JOURNAL OF ULTRASTRUCTURE RESEARCH 64, 14-22 (1978)

Catecholaminergic Salivary Glands in Gammarus pulex (Crustacea, Amphipoda): An Electron Microscopic and Microspectrofluorometric

Study R O L F ELOFSSON, 1 H A R R Y MYHRBERG, R O B E R T ARAMANT, OLLE LINDVALL, AND B E N G T FALCK

Departments of Zoology and Histology, University of Lund, Lund, Sweden Received February 8, 1978 The type of gland (salivary gland) described here for the amphipod Gammarus pulex belongs to the tegumental glands, which have different structural characteristics. The present type, called rosette gland, is common in some crustaceans and is located in the ventral half of the head. The functional unit is a lobule of gland cells with a central-draining duct. Ducts from groups of lobules conjoin and terminate on the body surface at different points around and in the mouth and mouth parts. With the fluorescence histochemical method of Falck and Hillarp, specific green fluorescence was discerned centrally in the lobules and was confined to the gland cells. The spectral characteristics of the fluorescence, as revealed by microspectrofluorometric analysis, indicated either a mixture of dopamine and a presumed new catechol compound or the presence of two tautomeric forms either of dopamine or of a new catechol compound. Evidence of new catechol compounds with similar spectral characteristics has previously been found in the sensory cells of some invertebrates. The fluorescence distribution within the lobule coincides with the presence, ultrastructurally, of large dense vesicles in the gland cells. These dense vesicles occur in the predominant cell type, also characterized by a smooth endoplasmic reticulum. The other cell type in the lobules differs ultrastructurally by possessing a rough endoplasmic reticulum and a different vesicle type. No innervation of the salivary gland was perceived.

In a survey of monoaminergic neurons in the crustacean nervous system using the fluorescence histochemical method of Falck and Hillarp (Falck, 1962; Falck et al., 1962), specific fluorescence was also obtained from glands situated ventrally in the headthorax region of Gammarus pulex (Aramant and Elofsson, 1976). These glands have the position and presumed function of salivary glands. Salivary glands of various insects (Whitehead, 1971; Klemm, 1972; Bland et al., 1973; Robertson, 1975), an exception being the blowfly (Oschman and Berridge, 1970), and mollusks (Matus, 1971a,b; Martin and Barlow, 1972; Arluison and Ducros, 1976) have been reported to have a monoaminergic innervation. Gammarus pulex seemed to

However, the results of the present ultrastructural study would seem to indicate a non-neuronal localization of the catecholamine: The fluorogenic compound is situated within the gland cells. MATERIALS AND METHODS The freshwater species Gammarus pulex de Geer was obtained from local ponds and streams in southern Sweden and was maintained in tanks in cold water (4°C) until use. Electron microscopy. Ventral halves of the heads were fixed in paraformaldehyde-glutaraldehyde (Karnovsky, 1965) for 3 hr and then postfixed in 2% osmium tetroxide for 1 hr. Cacodylate buffer was used throughout. Block staining was performed in 1% phosphotungstic acid and 0.5% uranyl acetate. Vestopal W was used as the embedding medium. Sections were placed on Formvar-coated grids and examined under Zeiss EM 10 and Philips EM 300 microscopes. Additional staining of the sections was done with uranyl acetate. Fluorescence microscopy. Tissue pieces containing head and thorax were freeze-dried and processed for visualization of biogenic monoamines according to the Falck-Hillarp fluorescence histochemical method (for details, see Bj6rklund et al., 1972b).

offer an analogous situation in crustaceans. ~To whom correspondence and requests for reprints should be addressed at: Department of Zoology, Helgonavhgen 3, University of Lund, S-223 62 Lund, Sweden. 14 0022-5320/78/0641-0014502.00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

CATECHOLAMINERGIC SALIVARY GLANDS Microspectrofluorometry. Paraffin sections conraining the glands, which were treated as described above, were used for microspectrofluorometric characterization of the fluorogenic compound(s). Excitation and emission spectra of the fluorophore were recorded with a modified Leitz microspectrofluorometer (BjSrklund et a l , 1972b). For further characterization of the fluorophore, the paraffin sections were exposed to HC1 vapor {10-30 min) or to NH3 vapor (10-30 rain} at room temperature. All spectra were corrected according to the procedure described by BjSrklund et al. (1968) and are expressed as relative quanta versus wavelength.

