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Ultrastructure of a neurosensory organ in a root-knot nematode
JOURNAL OF ULTRASTRUCTURE RESEARCH
56, 258-276 (1976)
Ultrastructure of a Neurosensory Organ in a Root-Knot Nematode WILLIAM P. WERGIN AND BURTON Y. ENDO Nematology Laboratory, Plant Protection Institute, USDA, A R S Beltsville Agricultural Research Center, Northeastern Region, BeltsviIle, Maryland 20705 Received December 29, 1975, and in revised form, May 25, 1976 Second stage larvae of the root-knot nematode, Meloidogyne incognita [(Kofoid and White) Chitwood], were examined to elucidate the u l t r a s t r u c t u r e of the amphids, which are paired lateral organs believed to function in chemosensory perception. The amphids were found to consist of supportive, nervous, and secretory tissues. The support tissue forms two cuticular canals and encircling "supporting cells," which extend from the external anterior region of the nematode posteriorly into the body of the larva. Each amphid is innervated by a group of axons t h a t become modified into cilia. Generally, seven of these cilia enter the amphidial canal and t e r m i n a t e n e a r the external pore; whereas, t h r e e to five "accessory" cilia r e m a i n external to the canal and t e r m i n a t e b e n e a t h the cuticle at the cephalic region. The secretory structures of the amphid consist of an amphidial "gland," a large irregularly shaped cell containing mitochondria, and a duct, an intercellular area h a v i n g a g r a n u l a r content. The physical associations and the functional significance of these structures are discussed.
Paired lateral organs on the lip region of nematodes were initially described in 1865 by Bastian using a light microscope (25). Later, the organs were named "amphids" by Cobb (7) who found that these structures were widely distributed in the phylum Nematoda and concluded that they probably functioned in sensory perception. By 1950, the results of numerous light microscopic studies had established that an amphid consisted of a group of ascending nerve fibers that entered a large cell termed the amphidial gland. Anterior to the gland, the fibers became less closely associated with one another in a region known as the amphidial pouch. Specialized ends of the fibers, called terminals, continued to penetrate the anterior portion of the nematode through a cuticular channel (6). Because the ends of the terminals, which were collectively called sensillae, were exposed to the outer environment of the nematode, the amphid was believed to function in chemosensory perception. Electron microscopic studies of the amphid have revealed the ultrastructure of the nerve fibers, which are referred to as the amphidial nerves (28), neurons (36,
37), dendritic nerve processes (14, 32), dendrites (1-3, 5, 16, 30, 31, 34, 38-42), and axons (19-24, 43, 44). The terminals are reported as neurons (36, 37); sensillar dendrites (31), dendritic processes (2, 30, 40-42), modified cilia (19-24, 32), ciliary elements (29), ciliary processes (9, 16, 17), cilia-like processes (1), and cilia (3--5, 14, 15, 18, 28, 30, 34, 38, 39, 43, 44). The postulated chemosensory function of amphids has received further support due to the structural similarities that exist between these structures in nematodes and chemosensory organs described in many insects. Recent investigations have documented the presence of accessory cilia (2, 3, 5, 9, 17, 24, 36, 38-42) and nerve processes (4, 22-24, 36, 38, 39, 42) associated with the amphid. Preliminary observations in our laboratory indicated that the accessory cilia originated from the amphidial nerve and that a highly developed nerve process has continuity with at least one axon of the nerve. Because these structures may provide additional insight into the functional role of the amphid, a detailed examination of this organ was undertaken in
258 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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the plant-parasitic nematode, Meloidogyne incognita [(Kofoid and White Chitwood]. MATERIALS AND METHODS Second stage larvae of Meloidogyne zncognita were obtained from egg masses collected from roots of infected tomato plants grown in a greenhouse soil bed. The larvae either were used to infect clover roots, or were mixed with w a r m molten 2% w a t e r agar, which t h e n was allowed to solidify, previously reported procedures were used to inoculate roots and to prepare tissues for transmission electron microscopic e x a m i n a t i o n (10). Briefly, the solidified a g a r t h a t contained nematodes was diced into 2- to 3-ram cubes and transferred to glass vials containing 3% glutaraldehyde in 0.05 M phosphate buffer, at 22°C, for chemical fixation of the larvae. Rinsing and postfixation in osmium tetroxide were also carried out in 0.05 M phosphate buffer (pH 6.8). Fixation for 1.5 h r was followed by washing in six changes of buffer over a period of 1 hr. The agar blocks t h e n were postfixed in 2% osmium tetroxide for 2 hr, dehydrated in an acetone series, and infiltrated with a low viscosity m e d i u m (33). Silver-gray sections of selected nematodes were cut on a Sorvall MT-2 ultramicrotome with a diamond knife and mounted on uncoated 75 x 300 mesh copper grids. The sections were stained with 2% aqueous uranyl acetate (10 min), t h e n with lead citrate (5 min). Thin sections were viewed in a Hitachi HU-11C operating at 75 kV with a 30/*m objective aperture, or a Philips 200 or 301 electron microscope operating at 60 kV with 20 b~m apertures. Larvae were prepared for scanning electron microscopic e x a m i n a t i o n by chemically fixing with 3% glutaraldehyde in 0.05 M phosphate buffer for 1.5 hr, at 22°C, d e h y d r a t i n g in a n ethanol series and critically point drying. The larvae were mounted on a l u m i n u m studs, coated with carbon t h a t was followed by gold-palladium and viewed with a Hitachi HHS-2R scanning electron microscope operating at 15 or 20 kV. RESULTS
The amphid is a structurally complex organ associated with supportive, nervous, and secretory tissues (Figs. 1 and 2). The following results are organized to correspond to these associations. I. SUPPORTIVE TISSUES
A. Cuticle An examination of second stage larvae
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with the scanning electron microscope reveals the amphids as two lateral pits in the cephalic region of the nematode. Electron opaque material is generally present within and external to the pits (Fig. 3). However, when this material is not present, measurements obtained with stereomicroscopy indicate that pits are 0.2/~m deep and appear as elongate openings 0.8 x 0.2 /~m (Fig. 4). Transmission electron microscopy of cross sections through the nematode, beginning at the bottom of the pits and proceeding posteriorly, shows the amphids as slit-like canals (Figs. 5 and 6) that gradually become rounded (Figs. 7-9) and extend about 12/~m below the anterior surface. The canals are limited by a tapered, circular sheath of amorphous material that is apparently continuous with the external cuticle of the nematode. This sheath delineates the amphidial canal, which constitutes the anterior segment of the amphid.
B. ~Supporting Cell" An elongate tubular cell, previously designated %upporting cell" by McLaren (22) encircles the amphidial canal (Figs. 8 and 9). The membranes of the encircling cell meet or are apposed, distolaterally to the canal, forming a tight junction (arrows, Figs. 8 and 9). This membrane configuration spans the length of the supporting cell, which extends from the base of the amphidial canal anteriorly to the cephalic framework. Cellular or membranous continuity between the supporting cell and the hypodermal tissue, which underlies the cephalic framework, could not be ascertained. No cellular organelles have been observed in the supporting cell, although microtubules and membrane-bounded vesicles having granular contents are frequently observed (Figs. 8 and 9). A more loosely aggregated granular material, not confined within vesicles, generally occurs also throughout the length of the cell.
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FIGs. 1-17. The sequence of figures begins at the anterior portions of t h e larvae and proceeds posteriorly into the nematodes. This sequence does not precisely parallel the organization presented in the text; however, we believe t h a t this sequential a r r a n g e m e n t will assist the reader in visualizing the i n t e r n a l structures of the amphid and in relating t h e i r complex associations. FIG. 1. A n illustration of a second stage l a r v a of M. incognita (a). Larvae are generally 400 tLm long; however, our study has concentrated on the anterior 20 t~m region illustrated in the dorsal (b) and sublateral (c) views. This region, containing t h e neurosensory organs or amphids, is innervated by nerves e m a n a t i n g from the nerve ring (a). In the text, Figs. 5 to 17 are a sequential series of micrographs t h r o u g h the amphidial regions illustrated in b and c. II. NERVOUS TISSUE
A. Axons The function of the amphid has not been experimentally determined. Whether an innervating element of this organ receives or transmits nerve impulses remains unknown. As a result, previous investigators have applied the terms "axon" and "dendrite" to the neural element. In our study, the element is designated an axon because this term conforms to the terminology used in a recent review (25) on sensory organs. The amphids are innervated by nervous tissue, which could be traced to the nerve ring of the nematode. In cross sections above the ring, two amphidial nerves, each consisting of a bundle of axons, lie
dorsolaterally (Fig. 1). The axons, which are irregularly shaped in cross sections, vary in diameter from 0.1 to 0.6 t~m but gradually become constricted and circular as they ascend and enter the base of the amphid (Figs. 2, 10-14). Each axon is limited by a membrane that frequently has a peripheral layer of granular material along its inner surface (Figs. 13 and 14). In addition to the granul ar material, the axons contain numerous mitochondria and longitudinally oriented microtubules (Figs. 2 and 16). Occasionally, particles resembling ribosomes can also be observed in these ascending structures.
