Journal of Nematology 28(2): 169-176. 1996. © The Society of Nematologists 1996. Position of Pharyngeal Gland Outlets in (Nemata)

A. COOMANS, I A. EYUALEM, 1'2 AND M. C. VAN DE VELDE 1

Abstract: In this study an attempt was made to determine the position of the outlets and nuclei of the pharyngeal glands in four monhysterid genera. Five Eumonhystera spp., seven Monhystera spp., and eight Monhystrella spp. were studied under the light microscope. Longitudinal sections of an undescribed Monhystera sp. and cross sections of Geomonhystera ckisjuncta were also studied under the scanning and transmission electron microscope, respectively. The results of the light microscopic studies were inconclusive about the position of the outlets but showed a number of nuclei in the basal part of the pharynx. The scanning and transmission electron microscopic studies revealed five pharyngeal glands and their outlets; their position was as follows: dorsal gland outlet at the base of buccal tooth, first pair of ventrosublateral gland outlets halfway along the pharynx, and second pair of ventrosublateral gland outlets close to the base of the pharynx. It is concluded that at least three, and possibly five, nuclei are in the basal part of the pharynx. This pattern, in the position of the outlets and nuclei, is similar to that in Caenorhabditi6 elegans (Maupas, 1900) Dougherty, 1953 and may well be the basic plan in the Class (including Secernentia as a subclass). Key words: Geomonhystera, Monhystera, , morphology, systematics, pharyn- geal gland outlet, pharynx, scanning electron microscopy, transmission electron microscopy, ultra- structure.

The number, shape, and position of near the base of the stoma; the ventrosub- pharyngeal glands, and especially the po- lateral (="subventral") glands never open sition of their nuclei and outlets, are im- at the anterior end of the pharynx. This portant features to establish phylogenetic arrangement is in contrast to Enoplida, relationships among at several where a pair of ventrosublateral glands of- taxonomic levels (2,4,5,10,13). Typically, ten opens through teeth in the stoma or at nematodes have three or five pharyngeal the anterior end of the pharynx, as the glands (one dorsal and one or two pairs of dorsal gland does. ventrosublateral glands). Maggenti (12) Monhysterida are usually classified as an considered the five glands to be an ances- order in Chromadoria (11,12). Chitwood tral character found in Enoplia and the and Chitwood (2) presented diagrams on presence of only three glands in Chroma- the position of pharyngeal nuclei in five doria (including Monhysterida) as derived. monhysterid genera (Desmolaimus de Man, Three glands in Secernentea was also con- 1880, Siphonolaimus de Man, 1893, Ter- sidered derived. However, he further schellingia de Man, 1880, Theristus Bastian, stated that five glands might be derived 1865, and Tripylium Cobb, 1920). The nu- from three glands by duplication of the clei of one dorsal and one pair of ventro- subventrals in some Chromadoria and Se- sublateral glands were shown in the poste- cernentea. On the other hand, examples rior region of the pharynx; the outlets of exist in some taxa, e.g., Dorylaimida, these glands were depicted at or close to where five glands have been reduced to the base of the stoma for the dorsal gland three (Longidoridae) (14). (all five genera), and close to the nerve ring According to Chitwood and Chitwood for the ventrosublateral glands (in Ter- (2), Chromadoria, such as Monhysterida, schellingia, Theristus, and Tripylium). This have only the dorsal gland opening at or pattern agrees with that of Chromadoria in general. Received for publication 15 May 1995. Riemann (13) described the pharyngeal 1 Zoology Institute, Ledeganckstraat 