Osmoregulation and FMRFamide-Related Peptides in the Salt Marsh Snail Melampus bidentatus (Say) (Mollusca: Pulmonata)

10 Pages·1999·4.2 MB·English

by  H R Khan

Reference: Bio/. Bull. 196: 153-162. (April 1999)
and FMRFamide-Related
Osmoregulation Peptides in
the Salt Marsh Snail Melampus bidentatus (Say)
(Mollusca: Pulmonata)
HAMID R. KHAN1'*. DAVID A. PRICE2, KAREN E. DOBLE2, MICHAEL J. GREENBERG2.
AND A.S.M. SALEUDDIN1
^Department ofBiology. York University, Toronto, Ontario, Canada M3J IPS: and
2Whitney Laboratory, University ofFlorida, St. Augustine. Florida 32086
Abstract. The pulmonate snail Melampus bidentatus oc- Introduction
cupies the high intertidal zone of salt marshes in a nearly
terrestrial environment. The hemolymph osmolarity of the Melampus bidentatus is a common amphibious pulmo-
snails collected in the field paralleled that of the adjacent nate snail. Its habitat is the high intertidal zone of salt
water and was affected by the tides and precipitation. The marshes, and it ranges from Nova Scotia, Canada, to the
snails initially gained or lost weight when submerged in coast ofthe GulfofMexico in Texas (Apley. 1970; Hilbish.
hypo- or hyperosmotic media, respectively, but returned to 1981). Melampus belongs to the primitive family Ellobiidae
their original weight after 24 h. The content oftheir immu- and is believed to be related to an ancestral marine strain
noreactive (IR)-FMRFmide-/\elated Peptide.v (FaRPs) was that colonized the intertidal habitat, giving rise to the land
measured in various tissues by radioirnmunoassay, and IR- snails and then to the freshwater snails (Morton, 1955:
FaRPs were found in every tissue analyzed. The subesopha- Russell-Hunter. 1978). Environmental factors such as tem-
geal part of the central nervous system (CNS) contained perature, salinity, and tides are extremely variable in the
more IR-FaRPs than the supraesophageal part, and the kid- high littoral habitat. But the adult snails are adapted to this
ney and the tissues ofthe reproductive tract contained more evaxrtiraetmioens,oafnd-c1a2nCsutrovi4ve0Cf.orssuebvemrearlgednacyes aitn t2e5m%pe-r1a0tu0r%e
CthNaSn,otkhiedrnepye,ripahnedralhetmisoslueysm.pThhewelreveelhsiogfheIrR-inFasRnPasilsintthhaet (sPeraiwcaet,er,19a8n0d;deMscicMcaathioonnata0nd% rReulsastievlel-hHuumnitdeirt,y f1o9r812)7.-3A6l-h
were immersed in higher concentrations ofseawater. Many
IR neurons are present in all ganglia ofthe CNS except the though adults ofM. bidentatus can endure a terrestrial life,
its planktonic veliger larvae are restricted to the aquatic
pwlietuhrianltghaengCliNaS, aannddIiRtsnceounrniteecstiavree teixstseunesisvheelayth.disTthreibuvtiesd- habTihtaet onafteusrtaularhiiessto(rRyusosfelMl-.Hubnitdeernteattauls.. s1u9g7g2e)s.ts that in
ceral nerve from the visceral ganglion is immunoreactive estuarine and semiterrestrial molluscs, osmotic and volume
and could be seen to innervate the kidney, which contains regulation must be particularly responsive. Moreover, scat-
IR-varicosities. An osmoregulatory role for the FaRPs is tered evidence suggests that the family of neuropeptides
suggested. related to FMRFamide is involved in this regulation. First.
