Oltrastructure of Phasmid Development in Meloidodera Floridensis and M

Journal of Nematology 22(3):362-385. 1990. © The Society of Nematologists 1990. Oltrastructure of Phasmid Development in Meloidodera floridensis and M. charis (Heteroderinae) 1 L. K. CARTA2 AND J. G. BALDWIN3

Abstract: Phylogenetic characters for Heteroderinae Luc. et al., 1988 are evaluated in Meloidodera which is believed to have primarily ancestral characters. Phasmid uhrastructure is observed in second-stage juveniles (J2), third-stage juvenile males, fourth-stage juvenile males, and fifth-stage males of Meloidodera floridensis and M. charis. Phasmid secretion occurs inside the egg before the J1-J2 molt. Before J2 hatch, concentric lamellar membranes occur within the sheath and socket cells. Some membranes become lamellae of the sheath cell plasma membrane; others become multilamellar bodies. During early molting, plasma membrane lamellae disappear and a distal dendrite segment appears in a rudimentary canal. After the molt, the distal dendrite is not present within the canal. The phylogenetic utility of phasmid features is discussed. In both species the ampulla shape and size between molts are stable features in juveniles and males. The posthatch J2 sheath cell receptor cavity may vary in a species specific manner, but comparative morphology requires precise timing after hatch. Key words: end apparatus, Heteroderinae, male development, Meloidoderafloridensis, Meloidodera charis, molting, multilamellar body, neuromorphology, phasmid, phylogeny, secretion, uhrastructure.

Phasmid sensory organs have useful stood if phasmid characters are tobe used taxonomic characters in many secernen- in an updated phylogenetic analysis. There tean nematodes (42) including the Heter- have been insufficient reliable characters oderinae Luc et al., 1988 (Heteroderidae to determine a phylogenetic pattern for sensu lato) (30). In a cladistic analysis of the Heteroderinae, which contains numer- the Heteroderinae, a question arose about ous new genera (2,3). the structure of the phasmid. Although the A few free-living, animal-parasitic and character states of the phasmid opening plant-parasitic nematode phasmids have had an apparent phylogenetic polarity of been examined at the uhrastructural level lens shape to pore shape, one species with (1,5,9,31,33,44,47). Phasmids have a cu- a pore had been grouped with otherwise ticle-lined opening that may be differen- similar species having a lens (14,15). This tiated into an ampulla, which is usually oc- problem was resolved when it was dem- cluded by a plug of electron-dense material onstrated with transmission electron mi- (Fig. 1). The ampulla leads to a narrow croscopy (TEM) that the so-called pore- cuticular canal contained within one or two shaped phasmid opening of Meloidodera socket cells which are developmentally de- charis Hopper, 1960 was really a lens sim- rived from the hypodermis and are con- ilar to, but smaller than, that of M. flori- nected to the hypodermis and the basal densis Chitwood et al., 1956 (2). The range region of the sheath cell by intercellular of variability needs to be completely under- junctions. These junctions have been described as tight junctions or belt desmo- Received for publication 4 August 1988. somes (38). The socket cells also surround l This research was supported in part by the National Sci- ence Foundation grant BSR-84-15627 and represents a part portions of the sheath cell. The sheath cell of the first author's Ph.D. dissertation. Mention of a product secretes an electron-dense substance with- or trade name does not constitute an endorsement of that product. in its extracellular receptor cavity which 2 Research Fellow, Division of Biology, California Institute loosely surrounds the distal ends of one or of Technology, Pasadena, CA 91125. s Associate Professor, Department of Nematology, Uni- two ciliary dendrites. From the sheath cell versity of California, Riverside, CA 92521. receptor cavity, the dendrites pass ante- The authors thank D. T. Kaplan, USDA, Orlando, Florida, riorly within the tightly surrounding arms for supplying cultures ofMeloidoderafloridensis, H. T. Simte, University of California, Riverside, for technical assistance of sheath cell cytoplasm; they are delin- with one of the many tested fixation procedures, and M. A. McClure, University of Arizona, Tucson, for access to un- eated from the cytoplasm by intercellular published research on Meloidodera charis. junctions. The distal region of the neuron 362 Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 363 !NI

F| A B Fro. 1. Schematic diagram of phasmids. A) Heterodera schachtii. B) Caenorhabditis elegans. N = neuron, P1 = ampullar plug, R = receptor cavity, Sh = sheath cell, So = socket cell, arrows = intercellular junctions. may terminate at the base of the receptor velopmental stages in M. charis to confirm cavity or extend into the canal. Phasmid its lens-like form and to test its homology neuron synapses were observed in Caeno- or nonhomology with pore-like phasmid rhabditis elegans Maupas, 1899 (48). character states. One currently debated test The phasmid is believed to be a che- of homology employs the rule that more moreceptor in contact with the external specialized characters occur late in devel- environment, despite the electron-dense opment. Therefore adults, including males, plug reported in most species. Although tend to have the most derived and there- developmentally related to the amphid (20, fore useful characters in a phylogenetic 21), its specific functions are still uncertain. analysis (2). Detailed morphology of this Because of this functional uncertainty, se- phasmid may provide a basis for comparing lection pressures for possible convergent the phasmids in Heterodera, Verutus, and evolution and phylogenetic reversals of its other genera where they appear to be par- features are difficult to evaluate in alter- tially or completely lost (8,35). native phylogenetic trees. The relative degree of development of the phasmid is as- MATERIALS AND METHODS sessed here in different stages to provide Meloidodera floridensis, the type species, possible functional insights. was chosen as a model for other species in In this study, the ampulla shape is also the genus because males will develop in examined in greater detail through the de- water without a host plant (21,45). Meloi- 364 Journal of Nematology, Volume 22, No. 3, July 1990 doderafloridensis was cultured in the green- for a phasmid study of Heterodera schachtii house on slash pine, Pinus elliotii. Feeder Schmidt, 1871 juveniles (1), and the other roots were placed in a mist chamber and was a modification of a method used for C. second-stage juveniles (J2) were collected e{egans (6). This modification included use every 2 to 3 days over a period of 3 weeks. of embedding capsule baskets with 25-#m These J2 were kept in tap water in Stender nylon mesh for transfers (12), 0.2 M cac- dishes for 2 months at 21.5-24.5 C and odylate buffer at 60 rather than 70 C, and transferred weekly to clean water. During puncture of the body after 1 hour of fix- this time a high proportion of the popu- ation with an eyeknife or electrochemically lation developed into immature third-stage sharpened tungsten needle (22). males (M3), immature fourth-stage males Specimens were embedded in 2% water (M4), and mature adult males (M5) as a agar, which was cut into blocks for proper direct result of starvation-induced male sex orientation, dehydrated in an acetone se- determination. Intermediate M3 and M4 ries, and infiltrated with Spurr's epoxy. Se- stages could be discerned by counting cu- rial sections were cut with a Sorvall 6000 ticles (21,45) and selecting individuals with ultramicrotome. Silver sections were placed the most distinct stylets. These stages were on formvar-coated 200-mesh hexagonal also selected from soil sievings. Individuals grids stained with uranyl acetate for 30 still in the molt phase were identified with minutes and Reynolds lead citrate for 5 the TEM for assessing the sequence of minutes. Specimens were viewed with a Hi- molting events by changes in body wall tachi H-600 transmission electron micro- morphology (4,43). scope at 75 kV. Meloidodera charis was collected from wild Voucher specimens of J2 and M5 are peonies, Peonia cal~ornica, in Badger Can- deposited in the University of California, yon, San Bernardino, California. Feeder Riverside Nematode Collection (UCRNC). root pieces were placed under mist for 5 days; males were selected and fixed 2 days RESULTS later. An Arizona population was grown Second-stage juvenile and adult male phas- on pinto bean, Phaseolus vulgaris (17), and raids: The phasmid is similar in form but J2 and early M5 stages were dissected from becomes smaller from the J2 to the M5 the roots. The M5 was identified by the male of both M. floridensis and M. charis presence of rudimentary spicules. First- (Figs. 2, 3). Height of a typical M5 phasmid stage juveniles (J1) and early J2 stages of from the ampulla to the top of the neuron the Arizona population were released from nucleus and diameter at the level of the the egg with a needle and identified by receptor cavity are about 60% of those of stylet morphology and degree of intestinal the J2. development. The J1 within the egg had The sheath cell of the phasmid is bound- obscure stylets and when released from the ed on the lateral side by the hypodermal egg, the J1 body did not completely seam cell; this boundary is indicated by straighten. Unfixed specimens, unlike fixed small intercellular junctions (Fig. 4A'). On specimens, swelled noticeably after about either side of the seam cell are two socket 1 hour. cells that wrap half way around the sheath Phasmids of at least three specimens from cell and meet near the middle of the in- each stage were examined. Seven individ- testinal side of the sheath cell (Fig. 4A). uals of various stages were observed during Nuclei of these socket cells may lie on the the molt. Ten specimens each of J2 and inner side of the sheath cell (Fig. 4A) or to M5 males were used to characterize and one side of the sheath and seam cells when reconstruct phasmids by photographing extensive lipid occurs in the intestine. cross sections and longitudinal sections at Canals of the phasmids on each side of intervals of 2-6 sections. Two methods of the body meet their sheath cell receptor fixation were employed; one had been used cavities on the subdorsal side of the body Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 365

