described and figured by different writers, so that we have a very good idea of his working equipment. He preferred the single lens, with a small glass of marked curvature, giving a small field but clearer definition than the compound microscope of Hooke. He made different microscopes to suit his purposes, having a range of magnifi- cation from 40 to 270 diameters. PRIMITIVE MICROSCOPES 103 One of Leeuwenhoek's originals exists at the University of Utrecht, and at my request Professor H. F. Nierstrasz photographed this instru- ment natural size. Three views of his photographs are shown in Fig. 6. Fig. 6. The Leeuwenhoek microscope in the University of Utrecht. Professor Nierstrasz. Photographed by The instrument has two small copper plates, perforated by an orifice in which the small, nearly spherical lens is inserted. In the original, the copper plates measure one inch broad and a little short of two inches long. The object-holder is represented in the lower right-hand figure as thrown to one side. By a vertical screw the object could be elevated or lowered, and by a transverse screw it could be brought near or removed farther from the lens and thus be brought into focus. In use, the instrument was held close before the eye (Fig. 7) against the light, and the object was viewed by transmitted light. Fig. 7. To show how the Leeuwenhoek microscope was held. (Petri.) 104 WILLIAM A. LOCY In some instances, however, the microscope was provided with a concave reflector (Fig. 8) similar to that used by Descartes, to illuminate the object by reflected light. Fig. 8. A Leeuwenhoek microscope provided with a concave reflector. (Petri.) Fig. 9 shows the way in which the microscope was arranged by Leeu- wenhoek to examine the circulation of blood in the transparent tail of a small fish or tadpole. The animal was placed in water in a slender glass Fig. 9. Leeuwenhoek 's arrangement for e.xamining circulation of the blood. tube, and the latter was held in a metallic frame to which a plate (marked D) was joined, carrying the magnifying glass. The latter is indicated in the circle above the letter D, near the tail-fin of the animal. The eye of the observer was applied close to the lens which was brought into position and adjusted by means of screws. Of the many discoveries of Leeuwenhoek, we can give only one example. This will be his observation of the bacteria; since it is the earliest account of bacteria accompanied with sketches, it is of especial interest. The PRIMITIVE MICROSCOPES 105 discovery of these minute forms was a feat of trained observation, and it is remarkable that Leeuwenhoek, with his primitive equipment, was able to see them and to describe them so clearly. One of his letters of 1681 indicates that he had seen bacteria at that date, but his formal description of them came in 1683. There can be no doubt from his sketches and descriptions that he saw the chief forms of bacteria — round, rod-shaped and spiral forms. His first observations on bacteria were communicated to The Royal Society of London, in a letter dated Sept. 17 (not 14), 1683, and published in the Philosophical Transactions for the year, 1684. A photograph of the cut published with his observations is shown in Fig. 10. It may be remarked in passing that the reproduction of the cut by Loffler, Petri, and others, is not quite facsimile and their quotations A ^ J) -Jt. '6f-3- M '"S:J'"JiL Fig. 10. Photograph of the original plate of bacteria as seen by Leeuwenhoek in 1683. (After Charles Singer, from the Philosophical Transactions, 1684.) do not correspond verbally with the text in the Philosophical Transactions. A few lines from the original publication in the Philosophical Transactions shows the objective quality of Leeuwenhoek's descriptions: "Tho my teeth are kept usually very clean, nevertheless when I view them with a Magnifying Glass, I find growing between them a little white matter as thick as wetted flour: in this substance tho I could not perceive any motion, I judged there might probably be living creatures. "l therefore took some of this flour and mixt it either with pure rain water wherein were no animals; or else with some of my Spittle (having no Air bubbles to cause a motion in it) and then to my great surprize perceived that the aforesaid matter contained very many small living 106 WILLIAM A. LOCY Animals, which moved themselves very extravagantly. The biggest sort had the shape of A (see the cut). Their motion was strong and nimble, and they darted themselves thro the water or spittle, as a Jack or Pike does thro the water. These were generally not many in number. The 2d. sort had the shape of B. These spun about like a top, and took a course sometimes on one side, as shown at C and D. They were more in number than the first. In the 3d. sort I could not well distinguish the Figure, for sometimes it seem'd to be an Oval, and other times a Circle. These were so small that they seem'd no bigger than E. and therewithal so swift, that I can compare them to nothing better than a swarm of Flies or Gnats, flying and turning among one another in a small space. Of this sort I believe there might be many thousands in a quantity of water no bigger than a sand tho the flower were but the 9th. part of the water or spittle containing them." "Besides these Animals there were a great quantity of streaks or threads of different lengths, but like thickness, lying confusedly together, some bent, and some straight as at F. These had no motion or life in them, for I well observed them, having formerly seen live-Animals in water of the same figure." Leeuwenhoek extended his observations to others: two women; a child of 8 years old; the spittle of "an old man that had lived soberly; and another old man who was a good fellow." The "meal" between the teeth of the old men "had a great many living Creatures, swimming nimbler than I had hitherto seen. The biggest sort were numerous, and as they moved, bent themselves like G. The other sorts of Animals were in great numbers insomuch that tho the meal were little, yet the water that it was mixt with seem'd to be all alive, there were also the long threads above mentioned." The figure marked "H" has very generally perplexed writers, and has been designated by some as a representation of those round bacteria which occur in packets of cubes (sarcinae), but