The Cuticular Ultrastructure of Paragordius Varius (Leidy, 1851) (Gordioidea: Chordodidae)

228 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY

Keppner, E. J. 1967. Fossor taxidiensis (Skinker, America Physalopterinae (Nematoda). Proc. 1935) n. comb, with a note on the genus Helm. Soc. Wash. 8: 63-64. Fossor Honess, 1937 (Cestoda: Taeniidae). . 1942. The Physalopterinae (Nematoda) Trans. Am. Microscop. Soc. 86: 157-158. of North American vertebrates. Sum. Doctoral . 1969a. Filaria taxidea n. sp. (Filario- Diss. Univ. Wise. 6: 88-91. idea: Filariidae) from the badger, Taxidea . 1943. New host records of nematodcs taxus taxus from Wyoming. Trans. Am. from Mustelidae (Carnivora). J. Parasit. 29: Microscop. Soc. 88: 581-588. 158-159. 1969b. Occurrence of Atriotaenia Pederson, E. D., and P. D. Leiby. 1969. procyonis and Miloneus mustelae in the Studies on the biology of Monordotaenia taxi- badger, Taxidea taxus (Schreber, 1778), in diensis, a taeniid cestode of the badger. J. Wyoming. J. Parasit. 55: 1161. Parasit. 55: 759-765. Law, R., and A. Kennedy. 1932. Parasites of Rausch, R. 1947. A redescription of Taeniu furbearing animals. Bull. Dept. Game and taxidiensis Skinker, 1935. Proc. Helm. Soc. Fisheries, Ontario 4: 1-30. Wash. 14: 73-75. Leiby, P. D. 1961. Intestinal helminths of some Skinker, M. S. 1935. Two new species of tape- Colorado mammals. J. Parasit. 47: 311. worms from carnivores and a redescription of Leidy, J. 1886. Notices of nematoid worms. Taenui laticollis Rudolphi, 1819. Proc. U.S. Proc. Acad. Nat. Sci. Philadelphia 38: SOS- Natl. Mus. 83: 211-220. SIS. Swanson, G., and A. B. Erickson. 1946. Alaria Miller, G. C., and R. Harkema. 1964. Studies taxideae n. sp., from the badger and other on the helminths of North Carolina verte- mustelids. J. Parasit. 32: 17-19. brates. V. Parasites of the mink, Mustela Walton, A. 1927. A revision of the nematodes vison Schreber. J. Parasit. 50: 717-720. of the Leidy collection. Proc. Acad. Nat. Sci. Morgan, B. B. 1941a. A summary of the Philadelphia 79: 49-163. Physalopterinae (Nematoda) of North Amer- Worley, D. E. 1961. The occurrence of Filaria ica. Proc. Helm. Soc. Wash. 8: 28-30. mortis Gmelin, 1790, in the striped skunk and . 1941b. Additional notes on North badger in Kansas. J. Parasit. 47: 9—11.

The Cuticular Ultrastructure of Paragordius varius (Leidy, 1851) (Gordioidea: Chordodidae)

JOHN E. ZAPOTOSKY Faculty of Zoology, College of Biological Sciences, Ohio State University, Columbus Ohio

ABSTRACT: The structure of the cuticle of an adult nematomorphan, Paragordius varius, has been examined by means of electron microscopy. Present studies reveal several distinct layers and structures previously unresolved within the cuticle. The cuticle apparently possesses morphologically similar structures and layers to those found in various nematode cuticles. The layers (named inwardly) are: ependyma, external cortical, internal cortical, areolar, fibrous, and basal lamella.

