Morphological Comparison and Taxonomic Utility of Copulatory Structures of Selected Nematode Species 1

Journal of Nematology 19(3):314-323. 1987. © The Society of Nematologists 1987. Morphological Comparison and Taxonomic Utility of Copulatory Structures of Selected Nematode Species 1

ABDALLAH RAMMAH ~ AND HEDWIG HIRSCHMANN 3

Abstract: Spicules of 9 Meloidoffyne, 2 Heterodera, 3 Globodera, and 12 other plant-parasitic, insect- parasitic, and free-living nematodes were excised and examined using scanning electron microscopy (SEM). Gubernacula of some of the species were also excised, and their structure was determined. The two spicules of all species examined were symmetrically identical in morphology. The spicule typically consisted of three parts: head, shaft, and blade with dorsal and ventral vela. The spicular nerve entered through the cytoplasmic core opening on the lateral outer surface of the spicule head and generally communicated with the exterior through one or two pores at the spicule tip. Spicules ofXiphinema sp. and Aporcelaimellus sp. were not composed of three typical parts, were less sclerotized, and lacked a cytoplasmic core opening and distal pores. Spicules of Aphelenchoides spp. had heads expanded into apex and rostrum and had very arcuate blades with thick dorsal and ventral edges (limbs). Gubernaculum shapes were stable within a species, but differed among species examined. The accessory structures of Hoplolaimus galeatus consisted of a tongue-shaped gubernaculum with two titillae at its distal end and a plate-like capitulum terminating distally in two flat, wing-like structures. A comparison of spicules of several species of Meloidogyne by SEM and light microscopy revealed no striking morphological differences. Key words: spicule, gubernaculum, capitulum, titillae, scanning electron microscopy, light microscopy, Aphelenchoides, Aporcelaimel~us, Belonolaimus, Dolichodorus, Globodera, Heterorhabditis, Het- erodera, Hoplolaimus, Meloidogyne, Mesorhabditis, PanagreUus, Tylenchorhynchus, Xiphinema.