later in the same paper, Leeuwen- hoek says that "H" represents scales of the outer skin (cuticula). It is worthy of note that bacteria were pictured before protozoa (which had been discovered by Leeuwenhoek in 1675), and if we except the poor picture of a shelled-protozoan, (Rotalia), by Robert Hooke, in 1665, they were, I believe, the first micro-organisms to be illustrated in printed pictures. Fig. 11 is a picture of Robert Hooke's compound microscope, made about 1660, and constituting the frontispiece of his Micrographia, which, in 1665, was published as the first book devoted expressly to microscopical observations. This shows the form to which the compound microscope had attained in the last part of the seventeenth century. Space does not permit us to follow its further development through the eighteenth and PRIMITIVE MICROSCOPES 107 nineteenth centuries. Hooke's Micrographia gave a real impetus to obser- vations with the microscope, especially in England. Among others, Neimiah Grew, the fellow countryman of Hooke, was stimulated by its publication to carry on his extensive observations on the microscopic structure of plants. The psychological influence of the use of the microscope was very great. By sharpening attention and directing it towards definite points, the powers of mental application were improved and impressions received through the sense of sight were made more exact. Now, perception through U- Fig. 11. Hooke's compound microscope (about 1660). From his Micrographia, after Carpenter. trained senses is the foundation of all scientific knowledge, and, as a matter of fact, we find the early workers with the microscope, Robert Hooke, Malpighi, Grew, and Leeuwenhoek, seeing nature more scientifi- cally and exactly than their predecessors. As Sachs remarks in his History of Botany: "Preception by the use of the optic nerve had to be accom- panied by conscious and intensive reflection, in order to make the object, which is observed only in part by the magnifying glass, clear to the mental eye in all the relation of the parts to one another and to the whole. Thus the eye armed with the microscope became itself a scientific instrument, which no longer hurried lightly over the object, but was subjected to severe discipline by the mind of the observer and kept to methodical work." Although there was started a period of more incisive observation, the early microscopes were very imperfect and it was not until their improve- ment in the first third of the nineteenth century that the full effect of their use was realized. AN ILLUMINATING DEVICE FOR MICROSCOPES By William A. Beck University of Dayton Introduction Much attention has been given by the builders of microscopes to the selection of the correct combination of objectives and oculars for the study of various preparations. The importance of proper illumination has also been appreciated and much thought and care has been expended upon this phase of microscopic methods. There are difficulties presented, however, because of the very nature of things, so that much remains to be desired in modes of illumination for certain fields of investigation. The device which I am bringing before the profession is an effort to improve the illumination. Modes of Illumination Bright Field Illumination. About 95% of the study of microscopic objects is done in a bright field. The entire field is lighted and the objects to be studied appear as colored objects, with the different tones severely bounded, each for itself being homogeneous within its own bounds. In extreme cases the objects stand out as silhouettes in their bright field. The very nature of the microscope has predetermined the development of this mode of illumination because of the relatively short working distance of the objectiv^es. Any other mode was too limited in its application, particularly during times when an intense and concentrated source of artificial illumination was impossible. The bright field illumination is obtained by allowing the light from the sun or some artificial source to fall directly or after diffusion, upon the mirror of the substage, whence it is directed, with or without the aid of a condenser, upon the subject under study, which must naturally be trans- parent or translucent. The light, thus directed, may be transmitted axially or obliquely or may be so thoroughly diffused by secondary reflecr tion, that the illumination no longer has the character of being directed. Dark Field Illumination. In contrast with this is Dark Field Microscopy. Ordinarily the field is actually very dark and the object is bright or, in some cases at least, the light is deflected into the objective and gives the impression of a white object on a dark background. In other cases the background is not ac- tually dark or of even tone, in fact it may be brighter than some tones of the image, but since this is due to secondary reflection under studied con- 108 AN ILLUMINATING DEVICE FOR MICROSCOPES 109 ditions, so as to bring the subject into better relief by the same methods, we might well consider this mode under the heading of Dark Field Micro- scopy. There are two principal cases. First when the objects are self-luminous i.e., phosphorescent, or when, by illumination with ultra-violet light, they become phosphorescent. Secondly, when the objects themselves do not emit light but which reflect or deflect the light, reaching them from some outside source, passes into the microscope. Different opaque objects having different reflecting powers, will under these conditions, produce widely different tones in the images produced. We might do well to borrow a term from Astronomy in describing them, where albedo is taken to mean the ratio of the quantity of light reflected to the quantity of light received. If one looks into the sky and notes the stars against the dark vault, he has a good illustration of the first case, i. e., of darkground illumination. If he regards the planets he has an illustration of the second case. My device pertains chiefly to the domain of Dark Field Microscopy and in particular to the Second Case. History of Illumination of Opaque Objects For progress in any field, the first step must always be the investigation of the principles involved. Later we find that development of knowledge in other fields are helpful in making proper