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Osmoregulation and FMRFamide related peptides in the salt marsh snail Melampus bidentatus (Say)(Mollusca: Pulmonata).

Introduction

Melampus bidentatus is a common amphibious pulmonate snail. Its habitat is the high intertidal zone of salt marshes, and it ranges from Nova Scotia, Canada, to the coast of the Gulf of Mexico in Texas (Apley, 1970; Hilbish, 1981). Melampus belongs to the primitive family Ellobiidae and is believed to be related to an ancestral marine strain that colonized the intertidal habitat, giving rise to the land snails and then to the freshwater snails (Morton, 1955; Russell-Hunter, 1978). Environmental factors such as temperature, salinity, and tides are extremely variable in the high littoral habitat. But the adult snails are adapted to this variation, and can survive for several days at temperature extremes of -12 [degrees] C to 40 [degrees] C, submergence in 25%-100% seawater, and desiccation at 0% relative humidity for 27-36 h (Price, 1980; McMahon and Russell-Hunter, 1981). Although adults of M. bidentatus can endure a terrestrial life, its planktonic veliger larvae are restricted to the aquatic habitat of estuaries (Russell-Hunter et al., 1972).

The natural history of M. bidentatus suggests that in estuarine and semiterrestrial molluscs, osmotic and volume regulation must be particularly responsive. Moreover, scattered evidence suggests that the family of neuropeptides related to FMRFamide is involved in this regulation. First, in a freshwater pulmonate snail, Helisoma duryi, FMRFamide causes water retention in the kidney in vitro (Saleuddin et al., 1992). Further, the level of immunoreactive, FMRFamide-related peptides (IR-FaRPs) in the kidney of Helisoma trivolvis kept in hypoosmotic medium is lower than that in the kidneys of snails kept in isosmotic medium (Madrid et al., 1994). The IR-FaRPs have also been localized in the kidneys of the terrestrial pulmonate snail Helix aspersa (Lehman and Price, 1987), and in the central nervous system (CNS) of the veligers of M. bidentatus (Moffett, 1992). Finally, the regulation of hypoosmotic cell volume by cardiac muscles of the clam Mercenaria mercenaria is potentiated by FMRFamide (Deaton, 1990).

Since M. bidentatus inhabits highly variable osmotic conditions, it is an appropriate species with which to further test the proposal that the FaRPs have a role in osmoregulation. In this paper, we have studied, both in the field and the laboratory, the influence of the ambient osmotic concentration on that of the hemolymph. We have then compared the levels of IR-FaRPs in the CNS, hemolymph, and kidney of snails maintained in media of different osmotic concentrations. We also report IR-FaRP-staining of neurites in the kidney, and of neurons in the CNS of adult snails.

Materials and Methods

Animals and media

During January to April, M. bidentatus adults (shell lengths, 10.0 [+ or -] 2.0 mm) were collected at random from an area 100 m x 20 m in a salt marsh on the Matanzas river estuary near Crescent Beach, Florida (29 [degrees] 40 [minutes] N: 81 [degrees] 13 [minutes] W). Experiments with the freshly collected snails were performed nearby, at the Whitney Laboratory of the University of Florida. At York University, the animals were maintained at 22 [degrees] C, 95% relative humidity, and a photoperiod of 14 h light to 10 h dark, in glass tanks (30 cm L x 15 cm W x 20 cm H) covered with nylon screens. The floor of each tank was covered with a layer of crushed oyster shells (poultry feed) graded in thickness from 0.5 cm at one end of the tank to 3.0 cm at the other. A volume of 50% artificial seawater (ASW; Instant Ocean, Aquarium Systems, Ohio) was added to cover about one-third of the crushed oyster shells. Slanted against the walls of each tank were 4-6 broken, irregularly shaped pieces of clay plant-pot (about 10 cm in diameter). Most snails crawled to terrestrial conditions above the water level on the walls of the tank and on the dark side of the moist pieces of clay plant-pot. Distilled water was added daily to maintain osmolarity and water level, and the snails were fed fish-food and boiled lettuce ad libitem. The snails were acclimated to laboratory conditions for at least 2 weeks before experimentation.

