Post-embryonic development of the reproductive system of Pneumonema tiliquae (Nematoda: Rhabdiasidae)

International Journal/or Parasilology Vol. 21, No. 5, pp. 53S547, Primed in Great Britain

1991 0

002~7519/91 $3.00 + 0.00 Pergamon Press plc I99 I AustralianSociety for Porarirology

POST-EMBRYONIC DEVELOPMENT OF THE REPRODUCTIVE SYSTEM OF PNEUikfONEiWA TILIQUAE (NEMATODA: RHABDIASIDAE) ROBERTJ.BALLANTYNE School of Science and Technology,

Charles Sturt University,

P.O. Box 588, Wagga Wagga,

New South Wales 2650, Australia

(Received 22 August 1990; accepted 28 February 1991) A~S~T~C~-BALLANTYNER. J. 1991. Post-embryonic development of the reproductive system of Pneumonema tiliquae (Nematoda: Rhabdiasidae). International Journalfor Parasitology 21: 535-547. The post-embryonic development of the reproductive system of both generations of Pneumonema tiliquae is described from fixed and stained specimens. In recently hatched first-stage larvae all the nuclei stained alike with males having 10 nuclei and females six. The development of the genital primordium during each stage is very precise in both generations; division of the nuclei begins 4-6 h after hatching. At moult 1 the male genital primordium is clearly organized into an anterior gonoduct area of eight nuclei and 12-13 spermatogonia. The anterior gonoduct nuclei arise from a common precursor cell (21) whereas one distal tip cell (dtc) and the germinal nuclei that form post-hatching have a common precursor cell origin (24). In the female, there are 20 nuclei at moult 1, all appearing essentially similar; in the early second stage these nuclei differentiate into 12 somatic and eight germinal nuclei; the somatic nuclei are considered to be of Zl and 24 origin. In the male the gonoduct nuclei grow, reflex and divide before joining to the rectum during the fourth stage. At moult 4 the system is fully formed with the testis containing fully developed secondary spermatocytes; sperm are produced and ejaculated in two phases. In the second-stage larvae the female primordium differentiates into a central gonoduct area with the germinal cells formed into distal opposed regions; subsequent development is rapid and during the fourth stage a small vulva joins the uteri to the body wall. Fertilized eggs develop and hatch in utero; recently hatched first-stage larvae usually have six nuclei in the genital primordium. At moult 2 the primordium has 12 somatic and two germinal nuclei. Once established in the lizard, the reproductive system develops rapidly with development similar to that seen in the free-living generation except the system is larger and contains about four times as many nuclei. Development ceases pre-sperm development if adults remain in the body cavity of the lizard. In the lungs the system continues to grow via increase in cell size with the anterior system folding and the relative position of the vulva changing from 57 to 44% ofthe body length as the adult increases in length. INDEX KEY WORDS: reproductive system.

Nematoda:

Rhabdiasidae;

Pneumonema;

post-embryonic

development;

construction of developmental patterns. It should be noted that in both these techniques it is the nucleus that is clearly visible, although good fixation and staining often show the complete cell. This study reports on the post-embryonic development of the reproductive system of the freeliving and parasitic generations of Pneumonema tiliquae using a staining technique.

INTRODUCTION

PAI (1928) was the first author to distinguish clearly between germinal and somatic nuclei in the genital primordium and to trace the derivatives of these nuclei throughout the development of the gonad of Turbatrix aceti (Rhabditoidea). Several authors (Nigon & Brun, I955; Hirschmann, 1962; Roman & Hirschmann, 1969; Hechler, 1970; Kimble & Hirsh, 1979; Sternberg KCHorvitz, 1981; Horvitz & Sternberg, 1982), studying either free-living or plant nematodes, have since added to our knowledge of the development of the reproductive system of nematodes. The techniques used to determine development are staining and standard light microscopy, and agar-slab slide culture and Nomarski microscopy. Staining requires that individual worms be killed, whereas agar slabs allow the development of live worms to be followed. The development of all worms is recorded either on film or via drawing-tube diagrams. These records become the basis for later analysis and

MATERIALS

AND METHODS

Materials and methods where appropriate are as described by Ballantyne (1991). Nomenclature and cell lineage charts follow the format as modified by Stemberg & Horvitz (1981) after Kimble & Hirsh (1979). In lineage diagrams anterior is drawn to the left and posterior to the right. Abbreviations are a, anterior; p, posterior; D, dorsal; V, ventral. Proximal and distal refer to the ends of the gonadal arm nearest and furthest, respectively from the opening to the outside. Most cells are named according to the sequence of divisions of their ancestral progenitors. Some blast cells are redefined by two capital letters, e.g. AL (anterior left) for ease of reference. 535

010 i 5a

dtc-i-

5b

7a

7b b

10

4

8a

3

r?

11

gb’

12

-

‘gb

8b FIGS. 1-12. Development of genital, spicule and gubemaculum primordia. 1-3, 9, 11. Genital primordium, ventral. 1. Ll recently hatched. 2. Ml. 3. M2. 9. M3. 11. M4.48b, 10, 12. Spicule and gubernaculum primordium. 4. Ll recently hatched. 5a. Pre-M 1.5b. As 5a, dorsal. 6. L2 after B has divided. 7a. Post-first division of s. 7b. As 7a, dorsal. 8a. As 7a with F divided. 8b. As 8a dorsal. 10. M3: spicule and gubemaculum nuclei. 12. Spicule (left) and gubemaculum. B, Spicule/gubemaculum precursor; cc, coelomocytes; DS, sperm duct precursors; dtc, distal tip cell nuclei; ED, ejaculatory duct precursors; ed, ejaculatory duct; edv, ejaculatory duct diverticulum; F, spicule precursor; gb, gubemaculum precursor; gb’, gubernaculum; lc, linker cell nucleus; rj, recta-intestinal junction; s, spicule precursor; sph, sphincter; spd, sperm duct.

