Russian Journal of Nematology, 1999, 7 (2), 105 - 1 14 The embryonic development of Pontonema vulgare (Enoplida: Oncholaimidae) with a discussion of nematode phylogeny Dmitrii A. Voronov Institute for Information Transmission Problems of Russian Academy of Sciences, Bolshoy Karehy per. 19, Moscow, 101447, Russia, e-mail: [email protected] Accepted for publication 20 July 1999 Summriry. The cell lineage in embryonic development of the marine nematode Pontonema vulgare (Enoplida, Oncholaimidae) was studied using cell labelling by fluorescent dye. After labelling at the two-cell stage, the border between labelled and unlabelled cells is different in different Lima bean and comma stage embryos crossing them in left-right, anterior-posterior, or intermediate direction.Therefore, in contrast to the majority of nematodes, P. vulgare has a variable type of development. However, as in other nematodes all the endoderm is derived from a single blastomere of the elght-cell stage, and the gastrulation process in P. vulgare is comparable to that in other nematodes. In general, the development of P. vulgare is similar to that in the previously studied Enoplus brevis (Enoplida, Enoplidae). Using the type of development, all nematodes can be placed in one of three major groups: 1, with variable cleavage (order Enoplida); 2, with invariant cleavage and anterior localization of endoderm material at the two-cell stage (remaining Enoplea); 3, with invariant cleavage and posterior localization of endoderm material at the two-cell stage (Chromadorea and Secernentea). This classification is independently supported by molecular phylogeny studies and by several anatomical characters. Key words: nematodes, embryonic development, cell lineage, cleavage, gastrulation, phylogeny. Study of nematode embryonic development is a This, together with untransparency of embryos due well-developed field of science with fundamental to high concentration of yolk granules, prevents results having been obtained with Caenorhabditis study of the enoplidan cell lineage by direct obser- elegans. However, until now its significance in nema- vation. However, application of the technique of tode phylogeny and taxonomy has been little recog- intracellular labelling by fluorescent dye can over- nized. This situation has arisen as a result of unequal come these problems. Cell labelling enables ob- study of development in different nematode taxa. servation to be made of the progeny of an arbitrarily However, some reports suggest that the cell lineage chosen blastomere in a succession of developmental and other fundamental characteristics of nematode stages. Moreover, the bright luminescence of an development may have utility for solving problems appropriate label provides information of the redis- of nematode phylogeny (Drozdovsky, 1975, 1977, tribution of cells within the embryo, e.g. in studies of the gastrulation process. 1978; Skiba & Schierenberg, 1992; Goldstein et a/., The cell tracing technique revealed the absence 1998; Voronov et al., 1998; Wiegner & Schierenberg, of fmed cell lineage in the enoplidan species, Enoplus 1998). Thus, Drozdovsky (1969, 1975, 1977, 1978) brevis (Enoplidae) (Voronov et al., 1986, Voronov demonstrated that the cell lineage of representatives & Panchin, 1995b, 1998). However, E. brevis is the of Enopleais markedly different from that in Se- only enoplidan species for which cell tracing experi- cernentea and in Chromadorea, and proposed an ments have been made. In the present study the cell embryological criterion for distinguishing between tracing technique was used to investigate the embry- these two branches of nematodes. onic development of the enoplidan Pontonema The cleavage pattern in the order Enoplida, in vulga~(Oncholaimidae). The results obtained and contrast to all previously studied nematodes, is highly their significance for nematode phylogeny are dis- variable (Cherdantsev et al., 1972; Malakhov, 1994). cussed. D. A. Voronov MATERIALS AND METHODS Specimens of the free-living marine littoral nema- tode Pontonema vulgare (Bastian, 1865) (Enoplida, Oncholaimidae) were collected from Kandalaksha Bay of the White Sea in the vicinity of the Kartesh Marine Biological Station of the Russian Academy of Sciences, and the White Sea Biological Station of Moscow State University. Uncleaved eggs were dis- sected from gravid females in filtered seawater. To observe both sides of the embryo, the eggs were placed on cover slips to which they stuck by their outer mucus sheath. During development eggs were kept at 15-16 "C. Fig. I. Some stages of P. vulgare development.Camera The fluorescent dye 5(6)-carboxyfluorescein lucididrawings of living embly6s. A: Interphase cytoplas- (CF) (Sigma, Mw 376.3) was iontophoretically mic movements of zygote; similar movements are also injected at the two- or eight-cell stage to visualize peculiar for interphase blastomeres; B: The rounded zygote before the first division of cleavage; two polar the progeny of labelled cells in the developing em- bodies are seen near the equator of the egg-shell; C: bryo. Microelectrodes used for injections