A,C,D 1pm B lOpm

LF FIG. 2. Reconstruction ofMeloidoderafloridensisJ2phasmids. A) Phasmid from lateral view including ciliary neuron (N) with microtubular zones. Am = ampulla, Ca = canal, Nu = nucleus, R = receptor cavity, Sh = sheath cell, Sot = socket cell 1, So~ = socket cell 2, arrows = intercellular junctions. B) Size and position of lens-shaped ampulla (Am) relative to lateral view of entire tail. Partial cross section at level of ampulla. C) Cross section of sheath cell (Sh). D) Three dimensional view from lateral field (LF) of canal (Ca) and the lens- shaped ampulla (Am) including cross section through ampulla.

(Fig. 5E). The sheath cell is surrounded by by the sheath cell cytoplasm also enclose two socket cells for much of its length. In- the enlarged dendrite base (Figs. 2, 3, 7D, tercellular junctions delineate the base of 10B). The neuron nucleus lies just anterior the sheath cell and socket cells (Figs. 2, 3, and lateral to the sheath cell nucleus. In 10B). Intercellular junctions surrounded the M5, the sheath cell nucleus lies be- 366 Journal of Nematology, Volume 22, No. 3,July 1990

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1 pm Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 367 tween the body wall muscles and the spic- parent pseudocoelom which is most evi- ule retractor and protractor muscles. dent at this time in development (Fig. 6A). Characteristics of the phasmid and body wall The basal lamina is adjacent to the inner during molting: During the early, early- lateral side of the narrow sheath cell. middle, late-middle, and late molting pe- In the late-middle molt, the old cuticle riods, a general pattern of development of begins to dissolve, and differentiation of parts of the phasmid is evident within the layers begins in the new cuticle. The neu- various stages. Early molt is characterized ron and canal may be turned 90 degrees by degradation of phasmid features and from their usual position (Fig. 7A). The muscles, early-middle molt by cuticular neuron at this time has rootlets in the en- separation from the enlarged interchordal larged dendrite base, and the distal seg- hypodermis, late-middle molt by hypo- ment of the dendrite is irregularly shaped dermal vesiculation and the beginning of and contains vesicles. cuticle layering and old cuticle resorption, During the late molt, newly differentiat- and late molt by hypodermal shrinkage and ed cuticle undergoes maturation of its lay- cuticular maturation. ers. A M. charis J1 within the egg during During the very early molt after root the late molt can be used to describe phas- penetration, smooth endoplasmic reticu- mid secretion characteristics. These secre- lum (SER) in M. charis J2 becomes promi- tions are also visible in slightly later molt nent in the hypodermis and in the degen- J2 individuals of both species. erating sheath cell (Fig. 5D). The receptor In late embryo-early J1, all of the sur- cavity secretion is present, but sheath cell faces of the socket and sheath cells are lamellae are degenerated. A small portion bounded by intercellular (probably tight) of the dendrite apparently buds into the junctions. The receptor cavity and neuron receptor cavity. There is no evidence of a of the J 1 are completely formed. The lin- canal or ampulla. ing of the wide canal and ampulla, how- During cellular enlargement of the ear- ever, is not completely deposited (Fig. ly-middle molt, the cuticle is completely 5A). A dark secretion occurs from the re- separated from the hypodermis and the ceptor cavity to the canal, ampulla, and outer layer of the new external cortex ap- around the external cuticle. A secretion of pears on the outer edge of the hypodermis. similar texture and density is continuous There is no evidence of a secreted elec- from the rectum to the body wall cuticle tron-dense plug. A dense inclusion body is as well. This secretion also occurs in pre- characteristically present adjacent to the hatch J2 of both species. The more mature socket cell nucleus (Fig. 6B). A distal seg- premolt cuticle of these J2 was an indica- ment of the dendrite, which may show sin- tion that the secretion probably had dis- glet microtubules (Fig. 6A', C, D), exists sipated from the outer cuticle. during the molt but is absent in stages after Ampulla: All fully developed stages have molts. At this time the exceptionally large an ampullar opening with a cuticular lining hypodermal cells contain extensive rough containing a dark plug. This internal cu- endoplasmic reticulum and some Golgi ticular lining is continuous with the outer complexes (Fig. 6B). The basal lamina as- cuticle. During certain periods of molting, sociated with muscle fields outlines the ap- there is no ampullar opening or plug. For

