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Salinity induced cytosomal morphogenesis in the oyster Crassostrea virginica (Gmelin)
Camp. Biochem. Phvsol. Vol. 85A, No. 3, pp. 513-522, 1986
0300-9629/86 $3.00 + 0.00 Pergamon Journals Ltd
Prmted in Great Biitain
SALINITY INDUCED CYTOSOMAL MORPHOGENESIS THE OYSTER CRASSOSTREA VIRGINICA (GMELIN)
IN
ANTHONYA. PAPARO Department
of Anatomy,
School
Illinois University, Carbondale, Sciences
Consortium,
of Medicine
and Department
of Zoology,
College of Science, Southern Marine Environmental Island, AL 36528, USA
IL 62901, USA and Marine Science Program, Dauphin
Island
Sea Lab.,
(Receitled I8 February
Dauphin 1986)
Abstract-l. Lamellar transformations of cytosomes, DOPA decarboxylase and lateral ciliary activity were measured in neuronal elements and/or ctenidia of the oyster, Crassostrea oirginica (Gmelin). 2. Rapid salinity changes, exposure to light, hypoxic conditions and perfusion with EGTA (a calcium chelator) and A-23187 (a calcium releasing agent) increase lamellar transformations. 3. This is accompanied by a decrease in decarboxylase and lateral ciliary activity. 4. The magnitude of the cilioinhibition and decarboxylase activity is directly related to the number of secondary conversions, habitat and reacclimation salinities.
INTRODUCTION
water, and the dish was placed in a holder fastened to the adjustable stage of a microscope. The ctenidium was seen to consist of numerous parallel ctenidial filaments. Three major types of ciliated cells were clearly distinguished: frontal, laterofrontal and lateral. This study was concerned with the rate of beating of lateral cilia which beat in such a way that metachronal waves appear to travel in opposite directions along the two sides of each ctenidial filament. The rate of ciliary beating in beats per second was estimated by synchronizing the rate of flashing of a calibrated, stroboscopic light used in place of the substage lamp, with the rate of beating of the cilia. Synchronization was achieved when the metachronal wave appeared to stand still. Measurements were made from dorsal to ventral border, and from left to right across the field, giving 12 sets of measurements per animal. The Petri dish was perfused with sea-water via a four channel variable speed pump with a flow rate of ca 0.5 ml/min. The planetary gear mechanism of this pump ensures minimum pulsing and stable drift-free flow permitting accurate measurements of ciliary movement. A positive displacement piston metering pump with micrometric adjustment permits rapid removal of solution from the Petri dish. A continuous flow across the dish can be maintained with constant temperature by means of stainless steel tubing which cools down the movable platform (ca I .tYC/O.S min) by means of a circulating water from a cooling system.
There is physiological evidence that lateral ciliated cell-system on the ctenidium is individually controlled (Spittstijsser, 1913; Setna, 1930; Paparo, 1972; Paparo and Dean, 1984). In addition, the central and peripheral nervous system of many molluscs are highly pigmented (Arvanitaki and Chalazonitis, 1960). Neurophysiologically, molluscan brain cytosomal pigment renders nerve cells sensitive to incident light (Kennedy, 1961; Paparo, 1985). Other experiments (Paparo and Murphy, 1976, 1978) have indicated that the rate of beating of lateral cilia on ctenidia can be influenced by radiation of certain wavelengths. The photo-response has been shown to be mediated by neuronal pigments (cytosomes) which are morphologically transformed into lamellar organelles that release sequestered calcium ions intracellularly (Brown et al., 1975; Paparo, 1985). The purpose of this study is to elucidate the effect of rapid change in salinity, in habitat oyster populations, on cytosomal transformations, decarboxylase activity and ciliary activity in neuronal tissues (both central and peripheral) and/or in the ctenidium.
MATERIALS
Drug perfusion
AND METHODS
Isolation of ctenidium and measurement
of ciliary activity
Oysters (Crussoslrea G-ginica Gmelin) were collected from natural subtidal beds on the north side of Dauphin Island, Alabama. The oysters were placed in standing sea-water aquaria with undergravel filters filled with Instant Ocean artificial sea-water at the appropriate salinities at 23-50°C for 3-5 days before use in a particular experimental procedure. Before each experiment, oysters of 5-8 cm in length were shucked by breaking the ligament with an oyster knife and cutting the adductor muscle at the points of insertion on the upper and lower valves. The whole tissue was maintained in a tray of clean aerated artificial sea-water at the particular habitat or reacclimation salinity and 22°C. The mantle was excised from one side to expose the ganglia, nerve and ctenidium. This preparation was pinned to rubber mats glued in the bottom of a Petri dish containing sea513
Drugs were added by replacing the content of the perfusion dish with the desired concentration of the drug in sea-water at 22”C, and perfusion was continued with drug solution. Drugs were washed out by repeating the procedure with sea-water. The following drugs were used: IOmM ethylene glycol-bis (amino-ethylether)-N; N’tetra-acetic acid, EGTA (a calcium chelator) and IO-’ M ionophore A-23187 prepared as 10m3 M stock solution in 0.25% dimethformamide and 0.75% ethanol (a calcium releasing agent). Electrical srimulation of branchial nerve For nerve stimulation, an electrode was placed on the branchial nerve at a point of its emergence from the visceral ganglion. A stimulator supplied electrical pulses with the following characteristics: 10.0 V, 5.0 biphasic pulses per second, 2 msec duration.
514
ANTHONY A.
Yellow pigment extraction and measurement Yellow pigment color of visceral ganglion and nerves of Crassostrea was examined in living preparations. Yellow neuronal pigments (cytosomes) were examined in squash preparations, cryostat sections, and under fluorescence microscopy. In latter investigation, ganglia were frozen in Freon 22, cooled by liquid nitrogen and lyophilized at 40°C at 1O-4 torr for 2 hr. The yellow autofluorescent neuronal pigment granules could then be visualized with a Leitz MPV II microscope equipped with a fluorescence module. The fluorophore that was regenerated by re-exposure to formaldehyde vapor at 80°C for I hr was due to monoamines, and could be readily distinguished from cytosomal pigment autofluorescence (Falck, 1962). The pigment was collected in a petroleum ether extract and the spectra recorded using a microfluorometric method. The measurements of the yellow pigment extracts were carried out on the Leitz MPVII system equipped with microspectrofluorometry and an automatic measuring beam interference graduated filter.