Spicules, a pair of copulatory structures Efforts have been made to elucidate the in the tail region of the male, vary consid- ultrastructure of spicules of free-living, an- erably in shape and size in different nema- imal-parasitic, and plant-parasitic nematode groups. Within each group their form todes (3,4,14,15,17-19). Detailed work by shows little variation. Although spicule transmission electron microscopy (TEM) length has often been used in nematode showed that spicules are innervated, func- identification, few species descriptions have tion as sense receptors, and generally are considered spicule structure or shape. The instrumental as intromittent organs to pass spicule has been used as a taxonomic char- sperm from the male to the female (3,4,14, acter at both generic and specific levels in 15,18,19). Three-dimensional reconstruc- some aphelenchids and cephalobids (11,16), tion of spicules has been based on light and spicule form and dimensions have been microscope (LM) observations, on TEM se- considered important in certain tylenchid rial cross sections, and on scanning elec- genera (10). tron microscopy (SEM) of exposed parts of Spicule structure is sometimes difficult protruded spicules. to interpret in whole nematode mounts be- To date, few observations have been re- cause details are often obscured or are be- ported on spicules completely isolated from yond the light microscope resolving power. the nematode body (7). The objective of this study was to make a detailed compar- Received for publication 2 September 1986. ison of excised spicules using SEM in order 1 Paper No. 10682 of the Journal Series of the North Car- olina Agricultural Research Service, Raleigh. This study was to define precisely the three-dimensional supported in part by the USAID Morocco Project No. 608- aspects of spicule structure in several taxa. 016, administered by the University of Minnesota-St. Paul. Part of the first author's Ph.D. thesis project. A light microscope comparison of spicules Assistant Professor, Department de Phytopathologie, In- of the four common species of Meloidogyne stitut Agronomique et Veterinaire Hassan II, B.P. 6202, Ra- bat-Instituts, Rabat, Morocco. was undertaken concurrently with SEM Professor, Department of Plant Pathology, North Caro- observations to determine whether any lina State University, Raleigh, NC 27695. We thank Drs. B. B. Brodie and L. I. Miller for providing features revealed by SEM can also be ob- specimens of Globodera spp. served in LM, and thus can be used in rou- 314 Nematode Copulatory Structures: Rammah, Hirschmann 315 tine taxonomic work. In addition, guber- case of Heterorhabditis, from Heliothis zea nacula of a few species examined were (Boddie) larvae. Live males of Meloido- excised in order to determine their exact gynidae were obtained by incubating shape. Like the spicules, these accessory washed root systems in moist chambers at copulatory structures appear to differ in room temperature. shape and size among taxa and, therefore, Spicules were excised by employing the can be useful diagnostically. same method used previously for nematode stylets (8). Five live males were transferred to a drop of 40% lactic acid (50% MATERIALS AND METHODS for glycerin-preserved males) on a small, Males of three populations each of the round, ringed coverslip which was at- four most common species of Meloido- tached to a microscope slide. After 5 min- gyne M. incognita (Kofoid and White) utes, the posterior end of each male was Chitwood, M. javanica (Treub) Chitwood, cut off using an eye knife or sharp dental M. arenaria (Neal) Chitwood, and M. hapla root canal file under a stereoscope at 60 x Chitwood--were selected. Males of one magnification. The spicules were pushed population each of M. spartinae (Rau and out of the spicule pouches and cleaned of Fassuliotis) Whitehead, M. microcephala Cliff attached muscle tissues inside the body cu- and Hirschmann, M. microtyla Mulvey, ticle before they were transferred to the Townshend and Potter, M. naasi Franklin, coverslip, where further cleaning was done and M. hispanica Hirschmann were also ex- as necessary. Each spicule was adhered to amined to obtain additional information the coverslip by pressing down on the spic- on spicule structure in the family Meloi- ule head. The lactic acid was removed with dogynidae. a very fine micropipette, and 2% formalin Males of Heterodera gIycines Ichinohe, H. was added several times to eliminate any schachtii Schmidt, Globodera rostochiensis traces of lactic acid. Excision of the two (Wollenweber) Mulvey and Stone, G. ta- spicules without their separation was done bacum virginiae (Miller and Gray) Stone, and by lowering the concentration of lactic acid Globodera sp. were used to compare spicule of live or glycerin-preserved specimens. morphology within the family Hetero- After 10-15 minutes, the formalin was ab- deridae. sorbed with filter paper and the spicules In addition, one population each of the were air dried and marked by glass rods. following species of free-living, plant-para- The coverslip was removed from the slide sitic, and insect-parasitic nematodes was and was attached to a stub. After coating examined for morphological features of with 250 ~t of gold or gold-palladium, the spicules and gubernacula: Aphelenchoides spicules were viewed and photographed us- fragariae (Ritzema Bos) Christie, Aphelen- ing a JEOL T 200 SEM at 25 kV. Obser- choides sp., Belonolaimus longicaudatus Rau, vations were made with the specimens per- Dolichodorus heterocephalus Cobb, Hoplolai- pendicular to the electron beam to avoid mus galeatus (Cobb) Sher, Tylenchorhynchus foreshortening of the image. At least 20 claytoni Steiner, Xiphinema sp., Aporcelai- spicules from each population were ob- mellus sp., Mesorhabditis spiculigera (Steiner) served and photographed at different po- Dougherty, Mesodiplogaster lheritieri (Mau- sitions for comparison. pas) Goodey, Panagrellus redivivus (Linn.) The same technique was used to excise Goodey, Heterorhabditis heliothidis (Khan, the gubernacula of some of the species. Brooks and Hirschmann) Poinar. For excision of spicules, males were used either live or fixed and preserved in glyc- OBSERVATIONS erin. They were obtained from appropri- Excised spicules usually lie laterally on ate host plants reared in the greenhouse, their inner or outer surface. Only spicules from soil samples, agar cultures, and in the lying in the same position were compared 316 Journal of Nematology, Volume 19, No. 3,July 1987