4---- FIG. 3. Reconstructionof Meloidoderafloridensis M5 phasmid. A) Phasmid from dorso-ventral view. Am = ampulla, Ca = canal, N = neuron, Nu = nucleus, R = receptor cavity, Sh = sheath cell, Sot = socket cell 1, So2 = socket cell 2, arrows = intercellularjunctions. B-E correspond to cross sections as follows: B) Neuronal region above neuron-sheathjunction. C) Lamellar-anastomosisdendrite zone of neuron (N) with vesicles (V) and receptor cavity (R). D) Transition zone of neuron. E) Middle segment of neuron. F) Longitudinalview of infolded membranes (IM) of socket cell (So) surrounding neuron at the molt. Ca = canal, N = neuron. 368 Journal of Nematology, Volume 22, No. 3, July 1990 example, the early molt postpenetration M. 9B). Ampullae in the M5 of both species charis J2 loses the old ampulla and canal, are smaller than in the J2. The lens shape and only the old neuron with its poorly becomes somewhat elongated, forming a formed receptor cavity remains (Fig. 5D). more rhomboid opening (Fig. 9B). In M. floriclensis and probably in M. charis, Canal and socket cells: The posterior ends middle molt M3, M4, and M5 have new of all socket cells form intercellular junc- canals and neurons, but no openings to the tions with the adjacent hypodermal and exterior. Late moltJ 1 and J2 of both species sheath cells. The canal of J2 of both species have rudimentary ampullar openings where is large and crescent shaped in cross section the cuticle is being deposited and the elec- (Fig. 5E). The M3 has an unusually short tron-dense plug is accumulating (Fig. 8D) canal, relative to the J2, and the M5 has to form the ampulla of the next juvenile the longest, narrowest canal (Figs. 2, 3). stage (Fig. 8E). Instead of the socket cell Late molt canals are wider than the canals directly depositing the ampullar cuticle, after the molt. vesicles from the active hypodermis (Fig. In prehatch M. floridensisJ2, both socket 8D) and possibly some plug secretion (Fig. cells are filled with double intracellular 8E) appear to form the ampullar cuticle. membranes. The socket cells are larger The final plug often contains a dark, crys- than in the posthatch J2. Near the junc- talline differentiation on its outer surface tional complexes defining the canal within (Fig. 8E). the socket cell, secretions associated with Ampullae of post-hatch M. floridensis J2 intracellular double membranes accumu- in cross section have the shape of a large late around the periphery of the canal. lens (Figs. 2, 8E, 9B). The incompletely These electron-dense secretions are closely formed ampullae of prehatch M. charis J2 associated with the developing cuticle dur- are approximately the size and shape of the ing this late phase of the molt (Fig. 8A). In large posthatch lens of M.floridensisJ2 (Fig. fully developed stages, dark material sim-

-----4 FIG. 4. Meloidoderafloridensis J2 phasmid cross sections. A) Posthatch J2 with two socket cell nuclei (SON1 and SoN~). From lateral view, socket cell 1 is adjacent to right side of hypodermal seam cell (Sc). Socket cell 1 encircles ampulla and lower canal in counterclockwise direction. Socket cell 2 surrounds upper canal and sheath cell in clockwise direction. G = glycogen, L = lipid, arrows = intercellular junctions. Inset A') Inter- cellular junctions of seam cell (arrows). B) Multivesicular body (MVB) and multilamellar body (MLB), adjacent to socket cell nucleus (SON).

FIG. 5. Meloidodera charis and M. floridensis canal and receptor cavity cross sections. A) M. charis J 1 with incompletely formed ampulla (Am). Dots indicate boundary between sheath cell (Sh) and receptor cavity (RC). Ca = canal, Cu = outer cuticle, SE = secretion, So = socket cell, SC = Seam cell. B) Prehatch J2 at base of sheath cell receptor cavity (R). DN = distal neuron, EA = end apparatus of canal, L = lamellae, P = pocket. C) Prehatch J2. Oval neuron region (N) with vesicles (V) near junction with sheath cell, L = lamellae, P = pocket, R = receptor cavity. D) Postpenetration J2. Neuron (N) with bud (arrow) in receptor cavity (R) of degenerating sheath cell (Sh). SER = smooth endoplasmic reticulum. E)M. floridensisJ2, crescent-shaped canal (Ca) extending from socket cell on right (So) to sheath cell (Sh) on left. Do = dorsal side.

FIG. 6. Meloidodera floridensis middle molt, phasmid cross sections. A) M2 molt. Interior storage region (In) containing lipid bounded by basal lamina (arrow) continuous with left subventral muscle field (LSVM). H = interchordal hypodermis, N = neuron, SC = seam cell, Sh = sheath cell, So = socket cell. Inset A') Ciliary distal zone of neuron (N). B) M3 molt. Sheath cell (Sh) with receptor cavity (R) and neuron (N) surrounded by socket cell (So) containing dense body (DB). Hypodermal seam cell (SC) containing circular Golgi complex (G). RER = rough endoplasmic reticulum. C) M2 molt. Socket cell (So) infolded on itself with intercellular junctions (arrow) forming a star-shape. Star-shaped junctions surround electron-dense double membrane of canal (Ca). Distal ciliary neuron region (N) and vesicles (V) within the canal. D) M3 molt. Socket cell (So) encircles distal neuron (N). Intercellular junctions (arrow) of infolded membranes of socket cell surround canal membrane (Ca). M = microtubules. Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 369 370 Journal of Nematology, Volume 22, No. 3, July 1990

N Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 371 372 Journal of Nematology, Volume 22, No. 3, July 1990 Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 373 ilar to the secretion during the molt also the transition zone of the dendrite (Fig. lines the cellular side of the canal cuticle 3D, E). Doublets of the transition zone of and ampulla. the cilium contain 13 + 11 fibrils. A ciliary In the J2 just after hatch, the cytoplasm segment close to the region where a basal of the socket cells appears to contain pri- body would be expected to occur contains marily glycogen and lipid (Fig. 4A). After eight doublets with tenuous connections to molts, the socket cells degenerate in all seven singlets (Fig. 7D). The middle seg- stages. A typical example is an aged M5 ment membrane of the cilium has eight specimen with a socket cell largely defined lobes (Fig. 3E). As the neuron approaches by the remnants of the surrounding inter- its junctional connection with the sheath cellular junctions and with little cellular cell, it becomes triangular to oval with cen- material around the distal base of the canal tral and lateral vesicles (Figs. 3C, 5C). (Fig. 7C). Often a dark deposit, similar to Above the tight junctions in the distal den- the substance of the ampulla plugs, is found drite base, vacuoles are usually found in at the base of this aging cell. intermolt dendrites (Fig. 10). At middle During middle molts, before any cuticle molt, the distal dendrite base contains or ampullar opening exists in any stage, a rootlets (Fig. 7A). rudimentary canal defined by a double During all observed molts, an extended membrane is visible (Figs. 3F, 6C, D). This dendrite is seen within the canal (Figs. 6C, double membrane is situated surprisingly D; 7A). The shape of this distal zone is close to the phasmid opening. The socket irregular, appearing to be in the process cell folds in on itself in the longitudinal of vesiculation (Figs. 6C, 7A). Above this plane (Fig. 6C, D). Horizontally the socket irregular region, the dendrite region in the cell forms a doughnut shape around the lower receptor cavity contains single mi- canal with a seam readily identified by an crotubules (Fig. 6A'). These distal regions intracellular junction. Microtubules are are absent in postmolt neurons. Then the visible on close inspection in the outer re- dendrite terminates at the beginning of the gion of the socket cell (Fig. 6D). The canal receptor cavity, just inside the end appa- contains the distal segment of the neuron ratus of the canal (Fig. 5B). In one male, for most of its length (Figs. 6C, D; 7A). determined to be a young M5 on the basis A cuticular extension of the canal exists of spicule maturity, the middle ciliary seg- at the beginning of the sheath cell receptor ment of the dendrite still extended slightly cavity. This canal extension or end appa- into the end apparatus of the canal. (Fig. ratus (Fig. 5B) appears to merge with a 12). sheath cell lamella before the cuticle is Receptor cavity and sheath cell: Phasmids completely laid down in prehatch individ- of prehatch J2 of M. floridensis are ex- uals. tremely large in cross section (Fig. 8B, C). Neuron: Dendritic ciliary regions in these The cross sections of sheath cells at the phasmids contain eight microtubular dou- level of the receptor cavities comprise an blets and 2-6 central singlets. This pattern unusually large (nearly 75%) area o£ the occurs between the middle segment and tail. Sheath cells have enormous numbers