application of these principles, in the attainment of a given end. The proper illumination of opaque ob- jects under microscopic examination is no exception. Early investigators appreciated that the principle of contrast was of the highest importance in rendering objects visible. The application of convex lenses for magnifying the objects was made by Roger Bacon as early as 1266. In 1610 Kepler devised the compound microscope with convex objective and convex ocular from which, in time, evolved the modern form. In this evolution, condensers, for lighting the objects, played a prominent part. In 1637 Descartes used a large parabolic mirror to direct sunlight upon the object. This mode of lighting showed the ob- ject more or less bright, on a dark background. His unwieldy apparatus gave way to more convenient microscopes and condensers, as progress was made in the various domains of science. It is interesting to note, however, how men have struggled down to the present time for a better application of a principle, recognized at an early period. Lister appreciated the fundamental difference between Bright Field Microscopy and Dark Field Microscopy in 1830. It was not until Zeiss in 1904 and Leitz in 1905 made practical applications, which were later discarded for more effective devices, so that the object is illuminated, by beams of light, in such a direction with reference to the axis of the objec- tive, that none of them can enter the objective directly and the light, going 110 WILLIAM A. BECK into the microscope, comes only from the objects themselves, so that they appear self-luminous on a dark ground. When the light is directed from below upon the object we do not expect to obtain a clear image of the upper surface of the object but are more concerned about discovering the existence of such objects and note their movements etc. The application of this principle has led to the wonderful developments in Ultra Microscopy. If the light is directed upon the object from above and the object is over a non-reflecting background, the object will appear bright in a dark field. From the time of Descartes, many efforts have been made to improve the mode of illumination from above. Reflectors were used to direct the light from the side onto the object, or again a "Bull's Eye" Condenser concentrated the light, from a specific direction, onto the object. The Lieberkuhn reflector which was devised in 1740 is not unlike Des- cartes' device, in principle, where a parabolic mirror directs the light onto the object. Since 1850 two additional devices for illuminating from above, have come into use. In 1852 Riddell suggested introducing the light from the side into the objective and reflecting it down upon the object. From this suggestion have resulted the various types of Vertical Illuminators. Some employ total reflection in a prism, to direct the light down through the objective, others a disk of glass and still others a mirror. A short time ago a new method was devised by Professor Alexander Silverman of the University of Pittsburgh. It consists of a circular electric filament lamp, Which surrounds the objective and shines down upon the object. The Illumination of an Object Under Study Up to the present only directed illumination was employed in the mi- croscopic study of opaque objects, to obtain a knowledge of their surface configuration. The directed light was either oblique to the optic axis of the system of lenses or approximately parallel, i.e., either oblique or vertical with respect to the surface under examination. We might then simply use the terms oblique or vertical, to designate these modes of illumination. Both are to be considered as directed illumination, because we have a pencil of light from a definite direction. Oblique Illumination. Oblique illumination can be obtained either by means of a reflector attached to the objective or by directing rays from a radiant, lying above the plane of the surface of the object. When a radiant is employed, for example an arc lamp, a tungsarc, or a filament lamp, a condensing lens is AN ILLUMINATING DEVICE FOR MICROSCOPES 111 usually interposed, between the light and the object, in order to concen- trate the light rays and to facilitate the proper placing of the beam. If we are dealing with a highly polished surface this mode of illumination has no value, because no rays can enter the objective according to the laws of regular reflection (Figure 1). Fig. 1 Illustrating the law of regular reflection and showing how oblique illumination on a flat surface cannot direct the light into the microscope. Fig. 2 Illustrating regular reflection on an irregular surface, showing how regions at the proper angle reflect an intense light into the tube of the microscope while others do not. points in close pro.ximity may produce defraction patterns. Many If the surface of the object illuminated is irregular or etched, the rays entering the objective from some points are very intense, while from others they may be entirely lacking and therefore do not express the relative albedo of the surface, nor give an impression of the plasticity of the object. This becomes clear at once from a study of figure 2. This figure also shows how certain regular markings in certain cases produce diffrac- tion effects. Diffraction patterns make it difficult to interpret the true structure. The greater the obliquity the greater the incident difficulties become, so that with high power objectives, where the free working distance is very small, this mode of illumination is entirely out of the question. Illuminating from several sides tends to correct somewhat the dif- ficulties referred to, in giving the body illuminated more of the character of a self luminous body, according to Huygens' impression, that each point illuminated becomes a new point source of disturbance. Then again dif- 112 WILLIAM A. BECK fraction patterns are eliminated. Complete annular illumination was attempted in the paraboliod illuminator which was very popular at one time. For correct illumination, the curvature of a reflector placed around the objective would have to vary for each working distance. These were later almost entirely superseded by the vertical illuminators. In the Silverman device, that has come into the field in relatively recent years, and which became popular immediately