The light microscopy of the adult cuticle of cuticle of all nematomorphans: defense, are- Paragordius varius was originally done by olar, fibrous, and pigmental layers. The pri- Montgomery (1903) and was reworked and mary aim of the present study is to reveal the supplemented by May (1919). May (1919) structure of the cuticle of gordioids by means of described the adult cuticle as consisting of an electron microscopy. outer homogenous layer with "protoplasmic connections" to the epidermis and with areolae Materials and Methods overlying a large fibrous layer. Later, Kirja- Adult specimens of Paragordius varius were iiova (1959) recognized four layers in the collected from the east fork of Clear Creek at

Abbreviations (all figures): a, areole; ab, ainembranous body; al, areolar layer; li, basal portion of cuticle; bl, basal lamella, C, cortical portion of cuticle; ec, external cortical layer; ep, ependyinal layer; fb, fibrillar bundle; H, hypoderrnis; ic, internal cortical layer; M, muscle; Ma, macroanal. Figure 1. A three-dimensional diagram of the adult cuticle of'. Paragordius vtnius. the Fallsville Wildlife Area, Highland Co., (1 hr); 1—3 (18 hr); and full strength resin Ohio. A female specimen was fixed for .12 hi- (29 hr). The resin was a modification of Luft's nt room temperature in 5% gluteraldehyde- embedding medium (20 ml Epon 812, 20 ml phosphate buffer at pH 7.4, was washed for 1M> Araldite 502, 60 ml dodecenylsuccinic anhy- hr in phosphate buffer, and while in this wash dride, and 2 ml dimethyl phthalate) (Geisy, was cut into pieces. The anterior end, posterior personal communication). Specimens in the end, and portions of the cuticle were removed embedding medium were placed in an oven at and used for the identification of the species. 75 C for 3 clays to effect polymerization of Lengths from the mid-portion of the body were the resin. Sections were cut with glass knives then placed in a phosphate-buffered 1% os- on either a Servall Porter-Blum MT-1 or MT-2 mium tetroxide solution at 4 C for 3 hr. microtome. Several "thick" sections (0.2- These were dehydrated in a graded series of 0.5 p. were attached to glass slides and stained ethanol. Following two changes of propylene with hot, aqueous 1% solution of Azure B for oxide (15 min each) the tissue was placed in preliminary evaluation of the cuticle with a a graded series of resin concentrations: 3 parts light microscope. Sections were mounted on propylene oxide—1 part resin (15 min); 1—1 200 mesh copper grids coated with 2% parlo-

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 38, NUMBER 2, JULY 1971 231 dion in amyl acetate solution. Sections were The areolar layer is bounded externally by counterstained with a saturated solution of the cortex and internally by the basal division uranyl acetate in 50% ethanol (Gibbons and of the cuticle. Within this layer three distinct Grimstone, 1960) and in. lead citrate prepared types of areolae have been observed: areolae according to Venable and Coggeshell (1965) or with large fibrils (Fig. 6), areolae with small Reynolds (1963). The cuticle was observed fibrils (Fig. 6) and areolae without fibrils in and photographed with an RCA electron micro- their lumen (Fig. 2). Hollow, branching fibrils scope Model EMU-3H at both 50 and 100 190 A and 115 A in diameter are located within kilovolts. the lumen of the areolae with large fibrils and those with small fibers, respectively (Fig. 6). Observations The apparent suspension of these fibrils within The general arrangement of the cuticle of the lumen suggests the presence of an osmio- Paragordius varius is seen in Figures 1 and 2. philic matrix within both bodies. Several pores The cuticle can be divided into three main opening to the exterior were observed on the areas: cortical, areolar, and basal. The cortical outer surface of the areolae without fibrils. area (cortex) can then be subdivided into an All three types of areolae share a similar ependymal, external cortical, and internal corti- enclosing lamellar morphology. Typically, cal layers, while the basal area may be sub- there are three osmiophilic lamellae surround- divided into a fibrous portion and a basal la- ing a body, each of these are in turn separated mella. by a nonstaining lamella (Fig. 6). Often a The ependymal layer, outermost layer of the fourth discontinuous lamella is found on the cortex or "garment" of the cuticle, consists of cortical side of the body. Where the bodies two strata: an outer, irregular osmiophilic lie close to the surface or receive projections stratum and an inner nonstaining stratum. The from the external cortical layer, the enclosing inner stratum (300-700 A thick) contains lamellae lose their distinct stratification and moderately dense lamellae (Fig. 3). the osmiophilic material appears to coalesce. Beneath the ependymal layer, the osmiophilic Mesiad to the areolae lies the thickest layer external cortical layer is characterized by its of the cuticle, the fibrous layer. This layer is many granular projections into the adjacent composed of several strata (16—19 observed in internal cortical layer. Many of the cylindrical this study), each stratum consisting of a single projections (300-600 A in diameter) appar- layer of parallel fibers (Figs. 1, 2). The layers ently end blindly within the internal cortical of fibers wind spirally around the body, each layer; while others appear to pass through the layer alternating in one of two directions (May, entire thickness of the internal cortical layer, 1919). The fibers also appear to be suspended or into the lamellar covering of the various are- in an osmiophobic matrix. Myelin figures (60— olae present in the cuticle. 130 A thick) are found between the layers of The lightly granular internal cortical layer fibers. Individual fibers are composed of a makes up the bulk of the cortex. Its internal granular substructure arranged in bands (Fig. boundary is well defined (Fig. 1), while the 4). outer border is obscured by the projections of Fibrillar bundles and macrocanals have been the external cortical layer. At the base of this observed traversing the fibrous and areolar layer, amembranous osmiophilic bodies are layers of the cuticle. The macrocanals open found aligned between and exteriad to the directly into the internal cortical layer and areolae. The amembranous bodies are ovoid to basally they apparently connect into the hypo- spherical in shape, composed of small granules, clermis. These large canal-like structures have and lack an enclosing membrane (Fig. 5). always been found to open between two areolae