Fro. 7. Meloidoderafloridensis and M. charis cross sections of phasmid. A) M. floridensis, M4 molt. Dendrite shifted 90 degrees from intermolt position, resulting in longitudinal orientation. Rootlet region (Rt) above ciliary base (CB). Distal ciliary region (DN) with neurotubules and vesicles. Intercellular junctions (arrow) separate sheath cell (Sh) from socket cell (So). Ca = canal, R = receptor cavity, V = vesicle. B) M. charis M5. Neuron (N) and receptor cavity (R) with multivesicle (MV). C) M. charis M5. Phasmid with neuron (N) and degenerate socket cells (So) and sheath cells (Sh) containing dense deposits. D) M. charis M5. Receptor cavity (R) with neuron and intercellular junctions (arrows). Neuron is near level of transition zone close to basal body and includes eight microtubule doublets (Db) and eight singlets. 374 Journal of Nematology, Volume 22, No. 3, July 1990 Phasmid Ultrastructure in Meloidodera sp.: Carta, 15aldwin 375 of closely apposed lamellar membranes as- 1 pm sociated with a secretion that is especially dense at the inflated inner tips (Fig. 8A- C). These tips surround a large receptor cavity that is circular below the level of the a b neuron (Fig. 8B). Near the level of the transition zone of the neuron, the receptor cavity is broadly fusiform (Fig. 8C). Membranes are in various forms of differentiation. At some points in development double membranes are essentially d A perpendicular to the long axis of the cell, 1 pm whereas at other points of development they are nearly parallel or circular. Some double membranes have distinct extracellular space between them, whereas others do not. Concentric membranes identical to a b those on the periphery of the sheath cell exist in a thin row of socket cell nuclei separating the two sheath cells (Fig. 8C). Concentric membranes are regularly spaced between the more-or-less perpendicular lamellae extending to the periphery of the cell. These concentric mem- 1t branes are occasionally expelled into the FIG. 9. Comparison of reconstructed receptor receptor cavity (Fig. 8B). The average size cavities and ampullae of Meloidoderafloridensis and M. of a membrane infolding is 0.03 t~m wide ch.aris. A) Receptor cavity (cross section) of M. charis x 0.15 ~m long in M. floridensis; in pre- J2 (a) and M5 (b) and iV/.floridensisJ2 (c) and M5 (d). Cellular details shown in Figures 2C and 5. B) Large hatch M. charis the infoldings surrounding lens ampulla (longitudinal section) of M. floridensis J2 the circular receptor cavity are slightly (a) in relation to elongated rhomboid ampulla in M5 thicker (0.04 x 0.13 #m) (Fig. 5B, C). Some (b). Small lens ampulla ofM. charisJ2. It is enlarged infoldings anastomose with others at var- in the prehatched J2 (c) and becomes reduced after hatch (d). ious points including the tips (Fig. 5B). Anastomosing lamellae may lie adjacent to the neuron (Fig. 5C) or adjacent to the cretion extends from the receptor cavity internal terminus of the canal at the re- through the wide canal and accumulates ceptor cavity (Fig. 5B). This canal region most densely at the outer rim of the large is also called the end apparatus. ampulla. Anastomosed lamellae lie adja- Characteristic oval, inflated pockets may cent to the end apparatus where the secre- contain secretions (Fig. 5B, C). These tion is more condensed than in the recep- pockets sometimes contain concentric tor cavity (Fig. 5B). membranes or vesicles. The membrane se- In the posthatch J2 in cross section, the

b-- FIG. 8. Meloidoderafloridensis J2 phasmid cross sections. A) Prehatch J2 canal (Ca) surrounded by dense secretion (arrows) from intracellular lamellar membranes (ILM) in socket cell (So). R = Receptor cavity. Inset A') Secretion concentrated at periphery of canal. B) Lamellar membranes (LM) and secretions (S) from sheath cell (Sh) in receptor cavity (R). C) Prehatch J2 receptor cavity (R). CM = concentric membranes, DN = distal neuron, SoN = socket cell nucleus, Sh = sheath cell. D) Prehatch J2 plug material (P1) filling ampullar (Am) region. Cuticular matrix (Cu) is deposited by hypodermis (H). E) Posthatch J2 ampulla (Am) with amorphous plug (Pl) and dark outer crystals (Cr). 376 Journal of Nematology, Volume 22, No. 3, July 1990

FIG. 10. Longitudinal sections of M5 phasmids. A) Meloidodera charis. B) Meloidoderafloridensis. Am = ampulla, Ca = canal, N = neuron, R = receptor cavity, Sh = sheath cell, So = socket cell, V = vacuole, arrows = intercellular junctions.