The kidney is embedded just under the dorsal surface of the mantle tissue, and the CNS is located under the skin, between the tentacles. Before dissection, the snails were chilled on ice for 5-10 min, which reduced their movements and mucus secretion; no other anesthetic was used. Deshelled snails were pinned on a dish lined with Sylgard (Dow Corning, Canada) and dissected in filtered isosmotic ASW. We adjusted the osmolarity of the ASW with distilled water to be isosmotic with that of the hemolymph, which varies with the medium or relative humidity (described later). The kidney and CNS were removed from a snail within 3-5 min, and were used for FaRP studies.

Osmotic conditions

Snails that had been maintained under terrestrial conditions (described above) were submerged for various periods (maximum, 4 days; minimum 3 h) in 10%, 50%, or 100% ASW (dilutions made with distilled water) in 250-ml plastic containers, each with a nylon screen. The levels of IR-FaRPs were measured (described later) after 24 h of osmotic treatments. Hemolymph was collected as follows. The snails were pricked in the head area with a sharp needle. Each animal was then quickly placed in a 0.5-ml polypropylene microcentrifuge tube with a hole in its bottom. This tube was then placed in a larger (1.5 ml) polypropylene microcentrifuge tube. The nested tubes and the snail were spun at 500 x g for 2-5 s. Then the inner tube containing the snail was removed, and the outer tube containing the hemolymph was spun for an additional 5 min. About 30-50 [[micro]liter] of hemolymph could be collected from each snail. The osmotic pressures of 10-[[micro]liter] samples were measured in a Wescor Model 5300 vapor pressure osmometer (Logan, Utah). After the bleeding, all of the snails fed and lived for many days thereafter. To measure the effects of osmotic water exchange on weight, the snails were weighed with a Mettler AE163 balance after the visible water and mucus had been removed from their shells and feet with adsorbent tissue.

Radioimmunoassay

Pulmonate molluscs contain two major classes of FaRPs: the tetra-FaRPs (FMRFamide and FLRFamide) and the hepta-FaRPs (XDP[F/Y]LRFamide, where the N-terminal residue X is pQ, S, N, or G). The hepta-FaRP analog with the glycyl residue occurs only in snails of the subclass Basommatophora (like M. bidentatus), and these animals lack the analog with the pyroglutamic acid (pQ) residue, which occurs only in stylommatophorans (Price et al., 1987a, b). To ensure that all of the FaRPs would be detected, we used S253 antiserum in the radioimmunoassay (RIA). The antiserum was raised to thyroglobulin-peptide (synthetic analog YGGFMRFamide) conjugate in a rabbit. It has high affinities for both FMRFamide and GDPFLRFamide, and was used in the assays at a dilution of 1:10,000. Iodinated pQYPFLRFamide was used as tracer (for details see: Price et al., 1990; Lesser and Greenberg, 1993).

The protocol of Madrid et al. (1994) was followed in processing the tissues. After dissection, each tissue sample was placed in a plastic tube (1.5 ml) containing HPLC grade acetone (1 part tissue: 4 parts acetone) and frozen immediately at -80 [degrees] C for at least 24 h. The acetone extracts were spun for 5 min at 5,000 x g, and the supernatants were collected and then dried in a Speed-Vac centrifuge. Each pellet was dissolved in 100 [[micro]liter] RIA buffer [0.01 M sodium phosphate with 1% bovine serum albumin (BSA), 0.9% sodium chloride, 0.01% merthiolate, and 0.025 M sodium EDTA]. Aliquots of 10 [[micro]liter] were placed in glass tubes to which 100 [[micro]liter] tracer (10,000 cpm) (in RIA buffer) and 100 [[micro]liter] diluted antiserum (in RIA buffer) were added. The tubes were stored at 4 [degrees] C overnight; on the following morning, 1.0 ml charcoal suspension (0.25% charcoal, 0.025% dextran, 0.01% merthiolate in 0.1 M sodium phosphate, pH 7.5) was added to each tube. After 10 min, the mixture was centrifuged at 2500 x g for 15 min at 4 [degrees] C, and the supernatant was counted in an LKB MiniGamma counter. All statistical comparisons were made with a one-way analysis of variance (ANOVA); when the ANOVA showed a significant difference, the effect was followed with the Bonferroni multiple comparison post test using a statistical software package (InStat, GraphPad Software, San Diego, California).