B. Nerve Process Approximately 10/zm from the anterior end of the nematode, a highly convoluted
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Amphidial Gland~ Fro. 2. Illustrations of longitudinal and cross sections t h r o u g h the amphid. The longitudinal view (a) is a simplified model i l l u s t r a t i n g continuity between the axons and t h e i r ciliary extensions. Accessory cilia, which ascend into the lateral sector of the cephalic region, are represented by the two cilia on the left. Three cilia are shown ascending the amphidial canal. The p a r e n t a l axon from one of these cilia is continuous with the nerve process. Drawings b, c, and d correspond to the mierographs in Figs. 9, 13, and 15, respectively. These illustrations correlate the spatial relationships between supporting, nervous, and secretory tissues
shown in cross sections with those illustrated in the longitudinal model (a). membrane process is associated with each bundle of ascending axons (Figs. 10-16). This process, which lies adjacent and dorsal to the axons, is about 10 tLm long and measures 2 to 3 ~m in diameter. It consists of tubular projections (Fig. 16) and one or more cilia (Figs. 15 and 16), all of which are delineated by a limiting membrane. The tubular projections resemble microvilli. They extend anteriorly and posteriorly 2 to 4 ~m from the center of the process (Fig. 16). As m a n y as 200 projections may lie parallel to one another in a densely packed arrangement (Figs. 12-14).
The projections are about 850/~ in diameter and contain several microfilaments (neurofilaments). One or more short cilia also extend outward from the center of the process (Fig. 16). These short segments, which lie central and parallel to the microvilli, can be easily distinguished from the latter by their larger diameters and characteristic arrangement of microtubules. Occasionally, the center of the process also contains rootlets, which are associated with the cilia, and mitochondria that are several times larger than those normally present in the axons.
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FIGS. 3 and 4. Scanning electron micrographs of the heads of second stage larvae. Electron opaque material (arrows, Fig. 3) is generally associated with and obscures the view of the amphidial pits. When this m a t e r i a l is absent (Fig. 4), stereo e x a m i n a t i o n reveals the pit (arrow) as an elongate opening, 0.8 x 0.2 ~m, t h a t is about 0.2 ~ m deep. Figures 3 and 4, x 20 000.
Careful examination of cross and longitudinal sections has failed to reveal the precise structural relationship t h a t exists between the nerve process and the ascending axons of the amphidial nerve. However, membrane continuity has been established between the process and at least one of the amphidial axons (Fig. 16).
C. Transitional Region between Axons and Their Ciliary Extensions Above the nerve process, the axons become modified into cilia t h a t extend toward the anterior region of the amphid (Figs. 2 and 17). The membranes of the axons are continuous with those of the cilia; however, the transitional regions between the two structures are characterized by the presence of tight junctions, vesicles and rootlets (Figs. 12-14, 17). The tight junctions, which appear as electron dense ~collars" around the terminal part of each
axon (arrows, Fig. 13), are formed from the limiting membrane of an axon and t h a t from either the adjacent nerve or the amphidial gland. Internally, this region of the axon contains numerous vesicles, resembling synaptic vesicles, t h a t frequently lie around a central rootlet (Fig. 17). The rootlet is an elongate fibrillar structure t h a t exhibits a cross-banding pattern with a major repeat distance of about 350/~. Immediately above the rootlets are the ascending cilia of the axons (Figs. 1, 2, 1012, 17). These cilia contain a peripheral ring of 6 to 8 microtubular doublets and 0 to 5 single inner microtubules t h a t are about 1 t~m in length. They separate the posterior parent axons from the anterior extensions of cilia which are more loosely organized and ascend toward the amphidial canal. Although the presence of distinct dynein arms has not been confirmed
FIG. 5. Cross section of the amphidial region of the nematode n e a r the bases of the lateral amphidial pits (P). Illustrated are the hexaradiate pattern of the cephalic framework, a ring of six labial sensory canals around a central stylet and four outer cephalic sensory organs (arrows). × 38 000. FIG. 6. Cross section through the anterior portion of an amphidial canal. At this level, the amphid (A) appears as a slit-like structure. Above the amphid lie the terminal portions of several accessory cilia (arrows) having sparse granular contents and an occasional microtubule. Two cephalic sensory organs (C) can be observed at the left and right of the amphidial slit. z 50 000. 263
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granular material in addition to the randomly spaced microtubules, which become less numerous in the anterior parts of these extensions (Figs. 7 and 8). The granD. Anterior Extensions of Cilia ular material tends to accumulate along The cilia, which arise from the axons, the inner surface of the limiting memmaintain their characteristic structure for branes of the modified cilia. about 1 t~m. Then their diameters enlarge 2. Cilia of the lateral sector (accessory 2- to 3-fold, the peripheral rings of micro- cilia). Generally, three to five accessory tubular doublets are lost and 4 to 12 ran- cilia, which lie lateral and parallel to the domly spaced, single microtubules appear amphidial canal, ascend toward the lateral and ascend these ciliary extensions. These cephalic sector (Figs. 7-9) and terminate structural features easily distinguish the beneath the hypodermis. The internal anterior extensions from the 1 t~m bases of structure of the cilia of the amphidial the cilia. canal and that of the accessory cilia are Each lateral bundle of cilia divides into similar. However, unlike the former, actwo distinct groups (Figs. 10 and 11). One cessory cilia are not closely associated in a group, generally consisting of seven cilia, symmetrical pattern and do not maintain enters and ascends the amphidial canal. a tubular shape along their length. As The second group, usually consisting of these cilia ascend, they become flattened three to five cilia, ascends externally and into broad sheath-like structures that conlaterally to the canal toward the lateral form to the constricting space that lies becephalic sector (Figs. 1 and 2). tween the amphidial canal and the outer 1. Cilia of the amphidial canal. Cross cuticle of the nematode. sections along the amphidial canal reveal that the ascending cilia are arranged in a III. SECRETORY TISSUE symmetrical hexaradiate pattern (Fig. 9). This pattern consists of six cilia equidis- A. Amphidial '~Gland" tantly spaced around a central cilium. The The term ~amphidial gland" was introcilia lose this symmetrical pattern, become duced by light microscopists to describe bulbous and terminate below the pit of the the cell that encircles the innervating eleamphidial opening (Figs. 1, 7, 8). ments of the amphid. In M. incognita, this The cilia contain sparsely scattered "gland" apparently corresponds to a large
on the subtubules, the general appearance of these amphidial cilia resembles that commonly observed in kinocilia.
FIGS. 7 and 8. Successive cross sections t h r o u g h the amphidial canal illustrating the tips of several cilia. The t e r m i n a l portions of the amphidial cilia become slightly bulbous at t h e i r ends and t e r m i n a t e at different levels near the anterior region of the canal. The amphidial canal is encircled by a '~supporting cell" whose m e m b r a n e forms a t i g h t junction (arrows) lateral to the canal. An anterior extension of the amphidial gland, which lies between the canal and the hypodermis (H), contains three flattened accessory cilia (C) h a v i n g microtubular singlets and doublets. The outer m e m b r a n e s t h a t appear around these modified cilia are actually continuous with the i n n e r m e m b r a n e of the amphidial gland. Figure 7, x 52 000; Fig. 8, x 52 000. FIG. 9. Two amphids of a nematode in a cross section t h r o u g h the midregion of the amphidial canal. Each canal contains seven cilia a r r a n g e d in a symmetrical pattern. Near the midregion, each cilium generally h a s 4 to 12 longitudinally oriented microtubules t h a t do not appear in any organized pattern. The amphidial canals are surrounded by supporting cells h a v i n g vesicles with g r a n u l a r contents. A t i g h t junction, formed by the apposing m e m b r a n e s of this cell, is indicated by the arrows. In addition to these structures, portions of the a m p h i d i a l glands, each containing three accessory cilia (C), can be observed. The accessory cilia are more flattened t h a n those found in the canal and frequently exhibit microtubular doublets at this level. Protractor muscles (P) of the stylet (S) lie between the lateral amphids. The amphid in the left portion of the micrograph has been illustrated in Fig. 2b. x 30 000.