PAPARO
Sections voltage.
were examined
under
TEM
at 50 KV operating
TEM autoradiographic localization Autoradiographic localization of [3H]leucine was performed on all neuronal tissues. These were incubated in 10m6 M [‘Hlleucine in sea-water at 22°C for 10 min. They were then washed in sea-water solution with six changes of solution, each for 5 min. They were fixed and embedded for TEM as described above, except that the fixing solutions were made hypertonic by addition of 9.0% sucrose. Under these conditions of fixation, movement of the [-‘H]leucine was halted. Thin sections were cut and placed on 200 mesh grids and coated with a photographic emulsion (diluted 1:6) by looping. The emulsion coated grids were exposed for 46 weeks in dry light-tight boxes in an atmosphere of 100% CO,. After photographic development for 3-5 min at 22”C, sections were stained with uranyl acetate and counterstained with lead citrate.
Dark/light preparations Dark-adapted ctenidium preparations were maintained under illumination of a photographic safe light with a dark red filter. In addition, isolated ganglia were incubated for various lengths of time in sea-water in total darkness. Illuminated ganglia were prepared by dissection from oysters in room light. The temperature of gangliacontaining solutions in light or darkness stayed at approx. 22°C. Anaerobic conditions Anaerobic conditions were maintained in a vessel containing eight times more sea-water than body weight of the oysters. The surface of the water was then covered with a paraffin oil layer of l-2 cm in thickness. Under such circumstances oxygen content of sea-water is used up and total anoxia sets in within 48 hr. The animals are considered as living up to the moment that they cease to react with movement upon influence of mechanical stimulation.
Differential and gradient centrifugation of neuronal elements Neuronal components of oysters were kept in ice-cold sea-water during dissection. Ganglia and nerves were blotted with filter paper and homogenization was carried out in 0.05 M sucrose in a glass tube fitted with a Teflon pestle to give a 10% tissue suspension. The supernatant fluid was collected and centrifuged again at 17,500 g for 30 min. The pellet (P) was saved for subfractionation by resuspending it in 0.05 M sucrose. This suspension was layered onto a discontinuous sucrose gradient containing 1.20 and 1.50 M sucrose. Centrifugation was at 50,OOOn for 2 hr. which yielded three fractions: PA, PB, PC. The PB pellet was transferred to 2.5% glutaraldehyde in 0.20 M S-collidine buffer, pH 7.8, for lf hr. Samples were then processed for TEM as described previously.
RESULTS
Measurement of decarboxylase activity
General organization of ctenidium and nervous system
DOPA decarboxylase activity assays were made using extracts of ganglia, connectives, and nerves in 5 ml homogenate (average 40mg), 0.85 ml of phosphate buffer, and 0.1 ml of 0.01 M DL-[IW] DOPA (total radioactivity, 1.5 x lo6 c.p.m.) were added to the test tube. For ctenidium assays 0. I ml of ctenidial homogenate (average 40 mg), 0.8 ml of phosphate buffer, and 0.1 M DL-[I14C] DOPA were added to the test tube. These test tubes were sealed with rubber covers, from each of which was suspended a well containing 0. I ml of 1 M KOH. The extracts were incubated at 38°C for 24min. The reaction was stopped with the introduction of 0.2ml of 1 M H,SO, solution through the rubber cover. The test tubes were shaken on a shaker for 55 min. The well containing the trapped 14C0, was then dipped in lOm1 of liquid scintillation medium and the radioactivity was determined in a liquid scintillation counter. Counts on heat-inactivated extracts were performed simultaneously.
The general organization of the ctenidium is shown in Fig. la,b. More detailed accounts may be found in Gray (1924, 1929), Lucas (1931a,b, 1932) and Paparo and Dean (1984). In cross-section each of two ctenidia (Gi) appears in the form of a “W” suspended from the dorsal margin of the oyster. The inner and outer surfaces (called lamellae) are made up of a large number of ctenidial filaments (GF) running lengthwise in the lamellae. The branchial nerve which arises from the visceral ganglion VG runs obliquely downward and backward to enter the afferent blood vessel (ABV) in the dorsal margin of the ctenidium axis. The valves are held in place via the anterior adductor (A Ad M) and posterior adductor (P Ad M) muscles. In addition to VG and BN, there are other elements of the nervous system: cerebral ganglion (CC), pedal ganglion (PG), cerebrovisceral connective (CVC), and cerebropedal connective (CPC). The VG is covered by a loose connective tissue sheath. Beneath the sheath, a closely adhering capsule completely encases the ganglion and is not removable without disrupting the ganglion. There is a rind of nerves, beneath the capsule, whose axonal processes form part of the fibrillar neuropil in the interior of the ganglion. The fibrillar neuropil constitutes the beginning of the BN (Fig. 2a). Clusters of nerve cells leave the VG and accompany the BN in its course to the ctenidium epithelium (Fig. 2b).
TEM preparation Samples were prepared for TEM by removing a valve and pouring the fixative (2.5% glutaraldehyde) directly onto the underlying tissues. After 15 min, ctenidia, ganglia, connectives and nerves were isolated. These tissues were fixed in 2.5% glutaraldehyde in 0.2 M S-collidine buffer, pH 7.8 (50~2000mOsm) for 2 hr. It was then rinsed in several changes of 0.2 M S-collidine buffer at 4°C for a period of 1 hr, postfixed for 1 hr in 1.0% osmium tetroxide in the same buffer, dehydrated in a graded series of ethanol, and embedded. Sections were stained with 5.0% uranyl acetate for 30 min. and counterstained with lead citrate for 5 min.
Salinity
induced
neuro-pigmental
transformations
in Crassostrea
Fig. I. (a) Dissected specimen showing the visceral ganglion (VG), branchial nerve (BN) and ctenidial lamella (CL). Arrow traces the BN from its emergence from the ventral surface of the VG along its course within the afferent branchiai blood vessel. x 30. (b) Diagram represents a cross-section of the entire animal on the half shell. Three ganglia are shown: cerebral (CG), visceral (VG) and pedal (PG). Additional components include: branchial nerve (BN) coursing with the afferent branchial blood vessel (ABV) on its way to the ctenidium (Gi) and ctenidial filaments (GF); cerebra-visceral connective (CVC), cerebropedal connective (CPC); and adductor muscles, anterior (A Ad M) and posterior (P Ad M). A-B indicates a typical section of the GF used in following the axons of the BN within the Gi. Nerve
cells and cytosomes
The nerve cells are unipolar and approximately oval in shape, measuring about 25 pm in length and from 10 to 17 pm in width. The contour of the nucleus is similar to that of the cell. Many of these nerve cells contain yellow pigment granules (cytosomes), giving the ganglion a yellow color. Golgi bodies were found to be constantly associated with cytosomes (Fig. 2~). Small vesicles, generated at the mature face of the Golgi apparatus, were observed in
close proximity and at times joined to the surface of cytosomes. The material in the small vesicles was similar in density to electron-dense material in the cytosomes. Autoradiographic studies ([3H]leucine) done at the EM level confirm that the Golgi apparatus plays an important role in the formation of cytosomes (Fig. 3a). Furthermore, the [3H]leucine label was also found associated with cytosomes in the axonal population of the BN (Fig. 3b). Structural detail of cytosomes was best preserved by using 2.5% glutaraldehyde in 0.2 M S-collidine,
516
ANTHOW A.