upon its appearance, the illuminant is attached directly to the objective. Here the illumination is about three fourths annular and consequently shows decided improve- ment over previous modes of illumination by the oblique method. There still remain these difficulties: the illumination is not completely annular. The angle of illumination cannot be varied at will. The intensity of il- lumination is limited. The light cannot be completely diffused. The light cannot be filtered at will. The life of the lamp is short. A considerable amount of undesirable heat is developed. Fig. 3 Showing a rough surface under vertical illumination. Note that only a strictly horizontal surface can return the light directly into the tube of the microscope. Diffraction patterns cannot produce images in the field. Vertical illumination is obtained by placing a reflecting surface in a mounted cell attached to the microscope, just above the objective. The reflector sends the illuminating beam of light through the objective, which acts as a condenser, concentrating the light rays into a bright spot of light, upon the surface of the object, at a point lying, appro.ximately, in the optic axis of the microscope. From the surface of the object, the rays are reflected back through the objective and form the image of the object in the usual manner. If the object to be studied is absolutely flat, it is evident that the rays reflected from the object might be considered as emitted by the object and these should form an image, that should give a correct impres- sion of the albedo of the various parts of the field. Furthermore, if there AN ILLUMINATING DEVICE FOR MICROSCOPES 113 are sections, that otherwise would be inclined to form diffraction patterns, the law of regular reflection would require that all reflected and diffracted rays fail to enter the objective, not being parallel to the optic axis. (Figure 1, central beam. See also Figure 3 where points lie in close proximity.) There is absolutely no question that this type of illuminator has rendered, and is still rendering, immeasurable service to science. For certain work it may never be replaced. When the object to be studied is not flat, the image immediately ceases to give the correct impression regarding albedo and the nature of the surface. The image needs interpretation. The investigator may be dealing with more or less highly polished surfaces and with areas, part of which, are polished, part rough and often studded with minute points. Some- times cracks or cleavage planes may cross the field. With ordinary etched surfaces, polished portions appear bright and etched surfaces less bright. The glare from the brighter surfaces, seeks to lessen the definition in the less bright fields. It is questionable if the detail of the mat surfaces are in any degree accurate. To demonstrate fissures, cleavage planes, depressions etc., it becomes necessary that the examination with the vertical illuminator be supplemented by oblique illumination and to study the direction of the shadows with respect to the radiant, remembering of course that in the image seen in the microscope, directions are completely reversed. The need of this study suggests a mode of oblique illumination, such that the light beam can be turned about the axis of the microscope, with least possible difficulty. The Illumination of Macroscopic Objects Since the principles for the correct illumination of objects, as we ob- serve them in every day life, with the unaided eve, are the same as for microscopic study, we might do well to give a little attention to the phe- nomenalprogress that has been made within the past decade by illuminating engineers in their proper sphere. Prominent among the scientists who are working for better illumination to obtain correct values and at the same time to save the eyes, is Professor C. E. Ferree of Philadelphia. He has made a series of tests of the eye, under different kinds of illumination, daylight, indirect lighting, semi- direct lighting and exposed filament lighting. He found that after three hours' work under day light, the eye lost practically nothing in seeing efficiency. Under indirect lighting the effect was almost the same; the eye was 91% efficient in seeing ability. Under exposed direct or semi-indirect lighting the loss in seeing efficiency was enormous; the remaining efficiency being only 25% with semi-direct and 14%, with direct. Science has conclusively demonstrated, that if we would preserve our eyesight and obtain correct values of the tones in a 114 WILLIAM A. BECK scene we must avoid, so far as possible, glare and direct illumination, be- cause the eye cannot endure excessive light. Momentary blindness results from intense light because the entire retina or a portion of it becomes paralysed. The result is that we do not perceive objects within the range of vision that should be seen normally. The greater the glare of any spot in the field, the more intense the illumination must be before the other portions can be perceived, which increase however, serves to render the "spot" more glaring, thus defeating the purpose. The Formation of an Image. If in the neighborhood of a luminous point P, there are refracting and reflecting bodies having an arbitrary arrangement, then, in general, there passes through any point in space, one and only one ray of light, i.e., the direction which it takes from P to P' is completely determined. Under given conditions, certain points may be formed, at which, two or more of the rays emitted by P intersect in a point P' which is called the optical image of P. If we have many points in our object under study, the summa- tion of all the images of these points will give a more or less accurate replica of the object, according as the numbers expressing rays, passing through the P's do or do not bear the same ratios to each other, as the numbers expressing the rays actually emitted by the conjugate points in the object. From what has been said of regular reflection, it is clear that we cannot form an adequate im.age of the reflecting object when it reflects regularly, but only of some other object that emits rays of light which come to us after reflection. When the light falls in a definite direction on an unpolished surface it is reflected in various directions. The amount of light going in a given