Early cuticle formation in an adenophorean nematode

InIernarionolJournoifor Parasitology Vol. 18, No. 6,pp. 793-801, 1988. Printed in Great Britain.

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00X-7519/88 $3.00 + 0.00 Pergomon Press plc 1988 Ausrrolian Sociezy for Parasrfology

EARLY CUTICLE FORMATION IN AN ADENOPHOREAN

NEMATODE

ANN PLATZER and EDWARD G. PLATZER Department of Biology and Nematology, University of California, Riverside, Riverside, CA 9252 1, U.S.A. (Received 27 October 1987; accepted 3 April 1988) Abstract--PLATzErt A. and PLATZER E. G. 1988. Early cuticle formation in an adenophorean nematode. International Journalfor Parasitology 18: 793-801. The ultrastructure of cuticular formation was studied during the first 8 days of development in Romnnomermis culicivorax. On day 1 the plasma membranes of

the blastomeres show no specialization. On day 2 short pseudopodial extensions from some of the outer cells begin to migrate over adjacent cells. Small spherical amembraneous structures, which may be connected by fine strands, are present external to the outer layer of cells. During day 3 many of the surface cells are polarized as plaques and minute projections occur on the apical plasma membranes. In addition these epidermal cells now have long pseudopodia which cover adjacent cells and interconnect by numerous desmosomes. On day 4 the apical epidermal membrane forms regularly aligned plicae which contain plaque material and closely associated ribosomes. Externally, there are two delicate membrane-like layers: only the inner one follows the contours of the plicae. On day 5 a distinct granular cuticle 1 (C-l) is present. In addition, the second cuticle (C-2) contains a cortical zone with radial lamellae. Distinct annulae now circumscribe the worm. The narrow C-l is firmly attached to a dark band (the future epicuticle) of C-2. A ‘foamy’ region lies between the epidermis and the cuticle and possibly represents an area of recently synthesized cuticular material. The epidermis contains projections from which cuticular material appears by exocytosis. At this time the cell body of the epidermal cell is internal. On day 6, a narrow osmiophilic clear band separates C-l from C-2. C-2 has a trilaminate epicuticle and a median zone appears. 0; day 7 regions of C- 1 are detached and the basal zone of C-2 is present with several lavers. Bv dav 8 C-l has molted and C-2 consists of an epicuticle, cortical zone, a median zone with two or more- indistinct layers, a basal zone consisting of five alternating dark and pale wavy fibrillar bands. A wide osmiophilic pale zone separates C-2 from the underlying epidermis. INDEX KEY WORDS: Romanomermis culicivorux; annula; apical membrane median and basal zones: cuticle: develooment: enicuticle; epidermis; integument; tion: pseudopodia; radial lamellae; ultrastructure. 1

INTRODUCTION

MATERIALS

AND METHODS

R. culicivorux was maintained in Culex pipiens as described earlier (Platzer & Stirling, 1978). Recently emerged female and male larvae were placed in 35 X 10 mm Petri plates containing spring water and maintained at 27°C. After sexual maturation, approximately 10 days postemergence, freshly laid eggs were collected once per day over a 3 h period for 9 days. The collected eggs were maintained in spring water at 27°C until preparation for electron microscopy. The embryos were collected and concentrated by centrifugation and then placed in vials constructed from 1.O cm ID glass tubing with 1.O iurn mesh screening on the bottom to facilitate subsequent transfers. To enhance penetration of fixatives embryos were placed in a 2% bleach solution for 13 min followed by three rinses in dechlorinated water (Sulston, Schierenberg, White & Thompson, 1983). The embryos were fixed for 2 h in 4”/, glutaraldehyde in 0.1 M-cacodylate buffer at room temperature, rinsed in buffer and postfixed in 1% osmium tetroxide in cacodylate buffer for 1.5 h at 4°C (Platzer & Platzer. 1985). The material was dehydrated in an ascending series of ethanol. Specimens were embedded in Spurrs Resin (Platzer & Platzer, 1985). Sections were taken at intervals along the length of the nematode. They were double stained with uranyl acetate followed by lead citrate and examined with a Philips 300 electron microscope.