pH 7.8, 1lOOmOsm. There were significant ultrastructural changes due to osmolality of fixative solutions. A 500 mOsM solution caused a disruption of the outer cytosomal limiting membrane while 1OOOmOsM seemed to reduce such distortions. The use of phosphate buffers in the presence of calcium in
PAPARO
sea-water caused precipitates throughout neuronal components. The limiting membrane exhibited a characteristic trilaminar appearance. The greatest number of immature cytosomal forms were observed in dark-treated preparations (Fig. 4a). The small cytosomes which
Fig. 2. (a) Photomicrograph of the visceral ganglion just beneath the capsule is a rind of nerve cellswhose axonal processes form part of the fibrillar neuropil-in the interior of the ganglion. x 300. (b) Nerve cells dissected from beneath the capsule of the visceral ganglion. Many of these cells contain yellow pigment granules (cytosomes) which give the ganglion a yellow color in the living preparation. x 600. (c) TEM illustrating the relationship routinely observed between the Golgi bodies (G), their vesicles (GVs) and maturing cytosomes (mc). A densely staining material present in GVs appears to be the same electron-dense material observed in the cytosome matrix. x 11,625.
Salinity induced neuro-pigmental
transformations
in Crassastrea
517
Fig. 3 (a) Autoradiograph of the branchial nerve, which takes origin from the neurophil of the viscerai ganglion, showing [)H]leucine label (I) over axonal profiles and cytosomes. x 17,250. (b) Autoradiograph of a nerve cell at the EM level showing incorporation of 13Hlleucine label ft) over the Golni bodies and cytosome. x 17:256
contained both electron-dense and a fine reticular material ranged from 0.3 to 0.5 ftm in diameter. The
larger cytosomes (2 1.Opm in diameter) which contained markedly osmiophilic material, were assumed to be the mature form (Bauer et al., 1977) of this organelle. These granules have also been observed in subcellular fractions obtained from VG and BN homogenates (Fig. 4b). These isolated preparations exhibit the same alterations, but in a more rapid manner, as previously described in the intact preparations. Lamellar or paracrystalline arrangements of electron material were observed in these organelles (Fig. 4~). The numerous paracrystalline arrays gave this organelle a mottled appearance. These intermediate cytosomes having this mottled configuration have been classified as “primary conversions” (Bauer el al., 1977). Membrane assembly appeared to involve the osmiophilic component and moderately electron-dense homogeneous material. At higher magnifications, this membranous material had a trilaminar appearance. The crystalline-like arrangement occurred first, followed by membrane formations.
Globular vesicular components were seen after initial membrane formation. These latter components may be formed by swelling of precipitous laminations scattered throughout the center of the cytosomes. Those cytosomes that exhibit complete membranous transformations are classified as “secondary conversions” (Bauer rt al., 1977). Percent lameliar type cytosomes and ciliary activity in habitat populations
The neuronal components and ctenidium of 5%0 S habitat oysters significantly (P < 0.001) contained the highest populations of lamellar type organelles. Light enhanced the frequency of lamellar conversions which was accompanied by a cilioinhibitory response (Fig. 5). Microspectroanalysis of cytosomal yellow pigment, decarboxylase and ciliary activities
Microspectrophotometric measurements of the yellow cytosomal pigment showed alterations in the
518
ANTHONYA.
PAPARO
Fig. 4. (a) TEM of branchial nerve within the afferent blood vessel showing cytosomes (7) within its axonal profiles. x 11,250. (b) Cytosomes (t) subjacent to the lateral ciliated cell. x 18,750.
absorption spectra for both changes in photoperiod and rapid salinity changes (Figs 6a,b and 7a). An increase in lamellar conversions is directly correlated to a decrease in decarboxylase and lateral ciliary activities (Fig. 7a,b,c). The 5%0 S habitat animals exhibited the lowest decarboxylase activity levels (P < 0.05) in the neuronal components and in the ctenidium during the control observations. This is correlated to lowest pre and post-stimulatory lateral ciliary rates of activity observed in 5%” S group.
While dark periods slightly increased enzymatic activities, light periods significantly reduced enzymatic (P < 0.001) and pre- and post-stimulatory ciliary activities (P < 0.001) in 5 and 15%” S experimental groups. The 30%0 S group showed only minor alterations in these activities in response to changes in the photoperiods. However, hypoxia (EGTA and A-23 187) significantly reduced both the enzymatic, basal and post-stimulatory levels of ciliary activities (Table 1).
Salinity induced neuro-pigmental transformations in
i0
l-5
Crassostrea
519
i0
Habitat Salinitir (Y’..) Tig. 5. The percentage of lamellar type cytosomes (bars) observed in the neuron as related to the average lateral ciliary rate of beating (line graph).