RESULTS

General Appearance and Fluorescence Microscopy The salivary gland of Gammarus pulex is composed of many small lobular units combined in loose aggregates occupying the ventral portion of the head below the nervous system (Fig. 1). Anteriorly, the gland extends to the antennar base and, posteriorly, it extends to the maxilliped and even projects down into the leg. The glandular tissue surrounds the esophagus and also follows the lateral body walls dorsally for some distance. The gland occupies a large area in the ventral head region; however, it is not a continuous organ, but rather an assembly of independent groups of lobular

7

F~G. 1. A schematic drawing of the head of Gamm a r u s p u l e x in sagittal view. The hatched area indicates the position of the salivary gland in the animal.

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units with each group having separate outlets. Upon treatment with the fluorescence histochemical method of Falck and Hillarp, a green fluorescence can be discerned in the central portion of each lobule (Fig. 2). It has never been observed to extend over the whole lobule. Only occasionally, spoke-like fluorescing threads radiate toward the periphery of the lobules. No sexual differences exist. Each lobule is drained by a thin duct that conjoints with those of other lobules to form a bundle of ducts terminating on the body surface (Fig. 2). There are several such bundles from the whole gland ending at different sites in the mouth region. Paired outlets are found in the oral cavity on both the labrum and the labium. The ducts of the glandular portion in the maxilliped run in a distal direction toward the tip of the leg. The ducts manifest a weak green fluorescence, at least in their proximal portions, and are thus traceable with the Falck-Hillarp method.

Electron Microscopy The cell population of the gland consists of two morphologically distinct types. One type, presumed here to contain the catechol compound, is very conspicuous (Fig. 3). The part of the cell constituting the periphery of the lobule is spongy in appearance with cytoplasm restricted to thin strands separated by large spaces (Fig. 4). This part of the cell contains the nucleus and a great number of Golgi apparatuses. Vesicles with moderately dense contents are found in different sizes and positions in and in close association with the Golgi apparatuses (Fig. 6). Only a short distance from the Golgi apparatuses, the large dense vesicles characteristic of the central part of the cell begin to appear, though few in number. The central portion of the gland cell has a continuous cytoplasm containing, among other things, an agranular (smooth), tubular-type endoplasmic reticulum. The most salient components of this part of the cell are the large (approximately 500 nm in

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ELOFSSON E T AL.

CATECHOLAMINERGIC SALIVARY GLANDS diameter; range: 200-800 nm), electrondense, m e m b r a n e - b o u n d vesicles (Figs. 4 and 5). Occasionally, cells of this t y p e are found where the dense vesicles are replaced by s o m e w h a t larger e m p t y vesicles. Although these vesicles simulate artifacts, other cell organelles are intact. We could not d e t e r m i n e w h e t h e r the finding is artifactual or of functional significance. T h e o t h e r cell t y p e differs b y having a continuous cytoplasm, which is very electron-dense owing to a tight, granular (rough) endoplasmic reticulum (Fig. 7). Golgi a p p a r a t u s e s are frequent. T h e whole cell is uniformly filled with large vesicles the d i a m e t e r s of which m e a s u r e as wide as four times those of the dense vesicles of the o t h e r cell type. T h e vesicular contents are always electron opaque. T h e y do not, however, obtain the vacuous a p p e a r a n c e exhibited b y the a b e r r a n t vesicles of the o t h e r cell type. T h e spherical m a s s of cells, designated here as a lobule, is drained f r o m its center by a duct f o r m e d f r o m special duct cells. T h e s e ramify profusely into the central p a r t of b o t h t y p e s of gland cells. T h e duct b r a n c h e s b e c o m e progressively thinner and end as alveolar-like bladders (Fig. 5). T h e ducts are c o m p l e t e l y filled with an electronopaque substance. We believe t h a t the electron-dense granules, p r e s u m e d to contain the catecholamine on liberation of the amine, a t t a c h to the thin ducts and the contents are t h e n released exocytotically. Although in sections one type of gland cell seems to d o m i n a t e one lobule, we h a v e not been able to establish with certainty if a lobule is comprised of a p u r e population of one cell type or a mixture of both. So far, no innervation of the gland has

been found. N e r v e the subesophageal cinity of the gland, the gland has been

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fibers originating f r o m ganglion run in the vib u t no connection with demonstrated.

