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A preliminary histochemical study on the labral glands of Daphnia obtusa (Crustacea, Cladocera)
Acta histochem. 88,175-181 (1990) VEB Gustav Fischer Verlag lena
Institute of Comparative Anatomyl), University of Ferrara, Ferrara, Italy, and Department of Animal Biology2), University of Modena, Modena, Italy
A preliminary histochemical study on the labral glands of Daphnia obtusa (Crustacea, Cladocera) By CRISTINA ZENI') and ANTONELLA FRANCHINI 2 ) With 7 Figures (Received November 17, 1989)
Summary The gland cells located in the upper lip of the c1adoceran Daphnia were studied by histochemical reactions to establish the chemical nature of some substances they synthesize. Neutral polysaccharides were found to be present, but acid glycosaminoglycans absent. Large amounts of proteins and ribonucleoproteins are also present, lipid substances were not detected. Immunohistochemical methods failed to reveal lX-amylase in the labral gland cells, though the enzyme was detected in the cells of the intestine. The secretion products of the labral gland cells are probably glycoproteins. The results are discussed in terms of the possible roles of these substances in the animal's physiology.
1. Introduction Far fewer histochemical, biological, and ultrastructural studies have been made on the buccal gland system of crustaceans than of that of other invertebrates, and only limited information is available on the labral glands of non-malacostracans crustaceans, i.e., branchiopods. Most histomorphological and histochemical studies date back 30 a or more (CUNNINGTON 1903; CANNON 1922, 1935; NICHOLSON and YONGE 1935; STERBA 1957; DORNESCO and STEOPOE 1958; FRYER 1962). Some more recent reports, however, have supplied ultrastructural details on the labral glands of harpacticoid copepods (GHARAGOZLOU-VAN GINNEKEN 1977) and daphnid cladocerans (ZAFFAGNINI and ZENI 1987). The upper lip of adult daphnids has 4 large cells on each side, arranged in pairs (CANNON 1922; STERBA 1957). The ultrastructural appearance of the cytoplasm is similar in 3 of the 4 cells (the anterior pair and cell A of the posterior pair) and differs in cell B of the posterior pair (ZAFFAGNINI and ZENI1987). The cells are the site of intense synthetic activity, and are in contact with a duct cell, whose narrow, cuticularized lumen appears to contain no secretion (ZAFFAGNINI and ZENI 1987). The presence of nerve fibres in contact with these gland cells has recently been demonstrated (ZENI and ZAFFAGNINI 1988). The probable function of the labral glands have been discussed, and some authors have hypothesized that in anostracans they may be responsible for the production of a sticky substance that might facilitate swallowing when brushed onto food particles (CANNON 1935; DORNESCO and STEOPOE 1958; FRYER 1983). Other authors have denied that there is any relationship between these gland cells and feeding, asserting instead that the glands have the same function as decapod tegumental glands, namely, the formation of the external cuticle (NICHOLSON and YONGE 1935). As for the labral glands of cladocerans, it has been hypothesized that their secretion is utilized for the food ingestion, as claimed for anostracans (CANNON 1922; STERBA 1957; FRYER 1962). 13'
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C. ZENI and A. FRANCHINI
They have also been hypothesized to have an endocrine function (ZAFFAGNINI 1964). The information currently available on tlIe chemical nature of their secretion is insufficient to identify tlIem as salivary glands, as suggested by GHARAGOZLOU-VAN GINNEKEN (1977) for the labral glands of harpacticoid copepods. This work represents a first attempt to histochemically characterize the material produced by gland cells of the labrum of Daphnia, in order to obtain information about their function.
2. Material and methods Adult females of Daphnia obtusa, collected in a pond at the Zoological Institute of Bologna University, were fixed in Bouin's fluid and 10% buffered formol and embedded in paraffin. Histomorphological stains (haematoxylineosin, Azan-Mallory) and histochemical reactions were applied to 5 to 7 flm sections to detect proteins and polysaccharides. Other animals were frozen with CO2 and sliced with a Cryocut. These sections were fixed in Baker's formol-calcium and tested for lipids. The following histochemical methods were employed. Proteins: The reaction with mercury-bromophenol blue after MAZIA, BREWER and ALFERT (1953), the Ninhydrin-Schiff reaction after YASUMA and IcHIKAWA (1953); MOREL and SISLEY'S reaction after GLENNER and LILLIE (1959); the reaction with DOD after BARNETT and SELIGMAN (1952); the performic acid-Schiff reaction after PEARSE (1951); the ferric ferricyanide method after CHtvREMONT and FREDERIC (1943), see (PEARSE 1985). Ribonucleoproteins: Azur A; gallocyanin chromalum; pyronine (PEARSE 1985). These reactions were also carried out against digestion with RNase (I mglml distilled water, I h at 37 QC). Polysaccharides: The PAS reaction; Alcian blue 8GX pH = 2.5 (PEARSE 1985). Lipids: The Sudan black B method after BAYLISS and ADAMS (1972); Oil Red 0 after LILLIE and ASHBURN (1943); Nile blue sulphate and BAKER'S (1946) acid haematein (see BANCROFT and STEVENS 1977). Immunocytochemical method for detection of ex-amylase, according to Hsu et aI. (1981), was done on sections Q obtained from animals fixed in GPA. Antihuman ex-amylase Sigma (1:250) was applied for 24 h at 4 C. Negative controls of the reaction omitted the primary antibody; positive controls utilized human submandibular gland sections. A few animals were fixed in 2.5 % glutaraldehyde in 0.1 molll cacodylate buffer at pH = 7.4 and postfixed in OS04 in the same buffer. After dehydration in alcohols and propylene oxide, the animals were embedded in toto in Araldite. Semithin sections, obtained with a Reichert OM U3 ultramicrotome, were stained with toluidine blue and PAS technique (LASCHI and GOVONI 1978). Ultrathin sections obtained using a diamond knife, placed on 100 mesh Formvar-coated aurum grids, were stained according to Thiery's (1967) method. The exposure to thiosemicarbazide (TSC) varied from I h to 3 d. The grids were observed with a Hitachi H-800 electron microscope at the Electron Microscopic Centre of Ferrara University.