in a freshwater pulmonate snail, Helisoma duryi, FMRF-
Received 7 January 1998; accepted 27 January- 1999. amide causes waterretention in the kidney in vitro (Saleud-
*To whom correspondence should be addressed. E-mail: hamid(S' din et al.. 1992). Further, the level of immunoreactive,
luring.sci.yorku.ca FMRFamide-related peptides (IR-FaRPs) in the kidney of
Abbreviations: One-letter abbreviations ofthe amino acids are used to Helisoma trivolvis kept in hypoosinotic medium is lower
astlabtuemtihne;pCeNptSi.decesnetqruaelncneesr.voAusSWs.ystaretmi;ficFiaalRPsse.awFatMeRrF;mnBiSdAe.-rbeolvianteedse/>reupm- than that in the kidneys ofsnails kept in isosmotic medium
tide.s; IM. incubation medium; IR. immunoreactive: NGS. normal goat (Madrid et al., 1994). The IR-FaRPs have also been local-
serum: PBS. phosphate-buffered saline: RIA. radioirnmunoassay. ized in the kidneys of the terrestrial pulmonate snail Helix
153
154 H. R KHAN ET AL.
aspersa (Lehman and Price, 1987), and in the central ner- later). The kidney and CNS were removed from a snail
vous system (CNS) of the veligers of M. bidentatns (Mof- within 3-5 min, and were used for FaRP studies.
fett, 1992). Finally, the regulation ofhypoosmotic cell vol-
ume by cardiac muscles ofthe clam Mercenaria mercenaria Osmotic conditions
is potentiated by FMRFamide (Deaton. 1990). Snails that had been maintained under terrestrial condi-
SinceM. bidentatns inhabits highly variable osmoticcon-
tions (described above) were submerged for various periods
dItinhteitophnirsso,ppoitaspiaselra,tnhaawtpeptrhhoeparvFieaaRtsePtsusdpiheeacdiv,eesbawoitrthohleiwnihnithcoehsmtfooireefldugrutalhanetdritotenhs.et (cAomSnatWxaiin(medurisml,u,tie4oancdshamysaw;ditemhiwniatihnmydulimsotni3llshec)drewieannt.e1r0T)%h,ien52l05e%0v,-elmsolropfl1a0sIt0Ri%-c
tliaobnoroantotrhya,ttohfetihneflhueemnocelyomfpthh.e Wamebiheanvteotshmeonticcomcpoanrceendtrtah-e FaRPsweremeasured (described later) after24h ofosmotic
treatments. Hemolymph was collected as follows. The
levels of IR-FaRPs in the CNS, hemolymph, and kidney of
snails were pricked in the head area with a sharp needle.
snails maintained in media of different osmotic concentra-
tions. We also report IR-FaRP-staining of neurites in the Each animal was then quickly placed in a 0.5-ml polypro-
kidney, and of neurons in the CNS of adult snails. ptuybleenewamsictrhoecnentprliafcuegdeitnubaelwairtghera(h1o.l5emiln)itspobloytptroomp.ylTehnies
microcentrifuge tube. The nested tubes and the snail were
Materials and Methods spun at 500 X g for 2-5 s. Then the inner tube containing
the snail was removed, and the outer tube containing the
Animals and media hemolymph was spun foran additional 5 min. About 30-50
alerneDgauthr1si0,n0g1m0.J0aXnu2ar02y.0mtmoimn)Aaprwsieallr,temMca.orlslbehicdtoeenndttaahttenrsMaanatddaounlmztsafsro(rsmihevalenlr oW/isedmsoocftoihrcemMprooeldsyeslmurpeh5s3c0oo0fulv1d0a-pbjouelrcosplarlmeepscsltueersdefworesormmeoemmaeectahesrusnrae(idlL.ogiTanhne,a
estuary near Crescent Beach, Florida (294()' N : 8113' W). mUtaanhy).dAafytsertthheereabflteeerd.inTg,oalmleaosfutrhee stnhaeilesfffeecdtsanodfloisvemdotfiocr
Experiments with the freshly collected snails were per- water exchange on weight, the snails were weighed with a
formed nearby, at the Whitney Laboratory ofthe University Mettler AE163 balance after the visible water and mucus
ofFlorida. At YorkUniversity, the animals were maintained had been removed from their shells and feet with adsorbent
at 22C, 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 tissue.