receptor cavity at certain levels may appear charis has a shorter Y-shaped cavity than slit-like or fusiform, but the cavity is essen- M. floridensis (Fig. 9A). tially Y shaped at the level of the ciliary Freshly extracted M. floridensis J2 show transition zone (Fig. 11). The Y has a slight some variation in form of sheath cell mem- inflation at the branch point. Meloidodera branes. Membranes of the lamellae are Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 377 continuous with the plasma membrane and closely packed together in M.floridensis than inflate to varying degrees (Fig. 11). The in M. charis (Figs. 7B, 10A, B). vesicles of the sheath cell are very small when double membranes are so closely apposed that lamellae are not formed be- DISCUSSION tween them (Fig. 11B, C). Another form The approximately 35 specimens ob- of sheath cell has highly inflated interla- served in this study mostly included the mellar spaces (Figs. 8D, E; llA) and con- readily accessible J2 and M5. Even within tains relatively large multivesicles, multi- these stages, minor variability occurred in lamellae, lamellar anastomoses, and dense degree of natural degradation, apparently cytoplasm. associated with age of nematode and phas- From the previous descriptions, an in- mids; however, the number of specimens ferred sequence of events can be summa- is sufficient to infer developmental trends rized for both species. Before hatching, and morphologically unique features. The membranes of lamellae are numerous and M3 and M4 stages and molts, while rep- may be either closed or inflated near the resented by relatively few specimens, have receptor cavity. Inflation is greatest just been essential to understand the overall before and after hatch. After hatch, the developmental sequence indicated by more receptor cavity flattens in on itself and pe- comprehensive observations in J2 and M5. ripheral membranes undergo vesiculation Phasmids in all the stages of Meloidodera and lysis. Lamellae anastomose at their tips, should be compared with other heterod- forming a smooth boundary to the recep- erine genera, including those in which the tor cavity. Inside the cell, reclosed mem- phasmid may be partially or completely lost branes form successive internal vacuoles. in adults (8,35). By the beginning of the molt, the inner Ampulla: The ampulla is the most stable membranes have been transformed or dis- feature of the phasmid in all stages. As- integrated. pects of both ampullar form as well as am- Four specimens were observed in the pullar plug content will be carefully con- ephemeral fourth stage. Only toward the sidered for phylogenetic value. Results of end of the molt, before the cuticle had dif- this study indicate that the cuticle-like ma- ferentiated, was the phasmid visible with trix surrounding the ampulla is formed at the neuron extending into the canal. In the the end of the molt from secretions of the other specimens where the cuticle was dif- hypodermis rather than the socket cell. ferentiated, no phasmid was apparent. The large lens-shaped ampulla ofM. flo- The early fifth-stage male has a sheath ridensis is smaller in later developmental cell with especially long lamellae (Fig. 12). stages, as well as after the molt in a given Some of these lamellae radiate around the stage. For instance, the smaller lens of M. large end apparatus which encloses the be- charis is considerably larger before hatch- ginning of the neuron at the junction of ing and appears similar in size and shape the canal and receptor cavity. Most adult to the ampulla of posthatch M. floridensis. male phasmids have a degenerated and Thus the ampulla of M. floridensis J2 may vacuolated sheath cell. The sheath cell and represent a developmentally earlier form receptor cavity of the adult are smaller than ofphasmid than that ofM. charis. A similar those of the J2 (Fig. 9a). The lamellae are phenomenon occurs among species of fi- generally perpendicular to the long axis of larial nematodes regarding the degree of the receptor cavity in cross section. Some- differentiation of cells present in the first- times, however, the lamellae appear stage microfilaria (24). roughly parallel to the sides of the oval or It is important to distinguish between Y-shaped receptor cavity (Fig. 7B). Occa- ampulla shape and ampulla size. Through sionally large multivesicles are released into development in these species, the opening this cavity. Lamellae are thinner and more becomes smaller, but its essential shape re- 378 Journal of Nematology, Volume 22, No. 3, July 1990

\ ~~• ~• r~ c Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 379 mains constant. The rhomboid ampulla of chemotaxonomy, as waxes are in insects M5 is basically an elongated lens shape. (46). It has been noted that the size of the Timing of the deposition of the plug se- ampulla is correlated with the relative sur- cretion on the surface of Meloidodera cu- face area of the membranes of sheath cell ticle at the end of the molt and timing of lamellae. In future studies, ampulla size may wax deposition from insect glands are sim- serve as a predictive index of lamellar ilar (49). Besides affecting cuticular per- membrane surface area in other phasmids. meability, lipids and waxes are believed to An understanding of the function of the protect insects from fungal attack (29). If lamellar surface may be helpful in future the phasmid, which is specific to Secernen- studies of the possible adaptive benefit of tea, definitively could be shown to secrete relatively large phasmid ampullae. lipid and wax onto the cuticle, aided per- AmpuUar plug content: Because of the re- haps by the trough-like lateral incisures ad- lease of lamellar membranes from the jacent to the ampulla, it would be consis- sheath cell into the receptor cavity, it is tent with Paramonov's hypothesis that soil possible that lipoproteins are components fungi present a selection pressure for rel- of the ampullar plug and the cuticle gly- atively impermeable cuticles in terrestrial cocalyx. In insects, lipoprotein deposits in secernentean nematodes (36). the pores of chemosensilla are believed to The secretion seen on the outer cuticle act as hydrophobic filters for lipid-soluble and in the ampulla of the first stage is prob- substances (18). Acetone aids in the pen- ably a common feature at the end of all etration of water-soluble precursors of the molts. This secretion may be a source of cobalt sulfide stain for sensory organs in the glycocalyx on the cuticle surface, be- insects (40). It also aids in the penetration lieved to be important in host and fungal of this stain into nematode phasmids (8), parasite recognition. Glycocalyx glycos- presumably by solubilizing the ampullar aminoglycans were proposed to be secret- plug. ed from nematode sensory organs (53). These plugs and the plugs observed in Acid mucopolysaccharides (glycosamino- other plant-parasitic genera (1,9,47) have glycans) have been histochemically detect- a darker, sometimes crystalline differentia- ed in the phasmid of an animal parasite tion on the outer surface of the ampullar (33). Glycosaminoglycans are believed to opening. This dark material has the ap- be processed in Golgi vesicles (38). There pearance of a highly condensed form of is nothing to preclude these substances wax found on the cuticles of some juvenile from being secreted in phasmid vesicles, insects (28,29). The filamentous liquid- along with lipids and proteins in the secre- crystal, lipid-water wax precursors in in- tion, although the relative proportions of sects also appear similar to the filamentous these substances may change with time. substructure of the ampullar plugs in Hop- There is a possible parallel in insects where lolaimus and Scutellonema (9). This struc- the outermost layer of the cuticle contains tural variation in ampullar plugs could in- a mucopolysaccharide cement mixed with dicate the possible usefulness in wax (29).