35, 9000 Gent, Bel- glum, and 2Bahir Dar Teachers' College, Addis Ababa Uni- glands of , a family of Mon- versity, Personal P.O. Box 223, Bahir dar, Ethiopia. hysterida. In Paralinhomoeus de Man, 1907 The authors acknowledge the technical assistance of R. V. Driessche for processing the samples, mounting the slides, and Anticyclus Cobb, 1920, the orifices of and making sections. one dorsal and two ventrosublateral glands 169 170 Journal of Nematology, Volume 28, No. 2, June 1996 are situated in the stoma region. In the positions of S1N (at level or just poste- Eleutherolaimus Filipjev, 1922, an addi- rior to nerve ring) and S2N (near base of tional pair of ventrosublateral glands with pharynx). orifices at the level of the nerve ring was described. Riemann (13) referred to a pa- MATERIALS AND METHODS per by zur Strassen (17) in which the latter Specimens for light microscope (LM) author described ventrosublateral gland and scanning electron microscope (SEM) openings close to the anterior end of the observations were fixed with hot (--+60 °C) pharynx in Siphonolaimus. On the basis of 4% formaldehyde. Nematodes were ex- these observations, Riemann (13) pro- tracted with centrifugal flotation from posed classifying Linhomoeidae and Si- freshwater sediment of Lake Tana, Ethio- phonolaimidae in Siphonolaimoidea and pia. Ludox (50% silicasol [SiO2] colloid so- to differentiate this superfamily from lution) was used as an extraction agent. Monhysteroidea by the position of the For SEM, nematodes were hand-sectioned pharyngeal gland outlets, among other longitudinally under a binocular micro- characters. This proposition was accepted scope; the longitudinal sections then were by Lorenzen (11). dried with the critical point drying proce- Jacobs et al. (9) described three pharyn- dure and attached to self-adhesive tape, geal gland cells (one dorsal and a pair of coated with gold, and studied under SEM, ventrosublateral) in Diplolaimella dievenga- according to the procedures of Eyualem tensis Jacobs, Van de Velde, Geraert & and Coomans (6). Nematodes for trans- Vranken, 1990, occurring at the base of mission electron microscopy (TEM) were the pharynx. The ventrosublateral nuclei isolated from "Sluice Dock" Ostend, a were referred to as S2N 1 and S2N 2 in the man-made marine lagoon near the Belgian text (p. 9-10) as well as on the illustration coast, and cultured according to Vranken (Fig. 10H,J) (9). Another pair of smaller et al. (16). Specimens were picked from but still prominent nuclei were located just petri dishes, cooled in an ice-bath to behind the nerve ring. These nuclei were stretch, and then killed and fixed in an ice- referred to as SIN 1 and SIN 2 in the text (p. cooled fixative composed of 1.5% acrolein, 10), where it was mentioned that "these 3% glutaraldehyde, and 1.5% paraformal- were first interpreted as gland nuclei"; but dehyde in 0.2 M sodium cacodylate buffer. SIN s and S1N 2 were still explained as "an- After fixation and rinsing, the specimens terior set of ventrosublateral gland nuclei" were post-fixed in 2% osmium tetroxide, in the legend to Fig. 10. Thus, there re- followed by en bloc staining in 2% uranyl mains some ambiguity about the ventro- acetate. Other details of the procedures of sublateral nuclei. nematode preparation for TEM were Jacobs and Heyns (7) described five pha- those described in Van de Velde and ryngeal glands in Monhystera coomansi Ja- Coomans (15). cobs & Heyns, 1992: one dorsal gland with nucleus (DN) at the base of the pharynx RESULTS and the outlet (DO) at the base of the Light microscopic observations on dif- stoma, a first pair of ventrosublateral ferent Monhysterids, viz. Monhystera cf. glands with nuclei (SiN)just in front of the gabaza Joubert & Heyns, 1980; M. somereni nerve