Description and developmental biology of the predatory diplogastrid Acrostichus nudicapitatus (Steiner, 1914) Massey, 1962 (Nematoda: Rhabditida)

See Table 1.

Female. Body slender, medium to large-sized; straight or slightly curved after fixation, tapering at both extremities. Cuticle with fine transverse striations and longitudinal ridges; lateral fields not demarcated. Lip region flattened to slightly truncate, continuous or demarcated by a slight depression from adjoining body. Lips amalgamated; labial sensilla slightly raised. Amphidial apertures elliptical, situated about 4 – 6 μm from anterior end of stoma. Cheilostom cuticularised, smaller than gymnostom, converging anteriorly with six faintly visible adradial plates. Stegostom anisotopic, anisomorphic. Dorsal metastegostomal wall with an anteriorly directed, upright to slightly arcuate tooth, each sub-ventral wall provided with a small tooth. Pharyngeal corpus muscular, 40 – 45 μm long; metacorpus ovoid, strongly developed with thickened lumen; isthmus distinctly differentiated from metacorpus, 15 – 19 μm long. Basal bulb small, glandular in nature, continuous with isthmus, 16 – 18 μm x 12 μm in dimension. Nerve ring encircling isthmus at 73 – 75 % of pharyngeal length from anterior end. Hemizonid usually conspicuous in basal bulb region. Secretory-excretory pore placed posterior to hemizonid or at 78 – 85 % of pharyngeal length from anterior end. Body at pharyngeal end ca 2.9 – 3.4 times labial diam. wide. Cardia 6 – 7 μm long. Intestine thin-walled, often with a bacterial pouch present in anterior part. Rectum ca 1.2 – 1.5 times anal body diam. long, with inconspicuous rectal glands. Reproductive system didelphic, amphidelphic; ovaries reflexed, usually reaching the level of vulva but occasionally crossing each other; anterior ovary on right side and posterior on left side of in-testine. Posterior genital branch usually larger as compared to anterior genital branch. Oocytes arranged in double row in proliferation zone of each ovary; proximal part of ovary separated from rest of ovary by a sphincter. Oviduct a narrow tube leading to wider, ovoid spermatheca. Uteri containing 1 – 2 eggs of 45 – 48 μm x 23 – 26 μm in dimension occasionally in early stage of segmentation. Older females contained as many as 4-6 intra-uterine eggs. Each uterus with a bilobed or kidney-shaped dorsal pouch connected to vagina, occasionally containing sperms. Vagina 10 – 12 μm, cuticularised, with thick lumen occupying about 1/3rd – 1/4th of corresponding body diam.; epiptygma present. Vulval lips slightly protruding. Distance between vulva-anus 0.6 – 0.8 times tail length. Tail long filiform with whip-like terminus.

Male. Similar to female in general appearance but shorter in length and strongly curved in posterior region. Testis single, laterally reflexed, reflexed part 45 – 52 μm long. Vas deferens continuous with a tapering ejaculatory duct joining with rectum to form cloaca. Spicules strongly-built, ridged, arcuate in proximal half or bent at 40 % of length proximally, 1.5 – 1.8 times anal body diam. long with rounded capitula, a distinct neck with small ventral protrusion/ blister in its ventral wall; dorsal wall of spicule without a spur or appendix and distal part deeply attenuated. Gubernaculum stout, heavily-built, 70 – 85 % of spicule length, proximally arcuate with a tapering end; dorsal wall partially without sclerotization; distal end with two pairs of short spines (in ventral view) that appear obtuse tubercles in lateral view. Tail demarcated into two parts; a short, conoid anterior part and a long whip-like posterior part. Genital papillae divided into three precolacal and six postcloacal pairs. v1 slightly longer than and close to subventral v2; v3d lateroventral; v4 subventral slightly posterior to cloaca; ad lateral, situated more than one cloacal body diam. posterior to cloaca; v5, v6 and v7 long, tubular, grouped together subventrally; pd posterior most, subdorsal in position. Copulatory muscles representing 5 – 6 pairs of broad bands. Phasmids pore-like, about 1.0 – 1.3 cloacal body diam. posterior to cloacal opening.

Samples containing Acrostichus nudicapitatus (Steiner, 1914) Massey, 1962 were collected from soil rich in decaying leaf litter close to a tree base near State Bank of India at Aligarh (Geographical coordinates: 27.88334 N, 78.07475 E.), Uttar Pradesh, India.