FIG. 1 1. Meloidoderafloridensis posthatch J2 sheath cell cross sections. A) Open membrane, open receptor cavity (R) form. Lamellar membranes (LM) entirely extracellular. Y bifurcation of cavity at dorsal (Do) end of sheath cell. Neuron (N) at level of transition zone. V = vesicles. B) Closed membrane, open receptor cavity (R) form. Lamellar membranes (LM) partially extracellular, mostly intracellular. Cavity open, just below bifurcation. Neuron (N) at level between middle segment and transition zone, slightly distal to neuron pictured in Figure 12. V = vesicles. C) Closed membrane, closed receptor cavity (R) form. Neuron (N) at transition zone level as in Figure 12. V = vesicles. 380 Journal of Nematology, Volume 22, No. 3, July 1990

FIG. 12. Meloidoderafloridensis young M5 cross section of phasmids. End apparatus (EA) visible around neuron (N) of right socket cell (So). Sp = spicules, SRM = spicule retractor muscle, Sh = sheath cell. Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 381

Socket cell and canal: The socket cell is one socket cell in Heterodera (1) may be a better developed during the molt than in- function of the relatively large canal in Me- termolt. At molt the transient lamellar loidodera rather than an independent phy- membranes provide secretory precursors logenetic character. Caenorhabditis elegans to the cuticle lining of the canal. J 1 have only hypodermis for this function, Cuticular components such as acid mu- J2 have one socket cell, and males have two copolysaccharides (glycosaminoglycans) socket cells associated with a long canal have been histochemically detected in (44). phasmids (33), and chondroitin sulfate and Neuron: The neuron represents the most hyaluronic acid are both components which continuous structure of the phasmid support collagen in the nematode cuticle throughout all stages, although its mor- (26). During formation of the canal in the phology varies during development. prehatch J2, socket cell membranes in- Only one ciliary dendrite is observed in fused with an electron-dense material ac- all developmental stages of Meloidodera cumulate at the outer edge of the canal phasmids, as in H. schachtii (1), and adult region. When each stage is fully developed, Scutellonema brachyurum (47). Two neurons occasional dark deposits appear along the occur in phasmids of the animal parasites outer edge of the cuticular canal. This could Dracunculus medinensis (33) and Dipetalo- be a remnant of the earlier dark membrane nema viteae (31) and the free-living nema- secretion, observed during molt, which had tode C. elegans (44). not become modified into the hyaline state In Meloidodera the dendrite terminates of the canal lining. in the receptor cavity between molts and The content of phasmid secretions may in the adult, but it has an extensive distal vary with developmental time. It would not zone and extends into the canal during the be surprising to see a large proportion of molt. The direction of development is from cuticle precursors in the premolt secretion a complete to a shortened dendrite. being followed by increasing amounts of Vacuoles in the enlarged dendrite base lipid before hatch or ecdysis. This is the of Meloidodera males are more apparent in case in dermal wax gland cells in insects stages after the molt than during the molt, which have alternate layering of cuticle and and a true basal body might be present only a wax complex (46). during the middle of the molt when root- Lamellae at the base of the sheath cell lets are present. The rarity of finding root- receptor cavity appear to form part of the lets and basal bodies in this and other stud- end apparatus of the canal within the re- ies of sensory neurons supports the notion ceptor cavity. A possible parallel is seen in of their rapid disappearance associated with insects where the end apparatus also ap- vacuolization and aging. pears to be constructed by the gland cell A small portion of the canal completely in class-3 epidermal glands (34,46). encircles the dendrite in the early M5; During the middle molt of M. floridensis however, this part of the canal is shorter M4, and probably during other stages, mi- than its counterpart in other nematodes crotubules are scattered in the socket cell. (24,31). Therefore, for some other nema- The surrounding cells and cuticle are re- todes the possible character state of neu- forming at this time, and these long tubules ron in the canal would not apply to the may be especially important for cellular fully developed stages of Meloidodera. support and morphological polarity during Morphological differences of the neu- development (11). Regular arrays of mi- ron, such as degree of vacuolization, ap- crotubules (11) are not seen in the stages parent shortening of the distal segment, between molts in Meloidodera as they are and position relative to the canal, may have in Heterodera J2 (1). functional significance. For example, a The presence of two socket cells in J2 shortened dendrite that ended in the re- and M5 phasmids of Meloidodera versus only ceptor cavity rather than in the canal was 382 Journal of Nematology, Volume 22, No. 3, July 1990 the only morphological aberration in Cae- loss may prove to be present in all molt to norhabditis OSM3 FITC staining mutants. intermolt transitions in plant parasites. While unable to detect salt and dauer pher- Thus the absence of the dendrite in the omone, the mutant neurons could still de- canal may be directly related to some para- tect carbon dioxide (37). Carbon dioxide sitic function, rather than to a relatively is an important signal for hatching and stable phylogenetic ancestry. molting (39). Thus it is conceivable that Sheath cell: The most outstanding fea- during the intermolt these shortened neu- tures of the sheath cells of Meloidodera rons respond primarily to a molting stim- phasmids are the extensive infolded la- ulus. mellae of the plasma membrane. These are Neuron function might also be associ- not reported in the sheath cell around the ated with its relative position in the chan- phasmid neuron of Caenorhabditis (44). In nel. The chemical microenvironment M. floridensis a few lamellae occur in the surrounding the distal cilium could be dif- anterior counterpart to the phasmid, called ferent in the receptor cavity or the canal. the amphid (unpubl.). However, only after In insects, the end apparatus and duct are host infection do amphids develop exten- believed to be physiological compartments sive lamellae or reticulations in Heterodera containing enzymes such as esterase for ex- (23). Although lamellae are not found in tracellular reactions in the final stage of lateral and caudal epidermal glands of most secretion from the epidermal glands (34). Adenophorea, extensive lamellae do occur Physiological compartmentalization is sug- in the lateral bacillary bands of the ade- gested in these results by the more con- nophorean animal parasitic trichurids, and densed secretion near the end apparatus they are believed to be osmotically impor- and the near absence of secretion in the tant (51). Lamellae are also found in only canal in intermolt stages. one of the three neurons of the spicules of Changes in length and position of the Meloidoclera, Verutus, and Heterodera (un- dendrite observed in these studies also oc- publ.). This suggests that highly developed cur at the beginning of the molt in insects lamellae may serve some function in ad- under the control of ecdysone. Although dition to protection of the neuron. Some this insect dendrite responds to stimula- idea of this lamellar function might be tion, its response amplitude is probably dif- gained from associations in other systems. ferent from its amplitude during the fully Extensive lamellae of similar proportions developed stage. This changed amplitude to the sheath cell of Meloidodera phasmids could be a compensation for the loss, dur- are visible in the salt receptors of the blow- ing molt, of the transepithelial potential fly (25), salt glands of various other organ- from the microvilli of the tormogen cell isms (7,10,13), and mosquito rectal glands surrounding the receptor cavity (16). (32). If the presence or absence of dendrites Because similar fixation techniques were in the canal proves to be a variable char- used, the variations in degree of inflation acter within the Heteroderinae or higher of lamellar membranes in this work appear taxa, this direction of development could to reflect developmental differences; how- support the absence of dendrites in the ca- ever, osmotic effects can influence the in- nal in adult males as a derived state. Lack- flation. Hypertonic solutions caused ex- ing a precise knowledge of function, we are treme interlamellar inflation in Scutellonema unable to identify possible selection pres- (47). Therefore special care must be taken sures for homoplasy (i.e., parallel evolu- in the comparative morphology of phas- tion). We have noted that the dendrite ap- mid sheath cells to standardize fixation pears to lose its distal region through procedures. vesiculation as in chemosensory organs of Lamellar membranes in Meloidodera are certain insects (52). This pattern of neuron highly labile from the end of the molt to Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 383