ring and outlets ($10) at the base of Allg~n, 1952; four undescribed Monhystera stoma, and a second pair of ventrosublat- spp.; Monhystrella macrura (de Man, 1880) eral glands with nuclei (S2N) between level Andr~ssy, 1981; M. lepidura altherri (Juget, of DN and nerve ring and outlets ($20) at 1969) Jacobs, 1987; six undescribed Mon- level of nerve ring. Jacobs et al. (8) rede- hystreUa spp.; Eumonhystera dispar (Bastian, scribed three Monhystera species and gave 1865) Andr~issy, 1981; E. filiformis (Bas- the same interpretation as Jacobs and tian, 1865) Andr~issy, 1981; E. pseudobul- Heyns (7), with the only difference being bosa (yon Daday, 1896) Andr~ssy, 1981; E. Pharyngeal Gland Outlets in Monhysterids: Coomans et al. 171 mwerazii (Meyl, 1957) Andr;issy, 1981; and f E. vulgaris (de Man, 1880), Andr/tssy, 1981, did not yield conclusive evidence about the exact position of the pharyngeal DO -- -- "'-~ gland outlets and nuclei. The dorsal gland I nucleus (DN) is situated near the base of the pharynx, and the cell extends to the stoma region. The ventrosublateral sectors are often vacuolar, which makes it difficult to locate the gland nuclei. However, a pair of nuclei in sublateral to lateral position was found in most specimens slightly be- hind the level of DN. A second pair of such nuclei was seen, though less frequently, anterior to the level of DN at less than two pharynx widths from pharynx base. Still other nuclei occur and, apart from DN, it is impossible to decide whether the above- mentioned nuclei are really S2N and S1N. Glandular tissue in the ventrosublateral sectors was observed up to the nerve ring (Fig. 1). -- -- f-,.,-,lmD Scanning electron microscopy of longi- $10 f " J tudinal sections of an undescribed Monhys- A tera sp. revealed five outlets (Fig. 2). The l ' dorsal outlet (DO) is a small (about 0.7- I Ixm-long) slit just behind the small dorsal I a tooth (Fig. 2B). A first pair of ventrosub- t f # lateral gland outlets ($10) occurs at 52- | I !-. 54% of the pharynx length, at the level of I the nerve ring (Fig. 2A,C); a second pair I ($20) occurs at 83-85% of the pharynx S1 " length (Fig. 2A,D). $10 and $20 are about S20-- t t 1-~m-long slits (Fig. 2). The tissue oppo- t site $20 is different from the remainder of DN the pharyngeal region. This area appears \'°.'°2 -.;' :% granular as opposed to the fibrillar (=muscular) overall appearance of the S2N pharynx (Fig. 2A). Transmission electron microscopy con- FIG. 1. Schematic representation of pharyngeal firmed the position of DO and $10 in glands in monhysterids. DN: Dorsal gland nucleus, DO: Dorsal gland outlet, S1N: Nuclei of the first pair Geomonhystera disjuncta (Bastian, 1865) Ja- of ventrosublateral glands, S~O: Openings of the first cobs, 1987 (Fig. 3A,B) and Diplolaimella di- pair of ventrosublateral glands, S2N: Nuclei of the evengatensis. In the posterior part of the second pair of ventrosublateral glands, $20: Outlets pharynx there are elaborate cytoplasmic of the second pair of ventrosublateral glands. areas composed of rough endoplasmic re- ticulum with wide cisternae and secretory over the entire width of both ventrosublat- granules. In the most basal part (at cardia eral sectors, occurs the cytoplasm of the level), the dorsal gland ends in a narrow second pair of ventrosublateral glands. In middorsal extension (Fig. 4A). To the left this cytoplasm are two nuclei, one each in and to the right of this extension, as well as the left and right adradial dorsal sectors; 172 Journal of Nematology, Volume 28, No. 2, June 1996