Immunocytochemistry

Isolated CNS and kidney tissues from animals in laboratory terrestrial condition were either embedded in paraffin and sectioned, or examined in whole mount. Sections were prepared as follows. The tissues were fixed for 12-18 h in modified Bouin-Hollande [7% picric acid, 2.5% copper acetate, 2% formaldehyde (freshly prepared from paraformaldehyde), and 1.5% glacial acetic acid, in phosphate-buffered saline (PBS) (0.2 M NaCl, 0.003 M KCl, 0.002 M K[H.sub.2]P[O.sub.4], 0.02 M [Na.sub.2]HP[O.sub.4], 0.001 M Ca[Cl.sub.2], 0.001 M Mg[Cl.sub.2], pH 7.5)], then dehydrated in graded ethanol solutions and embedded in paraffin. Serial sections (10 [[micro]meter]) were cut and mounted on coverslips (22 mm x 22 mm) coated with 0.5% gelatin, 0.5% chrome alum, and 0.01% formaldehyde. The deparaffinized sections were treated with 100% methanol and 0.1% [H.sub.2][O.sub.2] for 5 min, hydrated in a graded series of ethanol solutions, kept 30 min in an incubation medium (IM) consisting of PBS containing 2% Triton x 100 (Sigma Chem., St. Louis, MO), 5% normal goat serum (NGS), and 1% BSA. The sections were incubated for 1 h at 22 [degrees] C with hepta-FaRP-specific CK antiserum, which was raised in a rabbit to the peptide CKQD-PFLRFGK (a gift from Dr. G.A. Cottrell, University of St. Andrews, Scotland) (Cottrell et al., 1994). The primary antiserum was diluted 1:200 in IM. After many rinses in IM for 10 min, the sections were incubated for 1 h at 22 [degrees] C in fluorescein-conjugated goat antirabbit-serum (Sigma) diluted 1:80 in IM (secondary antibody). The coverslips were rinsed well in PBS and mounted on glass slides in 5% polyvinyl alcohol, 30% glycerol, and 0.1% phenylenediamine in PBS (mounting medium).

Whole mounts were prepared as follows. The tissues were treated with proteases before and after fixation (Longley and Longley, 1986). The tissues were first treated in 0.05% pronase in isosmotic saline for 10 min, then fixed in Bouin-Hollande (see above) for 12 h. After fixation, the tissues were rinsed in saline for 10 min, then treated in 0.1% trypsin in saline for 30 min, and rinsed well in saline for 10 min. A preincubation in IM for 6-12 h at 4 [degrees] C was followed by incubation in primary antibody (hepta-FaRP-specific CK antiserum; 1:200 in IM) for 24 h. After rinsing several times in 1% Triton X 100 in PBS for 1 h, the tissues were incubated in secondary antibody (1:80 in IM) for 6 h, followed by repeated rinses in IM. When whole-mount tissues were incubated for more than 60 min, this was done in the dark at 4 [degrees] C. The tissues were mounted in mounting medium (see above) between two coverslips (one rectangular, 60 mm x 22 mm; and one circular, 18 mm). To prevent tissue distortion, the weight of the coverslip was supported by four pieces of broken coverslip ([approximately]0.5 mm diameter). The larger coverslip was affixed with adhesive tape on an aluminum slide (36 mm x 80 mm x 1 mm) with a central 25-mm circular window. The smaller circular coverslip was placed face down in this window, so these preparations could be flipped for viewing from either side.