the completion of a given stage. Particu- Part of the source of the lamellar mem- larly at the end of the molt, their relative branes appears to be the extensive hyaline dimensions and shapes may prove to be lipid associated with socket cell nuclei be- species specific. For instance, large distal fore hatch. During the molts of later in- lamellar anastomoses, relatively thick la- termediate stages an electron-dense body mellae, and deep oval pockets are distinc- in the socket cell may represent another tive features of sheath membranes in M. form of lipid for the membranes which charis. Use of sheath cell membrane pat- reappear at the end of the molt. terns as a potential phylogenetic character Sheath cell lamellae are numerous dur- requires observations of a number of spec- ing the second half of the molt (]1 and J2) imens under precisely defined conditions but are absent during the beginning of the and life stages. molt (M3 through M5). Therefore they ap- Some unusual organelles are observed in pear to be in a cycle of degeneration be- sheath cells of Meloidodera. These include tween molts. This is borne out by the ap- multivesicular bodies, multilamellar bod- parent loss of much membrane material ies, and membrane-filled vesicles. Multi- from prehatch to posthatchJ2. Beyond the vesicular bodies were described in Dipeta- J2, lamellae become less numerous in suc- lonema phasmids (31). Very similar cessive intermolt stages as well, perhaps be- organelles have been seen in a hypodermal cause of decreasing lipid in these nonfeed- sensory organ of Chromadorina germanica ing stages. The sheath cell in the M5 has (Adenophorea) (27), in rectal glands of Me- considerably less membrane area than in loidogynejavanica (4), in the intestine of As- any juvenile. caris (41), in salt glands of invertebrates Loss of lamellae at molt, and lamellar and plants (10), and in insect dermal glands reduction in M3 and M4, may be function- (34). Membrane-filled vesicles are present ally associated with the general muscular in lateral hypodermal cells of Heterodera atrophy and inactivity at these develop- (50). Many studies suggest a lysosomal mental times. A well-developed osmotic function for these organelles (10,14); in detection ability, perhaps represented by some cases, however, these bodies are so these lamellae, may be more important to numerous it suggests a secretory function active than inactive stages. It was reported (34). Multivesicular bodies are present that secernentean nematodes with high when Golgi bodies are absent and might turgor pressure in a terrestrial environ- concentrate and sequester protein as Golgi ment had phasmids, whereas low turgor analogues (27). pressure marine nematodes lacked phas- Observations in Meloidodera phasmids mids (36). lend support to the proposed secretory and We have only begun to evaluate the phy- Golgi-like nature of these multivesicular logenetic consequences of the different bodies. Because of their similar shape to phasmid structures. Even tentative conclu- the concentric lamellar membranes in pre- sions regarding these features must wait hatch cells, multivesicular and multilamel- for observations on additional members of lar bodies may represent the remnants of the Heteroderinae and its putative phylo- lamellar membranes that did not reach the genetic outgroup, the Hoplolaimidae (3). extracellular boundary of the receptor cav- These observations, however, provide some ity. The lamellar membranes that did reach understanding of the degree of morpho- the receptor cavity showed distinctive Gol- logical and fine structural variations which gi-like behavior in their vesiculation at both are possible complexities of phasmids, as ends. However, the possible origin of con- well as speculations on function which may centric lamellae within the nucleus rather contribute to phylogeny. In light of the than surrounding it makes a true Golgi as- reports of phasmid disappearance in other signment questionable. genera (8), it is significant that the phasmid 384 Journal of Nematology, Volume 22, No. 3, July 1990 of Meloidodera is never completely lost dur- 16. Gnatzy, W., and F. Romer. 1980. Morpho- ing any stage. genesis of mechanoreceptor and epidermal cells of crickets during the last instar, and its relation to molting-hormone level. Cell Tissue Research 213:369- LITERATURE CITED 391. 1. Baldwin,J. G. 1985. Fine structure of the phas- 17. Hartman, K. M. 1978. The biology, host range mid of second stage juveniles of Heterodera schachtii. and occurrence ofMeloidodera charis in Arizona. M.S. Canadian Journal of Zoology 63:534-542. thesis, University of Arizona, Tucson. 2. Baldwin,J. G. 1986. Testing hypotheses of phy- 18. Hawke, S. D., and R. D. Farley. 1971. The logeny of Heteroderidae. Pp. 75-100 in F. Lamberti role of pore structures in the selective permeability and C. E. Taylor, eds. Cyst nematodes. New York: of antennal sensilla of the desert burrowing cock- Plenum Publishing Co. roach, Arenivaga sp. Tissue and Cell 3:665-674. 3. Baldwin, J, G., and L. P. Schouest, Jr. 1990. 19. Hedgecock, E. M., J. G. Culotti, J. N. Thom- Comparative detailed morphology of the Heterod- son, and L. A. Perkins. 1985. Axonal guidance mu- erinae Filip'ev & Schuurmans Stekhoven, 1941, sensu tants of Caenorhabditis elegans identified by filling sen- Luc et al. (1988): Phylogenetic systematics and revised sory neurons with fluorescein dyes. Developmental classification. Systematic Parasitology 15:81-106. Biology 111:158-170. 4. Bird, A. F. 1971. The structure of nematodes. 20. Herman, R. 1984. Analysis of genetic mosaics New York: Academic Press. of the nematode Caenorhabditis elegans. Genetics 108: 5. Bird, A. F. 1979. Uhrastructure of the tail re- 165-180. gion of the second stage preparasitic larva of the root 21. Hirschmann, H., and A. C. Triantaphyllou. knot nematode. International Journal of Parasitology 1973. Postembryogenesis of Meloidoderafloridensis with 9:357-370. emphasis on the development of the male. Journal of 6. Byard, E. H., w.J. Sigurdson, and R. A. Woods. Nematology 5:185-195. 1986. A hot aldehyde-peroxide fixation method for 22. Hubel, D. H. 1957. Tungsten microelectrode electron microscopy of the free-living nematode Cae- for recording from single units. Science 125:549- norhabditis elegans. Stain Technology 61:33-38. 550. 7. Carr, K. E., and P. G. Toner, editors. 1982. 23. Jones, G. M. 1979. The development of am- Cell structure: An introduction to biomedical elec- phids and amphidial glands in adult Syngamus trachea tron microscopy, 3rd ed. New York: Churchill Liv- (Nematoda: Syngamidae). Journal of Morphology 160: ingstone. 299-322. 8. Carta, L. K., andJ. G. Baldwin. 1990. Phylo- 24. Kozek, W.J., and T. C. Orihel. 1983. Ultra- genetic implications of phasmid absence in males of structure of Loa loa microfilaria. International Jour- three genera of Heteroderinae. Journal of Nematol- nal of Parasitology 13:19-43. ogy 22:386-394. 25. Larsen, J. R. 1962. The fine structure of the 9. Coomans, A., and A. DeGrisse. 1981. Sensory labellar chemosensory hairs of the blowfly, Phormia structures. Pp. 127-174 in B. M. Zuckerman and R. regina Meig. Journal of Insect Physiology 8:683-691. A. Rohde, eds. Plant parasitic nematodes, vol. 3 New 26. Lee, D. L. 1977. Physiology of nematodes, York: Academic Press. 2nd ed. New York: Columbia University Press. 10. Copeland, D. E., and A. T. Fitzjarrell. 1968. 27. Lippens, P. L. 1974. Uhrastructure of a ma- The salt absorbing cells in the gills of the blue crab rine nematode, Chromadorina germanica (Buetschli, (Callinectes sapidus Rathbun) with notes on modified 1874). II. Cytology of lateral epidermal glands and mitochondria. Zeitschrift fiir Zellforschung und Mi- associated neurocytes. Zeitschrift f/Jr Morphologie der kroskopische Anatomie 92:1-22. Tiere 79:283-294. 1 I. Dustin, P. 1978. Microtubules. New York'. 28. Locke, M. 1961. Pore canals and related struc- Springer-Verlag. tures in insect cuticle. Journal of Biophysical and Bio- 12. Eisenback, J. D. 1985. Techniques for pre- chemical Cytology 10:589-619. paring nematodes for scanning electron microscopy. 29. Locke, M. 1974. The structure and formation Pp. 79-105 in K. R. Barker, C. C. Carter, andJ. N. of the integument in insects. Pp. 123-213 in M. Rock- Sasser, eds. An advanced treatise on Meloidogyne, vol. stein, ed. The physiology of Insecta, vol. 6, 2nd ed. 2. Methodology. Raleigh: North Carolina State Uni- New York: Academic Press. versity Graphics. 30. Luc, M., A. R. Maggenti, and R. Fortuner. 13. Eisenbeis, G., and W. Wichard. 1977. Zur 1988. A reappraisal of Tylenchina (Nemata). 9. The feinstrukturellen Anpassung des Transportepithels am family Heteroderidae Filip'ev & Schuurmans Stek- Ventraltubus von Collembolen bei unterschiedlicher hoven, 1941. Revue de N6matologie 11:159-176. Salinit~it. Zoomorphologie 88:175-188. 31. McLaren, D.J. 1976. Nematode sense organs. 14. Ferris, V. R. 1979. Cladisticapproaches in the Pp. 195-265 in B. Dawes, ed. Advances in parasitol- study of soil and plant parasitic nematodes. American ogy, vol. 14. New York: Academic Press. Zoologist 19:1195-1215. 32. Meredith, J., and J. E. Phillips. 1973. Rectal 15. Ferris, V. R. 1985. Evolution and biogeog- uhrastructure in salt and freshwater mosquito larvae raphy of cyst-forming nematodes. European and in relation to physiological state. Zeitschrift fiir Zell- Mediterranean Plant Protection Organization Bul- forschung und Mikroskopische Anatomie 138:1-22. letin 15:123-129. 33. Muller, R., and D. S. Ellis. 1973. Studies on Phasmid Ultrastructure in Meloidodera sp.: Carta, Baldwin 385