Ten females and eight males on slide Acrostichus nudicapitatus (Steiner, 1914) Massey, 1962 SA39/ 1-10 deposited in the Nematode Collection, Department of Zoology, Aligarh Muslim University, Uttar Pradesh, India.

The present population shows conformity to Acrostichus nudicapitatus (Steiner, 1914) Massey, 1962 in most morphological and morphometric characters barring few minor differences viz., dorsal wall of spicules without a fine spur (vs a fine spur or appendix present); gubernaculum narrow and tapering proximally (vs proximal end relatively thicker); relatively smaller ‘c’ value (2.7 – 3.3 vs 2.9 – 4.3); smaller intra-uterine eggs (45 – 48 x 23 – 26 μm vs 50 – 63 x 25 – 36 μm) and smaller spicules (29 – 32 μm vs 32 – 40 μm in original population of A. nudicapitatus apudWeingartner, 1955). The present population although closely related to A. ponderosusMassey, 1962 shows some differences viz., greater ‘a’ value (22.6 – 24.1 vs 16.0); smaller ‘V’ value (38.3 – 42.0 vs 48.0) and larger gubernaculum (24 – 25 μm vs 22 μm). The two species, A. nudicapitatus and A. ponderosus show striking similarities with each other in most morphological and morphometric characters including the body length, nature of longitudinal lines, shape of lip region, nature of labial and cephalic sensilla, position of amphids; shape and size of stoma, uterine chamber and spicules and structure of gubernaculum (in having two pairs of distal conical processes or tubercles). A. nudicapitatus was described with fairly good number of specimens by Steiner (1914) while A. ponderosus was described by Massey (1962) on the basis of one male and one female. Based on their original descriptions, both can be differentiated by ‘a’ (20.8 – 28.0 vs 16.0) and ‘b’ (5.0 – 5.5 vs 7.0) values in females and by L (0.55 – 0.56 mm vs 0.50 mm) and ‘c’ (2.8 – 3.2 vs 5.0) values in males, respectively. The imprecisely drawn stoma (Fig. 8: Steiner, 1914) of A. nudicapitatus in original publication does not offer enough for comparison. Further, in A. ponderosus, the disparity in the number of precloacal genital papillae in males [two precloacal pairs (Fig. 1CapudMassey, 1962; Fig. 1DapudMassey, 1966) vs three precloacal pairs (Fig. 50FapudMassey (1974)] and the conspicuously broad and arcuate dorsal tooth (Kanzaki et al. (2009)Fig. 4C, D) in apparently flattened original specimens (Fig. 4A, B, F) vouch for a thorough study of the two representatives of the species which shows no subsequent reports. Gagarin (2002) reported two populations of A. nudicapitatus from sewage and cow manure with a wide range of values (a= 16 – 22, V= 36.2 – 44.3, spicule length= 32 – 39 μm and gubernaculum length= 22 – 31 μm) which nearly overlapped with the values of the single male and female specimens of A. ponderosus. On the basis of overlapping morphometrics and similar morphological features, Gagarin (2002), synonymized A. ponderosus with A. nudicapitatus considering the latter as senior synonym, however, he endorsed Andrássy’s (1984, 2005) views to consider them both under the genus Diplogasteritus Paramonov, 1952. The observations of Mahamood (2014) further indicate wide ranges in body length (552 – 722 μm), ‘a’ value (20.2 – 25.2), spicule (28 – 35 μm) and gubernaculum (22 – 25 μm) lengths of A. nudicapitatus. The unusual range of ‘b’ value (15.1 – 6.3) in the Table 1 (page no. 149) of above publication, however, appears to be due to oversight (hence corrected in Table 2 of present publication) while the statement about presence of circular amphids, also seems to be erroneous for diplogastrids. Keeping in view the conspicuous overlaps in morphometrics as well as morphological characteristics and in view of inadequate number of original specimens, the status of A. ponderosus with respect to A. nudicapitatus needs to be ascertained. Perhaps a molecular characterization and a holistic comparison may help when a population of the former species is reported in future.

Fig. 4

The extracted natural population of A. nudicapitatus showed the male: female sex ratio to be 1:1. Later single gravid female culture also showed equal proportion of males and females.