P. tiliquue: development of reproductive system

Other cells, e.g. anchor cell (ac), distal tip cell (dtc), linker celi (Ic) and vulva1 precursor cells (P5p, P6p, P7p), are named as in Caenorhabditis elegans (see. Sulston & Horvitz, 1977). Ll, L2, L3, and L4 are used for first- to fourth-stage larvae, and Ml, M2, M3 and M4 for moults 14. RESULTS Free-living males

The genital primordium of recently hatched Ll males was 55-60 pm long with 10 large nuclei (Fig. 1). All nuclei stained alike but the nucleus at each end was slightly larger, had a more conspicuous nucleolus and was surrounded by more cytoplasm. During Ll, the primordium increased slightly in length and 4-6 h after hatching, as Ml approached, the nucleus at each end divided unequally. The small nucleus at the anterior end migrated immediately along the dorsal surface of the primordium until it lay adjacent to the small nucleus at the posterior end. These two small nuclei (dtc) remained at the distal end of the primordium and were the only nuclei associated with the testis epithelium. The large nucleus (G) from the posterior end division formed part of the germinal cell lineage. The large nucleus from the anterior end division usually divided immediately producing two smaller sized nuclei. The sequence of divisions of these two nuclei was irregular although each nucleus produced an area of four cells. The anterior four nuclei (ED) were larger and stained lightly and were arranged one anterior (lc), two ventrally crosswise and one dorsally. The other four nuclei (DS) were smaller than ED and stained intensely and were arranged crosswise, two dorsally and two ventrally. The eight inner nuclei of the recently hatched primordium did not divide before maturing into primary spermatocytes during M3. Additional spermatogonia were produced during Ll-L3 from the G nucleus. At M 1 (Fig. 2), the primordium was 70-80 pm long with 12-13 germinal cells occupying most of the length. Division and development was continuous from early L2 to the end of M3; during this time the two dtc nuclei were often displaced laterally or dorsoventrally as the primordium grew. During L2, ED increased in length, then lc elongated laterally, causing ED and DS to reflex 180” and move posteriorly along the left side of the primordium during development. While ED increased in length, the four DS nuclei underwent a sequence of divisions producing 11-13 nuclei similar in appearance to the originals. At M2 (Fig. 3), the primordium was clearly differentiated into 27 gonoduct nuclei and a testis about 130 pm long with 15-20 spermatogonia. The gonoduct nuclei had completely reflexed and were differentiated into two areas, ED with 11 and DS with 16 nuclei. On closer examination, ED contained one lc, two large nuclei and eight slightly smaller nuclei; in DS the four nuclei adjacent to ED were slightly smaller than the remaining 12. Abutting the growing tip of the gonoduct were two small, intensely staining nuclei (cc); these were carried posteriorly by the growing tip

537

to the level of the intestinal constriction, at which point they remained and developed into what appeared to be coelomocytes. During L3, division occurred firstly in the eight smaller ED nuclei, then in the two large ED nuclei, resulting in a loss of a clear differentiation between ED and DS. As ED and DS divided, the ED cells moved mid-ventrally and grew rapidly towards the posterior end; lc divided once when adjacent to the rectointestinal junction joining the gonoduct area to the developing cloaca. The testis region became reflexed as the spermatogonia and gonoducts developed. At M3 (Fig. 9), the gonoduct end of the testis was reflexed to the level of the dtc nuclei and was 110-130 pm long with 23-27 spermatogonia. The proximal spermatogonia were almost fully formed primary spermatocytes and the chromosomes did not stain. On completion of M3, the gonoduct area was 110-130 pm long and divisible into three regions: anteriorly there were 25 nuclei, arranged dorsally and ventrally with the four nuclei adjacent to the spermatogonia elongated; a mid-region of 20 small nuclei arranged dorsally and ventrally; and posteriorly, a large region of 30-32 irregularly arranged nuclei. During L4 and without any further division or increase in length the gonoduct nuclei formed into ducts. The anterior 25 nuclei formed a short thinwalled sperm duct; eight nuclei from the median area formed a sphincter between the sperm and ejaculatory ducts, and the remaining 12 nuclei formed two anteriolateral diverticula of the ejaculatory duct. The posterior area formed the ejaculatory duct, usually extending laterally round the sphincter, increasing the length of the diverticula (Fig. 10). The ejaculatory duct and diverticula assumed a glandular appearance with a small lumen appearing in the diverticula. The 12 nuclei from the median region were not always distributed equally into each diverticulum and in the adult the diverticula varied in length and were often quite short. The proximal spermatocytes increased greatly in size lengthening the testis by about 50% before completing the first maturation division prior to M4. Immediately after M4 these spermatocytes underwent a second maturation division and the male was then ready to inseminate the female. Sperm were l&15 pm in diameter with a small nucleus containing either five, six or seven chromosomes in a rosette-like ring. Following sperm ejaculation the testis area shortened and lost its reflexed appearance. A second phase of sperm production occurred in the testis which depleted the germ cell line and worms began to die some 48-60 h after hatching. During Ll the small nuclei in the ventral cord anterior to the rectum divided, then during L2 the large nucleus (U) underwent several divisions. By L4 the ventral cord immediately anterior to the anus was packed with nuclei and some of these nuclei were probably involved in the widening of the ventral wall

538

dfc

dfC-

FIGS. 13-22. Develapment of genital and vufval primordia: free-living generation. 13. Ll recently hatched. I4a. MI, ventral. 14b. Ventral cord at Ml. 15. Early L2. 16. M2 completed. 17. M3. 18. Ventral cord of 17. 19. Late L4. 20. Ventral cord of 19. 2i. Young adult (ventral cord same scale as Figs. 13-20). 22. Adult vulva (nuclei lateral to vulva belong to uterine epithelium). ac, vulva1 linker cell nucleus; dtc, distal tip cell nucleus; Ep, ovarian epithelial precursor nucleus; e, ovarian epithelium; G, oocyte precursor; L, effette nuclei (see text); Od, oviduct precursor nucleus; od, oviduct; vc, ventral cord; P5p, etc., vulva1 nuclei (see text); AL, AR, DL, DR, PL, VL, VR, gonoduct blast cells (see text); a, anterior; p, posterior.