20cm H) coveredwith nylon screens. The floorofeach tank
Radioimmnnoassay
was covered with a layer of crushed oyster shells (poultry
feed) graded in thickness from 0.5 cm atone end ofthe tank Pulmonate molluscs contain two majorclasses ofFaRPs:
to 3.0 cm at the other. A volume of50% artificial seawater the tetra-FaRPs (FMRFamide and FLRFamide) and the
(ASW; Instant Ocean, Aquarium Systems, Ohio) was added hepta-FaRPs (XDP[F/Y]LRFamide, where the N-terminal
tocoveraboutone-thirdofthecrushedoystershells. Slanted residue X is pQ, S, N, or G). The hepta-FaRP analog with
against the walls ofeach tank were 4-6 broken, irregularly the glycyl residue occurs only in snails of the subclass
shaped pieces of clay plant-pot (about 10 cm in diameter). Basommatophora (like M. bidentatiis), and these animals
Most snails crawledto terrestrial conditions above the water lack the analog with the pyroglutamic acid (pQ) residue,
level on the walls of the tank and on the dark side of the which occurs only in stylommatophorans (Price et al,
moist pieces of clay plant-pot. Distilled water was added 1987a, b). To ensure that all of the FaRPs would be de-
daily to maintain osmolarity and water level, and the snails tected, we used S253 antiserum in the radioimnumoassay
were fed fish-food and boiled lettuce ad libitem. The snails (RIA). The antiserum was raised to thyroglobulin-peptide
were acclimated to laboratory conditions for at least 2 (synthetic analog YGGFMRFamide) conjugate in a rabbit.
weeks before experimentation. It has high affinities for both FMRFamide and GDPFLRF-
The kidney is embeddedjust under the dorsal surface of amide. and was used in the assays at a dilution of 1:10,000.
the mantle tissue, and the CNS is located under the skin, lodinated pQYPFLRFamide was used as tracer (for details
between the tentacles. Before dissection, the snails were see: Price et ai, 1990; Lesser and Greenberg. 1993).
chilled on ice for5-10 min, which reduced theirmovements The protocol of Madrid et al. (1994) was followed in
and mucus secretion; no other anesthetic was used. processing the tissues. After dissection, each tissue sample
Deshelled snails were pinned on a dish lined with Sylgard was placed in aplastic tube ( 1.5 ml)containing HPLC grade
(Dow Corning, Canada) and dissected in filtered isosmotic acetone (1 part tissue: 4 parts acetone) and frozen immedi-
ASW. We adjusted the osmolarity ofthe ASW with distilled ately at -80C for at least 24 h. The acetone extracts were
water to be isosmotic with that of the hemolymph, which spun for 5 min at 5,000 X g, and the supernatants were
varies with the medium or relative humidity (described collected and then dried in a Speed-Vac centrifuge. Each
OSMOREGULATION AND FMRFAMIDE IN A SNAIL 155
M
pellet was dissolved in 100 ju.1 RIA buffer |().()1 sodium 0.05% pronase in isosmotic saline for 10 min, then fixed in
phosphate with 1% bovine serum albumin (BSA), 0.9% Bouin-Hollande (see above) for 12 h. After fixation, the
sodium chloride. 0.01% merthiolate. and 0.025 M sodium tissues were rinsed in saline for 10 min, then treated in 0.1%
EDTA|. Aliquots of 10 /j.1 were placed in glass tubes to trypsin in saline for 30 min, and rinsed well in saline for 10
which 100 y,\ tracer ( 10.000 cpm) (in RIA buffer) and 100 min. A preincubation in IM for6-12 h at4C was followed
/Ltl diluted anti4seCrum (in RIA buffer) were added. The tubes by incubation in primary antibody (hepta-FaRP-specific CK
were stored at overnight: on the following morning. 1.0 antiserum; 1:200in IM) for24 h. Afterrinsing several times
ml charcoal suspension (0.25% charcoal, 0.025% dextran. in 1% Triton X 100 in PBS for 1 h, the tissues were
M
0.01% merthiolate in 0.1 sodium phosphate. pH 7.5) was incubated in secondary antibody (1:80 in IM) for 6 h,
added to each tube. After 10 min. the mixture was centri- followed by repeated rinses in IM. When whole-mount
fuged at 2500 X g for 15 min at 4C, and the supernatant tissues were incubated for more than 60 min. this was done
was counted in an LKB MiniGamma counter. All statistical in the dark at 4"C. The tissues were mounted in mounting
comparisons were made with a one-way analysis of vari- medium (see above) between two coverslips (one rectangu-
ance (ANOVA); when the ANOVA showed a significant lar. 60 mm X 22 mm; and one circular, 18 mm). To prevent
difference, the effect was followed with the Bonferroni tissue distortion, the weight of the coverslip was supported
multiple comparison post test using a statistical software by four pieces of broken coverslip (0.5 mm diameter).