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J FIG. 12. Cross section t h r o u g h the nematode illustrating the large area occupied by the amphids, which consist of the glands, ducts, and nerve tissues (cilia, axons, and nerve processes). The amphid in the lower portion of the micrograph contains several cilia, as well as t e r m i n a l portions of two axons (arrows). The r e m a i n i n g amphid consists of the axons except for a single cilium at the upper right. Approximately 200 microvilli are associated with each of the amphids, x 20 000. FIGS. 10 and 11. Successive cross sections of the amphid near the base of the amphidial canal. The canal (C) has apparently t e r m i n a t e d along the lower portion illustrated in Fig. 10 (arrows), and is no longer found in Fig. 11. The cilia present in the canal (Fig. 10) were sectioned above the ciliary segments shown in Fig. 11. These segments, which resemble true cilia, have an outer ring of 5 to 9 microtubular doublets and 0 to 6 i n n e r singlets. Four accessory cilia (AC), present in the amphidial gland (Fig. 10) merge with those from the canal (Fig.- 11). Numerous microvilli extending upward from the nerve process lie to the r i g h t of the cilia. Two types of m e m b r a n e configurations are associated with the microvilli. Concentric circles (arrows, left) result when the m e m b r a n e s of the microvilli are closely appressed to the inner m e m b r a n e of the amphidial gland. Alternatively, single m e m b r a n e bound microvilli appear when a group (G) collectively "invaginates into" the gland. In this case, each microvillus is limited by its own m e m b r a n e but the group is bounded by the i n n e r m e m b r a n e of the amphidial gland. G r a n u l a r m a t e r i a l of the duct can be observed around the cilia and the microvilli. Figures 10 and 11 x 42 000.
FIG. 13. Cross section of an amphid illustrated in Fig. 12. Tight junctions (arrows), which form from the apposition of adjacent membranes, can be observed around two of the axons ( u p p e r left). The terminal portion of an axon contains an occasional mitochondrion (M), longitudinally oriented microtubules, peripheral granular material and a central rootlet (R), which descends from the ciliary segment. Microfilaments or neurofilaments are present in each of the microvilli. This amphid is included in the illustration in Fig. 2c. × 52 000. 268
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irregularly shaped cell that extends from the anterior region of the amphidial nerve upward toward the lateral cephalic sector (Fig. 2). The cell encircles the cilia, the nerve process, and the accessory cilia (Figs. 10-13). The gland contains numerous mitochondria; however, neither nuclei nor other cellular organelles were encountered in this cell. Although the amphidial gland encircles the nerve process, its membrane m a y or may not tightly appose that of the microvillar projections. As a result, two types of membrane configurations can be observed in cross sections (Figs. 2, 10-15). When the two membranes are tightly apposed, a double membrane appears around each microvillus; the inner one is continuous with that of the nerve process, while the outer membrane belongs to the amphidial gland. When the membranes are not tightly apposed, a group of microvilli appear to project into the lumen of the gland. Cross sections of this association show a group of small circular membranes, delineating the microvilli, which are collectively enclosed by the membrane of the amphidial gland.
B. Amphidial Duct The amphidial duct consists of the intercellular space that lies between the membranes of the amphidial gland and those of either the nerve process, the amphidial axons or the amphidial cilia (Figs. 2, 1115). When these membranes are closely apposed, such as along the accessory cilia, the duct is constricted. However, the duct constitutes a significant dilated area, having granular contents, around the cilia and the nerve process. Although this granular material is accessible to the lumen of the amphidial canal, the extent to which it is secreted is not readily apparent. DISCUSSION I. AMPHIDIAL STRUCTURE
Our general observations relating to the innervation of the amphids by cilia that
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arise from the amphidial axons are similar to conclusions reported by other investigators. However, our results and interpretations relating to the nerve process, amphidial cilia, accessory cilia, and the amphidial duct differ in several respects from those previously reported.
A. Nerve Process Microvillar processes, termed ~'nerve processes," were initially illustrated by Bird (4). These processes extend distally from a nerve bulb in the amphidial pouch. Electron microscopic studies by Baldwin and Hirschmann reported a similar microvillous gland in males of Meloidogyne
incognita (2)andHeterodera glycines (3). However, these reports concluded that the microvilli were extensions of the amphidial gland and were physically distinct from the amphidial nerve bundle. In our study, the microvilli are neither a part of nor continuous with the amphidial gland. Alternatively, the microvilli, constitute processes whose membranes exhibit continuity with that of at least one, and possibly more, axons of the amphidial nerve. Therefore, the microvillous gland described by Baldwin and Hirschmann (2, 3), probably corresponds to the nerve process ofM. incognita described in our study. This conclusion parallels that of McLaren (22, 24) Ward et al. (36), and Wright (42) who have recently demonstrated nerve processes extending from the amphidial nerve axons in filaria (22), strongyles (24), Caenorhabditis elegans (36), and
Nippostrongylus brasiliensis (42). B. Cilia A basal body is a centriolar pinwheel that bears a cilium or a flagellum (13). It generally consists of a distal basal plate, a cylindrical wall, which is composed of nine groups of microtubular triplets, and a set of fibers or rootlets that extends into the subjacent cell (12, 13, 27). The term %asal body" has been used to describe the terminal regions of the amphidial cilia in nema-
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todes (14, 16, 30, 36, 41, 42). However, in these studies, one or more of the features generally associated with basal bodies are modified or absent. For example, in all cases the microtubular wall consists of a ring of doublets. Basal plates (16) and rootlets (36, 42) have been noted in only a few of these studies. Although the t e r m "basal body" has been used in investigations of sensory cilia in nematodes, the absence of basal plates, combined with the presence of a ring of microtubular doublets instead of triplets and one or more central singlets in these short segments are features normally associated with true cilia r a t h e r th an basal bodies. Therefore, in our study we have used the term "cilia" to refer to the transitional zones t hat exist between nerve axons and their anterior extensions. A discussion of the function of these cilia, which may act as kinocilia, is presented in Sect. II.
C. Cilia of the Lateral Sector (Accessory Cilia) In the past, an amphidial nerve was thought to consist of axons t hat undergo structural modification and enter the amphidial canal. However, recent studies of several tylenchids indicate t hat the amphidial nerve consists of axons which divide into two groups (9, 17). One group, consisting of seven axons, gives rise to %i1iary processes" t hat innervate the amphid; but the other "processes" remain outside and ventral to the amphidial canal. Two groups of amphidial ~dendrites" were described by Baldwin and Hirschmann in M.
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incognita (2) and in H. glycines (3). One group extends from the amphidial nerve into the canal. However, the other group of ~dendrites" either terminates in the amphidial gland or ascends and innervates other areas of the head including certain papillae. Similar divisions apparently occur in Necator americanus (24), C. elegans (36), a n d N . brasiliensis (42). In our examination of second stage larvae of M. incognita, three to five accessory cilia arising from axons of the amphidial nerve were consistently observed laterally to the amphidial canal. However, these cilia, which may correspond to the ciliary processes, dendrites or axons reported in the other studies, do not innervate either the cephalic or the labial papillae. Alternatively, they ascend into the lateral cephalic sectors, become flattened, and terminate. If this interpretation is correct, these accessory cilia could serve as tactoreceptors, thereby making the amphids structurally and functionally more complex than previously thought.
D. Amphidial Duct The amphidial duct is a well-established component of the amphid; however, it has only recently received attention in electron microscopic studies. In M. incognita males, the duct, which has been described as the lumen of the amphidial canal, contains the distal ends of the nerve processes and extends from above the ciliary regions of the dendrites to the orifice of the canal (2). In N. americanus, the term ~duct" also includes the area around the basal-
FIG. 14. Amphids in cross section. At this level, the amphidial axons are no longer enclosed by the gland. Within the group of microvilli, a cilium can be observed (arrow). This structure apparently is not associated with either the cilia t h a t enter t h e canal or the accessory cilia. It does not extend above the tips of the microvilli and terminates posteriorly in the nerve process. × 38 000. FIG. 15. An amphid in cross section illustrating the center of the nerve process. The process contains numerous mitochondria (M), as well as a ring of microtubular doublets, which represents the base of a cilium shown in Fig. 14. The nerve process and duct are bounded by the amphidial gland. The amphidial axons, which are grouped in the left portion of the micrograph, have an irregular outline, contain numerous mitochondria and microtubules, and are not encircled by the amphidial gland. This amphid is included in the illustration shown in Fig. 2d. × 40 000.
