Mesorhabditis paucipapillata (Paetzold, 1955)

Ahlawat, S. & Tahseen, Q., 2023, A comparative account of the embryonic lineage and gonad development in two species of Rhabditidae (Nematoda), Journal of Natural History 57 (17 - 20), pp. 1157-1177 : 1159-1167

Embryogenesis and gonad development in Mesorhabditis paucipapillata

Mesorhabditis paucipapillata , a bacterivore, collected from decaying organic matter from the Shekha Jheel wetland, reflected a lopsided sex ratio of female:male (ie 6:1) in a natural population. On rearing a single gravid female culture in the laboratory, the sex ratio changed to 2:1 with a relatively greater occurrence of males.

Embryogenesis ( Figures 1–3 )

The females of M. paucipapillata were oviparous, with eggs generally laid in the single-celled stage. The uterine tract usually accommodated 1– 2 eggs. Eggs beyond the four-celled stage of embryonation were not found in the uterine tract of gravid females. The eggs were smooth-shelled and elongate, measuring 50–61 µm × 22–32 µm, narrowing towards the poles with the anterior pole relatively wider. In freshly laid eggs (Po), the cytoplasm seemed to stick to the inner egg surface with no empty space in between. The pole of entry of the sperm ( Figure 2a ) marked the posterior end of the developing embryo. However, polarity could also be determined in the uterine tract in cases of delayed egg laying, where in two- to four-celled stage eggs the posterior pole pointed towards the vulva. The male pronucleus (marked by an arrow) advanced towards the egg pronucleus ( Figure 2a ) and both migrated to the centre of the egg followed by their fusion. Cytoplasmic streaming leading to pseudocleavage was observed soon after sperm entry, which ended with the retraction of the egg cytoplasm, leaving spaces along the sides and poles. The egg, with a prominent central nucleus and retracted cytoplasm, prepared for division.

First cleavage occurred 25–30 min after pronuclear fusion and was transverse, giving rise to larger anterior (AB) and smaller posterior (P1) blastomeres ( Figure 2b ). After 20–25 min, AB divided transversely into anterior ABa and posterior ABp and simultaneously P1 divided obliquely into EMS and P2 ( Figure 2c ). Later, ABa divided into ABar and ABal after 25–30 min ( Figure 2e ), followed by the division of ABp into ABpa and ABpp 20–30 min later to form the six-celled stage. The division of EMS into MS and E occurred after 10–15 min, followed by the division of P2 into C and P3 after 10–15 min to form the eight-celled stage ( Figure 2f ). The total time taken from egg-laying to the eight-celled stage was about 2.0–2.5 h. The 10- celled stage was formed 25–30 min later while the 12-celled stage developed subsequently after 25–35 min. The 16-celled stage was reached at about 3.0–3.5 h ( Figure 2g ), whereas morula was formed ( Figure 2h, i ) after 4–4.5 h since egg laying. The blastula stage developed ( Figure 2j ) about an hour after the formation of morula. Gastrulation started 6–6.5 h after first cleavage, with the ingression of endodermal cells, and led to the differentiation of anterior and posterior regions of the gut. The stage involved germ layer differentiation as well as cell proliferation and cell movement. Later, the invagination marked the ̍lima bean̾ stage ( Figure 2l ) and further deepening of the cleft resulted in the ̍comma̾ stage ( Figure 2m ), about 1.5–2.0 h and 2.5–3.0 h after gastrulation, respectively.

The embryo showed growth and elongation at a great pace and started organogenesis. The ̍tadpole̾-stage embryo ( Figure 2n ) was formed 8.5–10.0 h after egg laying. The embryo then showed, for the first time, a worm-like body with an anterior shallow depression. The plum stage ( Figure 2o ) was formed about 40–50 min after the ̍tadpole̾ stage and marked the commencement of embryonic movement. After 45–60 min of the ̍plum̾ stage, the embryo attained a ̍loop̾ shape or appeared two egg-folds long. The movement of the embryo increased during the ̍loop̾ stage which also marked the development of faint stomal walls as thin refractory outlines. The latter was followed by the early pretzel stage ( Figure 2p ) after 2.5–3.0 h, when the embryo attained 2.0–2.5 egg-fold length. The early pretzel stage lasted for 3.0–3.5 h and led to the late pretzel stage. During the latter stage, the intestine, pharynx and rectum were well formed and the tail narrowed to a fine terminus. The stomal outline thickened and was differentiated into stomal components. The length of the juvenile prior to hatching was approximately 2.5–3.0 times the egg length. The embryonic movement sped up during this stage, with circular movements supplemented with body twisting and turning. After about 3.5–4.0 h in the late pretzel stage, the juvenile exerted pressure against the shell due to continuous body movements and repeated labial explorations. The total embryonation time ( Figure 3 ) from the single-celled laid egg to hatching was 19–22 h at 25 ± 2°C.

Morphology of gonad development in the female ( Figures 4 , 5 )

The gonad development continued from J1 to J4 as a part of post-embryonic development. However, the growth patterns of the genital primordium of M. paucipapillata were found to be good markers to differentiate these stages. The configuration of nuclei/cells including those in the ventral cord at each developmental stage is given in Table 1 .

