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Discussion

We have described the ultrastructure of the C. elegans secretory-excretory system. This four-cell organ develops embryonically with the exception of the pore cell (designated G2p) which surrounds the excretory duct early in the second larval stage, replacing an embryonic pore cell, G1 (Sulston and Horvitz, 1977). The entire system is descended from the early embryonic stem cell, AB, which gives rise to primary ectoderm (von Ehrenstein and Schierenberg, 1980; Sulston, White, Thomson, Schierenberg, and von Ehrenstein, personal communication).

Functions which have been attributed to nematode excretory systems are (1) excretion of metabolic waste, (2) osmoregulation, (3) secretion of molting exsheathment fluid, and (4) secretion and export of hormones to target tissues. The excretory cell of C. elegans has a morphology consistent with any or all of these functions. The excretory canals are exposed to the pseudocoelom, and they form extensive gap junctions with the hypodermis. The energy required for active transport across canal membranes could be made available by the abundant mitochondria within the canal cytoplasm, and the surface area at which exchange may take place is increased by canalicular branching of the lumena. The exchange process conceivably could work in either direction, to excrete wastes into the canals or to resorb, perhaps selectively, substances from the lumena. Any substances collected in the excretory canals are presumably transported from the organism's body via the excretory sinus, through the excretory duct to the excretory pore. In addition, it is possible that substances may be excreted from the hypodermis directly through the cuticle, which is water permeable in all stages.

In other nematode species, osmoregulation is perhaps the best documented excretory function. Weinstein (1952), Croll et al., (1972), and Atkinson and Onwuliri (1981) observed that changes in the rate of excretory duct pulsation correspond with changes in the osmolarity of the environment. In general, the more sudden the change toward a hypotonic medium, the faster were the pulsations of the duct. Waddell (1968) observed excretory duct pulsation in L3 (infective stage) larvae of Stephanurus dentatus (pig kidney worm), but fluid from L4s or adults was excreted without duct pulsation. Excretory duct pulsation also has been observed in Panagrellus and Heterodera (Narang, 1972).

Under routine laboratory conditions, we were able to detect pulsation of the excretory duct only in dauer larvae. It is possible that only relatively fast rates of excretion involve duct pulsation. In C. elegans, a rapid change in water balance occurs at the L2-dauer molt, when radial shrinkage of the body results in an increased density of the dauer larva relative to the L2. Electron micrographs reveal that the volume of the hypodermis is preferentially reduced. The excretory system may be involved in the establishment or maintenance of this state. Thus, dauer larvae may employ excretory pulsing to maintain an appropriate water balance, whereas other stages of lower density may not need to do so. Additionally, the specialized dauer larva cuticle may be less permeable to excreted products, imposing a greater demand on the dauer excretory system.

We observed no structural features suggesting neuromuscular control of excretory duct pulsation in dauer larvae. Instead, the normally collapsed duct may be periodically forced open by internal pressure from the excretory cell. Once accumulating fluid has opened the duct along its entire length to the excretory pore, internal pressure would be quickly released, and the duct could collapse once again. The extensive folding of the duct cell plasma membrane around the duct may provide for the rapid expansion and contraction of the duct during excretory pulsing. Similar lamellar folds surrounding the excretory duct also have been observed in Panagrellus redivivus (Narang, 1972).

A second possible function of the duct cell membrane is exchange of materials between the duct contents and the duct cell cytoplasm. The duct cell may selectively reabsorb salts or other substances entering the duct. The lamellar membrane amplifies the cell surface area at the duct, and abundant mitochondria within the duct cell could provide the required energy for active transport. We would not propose such a function for the pore cell, because its plasma membrane lacks the lamellar structure.

The morphology of the gland cell obviously suggests that a large amount of material is synthesized within this cell, packaged into granules, secreted into the duct, and transported to the exterior of the worm along with fluid from the excretory sinus. The presence of secretory granules in all growing stages examined suggests that secretion is either constant or occurs at repeated intervals. At least some, if not all, of the secretory granules are membrane-bound. If they release their contents into the duct by fusing with the plasma membrane of the gland cell at the secretory-excretory junction, the apparent complexity of the secretory membrane at this point may simply reflect the accumulation of membrane in the restricted area which is entirely bound by a tight junction. We do not know whether the type of tight junction found in the C. elegans secretory-excretory system is functionally identical to the zonula occludens found in vertebrate epithelium. However, it meets the major morphological criteria established for tight junctions in vertebrates. Permeability of these structures to heavy metal tracers has not been tested.

The consistent morphology of the secretory membrane in animals of various developmental and physiological states suggests that this structure may be an organelle. Although some of the dense material associated with the secretory membrane may be deposited during fusion with the electron-dense granules, none of our micrographs actually show granules fusing with the secretory membrane, nor have we seen portions of the membrane budding from the cell. The secretory membrane retains its dense, convoluted, tubular morphology even in dauer larvae, which lack the secretory granules altogether. Examination of previously published light micrographs (Romanowski et al., 1971) indicates that a similar structure may exist in other nematodes as well.

Studies on Phocanema decipiens indicate that a molting hormone acts on the excretory gland to bring about release of a "molting fluid" through the excretory pore into the space between the old cuticle and the newly synthesized, underlying cuticle (Davey, 1966; Davey and Kan, 1968). A leucine aminopeptidase was identified as a component of the molting fluid, and it was proposed that such enzymes may be involved in release or partial degradation of the discarded outer cuticle. The morphology of the gland in C. elegans also is consistent with a role in secretion of molting enzymes. The results of laser ablation of the gland cell, duct cell, or excretory cell nucleus in L2 larvae, however, led Singh and Sulston (1978) to conclude that the C. elegans excretory system is not essential for molting. These workers observed the loosening of the older cuticle to begin at the tip of the head rather than at the excretory pore. Furthermore, the amount of secretory granules in the excretory gland does not cycle with the molts, but increases steadily during development (with the exception of the dauer stage), and the greatest number of granules are found in the adult. The gland cells of all feeding stages of C. elegans (including the adult) are PAF-positive, whereas the glands of starved animals and dauer larvae are not. The ultrastructural comparisons suggest that PAF staining is correlated with gland activity.

The gland may not be essential for development or reproduction in the laboratory. Nevertheless, it may function in many ways to enhance survival in the natural soil environment. One substance released by C. elegans during all growth stages is a pheromone that in crowded cultures enhances entry into, and inhibits exit from, the dauer larva stage (Golden and Riddle, 1982). This pheromone or other informational molecules could be secreted via the excretory duct.

The gland cell ultrastructure does not preclude the possibility that substances may be secreted into the pseudocoelom, either by direct diffusion across the plasma membrane, or by fusion of secretory granules with the plasma membrane at sites other than the secretory-excretory junction. In addition, secreted products could be transported throughout the body of the animal via the excretory canals.

The striking differences in gland cell morphology between dauer larvae and other developmental stages suggest that release or degradation of secretory granules accompanies dauer formation, and that secretory activity is not present in the dauer stage- It also seems likely that gland function is not required to initiate exit from the dauer stage. The elimination of secretory granules and vacuolation of the cell is correlated with the dauer state itself and not with nutritional condition. No cause and effect relationship between dauer formation and gland function, however, has yet been established. We plan to perform laser microsurgery experimerits, in which both gland cell nuclei will be destroyed during the first larval stage, in order to test the possible effects of cell ablation on dauer larva formation.

Adapted by Yusuf KARABEY for WORMATLAS, 2003