direc- tion for the formation of an image depends upon the position of the image with respect to the position of the illuminant. It is clear then that the number of rays that combine to form an image, depends upon the particu- lar position of the images, as well as upon the nature of the surface and the albedo of the object. This varying factor, of position, offers serious difficulty in the formation of a correct image. The thought naturally arises in the mind, that we attempt to illuminate equally from all sides in order that only the nature of the surface and the albedo shall determine the image. Reflection of diffused light, produces not only correct form and tone, but is also selective, so that we maintain correct color value. The rich red petal of the geranium thus illuminated by white light, reflects diffusely in all directions only the red rays. In order to form correct images it follows from the above discussion that direct illumination must be avoided and diffused illumination employed, whenever possible. AN ILLUMINATING DEVICE FOR MICROSCOPES 115 Illuminating engineers corrugate reflectors and give the walls a rough finish rather than an even finish to avoid regular reflection. All their efforts are directed in distributing the light energy, like a fine spray, throughout the entire room. We are all familiar with the excellent results obtained by the modern modes of illumination. Fig. 4 A study under Northern Sky illumination. Note the exactness of detail and the plastic- ity. The snow helps to diffuse and illuminate, so that the illumination is practically annular and diffuse. Copyright by John Mathews. Photographers have long ago known that they obtain their best pic- ture by the evenly distributed illumination from a northern sky Figure 4. The commercial photographers recognize the importance of completely annular illumination and diffuse illumination and employ methods in which they obtain so called "cross-fire" of light, by which term they mean, that the light comes from all directions with almost equal intensity. 116 WILLIAM A. BECK To illustrate the differences between obliquely directed illumination and vertical illumination and "cross-fire" illumination a bust of Schiller, which was painted with a dull even white, was photographed under the three conditions. Just as was expected marked differences were noted. The picture that was taken with the aid of oblique illumination, was so badly distorted by extreme contrasts and heavy shadows that it gave no impression whatever of Schiller's physiognomy. The vertical illumination produced a picture that was more even and lacked that extreme contrast, in fact was flat. The greatest difticulty with the picture was that it gave a false impression of the real appearance ot the subject, because elevations Fig. 5 This diagram illustrates how the light, from vertical iUumination upon an elevation, fails to return to the observer or the instrument. were dark and depressions were bright. This is just as it should be. A hill would cause the light to be dispersed while the valley would return too much of the light. This is clearly shown in figures 5 and 6. The third picture was of the nature of those ordinarily produced by portrait photog- raphers, marked by correctness of form and surface variations and a remarkable plasticity. The chief aim in view in designing the new type of illuminator was to obtain a diffuse or "cross-fire" illumination for microscopic objects, of a sufficient intensity to obtain in pictures, of the same relative value as in the third case of the photography of the bust. The Illuminating Device The device consists of a glass medium which completely surrounds the object under study (Figure 7). The light enters at the ground surface A, is totally reflected at the surface C, and is finally refracted at the surface D, and thus directed from all sides upon the object under study. The object rests upon a stage Ei, which is adjustable, so as to allow a variation AN ILLUMINATING DEVICE TOR MICROSCOPES 117 in illumination. The illuminator can be made of any convenient size. The one which I actually employ and which I recommend for most work, is so designed as to fit the sleeve on the substage, which ordinarily receives the Abbe condenser. Any convenient light source may be employed. For ordinary study, a common filament lamp or the microscope lamp is satisfactory. With the gas-filled lamp, one can obtain greater intensity. Just as our modern illuminating engineers seek a great quantity of light very finely distributed, so also it is very advantageous to use an arc lamp in connection with the illuminator, distributing the intense light very evenly. The illuminator allows the microscopist to employ practically any intensity of light he desires. Fig. 6 This diagram illustrates how the valley returns too much of the light under vertical illumination and causes the values of the field to be reversed. Plane parallel light is ordinarily to be recommended, which may be obtained by placing a lens in such a position that the light source is in the principal focus of the lens. By varying the position of the lens, one can readily vary the nature of the illumination and in a very interesting man- ner, simulate "spot-lighting." Direct or diffused sun-light can be em- ployed as well as an artificial light source. The light may be allowed to fall immediately upon the surface Ai or to be reflected, from the mirror of the substage, onto that surface. If the mirror is employed one can "spot out" certain regions of the subject from one side or the other by simply turning the mirror. This makes it very easy to study the nature of a given object by means of shadows. The angle which the side Di makes with the vertical, determines the deviation of the beam of light from the horizontal as it passes from Ci to Di- The deviation rnust be such that the entire field to be studied by the 118 WILLIAM A. BECK a E, Fig. 7 Longitudinal section of the illuminating device and the plan of the same. Bi is the glass medium mounted in a metal sleeve. The light enters the medium at \\ and is totally reflected at the surface Ci and thus directed to the surface Di. From Di the light deviates from the horizontal direction and is directed to the stage Ei. The entire piece is inter- changeable with the .'