A B

ventral ve hd hd t sh r-'-; sh

1 ve dorsal F1G. 1. Generalized lateral diagrams of tylenchid spicules. A) Inner surface. B) Outer surface, cc = cytoplasmic core opening; bl = blade; hd = head; sh = shaft; ve = velum. in all SEM studies. No morphological dif- low the head and widens towards the blade. ferences were noticed between spicules ex- The blade is arcuate and curved ventrally. cised from live or glycerin-preserved males. Wide where it merges with the shaft, it Also, in all taxa examined, the two spicules narrows considerably after a short distance of each male were morphologically iden- posteriorly and tapers progressively to- tical exhibiting a mirror image relation- wards the tip. The two wing-like vela can ship. be clearly observed on the inner surface of Basic spicule morphology was similar in the spicule blade (Fig. 2E, G, I). The in- all nematodes examined (Fig. 1) except ward curvature of the vela and lateral out- Dorylaimida and Aphelenchida. In gen- ward curvature of the blade result in a ca- eral, each spicule is a tubular structure con- nal-like shape of the spicule. The blade tip sisting of a sclerotized cuticular covering is simple, with two pores to the exterior. and a central cytoplasmic core containing When the two spicules of a male are excised nerve tissues. Three parts can generally be as a whole in their natural position, the distinguished: a cylindrical head, a cylin- dorsal and ventral vela overlap, forming a drical shaft, and a more flattened blade channel that insures sperm transmission with two sclerotized, wing-like projections during copulation (Fig. 2A, B). (the vela), one extending toward the dorsal SEM observation of spicules of the most and the other toward the ventral side. The common species of root-knot nematodes-- vela are more conspicuous when the spic- M. incognita, M. javanica, 34. arenaria, and ule is observed from the inner surface. The M. hapla (Fig. 2C-F)--revealed no major cytoplasmic core opening is usually situ- morphological differences useful in rou- ated on the lateral outer surface of the spic- tine species identification. Slight differ- ule head. There may be one or two small ences in shape and size of the head and in pores at the distal tip of each spicule. blade length and curvature are difficult to Meloidogyne spp. (Fig. 2): Each spicule is observe with LM. Spicules ofM. spartinae, composed of a short, cylindrical, distinctly however, can be distinguished from those demarcated head with circular cytoplasmic of other species using SEM as well as LM. core opening, a broad shaft, and a tapering They have a more ventrally curved and blade. Variations of head size and shape markedly set off head and a broad shaft were observed between and within species. ending in a curved tip (Fig. 2H). The cytoplasmic core opening is oriented Heterodera and Globodera spp. (Fig. 3): The slightly laterally on the outer surface. The spicules of H. schachtii (Fig. 3A, B) and H. shaft can be well defined (Fig. 2G, H). It glycines (Fig. 3C, D) are morphologically starts with a constriction immediately be- similar. The short, cylindrical head is swol- Nematode Copulatory Structures: Rammah, Hirschmann 317

S~ i •

FIc. 2. SEM photographs of spicules of males ofMeloidogyne spp. A) M. hispanica. B, C) M. javanica. D) M. incognita. E) M. hapla. F) M. arenaria. G) M. naasi. H) M. spartinae. I) M. microtyla. J) M. microcephala. A, B) Spicules not separated. C, D, F, H, J) Outer aspect. E, G, I) Inner aspect. All scales 5 um (D same scale as A; C, E-J same scale as B). 318 Journal of Nematology, Volume 19, No. 3, July 1987

A B

". • ,, jj

C D

E F

~ .fc~_ ~~

FIG. 3. SEM photographs of spicules of males of Heterodera spp. and Globodera spp. A, B) H. schachtii. C, D) H. glycines. E, F) G. rostochiensis. G) G. tabacum virginiae. H) Globodera sp. B, D, F, H) Outer aspect. A, C, E, G) Inner aspect. Scale 5 #m. len and distinctly bent ventrally. The cy- the other parts. The blade with its vela is toplasmic core opening is situated slightly smoothly curved ventrally and terminates outward on the lateral side of the head. with a bifid tip. The spicules of G. rosto- The shaft is cylindrical and distinct from chiensis (Fig. 3E, F), G. tabacum virginiae (Fig. Nematode Copulatory Structures: Rammah, Hirschmann 319

Y.y :.fx/

FIG. 4. SEM photographs of spicules and gubernacula of males of selected plant-parasitic nematodes. A) Spicule ofHoplolaimus gaIeatus. B) Gubernaculumand capitulum ofH. galeatus. C, D) Dolichodorus heterocephalus. E, F) Tylenchorhynchus claytoni. F) Spicules and gubernaculum. G) Belonolaimus longicaudatus. H) Xiphinema sp. I) Aphelenchoidesfragariae. J) Aphelenchoides sp. A, C, J) Outer aspect. D, E, G, H) Inner aspect. F, I, J) Spicules not separated. All scales 5/~m (C, D, G same scale as A; F, J same scale as E; I same scale as B). 320 Journal of Nematology, Volume 19, No. 3,July 1987