Effects of illumination upon other organelles Our laboratory has studied the effects of illumination using similar light intensities upon mitochondria, nuclear material, and endoplasmic reticulum at the LM and TEM levels. No changes were observed in the nonilluminated state. Furthermore, even in the case of the Golgi apparatus which is intimately associated with cytosomes, illumination does not render morphological transformations. DISCUSSION
The neurons of many molluscs contain large populations of yellow pigment granules, called cytosomes by Nolte et al. (1965). The localization of neuronal pigments within discrete granules in neuronal cytoplasm raises the question of the nature and function of these granules. Baker (1963) raised the possibility that molluscan cytosomes belonged to the lysosomal group of organelles, and Lane (1966) has provided evidence for the presence of known lysosomal enzymes in the cytosomes of several snail species. The presence of neuronal pigments in the neurons of vertebrates in lysosome-like granules is also known (Novikoff, 1967) and these are often termed lipofuchsin granules. If the pigment-containing cytosomes of snail neurons are organelles of the lysosomal group then the carotenoprotein may be accumulations of so-called “aging substances”. Evidence discussed by Benjamin and Walker (1971) indicates a more functional role for molluscan neuronal pigments. This evidence is summarized as follows: (a) neuropigments are present in newly hatched Lymnea, (b) pigments are not simply accumulated in constant amounts in all ganglia but are distributed in varying proportions between them, and (c) in one case a carotenoprotein-mediated sensitivity to light is known to be functionally important in a behavioral shadow response. It could be that the
latter case is a specialized one but experiments on several molluscan species show that neuronal photosensitivity is widespread. The immature cytosomes appear to begin as small vesicles containing a fine homogeneous matrix. The origin of these initial vesicles could not be determined. The osmiophilic material contained within mature cytosomes apparently is generated by Golgi apparatus which is often observed to be joined to cytosomes. Brief periods of illumination (I 5 min) produce intermediate cytosomes which contain paracrystalline arrays. Illumination of greater duration (I 20 min) produces membranous organelles which were previously reported by Henkart (1975), Brown et al. (1975), Paparo and Murphy (1976, 1978), and Zs.-Nagy (1969, 197la,b). The globular vesicular arrays observed in this study were also described by Henkart (1975) in Aplysia. The fate of the fully converted membranous cytosomes (secondary conversions) appears to be the dissociation into vesicles which can be seen throughout the cell cytoplasm. It was not possible to determine if these vesicles could be resorbed and recycled into new cytosomes. There is relatively little endoplasmic reticulum within cytosomal fields which could possibly indicate that the reconstitution of mature cytosomes from vesicles could occur without requiring protein synthesis. The data in this study confirm previous findings that visible light (h = 500 nm) can alter cytosomes from dense to lamellar-type granules with subsequent decrease in decarboxylase and ciliary activities (Paparo, 1985). The results show that lateral ciliary activity is dependent on the decarboxylase activity. The distribution and amounts can be altered with salinity changes, photoperiod, hypoxia and perfusion with exogenous EGTA and A-23187. It appears that the ultimate source of the decarboxylase enzyme in the ctenidium is the VG. The enzymatic activities in the oyster seem to be largely confined to the VG, but
ANTH~NVA.
520
PAPARO
o--_-o 5%
30-h
zt+
Rapid
IS*%
c- a
15YI Ra@dsalinity
t 500
longlh
I
f&o
550 Wove
salinity+Light
i, _
hlinity
-
I
450
HabiIct, salinity+oarit
!i~..tbbir.t
6-d
I 650
(nm)
Fig. 6. (a) Microphotospectroanalysis of the cytosomal pigment in 5% habitat oysters. Values in parentheses are the beats per second at the particular spectral band. (b) Microphotospectroanalysis of the cytosomal pigment in 15%~S rapidly reacclimated oysters. These data represent a rapid salinity change of oyster populations as appears in Fig. 6a. Values in parentheses are bets per second at particuiar spectral maxima.
may also occur in significant amounts in the CG, Pg, CPC, CVC, BN and neuronal components (BN axons and NC) within the ctenidium. In addition, this study follows the sequential changes in cytosomal morphogenesis during light and electrical frequency stimulation. Brown et al. (1975) have shown that ultrastructural change of lamellartype granules is accompanied by an increase in intracellular [calcium]. The EGTA-induced lamella configuration results of this study confirm the data obtained by Sugaya and Onozuka (1978a,b). In addition, the latter authors found that cytosomes can release calcium and ultrastructurally go from a dense to lamellar-type granule. In another experiment, our laboratory has shown that the release of the cilioinhibitory neurotransmitter dopamine is directly related to the amount of free intracellular calcium in the BN axons (Paparo and Murphy, 1979). Taken
together the findings presented here suggest the following sequence of events: (1) induction of granule conversion (salinity changes, photoperiod etc.), (2) release of calcium from granule, (3) exocytosis of dopamine-containing vesicles from BN axons, (4) stimulation of calcium influx into the lateral ciliated cell by dopamine, and (5) ciiioinhibition. Both EGTA and A-23187 enhance the secondary conversion process and further exacerbate the cilioinhibition. This ciliary shut-down could conceivably enable the oyster to cease the filtering process under stressful conditions. It has been shown that ability of certain molluscs to tolerate extreme anoxic conditions is directly related to the presence of cytosomal carotenoids (Kerpel-Fronius and Zs.-Nagy, 1973). The carotenoids can serve as an intrinsic cytosomal electron acceptor substance (which is activated during the conversion process) which may substitute as the
Salinity induced neuro-p&mental transformations in Crussostrea
Fig. 7. (a) Frequency of lamellar type cytosomes as related to rapid salinity change and light exposure. (b) Percent inhibition of the decarbaxylase activity as related to rapid salinity change and light exposure. (c) Percent inhibition of lateral ciliary activity as related to rapid salinity change and light exposure.
electron acceptor function of molecular oxygen for a considerable time. This would maintain the ATPregenerating process even in anoxia (Zs.-Nagy, 197Ib; Zs.-Nagy and &mini, I972a,b). SUMMARY
Cytosomes could serve as temporary ion-buffering systems (an extension of the transport system of the neurolemma) capable of local and transient sequestration of certain ions to control microscopic disparities in solute composition within neuronat
521
ANTHONYA.
522
cytoplasm. These organelles are capable of responding to photic and chemical stimuli by cytostructural, biophysical and bioelemental alterations in their matrical and pigmental components. This study elucidates a mechanism for the role of the cytosome in the neuro-regulatory process controlling ctenidium ciliary activity.
Acknowledgemenrs-The author wishes to thank Dauphin Island Sea Laboratory for the use of wet-laboratory facilities, and the Center for Electron Microscopy at Southern Illinois University for the use of its TEM and SEM equipment. A. Paparo would like to personally thank the School of Medicine, Southern Illinois University for granting him several “Off-Campus Assignments” to complete this work.
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Brown A. M.. Baur P. S. and Tulev F. H. (1975) Phototransduction in Aptysia neurons: primary event is calcium release from pigmented granules. Science 188, 157-160. Falck B. (1962) Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol. stand. 56, 197 (abstract). Gray J. (1924) The mechanism of ciliary movement. IV. The regulation of ciliary activity to oxygen consumption. Proc. R. Sot. Land., Ser. B, 96, 95-114.