\bbt; condenser. AN ILLUMINATING DEVICE FOR MICROSCOPES 119 determined objective, be sufficiently illuminated after the objective is in the position of the correct working distance. The dispersion of the light depends upon the refractive angle of the device which is given by the angle which the face Di makes with the verti- FiG. 8 Another form of the illuminator. This one is made to fit the large opening on the stage of the microscope when the entire mechanical stage is removed. Note the three different refracting angles where the light leaves the glass medium. The cone Ai converts the solid cyhnder of light into a hollow cylinder of light. This form can be made in one or more pieces. The light is received at Ao, transmitted at Bj, reflected at C2, again reflected at D2 and refracted at the surfaces Ej.Fj and G2, falling at different angles, upon the subject resting on H2. cal. This angle is always small and the angular dispersion in consequence is negligible, so that we have a condition of achromatism without special correction. 120 WILLIAM A. BECK After a careful study of these values was made for the various objec- tives, it was found possible to have three refractive angles which would give highest efficiency for the various objectives that can be employed. If it is possible for the makers of objectives, to narrow down somewhat the diameter of the lower portion of high power objectives, there is no reason why this modification, should not make it possible to study any opaque object at the highest diameter of magnification, by this method of illumi- nation. To increase still further the intensity of illumination, a special design for the lower portion of the device was made to convert the solid cylinder of light, into a hollow cylinder of light, by means of a reflecting cone and totally reflecting surface. These modifications gave rise to the form shown in figure 8. It is not necessary that the transparent medium be of one piece as the diagram indicates. Furthermore the arrangement for a variable inner stage is not shown. Finally the new device can be arranged in two units. One portion resembling the illuminator shown in figure 7, and the other representing the lower portion in figure 8, by means of which the solid cylinder of light is converted into a hollow cylinder of light. The light can be diffused by placing a ground glass between the source and the illuminating device or by placing a diffusion medium around the object which receives the light from the surface Di, (figure 7). For certain work it is desirable to filter the light. Regular filters can , be interposed between the source of light and the illuminator. It is to be noted particularly that by this method we obtain completely annular illumination and that we can without difficulty obtain diffuse illumination resembling the illumination which photographers seek from the northern sky or such as is obtained by the devices employed by com- mercial photographers. If light shadows are desired one can very easily increase the illumination in a specific direction. The device can be used simultaneously with the vertical illuminator. In an interesting experiment, both were set into position and first the light from the one and then light from the other was employed. It was interesting to note a complete reversal of the tones as explained above and indicated in figures 5 and 6. The device was originally designed for the study of opaque objects but it can be used to advantage for the study of transparent objects by placing an uneven reflector, as a piece of filter paper, on the stage E and place the preparation, in the ordinary way, upon the regular stage of the microscope. The illumination thus obtained is remarkable for its softness, clearness and definition. One can study with less eye-strain when employing this method and the contrast is sharp, even though the light is not intense. This is just as it should be, according to the principles outlined above. It is to be hoped that this method of illumination will be helpful in various fields of microscopic investigations and that the method itself here outlined in principle, will be further developed. AX ILLUMIXATIXG DEVICE FOR MICROSCOPES 121 Explanation of Plate Fig. 1. Brassica juncea "wild mustard." Fig. 2. Linen cloth. Illumination evenly diffused. Fig. 3. Scales on wing of butterfly. Fig. 4. Potassium iodide isometric crystals showing accretion and cleavage planes. Fig. 5. Bottle cork. Sunlight was used. Illumination evenly diffused. Fig. 6. Shagreen of Squalus acanthias showing placoid scales. Evenly diffused illumina- tion. ABNORMAL SPECIMENS OF HELODRILUS CALIGINOSUS TRAPEZOIDES (DUCES) AND HELODRILUS ROSEUS (SAVIGNY)i By Bess R. Green University of Illinois Introduction Comparatively few abnormal earthworms have been described, al- though variations in both external and internal structures are of common occurrence among the various species of Lumbricidae. The importance of the study of abnormal development in earthworms lies in the application of the knowledge thus obtained to the relations that exist in normal develop- ment. In normal development growth proceeds in such a way as to bring about the formation of similar structures in the right and left halves of the same somite. Occasionally the normal method of procedure is inter- fered with and an asymmetrical arrangement of the organs results. The organs develop from paired germ-bands which arise practically inde- pendently on the right and left sides of the body thus making asymmetrical relations between the organs of the two sides possible. In abnormal worms the organs may be present in unequal numbers on the two sides, or the members of a pair of organs many develop in different somites on the right and left sides. In many specimens there is also a lack of certain correlations between external and internal structures that occur in normal individuals. Variations other than those involving asymmetrical relations are of two types. Abnormal specimens in which organs vary but slightly either forward or backward from their normal positions constitute one group or type. The second group is composed of worms in which the organs appear in other than normal positions due to a doubling of somites in the anterior part of the body. There may also be a lack of symmetry between the organs of the right and left sides in either group. In a recent paper Pro- fessor F. Smith (1922) described abnormal specimens of H. subrubicundus and H. tenuis which illustrate the first type of variation, and this paper treats of two examples of the second type found in H. caliginosus trape- zoides and //. roseus. A detailed discussion of the literature dealing with abnormalities in earthworms will be undertaken later in a more extensive paper. At present there is not sufficient data to make such a discussion of any great value. ' Contribution from the Zoological Laboratory of the University of Illinois, No. 219. 