Figure 2. Electron photomicrograph of a cross section through the body wall x 7,300. Figure 3. Cross section through the cortical layer of the cuticle. The arrow indicates the moderately dense lamellae x 123,750.

'?£&$$!& - :• ' ..•:•*. -JS? •••;•-. tyti 1

v^: .^r .,/, .' . -^• **-.«! • -• -• * .A*

4§f•.j»K • '. » •-,.*•«> _• . -c .

pltlfK

Table 1. A comparison of the suggested divisions for the cuticle of Paragordius varius to the cuticle of Neinatodes.

Present Study May & Montgomery Kirjanova (P. varius) Nematodes (Lee, 1967)

Homogenous layer Defensive layer Cortical layer Cortical layer epenclymal layer outer membrane internal cortical layer internal cortical layer Areolar layer Areolarnal layer Areolar layer Matrix layer Basal layer Basal layer Fibrous cuticle Support layer fibrous layer fibrous layer Pigmental layer basal lamella basal lamella and also are always located beneath a tubercle The outermost area of gordioid cuticle, called (Fig. 6). The macrocanals are double-walled the homogenous layer by Montgomery (1903) and are generally of a larger size (1370-2740 and May (1919) and called defensive layer by A in diameter) than the fibrillar bundles (830- Kirjanova (1959), has been resolved into three 1900 A in diameter). The fibrillar bundles distinct sublayers: ependymal, external corti- (Fig. 4) are composed entirely of small fibrils cal, and internal cortical. of the same nature as are found in the basal Although the outermost layer of nematode lamella and apparently lack a lumen. Distally, cuticle is variously named, the ependymal layer the fibrillar bundles connect into the lower of P. varius corresponds best, by virtue of its border of the internal cortical layer where they morphology, to the cuticle-hypodermis mem- branch to form a fine reticulum of fibrils. brane of Nematospiroides dubius. The external Some of the bundles have been observed to surface of JV. dubius is limited by a triple- connect into the lamellar covering of the are- layered membrane, 100 to 135 A thick. Ex- olae. Proximally, the fibrillar bundles appar- tending from the outer leaflet is a filamentous ently arise directly from the basal lamella zone (Bonner, Menefee, and Etges, 1970). (Fig. 4). The corresponding area in P. varius has been The basal lamella is composed of fibrils and named ependyma in order to avoid confusion lies directly over the wavy border of the hypo- with the interface between the adult cuticle dermis. The upper portions of the hypodermis and the hypodermis. possesses many hemidesmosomes. Several The cortical sublayers of P. varius have a groups of fibrils, arising from the hemi- similar osmic staining reaction and positioning desmosomes were seen to traverse the hypo- to that reported for Meloidogyne javanica by dermis. Bird and Rogers (1965). The granular material in the lower portion of the "homogenous" Discussion cuticle reported by May (1919) and the amem- Inglis (1964b) states that "nematode cu- branous osmiophilic bodies observed here are ticle is best considered as a three layered sys- apparently the same. They are of the same tem liable to modification and elaboration shape (spherical) and location (interareolar). around, or in association with a system of No rod-shaped bodies (Montgomery, 1903) were punctation canals." Although the cuticle of found. These amembranous bodies appear to P. varius is distinct from any given nematode, be composed by small granules and have an it morphologically fits into the generalized pat- osmiophilic staining similar to that of the ex- tern stated above (see Table 1). ternal cortical layer. In the course of preparing