First-stage juvenile. The genital primordium of the first-stage juvenile ( Figure 4a ) of M. paucipapillata was sexually undifferentiated, located at 51.8–55.1% of the body length from the anterior end. In the ventral view, the genital primordium appeared slightly obliquely oriented to the main body axis. It contained two large central germinal (Z2, Z3) and two relatively smaller polar somatic cells (Z1, Z4). The primordium in all J 1 juveniles showed a fixed number of primordial cells, without any sex differentiation. P(4–8).p were located posterior to P11.p. A series of 12 ectoblasts – P(1–12).p, were observed at regular intervals along the mid-ventral line.

Second-stage female juvenile. The spindle-shaped genital primordium ( Figures 4b , 5d ) was located at 52.6–54.9% of the body length from the anterior end. Further proliferation of germinal cells (Z2, Z3) was not observed. The sexually undifferentiated second-stage juvenile possessed a primordium similar to that of the first-stage juvenile. Initially, the primordium of J2 exhibited a mixed arrangement of somatic and germinal cells appearing as descendants of Z1/Z4 and Z2/Z3, respectively. However, during the moulting period, the localisation of cells and the resulting primordial growth indicated the future sex of the juvenile. The two polar cells Z1/Z4 gave rise to 2–6 descendants with a distal tip cell (DTC) at the anterior end and a cluster of somatic cells with an anchor cell (AC) towards the posterior end of the primordium. A slightly extended posterior end of the primordium with proliferating somatic cells indicated the future zone of elongation. The vulval precursor cells extended posteriorly to the elongating primordium, representing the presumptive equivalence group. The cells looked prominent, with slightly larger nuclei compared to other ventral cord cells.

Third-stage female juvenile. The primordium of third-stage female juvenile of M. paucipapillata was located at 51.4–51.9% of the body length from the anterior end. The primordium showed a posterior growth and extended as a result of the proliferation and placement of Z4 descendants ( Figures 4e , 5f, h ). The somatic cells were mostly localised in the posterior-elongating arm with a proximal-most AC. The latter was a transient cell that primarily functioned to lead the growth of the gonad towards the posterior vulva. The primordium during the third stage contained a range of 2–6 germinal and 8–24 somatic cells, including DTC, a single, large somatic cell located at the anterior tip of the primordium. The latter formed a close-fitting cap over the distal-most germinal cells. The moulting period marked the widening of the posterior end of the primordium due to the proliferation of somatic cells. The vulval precursor cells P(4–8).p were placed at about 70–75% of the body length from the anterior end, just close to the posteriorly-extending conduct (bearing a DTC-like AC) ( Figure 5h ).

Fourth-stage female juvenile. The fourth-stage female juveniles possessed a muchelongated genital primordium ( Figure 4i ), extending from 50.7–52.0% to 70–75% of the body length from the anterior end. The primordium showed extensive elongation posteriorly. The AC was found at the extreme posterior end. The posterior end reaching the level of vulval precursor cells widened significantly. The latter showed the location of the future uteri and vagina. Of the five vulval precursor cells P(4–8).p, the central ones P(5–7).p were aligned in the region at the rear end of the gonoduct to give rise to descendants that actively participated in vulva formation. P6.p assumed the primary fate, P5.p and P7.p the secondary fate and the remaining cells executed tertiary lineages. P(5–7).p form the vulva after migrating to a posterior position; however, the Pn.p cells are not equivalent in their potential to form the vulva. The cells forming the vulva appear to be arranged in six rings, stacked one above the other. In the moulting stage, the rear anterior end of the primordium had the germinal cells reflexed, thus differentiating into the germinal part/ovary containing germinal cells, while the remaining gonoduct formed the somatic gonad. The vulva formation along with the differentiation of the somatic gonad ( Figures 4i , 5j, l ) was completed with the termination of moulting.

Adult female. The females of M. paucipapillata possessed a mono-prodelphic reproductive system with a reflexed ovary ( Figures 4k , 5i, k ). The entire reproductive system was divided into the germ line and the somatic part. The germ line is constituted of the germinal cells in different stages of development, while the somatic part included the oviduct, spermatheca, uterus, vagina and vulva besides the DTC and gonadal sheath. The genital tract of M. paucipapillata was observed to have a slender ovary with a DTC at the end followed by two tiers of germinal cells in alternate arrangement. The differential size of sperm in the proximal part of the oviduct and spermatheca indicated the hermaphrodite sperm and those transferred by males. The spermatheca continued into a much narrower region (ie the glandular uterine part). The muscular part of the uterus was wider with thick walls. The vagina was simple, slightly anteriorly directed with radially ridged vulval lips ( Figure 5p ).