package (InStat. GraphPad Software, San Diego, Cali- The larger coverslip was affixed with adhesive tape on an
fornia). aluminum slide (36 mm X 80 mm X 1 mm) with a central
25-mm circular window. The smaller circularcoverslip was
Immunocytochemistry placed face down in this window, so these preparations
could be flipped for viewing from either side.
Isolated CNS and kidney tissues from animals in labora- For controls, either pre-immune rabbit serum (1: 200 in
tory terrestrial condition were either embedded in paraffin IM) or IM alone was used in the primary incubation; in
and sectioned, or examined in whole mount. Sections were either case, the tissues were then stained with secondary
prepared as follows. The tissues were fixed for 12-18 h in antibody as described above. The controls showed no stain-
modified Bouin-Hollande [7% picric acid. 2.5% copper ing. The specimens were viewed and photographed with
acetate. 2% formaldehyde (freshly prepared from parafor- either a Leitz epifluorescence microscope or a Bio-Rad
maldehyde), and 1.5% glacial acetic acid, in phosphate- MRC 600 confocal microscope. The IR-FaRP cells were
M M M
buffered saline (PBS) (0.2 NaCl. 0.003 KC1, 0.002 mapped from the serial sections and from whole-mount
KH2PO4. 0.02 M Na2HPO4, 0.001 M CaCL. 0.001 M images, and were measured with the calibration marker
MgCL, pH 7.5)], then dehydrated in graded ethanol solu- from the confocal images.
tions and embedded in paraffin. Serial sections (10 ^im)
were cut and mounted on coverslips (22 mm X 22 mm) Results
coated with 0.5% gelatin. 0.5% chrome alum, and 0.01%
Osmoregulation
formaldehyde. The deparaffmized sections were treated
with 100% methanol and 0.1% H2O2 for 5 min. hydrated in The high-tide location ofthe marsh where M. bidentatus
a graded series of ethanol solutions, kept 30 min in an was collected has an uneven surface with a shallow slope,
incubation medium (IM) consisting of PBS containing 2% and contains abundant decaying organic matter ofplant and
Triton X 100 (Sigma Chem., St. Louis, MO), 5% normal animal origin. The osmolarity and pH of the waters in the
goat serum (NGS). and 1% BSA. The sections were incu- habitatvariedwith timeand location intheobservation area.
bated for 1 h at 22C with hepta-FaRP-specific CK anti- During the sunny days of April, the average osmoiarities
serum, which was raised in a rabbit to the peptide CKQD- were 1200 26 mosm/kg H2O (mean standard error of
PFLRFGK (a gift from Dr. G.A. Cottrell. University of St. mean, n = 50) (range = 1015-1650mosm/kg FLO), and the
Andrews. Scotland) (Cottrell et al, 1994). The primary pH ranged from 6.0-7.2: at 1200-1400 h, the ambient air
antiserum was diluted 1:200 in IM. Aftermany rinses in IM temperature was 31 4C, and the soil surface and water
for 10 min, the sections were incubated for 1 h at 22C in temperatures were 34.2 1.3C. At the same time, the tidal
fluorescein-conjugated goat antirabbit-serum (Sigma) di- seawater coming through the Matanzas inlet had a consis-
luted 1:80 in IM (secondary antibody). The coverslips were tent osmolarity of980 5 mosm/kg FLO. a temperature of
rinsed well in PBS and mounted on glass slides in 5% 22 2C, and a pH of7.5 0.1. The variables that altered
polyvinyl alcohol. 30% glycerol, and 0.17r phenylenedi- the osmoiarities ofthe water in the habitat and hemolymph
amine in PBS (mounting medium). of snails were tide, temperature, and precipitation. Most
Whole mounts were prepared as follows. The tissues snails in the field were out of the water, on grass stems or
were treated with proteases before and after fixation (Long- higher points of the uneven ground, while a much smaller
ley and Longley, 1986). The tissues were first treated in number were creeping and feeding underwater. The osmo-
156 H R, KHAN ET AL.
larities ofthe hemolymph of snails from different locations Table I.