P. tiliquue: development

of the rectum at M4. By M4 the spicules, gubernaculum and genital papillae were fully formed. Formation of spicules and gubernaculum

Recently hatched Ll males had a primordium of four nuclei dorsal to the rectum; two of these nuclei (F, B) were enlarged with the posterior one (B) the larger (Fig. 4). During Ll B enlarged (Figs. 5a, b) and divided transversely at Ml giving two similar nuclei (s, gb, Fig. 6). The posterior nucleus (gb) divided late in L2 to form gubernacular nuclei. The anterior nucleus (s) enlarged and divided longitudinally to give two nuclei (Figs. 7a, b) which subsequently divided to form nuclei for spicule development. The other enlarged nucleus (F) of Ll often divided at this stage (Figs. 8a, b) and appeared to contribute to the spicules. The two small nuclei of the original primordium remained unchanged during Ll . By M2 the rectal area was a mass of dividing nuclei and it was impossible to determine the exact fate of dividing nuclei. By M3 (Fig. lo), the nuclei were in three groups corresponding to the positions of the future spicules and the gubernaculum. During L4 the two groups of spicule nuclei became laterally compressed into the shape of spicules and the actual spicules formed on the inside of the nuclei. The gubemacular nuclei were dorso-ventrally compressed and the gubemaculum formed on the ventral side of the nuclei. The spicules and gubernaculum were fully formed prior to M4 (Fig. 12). Divisions in the gonad and spicule primordia were not completely synchronous during each stage, with spicule development often lagging slightly behind gonad development. Free-living females

The genital primordium of recently hatched Ll females was about 35 pm long with six large nuclei (Fig. 13). All nuclei stained alike, but the nucleus at each end was slightly larger, had a more conspicuous nucleolus and was surrounded by more cytoplasm. Nuclear division began late in Ll; prior to this there had been an increase in length of the primordium from 35 to 55 pm and a gradual differentiation of the inner four nuclei. The inner four nuclei underwent a rapid sequence of divisions producing 16 nuclei. The two end nuclei divided once when there were 12 inner nuclei. At Ml, the primordium was 55-60 pm long with 20 nuclei; all nuclei stained alike, but varied in size and planes of orientation. After Ml the primordium increased in length to about 75 pm and the nuclei could be differentiated into 12 somatic and eight germinal nuclei (Fig. 14). The anterior end of the primordium was growing ventrolaterally to the right and the posterior end ventrolaterally to the left of the intestine. Nuclear division and growth began after organization of the nuclei into the future gonoduct areas (Fig. 15), and continued until shortly after M2 when the primordium was about 150 ,um long. The main activity occurred in the mid-

of reproductive

system

539

region producing a compact area of dorsal and ventral somatic nuclei. The germinal nuclei separated into two distinct areas at each end of the developing primordium (Fig. 16). The nuclei exhibited a characteristic pattern and were destined for particular areas in the adult reproductive system. The pattern from the anterior end consisted of: one (5 ,um) indistinct nucleus (dtc); five to six large (10 pm) lightly staining germinal nuclei (G); four small (3 pm) intensely staining epithelial nuclei (e); two (5 pm) intensely staining epithelial precursor nuclei (Ep); two large (10 pm) lightly staining oviduct precursor nuclei (Od); four small (34 pm) intensely staining nuclei (L); 11 dorsal and ventral nuclei varying slightly in size and staining intensity, these nuclei were to form the uteri; then a repeat of the anterior pattern, four L, two Od, two Ep, four e, five to six G and one dtc nuclei. During L3, division and growth were continuous. Early in L3 when the primordium was 200-225 ,um long, the distal ends of the ovaries reflexed 180”and the reflexed ends grew dorso-laterally in the opposite direction. When the ovaries reflexed, the uterine nuclei had started dividing; the ventral cord nuclei were differentiating to form the future vulva; the oviduct precursor nuclei (Od) had divided four times and each future ovary contained six to 10 oogonia. The gonoduct nuclei continued to divide and additional oogonia were produced by division of the germinal nuclei. Most oogonia divided before maturing into oocytes. At the completion of M3 (Fig. 17), there were 1620 uterine nuclei in a short (50 pm) compact area; the ac nucleus was situated at the base of the developing vulva; the Od nuclei had produced a double column of 24 nuclei in each oviduct; the L nuclei remained between the oviduct and uterine nuclei; the reflexed tip of each ovary had reached the level of the L nuclei and contained 18-21 oogonia with the proximal ones enlarged, forming a single row. The epithelial precursor nuclei remained at the distal end of the oviduct nuclei and there were nine nuclei surrounding the developing oogonia. During L4 the ac nucleus divided twice giving four cells which abutted and joined the uteri to the four cells of the developing vulva. The remaining uterine nuclei did not divide again but the cells increased in size, increasing the length of the uteri. The four L nuclei of each system moved into the centre of the nuclear column as the uterus increased in length. Each oviduct increased in length by nuclear division producing 44 48 nuclei (Fig. 19). The increase in length of the uteri and oviduct straightened the entire ovary area of each system; at this stage the chromosomes of the proximal oogonia stained. As more oogonia enlarged, the proximal end of each ovary became reflexed again and the oviducts began to fold; by this stage the chromosomes of the proximal oogonia did not stain. By L4, division had essentially ceased in the germinal areas of the ovary. During M4 the vulva was lined with cuticle and a lumen formed in each uterus; the four L nuclei formed