Morphology of gonad development in the male ( Figure 4 )

The development of the males started from an ovoid to spindle-shaped primordium in the first-stage juvenile without any sexual differentiation ( Figure 4a ) and was located 52.6– 54.9% of the body length from the anterior end. Further proliferation of germinal cells (Z2, Z3) was observed with the number of cells ranging from 2–4 that represented the germ line descendants. The sexually undifferentiated second-stage juvenile possessed a primordium similar to the first-stage juvenile. However, during the late J2 stage, the primordial growth indicated the future sex of juvenile. Of the two polar cells Z1/Z4, 4–12 descendants were generated with a DTC at the posterior end and a cluster of somatic cells with a linker cell towards the anterior end of primordium. The anterior end of the primordium showed an aggregation of somatic cells, indicating its future zone of elongation ( Figure 4b ).

Third-stage male juvenile. Third-stage male juveniles possessed a primordium situated at 47.3–51.3% from the anterior end and consisted of 2–6 germinal and 8–16 somatic cells including two DTCs at the distal end and a linker cell at the proximal end. The primordium showed anterior proliferation of the somatic cells ( Figure 4d ) and eventually, during the moulting phase, a flexure was present with the linker cell taking a lead in its posterior elongation. Two dense cellular aggregates were observed on each dorsolateral side of the rectum contributing to the development of the spicules and associated structures ( Figure 4f ).

Fourth-stage male juvenile. The fourth-stage male juveniles possessed a primordium at 47.4–53.9% of body length from the anterior end. The genital primordium showed a small reflexed part showing the germ line of the future gonad and a considerably longer tube elongating posteriorly towards the cloaca ( Figure 4h ). At the termination of the moulting process, the number of germinal cells increased from 6–18 in the germinal part or testis with two somatic DTCs tightly fitting at the distal tip. There were 20–52 somatic cells destined to take part in forming the somatic gonad with differentiation of the vas deferens, seminal vesicle and ejaculatory duct. The linker cell was proximally located, indicating the direction of gonad elongation. During the moulting phase, the gonoduct nearly reached the rectum, with the linker cell facilitating a connection with the rectum to form the cloaca. The stage marked the continuity of the gonoduct and the cloaca. The spicular precursor cells were approximately 18–34 in number, contributing to the development of spicules and the gubernaculum as hyaline structures during moulting. The bursa, together with the genital papillae, was the last structure to develop from special hypodermal cells called ray precursor cells.

Adult male. The male possessed a monorchic reproductive system with a reflexed testis ( Figure 4j ). The entire reproductive system could be divided into germ line and somatic parts. The germ line constituted of the germinal cells contained in the testis while the somatic part included the seminal vesicle, vas deferens, ejaculatory duct, cement glands/ejaculatory glands and cloaca besides the DTCs and gonadal sheath. The testis showed two DTCs at the distal end ( Figure 4j ) followed by the germ cells in different stages of development. The proximal part of the testis containing spermatocytes gradually widened into the seminal vesicle. The latter was filled with spermatids. The seminal vesicle showed greater diameter than either the testis or the posteriorly located vas deferens. The vas deferens showed the opening of a pair of ejaculatory glands or cement glands; and the posterior part, often referred to as the ejaculatory duct, joined with the rectum to form the cloaca. The accessory reproductive organs, spicules and gubernaculum were lodged in paired pockets in the cloacal chamber.

Figures

Figure 1. Early embryonic cell lineages in Mesorhabditis paucipapillata and Cephaloboides anisospiculus as compared to Caenorhabditis elegans.

Figure 2. Embryonic stages in Mesorhabditis paucipapillata. a: Egg showing pronuclei before fusion (arrow pointing to the male pronucleus); b: two-celled stage; c: four-celled stage; d: rhomboid arrangement of blastomeres; e: five-celled stage; f: eight-celled stage; g: sixteen-celled stage; h, i: morula stage; j: blastula stage; k: gastrula stage; l: lima bean stage; m: comma stage; n: tadpole stage; o: plum stage; p: pretzel stage. Scale bar = 10 µm.

Figure 3. Duration of landmark stages during embryonic development of Mesorhabditis paucipapillata and Cephaloboides anisospiculus.

Figure 4. Gonad development in Mesorhabditis paucipapillata. Genital primordium a–e, h, i. a: Firststage juvenile; b: moulting second-stage male juvenile; c: moulting second-stage female juvenile; d: third-stage male juvenile; e: third-stage female juvenile; f: tail of third-stage male juvenile showing spicular primordium; g: tail of third-stage female juvenile; h: fourth-stage male juvenile; i: fourth-stage female juvenile; j: adult male gonad; k: adult female gonad. Scale bars = 10 µm.

Figure 5. Schematic and LM micrograph showing sequence of vulva formation in Cephaloboides anisospiculus (a, c, e, g, i, k, m) and Mesorhabditis paucipapillata (b, d, f, h, j, l, n). C, D: Second-stage juvenile showing 12 Pn.ps; e, f: third-stage juvenile showing 12 vulval precursor cells with five cells forming vulval prominence group; i, j: fourth-stage juvenile showing vulval prominence group aggregating at vulval site; k, l: moulting fourth-stage juvenile with vulval precursor cells; m, n: vulval region: o, p: SEM of vulval region. Scale bars = 10 µm.