Dracunculus medinensis (Linnaeus). III. Structure of 44. Sulston,J. E., D. G. Albertson, andJ. N. Thom- the phasmids in the first-stage larva. Journal of Hel- son. 1980. The Caenorhabditis elegans male: Postem- minthology 47:27-33. bryonic development of nongonadal structures. De- 34. Noirot, C., and A. Quennedey. 1974. Fine velopmental Biology 78:542-576. structure of insect epidermal glands. Annual review 45. Triantaphyllou, A. C., and H. Hirschmann. of Entomology 19:61-80. 1973. Environmentally controlled sex expression in 35. Othman, A. A. 1985. Comparison of char- Meloidoderafloridensis. Journal of Nematology 5:181- acters with SEM for systematics and phylogeny of 185. Heteroderidae (Nematoda: Tylenchida). Ph.D thesis, 46. Waku, Y. 1982. Fine structure of the insect University of California, Riverside. wax glands. Pp. 113-116 in H. Akai, R. C. King, and 36. Paramonov, A. A. 1954. On the structure and S. Morohoshi, eds. The ultrastructure and function- function of the phasmids. Trudy Gelmintologiche- ing of insect cells. The Society for Insect Cells, Tokyo. skaya Lab Akademiya Nauk SSR 7:19-49. 47. Wang, K. C., and T. A. Chen. 1985. Ultra- 37. Perkins, L. A., E. M. Hedgecock,J. N. Thom- structure of the phasmids of Scutellonema brachyurum. son, andJ. G. Culotti. 1986. Mutant sensory cilia in Journal of Nematology 17:175-186. the nematode Caenorhabditis elegans. Developmental 48. White, J. G., E. Southgate,J. N. Thomson, and Biology 117:456-487. S. Brenner. 1986. The structure of the nervous sys- 38. Porter, K. R., and M. A. Bonneville. 1973. tem of the nematode Caenorhabditis elegans. Philo- Fine structure of cells and tissues, 4th ed. Philadel- sophical Translations of the Royal Society of London phia: Lea and Febiger. Biological Sciences 314:1-340. 39. Rogers, W. P., and T. Petronijevic. 1982. The 49. Wigglesworth, V. B. 1976. The distribution of infective stage and the development of nematodes. lipid in the cuticle of Rhodnius. Pp. 89-106 in H. R. Pp. 3-28 in L. E. A. Symons, A. D. Donald, andJ. K. Hepburn, ed. The insect integument. New York: El- Dineen, eds. Biology and control of endoparasites. sevier Publishing Co. New York: Academic Press. 50. Wisse, E., and W. Th. Daems. 1968. Electron 40. Schafer, R., and T. V. Sanchez. 1976. The microscopic observations on second stage larvae of nature and development of sex attractant specificity the potato root eelworm Heterodera rostochiensis. Jour- in cockroaches of the genus Periplaneta. Journal of nal of Ultrastructure Research 24:210-231. Morphology 149:139-158. 51. Wright, K. A. 1980. Nematode sense organs. 41. Sheffield, H. G. 1964. Electron microscope Pp. 237-295 in B. M. Zuckerman, ed. Nematodes as studies on the intestinal epithelium of Ascaris suum. biological models, vol. 2. New York: Academic Press. Journal of Parasitology 50:365-379. 52. Zacharuk, R. Y. 1980. Ultrastructure and 42. Siddiqi, M. R. 1978. The unusual position of function of insect chemosensilla. Annual Review of the phasmid of Coslenchus costatus and other Tylen- Entomology 25:27-47. chidae. Nematologica 24:449-455. 53. Zuckerman, B. M., and H.-B. Jansson. 1984. 43. Singh, R. N., and J. E. Sulston. 1978. Some Nematode chemotaxis and possible mechanisms of observations on moulting in Caenorhabditis elegans. host/prey recognition. Annual Review of Phytopa- Nematologica 24:63-71. thology 22:95-113.