Figure 4. Cross section through the inner portion of the basal area of the cuticle and upper portion of the hypodermis. Arrow indicates hemidesmosomes x !>7,750. Figure 5. Section through the lower portion of the internal cortical layer x 97,500.

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 38, NUMBER 2, JULY 1971 235 slices of cuticle for species identification, it was of the tubercles on the posterior end of males observed the cuticular pigmentation resides in may better serve to clarify the function of these the upper or cortical area. Since the immature structures. or developing cuticle is white, these bodies may The basal division of nematode cuticle varies represent the residue of a "tanning" compound from the complete absence of fibers, reported secreted into the cuticle. for the adults of P. decipiens by Davey (1965) The areolar layer which is bounded exter- and Eiichromadora vulgaris by Watson (1965) nally by the cortex and internally by the basal to as many as eight layers in some ascarids layer, corresponds by this position to the matrix (Hyman, 1951). In general the larger nema- layer of nematode cuticle. No structures re- todes possess these layers while the smaller ported in nematode cuticle apparently correlate do not (Lee, 1967). In P. varius, the number directly with the areolae of P. varius. Still, the of fibrous layers present exceeds that reported nematodes Neochromadoria sp., and Chroma- for any nematode. May (1919) has recorded dor ella sp. show cuticular differentiations (lat- as many as twenty-four present in the cuticle of eral bars and hexagonal blocks) arising as modi- P. varius, while Montgomery (1903) has re- fications of canals (Inglis, 1964b). Three kinds ported only eleven. This apparent disparity is of areolae, rather than the one type previously probably a phenomenon of the area of the body reported, have been demonstrated. Nonstained examined. May (1919) reports forty-five layers cuticle prepared for light microscopy (cleared in the mid-body and thirty in the posterior of and mounted in glycerine or Hoyer's media) G. robustus. The corresponding spiral fiber shows only one type of areole. While prepara- system of nematodes allows the anisometeric tions stained with osmium, azure B blue or stretching (ability to stretch antero-posteriorly azan's stain reveal both stained and non-stained but not radially) of the cuticle and is usually areolae. considered a refinement of large, highly May (1919) cites the presence of "proto- evolved forms (Inglis, 1964b). plasmic strands" running from the hypodermis The basal lamella corresponds also to the to the cuticular surface, usually between the pigmental layer of Kirjanova (1959) and to areolae, and cites Vejdovsky's observation of the basal lamella of nematode cuticle (Lee, these strands in relation to the areolae of other 1987). In the sections observed no pigments or gordioids. There were several suggestions of crystals were found. As stated earlier, most of fibrillar bundles attaching into the lamellar the cuticular pigments appear to reside in the covering of the areolae. Inglis (1964a) indi- cortical layers. cates two types of canals or "strands," massive The hypodermal arrangement of hemi- fibrillar elements and punctation canals in the desmosomes and traversing fibrils in P. varius lips of the nematodes Dujardinascam sp., Por- corresponds to that reported for Nippostrongy- rocaecum sp., and Angiisticaecum sp. Two lus brasiliensis by Lee (1970). It is suggested corresponding types of structures have also that this arrangement allows the indirect been demonstrated in this study. The larger attachment of the muscles to the cuticle. macrocanals appear more canal-like, while the Although the cuticle of P. varius is remark- fibrillar bundles appear to be entirely composed ably similar to nematode cuticle, other gordioid of fibers. The insertion of the fibrillar bundles characters still warrant the present retention into the basal lamella and upper portions of the of the gordiacea as a group separate from the cuticle suggests a possible anchoring function nematocla. in the adult. It has been suggested (Hyman, 1951) that Acknowledgments the cuticular ornamentation of gordioids may be Appreciation to Dr. John L. Crites and Dr. sensory in nature. This work does not support Wayne B. Parrish for their advice and help in this contention. However, a closer examination the operation of the electron microscope.