Embryogenesis (Fig. 3, 4):The females were oviparous and the eggs were laid in single-celled or two-celled stage, 90 – 160 min after fertilization. The uterine tract was observed to accommodate rarely as many as 4 – 6 eggs. The eggs were smooth-shelled, oval in shape measuring 45 – 48 x 23 – 26 μm in dimension. The pole of entry of sperm marked the posterior end of the developing embryo. Soon after the sperm’s entry, the cytoplasmic streaming led to formation of false cleavage furrow. During the event, the sperm pronucleus migrated to the centre of egg where it finally fused with the egg pronucleus. The first cleavage furrow was formed perpendicular to the longitudinal embryonic axis after 10 – 15 min of pronuclear fusion. The resulting blastomeres were unequal with anterior one (AB) slightly larger than the posterior (P1). At the commencement of the second cleavage division the cytoplasm of cell AB showed streaming. Thus, the second oblique cleavage after 10 – 15 min gave rise to cells ABa (anterior) and ABp (posterior) blastomeres. The blastomere P1 after 5 – 10 min, also divided obliquely to form EMS and P2 resulting into four-celled stage (Fig. 3B). Simultaneously within 10 – 15 min ABp transversely divided into ABpa and ABpp blastomeres (Fig. 3C) followed by division of EMS into MS and E (Fig. 3D) after 15 – 20 min. P2 divided into C and P3 (Fig. 4) after 15 – 20 min. The eight-celled stage was reached 90 – 110 min after egg laying followed by ten-celled stage after 100 – 120 min. Morula (Fig. 3F) representing a loose mass of cells was formed after 1.5 – 2.5 h of ten-celled stage. Blastula (Fig. 3G, H) reached 2.5 – 3.0 h of morula formation. Gastrulation (Fig. 3I) started after 1.0 – 1.5 h marking the germ layer differentiation. The stage involved the differentiation of ectoderm as an outer layer covering inner layer and an inconspicuous blastocoel. The ingression of endodermal cells led to the differentiation of anterior and posterior regions of the gut. The elongating embryo showed invagination along longitudinal axis followed by a depression slightly away from the centre of egg. The invagination marked the ‘Lima bean’ stage (Fig. 3J) 50 – 60 min after the initiation of gastrulation and further deepening of the cleft resulted into ‘Comma’ stage subsequently after 40 – 50 min (Fig. 3K). The embryo then prepared for organogenesis and acquired a worm-like body in ‘tadpole’ stage (Fig. 3L) after about 40 – 50 min of comma stage, with an anterior shallow depression representing the presumptive stoma. Plum stage (Fig. 3M) followed after about 1.0 – 1.5 h with the embryo showing first sign of movement which became more frequent later. Loop stage (Fig. 3N) was attained subsequently after about 30 – 40 min from plum stage when the embryo acquired almost two egg-folds length and moved continuously inside the shell in the anterior-posterior direction. The vermiform embryo exhibited a broader anterior end with small stomal cavity and a long pointed tail showing its posterior end. The loop stage was followed by pretzel stage after about 3 – 3.5 h, in which the stoma, pharynx, intestine, tail and rectum were differentiated. Continuous movements involving body rotation and turning were observed in the embryo during this stage with the embryo attaining 4.0 – 4.5 egg-fold length. About 3.0 – 3.5 h after the commencement of pretzel stage (Fig. 3O, P), the stoma is differentiated into cheilostom, gymnostom and stegostom while other structures viz., pharynx, genital primordium, rectum and tail were fully formed. After the completion of organogenesis, the juvenile underwent a moult within the egg shell (Fig. 3P) before finally moving out of the shell. As a result of the increased plasticity due to excessive pressure caused by the body movements as well as labial probations of the juvenile, the egg shell became very thin and finally ruptured in the form of a slit. However, the second-stage juvenile hatched out of the egg shell in several attempts 3 – 4 h after completion of organogenesis. The total embryonation time from single-celled stage to hatching was 20 – 25 h at 25 ± 2 °C.

Fig. 3

Gonad development (Fig. 5):During post-embryogenesis the gonad development continued from second stage juvenile (J2) to fourth stage juvenile (J4). The second stage juvenile showed a primordium similar to the first stage juvenile which remains in the egg shell. The juvenile stages could not be differentiated exclusively on morphometrics due to overlap of values. However, the growth patterns of the genital primordium served good marker to differentiate these stages.