Gray J. (1929) The mechanism of ciliary movement. Am. Nat. 63, 68-8 1. Henkart M. (1975) Light induced changes in Aplysia neurons. Science 188, 155-157. Kennedy D. (1961) Neural photo-reception in lamellibranch mollusc. J. gen. Physiol.-44, 277-299. Kerpel-Fronius S. and Zs.-Nagy I. (1973) Electron microscopic demonstration of energy production in molluscan neurons. Acta Biol. Acad. Sci. Hung. 24, 83-90. Lane N. J. (1966) The fine structural localization of phosphatases in neurosecretory cells within the gangha of certain snails. Am. Zoo/. 6, 139-157. Lucas A. M. (1931a) An investigation of the nervous system as a possible factor in the regulation of ciliary activity in the lamellibranch gill. J. Morphol. 51, 147-193. Lucas A. M. (193 1b) The distribution of the branchial nerve in Mytilus edulis and its relation to the problem of
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Paparo A. and Murphy J. A. (1979) The regulation of intracellular calcium and the release of neurotransmitters in the mussel, Mytilus edulis. Comp. Biochem. Physiol. 66, 5 17-520. Setna S. B. (1930) Neuromuscular mechanism of the gill of Pecten. Q. J. Microsc. Sci. 73, 365-392.
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0300-9629/86 $3.00 + 0.00 Pergamon Journals Ltd
Prmted in Great Biitain
SALINITY INDUCED CYTOSOMAL MORPHOGENESIS THE OYSTER CRASSOSTREA VIRGINICA (GMELIN)
IN
ANTHONYA. PAPARO Department
of Anatomy,
School
Illinois University, Carbondale, Sciences
Consortium,
of Medicine
and Department
of Zoology,
College of Science, Southern Marine Environmental Island, AL 36528, USA
IL 62901, USA and Marine Science Program, Dauphin
Island
Sea Lab.,
(Receitled I8 February
Dauphin 1986)
Abstract-l. Lamellar transformations of cytosomes, DOPA decarboxylase and lateral ciliary activity were measured in neuronal elements and/or ctenidia of the oyster, Crassostrea oirginica (Gmelin). 2. Rapid salinity changes, exposure to light, hypoxic conditions and perfusion with EGTA (a calcium chelator) and A-23187 (a calcium releasing agent) increase lamellar transformations. 3. This is accompanied by a decrease in decarboxylase and lateral ciliary activity. 4. The magnitude of the cilioinhibition and decarboxylase activity is directly related to the number of secondary conversions, habitat and reacclimation salinities.
INTRODUCTION
water, and the dish was placed in a holder fastened to the adjustable stage of a microscope. The ctenidium was seen to consist of numerous parallel ctenidial filaments. Three major types of ciliated cells were clearly distinguished: frontal, laterofrontal and lateral. This study was concerned with the rate of beating of lateral cilia which beat in such a way that metachronal waves appear to travel in opposite directions along the two sides of each ctenidial filament. The rate of ciliary beating in beats per second was estimated by synchronizing the rate of flashing of a calibrated, stroboscopic light used in place of the substage lamp, with the rate of beating of the cilia. Synchronization was achieved when the metachronal wave appeared to stand still. Measurements were made from dorsal to ventral border, and from left to right across the field, giving 12 sets of measurements per animal. The Petri dish was perfused with sea-water via a four channel variable speed pump with a flow rate of ca 0.5 ml/min. The planetary gear mechanism of this pump ensures minimum pulsing and stable drift-free flow permitting accurate measurements of ciliary movement. A positive displacement piston metering pump with micrometric adjustment permits rapid removal of solution from the Petri dish. A continuous flow across the dish can be maintained with constant temperature by means of stainless steel tubing which cools down the movable platform (ca I .tYC/O.S min) by means of a circulating water from a cooling system.
There is physiological evidence that lateral ciliated cell-system on the ctenidium is individually controlled (Spittstijsser, 1913; Setna, 1930; Paparo, 1972; Paparo and Dean, 1984). In addition, the central and peripheral nervous system of many molluscs are highly pigmented (Arvanitaki and Chalazonitis, 1960). Neurophysiologically, molluscan brain cytosomal pigment renders nerve cells sensitive to incident light (Kennedy, 1961; Paparo, 1985). Other experiments (Paparo and Murphy, 1976, 1978) have indicated that the rate of beating of lateral cilia on ctenidia can be influenced by radiation of certain wavelengths. The photo-response has been shown to be mediated by neuronal pigments (cytosomes) which are morphologically transformed into lamellar organelles that release sequestered calcium ions intracellularly (Brown et al., 1975; Paparo, 1985). The purpose of this study is to elucidate the effect of rapid change in salinity, in habitat oyster populations, on cytosomal transformations, decarboxylase activity and ciliary activity in neuronal tissues (both central and peripheral) and/or in the ctenidium.
MATERIALS
Drug perfusion
AND METHODS
Isolation of ctenidium and measurement
of ciliary activity
Oysters (Crussoslrea G-ginica Gmelin) were collected from natural subtidal beds on the north side of Dauphin Island, Alabama. The oysters were placed in standing sea-water aquaria with undergravel filters filled with Instant Ocean artificial sea-water at the appropriate salinities at 23-50°C for 3-5 days before use in a particular experimental procedure. Before each experiment, oysters of 5-8 cm in length were shucked by breaking the ligament with an oyster knife and cutting the adductor muscle at the points of insertion on the upper and lower valves. The whole tissue was maintained in a tray of clean aerated artificial sea-water at the particular habitat or reacclimation salinity and 22°C. The mantle was excised from one side to expose the ganglia, nerve and ctenidium. This preparation was pinned to rubber mats glued in the bottom of a Petri dish containing sea513
Drugs were added by replacing the content of the perfusion dish with the desired concentration of the drug in sea-water at 22”C, and perfusion was continued with drug solution. Drugs were washed out by repeating the procedure with sea-water. The following drugs were used: IOmM ethylene glycol-bis (amino-ethylether)-N; N’tetra-acetic acid, EGTA (a calcium chelator) and IO-’ M ionophore A-23187 prepared as 10m3 M stock solution in 0.25% dimethformamide and 0.75% ethanol (a calcium releasing agent). Electrical srimulation of branchial nerve For nerve stimulation, an electrode was placed on the branchial nerve at a point of its emergence from the visceral ganglion. A stimulator supplied electrical pulses with the following characteristics: 10.0 V, 5.0 biphasic pulses per second, 2 msec duration.
514
ANTHONY A.