FIG. 16. Longitudinal section through a portion of an amphid. A short cilium (C), which terminates in the center of the process, lies among the anterior microvilli. The continuity of an axon (arrow) can be traced to a ciliary segment and finally to its anterior extension. × 22 000. 272
FIG. 17. Longitudinal section t h r o u g h a few axons and t h e i r anterior ciliary segments. Microtubular doublets (upper arrows) end and a rootlet (R) begins at the t e r m i n a l portion of the axon. This region is also characterized by the presence of vesicles, resembling synaptic vesicles, t h a t lie along the rootlet and by t i g h t iunctions (lower arrows), which interconnect the m e m b r a n e s of adjacent axons. × 72 000. 273
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body-like region of the cilia and the termi- from the bases of these cilia into the antenal portions of the axons (24). In addition, rior portions of their respective axons. invaginations, called collecting ducts, ex- These regions of the axons are intercontend into the amphidial gland. These col- nected with tight junctions. Thus, the lecting ducts observed by McLaren (24) in short ciliary segments, having a structure N. americanus apparently correspond to closely resembling kinocilia, are free to the amphidial pouch described by Baldwin undulate along their length, but are apand Hirschmann (2) in M. incognita. In parently anchored by rootlets extending our studies of second stage larvae of M. into axons that are tightly interconnected incognita, the lumen of the amphidial by tight junctions. These features would be canal is continuous with and indistin- expected and required for kinocilia. Such guishable from an amphidial pouch. Con- movement by the ciliary segments could sequently the ~duct" refers to an intercel- convey materials through the amphidial lular space surrounding the anterior re- canal between the amphidial duct and the gion of the axons, the nerve process, cilia external surface of the nematode. Future and their modified anterior extensions. studies must demonstrate the presence of The duct, which has no membranes or or- subtubular arms and ATPase activity in ganelles, contains homogeneous granular amphidial cilia in order to support this material. However, secretory activity that hypothesis. would account for the origin of this material has not been observed in the amphid- B. Nerve Process ial gland during our study. The microvilli, which extend from the nerve process in M. incognita, are strucII. AMPHIDIAL FUNCTION turally similar to the numerous processes A. Cilia found on various epithelial cells whose The term "cilia" has been widely used to principal function is absorption (11). Ifmidesignate the anterior extensions of the crovilli functioned similarly in the nerve axons that ascend the amphidial canal. process, they would allow the nerve However, in our examination of M. incog- axon(s), which are continuous with the nita, only the 1 t~m transitional segments process, to absorb material from the amlocated between the axons and their ante- phidial duct. By acting in this manner, an rior extensions have a structure similar to axon could effectively monitor the secrethat of true cilia, which are characterized tions produced by the amphidial gland. by a 9 + 2 axoneme with dynein arms on This possible function is incorporated into subtubules a. The amphidial segments de- the following model. viate from kinocilia only in the arrangement of microtubules in the axoneme and C. Possible Functional Model For many years, the amphids were bein the confirmed presence of arms on subtubules a. lieved to be chemosensory organs because In addition to the structural similarity they were innervated by structures that between kinocilia and the ciliary seg- were exposed to the exterior environment ments, the location of rootlets and junc- through a pore in the cuticle. Recent retions in the latter may be functionally sig- ports, summarized by Croll (8) and Mcnificant. The cilia, which lie below the Laren (25), indicate that nematodes reamphidial canal, are unconfined along spond to numerous stimuli including CO2 their length and are loosely surrounded by gradients, oxidizing and reducing agents, the granular material in the amphidial amino acids, specific anions and cations, duct. Furthermore, rootlets, whose func- basic pH, cyclic AMP, temperature gration is generally thought to anchor, extend dients, and light. The amphids are the
NEMATODE NEUROSENSORY ORGAN
organs believed to play a role in the perception of these stimuli. In addition to its role as a sensory receptor, investigators of several species indicate that the amphid may also have a secretory function (3, 24, 35). In our study of the amphid, the presence of a gland, a duct with granular contents, and electron dense material near the cuticular opening, suggests that in second stage larvae this organ may be involved in secretion. The mechanism controlling amphidial function is not fully understood. However, McLaren (24) has recently proposed that the anterior extension of the cilium, together with the axon, constitute a receptor-effector cell. In her model, the anterior extension would initially have a sensory function, detecting changes in the external environment. Its axon would have a motor function and could activate secretory activity in the gland. The sensory cilia could then monitor the output of secretions and the motor apparatus would regulate the secretory activity. In our study, the presence of the accessory cilia and a highly developed nerve process, which is associated with at least one axon of the amphidial nerve, suggests possible modifications in this model. Namely, one or more of the cilia could function as chemoreceptors as proposed by McLaren; but the accessory cilia, which terminate in the cephalic sectors, might function also as tactoreceptors. Thus, either chemical or physical excitation could stimulate secretion by the amphidial gland. Secretions by the gland would pass over the microvilli of the nerve process on their way to the base of the duct. The nerve process could then act as a monitor of the secretory activity of the gland. Upon reaching the lumen of the duct, undulations by the cilia could move secretions upward through the amphidial canal. Thus, the amphid could be envisioned as a sensory organ capable of secreting closely monitored materials in response to chemical and tactile stimuli. Perhaps future studies on the ultrastruc-
275
ture of the amphid during the parasitic stage of the nematode and on the chemical nature of amphidial secretions will enable us to clarify the structure and function of this organ. The authors are grateful to Mrs. Sharon A. Ochs for providing technical assistance and to Mr. Robert B. Ewing for illustrating Figs. 1 and 2. REFERENCES 1. BALDWIN, J. G., AND HIRSCHMANN, H., J. Nematol. 4, 219 (1972). 2. BALDWIN, J. G., AND HIRSCHMANN, H., J. Nematol. 5, 285 (1973). 3. BALDWIN, J. G., AND HIRSCHMANN, H., J. Nematol. 7, 40 (1975). 4. BIRD, A. F., The Structure of Nematodes, 318 pp. Academic Press, New York, 1971. 5. BURR, A. H., AND BURR, C., J. Ultrastruct. Res. 51, 1 (1975). 6. CHITWOOD, B. G., AND CHITWOOD, M. B., A n Introduction to Nematology, Sect. I, 213 pp. Monumental Printing, Baltimore, Md., 1950. 7. CoBs, N. A., J. Wash. Acad. Sci. 3, 145 (1913). 8. CROLL, N. A., The Behavior of Nematodes, 117 pp. St. Martin's Press, New York, 1970. 9. DE GRISSE, A. W., LIPPENS, P. L., AND COOMANS, A., Nematologica 20, 88 (1974). 10. ENDO, B. Y., AND WERGIN, W. P., Protoplasma 78, 365 (1973). 11. FAWCETT, D. W., The Cell, 448 pp. Saunders, Philadelphia, 1966. 12. FREY-WYSSLING, A., AND MI)HLETHALER, K., U1trastructural Plant Cytology, 377 pp. Elsevier, New York, 1965. 13. FULTON, C., in REINERT, J. (Ed.), Origin and Continuity of Cell Organelles, Centrioles, p. 170. Springer-Verlag, New York, 1971. 14. HIRUMI, H., AND CHEN, T., Phytopathology 58, 1053 (1968). 15. KOZEK, W. J., J. Parasitol. 54, 838 (1968). 16. LIPPENS, P. L., COOMANS, A., DE GRISSE, A. T., AND LAGASSE,A., Nematologica 20, 242 (1974). 17. LIPPENS, P. L., AND DE GRISSE, A. T., Biol. Jb. Dodonaea 41, 147 (1973). 18. L6PEz-ABELLA, D., JIMI~NEz-MILL.AN~ F., AND GARCIA-HIDALGO, F., Nematologica 13, 283 (1967). 19. MCLAREN, D. J., Trans. Roy. Soc. Trop. Med. Hyg. 63, 290 (1969). 20. MCLAREN, D. J., Trans. Roy. Soc. Trop. Med. Hyg. 64, 191 (1970). 21. MCLAREN, D. J., Parasitology 65, 317 (1972). 22. McLAREN, D. J., Parasitology 65, 507 (1972). 23. McLAREN, D. J., Proc. Int. Congr. Parasitol. B~ 3rd 447 (1974).
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24. MCLAREN,D. J., Int. J. Parasitol. 4, 25 (1974). 25. MCLAREN, D. J., in DAWES, B. (Ed.), Advances in Parasitology, Vol. 14. Academic Press, London, in press. 26. MCLAREN, D. J., BURT, J. S., AND OGILVIE, B. M., Int. J. Parasitol. 4, 39 (1974). 27. NOVIKOFF,A. B., AND HOLTZMAN,E., Cells and Organelles, 337 pp. Holt, Rinehard, & Winston, 1970. 28. POINAR, G. O., AND LEUTENEGGER, R., J. Parasitol. 54, 340 (1968). 29. RASKI, D. J., JONES, N. O., AND ROGGEN, D. R., Proc. Helminth. Soc. Wash. 36, 106 (1969). 30. ROGGEN,D. R., RASKI, D. J., AND JONES, N. O., Science 152, 515 (1966). 31. ROGGEN,D. R., RASKI, D. J., AND JONES, N. O., Nematologica 13, 1 (1967). 32. Ross, M. M. R., Science 156, 1494 (1967). 33. SPURR, A. R., J. Ultrastruct. Res. 26, 31 (1969).
34. STORCH, V., AND RIEMANN, F., Z. Morph. Tiere 74, 163 (1973), 35. THORSON,R. E., J. Parasitol. 42, 26 (1956). 36. WARD, S., THOMSON, N., WHITE, J. G., AND BRENNER, S., J. Comp, Neurol. 160, 313 (1975). 37. WARE, R. W., CLARK, D., CROSSLAND,K., AND RUSSELL, n. L., J. Comp. Neurol. 162, 71 (1975). 38. WERGIN, W. P,, AND ENDO, B. Y., Proc. Int. Congr. Plant Pathol., 2nd No. 0208 (1973). 39. WERGIN, W. P., AND ENDO, B. Y., Proc. Int. Congr. Parasitol. B, 3rd 444 (1974). 40. WRIGHT, K. A., Proc. Int. Congr. Parasitol. B, 3rd 446 (1974). 41. WRIGHT,K. A., Canad. J. Zool. 52, 1207 (1974). 42. WRIGHT, K. A., Canad. J. Zool. 53, 1131 (1975). 43. YUEN, P. H., Canad. J. Zool. 45, 1019 (1967). 44. YUEN, P. n . , Nematologica 14, 554 (1968).