on cuticle formation and molting within the nematode egg and the role of the epidermis in this process (Bird, 1977). It is generally agreed that the cuticle is essentially extra-epidermal in origin (Bonner & Weinstein, 1972; Bird, 1977) although Lee (1970) reported that a new membrane forms beneath the epidermal membrane. In past studies, difficulties in interpreting the ultrastructural events of cuticle formation in all but the first molt (Bonner & Weinstein, 1972) have been complicated by the presence of an overlying cuticle. This problem arises because it is difficult to differentiate between areas of old and new cuticle (Bird, 1977). Also fixing and embedding nematodes have been difficult and this contributed to the lack of understanding of early cuticular development. The purpose of this research is to describe the early aspects of cuticular formation in Horruuzornermis culicivorux before hatching, emphasizing the development of the first two cuticles. Also the role of the epidermis, in particular its apical membrane specializations, in this event will be discussed. THERE

specializations; cortical plaque; plicae; polariza-

is little information

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RESULTS The term 'epidermis' will be used in conformity with current literature (Lee, 1977; Bird, 1984) and is synonymous with ‘hypodermis’. The major events occurring during the development of the first two cuticles and molt are summarized in Fig. 5 which is a diagrammatic depiction of the cuticle development compiled from a series of electron micrographs. uuy 1 During day 1 the embryo develops from a onecelled zygote to an early gastrula. Before cleavage the yolk platelets of the homogeneous ovum redistribute so that the zygote has a pale and a dark zone. Cleavage results in blastomeres laden with variable amounts of yolk. Initially the plasma membrane is smooth surfaced, poorly defined and a basement membrane is not visible. Small desmosomes occur on the lateral surfaces near the apical regions and also at irregular intervals between internal blastomeres. A string of desmosomes with an adjacent cluster of microtubules occurred between two blastomeres which were undergoing cytokinesis (Fig. 1A). Duy 2 As gastrulation proceeds the embryo elongates and becomes dorsoventrally flattened with a pointed tail and blunt cranial end. Flexure brings the two ends into varying degrees of juxtaposition, thus the embryos are commonly referred to as the ‘comma’ stage followed by the ‘two-fold’ stage (Sulston ef al., 1983). The outer plasma membrane is simple and smooth and occasionally short pseudopodia are beginning to migrate over adjacent cells and are held in place by desmosomes (Fig. 1 B). Rarely, outblebbings with thickened membranes are noted. These cells are probably presumptive epidermal cells. At irregular intervals external to the outer apical membranes are spherical amembraneous structures with filamentous projections, the outline of which is reminiscent of ‘mossy bryophores’ (Fig. 1C). The filaments may interconnect adjacent ‘bryophores’ or appear to attach to an apical membrane (Fig. 1D, day 3). These structures were noted from day 2 to only the very immature larvae on day 4. Day 3

The 3-day old embryo is sausage- or pretzel-shaped and exhibits intermittent jerky movements. Surface cells become distinctly polarized as long pseudopodia; tiny blebs and plaques appear on the apical

E.G. PLATZER surface (Figs. 2A and 2B). These are the first morphological criteria which identify them as epidermal cells. On their apical side long pseudopodia (future intercordal segments of the epidermal cells) have migrated over some adjacent cells and make contact with adjacent pseudopodia (Figs. 2A, 2B). They are held together by long and numerous desmosomes, which may lie parallel to the surface of the embryo. Septate junctions are also present. The extensions contain microtubules, small yolk platelets, free ribosomes, and rough endoplasmic reticulum (RER). The presence of dense plaques and minute blebs foreshadow cuticle formation. Each irregularly spaced bleb is capped by plaque material and from here a cloud of indistinct microfilaments may extend externally and obscure the outline of the membranes (Figs. 2A, 2B). In general the apical plasma membranes, in contrast to the basal membranes of the epidermal cells, are rarely distinct during cuticular development. especially where active synthesis is noted (Fig. 2A). uuy 4 The larvae are active and display about 1.5 coils inside the egg shell. The blebs seen on day 3 are now regularly aligned projections or plicae. They are more wavy and widely spaced, in longitudinal section (Fig. 2C) and more angular and closer together in cross section (Fig. 2D). Most plicae are capped by plaque material. Ribosomes are abundant in these areas and may extend into and attach to the plaque material of the plicae. Cuticle development first appears during day 4 as one or two (Fig. ZC) fuzzy membrane-like layers external to the epidermal plasma membrane. Only the inner one follows the contours of the epidermal projections. Day 5 c-1 consists

