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 mi- croscopy, 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 nema- tode 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 nema- tode stylets (8). Five live males were trans- ferred 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 = cyto- plasmic 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