56, 258-276 (1976)
Ultrastructure of a Neurosensory Organ in a Root-Knot Nematode WILLIAM P. WERGIN AND BURTON Y. ENDO Nematology Laboratory, Plant Protection Institute, USDA, A R S Beltsville Agricultural Research Center, Northeastern Region, BeltsviIle, Maryland 20705 Received December 29, 1975, and in revised form, May 25, 1976 Second stage larvae of the root-knot nematode, Meloidogyne incognita [(Kofoid and White) Chitwood], were examined to elucidate the u l t r a s t r u c t u r e of the amphids, which are paired lateral organs believed to function in chemosensory perception. The amphids were found to consist of supportive, nervous, and secretory tissues. The support tissue forms two cuticular canals and encircling "supporting cells," which extend from the external anterior region of the nematode posteriorly into the body of the larva. Each amphid is innervated by a group of axons t h a t become modified into cilia. Generally, seven of these cilia enter the amphidial canal and t e r m i n a t e n e a r the external pore; whereas, t h r e e to five "accessory" cilia r e m a i n external to the canal and t e r m i n a t e b e n e a t h the cuticle at the cephalic region. The secretory structures of the amphid consist of an amphidial "gland," a large irregularly shaped cell containing mitochondria, and a duct, an intercellular area h a v i n g a g r a n u l a r content. The physical associations and the functional significance of these structures are discussed.
Paired lateral organs on the lip region of nematodes were initially described in 1865 by Bastian using a light microscope (25). Later, the organs were named "amphids" by Cobb (7) who found that these structures were widely distributed in the phylum Nematoda and concluded that they probably functioned in sensory perception. By 1950, the results of numerous light microscopic studies had established that an amphid consisted of a group of ascending nerve fibers that entered a large cell termed the amphidial gland. Anterior to the gland, the fibers became less closely associated with one another in a region known as the amphidial pouch. Specialized ends of the fibers, called terminals, continued to penetrate the anterior portion of the nematode through a cuticular channel (6). Because the ends of the terminals, which were collectively called sensillae, were exposed to the outer environment of the nematode, the amphid was believed to function in chemosensory perception. Electron microscopic studies of the amphid have revealed the ultrastructure of the nerve fibers, which are referred to as the amphidial nerves (28), neurons (36,
37), dendritic nerve processes (14, 32), dendrites (1-3, 5, 16, 30, 31, 34, 38-42), and axons (19-24, 43, 44). The terminals are reported as neurons (36, 37); sensillar dendrites (31), dendritic processes (2, 30, 40-42), modified cilia (19-24, 32), ciliary elements (29), ciliary processes (9, 16, 17), cilia-like processes (1), and cilia (3--5, 14, 15, 18, 28, 30, 34, 38, 39, 43, 44). The postulated chemosensory function of amphids has received further support due to the structural similarities that exist between these structures in nematodes and chemosensory organs described in many insects. Recent investigations have documented the presence of accessory cilia (2, 3, 5, 9, 17, 24, 36, 38-42) and nerve processes (4, 22-24, 36, 38, 39, 42) associated with the amphid. Preliminary observations in our laboratory indicated that the accessory cilia originated from the amphidial nerve and that a highly developed nerve process has continuity with at least one axon of the nerve. Because these structures may provide additional insight into the functional role of the amphid, a detailed examination of this organ was undertaken in
258 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
NEMATODE NEUROSENSORY ORGAN
the plant-parasitic nematode, Meloidogyne incognita [(Kofoid and White Chitwood]. MATERIALS AND METHODS Second stage larvae of Meloidogyne zncognita were obtained from egg masses collected from roots of infected tomato plants grown in a greenhouse soil bed. The larvae either were used to infect clover roots, or were mixed with w a r m molten 2% w a t e r agar, which t h e n was allowed to solidify, previously reported procedures were used to inoculate roots and to prepare tissues for transmission electron microscopic e x a m i n a t i o n (10). Briefly, the solidified a g a r t h a t contained nematodes was diced into 2- to 3-ram cubes and transferred to glass vials containing 3% glutaraldehyde in 0.05 M phosphate buffer, at 22°C, for chemical fixation of the larvae. Rinsing and postfixation in osmium tetroxide were also carried out in 0.05 M phosphate buffer (pH 6.8). Fixation for 1.5 h r was followed by washing in six changes of buffer over a period of 1 hr. The agar blocks t h e n were postfixed in 2% osmium tetroxide for 2 hr, dehydrated in an acetone series, and infiltrated with a low viscosity m e d i u m (33). Silver-gray sections of selected nematodes were cut on a Sorvall MT-2 ultramicrotome with a diamond knife and mounted on uncoated 75 x 300 mesh copper grids. The sections were stained with 2% aqueous uranyl acetate (10 min), t h e n with lead citrate (5 min). Thin sections were viewed in a Hitachi HU-11C operating at 75 kV with a 30/*m objective aperture, or a Philips 200 or 301 electron microscope operating at 60 kV with 20 b~m apertures. Larvae were prepared for scanning electron microscopic e x a m i n a t i o n by chemically fixing with 3% glutaraldehyde in 0.05 M phosphate buffer for 1.5 hr, at 22°C, d e h y d r a t i n g in a n ethanol series and critically point drying. The larvae were mounted on a l u m i n u m studs, coated with carbon t h a t was followed by gold-palladium and viewed with a Hitachi HHS-2R scanning electron microscope operating at 15 or 20 kV. RESULTS
The amphid is a structurally complex organ associated with supportive, nervous, and secretory tissues (Figs. 1 and 2). The following results are organized to correspond to these associations. I. SUPPORTIVE TISSUES
A. Cuticle An examination of second stage larvae
259
with the scanning electron microscope reveals the amphids as two lateral pits in the cephalic region of the nematode. Electron opaque material is generally present within and external to the pits (Fig. 3). However, when this material is not present, measurements obtained with stereomicroscopy indicate that pits are 0.2/~m deep and appear as elongate openings 0.8 x 0.2 /~m (Fig. 4). Transmission electron microscopy of cross sections through the nematode, beginning at the bottom of the pits and proceeding posteriorly, shows the amphids as slit-like canals (Figs. 5 and 6) that gradually become rounded (Figs. 7-9) and extend about 12/~m below the anterior surface. The canals are limited by a tapered, circular sheath of amorphous material that is apparently continuous with the external cuticle of the nematode. This sheath delineates the amphidial canal, which constitutes the anterior segment of the amphid.
B. ~Supporting Cell" An elongate tubular cell, previously designated %upporting cell" by McLaren (22) encircles the amphidial canal (Figs. 8 and 9). The membranes of the encircling cell meet or are apposed, distolaterally to the canal, forming a tight junction (arrows, Figs. 8 and 9). This membrane configuration spans the length of the supporting cell, which extends from the base of the amphidial canal anteriorly to the cephalic framework. Cellular or membranous continuity between the supporting cell and the hypodermal tissue, which underlies the cephalic framework, could not be ascertained. No cellular organelles have been observed in the supporting cell, although microtubules and membrane-bounded vesicles having granular contents are frequently observed (Figs. 8 and 9). A more loosely aggregated granular material, not confined within vesicles, generally occurs also throughout the length of the cell.
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Median -- Nerve
phageat i!
t - -
I
AmphidialNerve B u n d l e s - - J
b
e
FIGs. 1-17. The sequence of figures begins at the anterior portions of t h e larvae and proceeds posteriorly into the nematodes. This sequence does not precisely parallel the organization presented in the text; however, we believe t h a t this sequential a r r a n g e m e n t will assist the reader in visualizing the i n t e r n a l structures of the amphid and in relating t h e i r complex associations. FIG. 1. A n illustration of a second stage l a r v a of M. incognita (a). Larvae are generally 400 tLm long; however, our study has concentrated on the anterior 20 t~m region illustrated in the dorsal (b) and sublateral (c) views. This region, containing t h e neurosensory organs or amphids, is innervated by nerves e m a n a t i n g from the nerve ring (a). In the text, Figs. 5 to 17 are a sequential series of micrographs t h r o u g h the amphidial regions illustrated in b and c. II. NERVOUS TISSUE
A. Axons The function of the amphid has not been experimentally determined. Whether an innervating element of this organ receives or transmits nerve impulses remains unknown. As a result, previous investigators have applied the terms "axon" and "dendrite" to the neural element. In our study, the element is designated an axon because this term conforms to the terminology used in a recent review (25) on sensory organs. The amphids are innervated by nervous tissue, which could be traced to the nerve ring of the nematode. In cross sections above the ring, two amphidial nerves, each consisting of a bundle of axons, lie
dorsolaterally (Fig. 1). The axons, which are irregularly shaped in cross sections, vary in diameter from 0.1 to 0.6 t~m but gradually become constricted and circular as they ascend and enter the base of the amphid (Figs. 2, 10-14). Each axon is limited by a membrane that frequently has a peripheral layer of granular material along its inner surface (Figs. 13 and 14). In addition to the granul ar material, the axons contain numerous mitochondria and longitudinally oriented microtubules (Figs. 2 and 16). Occasionally, particles resembling ribosomes can also be observed in these ascending structures.