of a single zone which is narrow, granular and exhibits annulae in longitudinal section. Membranes are difficult to discern (Figs. 3A, 3B, 3C). C-2 consists of an epicuticle band, a cortical zone and a ‘foamy’ zone. C-2 ranges from 125 to 250 nm in thickness when relaxed and stretched, respectively (Fig. 3A). The epicuticle, which is rarely trilaminar. is often confluent with the outer C-l. The epicuticle is attached to the cortical zone at the groove of each annula. The cortical zone is wide, finely granular with a more electron dense peripheral area. In contracted areas, viewed in longitudinal profile, the lateral regions of each peak have separated from the epicutitle leaving electron clear regions. This phenomenon

FIG. 1. (A) Day 1 embryo, four cell stage. Parts of two cells, which are undergoing cytokinesis. contain a cluster of microtubules (Mt) and a number of desmosomes (D). Yolk platelets (Y) vary in size and electron density. Cells are not specialized and a cuticle is absent. Bar equals 1.0 pm. (B) Day 2 embryo. Portions of three adjacent cells are shown. Two pseudopodia (P) are migrating over adjacent cells. Desmosomes (D) interconnect adjacent plasma membranes. Bar equals 0.5 pm. (C) Day 2 embryo. Portions of two adjacent cells are shown. Small spherical amembraneous structures (S) are present external to apical membranes. Yolk platelets (Y). nucleus (N). Bar equals 1 .O pm. (D) Day 3 embryo. Higher magnification of spherical amembraneous structures (S) with filamentous projections (arrows) which may interconnect adjacent cells and also attach to lateral apical membranes (arrow). They are present from days 2-4. Pseudopodium (P), nucleus (N). Bar equals 0. I pm.

Development of nematode cuticle

FIG. 1.

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FIG. 2.

Development of nematode cuticle is minimal

on convex surfaces. The cortical zone contains regular arranged radiating striae (seen in cross section) which are alternating thick and thin striae and extend from the epidermis to the epicuticle (Fig. 3B). Glancing surface views show that these striations are actually lamellae and attached to one another (Fig. 3B) (Popham & Webster, 1978). Beneath the lamellae are plicae and ribosomes which are aligned coincident to the cortical lamellae. Rarely a few fine strands may be seen extending from the ribosomes through the plaques to the lamellae. In longitudinal profile, a narrow ‘foamy’, possibly newly synthesized, zone rests on the epidermis (seen more clearly at day 6, Fig. 4E). Both cuticles display annulae, which circumscribe the worm, are in register and firmly attach to one another at the grooves. The spacing of the annulae range from 525 nm (concave surface) to 725 MI (convex surface) depending on the degree of stretch. The epidermal cell (and also the muscle cell) is now bipartite (see Fig. 3C, day 6) and its cell body with a heterochromatic nucleus has migrated internally. From the cell body radiates a stout stalk with encompassing pseudopodia (intercordal segments). These intercordal segments are fortified by close junctions and septate junctions. There is no indication that these cells are syncytial. The epidermis reflects active synthesis as abundant cisternae of RER lead from the nucleus and extend into the circumferential arms (see Figs. 4B, 4C, day 6). Several types of vesicles are present, some of which are membrane bound and in a cluster (see Figs. 3C, 4A, day 6). The Golgi apparatus was difficult to see. In longitudinal profile the plicae are prominent, often irregular in outline, frequently in register with the cuticular annulae. Projections are more minimal in the convex region (Fig. 3A). In contrast plicae observed in cross section are regularly outlined and aligned like brickwork and filled with plaque material (Fig. 3B). Ribosomes may accumulate in linear fashion coincident with the above plicae and striated lamellae. Day 6 Nematodes are very active in the egg. C-l is approximately 24.5 nm thick and there is a narrow osmiophilic clear zone between it and the epicuticle (Fig. 4A). It retains its annular configuration during all stages examined. The epicuticle is distinctly trilaminar (12 nm thick). C-2 now contains a median zone which