and conditions and during high and low tides were mea- Immunoreactive FaRPcontentdetectedbyS253antisemm in various
sured within 30 min after collection and compared with the tissuesofanimalsfrom terrestrialcondition in laboratory
osmolarities ofthe adjacent media. The hemolymph osmo-
larities ranged from 1 100 to 1500 mosm/kg H2O and ap-
peared to parallel that ofthe adjacent medium. A few hours
ofheavy rainfall reduced the osmolarity ofthe water in the
habitat to 750 40 mosm/kg H O; the hemolymph osmo-
2
larities also declined to 850 50 mosm/kg H2O. Most
snails in the laboratory crawled to the dry parts ofthe tank
(see methods section) and stayed away from the water; thus
they sought terrestrial conditions. Butafew snails were also
seen feeding and crawling underwater. The hemolymph
osmolarity of the laboratory snails that sought terrestrial
conditions was much lower (450 25 mosm/kg H O) than
2
that of field snails (1200 100 mosm/kg H O) collected
2
away from the water. When laboratory snails from terres-
trial conditions were held for24 h in 10% or50% ASW, the
osmolarity ofthe hemolymph paralleled, and was about 150
mosm/kg H2O hyperosmotic to, that of the medium. Snails
in 10% and 50%- ASW gained weight during the first 3-6h,
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
(Fig. 1).
Levels ofIR-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
Weight changes
100%ASW
50% ASW
10%ASW
7.5-
<u
OSMOREGULATION AND FMRF.uiiDE IN A SNAIL 157
IR-FaRP in CNS
5,40-
158 H. R. KHAN ET AL.
Ptn arranged in anterior and posterior groups in both ganglia
(Figs. 7. 14). Numerous IR-neurites emanate from the CNS
into the surrounding connective tissue and appear to termi-
nate in varicosities (Fig. 15).
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 al-
ways submerged, suggesting that they crawl in and out of
water intermittently. The osmotic concentration of the he-
molymph was correlated with that of the adjacent aqueous
medium. Moreover, IR-FaRPs are present in the CNS, kid-
ney, and hemolymph, and the levels of these peptides are
also correlated with the osmotic concentration ofthe ambi-
ent medium.
Osmoregulation and kidney
When the osmolarity ofthe external medium changes, the
hemolymph osmolarityofM. bidentatusalsochanges due to
inward and outward movements of water from its body.
Such conditions must be offset by various adjustments:
regulation ofurine production by its kidney is one ofthem.
The IR-staining ofthe 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 othersmall
areas that are probably bundles of smooth muscles. The
smooth muscles of the kidney of the basommatophoran
snail Helisoma duiyi are innervated by FMRFamide-immu-
noreactive axons (Saleuddin etai. 1992). In this species the
/'/( 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
Figure 7. Showing the schematic distribution of the IR-cells in the smooth muscles in various species. They are also known to
CNS. BG. buccal ganglia; CBC. cerebro-buccal connective; CC, cerebral regulate salivary glands and ion channels (Bulloch et al.,
commissure; Cn, cutaneous nerve; CPC. eerebro-pedal connective; CPIC. 1988; Green et ai, 1994; Price and Greenberg, 1994;
cerebro-pleural connective; DB, dorsal bodies; LCG, left cerebral gan-
glion; LL.lateral lobe; Ln, labial nerves; LPdG,leftpedalganglion; LP1G. Lingueglia et al.. 1995). In the pulmonate kidney they are
left pleural ganglion; LPrG, left parietal ganglion; On, optic nerve; PC, probably involved in regulating smooth muscle contractions
pedalcommissure;Pdn.pedalnerve;Ptn,peritentacularnerve;Prn.parietal forperistalsis and the production ofhydrostatic pressure for
nerves; PVn, parieto-visceralnerve; RCG. right cerebral ganglion; RPdG. urine formation, as well as in regulating ion channels for
right pedal ganglion; RP1G, right pleural ganglion; RPrG. right parietal selective secretion and reabsorption of ions from urine.
ganglion; SPC. sub-pedal commissure; Tn, tentacularnerve; VG. visceral
ganglion; Vn, visceral nerve. Not to scale.