Yellow pigment extraction and measurement Yellow pigment color of visceral ganglion and nerves of Crassostrea was examined in living preparations. Yellow neuronal pigments (cytosomes) were examined in squash preparations, cryostat sections, and under fluorescence microscopy. In latter investigation, ganglia were frozen in Freon 22, cooled by liquid nitrogen and lyophilized at 40°C at 1O-4 torr for 2 hr. The yellow autofluorescent neuronal pigment granules could then be visualized with a Leitz MPV II microscope equipped with a fluorescence module. The fluorophore that was regenerated by re-exposure to formaldehyde vapor at 80°C for I hr was due to monoamines, and could be readily distinguished from cytosomal pigment autofluorescence (Falck, 1962). The pigment was collected in a petroleum ether extract and the spectra recorded using a microfluorometric method. The measurements of the yellow pigment extracts were carried out on the Leitz MPVII system equipped with microspectrofluorometry and an automatic measuring beam interference graduated filter.
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Sections voltage.
were examined
under
TEM
at 50 KV operating
TEM autoradiographic localization Autoradiographic localization of [3H]leucine was performed on all neuronal tissues. These were incubated in 10m6 M [‘Hlleucine in sea-water at 22°C for 10 min. They were then washed in sea-water solution with six changes of solution, each for 5 min. They were fixed and embedded for TEM as described above, except that the fixing solutions were made hypertonic by addition of 9.0% sucrose. Under these conditions of fixation, movement of the [-‘H]leucine was halted. Thin sections were cut and placed on 200 mesh grids and coated with a photographic emulsion (diluted 1:6) by looping. The emulsion coated grids were exposed for 46 weeks in dry light-tight boxes in an atmosphere of 100% CO,. After photographic development for 3-5 min at 22”C, sections were stained with uranyl acetate and counterstained with lead citrate.
Dark/light preparations Dark-adapted ctenidium preparations were maintained under illumination of a photographic safe light with a dark red filter. In addition, isolated ganglia were incubated for various lengths of time in sea-water in total darkness. Illuminated ganglia were prepared by dissection from oysters in room light. The temperature of gangliacontaining solutions in light or darkness stayed at approx. 22°C. Anaerobic conditions Anaerobic conditions were maintained in a vessel containing eight times more sea-water than body weight of the oysters. The surface of the water was then covered with a paraffin oil layer of l-2 cm in thickness. Under such circumstances oxygen content of sea-water is used up and total anoxia sets in within 48 hr. The animals are considered as living up to the moment that they cease to react with movement upon influence of mechanical stimulation.
Differential and gradient centrifugation of neuronal elements Neuronal components of oysters were kept in ice-cold sea-water during dissection. Ganglia and nerves were blotted with filter paper and homogenization was carried out in 0.05 M sucrose in a glass tube fitted with a Teflon pestle to give a 10% tissue suspension. The supernatant fluid was collected and centrifuged again at 17,500 g for 30 min. The pellet (P) was saved for subfractionation by resuspending it in 0.05 M sucrose. This suspension was layered onto a discontinuous sucrose gradient containing 1.20 and 1.50 M sucrose. Centrifugation was at 50,OOOn for 2 hr. which yielded three fractions: PA, PB, PC. The PB pellet was transferred to 2.5% glutaraldehyde in 0.20 M S-collidine buffer, pH 7.8, for lf hr. Samples were then processed for TEM as described previously.
RESULTS
Measurement of decarboxylase activity
General organization of ctenidium and nervous system
DOPA decarboxylase activity assays were made using extracts of ganglia, connectives, and nerves in 5 ml homogenate (average 40mg), 0.85 ml of phosphate buffer, and 0.1 ml of 0.01 M DL-[IW] DOPA (total radioactivity, 1.5 x lo6 c.p.m.) were added to the test tube. For ctenidium assays 0. I ml of ctenidial homogenate (average 40 mg), 0.8 ml of phosphate buffer, and 0.1 M DL-[I14C] DOPA were added to the test tube. These test tubes were sealed with rubber covers, from each of which was suspended a well containing 0. I ml of 1 M KOH. The extracts were incubated at 38°C for 24min. The reaction was stopped with the introduction of 0.2ml of 1 M H,SO, solution through the rubber cover. The test tubes were shaken on a shaker for 55 min. The well containing the trapped 14C0, was then dipped in lOm1 of liquid scintillation medium and the radioactivity was determined in a liquid scintillation counter. Counts on heat-inactivated extracts were performed simultaneously.
The general organization of the ctenidium is shown in Fig. la,b. More detailed accounts may be found in Gray (1924, 1929), Lucas (1931a,b, 1932) and Paparo and Dean (1984). In cross-section each of two ctenidia (Gi) appears in the form of a “W” suspended from the dorsal margin of the oyster. The inner and outer surfaces (called lamellae) are made up of a large number of ctenidial filaments (GF) running lengthwise in the lamellae. The branchial nerve which arises from the visceral ganglion VG runs obliquely downward and backward to enter the afferent blood vessel (ABV) in the dorsal margin of the ctenidium axis. The valves are held in place via the anterior adductor (A Ad M) and posterior adductor (P Ad M) muscles. In addition to VG and BN, there are other elements of the nervous system: cerebral ganglion (CC), pedal ganglion (PG), cerebrovisceral connective (CVC), and cerebropedal connective (CPC). The VG is covered by a loose connective tissue sheath. Beneath the sheath, a closely adhering capsule completely encases the ganglion and is not removable without disrupting the ganglion. There is a rind of nerves, beneath the capsule, whose axonal processes form part of the fibrillar neuropil in the interior of the ganglion. The fibrillar neuropil constitutes the beginning of the BN (Fig. 2a). Clusters of nerve cells leave the VG and accompany the BN in its course to the ctenidium epithelium (Fig. 2b).
TEM preparation Samples were prepared for TEM by removing a valve and pouring the fixative (2.5% glutaraldehyde) directly onto the underlying tissues. After 15 min, ctenidia, ganglia, connectives and nerves were isolated. These tissues were fixed in 2.5% glutaraldehyde in 0.2 M S-collidine buffer, pH 7.8 (50~2000mOsm) for 2 hr. It was then rinsed in several changes of 0.2 M S-collidine buffer at 4°C for a period of 1 hr, postfixed for 1 hr in 1.0% osmium tetroxide in the same buffer, dehydrated in a graded series of ethanol, and embedded. Sections were stained with 5.0% uranyl acetate for 30 min. and counterstained with lead citrate for 5 min.