540

R. J. BALLANTYNE

into a small mass and moved into the lumen of the uterus; a lumen began to form in the proximal end of each oviduct, and one of the Ep nuclei often divided increasing the e nuclei to 10. In the adult, the vulva did not increase in size and in~mination occurred imm~iately after M4. The uteri contained sperm before a lumen was fully formed in each oviduct. The oviduct cells formed into a long and folded thin-walled duct and the uterus-oviduct junction became difficult to distinguish. The L nuclei remained as a small mass in the lumen of each uterus. The proximal oocytes increased in size and moved singly into the oviducts. At this stage the chromosomes stained. Sperm penetrated the oocytes and a single reductional division followed. In developing eggs, the polar body was outside the vitelline membrane. Eggs developed and Ll hatched in utero. Females that did not produce Ll lacked sperm in the reproductive tract and during their life span of 12-l 5 days the oocytes enlarged packing the uteri, oviducts and ovaries. Formation of vulva in the free-living females The ventral cord of recently hatched Ll contained about 18 small nuclei. During the late Ll the nuclei divided and differentiated into a few large and numerous small nuclei. At Ml (Fig. 14b), there were three large nuclei (PSp, P6p, P7p) over the genitai primordium with five to six small nuclei between adjacent large nuclei. The nuclei did not alter during L2 but toward the middle of L3 the three large nuclei were closer together over the centre of the primordium. The middle large nucleus divided and the two daughter nuclei (Pfipa, P6pp) invaginated towards the ac nucleus of the developing uteri. At M3 (Figs. 17, 18) there were : P6pa and P6pp deep in the cord against the uteri; two smaller nuclei (PSpp, P7pa) ventral to P6pa and P6pp; two larger lightly staining nuclei (PSpa, P7pp) anterior and posterior to P6pa and P6pp; and surface nuclei dividing or divided. Prior to M4 (Figs. 19,20) the future vulva consisted of: four progeny of P6p, at the corners of a square against the four progeny of ac; two lightly staining nuclei (PSpa, P7pp) ventral to and anterior and posterior to the P6 nuclei in the midline; three or four irregularly arranged nuclei (derived from P5pp and P7pa) ventral to PSpa and P7pp; and surface nuclei. After M4 (Figs. 21, 22) the vulva consisted of a sphincter of four nuclei at the uterine junction, three or four nuclei ventral to but not included in the sphincter, and surface nuclei. The two lightly staining nuclei assumed to be PSpa and P7pp were not evident and presumably died during L4. Parasitic generation Recently hatched Ll from the uteri of free-living females did not stain effectively. The smallest genital primordium observed was 17 pm long with four nuclei. In later Ll the genital primordium was 22-26

pm long with two large and four slightly smaller nuclei (Fig. 23). During M 1 the four smaller nuclei divided to give eight nuclei, while the two large nuclei (G) remained in the mid-region (Fig. 24). After L2 escaped from the female cuticle, four of the eight nuclei divided increasing the number to 12, and the two G nuclei moved toward the ends of the primordium (Fig. 25). At this stage M2 occurred. The genital primordium of ensheathed L3 was 24 30 pm long and contained 14 nuclei (12 somatic and two germinal, G) (Figs. 26, 27). The position and shape of all nuclei altered slightly with age. The divisional pattern of the somatic nuclei from LI to L3 appeared to be similar to that seen in the free-living female during M 1. Three small nuclei occurred outside the primordium, arranged two left and one right laterally in the mid-region (Fig. 27). These nuclei were seen clearly only in old L3 and may have been coelomocytes. In the lizard, nuclear division began on day 5 when the primordium was SO-55 pm long (Fig. 28). Development was rapid and within 24 h at M3 the primordium was 180-200 pm long with the germinal nuclei in the distal ends separated by an area 65-75 pm long (Fig. 29). The nuclei showed a similar pattern to that of the free-living female after M2 (Fig. 16), except for a variation in nuclear number in some areas. There were one dtc, four to five G, six e, four Ep, two Od, and four L nuclei in each system, plus 21 (including ac) uterine nuclei. In the free-living female there were two Ep and 11 uterine nuclei. During the early L4, all the uterine nuclei except ac divided producing 80 nuclei without any increase in length of the area. The oviduct and ovarian nuclei began to divide and the oviduct precursor nuclei remained adjacent to the ovarian epithelial precursor nuclei. Each oviduct area was 40-50 pm long and contained 2630 nuclei; each ovary was 95-110 pm and had 1620 epithelial nuclei and four to five germinal nuclei adjacent to the dtc nucleus. On completion of the above, ac divided twice joining the uteri to the developing vulva; the remaining uterine nuclei did not divide again but the cells increased in size, increasing the length of the uterine area from 5565 pm to 150-170 ,um and forming two opposed uteri each 75-85 pm long. As the uterine area lengthened the distal ends of the ovaries reflexed dorso-ventrally and grew in the opposite direction (Fig. 32). Once the ovary was reflexed, the ovarian epithelial nuclei divided rapidly producing a narrow column of cells. The L nuclei of each system remained between the uterus and oviduct; in some specimens, some of the nuclei divided producing up to eight nuclei. As the uterine area lengthened the L nuclei moved into the centre of the gonoduct cell column forming a distinct cellular body. While the adult buccal capsule was forming during M4, the vulva became lined with cuticle and a lumen was noticeable in each uterus. The reproductive system continued to grow and nuclear division oc-