B. Nerve Process Approximately 10/zm from the anterior end of the nematode, a highly convoluted
261
NEMATODE NEUROSENSORY ORGAN
Accessow Cili~
Amphidial C~n~l
o Junction
M;er~v;ll;
Axons a
Amphidial Gland~ Fro. 2. Illustrations of longitudinal and cross sections t h r o u g h the amphid. The longitudinal view (a) is a simplified model i l l u s t r a t i n g continuity between the axons and t h e i r ciliary extensions. Accessory cilia, which ascend into the lateral sector of the cephalic region, are represented by the two cilia on the left. Three cilia are shown ascending the amphidial canal. The p a r e n t a l axon from one of these cilia is continuous with the nerve process. Drawings b, c, and d correspond to the mierographs in Figs. 9, 13, and 15, respectively. These illustrations correlate the spatial relationships between supporting, nervous, and secretory tissues
shown in cross sections with those illustrated in the longitudinal model (a). membrane process is associated with each bundle of ascending axons (Figs. 10-16). This process, which lies adjacent and dorsal to the axons, is about 10 tLm long and measures 2 to 3 ~m in diameter. It consists of tubular projections (Fig. 16) and one or more cilia (Figs. 15 and 16), all of which are delineated by a limiting membrane. The tubular projections resemble microvilli. They extend anteriorly and posteriorly 2 to 4 ~m from the center of the process (Fig. 16). As m a n y as 200 projections may lie parallel to one another in a densely packed arrangement (Figs. 12-14).
The projections are about 850/~ in diameter and contain several microfilaments (neurofilaments). One or more short cilia also extend outward from the center of the process (Fig. 16). These short segments, which lie central and parallel to the microvilli, can be easily distinguished from the latter by their larger diameters and characteristic arrangement of microtubules. Occasionally, the center of the process also contains rootlets, which are associated with the cilia, and mitochondria that are several times larger than those normally present in the axons.
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FIGS. 3 and 4. Scanning electron micrographs of the heads of second stage larvae. Electron opaque material (arrows, Fig. 3) is generally associated with and obscures the view of the amphidial pits. When this m a t e r i a l is absent (Fig. 4), stereo e x a m i n a t i o n reveals the pit (arrow) as an elongate opening, 0.8 x 0.2 ~m, t h a t is about 0.2 ~ m deep. Figures 3 and 4, x 20 000.
Careful examination of cross and longitudinal sections has failed to reveal the precise structural relationship t h a t exists between the nerve process and the ascending axons of the amphidial nerve. However, membrane continuity has been established between the process and at least one of the amphidial axons (Fig. 16).
C. Transitional Region between Axons and Their Ciliary Extensions Above the nerve process, the axons become modified into cilia t h a t extend toward the anterior region of the amphid (Figs. 2 and 17). The membranes of the axons are continuous with those of the cilia; however, the transitional regions between the two structures are characterized by the presence of tight junctions, vesicles and rootlets (Figs. 12-14, 17). The tight junctions, which appear as electron dense ~collars" around the terminal part of each
axon (arrows, Fig. 13), are formed from the limiting membrane of an axon and t h a t from either the adjacent nerve or the amphidial gland. Internally, this region of the axon contains numerous vesicles, resembling synaptic vesicles, t h a t frequently lie around a central rootlet (Fig. 17). The rootlet is an elongate fibrillar structure t h a t exhibits a cross-banding pattern with a major repeat distance of about 350/~. Immediately above the rootlets are the ascending cilia of the axons (Figs. 1, 2, 1012, 17). These cilia contain a peripheral ring of 6 to 8 microtubular doublets and 0 to 5 single inner microtubules t h a t are about 1 t~m in length. They separate the posterior parent axons from the anterior extensions of cilia which are more loosely organized and ascend toward the amphidial canal. Although the presence of distinct dynein arms has not been confirmed
FIG. 5. Cross section of the amphidial region of the nematode n e a r the bases of the lateral amphidial pits (P). Illustrated are the hexaradiate pattern of the cephalic framework, a ring of six labial sensory canals around a central stylet and four outer cephalic sensory organs (arrows). × 38 000. FIG. 6. Cross section through the anterior portion of an amphidial canal. At this level, the amphid (A) appears as a slit-like structure. Above the amphid lie the terminal portions of several accessory cilia (arrows) having sparse granular contents and an occasional microtubule. Two cephalic sensory organs (C) can be observed at the left and right of the amphidial slit. z 50 000. 263
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NEMATODE NEUROSENSORY ORGAN
265
granular material in addition to the randomly spaced microtubules, which become less numerous in the anterior parts of these extensions (Figs. 7 and 8). The granD. Anterior Extensions of Cilia ular material tends to accumulate along The cilia, which arise from the axons, the inner surface of the limiting memmaintain their characteristic structure for branes of the modified cilia. about 1 t~m. Then their diameters enlarge 2. Cilia of the lateral sector (accessory 2- to 3-fold, the peripheral rings of micro- cilia). Generally, three to five accessory tubular doublets are lost and 4 to 12 ran- cilia, which lie lateral and parallel to the domly spaced, single microtubules appear amphidial canal, ascend toward the lateral and ascend these ciliary extensions. These cephalic sector (Figs. 7-9) and terminate structural features easily distinguish the beneath the hypodermis. The internal anterior extensions from the 1 t~m bases of structure of the cilia of the amphidial the cilia. canal and that of the accessory cilia are Each lateral bundle of cilia divides into similar. However, unlike the former, actwo distinct groups (Figs. 10 and 11). One cessory cilia are not closely associated in a group, generally consisting of seven cilia, symmetrical pattern and do not maintain enters and ascends the amphidial canal. a tubular shape along their length. As The second group, usually consisting of these cilia ascend, they become flattened three to five cilia, ascends externally and into broad sheath-like structures that conlaterally to the canal toward the lateral form to the constricting space that lies becephalic sector (Figs. 1 and 2). tween the amphidial canal and the outer 1. Cilia of the amphidial canal. Cross cuticle of the nematode. sections along the amphidial canal reveal that the ascending cilia are arranged in a III. SECRETORY TISSUE symmetrical hexaradiate pattern (Fig. 9). This pattern consists of six cilia equidis- A. Amphidial '~Gland" tantly spaced around a central cilium. The The term ~amphidial gland" was introcilia lose this symmetrical pattern, become duced by light microscopists to describe bulbous and terminate below the pit of the the cell that encircles the innervating eleamphidial opening (Figs. 1, 7, 8). ments of the amphid. In M. incognita, this The cilia contain sparsely scattered "gland" apparently corresponds to a large
on the subtubules, the general appearance of these amphidial cilia resembles that commonly observed in kinocilia.
FIGS. 7 and 8. Successive cross sections t h r o u g h the amphidial canal illustrating the tips of several cilia. The t e r m i n a l portions of the amphidial cilia become slightly bulbous at t h e i r ends and t e r m i n a t e at different levels near the anterior region of the canal. The amphidial canal is encircled by a '~supporting cell" whose m e m b r a n e forms a t i g h t junction (arrows) lateral to the canal. An anterior extension of the amphidial gland, which lies between the canal and the hypodermis (H), contains three flattened accessory cilia (C) h a v i n g microtubular singlets and doublets. The outer m e m b r a n e s t h a t appear around these modified cilia are actually continuous with the i n n e r m e m b r a n e of the amphidial gland. Figure 7, x 52 000; Fig. 8, x 52 000. FIG. 9. Two amphids of a nematode in a cross section t h r o u g h the midregion of the amphidial canal. Each canal contains seven cilia a r r a n g e d in a symmetrical pattern. Near the midregion, each cilium generally h a s 4 to 12 longitudinally oriented microtubules t h a t do not appear in any organized pattern. The amphidial canals are surrounded by supporting cells h a v i n g vesicles with g r a n u l a r contents. A t i g h t junction, formed by the apposing m e m b r a n e s of this cell, is indicated by the arrows. In addition to these structures, portions of the a m p h i d i a l glands, each containing three accessory cilia (C), can be observed. The accessory cilia are more flattened t h a n those found in the canal and frequently exhibit microtubular doublets at this level. Protractor muscles (P) of the stylet (S) lie between the lateral amphids. The amphid in the left portion of the micrograph has been illustrated in Fig. 2b. x 30 000.