Fro. 2. bLM photographs of Monhystera sp.. A) Longitudinal section of pharynx. Arrow-heads point to outlets of both pairs of ventrosublateral glands, arrows point to glandular tissue at $20 level. B) Stoma showing dorsal tooth (black arrow) and dorsal gland outlet (white arrow). C) Outlet of a ventrosublateral gland of the first pair (arrow). D) Outlets of second pair of ventrosublateral glands (arrows). Scale bars equal 10 ~m in A, 1 I~m in B-D. Pharyngeal Gland Outlets in Monhysterids: Coomans et al. 173

FIG. 3. TEM photographs of Geomonhystera disjuncta. A) Dorsal gland outlet (arrow). B) First pair of ven- trosublateral gland outlets (arrowheads). C) Gland nuclei at base of pharynx. DN: dorsal gland nucleus; N.R.: = nerve ring; S2N: nuclei of second pair of ventrosublateral glands. Scale bars equal 0.5 ~m in A and B, 1 p.m in C. 174 Journal of Nematology, Volume 28, No. 2, June 1996

FIG. 4. Geomonhysteradisjuncta. A) Cross-section at base of pharynx showing the two subdorsally located SN. B) Cross-section at level of DN, arrows indicate boundary between ventrosublateral gland tissue and dorsal gland tissue. CA: cardia; DG: dorsal gland extension; DN: dorsal gland nucleus; SN: ventrosublateral gland nuclei. Bar equals 1 p,m. Pharyngeal Gland Outlets in Monhysterids: Coomans et al. 175 they represent the S2N (Figs2 3C; 4A). and then may occupy other sectors than More anteriorly, the cytoplasm of the sec- the one to which they belong (3,14); this ond pair of ventrosublateral glands is branching also was seen in our TEM ob- shifted ventrally due to a considerable ex- servations. Therefore, it is clear that the tension of the dorsal gland, which now oc- only reliable criterion for determining the cupies the whole dorsal sector and the lat- outlets is the position of the outlets them- eral parts of the ventrosublateral sectors. selves, not the presence of glandular tissue At this level DN is found (Figs. 3C; 4B). on its own. For this reason the interpreta- tion of Riemann (13) for some Monhyster- DISCUSSION ida should be checked and that of Jacobs and Heyns (7) should be corrected on the The observations reported here are in basis of the information presented here. agreement with some of the literature on The pharyngeal gland pattern described Monhystera (7,8) for the presence of five above may be the typical plan for Monhys- pharyngeal glands and the position of DO terida. This arrangement is similar to that and DN, but not for the other details. described for Caenorhabditis elegans by A1- There is agreement withJacobs et al. (9) in bertson and Thomson (1), and DO at base the position of 82N in Diplolaimella, despite of stoma, $10 at base of metacorpus, and some ambiguity in that study about the $20 and all nuclei (DN and two pairs of ventrosublateral nuclei and the number of SN) in the basal bulb. Chitwood and Chit- pharyngeal glands. wood (2) described only three glands in The three outlets and nuclei described Rhabditis. The pattern with the DO close to by Chitwood and Chitwood (2) for Mon- or inside the stoma, the $10 about halfway hysterida (DO at or close to the stoma, SO along the pharynx, the $20 at the base of close to the level of the nerve ring, DN and the pharynx, and all gland nuclei in the a pair of SN at the base of the pharynx) posterior region of the pharynx may well were found in this study. To our knowl- be the basic pattern for the Class Chroma- edge, the two additional outlets in the pos- dorea (including the subclass Secernentia terior region of the pharynx have not been in our interpretation) as opposed to the reported previously in any study of Mon- Class ~Enoplea with the DO and S10 in or hysterida. close to the stoma. If this pattern is ac- On the basis of our combined observa- cepted as typical, then taxa still classified tions from LM, SEM, and TEM, we de- under Chromadorea but with an Enoplea scribe the glandular system of Monhystera pattern of pharyngeal gland outlets (e.g., as follows: DO at base of dorsal