8

, 4

a

542

R.J. BALLANTYNE

curred in the oviduct and ovaries epithelium. When the L3 and L4 cuticular sheathes were shed by days 7-8 (Fig. 34), each uterus was 110-120 pm long; the L nuclei had formed into a distant mass in the distal ends of the uteri and the nuclei still stained intensely; each oviduct was 90-100 ,um long with about 100 nuclei, folding on themselves in the mid-region to form the future sperm reservoir; and each ovary consisted of a non-reflexed proximal area S&60 pm long and a reflexed distal area 300-400 pm long with 105 epithelial nuclei and 10-16 germinal nuclei adjacent to the dtc nucleus. In young adults the system continue to grow and nuclear division occurred in the oviduct and the ovary. If immature adults remained in the body cavity the germinal nuclei stopped dividing when there were 2830 nuclei in an area 120-140 pm long adjacent to the dtc nucleus of each gonad. If immature adults were to enter the lungs, the germinal nuclei continued dividing and when the germinal area was 180-240 pm long sperm began to form from the proximal gametocytes. These gametocytes underwent two divisions without an increase in cell size and sperm were about 5 pm in diameter with either five, six or seven chromosomes in a rosette-like ring. In immature adults in the body cavity there were two complete reproductive systems. Each system consisted of: uterus 150-160 pm long; folded oviduct 160-190 pm long (total length 240-300 pm) containing about 160 nuclei; ovary with about 135 epithelial nuclei and 28-30 germinal cells, formed into a non-reflexed area 60-80 ,um, and a reflexed distal area 400-600 pm long. In the lungs division occurred only in the germinal nuclei, and increase in size of each area occurred through increase in cell size. Once in the lungs (days 12-14) oocytes began to form adjacent to the developing sperm. The oocyte development moved the sperm down the ovary for storage in the folded oviduct. Oocytes increased in size as they moved down the ovary and underwent a single maturation division after being fertilized as they moved through the sperm in the oviduct. In developing eggs the polar body was outside the viteiline membrane. In young adults it was common for the first few gametocytes not to divide to produce sperm but to continue to enlarge as they moved down the ovary and eventually enter the uteri as unfertilized ova. Ova production and fertilization continued until the uteri were full of eggs. By this stage the synapse zone had switched to sperm determination and a new batch of sperm formed and moved down the ovary to the oviduct. Throughout the life of the worm sperm and ova were determined alternately in the synapse zone. When all the eggs in the uteri contained fully formed Ll the uteri were emptied and another cycle of egg development began in each uterus. When the leading oocytes were fertilized the anterior and posterior reproductive systems were of similar size and the vulva was at 57% of the body length. As the adult grew the ovary increased in size,

the germinal zone increasing to 350-375 pm long, and the synapse zone was 60-80 pm long. The anterior ovary was forced to fold on itself as expansion into the forebody was restricted by the body muscle that developed in association with the body spines. The posterior ovary remained reflexed and extended almost to the anus. During adult growth the posterior half of the body lengthened and the relative position of the vulva changed from 57 to 44% of the body length. Formation of vulva in parasitic females

During the late Ll the nuclei of the ventral cord divided as in the free-living female, i.e. into a few large and numerous small nuclei. There was no further development until the parasitic L3 when the cord increased in size and the large nuclei became prominent in the mid-body region. As the nuclei of the genital primordium began to divide there were three large and four small nuclei situated above the primordium in the ventral cord (Fig. 28). By M3 (Figs. 30, 31), the nuclear arrangement was similar to M3 freeliving females except that the P5a and P7p nuclei were dividing. During the early L4 (Figs. 32, 33) the vulva1 area enlarged rapidly without an increase in nuclear division. The area contained the following nuclei: four derived from P6; two lightly staining from P5a and P7p; four mid-cord from P5p and P7a; two lightly staining from P5a and P7p; and surface nuclei. Anterior and posterior to the above nuclei were other nuclei, both dorsally and ventrally which tapered toward the developing vulva. Deep in the cord, anterior and posterior to the developing vulva, were four small nuclei (Fig. 33, un). At M4 the vulva consisted of two lateral compact units, each with five nuclei. Anterior and posterior to the opening, the nuclei were irregularly arranged into groups; lateral to these groups were nuclei which may have been the four small nuclei (un) in Fig. 33. In immature adults (Fig. 34) the vulva was a transverse slit-like opening 20 pm wide and 25 pm deep. The anterior and posterior nuclei were irregularly arranged into structures resembling rectal glands (vg). These structures varied in number from two to four and contained one to four nuclei. There were muscle-like fibres in each corner equivalent in position to the four small nuclei (un) in Fig. 33. Details of the cells in the vulva of adult worms were difficult to determine as eggs distended and obscured the area. DISCUSSION

The results reported in this paper were completed in 1970 prior to the extensive studies on Caenorhabditis e&guns by Kimble & Hirsh (1979) and Panagrellis redivivus by Sternberg & Horvitz (1981) where they used agar-slab cultures and Nomarski microscopy. The Carnoy’s fixation and acetic orcein staining technique used in this study produced specimens where it was easy to distinguish nuclei in different phases of development and many specimens retained