122 SPECIMENS OF HELODRILUS CALIGINOSUS 123 The specimens here described are from Professor F. Smith's collection of abnormal earthworms. These worms were found in the banks of a stream at Urbana, Illinois. HeLODRILUS CALIGINOSUS TRAPEZOIDES (DUGES) The description of this specimen is based on the study of sagittal sections of the first thirty-three somites. The worm resembles the normal H. caliginosus trapezoides in the general appearance of the somites and clitellum, in the relative positions of the setae, and in the presence of paired, ventral, glandular pads on three of the anterior somites. External Characters. — This worm which appears to have been injured at the posterior end measures 11.1 cm. in length and there are 130 somites present. There is no evidence of somites that are doubled on one side and not on the other. The setal arrangement in somites 24 to 30 is indicated by the formula aa:ab:bc:cd = 11:1.5:7.2:1. This is about the usual arrange- ment for the setae in the species. The saddle-shaped clitellum extends over somites 32-37 and is very slightly developed on 31 and 38. In a normal worm it commences on 27, five somites farther forward, and extends posteriorly over eight or nine somites. The tubercula pubertatis of the right side is continuous over three somites, 34-36, and the other one involves three and one third somites, 2/3 33-36. In a normal individual they are present on somites 31-33. They have the usual relation to the clitellum since they end posteriorly on the somite immediately in front of the one on which the clitellum ends. The glandular papillae which usually include the ventral setae of somites 9, 10, and 11 are found in this specimen on 10, 11, and 12 on the right side and on 11, 12, and 13 on the left. The setae included by these papillae are modified and are about twice as long as ordinary setae and are much more slender. The first dorsal pore is present on 14/15 which is five somites posteriad of its usual position. A spermiducal pore surrounded by a glandular papilla is present on 18 on the right side. A similar papilla is present on each of somites 20 and 21 on the left side and surrounds what appears to be a spermiducal pore on each of these somites. Normally there is a pair of pores on somite 15. An oviducal pore is present on 17 on the right side, and there is one on 19 on the left. These positions are respectively three and five somites posteriad of the normal positions of the pores. On the right side, sperma- thecal pores are present on 11/12 and 12/13, two somites posteriad of their usual positions. The spermathecal pores of the left side are on 14/15, 15/16, and 16/17. The first two pores are situated five somites posteriad of their usual positions, 9/10 and 10/11. Internal Characters. — Most of the reproductive organs of the right side of the worm are present in normal numbers but vary from their usual positions (Figs. 2 and 3). Spermaries are present in 12 and 13; sperm 124 BESS R. GREEN sacs bearing the usual relations to these organs are in 11, 12, 13, and 14; and spermathecae are included within the septa 11/12 and 12/13. These organs are situated two somites posteriad of their respective positions in a normal worm. The spermiducal pore is on somite 18, which is three somites posteriad of its normal position. Likewise the ovary, in 16, and the oviducal pore, on 17, are present three somites posteriad of their normal positions. The separation of the most posterior spermary and the ovary by two somites indicates a probable doubling of what would be somite 12 in a normal worm. Further evidence of the doubling of this somite is shown by the presence of two vessels uniting the lateral longi- tudinal vessel with the dorsal vessel, one in 14 and one in 15; while in normal worms there is but one such vessel, in 12. A pouch of the calciferous gland, normally in 10, is present in 12 on the right side of this specimen. It bears the usual relation to the first spermary being included in the same somite with it. The reproductive organs on the left side are found five somites pos- teriad of their respective, normal positions. Supernumerary organs in excess of the normal number will be mentioned in connection with the accountof certain reproductive organs and of the "hearts." The spermaries and spermiducal funnels, usually in 10 and 11, are present in 15, 16, and an additional one of each in 17. All three of these spermaries are equally developed. The sperm duct could not be traced to the exterior. Sperm sacs are present in the posterior part of 14, 15, and 16, communicating with somites next posterior; and in the anterior part of 16, 17, and 18, communicating with somites next anterior. An ovary and an oviducal funnel are present in 18. The oviduct extends from the oviducal funnel to the oviducal pore on 19. The usual position of the ovary and oviducal funnel is in 13 and that of the oviducal pore on 14. The spermathecae, three in number, are included within septa 14/15, 15/16, and 16/17. Normally there are but two spermathecae present, within septa 9/10 and 10/11. The pouch of the calciferous gland is very slightly perceptible, if present at all, in 14. The position of the pouch is not in conformity with its usual relation to the most anterior spermary. Ordinarily these two structures are present in the same somite. There are seven "hearts" on the right side and nine on the left. The most posterior "hearts," which are usually in somite 11, are in 17 on the left and in 13 on the right side. As above mentioned two vessels arise from the dorsal vessel in somites 14 and 15 on the right side and join to form a single lateral longitudinal vessel. A lateral longitudinal vessel was not seen on the left side. Normally a pair of these vessels joins the dorsal