3G), and Globoclera sp. (Fig. 3H) are iden- are distinct in this spicule, the head and tical and show gross morphology similar to boat-shaped blade. This spicule seems to that ofHeterodera spp., except that the head be less sclerotized than tylenchid spicules. is not as much bent ventrally and the blade The edges appear more solid than other tapers progressively toward a single tip. parts, and longitudinal grooves run Hoplolaimus galeatus (Fig. 4A, B): The two throughout the spicule length. No cyto- spicules are morphologically similar. Each plasmic core opening was observed. spicule has a tubular head with cytoplasmic Aphelenchoides spp. (Fig. 4L J): Spicules of core opening (Fig. 4A). The shaft is cylin- Aphelenchoides fragariae and Aphelenchoides drical and not clearly separated from the sp. are thorn shaped and smaller than spic- head. The slightly arcuate blade has two ules of all other species studied. A. fragariae well-expanded vela. The ventral velum has spicules with pronounced apex and ros- widens immediately anterior to the round- trum (Fig. 4I) and a markedly curved blade. ed tip. The blade comprises the solidly sclerotized The gubernaculum (Fig. 4B) is tongue dorsal and ventral limbs connected by a shaped and centrally concave with two thin bridge of less sclerotized material. The curved sides expanding laterally. Its prox- dorsal limb is longer than the ventral, and imal part is simple with slightly curved sides. its tip is hooked ventrally. The spicules of Distally it terminates in a thick, rectangular Aphelenchoides sp. (Fig. 4J) are similar to plate with one horn-like titilla projecting those of A. fi'agariae but less curved and from each side. The proximal part of the sclerotized. The cytoplasmic core opening capitulum is a simple plate that tapers dis- located at the outer surface of the apex is tally and terminates in two small flat wings distinctly visible (Fig. 4J). which fit into the middle of the concave Mesorhabditis spiculigera (Fig. 5A, B): Ven- center of the gubernaculum. trolateral and dorsal views of the spicules Dolichoclorus heterocephalus (Fig. 4C, D): show that they are completely fused at their The slightly arcuate spicule has a tubular distal ends (Fig. 5A) and connected over head that is continuous with a long shaft much of the dorsal velum length (Fig. 5B). (Fig. 4C). The distal half of the blade curves The small, globular head is set off and toward a sharply pointed tip. The ventral slightly curved laterodorsally. The short velum appears to be long and considerably shaft is well demarcated from the blade curved inward (Fig. 4D). The distal plate whose ventral velum is curved inward. The of the gubernaculum is parallel to the chord tip is long and pointed. The gubernaculum of the spicule and bears two titillae (Fig. (Fig. 5A), which becomes dislodged from 4D). its original position when the spicules are Tylenchorhynchus claytoni (Fig. 4E, F): The excised, is boat shaped and about two-thirds spicule consists of a slightly globular head, of the spicule length. a tubular, distinct shaft, and a crescent- Mesodiplogaster lheritieri (Fig. 5C, 19): The shaped blade ending in an acute tip (Fig. inside aspect of the spicule shows a short, 4E). The broad ventral velum becomes very globular head, constricted shaft, and ar- thin distally. The large gubernaculum is cuate blade with two vela (Fig. 5D). The enlarged distally, with raised sides, and is head is markedly set off from the short deeply curved proximally (Fig. 4F). shaft by a deep constriction and has a large Belonolaimus longicaudatus (Fig. 4G): The circular cytoplasmic core opening which is spicule is arcuate and deeply curved me- situated ventrolaterally (Fig. 5C). The spic- dially. The swollen head merges into a short ule tip is pointed. The gubernaculum, tri- shaft continuous with a blade provided with angular with oblique dorsal projection, dis- two pronounced vela and ending in a bifid tally encloses the spicule tips (Fig. 5D). tip. The distal one-fifth is narrow and Panagrellus redivivus (Fig. 5E, F): Spic- slightly bent dorsally. ules of this nematode are peculiar in shape Xiphinema sp. (Fig. 4H): Only two parts (Fig. 5E). The head is curved and bends Nematode Copulatory Structures: Rammah, Hirschmann 321