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is granular and only faintly annular (Fig. 4A). Vesicles are numerous in the cortical arms of the epidermis. Day 7 C-l is detached in areas (Figs. 4B, 4C, 4D and 4E). C-2 now contains a basal zone which may or may not consist of three alternating pale and dark undulating bands which are in concord with the above cortical and median zones and epicuticle (compare Figs. 4D and 4E). Osmiophilic dark blotches often punctuate the peripheral grooves and delineate this basal region from the above median zone. The ‘foamy’ zone rests on the epidermis. Day 8 Many nematodes are hatching. The first cuticle has molted (Fig. 4F). The second cuticle is about 550 nm thick. A thin osmiophilic clear layer usually subdivides the cortical and median zones. Radial lamellae do not extend beyond the cortical zone. A finely granular median zone is indistinctly subdivided into two or more layers. The basal zone now contains five alternating osmiophihc dark and pale sublayers which appear to be a condensation of filamentous material. C-2 appears to be separating from the underlying epidermis since the narrow ‘foamy’ band is replaced by a wide osmiophilic clear layer studded with granules and tine strands. The epidermis frequently contains regular protuberances which are in register with the annulae of the above cuticle. The peak of each outpocketing is denser on its cytoplasmic face. DISCUSSION The eggs of R. culicivorax are laid in fresh water where the developing embryo is protected by the egg shell during the first 8 days. Thus it is not surprising that C-l is thin and ephemoral. Similarly, Meloidogym javanica a phytoparasitic nematode, has only a trilaminar epicuticle during its initial development in the egg shell (Bird, 197 1). In contrast, the first cuticle of T. spiralis, although thin, contains an epicuticle plus three zones (Bruce, 1970; Kozek, 1971). In this case, the first stage larva requires protection from adversities of the host environment in the blood stream and subsequent residence in the muscle of the vertebrate host. C-2 of R. culicivorax is thicker and contains cortical, median and basal zones which protect it when it hatches and enters the environment on day 8 and also

FIG. 2. (A) Day 3 embryo. A long pseudopodium (P) has migrated over two cells. Apical plasma membrane (A) contains tiny plaques (arrowheads) and filamentous material (F) extends externally. Basal and adjacent internal membranes (I) are more distinct than apical ones. Bar equals 0.1 ,um. (B) Day 3 embryo. A pseudopodium (P) contains tiny blebs (B) which are capped by tiny plaques (arrowheads). Filamentous material (F) is external to plasma membrane. Yolk platelets (Y). Bar equals 0.1 pm. (C) Day 4 embryo. Longitudinal section showing a pseudopodial extension (P) of an epidermal cell. The apical membrane contains wavy regularly aligned plicae (K) which are capped by plaque material (arrowheads). Early cuticular development is present. Two fuzzy membrane-like layers (C-l, C-2) are evident external to the apical membrane. Only the inner one follows the contours of the plicae at this stage. Bar equals 0.1 pm. (D) Day 4 embryo. Transverse sections showing the more tightly packed and smaller plicae (K) on the apical membrane of an epidermal pseudopodium. Each peak is capped by plaque material (arrowheads). Since only one external zone (C-l) is present this embryo is at an earlier stage than (C). Bar equals 0.1 pm.

FIG. 3. (A) Day 5 larva. Longitudinal section showing early cuticular development. Cuticle I (C-l) is narrow, granular. and adherent to the epicuticular band (E) of C-2. A cortical zone (c) of C-2 is present. A pale foamy-zone (z) rests on the epidermis. Annular length varies, depending on degree of stretch. Pseudopodium (P), muscle (M). Bar equals 0.1 ,Um. (B) Day 5 larva. Glancing section of cuticle illustrating the radial striations (L) which occur in the cortical zone of C-2. The striations are interconnected lamellae (arrows). Three rows of transversely arranged plicae (K) are in a brick-like configuration and contain dense plaques (arrowheads). Cuticle-l (C-l). Bar equals 0.1 pm. (C) Day 6 larva in cross section. The epidermal cells (H) are bipartite and filled with RER (R). A long stalk passes externally and contains, in addition to RER, a cluster of small vesicles (V). Egg shell (ES), nucleus (N). Bar equals 1 .O pm.