For controls, either pre-immune rabbit serum (1:200 in IM) or IM alone was used in the primary incubation; in either case, the tissues were then stained with secondary antibody as described above. The controls showed no staining. The specimens were viewed and photographed with either a Leitz epifluorescence microscope or a Bio-Rad MRC 600 confocal microscope. The IR-FaRP cells were mapped from the serial sections and from whole-mount images, and were measured with the calibration marker from the confocal images.

Results

Osmoregulation

The high-tide location of the marsh where M. bidentatus was collected has an uneven surface with a shallow slope, and contains abundant decaying organic matter of plant and animal origin. The osmolarity and pH of the waters in the habitat varied with time and location in the observation area. During the sunny days of April, the average osmolarities were 1200 [+ or -] 26 mosm/kg [H.sub.2]O (mean [+ or -] standard error of mean, n = 50) (range = 1015-1650 mosm/kg [H.sub.2]O), and the pH ranged from 6.0-7.2; at 1200-1400 h, the ambient air temperature was 31 [+ or -] 4 [degrees] C, and the soil surface and water temperatures were 34.2 [+ or -] 1.3 [degrees] C. At the same time, the tidal seawater coming through the Matanzas inlet had a consistent osmolarity of 980 [+ or -] 5 mosm/kg [H.sub.2]O, a temperature of 22 [+ or -] 2 [degrees] C, and a pH of 7.5 [+ or -] 0.1. The variables that altered the osmolarities of the water in the habitat and hemolymph of snails were tide, temperature, and precipitation. Most snails in the field were out of the water, on grass stems or higher points of the uneven ground, while a much smaller number were creeping and feeding underwater. The osmolarities of the hemolymph of snails from different locations and conditions and during high and low tides were measured within 30 min after collection and compared with the osmolarities of the adjacent media. The hemolymph osmolarities ranged from 1100 to 1500 mosm/kg [H.sub.2]O and appeared to parallel that of the adjacent medium. A few hours of heavy rainfall reduced the osmolarity of the water in the habitat to 750 [+ or -] 40 mosm/kg [H.sub.2]O; the hemolymph osmolarities also declined to 850 [+ or -] 50 mosm/kg [H.sub.2]O. Most snails in the laboratory crawled to the dry parts of the tank (see methods section) and stayed away from the water; thus they sought terrestrial conditions. But a few snails were also seen feeding and crawling underwater. The hemolymph osmolarity of the laboratory snails that sought terrestrial conditions was much lower (450 [+ or -] 25 mosm/kg [H.sub.2]O) than that of field snails (1200 [+ or -] 100 mosm/kg [H.sub.2]O) collected away from the water. When laboratory snails from terrestrial conditions were held for 24 h in 10% or 50% ASW, the osmolarity of the hemolymph paralleled, and was about 150 mosm/kg [H.sub.2]O hyperosmotic to, that of the medium. Snails in 10% and 50% ASW gained weight during the first 3-6 h, but over 24 h, gradually returned towards their original weight. In contrast, snails kept in 100% ASW for 24 h first lost weight and then returned towards their original weight [ILLUSTRATION FOR FIGURE 1 OMITTED].

Levels of IR-FaRPs

Immunoreactive-FaRPs were detected in every tissue studied; the CNS had higher levels than the other organs. The supraesophageal portion of the CNS (the buccal and cerebral ganglia) had lower levels of IR-FaRPs than the subesophageal portion (the pedal, parietal, and visceral ganglia) (Table I).
Table I.

Immunoreactive FaRP content detected by S253 antiserum in various
tissues of animals from terrestrial condition in laboratory

Tissue                                     IR-FaRP content (picomol)