Salinity
induced
neuro-pigmental
transformations
in Crassostrea
Fig. I. (a) Dissected specimen showing the visceral ganglion (VG), branchial nerve (BN) and ctenidial lamella (CL). Arrow traces the BN from its emergence from the ventral surface of the VG along its course within the afferent branchiai blood vessel. x 30. (b) Diagram represents a cross-section of the entire animal on the half shell. Three ganglia are shown: cerebral (CG), visceral (VG) and pedal (PG). Additional components include: branchial nerve (BN) coursing with the afferent branchial blood vessel (ABV) on its way to the ctenidium (Gi) and ctenidial filaments (GF); cerebra-visceral connective (CVC), cerebropedal connective (CPC); and adductor muscles, anterior (A Ad M) and posterior (P Ad M). A-B indicates a typical section of the GF used in following the axons of the BN within the Gi. Nerve
cells and cytosomes
The nerve cells are unipolar and approximately oval in shape, measuring about 25 pm in length and from 10 to 17 pm in width. The contour of the nucleus is similar to that of the cell. Many of these nerve cells contain yellow pigment granules (cytosomes), giving the ganglion a yellow color. Golgi bodies were found to be constantly associated with cytosomes (Fig. 2~). Small vesicles, generated at the mature face of the Golgi apparatus, were observed in
close proximity and at times joined to the surface of cytosomes. The material in the small vesicles was similar in density to electron-dense material in the cytosomes. Autoradiographic studies ([3H]leucine) done at the EM level confirm that the Golgi apparatus plays an important role in the formation of cytosomes (Fig. 3a). Furthermore, the [3H]leucine label was also found associated with cytosomes in the axonal population of the BN (Fig. 3b). Structural detail of cytosomes was best preserved by using 2.5% glutaraldehyde in 0.2 M S-collidine,
516
ANTHOW A.
pH 7.8, 1lOOmOsm. There were significant ultrastructural changes due to osmolality of fixative solutions. A 500 mOsM solution caused a disruption of the outer cytosomal limiting membrane while 1OOOmOsM seemed to reduce such distortions. The use of phosphate buffers in the presence of calcium in
PAPARO
sea-water caused precipitates throughout neuronal components. The limiting membrane exhibited a characteristic trilaminar appearance. The greatest number of immature cytosomal forms were observed in dark-treated preparations (Fig. 4a). The small cytosomes which
Fig. 2. (a) Photomicrograph of the visceral ganglion just beneath the capsule is a rind of nerve cellswhose axonal processes form part of the fibrillar neuropil-in the interior of the ganglion. x 300. (b) Nerve cells dissected from beneath the capsule of the visceral ganglion. Many of these cells contain yellow pigment granules (cytosomes) which give the ganglion a yellow color in the living preparation. x 600. (c) TEM illustrating the relationship routinely observed between the Golgi bodies (G), their vesicles (GVs) and maturing cytosomes (mc). A densely staining material present in GVs appears to be the same electron-dense material observed in the cytosome matrix. x 11,625.
Salinity induced neuro-pigmental
transformations
in Crassastrea
517
Fig. 3 (a) Autoradiograph of the branchial nerve, which takes origin from the neurophil of the viscerai ganglion, showing [)H]leucine label (I) over axonal profiles and cytosomes. x 17,250. (b) Autoradiograph of a nerve cell at the EM level showing incorporation of 13Hlleucine label ft) over the Golni bodies and cytosome. x 17:256
contained both electron-dense and a fine reticular material ranged from 0.3 to 0.5 ftm in diameter. The
larger cytosomes (2 1.Opm in diameter) which contained markedly osmiophilic material, were assumed to be the mature form (Bauer et al., 1977) of this organelle. These granules have also been observed in subcellular fractions obtained from VG and BN homogenates (Fig. 4b). These isolated preparations exhibit the same alterations, but in a more rapid manner, as previously described in the intact preparations. Lamellar or paracrystalline arrangements of electron material were observed in these organelles (Fig. 4~). The numerous paracrystalline arrays gave this organelle a mottled appearance. These intermediate cytosomes having this mottled configuration have been classified as “primary conversions” (Bauer el al., 1977). Membrane assembly appeared to involve the osmiophilic component and moderately electron-dense homogeneous material. At higher magnifications, this membranous material had a trilaminar appearance. The crystalline-like arrangement occurred first, followed by membrane formations.
Globular vesicular components were seen after initial membrane formation. These latter components may be formed by swelling of precipitous laminations scattered throughout the center of the cytosomes. Those cytosomes that exhibit complete membranous transformations are classified as “secondary conversions” (Bauer rt al., 1977). Percent lameliar type cytosomes and ciliary activity in habitat populations
The neuronal components and ctenidium of 5%0 S habitat oysters significantly (P < 0.001) contained the highest populations of lamellar type organelles. Light enhanced the frequency of lamellar conversions which was accompanied by a cilioinhibitory response (Fig. 5). Microspectroanalysis of cytosomal yellow pigment, decarboxylase and ciliary activities
Microspectrophotometric measurements of the yellow cytosomal pigment showed alterations in the
518
ANTHONYA.
PAPARO
Fig. 4. (a) TEM of branchial nerve within the afferent blood vessel showing cytosomes (7) within its axonal profiles. x 11,250. (b) Cytosomes (t) subjacent to the lateral ciliated cell. x 18,750.
absorption spectra for both changes in photoperiod and rapid salinity changes (Figs 6a,b and 7a). An increase in lamellar conversions is directly correlated to a decrease in decarboxylase and lateral ciliary activities (Fig. 7a,b,c). The 5%0 S habitat animals exhibited the lowest decarboxylase activity levels (P < 0.05) in the neuronal components and in the ctenidium during the control observations. This is correlated to lowest pre and post-stimulatory lateral ciliary rates of activity observed in 5%” S group.
While dark periods slightly increased enzymatic activities, light periods significantly reduced enzymatic (P < 0.001) and pre- and post-stimulatory ciliary activities (P < 0.001) in 5 and 15%” S experimental groups. The 30%0 S group showed only minor alterations in these activities in response to changes in the photoperiods. However, hypoxia (EGTA and A-23 187) significantly reduced both the enzymatic, basal and post-stimulatory levels of ciliary activities (Table 1).
Salinity induced neuro-pigmental transformations in
i0
l-5
Crassostrea
519
i0
Habitat Salinitir (Y’..) Tig. 5. The percentage of lamellar type cytosomes (bars) observed in the neuron as related to the average lateral ciliary rate of beating (line graph).