P. tiliquae: development

their staining quality so it was possible to re-examine specimens to verify developmental patterns. P. tifiquue is an unusual animal as it has a distinct alternation of generations where the parasitic adult in the form of a female alternates with a morphologically distinct free-living generation of adult males and females. The sequence of events in this alternation shows a precision not usually found in other nematodes. In the parasitic form, sperm and ova are alternately determined in the synapse zone of the gonad; eggs remain in utero until they all contain fully developed Ll; once hatched Ll must pass from the lizard before the onset of cell division in the genital primordium, if not they die; excess moisture kills the free-living generation; males must inseminate females immediately after M4; Ll from the free-living generation hatch in utero and eating out of the female is necessary for development; gonad development stops in immature adults when suppressed in the body cavity by the presence of adults in the lungs (Ballantyne, 1991). The alternate production of sperm and ova in the gonad appears to occur in all rhabdiasid genera (Ballantyne, unpublished). Runey, Runey & Lauter (1978) in describing gametogenesis in Rhabdius ranae from fixed specimens did not observe this alternation. Although the two generations appear morphologically different the programme of gonadogenesis in the female forms is remarkedly similar. The free-living form has an even development from Ll to mature adult with minimal development occurring after M4, whereas the parasitic form develops in bursts with a lot of growth in the adult. In the female the number of somatic nuclei at Ml in the free-living generation corresponds to the number seen at M2 in the parasitic generation, and the number at M2 free-living corresponds to M3 parasitic except that in the parasitic form there are twice as many ovarian epithelial precursor nuclei (Ep) and twice as many uterine nuclei. The overall process of gonadogenesis in P. tiliquae is similar to that reported for C. eZegans and P. redivivus (Kimble & Hirsh, 1979; Stemberg & Horvitz, 1981) but there are some interesting differences, namely the number of nuclei in the genital primordium when Ll hatch and the relationship of the nuclei to the Z14 system; the possible absence of the Y (ganglion) cell in the Ll male; the division of lc (male linker cell) and ac (vulva1 linker cell) in gonoduct development; the presence of and elimination of the L nuclei in the females; the small vulva in the free-living generation; the total number of somatic nuclei produced; and the nature of the actual gonoducts and the method of gamete formation. Recently hatched Ll males and females are readily distinguished as the male has 10 nuclei in the genital primordium, compared with six in females; the B and F nuclei in the accessory primordium dorsal to the rectum are enlarged in the male and the ventral cord anterior to the rectum in the male has one enlarged

of reproductive

system

543

nucleus. There is a difference in the position of the coelomocytes posterior to the genital primordium but nothing as distinct as the situation in C. elegans (see Sulston & Horvitz, 1977). The single enlarged nucleus anterior to the rectum in the male is interesting as in both C. eIegans and P. redivivus there were two enlarged nuclei, U which divided to join the gonoduct to the cloaca and Y which formed ganglia (Sulston & Horvitz, 1977; Sternberg & Horvitz, 1982). In P. tiliquae the enlarged nucleus undergoes extensive division and the cells produced appear to be involved in the joining of the gonoduct to the rectum and in the widening of the rectum into a cloaca, therefore, it would follow that the original nucleus is equivalent to U and the Y (ganglion) cell is absent. A reason for the Y cell being absent is not apparent as U and Y exist in both C. eiegans which has bursal rays and P. redivivus which has similar papillae and tail organization to P. tifiquae. The females of all three species have both U and Y nuclei. The B and F nuclei of the primordium dorsal to the rectum undergo extensive division to form the spicules and gubernaculum and do not appear to play any role in the formation of the cloaca as perceived by Sulston & Horvitz (1977). In some free-living females the B and F nuclei began to develop and in the parasitic female the nuclei enlarged and during M3 a lumen formed within the primordium (Ballantyne, 1991). The spicules and the gubernaculum are synthesized internal to their nuclear primordium and it appears that these nuclei are responsible for the shape of the spicules and gubernaculum. The sex muscles may be required for proper morphogenesis of the spicules (Sulston, Albertson & Thomson, 1980), but their influence must be indirect as the muscles attach to the distal end of the formed spicules. The developing female gonad of both generations has four cells (L nuclei) determined at M2 free-living and M3 parasitic. These nuclei move into the lumen of the gonoduct at M4 and are lost from the system. These cells may be equivalent to the crustaformeria (Wu, 1967), the area responsible for egg shell formation. The elimination of these cells into the uterus is unusual as they could remain in the tract wall or undergo programmed cell death and not be involved in development. C. elegans has six cells at the junction of the spermatheca (oviduct) and uterus (Kimble & Hirsh, 1979). Two of the six cells formed a plug between the lumens of the uterus and the spermatheca during L4 and in the adult the cytoplasm of the cells protruded into the uterine lumen. The nuclei of these cells were present until the first eggs squeezed through the junction; unfortunately the subsequent fate of the nuclei was not determined. The time for development of the free-living generation from recently hatched Ll to adults in P. tiliquae is similar to that found in C. elegans and P. redivivus (Hirsh, Oppenheim & Klass, 1976; Sulston & Horvitz, 1981), but adult development is very different. In P. tiliquae the male is short lived and produces

R.J.

544

BALLANTYNE

ZI

l-5

Zla

ZIP

Od

L

Od

LL

appa

L

Z4a

240

Vk-----l i PL

L

ap pa

L

L

Od

i

dtC

.MI

Od

Fm. 35. Free-living female lineage pattern to post-M2. Zl4, gonad precursor cells; a, anterior; p, posterior; aa, anterior derivative from previous anterior derivative; ap, posterior derivative from previous anterior derivative; pa, anterior derivative from previous posterior derivative; pp, posterior derivative from previous posterior derivative; ac, anchor cell; dtc, distal tip cell; AL, AR, DL, DR, PL, PR, VL, VR, gonoduct blast cells; A, anterior; D, dorsal; P, posterior; V, ventral; L, left; R, right; e, epithehal cell of ovary; Ep, epithelial precursor cell of ovary; L, cell at oviduct-uterus junction; Od, oviduct precursor cell; M 1, first moult.