Microspectrofluorometry T h e spectral analysis gave no unequivocal picture of the fluorogenic compound. T h e emission m a x i m u m in all cases was situated at 475 n m (470-480 nm), b u t the excitation m a x i m u m directly after formaldehyde t r e a t m e n t was either at 380 n m with a shoulder at 410 n m or at 410 n m with a shoulder at 380 n m (Fig. 8). After exposure of the paraffin sections to HC1 v a p o r for 10 to 30 min, those fluorophores with an original excitation m a x i m u m at 380 n m showed only m i n o r spectral changes (the shoulder at 410 n m disappeared), whereas those originally situated at 410 n m shifted to 380 n m and the m a x i m u m p e a k r e m a i n e d t h e r e even after 30 m i n of exposure. F l u o r o p h o r e s showing an excitation m a x i m u m at 380 n m directly after formaldehyde t r e a t m e n t shifted to 410 n m after exposure of the paraffin sections to NH~ v a p o r for 10 to 30 min. A similar change took place w h e n sections t r e a t e d with HC1 v a p o r s u b s e q u e n t l y were exposed to NH3 vapor. I f the excitation m a x i m u m directly after f o r m a l d e h y d e t r e a t m e n t was at 410 nm, no further shift in the p e a k was induced b y NH3 t r e a t m e n t . However, the shoulder at 380 n m disappeared. N e i t h e r acidification nor alkalinization changed the emission spectrum. DISCUSSION T h e t y p e of gland described in this investigation is c o m m o n in crustaceans (Farkas, 1927; Yonge, 1932; Gorvett, 1946) and be-

FrG. 2. A demonstration of the green fluorescence in the salivary gland. The light dots to the left are the fluorescent central part of some lobules. White lines indicate the extent of the lobule. Arrows indicate a number of ducts from the lobules. Scale bar = 20 tam. Inset: One collection of duct outlets on the labium; scale bar = 20 tam.

FIG. 3. An electron micrograph survey of a salivary gland lobule presumed to contain catecholamine. The peripheral spongy region contains some nuclei. The central portion is filled with dense vesicles. Arrow indicates the beginning of the duct draining the lobule. Scale bar = 8 tam.

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ELOFSSON E T AL.

CATECHOLAMINERGIC SALIVARY GLANDS longs to a category called tegumental glands. Tegumental glands can appear in various forms; for instance, Gorvett (1946) found five (or six) structurally different types in isopods. One of these types, the rosette gland, is common in both decapods and isopods and is the one described here in Gammarus pulex. It is presumed to function as a salivary gland. It is confined to the head and mouth parts, and the condition in the amphipod G. pulex resembles in this respect the condition in the isopods. The main morphological features of the rosette glands are well delineated under a light microscope (Farkas, 1927; Gorvett, 1946), and their gross morphology has also been verified in the present ultrastructural investigation. The additional structural knowledge ascertained by electron microscopy includes, inter alia, the presence of two morphological types of rosette gland cells. One type is characterized by a granular endoplasmic reticulum and large electron-transparent vacuoles, which occur throughout the cytoplasm. It is suggested that this cell type produces a proteinaceous secretion. The other cell type has a spongy, agranular endoplasmic reticulum and electron-dense vesicles, which are peculiarly concentrated in the centrally disposed part of the cell in the lobule. The distribution pattern displayed by these dense vesicles coincides with that of the fluorogenic compound in the lobules detected with the fluorescence histochemical method. The intensely fluorescent central part of each lobule would have to have an abundant supply of nerves if the fluorescence were due to a catecholaminergic nerve supply to the gland. Ultrastrucrurally, however, no nerves have been found. Thus a catecholaminergically inner-

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vated salivary gland, as reported for various insects and mollusks (Whitehead, 1971; Matus, 1971 a,b; Martin and Barlow, 1972; Klemm, 1972; Bland et al., 1973; Robertson, 1975; Arluison and Ducros, 1976), does not seem to exist in crustaceans. Hence, it is reasonable to assume that the dense vesicles, present in the gland cells in the central part of the lobules, store the fluorogenic compound. The catecholamines should accordingly be situated inside the gland cells per se. A similar situation was indicated by Matus (1971 a,b) in the posterior salivary gland of octopus in which, apart from a monoaminergic innervation, some cells also contained very large dense vesicles (3 ttm), which were thought to store indoleamines and catecholamines. The storage site of biogenic amines in nervous and non-nervous tissue appears under an electron microscope as dense (core) vesicles. The size within the nervous tissue is 40-60 nm in diameter for dense-core vesicles, but larger vesicles having a diameter as large as 250 nm can be suspected to contain a monoamine (HSkfelt, 1968; Bloom, 1972). Non-nervous tissue, such as the adrenal medulla, has considerably larger vesicles: 200-350 nm for noradrenaline and 50-150 nm for adrenaline (Benedeczky et al., 1966). In comparison with these measurements, the vesicles of the gland cells in Gammarus pulex are still larger but are morphologically similar in other respects. T h e y are, on the other hand, much smaller than the dense vesicles (3 ttm) in the posterior salivary gland of octopus mentioned above (Matus, 1971 b). The microspectrofluorometric characterization of the fluorophores showed interesting spectral characteristics of the fluorescence in the gland. The emission maximum at 475 nm coincides with that of a group of