Buccal ganglia(*)                              1.2 [+ or -] 0.40a
Cerebral ganglia(*)                            4.0 [+ or -] 0.70a
Pedal ganglia(*)                              19.1 [+ or -] 7.0 b
Visceral and parietal ganglia(*)             13.50 [+ or -] 6.0 b
Heart(*)                                      1.07 [+ or -] 0.40a
Hemolymph (50 [[micro]liter])                 0.35 [+ or -] 0.10a
Buccal mass                                   0.20 [+ or -] 0.07a
Foot                                          0.22 [+ or -] 0.09a
Digestive gland                               0.36 [+ or -] 0.08a
Intestine                                     0.12 [+ or -] 0.03a
Kidney                                        3.45 [+ or -] 0.36a
Mantle                                        1.28 [+ or -] 0.20a
Ovotestis                                     1.10 [+ or -] 0.60a
Penis complex                                 3.55 [+ or -] 0.08a
Skin                                          0.40 [+ or -] 0.20a
Vulva/vagina complex                          4.20 [+ or -] 2.00a

For tissues marked with an asterisk (*), which are very small and
difficult to handle for weighing, the IR-FaRP content is expressed
as picomole/tissue; in all other tissues the content is expressed as
picomole/mg tissue (n = 5; mean [+ or -] standard error of mean).
The values followed by different letters (a or b) are significantly
different (P [less than] 0.05. F = 5.05) from each other, whereas
the values sharing same letters (a or b) are not.


The levels of IR-FaRP in the CNS, hemolymph, and kidney varied with the ambient osmotic conditions. In all three tissues, the levels of IR-FaRPs were correspondingly reduced in lower salinities, and the highest levels were always in terrestrial conditions. The CNS of animals submerged in 10% ASW for 24 h had levels of IR-FaRPs that were 80% lower than those in the terrestrial condition [ILLUSTRATION FOR FIGURE 2 OMITTED]. In hemolymph, the 10% ASW condition reduced the levels of IR-FaRPs by 77% compared to the terrestrial condition [ILLUSTRATION FOR FIGURE 3 OMITTED]. In kidney tissues, the 10% ASW condition reduced IR-FaRP levels by 64% compared to the terrestrial condition [ILLUSTRATION FOR FIGURE 4 OMITTED].

Immunocytochemistry

Like other dextral gastropods, M. bidentatus retains only its right kidney. A whole mount of the kidney reveals IR-staining of axons with varicosities; moreover, such staining and varicosities are restricted to the kidney, and none are present in the surrounding mantle tissue [ILLUSTRATION FOR FIGURE 5 OMITTED]. Longitudinal sections of the kidney also show localized IR-staining at the periphery [ILLUSTRATION FOR FIGURE 6 OMITTED]. Some aggregation of varicosities appear as intense staining. No IR-cell bodies were seen in the kidney.

The distribution of IR-neurons in the CNS is mapped in Figure 7. All commissures, connectives, and major nerves in the CNS display IR-fibers, and all ganglia except the pleurals contain IR-cell bodies. Visceral and right parietal ganglia contain both large (20 [+ or -] 5 [[micro]meter]) and small (10 [+ or -] 5 [[micro]meter]) IR-neurons, whereas mostly small and only a few large IR-neurons are found in the cerebral and pedal ganglia [ILLUSTRATION FOR FIGURES 7-9, 12 OMITTED].

Cerebral ganglia. Two groups of large (20 [+ or -] 5 [[micro]meter]) and small (10 [+ or -] 5 [[micro]meter]) IR-neurons are present at the anterior and posterior location of the cerebral commissure [ILLUSTRATION FOR FIGURES 7-9 OMITTED]. The middle part of each cerebral ganglion contains a large (20 [+ or -] 5 [[micro]meter]) IR-cell surrounded by small IR-cells [ILLUSTRATION FOR FIGURE 9 OMITTED]. In the lateral lobes, 2-4 large (20 [+ or -] 3 [[micro]meter]) cells and 8-12 small (12 [+ or -] 4 [[micro]meter]) IR-cells are seen [ILLUSTRATION FOR FIGURES 7, 10 OMITTED].

Buccal ganglia. In each buccal ganglion, 3 intermediate sized (18 [+ or -] 2 [[micro]meter]) IR-cells, and many IR-fibers and varicosities are seen [ILLUSTRATION FOR FIGURE 11 OMITTED].

Pedal ganglia. Groups of 6-10 small IR-neurons are seen at the periphery of each pedal ganglion [ILLUSTRATION FOR FIGURES 7, 12 OMITTED].