Osmoregulation and IR-FaRP levels
Buccal ganglia. In each buccal ganglion. 3 intermediate The CNS and hemolymph displayed the highest and
sized (18 2 /xnii IR-cells, and many IR-fibers and vari- lowest levels of IR-FaRPs, respectively. Organs containing
cosities are seen (Fig. 111. involuntary muscles, such as the reproductive tracts and
Pedalganglia. Groups of6-10small IR-neurons are seen kidney, have high levels of IR-FaRPs. These organs appar-
at the periphery of each pedal ganglion (Figs. 7, 12). ently receive FaRPs through direct innervation by neurons
Parietal ami visceral ganglia. More large and small IR- from the CNS. Thus, in these target tissues, the FaRPs may
cells were seen in the right parietal andvisceral gangliathan act as neuromodulators or as paracrine agents. Numerous
inanyotherganglion (Figs. 7, 13). The left parietal ganglion IR-FaRP processes terminating in varicosities in the con-
contains only small IR-neurons (Fig. 7). The IR-cells are nective tissue around the CNS may constitute a diffuse
OSMOKMiH .\IION AM) I-MRI-\\IIIM IN A SNA11 159
Figure 8. Groups of IR-cells (large arrows! in a cross section ofthe left and right cerebral ganglia; thin
arrows point to IR-material in the commissure (cm) and neuropile. Scale bar: 100 fj.ni.
Figure 9. Optical section ofthe left cerebral ganglion showing a large IR-cell (large arrow) among small
IR-FMRFamide cells and their neurites (small arrows). Scale bar: 20 /urn.
neurohemal area and account for the hemolymph IR-FuRP sodium-influx-stimulating peptides are present in this snail
levels. (De With et al., 1994). In Aplysia califomica, R15al neu-
The levels of IR-FaRPs in the CNS, hemolymph, and ropeptide causes water retention, thus having an osmoreg-
kidney varied under different osmotic conditions. The IR- ulatory role (Weiss et al., 1989). Additionally, in this spe-
FaRP levels were reduced in hypoosmotic conditions com- cies various kidney functions are modulated by L10 and
pared with those in hyperosmotic or terrestrial conditions. LUQ neurons, and the latter neurons are immunoreactive to
Terrestrial or hyperosmotic salt-water conditions produced FMRFamide (Koester and Alevizos, 1989; Giardino et al.,
threats of desiccation and salt loading in snails, whereas 1996). Su,ch systems include increased circulation ofhemo-
hypoosmotic conditions had the opposite effect. The lymph forultrafiltration, subsequent secretion andreabsorp-
changes ofIR-FaRPlevels in thetissues ofM. bidentatusby tion ofions andorganic matters, andexpulsion offinal urine
osmotic conditions may reflect osmoregulation by the snail. (Khan and Saleuddin, 1979). Bioassay studies assessing the
Since IR-FaRP levels increased in the CNS, hemolymph. effects of FaRPs on kidney functions will enhance under-
and kidney under conditions ofincreased water losses such standing of hormonal control of osmoregulation.