Development

of nematode

FIG. 4.

cuticle

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A. PLArzaa

DAY 3

and E. G. PLATZER

DAY 4

DAY 6

DAY 7

DAY 6

FIG. 5. A diagrammatic representation of cuticle development from days 3 to 8. On day 3 the epidermal cells are distinctly polarized. Long pseudopodia (P), which interconnect by desmosomes (D), occur apically. Plaques (Q) and tiny blebs (B) are on the apical membranes. Cuticle is not present. On day 4 regularly aligned epidermal projections (plicae) (K) are visible in longitudinal and cross sections. Plicae are capped by plaques (Q). Precuticle development is noted as one to two membranes, the anlage of C-l and anlage of the epicuticle (C-2), appear external to the epidermis. A distinct and granular C- 1 appears on day 5. C-2 contains an epicuticular band (E) and a cortical zone (c) with radial striations. C-l and C-2 are annulated and in register. A ‘foamy’zone (z) is present. Pseudopodium (P). On day 6, C- 1 is loosely attached to the epicuticle (E). C-2 contains a trilaminar epicuticle (E) and a median zone (m) appears between the cortical (c)and ‘foamy’ (z) zones. Pseudopodium (P). Molting of C-l is initiated on day 7. C-2 contains a trilaminar cuticle (E), cortical (c), median (m) layered basal (b) and ‘foamy’ (z) zones. On day 8 the larvae are hatching and C-l has been molted. The ‘foamy’ zone has been replaced by a wide osmiophilic clear zone (*) where apolysis is being initiated. Epicuticle (E), cortical zone (c), median zone (m), basal zone (b) and pseudopodium (P) are present.

FIG. 4. (A) Day 6 larva. Longitudinal sections of integument. The C-l is loosely attached to the epicuticle (E) which is now distinctly trilaminar. C-2 contains cortical (c) and median (m) zones. The apical epidermis, which may or may not be in register with the annulae contains plicae (K) and plaques (arrow). A foamy zone is present (z) but not distinct in this photograph. The intercordal segment contains ribosomes and two clusters of vesicles (V). Bar equals 0.1 pm. (B) Day 7 larva. Transverse section (slightly oblique). C-l is detached over a large area. C-2 is not clearly delineated. The epidermis contains stacks of rough endoplasmic reticulum (R). A few plicae (arrowheads) on the epidermal apical membranes are evident. Muscle(M) is abundant. Epidermal nuclei (N) are heterochromatic. Bar equals 5.0 pm. (C)Day 7 larva. Higher magnification of(B). The epidermis contains stacks of RER (R) which are in close association to a nucleus (N). Plicae (arrowheads), muscle (M). Bar equals 0.5 pm. (D) Day 7 larva. Longitudinal section of the cuticle. C-l is detached in a few areas. Epicuticle (E) is trilaminar. C-2 consists of granular cortical (c), pale media (m) and wavy basal zone (b). Beneath is a ‘foamy’ area (z) above the epidermis. Bar equals 0.1 ,um. (E) Late day 7 larvae. Longitudinal section. Faint longitudinal striations are present in the medial zone (m). The basal zone (b) is annulated, multi-layered and shows distinct longitudinal striations. A foamy zone (z) is still present beneath the basal zone. Epicuticle (E), cuticle-l (C- 1), cortical zone (c). Bar equals 0.1 pm. (F) Day 8 larva. Longitudinal section. Cuticle-l is absent, cuticle-2 is like that of(E) except that the foamy zone has been replaced by a wide clear area with some granulation. Apolysis (*) thus appears to be initiated. Bar equals 0.5 pm.