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NEMATODE NEUROSENSORY ORGAN
267
J FIG. 12. Cross section t h r o u g h the nematode illustrating the large area occupied by the amphids, which consist of the glands, ducts, and nerve tissues (cilia, axons, and nerve processes). The amphid in the lower portion of the micrograph contains several cilia, as well as t e r m i n a l portions of two axons (arrows). The r e m a i n i n g amphid consists of the axons except for a single cilium at the upper right. Approximately 200 microvilli are associated with each of the amphids, x 20 000. FIGS. 10 and 11. Successive cross sections of the amphid near the base of the amphidial canal. The canal (C) has apparently t e r m i n a t e d along the lower portion illustrated in Fig. 10 (arrows), and is no longer found in Fig. 11. The cilia present in the canal (Fig. 10) were sectioned above the ciliary segments shown in Fig. 11. These segments, which resemble true cilia, have an outer ring of 5 to 9 microtubular doublets and 0 to 6 i n n e r singlets. Four accessory cilia (AC), present in the amphidial gland (Fig. 10) merge with those from the canal (Fig.- 11). Numerous microvilli extending upward from the nerve process lie to the r i g h t of the cilia. Two types of m e m b r a n e configurations are associated with the microvilli. Concentric circles (arrows, left) result when the m e m b r a n e s of the microvilli are closely appressed to the inner m e m b r a n e of the amphidial gland. Alternatively, single m e m b r a n e bound microvilli appear when a group (G) collectively "invaginates into" the gland. In this case, each microvillus is limited by its own m e m b r a n e but the group is bounded by the i n n e r m e m b r a n e of the amphidial gland. G r a n u l a r m a t e r i a l of the duct can be observed around the cilia and the microvilli. Figures 10 and 11 x 42 000.
FIG. 13. Cross section of an amphid illustrated in Fig. 12. Tight junctions (arrows), which form from the apposition of adjacent membranes, can be observed around two of the axons ( u p p e r left). The terminal portion of an axon contains an occasional mitochondrion (M), longitudinally oriented microtubules, peripheral granular material and a central rootlet (R), which descends from the ciliary segment. Microfilaments or neurofilaments are present in each of the microvilli. This amphid is included in the illustration in Fig. 2c. × 52 000. 268
NEMATODE NEUROSENSORY ORGAN
irregularly shaped cell that extends from the anterior region of the amphidial nerve upward toward the lateral cephalic sector (Fig. 2). The cell encircles the cilia, the nerve process, and the accessory cilia (Figs. 10-13). The gland contains numerous mitochondria; however, neither nuclei nor other cellular organelles were encountered in this cell. Although the amphidial gland encircles the nerve process, its membrane m a y or may not tightly appose that of the microvillar projections. As a result, two types of membrane configurations can be observed in cross sections (Figs. 2, 10-15). When the two membranes are tightly apposed, a double membrane appears around each microvillus; the inner one is continuous with that of the nerve process, while the outer membrane belongs to the amphidial gland. When the membranes are not tightly apposed, a group of microvilli appear to project into the lumen of the gland. Cross sections of this association show a group of small circular membranes, delineating the microvilli, which are collectively enclosed by the membrane of the amphidial gland.
B. Amphidial Duct The amphidial duct consists of the intercellular space that lies between the membranes of the amphidial gland and those of either the nerve process, the amphidial axons or the amphidial cilia (Figs. 2, 1115). When these membranes are closely apposed, such as along the accessory cilia, the duct is constricted. However, the duct constitutes a significant dilated area, having granular contents, around the cilia and the nerve process. Although this granular material is accessible to the lumen of the amphidial canal, the extent to which it is secreted is not readily apparent. DISCUSSION I. AMPHIDIAL STRUCTURE
Our general observations relating to the innervation of the amphids by cilia that
269
arise from the amphidial axons are similar to conclusions reported by other investigators. However, our results and interpretations relating to the nerve process, amphidial cilia, accessory cilia, and the amphidial duct differ in several respects from those previously reported.
A. Nerve Process Microvillar processes, termed ~'nerve processes," were initially illustrated by Bird (4). These processes extend distally from a nerve bulb in the amphidial pouch. Electron microscopic studies by Baldwin and Hirschmann reported a similar microvillous gland in males of Meloidogyne
incognita (2)andHeterodera glycines (3). However, these reports concluded that the microvilli were extensions of the amphidial gland and were physically distinct from the amphidial nerve bundle. In our study, the microvilli are neither a part of nor continuous with the amphidial gland. Alternatively, the microvilli, constitute processes whose membranes exhibit continuity with that of at least one, and possibly more, axons of the amphidial nerve. Therefore, the microvillous gland described by Baldwin and Hirschmann (2, 3), probably corresponds to the nerve process ofM. incognita described in our study. This conclusion parallels that of McLaren (22, 24) Ward et al. (36), and Wright (42) who have recently demonstrated nerve processes extending from the amphidial nerve axons in filaria (22), strongyles (24), Caenorhabditis elegans (36), and
Nippostrongylus brasiliensis (42). B. Cilia A basal body is a centriolar pinwheel that bears a cilium or a flagellum (13). It generally consists of a distal basal plate, a cylindrical wall, which is composed of nine groups of microtubular triplets, and a set of fibers or rootlets that extends into the subjacent cell (12, 13, 27). The term %asal body" has been used to describe the terminal regions of the amphidial cilia in nema-
270
WERGIN AND ENDO
NEMATODE NEUROSENSORY ORGAN
todes (14, 16, 30, 36, 41, 42). However, in these studies, one or more of the features generally associated with basal bodies are modified or absent. For example, in all cases the microtubular wall consists of a ring of doublets. Basal plates (16) and rootlets (36, 42) have been noted in only a few of these studies. Although the t e r m "basal body" has been used in investigations of sensory cilia in nematodes, the absence of basal plates, combined with the presence of a ring of microtubular doublets instead of triplets and one or more central singlets in these short segments are features normally associated with true cilia r a t h e r th an basal bodies. Therefore, in our study we have used the term "cilia" to refer to the transitional zones t hat exist between nerve axons and their anterior extensions. A discussion of the function of these cilia, which may act as kinocilia, is presented in Sect. II.
C. Cilia of the Lateral Sector (Accessory Cilia) In the past, an amphidial nerve was thought to consist of axons t hat undergo structural modification and enter the amphidial canal. However, recent studies of several tylenchids indicate t hat the amphidial nerve consists of axons which divide into two groups (9, 17). One group, consisting of seven axons, gives rise to %i1iary processes" t hat innervate the amphid; but the other "processes" remain outside and ventral to the amphidial canal. Two groups of amphidial ~dendrites" were described by Baldwin and Hirschmann in M.
271
incognita (2) and in H. glycines (3). One group extends from the amphidial nerve into the canal. However, the other group of ~dendrites" either terminates in the amphidial gland or ascends and innervates other areas of the head including certain papillae. Similar divisions apparently occur in Necator americanus (24), C. elegans (36), a n d N . brasiliensis (42). In our examination of second stage larvae of M. incognita, three to five accessory cilia arising from axons of the amphidial nerve were consistently observed laterally to the amphidial canal. However, these cilia, which may correspond to the ciliary processes, dendrites or axons reported in the other studies, do not innervate either the cephalic or the labial papillae. Alternatively, they ascend into the lateral cephalic sectors, become flattened, and terminate. If this interpretation is correct, these accessory cilia could serve as tactoreceptors, thereby making the amphids structurally and functionally more complex than previously thought.
D. Amphidial Duct The amphidial duct is a well-established component of the amphid; however, it has only recently received attention in electron microscopic studies. In M. incognita males, the duct, which has been described as the lumen of the amphidial canal, contains the distal ends of the nerve processes and extends from above the ciliary regions of the dendrites to the orifice of the canal (2). In N. americanus, the term ~duct" also includes the area around the basal-
FIG. 14. Amphids in cross section. At this level, the amphidial axons are no longer enclosed by the gland. Within the group of microvilli, a cilium can be observed (arrow). This structure apparently is not associated with either the cilia t h a t enter t h e canal or the accessory cilia. It does not extend above the tips of the microvilli and terminates posteriorly in the nerve process. × 38 000. FIG. 15. An amphid in cross section illustrating the center of the nerve process. The process contains numerous mitochondria (M), as well as a ring of microtubular doublets, which represents the base of a cilium shown in Fig. 14. The nerve process and duct are bounded by the amphidial gland. The amphidial axons, which are grouped in the left portion of the micrograph, have an irregular outline, contain numerous mitochondria and microtubules, and are not encircled by the amphidial gland. This amphid is included in the illustration shown in Fig. 2d. × 40 000.