vessel in the somite next posterior to the one in which the most posterior "hearts" are found. specimen's of helodrilus caliginosus 125 Helodrilus roseus (Savigny) The following description is based on transverse sections of the first twenty-three and twenty-four somites and on frontal sections of the ventral half of somites 24 and 25 to 35 and 37. The fact that the sperma- thecal pores of this worm are located near the mid-dorsal line eliminates from consideration all species of this part of the country except H. foetidus and H. roseus. Since this worm does not have the bands of color which are characteristic of H. foetidus, it is assumed to be an abnormal specimen of H. roseus. External Characters. — On account of the doubling of certain somites on one side and not on the other the somites have been numbered on both sides independently. Posterior to 12 the left half of each somite bears a different number from that of the right half. The relative positions of the various organs are shown in the accompanying diagram. The location of the diflFerent organs indicates a probable doubling on both sides of most of the somites in the anterior part of the worm. Four somites, 13, 34, 39, and 66, are double on the left side; and 100 and 111 are double on the right side. The total number is 162 on the right side and 164 on the left. The maximum number recorded for a normal worm of this species is 150. The setae bear the usual relations to each other. Setae c and d on 16, 18, 21, 22, and 23 on the left side, on 20 and 21 on the right side, and setae a and b on 16 on the right side are about twice as long as the ordinary setae. Glandular swellings surround these setae. In normal worms setae of one or more bundles of 9, 10, 12, or 13 may be similarly modified. The clitellum, which is not very pro- nounced, is saddle-shaped and does not include the ventral setae. It is developed on fifteen somites, while in normal specimens it usually includes but eight. These somites are identical for both sides, although the somite numbers differ on the right (45-59) and left (48-62) sides. Each of the tubercula pubertatis is divided into two distinct areas. The one on the left side includes a smaller part which extends over the posterior two thirds of 54 and the anterior one half of 55, and a larger part which extends from the posterior two thirds of 56 to about the middle of 59. Of the one on the right side, the larger part is more anterior reaching from the posterior two thirds of 52 to the middle of 55; and the smaller part extends from the anterior margin of 57 to the middle of 58. In the normal worm the ridge of each side is continuous and extends over but three somites, 29-31. Glandular swellings are present surrounding setae ab on somites 27 and 28 of both right and left sides of the worm. On external examination there appeared to be a pore in the middle of each swelling, but a study of sections of this region showed but a single pore on each side situated on the anterior of the two glandular swellings. These are the spermiducal pores, present on 27 of each side. In a study of the sections two pairs 126 BESS R, GREEN of oviducal pores were found on 24 and 25 of both right and left sides. These pores are close to setae ab and are very inconspicuous. The sper- mathecal pores of the right side are present on 16/17-19/20, and those of the left side are on 15/16 and 17/18-19/20. Internal Characters.— Evidence of the doubling of somites is shown by the presence of twice the normal number of spermaries, spermathecae, and ovaries, and also by the large number of "hearts" that are found in this specimen (Figs. 1 and 2). All four spermathecae of each side are normal in form, and their pores are situated in the usual position near the mid-dorsal line. Spermaries and spermathecae of the same side are present in the same somites, 17-20 on the right side and 16, 18, 19, and 20 on the left. This is the usual relation of these organs. This relation is shown very clearly on the left side; where the first spermary is separated by one somite from the second, and the spermathecae of that side are likewise separated. Another striking relation is shown in the development of lateral pouches from the calciferous gland. Normally a pouch develops on either side of the oesophagus in the somite in which the most anterior pair of spermaries develops. In this specimen the first spermary on the left side is in 16, and a calciferous gland pouch is also present in that somite. The second spermary, the first of a series of three, is in somite 18, and a calciferous gland pouch is also present in that somite. On the right side, the spermaries are present in somites 17-20, and a pouch is found in the somite with the most anterior spermary. A third relation is shown by the relative positions of the sperm sacs and the spermaries. It is usual in this species for two sperm sacs to develop from the septum which bears the second spermary, one from the anterior face and one from the posterior face. This relation exists in this specimen in connection with the' septum numbered 18/19 on the left and 17/18 on the right. This septum bears the second spermary on the right side and the second of the series of three on the left side. The spermiducal funnels show normal relations to the spermaries and sperm ducts. Two well developed ovaries are present on both sides, each having the usual relations to an oviducal funnel, ovisac, oviduct, and oviducal pore. The ovaries on each side are situated in the third and fourth somites posteriad of the somite which contains the most posterior spermary of that side. Eleven "hearts" are present on each side of the alimentary canal in somites 10 to 12 inclusive. The "heart" in somite 12 on the left side is very small. The most posterior "heart" on each side is in the same somite with the most posterior spermary of that side. This is the usual relation of these organs. A lateral longitudinal vessel unites with the dorsal vessel on either side in the usual position, which is in the somite ne.xt posteriad of the one in which the most posterior "heart" is found. SPECIMENS OF HELODRILUS CALIGINOSUS 127 10 II II 10 If II II II 11 H . II 15 0 n rr ' A A II 15 . ^ // n/ 0 .... ^i'\ \>,^, ft "^ l^ ^,1 r.,^, (5^ st---- ^ 0 ¥a ,^) l^^ ss ,,0 ) ^ b^ 20 dv — ^^. 1 b V © os--- -u/ @ ® V . . 25 uj s- Y ...