Figure 6. Photomicrograph through tubercle, x 26,000.

Literature Cited worms (Nematomorpha-Gordioidea) [In Rus- sian]. Zool. Zh. 38: 509-519. Bird, A. F., and G. E. Rogers. 1965. Ultra- structure of the cuticle and its formation in Lee, D. L. 1967. The structure and composition Meloidogyne javanica. Nematologica 11: 224- of the helminth cuticle. In Advances in Para- 230. sitology. Academic Press, New York. pp. 187-254. Bonner, T. P., M. G. Menefee, and F. J. Etges. 1970. Ultrastructure of cuticle formation in . 1970. Molting in Nematodes: The for- a parasitic nematode, Nematospiroides dubius. mation of the adult cuticle during the final Z. Zellforsch. 104: 193-204. moult of Nippostrongyhis brasiliensis. Tissue Davey, K. G. 1965. Molting in a parasitic & Cell 2(1): 139-153. nematode, Phocanema decipiens. I. Cytologi- May, H. G. 1919. Contributions to the life cal events. Canad. J. Zool. 43: 997-1003. histories of Gordius robustus Leidy and Para- Gibbons, I. R., and A. V. Grimstone. 1960. gordius variits (Leidy). 111. Biol. Mono. 5: On flagellar structure in certain flagellates. 127-238. J. Biophy. Biochem. Cytol. 7: 697. Montgomery, T. H. 1903. The adult organiza- Hyman, L. H. 1951. The Invertebrates: Acantho- tion of Paragordius varius (Leidy). Zool. cephala, Aschelminthes, and Entoprocta. The Jahrb., Anat. 18: 378-474. Pseudocoelomate Bilataria. Vol. III. McGraw- Reynolds, E. S. 1963. The use of lead citrate Hill Book Co., New York. 572 pp. at high pH as an electron-opaque stain in Inglis, W. G. 1964a. The comparative anatomy electron microscopy. J. Cell Biol. 17: 208. of the ascaridoid cuticle (Nematoda). Bull. Venable, J. H., and R. Coggeshell. 1965. A Soc. Zool. de France. 89: 317-338. simplified lead citrate stain for use in elec- . 1964b. The structure of the nematode tron microscopy. J. Cell Biol. 25: 407. cuticle. Proc. Zool. Soc. Loncl. 143: 465- Watson, B. D. 1965. The fine structure of the 502. body wall in a free-living nematode Euchro- Kirjanova, E. S. 1959. Concerning the per- madera vulgaris. Quart. J. Microscop. Sci. meability of the cuticle of freshwater hair- 106: 75-81.

Investigations on the Trematode Fauna of Hyderabad, A.P. Part II. Parasites of Birds—(C). Psilochasmus singhi sp. n. from a Common Whistling Teal, Dendrocygna javanica

G. P. JAISWAL AND M. R. A. HUMAYUN Department of Zoology, University College of Science, Osmania University, Hyderabad-7 (A.P.) India

ABSTRACT: Ps&ochasmus singhi sp. n., is described from the Whistling Teal, Dendrocygna javanica from Hyderabad, A.P., India and compared with the other three previously described Indian forms and also with a Russian and a W. German species. It differs from all other species of the genus in the position of its genital pore, disposition of the ovary and vitellaria, structure of the esophagus, and the prin- cipal measurements of the body.

The genus Psilochasmus was established by intestine of the white-eyed Pochard, Liihe in 1909 with (1). P. oxyuris (Creplin, Fuligula mjroka in Russian Turkestan. 1825) as its type species. In addition to it the (3). P. agilis Travassos, 1921 from Poecilonetta following 9 forms have been described so far bahamensis in Brazil. from different parts of the world:— (4). P. lecithosus Otte, 1926 from the intestine (2). P. longicirratus Skrjabin, 1913 from the of domestic duck, in Latvia.