Effects of illumination upon other organelles Our laboratory has studied the effects of illumination using similar light intensities upon mitochondria, nuclear material, and endoplasmic reticulum at the LM and TEM levels. No changes were observed in the nonilluminated state. Furthermore, even in the case of the Golgi apparatus which is intimately associated with cytosomes, illumination does not render morphological transformations. DISCUSSION
The neurons of many molluscs contain large populations of yellow pigment granules, called cytosomes by Nolte et al. (1965). The localization of neuronal pigments within discrete granules in neuronal cytoplasm raises the question of the nature and function of these granules. Baker (1963) raised the possibility that molluscan cytosomes belonged to the lysosomal group of organelles, and Lane (1966) has provided evidence for the presence of known lysosomal enzymes in the cytosomes of several snail species. The presence of neuronal pigments in the neurons of vertebrates in lysosome-like granules is also known (Novikoff, 1967) and these are often termed lipofuchsin granules. If the pigment-containing cytosomes of snail neurons are organelles of the lysosomal group then the carotenoprotein may be accumulations of so-called “aging substances”. Evidence discussed by Benjamin and Walker (1971) indicates a more functional role for molluscan neuronal pigments. This evidence is summarized as follows: (a) neuropigments are present in newly hatched Lymnea, (b) pigments are not simply accumulated in constant amounts in all ganglia but are distributed in varying proportions between them, and (c) in one case a carotenoprotein-mediated sensitivity to light is known to be functionally important in a behavioral shadow response. It could be that the
latter case is a specialized one but experiments on several molluscan species show that neuronal photosensitivity is widespread. The immature cytosomes appear to begin as small vesicles containing a fine homogeneous matrix. The origin of these initial vesicles could not be determined. The osmiophilic material contained within mature cytosomes apparently is generated by Golgi apparatus which is often observed to be joined to cytosomes. Brief periods of illumination (I 5 min) produce intermediate cytosomes which contain paracrystalline arrays. Illumination of greater duration (I 20 min) produces membranous organelles which were previously reported by Henkart (1975), Brown et al. (1975), Paparo and Murphy (1976, 1978), and Zs.-Nagy (1969, 197la,b). The globular vesicular arrays observed in this study were also described by Henkart (1975) in Aplysia. The fate of the fully converted membranous cytosomes (secondary conversions) appears to be the dissociation into vesicles which can be seen throughout the cell cytoplasm. It was not possible to determine if these vesicles could be resorbed and recycled into new cytosomes. There is relatively little endoplasmic reticulum within cytosomal fields which could possibly indicate that the reconstitution of mature cytosomes from vesicles could occur without requiring protein synthesis. The data in this study confirm previous findings that visible light (h = 500 nm) can alter cytosomes from dense to lamellar-type granules with subsequent decrease in decarboxylase and ciliary activities (Paparo, 1985). The results show that lateral ciliary activity is dependent on the decarboxylase activity. The distribution and amounts can be altered with salinity changes, photoperiod, hypoxia and perfusion with exogenous EGTA and A-23187. It appears that the ultimate source of the decarboxylase enzyme in the ctenidium is the VG. The enzymatic activities in the oyster seem to be largely confined to the VG, but
ANTH~NVA.
520
PAPARO
o--_-o 5%
30-h
zt+
Rapid
IS*%
c- a
15YI Ra@dsalinity
t 500
longlh
I
f&o
550 Wove
salinity+Light
i, _
hlinity
-
I
450
HabiIct, salinity+oarit
!i~..tbbir.t
6-d
I 650
(nm)
Fig. 6. (a) Microphotospectroanalysis of the cytosomal pigment in 5% habitat oysters. Values in parentheses are the beats per second at the particular spectral band. (b) Microphotospectroanalysis of the cytosomal pigment in 15%~S rapidly reacclimated oysters. These data represent a rapid salinity change of oyster populations as appears in Fig. 6a. Values in parentheses are bets per second at particuiar spectral maxima.
may also occur in significant amounts in the CG, Pg, CPC, CVC, BN and neuronal components (BN axons and NC) within the ctenidium. In addition, this study follows the sequential changes in cytosomal morphogenesis during light and electrical frequency stimulation. Brown et al. (1975) have shown that ultrastructural change of lamellartype granules is accompanied by an increase in intracellular [calcium]. The EGTA-induced lamella configuration results of this study confirm the data obtained by Sugaya and Onozuka (1978a,b). In addition, the latter authors found that cytosomes can release calcium and ultrastructurally go from a dense to lamellar-type granule. In another experiment, our laboratory has shown that the release of the cilioinhibitory neurotransmitter dopamine is directly related to the amount of free intracellular calcium in the BN axons (Paparo and Murphy, 1979). Taken
together the findings presented here suggest the following sequence of events: (1) induction of granule conversion (salinity changes, photoperiod etc.), (2) release of calcium from granule, (3) exocytosis of dopamine-containing vesicles from BN axons, (4) stimulation of calcium influx into the lateral ciliated cell by dopamine, and (5) ciiioinhibition. Both EGTA and A-23187 enhance the secondary conversion process and further exacerbate the cilioinhibition. This ciliary shut-down could conceivably enable the oyster to cease the filtering process under stressful conditions. It has been shown that ability of certain molluscs to tolerate extreme anoxic conditions is directly related to the presence of cytosomal carotenoids (Kerpel-Fronius and Zs.-Nagy, 1973). The carotenoids can serve as an intrinsic cytosomal electron acceptor substance (which is activated during the conversion process) which may substitute as the
Salinity induced neuro-p&mental transformations in Crussostrea
Fig. 7. (a) Frequency of lamellar type cytosomes as related to rapid salinity change and light exposure. (b) Percent inhibition of the decarbaxylase activity as related to rapid salinity change and light exposure. (c) Percent inhibition of lateral ciliary activity as related to rapid salinity change and light exposure.
electron acceptor function of molecular oxygen for a considerable time. This would maintain the ATPregenerating process even in anoxia (Zs.-Nagy, 197Ib; Zs.-Nagy and &mini, I972a,b). SUMMARY
Cytosomes could serve as temporary ion-buffering systems (an extension of the transport system of the neurolemma) capable of local and transient sequestration of certain ions to control microscopic disparities in solute composition within neuronat
521
ANTHONYA.
522
cytoplasm. These organelles are capable of responding to photic and chemical stimuli by cytostructural, biophysical and bioelemental alterations in their matrical and pigmental components. This study elucidates a mechanism for the role of the cytosome in the neuro-regulatory process controlling ctenidium ciliary activity.
Acknowledgemenrs-The author wishes to thank Dauphin Island Sea Laboratory for the use of wet-laboratory facilities, and the Center for Electron Microscopy at Southern Illinois University for the use of its TEM and SEM equipment. A. Paparo would like to personally thank the School of Medicine, Southern Illinois University for granting him several “Off-Campus Assignments” to complete this work.
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