FIG. 4. An enlargedviewof the transition zonebetweenthe peripheral spongypart of the catecholaminergic lobule cells and the central part containing dense vesicles. Scale bar = 4/tm. FIG. 5. Detail of the central part of a catecholaminergiclobulecell showingthe fine branches of the draining duct (arrows) and the dense vesicles. Scale bar = 0.5/tm.

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ELOFSSON E T AL.

CATECHOLAMINERGIC SALIVARY GLANDS O), 1.o O~. max

O.s-

3~0

400

450

450

500

550

600

650 nm

Fro. 8. Excitation (left) and emission (right) spectra of the formaldehyde-induced fluorescence in the salivary glands of Gammarus pulex. The two characteristic types of excitation spectra are represented (compare with text).

compounds characterized on the basis of their emission peak maxima (Bj6rklund et al., 1971) and comprises the catecholamines, DOPA, and cysteinyldopa (emission maxima between 475 and 490 nm). However, the excitation spectra do not directly agree with the presence of any of these compounds. The excitation spectra obtained immediately after formaldehyde treatment, with peak maxima at either 380 or 410 nm and with a shoulder at the other wavelength, could indicate a mixture of two fluorophores (with separate excitation maxima at 380 and 410 nm, respectively) or a mixture of two tautomeric forms of the same fluorophore (for a detailed discussion, see Elofsson et al., 1977). The shift in excitation peak maxima of the gland fluorophores after treatment with HC1 and NH3 vapor (to 380 and 410 nm, respectively) agrees well with the pH-dependent tautomerism displayed by the catecholamine fluorophores (main excitation peak at 370 nm at acid pH and at 410 nm at neutral and alkaline pH) (Corrodi and Hillarp, 1964; Jonsson, 1966; Bj6rklund et al., 1968, 1972a). The fluorescence could thus be attributed to, e.g., dopamine, the fluorescence

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being a mixture of the two tautomeric forms of fluorophore (for further discussion, see Aramant, in preparation). However, because it has been demonstrated for the dopamine fluorophore that the form having an excitation maximum at 410 nm predominates in the pH range between 6 and 10 (Jonsson, 1966), a major peak at 370-380 nm would require a rather low pH in the cell. One must therefore consider the possibility that the compound present in the salivary gland is a hitherto unknown catechol compound probably identical to that believed to be present in the A r t e m i a cavity receptor organ (Elofsson et al., 1977). This compound also exhibits a pH-dependent tautomerism; but if it is the only one present in the cells (having a pH about 7), it is probably transformed to the acid form at a higher pH than is the dopamine fluorophore. Of course, the possibility cannot be ruled out that this unknown compound is exclusively in the acid form (maximum at 380 nm) and that dopamine is also present, its fluorophore being in the neutral form (maximum at 410 nm). Obviously, chemical identification of the fluorogenic compound or compounds is needed. The biological significance of this catecholaminergic secretion is obscure. Several functions are plausible. One could be analogous to that suggested for mammals, namely, that salivary gland parenchyma has an important function in the removal and inactivation of catecholamines much as do the pericytes or endothelial cells or both in the brain capillaries (Hamberger and Masuoka, 1965). And~n et al. (1963) presented evidence of an extraneuronal uptake of catecholamines in rat salivary glands. There are strong indications of a localization of extraneuronally bound noradrenaline to parenchymal cells of the submaxillary and sublingual glands (Almgren and

FIG. 6. A Golgi apparatus in the spongy part of a catecholaminergic lobule cell with several vesicles of variable density. Scale bar = 0.5 ttm. FIG. 7. Details of the noncatecholaminergic cell type in the salivary gland, showing the typical rough endoplasmic reticulum and the large transparent vesicles. Scale bar = 0.5 ttm.

ELOFSSON E T AL.