23-27 germinal cells compared to some 500 in C. elegans (Kimble & White, 1981). The free-living female has a very limited divisional sequence in the germinal cell lineage and produces less than 30 oocytes compared with about 1000 in C. eleguns (Kimble & White, 198 l), the vulva is small and the development in ufero of eggs and larvae appears obligatory. This obligatory development is different to all other nematodes where matricidal hatching (Luc, Taylor & Netscher, 1979) usually occurred only toward the end of the reproductive life of the female and in all cases the vulva was enlarged. Duggal (197X) suggested that in P. redivivus the developing L 1 produced a stimulus

which induced the uterus to contract and expel the Ll. The vulva in P. tiliguae consists of seven or eight nuclei and is functional for insemination but does not enlarge further and appears too small for eggs and Ll to pass through. Stemberg & Horvitz (1982) found that both C. efegans and P. redivivw produced a functional vulva (mate and release eggs or larvae) with as few as eight and 10 cells instead of the normal 22 and 20 cells, respectively. The small vulva coupled with the need for P. tiliquae larvae to feed on parent tissue would suggest that the uterine release stimuli have been lost and larval metabolism has been altered to depend on the parent environment and not on the culture environment.

P. tiliquae: development

,

11

of reproductive

system

545

8 Germinal

g,,,

Zla

ZlPP

?7!&kh DS

.MI

.M2

30-32 EJaculatory

FIG. 36. Free-living

duct

6 0 Dtverticula

6

sphincter

(8)

25 Sperm

.M3 duct

male lineage pattern from IO-cell stage to M3. For abbreviations see caption for Fig. 35, plus G, germinal cell precursor; Ic, linker cell; ED, DS, ejaculatory duct and sperm duct precursor cells.

The three specialized somatic cells, namely the uterine anchor cell (ac), the ovarian distal tip cell (dtc) and the testis linker cell (lc) are similar to those found in C. elegans and P. redivivus except that ac and lc both divide and form part of the gonoduct whereas in C. elegans and P. redivivus they underwent cell death and were thought to be eliminated (Kimble & Hirsh, 1979; Sternberg & Horvitz, 1981). The two dtc nuclei in the male do not play the directional role they perform in the female and are the only nuclei associated with the sheath surrounding the developing spermatogonia. In the females of both generations once the gonoducts are complete the dtc nuclei undergo chromatin elimination which appears to be a mechanism to enable the cell to lose redundant information (Wharton, 1986). Kimble & Hirsh (1979) and Sternberg & Horvitz (1981) showed that in C. elegans and P. redivivus, where recently hatched Ll had four cells (Z14) in the genital primordium, the somatic cells arose from Zl and 24 and germinal cells arose from 22 and 23. In P. tiliquae the genital primordium of recently hatched Ll has six nuclei in the females and 10 in the males and all nuclei stain alike, although the end nuclei are slightly larger. The divisional sequence of these nuclei was followed and Figs. 35 and 36 show the lineage pattern from Ll until M2. In the free-living female (Fig. 35) the lineage pattern from the six cell stage is similar to C. elegans and P. redivivus (Stemberg & Horvitz, 1981). It is proposed that of the six nuclei in the Ll, the inner two are equivalent to 22 and 23 and the two at each end arise from Zl and 24. Based on this hypothesis the divisional sequence of the four somatic is as follows: Zla divides asymmetrically to give dtc and AR; AR gives rise to the anterior right half of the ovary and

oviduct epithelium and one L nucleus; Zlp divides to give AL, DL, ac and VL; AL gives rise to the anterior left half of the ovary and oviduct epithelium and one L nucleus; DL divides to form the dorsal left half of the uteri and two L nuclei; the anchor cell (ac) divides during L4 to join the uteri to the vulva; VL divides to form the ventral left half of the uteri. Zlp and Z4a are obliquely arranged (Fig. 13) with Zlp progeny forming the anterior and left cells of the ovary, oviduct and left cells of the uteri and 24 progeny forming the posterior and right cells of the ovary, oviduct and right cells of the uteri. The divisional pattern in Z4a mirrors Zlp except that there is no ac nucleus; the pattern in Z4p mirrors Zla. In the free-living male (Fig. 36) the overall lineage pattern is similar to C. eZegans and P. redivivus (Sternberg & Horvitz, 1981) except for the origins of the somatic and germinal nuclei. The anterior nucleus of the recently hatched Ll gives rise to all the somatic nuclei except for one dtc nucleus. If the anterior nucleus was equated to Zl (Fig. 36) it would follow that the posterior nucleus be designated 24. 24 then gives rise to one somatic (dtc) and all the germinal cells except for the eight germinal already present in the Ll This hypothesis would suggest that the somatic and germinal cells in P. tiliquae males have a common origin. Pai (1928) considered in Turbatrix aceti that the P4 cell divided giving rise to the somatic cell lineage (S5) and the germinal cell lineage (P5). Sulston, Schierenberg, White &Thomson (1983) have since shown that in C. eleguns and T. aceti P4 gave rise to the germinal cell lineage (Z2,23) and the somatic cell lineage (Zl , Z4) arises from the MS founder cell (pl origin). To determine the exact origin of the nuclei in recently hatched Ll of P. tiliquae would be difficult as the development occurs within the uterus of the parasitic adult.

546

R.J.