as in terrestrial or 100% ASW conditions, the FaRPs may
have an antidiuretic function. //; vitro culture of the kidney Location ofIR-FaRP cells
tissues of Helisoma with synthetic FMRFamide increased
its intracellular water uptake, and FMRFamide has been The neurosecretory cells of the CNS of M. bidentatus
suggested to have an antidiuretic role (Saleuddin et al., have been described by using histochemical staining (Price,
1992; Madrid et al, 1994). Antidiuretic and diuretic activ- 1977; Ridgway, 1987). The IR-FaRP cells described in this
itiesofFMRFamide and GDPFLRFamide respectively have paperare some ofthese neurosecretory cells; theirpattern of
been demonstrated in nephridia of a leech (Salzet et al., distribution is basically similar to that ofother basommato-
1994). Basommatophoran pulmonates such as Melampus phoran pulmonates such as L\mnaea stagnalis and Heli-
contain GDPFLRFamide (unpub. obs., D. A. Price). Both soma duryi (Schot and Boer. 1982; Buckett et al., 1990;
tetra- and hepta-FaRPs are present in the osmoregulatory Murphy etal., 1985; Saleuddin et al.. 1992). In the cerebral
tissues of Helisoma (Madrid et al., 1994). The tetra- and ganglia ofM. bidentatus, the IR-cells are present in groups
hepta-FaRPs are expressed in exclusive neurons and may located anteriorly and posteriorly. Similar groups are also
have different actions in a target tissue (Greenberg and seen in the cerebral ganglia of H. duryi near the endocrine
Price. 1992; Benjamin and Burke, 1994; Price and Green- growth-regulating mediodorsal cells and the ovulation-reg-
berg, 1994). The precise mechanism ofthe tetra- and hepta- ulating caudodorsal cells. In the latter species, however, an
FaRP actions in molluscan kidneys awaits future studies. In additional large group of small IR-cells is seen in the left
molluscs, osmoregulation by the kidney appears to be a cerebral extension; such cells are absent in the correspond-
combined effect of many physiological systems regulated ing location in M. bidentatus. The lateral lobes of the
by different hormones. In Lymnaea stagnalis, a neuropep- cerebral ganglia of M. bidentatus contain several large and
tide that stimulates sodium uptake by the skin has been small IR-cells, whereas those ofH. duryi contain only two
sequenced; furthermore, axons that release immunoreactive IR-cells. and those of L. stagnalis have none (Saleuddin et
160 H. R. KHAN ET AL.
Saleuddin et /., 1994; Saleuddin and Ashton, 1996). In the
buccal ganglia of M. bidentatus, three IR-cells have been
seen; in those ofH. dur\i two cells occur: and in those of//.
Figure 10. Optical section ofthe right lateral lobe and a partofright
cerebral ganglion (eg) showing large (large arrows) and small (small
arrows) IR-cells. Note that the large cells are intensely stained and the
smallcellsarelessintenselystained;thecellscanberecognizedbythelack
of staining in their nuclei. Orientation of the tissue is shown by the two
axes showing anterior(A) to posterior (P). and left (L) to right (R) sides.
Scale bar: 20 jum.
Figure 11. Three cells (arrow heads) and many IR-neurites and vari-
cosities (thin arrows) are shown in the whole mount of right buccal
ganglion. Orientation ofthe tissue is shown by the axes showing anterior
(A)Fitgouproest1e2r.iorC(rPo)s.sasnedctlieoftn(oLf)thtoepriegdhatl(gRa)ngsliidaess.hSocwailnegbaar:sm5al0l/uIrRn-.cell midFirgeugrieon13o.fthCerorisgshtsepcatriieotnalof(pt)hegaanngtleiraiosrhroewgiinongoIfR-tcheellvsis(ctehrianla(rvr)owasn)d.
(large arrow) and many IR-neurites (thin arrows), ao, aorta. Scale bar: Scale bar: 50 p.m.
250 fim. Figure 14. Cross section of the mid region of the visceral (v) and
posterior region of the right parietal (p) ganglia showing IR-cells (thin
arrows). Scale bar: 50 /jm.
ul.. 1992; Schot and Boer, 1982). The lateral lobes of L Figure 15. Whole mount of the cerebral commissure (com) showing
stagnalis and H. diuyi are known to regulate both growth (eaxrtreonwssi)v.eOIrRi-ennetuartiitoensoafntdhevatriiscsouseitiisesshwoiwthninbythtehecotnwnoecatxievsesihnowadijnagceannttearrieora
and reproductive centers of the CNS (for review, see (A)toposterior(P). and left (L) toright(R) sides. Scalebar: 50 ^m.
OSMOREGULATION AND FMRFAMIDE IN A SNAIL 161
trivalvis about 30 smaller cells have been seen (Murphy et Greenberg, M. J., and I). A. Price. 1992. Relationships among the
ul., 1985: Saleuddin et al.. 1992). The IR-cells in the pari- FMRFamide-like peptides. ProK. Bruin Kes. 92: 25-37.
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hemolymph that perfuses the CNS. The FaRPs may also Lingtuheeglsniaai,l HE.e,liGx.asCphearsmap.igJ.nyEx,p.M.BioLla.zd17u8n:sk2i0,5-a2n3d0.P. Barbry. 1995.
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