FIG. 16. Longitudinal section through a portion of an amphid. A short cilium (C), which terminates in the center of the process, lies among the anterior microvilli. The continuity of an axon (arrow) can be traced to a ciliary segment and finally to its anterior extension. × 22 000. 272
FIG. 17. Longitudinal section t h r o u g h a few axons and t h e i r anterior ciliary segments. Microtubular doublets (upper arrows) end and a rootlet (R) begins at the t e r m i n a l portion of the axon. This region is also characterized by the presence of vesicles, resembling synaptic vesicles, t h a t lie along the rootlet and by t i g h t iunctions (lower arrows), which interconnect the m e m b r a n e s of adjacent axons. × 72 000. 273
274
WERGIN AND ENDO
body-like region of the cilia and the termi- from the bases of these cilia into the antenal portions of the axons (24). In addition, rior portions of their respective axons. invaginations, called collecting ducts, ex- These regions of the axons are intercontend into the amphidial gland. These col- nected with tight junctions. Thus, the lecting ducts observed by McLaren (24) in short ciliary segments, having a structure N. americanus apparently correspond to closely resembling kinocilia, are free to the amphidial pouch described by Baldwin undulate along their length, but are apand Hirschmann (2) in M. incognita. In parently anchored by rootlets extending our studies of second stage larvae of M. into axons that are tightly interconnected incognita, the lumen of the amphidial by tight junctions. These features would be canal is continuous with and indistin- expected and required for kinocilia. Such guishable from an amphidial pouch. Con- movement by the ciliary segments could sequently the ~duct" refers to an intercel- convey materials through the amphidial lular space surrounding the anterior re- canal between the amphidial duct and the gion of the axons, the nerve process, cilia external surface of the nematode. Future and their modified anterior extensions. studies must demonstrate the presence of The duct, which has no membranes or or- subtubular arms and ATPase activity in ganelles, contains homogeneous granular amphidial cilia in order to support this material. However, secretory activity that hypothesis. would account for the origin of this material has not been observed in the amphid- B. Nerve Process ial gland during our study. The microvilli, which extend from the nerve process in M. incognita, are strucII. AMPHIDIAL FUNCTION turally similar to the numerous processes A. Cilia found on various epithelial cells whose The term "cilia" has been widely used to principal function is absorption (11). Ifmidesignate the anterior extensions of the crovilli functioned similarly in the nerve axons that ascend the amphidial canal. process, they would allow the nerve However, in our examination of M. incog- axon(s), which are continuous with the nita, only the 1 t~m transitional segments process, to absorb material from the amlocated between the axons and their ante- phidial duct. By acting in this manner, an rior extensions have a structure similar to axon could effectively monitor the secrethat of true cilia, which are characterized tions produced by the amphidial gland. by a 9 + 2 axoneme with dynein arms on This possible function is incorporated into subtubules a. The amphidial segments de- the following model. viate from kinocilia only in the arrangement of microtubules in the axoneme and C. Possible Functional Model For many years, the amphids were bein the confirmed presence of arms on subtubules a. lieved to be chemosensory organs because In addition to the structural similarity they were innervated by structures that between kinocilia and the ciliary seg- were exposed to the exterior environment ments, the location of rootlets and junc- through a pore in the cuticle. Recent retions in the latter may be functionally sig- ports, summarized by Croll (8) and Mcnificant. The cilia, which lie below the Laren (25), indicate that nematodes reamphidial canal, are unconfined along spond to numerous stimuli including CO2 their length and are loosely surrounded by gradients, oxidizing and reducing agents, the granular material in the amphidial amino acids, specific anions and cations, duct. Furthermore, rootlets, whose func- basic pH, cyclic AMP, temperature gration is generally thought to anchor, extend dients, and light. The amphids are the
NEMATODE NEUROSENSORY ORGAN
organs believed to play a role in the perception of these stimuli. In addition to its role as a sensory receptor, investigators of several species indicate that the amphid may also have a secretory function (3, 24, 35). In our study of the amphid, the presence of a gland, a duct with granular contents, and electron dense material near the cuticular opening, suggests that in second stage larvae this organ may be involved in secretion. The mechanism controlling amphidial function is not fully understood. However, McLaren (24) has recently proposed that the anterior extension of the cilium, together with the axon, constitute a receptor-effector cell. In her model, the anterior extension would initially have a sensory function, detecting changes in the external environment. Its axon would have a motor function and could activate secretory activity in the gland. The sensory cilia could then monitor the output of secretions and the motor apparatus would regulate the secretory activity. In our study, the presence of the accessory cilia and a highly developed nerve process, which is associated with at least one axon of the amphidial nerve, suggests possible modifications in this model. Namely, one or more of the cilia could function as chemoreceptors as proposed by McLaren; but the accessory cilia, which terminate in the cephalic sectors, might function also as tactoreceptors. Thus, either chemical or physical excitation could stimulate secretion by the amphidial gland. Secretions by the gland would pass over the microvilli of the nerve process on their way to the base of the duct. The nerve process could then act as a monitor of the secretory activity of the gland. Upon reaching the lumen of the duct, undulations by the cilia could move secretions upward through the amphidial canal. Thus, the amphid could be envisioned as a sensory organ capable of secreting closely monitored materials in response to chemical and tactile stimuli. Perhaps future studies on the ultrastruc-
275
ture of the amphid during the parasitic stage of the nematode and on the chemical nature of amphidial secretions will enable us to clarify the structure and function of this organ. The authors are grateful to Mrs. Sharon A. Ochs for providing technical assistance and to Mr. Robert B. Ewing for illustrating Figs. 1 and 2. REFERENCES 1. BALDWIN, J. G., AND HIRSCHMANN, H., J. Nematol. 4, 219 (1972). 2. BALDWIN, J. G., AND HIRSCHMANN, H., J. Nematol. 5, 285 (1973). 3. BALDWIN, J. G., AND HIRSCHMANN, H., J. Nematol. 7, 40 (1975). 4. BIRD, A. F., The Structure of Nematodes, 318 pp. Academic Press, New York, 1971. 5. BURR, A. H., AND BURR, C., J. Ultrastruct. Res. 51, 1 (1975). 6. CHITWOOD, B. G., AND CHITWOOD, M. B., A n Introduction to Nematology, Sect. I, 213 pp. Monumental Printing, Baltimore, Md., 1950. 7. CoBs, N. A., J. Wash. Acad. Sci. 3, 145 (1913). 8. CROLL, N. A., The Behavior of Nematodes, 117 pp. St. Martin's Press, New York, 1970. 9. DE GRISSE, A. W., LIPPENS, P. L., AND COOMANS, A., Nematologica 20, 88 (1974). 10. ENDO, B. Y., AND WERGIN, W. P., Protoplasma 78, 365 (1973). 11. FAWCETT, D. W., The Cell, 448 pp. Saunders, Philadelphia, 1966. 12. FREY-WYSSLING, A., AND MI)HLETHALER, K., U1trastructural Plant Cytology, 377 pp. Elsevier, New York, 1965. 13. FULTON, C., in REINERT, J. (Ed.), Origin and Continuity of Cell Organelles, Centrioles, p. 170. Springer-Verlag, New York, 1971. 14. HIRUMI, H., AND CHEN, T., Phytopathology 58, 1053 (1968). 15. KOZEK, W. J., J. Parasitol. 54, 838 (1968). 16. LIPPENS, P. L., COOMANS, A., DE GRISSE, A. T., AND LAGASSE,A., Nematologica 20, 242 (1974). 17. LIPPENS, P. L., AND DE GRISSE, A. T., Biol. Jb. Dodonaea 41, 147 (1973). 18. L6PEz-ABELLA, D., JIMI~NEz-MILL.AN~ F., AND GARCIA-HIDALGO, F., Nematologica 13, 283 (1967). 19. MCLAREN, D. J., Trans. Roy. Soc. Trop. Med. Hyg. 63, 290 (1969). 20. MCLAREN, D. J., Trans. Roy. Soc. Trop. Med. Hyg. 64, 191 (1970). 21. MCLAREN, D. J., Parasitology 65, 317 (1972). 22. McLAREN, D. J., Parasitology 65, 507 (1972). 23. McLAREN, D. J., Proc. Int. Congr. Parasitol. B~ 3rd 447 (1974).
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24. MCLAREN,D. J., Int. J. Parasitol. 4, 25 (1974). 25. MCLAREN, D. J., in DAWES, B. (Ed.), Advances in Parasitology, Vol. 14. Academic Press, London, in press. 26. MCLAREN, D. J., BURT, J. S., AND OGILVIE, B. M., Int. J. Parasitol. 4, 39 (1974). 27. NOVIKOFF,A. B., AND HOLTZMAN,E., Cells and Organelles, 337 pp. Holt, Rinehard, & Winston, 1970. 28. POINAR, G. O., AND LEUTENEGGER, R., J. Parasitol. 54, 340 (1968). 29. RASKI, D. J., JONES, N. O., AND ROGGEN, D. R., Proc. Helminth. Soc. Wash. 36, 106 (1969). 30. ROGGEN,D. R., RASKI, D. J., AND JONES, N. O., Science 152, 515 (1966). 31. ROGGEN,D. R., RASKI, D. J., AND JONES, N. O., Nematologica 13, 1 (1967). 32. Ross, M. M. R., Science 156, 1494 (1967). 33. SPURR, A. R., J. Ultrastruct. Res. 26, 31 (1969).
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