~ALLANTYNE

In P. tiliquae males, like C. elegans (Kimble & Hirsh, 1979) the initial division of nuclei that may equate to Zl and 24 produces the dtc nuclei. The Zl derived dtc nucleus migrates to the posterior end of the primordium and lies adjacent to the 24 derived dtc nucleus. These two nuclei are the only nuclei of the testis epithelium. In P. redivivus the nuclei equivalent to the ones producing the two dtc nuclei in P. tiliquae and C. elegans underwent a further division (Steinberg & Horvitz, 1981). The two Zl derivatives migrated to the mid-region of the primordium to form part of the sheath surrounding the spermatocytes. One of the two 24 derivatives formed a dtc nucleus while the other one migrated anteriorly to form part of the sheath surrounding the spermatocytes. Sternberg & Horvitz (1981) labelled the non-dtc nuclei sv, and indicated that they formed part of the seminal vesicle although showing them surrounding the developing germinal nuciei. This variation in dtc nuclear development in P. redivivus is interesting as it gives the sheath surrounding the developing spermatocytes three nuclei, whereas in P. tiliquae and C. elegans there are no nuclei in the actual sheath that surrounds the spermatocytes. The germinal cell development in C. elegans varies from animal to animal (Kimble & White, 1981). This is in stark contrast to P. tiliq~ae where the pattern is precise and shows no variation from animal to animal. In P. tiliquae the germinal nuclei are large and almost in linear order throughout the length of the gonad. In C. elegans the distal end of the gonad consists of a cytoplasmic core surrounded by numerous nuclei and individual cells do not form untii the cytoplasm reaches the proximal arm of the ovary (Hirsh et aZ., 1976). Studies on the post-embryonic development of nematodes, especially on the reproductive system, should provide information on the relationships within and between various nematode groups. The initial divisions and establishment of cell lineages appear to be similar in all nematodes studied. The further division of these lineages into sub-lineages appears to be where various genera and species will vary. An analysis of the early lineages and the manner in which subsequent sub-lineages are formed and how the cells of the various areas are reunited should provide valuable information for phylogenetic studies. Acknowledgemenfs-I am indebted to many people and institutions for their assistance with this study. The bench work was carried out in the Parasitology Department of the University of Queensland with the aid of Department funds and a Commonwealth Postgraduate Award (196% 1970). The Zoology Department of the Australian National University provided o&e space and library facilities during my tenure of a Visiting Fellowship in 1989 and Charles Sturt University supported my PEP Leave in 1989. I thank John Pearson, Warwick Nicholas and Lesley Ballantyne for their assistance with various drafts of the manuscript.

REFERENCES BALLANTINE R. J. 1991. Life history and development of Pneumonema tiliquae (Nematoda: Rhabdiasidae). Inrernational Journalfor Parasitology 21: 521-533. DUGGALC. L. 1978. Initiation of copulation and its effect on oocyte production and life span of adult female Punagr&s r&iivivus. NematoIogi& 24: 266-276. HECHLERH. C. 1970. Reproduction, chromosome number, and postembryonic development of Panagreihu redivivus (Nematoda: Cephalobidae). Journal of Nematology 2: 355361. HIR~CHMANNH. 1962. The life cycle of Diry1encha.r triformis (Nematoda: Tylenchida) with emphasis on postembryonic development. Proceedings of the He~minthologica~ Society of w~h~gtoa 29: 30-43. HIRSH D., OPPENHEIM D. & KLAUSM. 1976. Development of the reproductive system of Caenorhabditis elegans. Developmental Biology 49: 2O(t2 19. HORVITZH. R. & STERNBERGP. W. 1982. Nematode postembryonic cell lineages. Journal of Nematology 14: 240248. KIMBLEJ. E. & HIRSH D. 1979. The postembryonic cell lineages of the hermaphrodite and male gonads of Caenorhabditis eleguns. Developmental Biology 70: 396

417. KIMBLEJ. E. &WHITE J. G. 1981. On the control of germ cell development in Caenorhabditis elegans. Developmental Biology 81: 208-219. Luc M., TAYLORD. P. & NETSCHER C. 1979. On endotokia

mat&da and intrauterine development and hatching in nematodes. Nematolgica 25: 268-274. NIGON V. & BRUN J. 1955. L’evolution des structures nucleares

darts l’ovogenese

de

Caenorhabditis

elegans

Maupas 1890. Chromosoma 7: 129-165. PAI S. 1928. Die Phasen des Lebenscyclus der Anquiihda aceti und experimentell-morphologische Ehrbg. ihre Reeinthrssunn. Zeitschrift Er W~senschaftIiehe Zooiogie 131: 293-344: - I ROMAN J. & HIRSCHMANNH. 1969. Embryogenesis and postembryogenesis in species of Pratylenchus (Nematoda: Tylenchidae). Proceedings of the Helminthological Society of Washington 36: 164174. RUNEY W. M., RUNEY G. L. & LAUTER F. H. 1978. Gametogenesis and fertilisation in Rhabdias ranae Walton 1929: 1. The parasitic he~aphrodite. Journal of Parasitology 64: 1008-1014. STERNBERGP. W. 8~ HORVITZH. R. 1981. Gonadal cell lineages of the nematode Panagreliis redivivus and im-

plications for evolution by the modification of cell lineage. Developmental Biology 88. 147-166. STERNBERGP. W. & HORVITZH. R. 1982. Postembryonic nongonadal cell lineages of the nematode PanagreiIis redivivus: description and comparison with those of Caenorhabd~t~ elegatts. Develop~tai Biology 93: 181-

205. SULSTONJ. E. & HORVITZH. R. 1977. Postembryonic cell lineages of the nematode, Caenorhabditis elegans. Developmental Siology 56: 110-l 56. SULSTONJ. E., ALBERTSON D. G. & THOMSON J. N. 1980. The Cuenorhabdit~ eleguns male: postembryonic development of nongonadal structures. Develop~ntal Biology 78: 542576. SULSTONJ. E. & HORVITZH. R. 1981. Abnormal cell lineages in mutants of the nematode Caenorhabditis elegans. Developmental Biology 82: 41-55. SULSTONJ. E., SCHIEREN~ERG E., WHITEJ. G. &THOMSONJ. N.

P. tiliquae: development 1983. The embryonic cell lineage of the nematode Caenorhabditis elegans. Developmental Biology 100:64-l 19. WHARTOND. A. 1986. A Functional Biology of Nematodes. Croom Helm, London. WIJ L-Y. 1967. Anguina calumagrostis, a new species from

of reproductive

system

541

grass, with an emendation of the generic characters for the genera Anguina Scopoli, 1977 and Ditylenchus Filipjev, 1936 (Tylenchidae: Nematoda). Canadian Journal of Zoology 45: 1003-1010.