buccal Linhomoeidae, Desmoscolecida) do not tooth or at the base of the stoma when a belong in Chromadorea. However, it is ad- dorsal tooth is absent; $IO at level of nerve visable to make such transfers only after ring, S1N in posterior pharynx region at adequate observations on these taxa are level of the slight basal expansion (almost performed. two pharynx widths anterior to pharynx base); $20 at 83-85% of pharynx (i.e., LITERATURE CITED slightly posterior to S1N ), S2N slightly be- hind level of DN. This arrangement is in 1. Albertson, D. G., andJ. N. Thomson. 1976. The agreement with the generally accepted pharynx of Caenorhabditis elegans. Philosophical Transactions of the Royal Society, London 275:299- idea that SO never open at the anterior 325. end of the pharynx in Chromadoria, but at 2. Chitwood, B. G., and M. B. Chitwood. 1950. In- variance with observations by zur Strassen troduction to nematology. Baltimore: University Park (17), Riemann (13), and Jacobs and Heyns Press. (7). 3. Coomans, A. 1985. A phylogenetic approach to the classification of the Longidoridae (Nematoda: Other authors have asserted that uninu- Dorylaimida). Agriculture, Ecosystems and Environ- cleate pharyngeal glands may be branched ment 12:335-354. 176 Journal of Nematology, Volume 28, No. 2, June 1996

4. Coomans, A., and P. A. A. Loof. 1970. Mor- 12. Maggenti, A. 1981. General hematology. New phology and of Bathyodontina (Dorylaim- York: Springer Verlag. ida). Nematologica 16:180-196. 13. Riemann, F. 1977. Oesophagusdr~sen bei Lin- 5. De Coninck, L. 1965. Classe des N6matodes, homoeidae (Monhysterida, Siphonolaimoidea). Bei- Syst6matique des N6matodes et sous-classe des Ade- trag zur Systematik freilebender Nematoden. Ver6f- nophorea. Trait6 de Zoologie 4:586-681. fentlichungen des Instituts fiir Meeresforschung in 6. Eyualem, A., and A. Coomans. 1995. Freshwa- Bremerhaven 16:105-116. ter nematodes of the Gal~ipagos. Hydrobiologia 299: 14. Robertson, M. W., and U. Wyss. 1979. Obser- 1-51. vations on the ultrastructure and function of the dor- 7. Jacobs, L. J., andJ. Heyns. 1992. Morphology of sal oesophageal gland cell in Xiphinema index. Nema- Monhystera coomansi sp. n. from Algeria (Nematoda: tologica 25:391-396. Monhysteridae). Nematologica 38:1-21. 15. Van de Velde, M. C., and A. Coomans. 1991. The ultrastructure of the buccal cavity of the mon- 8. Jacobs, L.J., J. Heyns, and A. Coomans. 1994. hysterid nematodes Geomonhysteradisjuncta and oiplo- Redescription of three South African Monhystera spe- laimella dievengatensis. Revue de N~matologie 14:133- cies (Nematoda). Suid-Afrikaans Tydskrif Dierkunde 146. 29:62-69. 16. Vranken, G., D. Van Brussel, R. Vanderhae- 9. Jacobs, L.J., M.C. Van de Velde, E. Geraert, ghen, and C. Heip. 1984. Research on the develop- and G. Vranken. 1990. Description of Diplolaimella ment of a standardized ecotoxicological test on ma- dievengatensis sp. n. (Nematoda: Monhysterldae). rine nematodes. I. Culturing conditions and criteria Nematologica 36:1-21. for two monhysterids, Monhystera di.sjuncta and Mon- 10. Loof, P. A. A., and A. Coomans. 1970. On the hystera microphthabna. Pp. 159-184 in Persoone G., E, development and location of pharyngeal gland nuclei Jaspers, and C. Claus, eds. Ecological testing for the in the Dorylaimina. Zeszyty Problemowe Postepow marine environment, vol. 2. Bredene, Belgium: State Nauk Rolniczych 92:79-161. University of Ghent and Institute of Marine Scientific 11. Lorenzen, S. 1981. Entwurf eines phyloge- Research. netischen Systems der freilbenden Nematoden. Ver- 17. zur Strassen, O.L. 1904. Anthraconema, eine 6ffentlichungen des Instituts for Meeresforschung in neue Gattung freilebender Nematoden. Zoologische Bremerhaven, Supplement 7:1472. Jahrbiicher, Supplements 7:301-343.