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Jonason, 1971). A fluorescence histochemical study (Hamberger et al., 1967) revealed extraneuronally bound catecholamines in the salivary gland parenchyma, although high amine concentrations in the incubation medium had to be used. In the case of Gammarus pulex, however, the removal of the catecholamines should be combined with transport from the salivary gland to the oral cavity. It was shown that the fluorescence in the ducts is specific, suggesting that no degradation of the fluorogenic compounds occurred prior to or after release from the gland cells. The possibility that the substances function as a pheromone is reproduction can be ruled out because no sexual differences were found. It has recently been found that catecholamines may influence the receptors of a snail believed to be involved in chemoreception (Salanki, personal communication). This mechanism would provide a time-protracted influence on food selection and dietary changes. It is, of course, possible that the catecholaminergic secretion may have a more direct action on the food and its degradation, although no evidence is available to support this. Also, any kind of toxic influence from the catecholamines or their metabolic products is plausible. In conclusion, the role of the salivary secretion is probably to be sought within a wide range of functions. This investigation has been s pported by grants from the Swedish Natural Science Research Council (2769-009) and the Swedish Medical Research Council (04X-712). We are indebted to Mrs. Rita Wall~n for her skillful technical assistance. REFERENCES ALMGREN, O., AND JONASON, J. (1971) NaunynSchmiedebergs Arch. Pharmakol. 270, 289-309. ANDI~N, N.-E., CARLSSON, A., AND WALDECK, B.

(1963) Life Sci. 2, 889-894. ARAMANT, R., AND ELOFSSON, R. (1976) Cell Tissue Res. 170, 231-251. ARLUISON, M., AND DUCROS, C. (1976) Tissue Cell 8, 61-72. BENEDECZKY, I., PUPPI, A., TIGYI, A., AND LISSAK, K. (1966) Nature (London) 209, 592-594. BJ(~RKLUND, A., EHINGER, B., AND FALCK, B. (1968) J. Histochem. Cytochem. 16, 263-270. BJORKLUND, A., EHINGER, B., AND FALCK, B. (1972a) J. Histochem. Cytochem. 20, 56-64. BJORKLUND, A., FALCK, B., AND OWMAN, Ch. (1972b) in RALL, Z. E., AND KOPIN, I. J. (Eds.), Methods of Investigative and Diagnostic Endocrinology: Vol. 1: The Thyroid and Biogenic Amines, pp. 318-368. North-Holland, Amsterdam. BJORKLUND, A., FALCK, B., AND STENEVI, U. (1971) Progr. Brain Res. 34, 63-73. BLAND, K. P., HOUSE, C. R., GINSBOURG, B. L., AND LASZLO, I. (1973) Nature New Biol. 244, 26-27. BLOOM, F. E. (1972) in BLASCHKO,H., AND MUSCOLL, E. (Eds.), Handbook of Experimental Pharmacology: Vol. 33: Catecholamines, pp. 46-78. SpringerVerlag, Berlin/Heidelberg/New York. CORRODI, H., AND HILLARP, N.-A. (1964) Helv. Chim. Acta 47, 911-918. ELOFSSON, R., FALCK, B., LINDVALL, O., AND MYHRBERG, H. (1977) Cell Tissue Res. 182, 525-536, FALCK, B. (1962) Acta Physiol. Scand. 56(Suppl. 197), 1-25. FALCK, B., HILLARP, N.-/~., THIEME, G., AND TORP, A. (1962) J. Histochem. Cytochem. 10, 348-354. FARgAS, B. (1927) Zool. Jahrb. Anat. 49, 1-56. GORVETT, H. (1946) Quart. J. Microse. Sci. N.S. 87, 209-235. HAMBERGER, B., AND MASUOKA, D. (1965) Acta Pharmaeol. Toxicol. 22, 363-368. HAMBERGER, B., NORBERG, K.-A., AND OLSON, L. (1967) Acta Physiol. Scand. 69, 1-12. H(~KFELT, T. (1968) Z. ZeUforsch. 91, 1-74. JONSSON, G. (1966) Acta Chem. Scand. 20, 2755-2762. KARNOVSK¥, M. J. (1965) J. Cell Biol. 27, 137A. KLEMM, N. (1972) Comp. Biochem. Physiol. 43A, 207-211. MARTIN, R., AND BARLOW, J. (1972) Z. Zellforsch. 122, 16-30. MATUS, A. I. (1971a) Tissue Cell 3, 389-394. MATUS, A. I. (1971b) Z. ZeUforsch. 122, 111-121. OSCHMAN, J. C., AND BERRIDGE, M. J. {1970) Tissue Cell 2, 281-310. ROBERTSON, H. A. (1975) J. Exp. Biol. 63, 413-419. WHITEHEAD, A. W. (1971) J. Morphol. 135, 483-506. YONGE, C. M. {1932) Proc. Roy. Soc. London B 11, 298-329.