Development

of nematode

for initial residence inside the hemocoel of the mosquito larva. The early appearance of radiating striae or lamellae when the first zone of C-2 is laid down suggests that the lamellae may act as scaffolding for retention of body form. Surface epithelial cells are actively involved in cuticular formation. On day 3 the epidermal cells are polarized and early cuticle development is foreshadowed by the presence of pseudopodia containing plicae, plaques, and filamentous material. These cells resemble vertebrate fibroblasts morphologically (elongate, numerous RER, pseudopodia) and physiologically (synthesis of collagen, Bird, 1971; Platzer & Platzer, 1985). The presence of plasma membrane plaques is an interesting similarity which exists between the development of the cuticle of R. culicivorux and that of the insect (Locke, 1976) and the lobster (Arsenault, Caste11 & Ottensmeyer, 1984). Locke has studied extensively epidermal involvement in exoskeleton deposition in the insect (Locke, 1976; Locke & Huie, 1979). He described plaques close to the sites where the cuticle first appears and thus considers them ‘to have a role in their synthesis and/or deposition and orientation.’ In this present study on R. culicivorax, similar plasma membrane plaques were first observed before the presence of the first cuticle and also during the deposition of the second cuticle during days 5-7. Plaques may be involved in cuticular development in other nematodes. Half desmosomes and desmosomes have been described by Bruce (1970) as holding the epidermis to the overlying cuticle in T. spiralis (see plate 1A and compare to our plate No. 21)) and by Grootaert & Lippens (1974) in A~orcelai~~~l~s. Since the half desmosomes resemble plaques after uranyl acetate staining, it may be necessary to use bismuth stain, which stains plaques and not hemidesmosomes (Locke & Huie, 1977) during the examination of precuticle development to resolve this discrepancy. The presence of lamellae in other nematodes has been doc~ented by Bird (1971, p. 64). Although C-2 is not shed until the parasite phase, there is morphological evidence that apolysis, or separation of epidermis from old cuticle, has been initiated before hatching and is similar to apolysis as documented in insects (Locke & Huie, 1979). In R. cuticivorax there is no apparent resorption of either C-l or C-2 prior to molting. No resorption of shed cuticle was observed by Lee (1970) in ~~~~ostrongylus brasiliensis. However, Bird (1977) reported partial resorption of shed cuticle in Ascaris hnbricoides and M. juvanica. Acknawfedgemenrs-The authors wish to thank Dr Alan Bird for reading the manuscript during its preparation and for his helpful suggestions. The patient and diligent assistance of Mrs Betty Pennington is gratefully acknowledged. This project was iupported in pa;t by reiearch grant-AII57 I7 from NIAID, U.S. Public Health Service, National Institute of Health.

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REFERENCES ARSENAULT A. L., CASTELL J. D. & OTTENSMEYER F. P. 1984. The dynamics of exoskeletal-epidermal structure during molt in juvenile lobster by electron microscopy and electron spectroscopic imaging. Tissue and Cell 16: 93-

106. BIRD A. F. 197 1. The Structure of Nemutodes. Academic Press, New York. BIRD A. F. 1977. Cuticle formation and moulting in the egg of Meloidogyne juvanica (Nematoda). Parasitology -74: 149-152. BIRD A. F. 1984. Nematoda in Biolow Intewment, .__ ofthe Vol. I, Znvertebrates (Edited by RBREITER-H&N J., MATOLTSY A. G. & RKHARDS K. S.I., DD. ** 212-233. Springer, New York. BO~YNERT. P. & WEINSTEIN P. P. 1972. Ultrastructure of the hypodermis during cuticle formation in the third molt of the nematode Nippostrongylus brasiliensis. Zeitschrifi &er Zellforschung und Mikroskopische Anatomie 126: 17-24. BRUCE R. G. 1970. Trjchine~~u spiralis: fine structure of body wall with special reference to formation and rnoulting of cuticle. Experimental Parusitology 28: 499-5 1 1. GROOTAERT P. & LIPPENS P. L. 1974. Some ultrastructural changes in cuticle and hypodermis of Aporcelaimeilus duriig the first mouli _(Nematoda:D&ylaimoidea). Zeitschrifr fuer Morpholoxie . _ und Okologie der Tiere 79: X9-28i. ’ Koz~c W. J. 1971. The molting pattern in Tr~chine~~a spiralis-11. An electron microscope study. Jouri~a~ of Parasitology 57: 1027-1038. LEE D. L. 1970. Moulting in nematodes: the formation of the adult cuticle during