were made Two-cell stage, the usual arrangement of blastomeres along of glass with inner filaments (Clark Electromedical the longitudinal egg-shell axis; D: The oblique disposition Instruments, GC100F-15) and backfilled with injec- of blastomeres at the two-cell stage; E, F, and G: Examples tion solution. Single blastomeres were injected using of blastomere distribution at the four-cell stage (E, rhom- aseveral seconds pulse of 1-5 nA negative current bus; F, tetrahedron; G, T-shaped figure); H: The begin- ning of ventral cleft formation in ventral view with the through 30-50 MegR glass microelectrodes filled ventral cleft seen as an elliptical depression on the ventral with 3% solution of potassium carboxyfluorescein in side of the embryo (stipled); I: Lima bean stage in left distilled water. Injections were performed using a lateral view with the anterior at the top; J: Comma stage fluorescent microscope (ML-2, LOMO, Russia) in left lateral view with the anterior at the top. Scale bar - with a filter set optimized for fluorescein. The 100 om. development of the labelled embryos was traced from the beginning to the comma stage, when the pattern The early cleavage divisions occur approximately of label distribution gradually became unclear due every six hours. They are usually (up to the 16-cell to the loss of CF by differentiating cells. stage) synchronous, but sometimes delays of divi- Drawings were made with a camera lucida. Lllus- sions of some blastomeres were observed. These trations of labelled embryos were taken on Kodak delays can last up to 2-3 hours, but do not perturb Gold 400 film or ahigh sensitivity video-camera further development. (DeltaTex, Russia). At the 2 to 16-cell stage blastomeres are indistin- guishable. Early blastomeres are approximately equal, RESULTS but sometimes slight irregular differences in their size are visible. The spatial-temporal pattern of early Only a brief general description of P. vulgare cleavage varies between different embryos, e.g. blas- development is presented as it has been reported tomeres at the two-cell stage are usually located along previously (Cherdantsev et al., 1972; Malakhov & the longitudinal egg-shell axis, but sometimes they Cherdantsev, 1975; Malakhov, 1994). occur at a random angle to the axis (Fig. lC, D). The zygote diameter in P. vulgare eggs is about Blastomeres at the four-cell stage can be arranged as 130 pm. Egg-shell shape is ellipsoidal, often visibly a tetrahedron, rhombus, quadrate, or T-shaped asymmetrical (Fig. I), and the egg-shell is large configuration (Fig. lE, F, G). These configurations providing a large free space for the embryo. Before can transform from one to another due to interphase cleavage, two polar bodies are formed. Their posi- motility of the blastomeres. tion, relative to the longitudinal egg-shell axis, varied During the course of later development it is in difTerent eggs from polar to equatorial. possible to distinguish the stages similar to that in The interphase zygote has prominent cytoplasmic other nematodes (Fig. IH, I, J), i.e., ventral cleft movement (Fig. IA), which cease prior to mitosis formation (beginning at about 90 hours at 15-16 "C), (Fig. 1B). Similar movements are characteristic for Lima bean stage (140 hours), comma stage (170 interphase blastomeres, and these can interfere with hours). The first stage juvenile hatches after approxi- direct observation of cell lineage. mately 30 days of development. The dorsal-ventral Development of Pontonema vulgare Fig. 2. The distribution of descendants of the first two blastomeres in the comma stage embryos of P. vulgare. These embryos were labelled by carboxyfluorescein (CF) at the two-cell stage in an arbitrarily chosen blastomere. Anterior at the left in ventral view (capital letters) and dorsal view (lower case letters). Initial position of injected blastomere at the two-cell stage was at the right in A, a, B, b and at the left in C, c: A, a, the transverse border between descendants of two-cell stage blastomeres; B, b, the oblique border between descendants of two-cell stage blastomeres; C, c, the label is approximately evenly distributed along the longitudinal embryo axis and therefore the fmt two blastomeres can not be designated as being "anterior" or "posterior". In this embryo the main part of the surface cells is labelled. In the embryo A, a the mid-gut was stained and their fluorescence is clearly seen through unstained epithelial cells at the dorsal side of the embryo (a, asterisk). In B, b and C. c the mid-gut was not stained and it was seen as a darkmass under the dorsal surface (b, c, asterisk). Scale bar - 100 pm. polarity of the embryo is evident after the beginning and also within the embryo was used). Therefore, in of gastrulation, the direction of the anterior-poste- different eggs of P. vulgare the first two blastomeres rior axis is visible after the ventral clef? stretching, give rise to different parts of the embryo, ranging and the polarity of the anterior-posterior axis is from anterior-posterior to left-right distribution with discernible only at the Lima bean stage.