Parietal and visceral ganglia. More large and small IR-cells were seen in the right parietal and visceral ganglia than in any other ganglion [ILLUSTRATION FOR FIGURES 7, 13 OMITTED]. The left parietal ganglion contains only small IR-neurons [ILLUSTRATION FOR FIGURE 7 OMITTED]. The IR-cells are arranged in anterior and posterior groups in both ganglia [ILLUSTRATION FOR FIGURES 7, 14 OMITTED]. Numerous IR-neurites emanate from the CNS into the surrounding connective tissue and appear to terminate in varicosities [ILLUSTRATION FOR FIGURE 15 OMITTED].

Discussion

In both the field and the laboratory, most individuals of M. bidentatus were emergent, living under semiterrestrial conditions. But some members of the population were always submerged, suggesting that they crawl in and out of water intermittently. The osmotic concentration of the hemolymph was correlated with that of the adjacent aqueous medium. Moreover, IR-FaRPs are present in the CNS, kidney, and hemolymph, and the levels of these peptides are also correlated with the osmotic concentration of the ambient medium.

Osmoregulation and kidney

When the osmolarity of the external medium changes, the hemolymph osmolarity of M. bidentatus also changes due to inward and outward movements of water from its body. Such conditions must be offset by various adjustments: regulation of urine production by its kidney is one of them. The IR-staining of the kidney was seen as a diffuse pattern on the kidney cells, as an intense localized pattern in areas with neural arborizations and varicosities, and in other small areas that are probably bundles of smooth muscles. The smooth muscles of the kidney of the basommatophoran snail Helisoma duryi are innervated by FMRFamide-immunoreactive axons (Saleuddin et al., 1992). In this species the in vitro contraction of kidney tissues was influenced by FaRPs (unpubl. obs., A.S.M. Saleuddin). The FaRPs are well known for their contractile activity on visceral and smooth muscles in various species. They are also known to regulate salivary glands and ion channels (Bulloch et al., 1988; Green et al., 1994; Price and Greenberg, 1994; Lingueglia et al., 1995). In the pulmonate kidney they are probably involved in regulating smooth muscle contractions for peristalsis and the production of hydrostatic pressure for urine formation, as well as in regulating ion channels for selective secretion and reabsorption of ions from urine.

Osmoregulation and IR-FaRP levels

The CNS and hemolymph displayed the highest and lowest levels of IR-FaRPs, respectively. Organs containing involuntary muscles, such as the reproductive tracts and kidney, have high levels of IR-FaRPs. These organs apparently receive FaRPs through direct innervation by neurons from the CNS. Thus, in these target tissues, the FaRPs may act as neuromodulators or as paracrine agents. Numerous IR-FaRP processes terminating in varicosities in the connective tissue around the CNS may constitute a diffuse neurohemal area and account for the hemolymph IR-FaRP levels.