FiG. 5. SEM photographs of spicules and gubernacula of free-living and insect-parasitic nematodes. A, B) Mesorhabditis spiculigera. A) Fused spicules, ventrolateral, and gubernaculum. B) Fused spicules, dorsal. C, D) Mesodiplogaster lheritieri. D) Spicule and gubernaculum. E, F) Panagrellus redivivus. F) Gubernaculum, ventral. G) Heterorhabditis heliothidis. H) Aporcelaimellus sp. C, E, G) Outer aspect. D, H) Inner aspect. All scales 10 t~m (B-D same scale as A; G same scale as E). 322 Journal of Nematology, Volume 19, No. 3, July 1987 ventrally, forming a hook. A large cyto- from fresh and preserved material without plasmic core opening is situated laterodor- creating unacceptable artifacts. sally on the outer lower part of the head~ Spicules are variable among taxa in size The blade is curved, with its distal one- and shape and have been shown to be use- third bent almost to a 90-degree angle and ful in identifying some members of Rhab- terminating in a bifid tip. The dorsal edge ditida (11), Aphelenchida (16), and a few of the blade appears thick; the nerve tissue genera of Tylenchida (10). The original probably passes through it. The large ven- illustrations of Chitwood (2) depict some tral velum is membranous and soft and differences in spicule size between males of flexible. The large, shallow trough-shaped the four most common species of root-knot gubernaculum has thick incurved edges nematodes. The SEM and LM observa- (Fig. 5F). tions of spicules in our studies, however, Heterorhabditis heliothidis (Fig. 5G): The showed no striking morphological differ- spine-shaped spicule is straight to slightly ences that could be used to distinguish these curved, with small head and pointed tip. four Meloidogyne species. Also, the mor- The shaft is short and continuous with the phology of spicules of other amphimictic blade tapering progressively toward the tip. and parthenogenetic species seemed to be The cytoplasmic core opening is located very similar. Only M. spartinae had slightly terminally at the proximal end in the di- different spicules exhibiting a more curved rection of the spicule axis. head and blade tip. We also found that the Aporcelaimellus sp. (Fig. 5H): The differ- morphology of spicules of H. glycines and ent parts of this large, slightly sclerotized H. schachtii is similar and cannot be used as spicule cannot be distinguished easily. No a differentiating character. In addition, no cytoplasmic core opening was observed. morphological differences were found be- The edges extend the length of the spicule tween the selected Globoclera species. The and become more solid and thick at its dis- blade tip can be used to distinguish be- tal end. The tip is truncate. tween males of Heterodera and Globodera species, as reported previously (4). DISCUSSION Individual spicules of Hoplolaimus ga- Scanning electron microscopy has re- leatus, Pratylenchus penetrans, and Tylenchu- cently become an important tool in nema- lus semipenetrans have been reported to be tology for revealing relatively stable mor- unequal in length (14,15,19). This dissim- phological characters, some of which have ilarity may be observed only in the position subsequently been observed with LM and of the protruded, exposed parts of spicules, thus shown to be of practical value for not when they are retracted. Our study species identification. Such characteristics showed that males of all species examined include in particular the head and stylet have morphologically identical spicules that morphology in plant-parasitic Tylenchida exhibit mirror image relationships. and Aphelenchida (8,9,13). Spicules of the Aphelenchoides species ex- Few studies have used TEM and SEM to amined consist of a head with apex and clarify spicule morphology and function rostrum and a blade with thick dorsal and (1,3-5,10,14,15,17-19), and SEM studies ventral edges (limbs). Our SEM observa- concerned only the protruded parts of spic- tions support earlier TEM observations (3) ules (3,4,6,14,17). The three-dimensional that the spicular nerve enters through the aspect of spicules has been reconstructed apex of the spicule head and proceeds to from TEM serial sections (18) which is a the tip along the outer dorsal surface. time-consuming method and less precise. Spicules have been described as tubular The method employed here has been structures formed of sclerotized cuticle en- shown to be very satisfactory for SEM ob- closing a cytoplasmic core (12). Our observations. Spicules of different nematode servations confirmed that spicules of all species have been successfully dissected species examined, except those of the Nematode Copulatory Structures: Rammah, Hirschmann 323 dorylaimids Xiphinema sp. and Aporcelai- Zuckerman and R. A. Rohde, eds. Plant parasitic mellus sp., are solid and hard sclerotized nematodes, vol. 3. New York: Academic Press. 