Fig. 5

In the egg shell, the first-stage juvenile showed an obliquely-oriented primordium containing two large central germinal (Z2, Z3) and two relatively smaller somatic cells (Z1, Z4) at poles. The genital primordium was similar in all first stage juveniles with fixed number of primordial cells and was sexually undifferentiated.

The primordium (Fig. 5A) of second stage juvenile (J2) that hatched out of the egg shell, was located at 42.2 – 44.3 % from anterior end and was similar (Fig. 5B, C) to that of first stage juvenile and identical in all juveniles that were to develop into males or females. However, during the moulting period, the localization of cells and the resulting primordial growth indicated the future sex of juvenile. In second stage juvenile destined to develop into female, by the end of moulting, the cells present in the genital primordium included two distal tip cells (DTCs) helpful in gonad elongation and germ-line patterning to form all future somatic gonad cells; and 4 germinal cells ready to form the future germline of the female gonad. In second stage moulting juveniles destined to develop into males, the two DTCs were positioned at the posterior distal end and the somatic cells showed an anterior placement in the primordium indicating its future direction of elongation. Third-stage female juveniles of A. nudicapitatus possessed a much developed genital primordium, present at 42.1 – 47.2 % of body length from anterior end. The primordium (Fig. 5D, E) showed a bidirectional growth and elongated anteriorly, as well as posteriorly, as a result of cellular proliferation. The genital primordium contained germinal and somatic cells including two DTCs at the anterior and posterior extremes. Some of the germinal cells migrated to the ends of the elongating arms while the somatic cells largely remained in the centre (Fig. 5E) along with few germinal cells and an anchor cell (AC). The latter was a transient cell that primarily functioned to indicate the site for vulva formation and helped in vulva patterning. The moulting period marked the proliferation of somatic cells in the centre of primordium resulting in its extension. Ventral hypodermal chord cells, differentiated into precursors for the vulva and aggregated on the ventral aspect of primordium at 45 – 55 % of body length from anterior end (Fig. 5E).

The genital primordium of third-stage male juveniles was located at 42.7 – 45.5 % of body length from anterior end. It consisted of germinal and somatic cells including two DTCs at the distal end and a linker cell at proximal end. The primordium (Fig. 5H) showed anterior growth due to proliferation of the somatic cells and eventually developed a flexure (Fig. 5J) during the moulting period. Two dense cellular aggregates were observed each on dorso-lateral side of rectum representing the precursors of the spicules and associated structures (Fig. 5K).

Fourth-stage female juveniles possessed a much elongated genital primordium located at 37.7 – 39.1 % of body length from anteriorend. It consisted of two primordial arms directed in anterior and posterior directions (Fig. 5F). The arms represented the germline while the central broader part showed the somatic parts of the future reproductive system. The part between the presumptive oviduct and uteri showed germinal cells that proliferated later to mark spermatogenesis in the hermaphrodite juvenile. The area later served to store the spermatids. The anchor cell (AC) located in the centre of primordium (Fig. 5E) and morphologically distinct from all other somatic cells, played a significant role in vulva induction. The cell also played important role in signaling to other ventral uterine cells and finally generating the connection between the uterus, vagina and the outside environment. Of the 12 VPCs (vulval precursor cells) the central three (Fig. 5F) divided to give rise to up to 16 descendants that actively participated in vulva formation. The rest of the precursor cells disappeared in late stage due to programmed cell death. In the moulting stage, the two extreme ends of the primordium containing germinal cells reversed forming the germ line/ ovaries while the remaining gonoduct formed the somatic gonad. The vulva formation along with the differentiation of the somatic parts of the gonad was completed with the termination of moulting (Fig. 5G).

The fourth-stage male juveniles possessed a genital primordium at 36.1 – 37.4 % of body length from anterior end. The genital primordium showed a reflexed part showing the germline of the future gonad with germinal cells contained in and a considerably longer tube elongating posteriorly. The somatic tube contained proliferating somatic cells with the linker cell placed ahead to join with the rectum to form cloaca during late moulting stage. The spicular precursor cells showed more compaction (Fig. 4K, L) and formed the spicules and gubernaculum in the late fourth stage initially as refractory lines. The genital papillae were the last structures to develop by special hypodermal cells known as papillae precursor cells. The caudal region of male juvenile distinctly differed from that of the female juvenile (Fig. 5M).