The levels of IR-FaRPs in the CNS, hemolymph, and kidney varied under different osmotic conditions. The IR-FaRP levels were reduced in hypoosmotic conditions compared with those in hyperosmotic or terrestrial conditions. Terrestrial or hyperosmotic salt-water conditions produced threats of desiccation and salt loading in snails, whereas hypoosmotic conditions had the opposite effect. The changes of IR-FaRP levels in the tissues of M. bidentatus by osmotic conditions may reflect osmoregulation by the snail. Since IR-FaRP levels increased in the CNS, hemolymph, and kidney under conditions of increased water losses such as in terrestrial or 100% ASW conditions, the FaRPs may have an antidiuretic function. In vitro culture of the kidney tissues of Helisoma with synthetic FMRFamide increased its intracellular water uptake, and FMRFamide has been suggested to have an antidiuretic role (Saleuddin et al., 1992; Madrid et al., 1994). Antidiuretic and diuretic activities of FMRFamide and GDPFLRFamide respectively have been demonstrated in nephridia of a leech (Salzet et al., 1994). Basommatophoran pulmonates such as Melampus contain GDPFLRFamide (unpub. obs, D. A. Price). Both tetra- and hepta-FaRPs are present in the osmoregulatory tissues of Helisoma (Madrid et al., 1994). The tetra- and hepta-FaRPs are expressed in exclusive neurons and may have different actions in a target tissue (Greenberg and Price, 1992; Benjamin and Burke, 1994; Price and Greenberg, 1994). The precise mechanism of the tetra- and hepta-FaRP actions in molluscan kidneys awaits future studies. In molluscs, osmoregulation by the kidney appears to be a combined effect of many physiological systems regulated by different hormones. In Lymnaea stagnalis, a neuropeptide that stimulates sodium uptake by the skin has been sequenced; furthermore, axons that release immunoreactive sodium-influx-stimulating peptides are present in this snail (De With et al., 1994). In Aplysia californica, R15[Alpha]1 neuropeptide causes water retention, thus having an osmoregulatory role (Weiss et al., 1989). Additionally, in this species various kidney functions are modulated by L10 and LUQ neurons, and the latter neurons are immunoreactive to FMRFamide (Koester and Alevizos, 1989; Giardino et al., 1996). Such systems include increased circulation of hemolymph for ultrafiltration, subsequent secretion and reabsorption of ions and organic matters, and expulsion of final urine (Khan and Saleuddin, 1979). Bioassay studies assessing the effects of FaRPs on kidney functions will enhance understanding of hormonal control of osmoregulation.

Location of IR-FaRP cells

The neurosecretory cells of the CNS of M. bidentatus have been described by using histochemical staining (Price, 1977; Ridgway, 1987). The IR-FaRP cells described in this paper are some of these neurosecretory cells; their pattern of distribution is basically similar to that of other basommatophoran pulmonates such as Lymnaea stagnalis and Helisoma duryi (Schot and Boer, 1982; Buckett et al., 1990; Murphy et al., 1985; Saleuddin et al., 1992). In the cerebral ganglia of M. bidentatus, the IR-cells are present in groups located anteriorly and posteriorly. Similar groups are also seen in the cerebral ganglia of H. duryi near the endocrine growth-regulating mediodorsal cells and the ovulation-regulating caudodorsal cells. In the latter species, however, an additional large group of small IR-cells is seen in the left cerebral extension; such cells are absent in the corresponding location in M. bidentatus. The lateral lobes of the cerebral ganglia of M. bidentatus contain several large and small IR-cells, whereas those of H. duryi contain only two IR-cells, and those of L. stagnalis have none (Saleuddin et al., 1992; Schot and Boer, 1982). The lateral lobes of L. stagnalis and H. duryi are known to regulate both growth and reproductive centers of the CNS (for review, see trivolvis about 30 smaller cells have been seen (Murphy et al., 1985; Saleuddin et al., 1992). The IR-cells in the parietal and visceral ganglia of M. bidentatus are arranged in anterior and posterior groups, which appear to be similar to those in H. duryi and L. stagnalis (Schot and Boer, 1982; Buckett et al., 1990; Saleuddin et al., 1992). The location of IR-cells in the bassommatophoran pulmonates such as H. duryi and L. stagnalis is close to or within important endocrine centers that regulate growth and reproduction, and the lateral lobes of the cerebral ganglia that regulate both growth and reproduction. Numerous IR-fibers emanate from the CNS and terminate as varicosities in the nearby connective tissue, suggesting that the FaRPs are released into the hemolymph that perfuses the CNS. The FaRPs may also participate in regulating other endocrine centers of the CNS.

Acknowledgments

This study was supported by grants from York University (to HRK), NSERC, Canada (to ASMS), and NIH, USA (to MJG). We thank Dr. B. G. Loughton, York University, for his helpful comments on the manuscript and Ms. Mary-Lou Ashton, York University, for her technical help.

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Author:Khan, Hamid R.; Price, David A.; Doble, Karen E.; Greenberg, Michael J.; Saleuddin, A.S.M.
Publication:The Biological Bulletin
Date:Apr 1, 1999
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