6. Duggal C. L. 1978. Copulatory behavior of male structures. Dorylaimid spicules appear to Panagrellus redivivus. Nematologica 24:257-268. be formed of less sclerotized material, and 7. Eisenback, J. D. 1985. Techniques for prepar- a cytoplasmic core opening was not de- ing nematodes for scanning electron microscopy. Pp. tected. The presence of a cytoplasmic core 79-105 in K. R. Barker, C. C. Carter, andJ. N. Sasser, eds. An advanced treatise on Meloidogyne, vol. 2. Meth- opening in most nematodes studied indi- odology. Raleigh: North Carolina State University cates the sensory nature of spicules and Graphics. their probable function as tactile or che- 8. Eisenback, J. D., and H. Hirschmann. 1982. moreceptive organs. In the case of Dory- Morphological comparison of stylets of male root- knot nematodes (Meloidogyne spp). Scanning electron laimida, the supplementary organs on the microscopy 2:837-843. ventral side of the male tail may play the 9. Eisenback, J. D., H. Hirschmann, J. N. Sasser, role of finding females and locating the and A. C. Triantaphyllou. 1981. A guide to the four most common species of root-knot nematodes (Me- vulva. loidogyne spp.), with a pictorial key. A cooperative pub- The excised gubernacula are of similar lication of the Departments of Plant Pathology and hard, sclerotized material as the spicules. Genetics, North Carolina State University, and the The three-dimensional aspect of the gu- United States Agency for International Develop- ment. Raleigh: North Carolina State University bernaculum ofH. galeatus and its morpho- Graphics. logical features are shown here for the first 10. Geraert, E., and A. De Grisse. 1981. The male time. The gubernacula of H. galeatus and copulatory system in tylenchid taxonomy (Nemato- D. heterocephalus have similar gross mor- da). Nematologica 27:432-442. 11. Hechler, H. C. 1971. Taxonomic notes on phology with respect to the presence of a four species ofPanagrellus Thorne (Nematoda: Ceph- capitulum. The space existing between the alobidae). Journal of Nematology 3:227-237. distal part of the capitulum and the gu- 12. Hirschmann, H. 1971. Comparative mor- bernaculum may allow for the sliding of phology and anatomy. 3. Digestive system (C. Intes- tine. D. Posterior gut). Pp. 38-42 in B. M. Zucker- the dorsal vela of the spicules when they man, W. F. Mai, and R. A. Rohde, eds. Plant parasitic are protruded. The capitulum is probably nematodes. New York: Academic Press. attached to the gubernaculum on its dorsal 13. Hirschmann, H. 1983. Scanning electron mi- side and plays a role in directing sperm to croscopy as a tool in nematode taxonomy. Pp. 95- 111 in A. R. Stone, H. M. Platt, and L. F. Khalil, eds. the channel formed by the spicules during Concepts in nematode systematics. Systematics As- copulation. sociation Special Volume No. 22. London and New York: Academic Press. 14. H6gger, Ch., and G. W. Bird. 1974. Second- LITERATURE CITED ary male sex characteristics of Hoplolaimus galeatus. Journal of Nematology 6:12-16. 1. Bird, A. F. 1976. The development and orga- 15. Natasasmita, S., and A. De Grisse. 1978. The nization of skeletal structures in nematodes. 2. Cop- ultrastructure of the spicule region in Tylenchulus semi- ulatory spicules. Pp. 108-114 in N. A. Croll, ed. The penetrans (Nematoda: Tylenchulidae). Mededelingen organization of nematodes. New York: Academic van de Faculteit Landbouwwetenschappen, Rijksu- Press. niversiteit, Gent 43:779-794. 2. Chitwood, B. G. 1949. Root-knot nematodes. 16. Nickle, W. R. 1970. A taxonomic review of Part 1. A revision of the genus Meloidogyne, Goeldi, the genera of the Aphelenchoidea (Fuchs, 1937) Thorne, 1887. Proceedings of the Helminthological Society of 1949 (Nematoda: Tylenchida). Journal of Nematol- Washington 16:90-104. ogy 2:375-392. 3. Clark, S. A., and A. M. Shepherd. 1977. Struc- 17. Rodriguez-M., R., and A. H. Bell. 1978. Ex- ture of the spicules and caudal sensory equipment in ternal morphology of the spicules of Trichodoridae. the male of Aphelenchoides blastophthorus (Nematoda: Journal of Nematology 10:127-132. Tylenchida, Aphelenchina). Nematologica 23:103-111. 18. Wang, K. C., and T. A. Chen. 1985. Ultra- 4. Clark, S. A., A. M. Shepherd, and A. Kempton. structure of secondary male sex organs of Scutellonema 1973. Spicule structure in some Heterodera spp. Ne- brachyurum. Journal of Nematology 17:435-444. matologica 19:242-247. 19. Wen, G. Y., and T. A. Chen. 1976. Ultra- 5. Coomans, A., and A. De Grisse. 1981. Sensory structure of the spicules of Pratylenchus penetrans. structures. 3. Genital sensilla. Pp. 159-164 in B. M. Journal of Nematology 8:69-73.