3. Results On tlIe basis of histochemical reactions, tlIe gland cells of the labrum of D. obtusa reveal to contain proteins and ribonucleoproteins (Fig. 1). The histochemical procedures for detecting tlIe -NH z, -SH, and -S-S- groups or the aminoacid tyrosine in tlIe proteins, which might have supplied information on protein types, all produced similar positive results. It was therefore impossible to demonstrate whether any radical or aminoacid predominated. RNase digestion shows that much of tlIe cytoplasmic contents of the labral gland cells are ribonucleoprotein materials (Fig. 2). The cytoplasm of tlIe labral glands positively reacts to the PAS technique indicating tlIe presence of polysaccharides (Figs. 3, 4). On the contrary, tlIe Alcian blue technique at pH = 2.5 gave negative results. At the ultrastructural level, the Thiery's reaction showed tlIe presence of much glycogen in tlIe cytoplasm of tlIe anterior gland cells, in clothing of
Histochemistry of the labral glands of Daphnia
177
a
2 Fig. 1. Micrograph of the labral glands of Daphnia obtusa. a anterior pair; p posterior pair. Azur A. X 160. Fig. 2. Micrograph of the labral glands of D. obtusa. a anterior pair; p posterior pair. RNase digestion/Azur A. x 160. Fig. 3. Micrograph of the labral glands of D. obtusa. a anterior pair; p posterior pair. PAS. x 160. Fig. 4. Micrograph of the labral gland of D. obtusa. a portion of cytoplasm of an anterior gland cell; p posterior pair. It is visible the large cell A and a little part of the cell B (at the right bottom). PAS on semithin section. x 160.
probably be related to the greater compactness of the RER and to the major amount of glycogen observed by the electron microscope. All the histochemical reactions carried out to detect lipid substances (neutral and acid lipids, phospholipids) were negative. All the results of the histochemical tests are reported in Table I. The procedure for the immunocytochemical detection of
Table I. Histochemical reactions on the labral glands of D. obtusa. Bromophenol blue Ninhydrin-Schiff Morel-Sisley DDD Performic acid-Schiff Chevremont-Frederic AzurA RNase/ Azur A Gallocyanin chromalum RNase/Gallocyanin Pyronine RNase/Pyronine PAS Alcian blue, pH = 2.5 Sudan black B Oil redO Nile blue sulphate Acid haematein Key:
+ positive; ++
very positive;
+
+ + + ±
+
++
++ ±
++ ± ++
± weakly positive; - negative reaction.
178
C. ZENI and A. FRANCHINI
4. Discussion Histochemical investigation of the labral glands of D. obtusa has demonstrated the presence of large amounts of protein substances in their cytoplasm. These findings are consistent with the abundance of RER observed in these cells at the ultrastructural level (ZAFFANGNINI and ZENI 1987). Much of the protein matter is associated with RNA, as previously reported by ZAFFAGNINI (1964), and confmned here by RNase digestion. The lack of cellular polarity permits no differentiation of the structural proteins from the secretory proteins. The secretion of the labral glands of non-malacostracan crustaceans (mystacocarids and copepods) is rich in polysaccharides (POCHON-MASSON et al. 1975; GHARAGOZLOU-VAN GINNEKEN 1977). In this context are included the labral glands of Daphina whose cytoplasm contains neutral polysaccharides. CANNON (1922) instead reports that the labral glands of Simocephalus lack mucins, and STERBA (1957) states that the labral glands of Daphnia are HOTCHKIss-negative. The Thiery's reaction shows that the glycogen is absent in the cell A. Therefore the PAS positivity of this cell should be due to different polysaccharides. We are not been able [also with exposure to TSC of 72 h as suggested by Thiery (1967»), to show other carbohydrates. This method, however, is considered not very sensitive for demonstration of glycoproteins (CANTIN and BENCHIMOL 1975). The secretion of the labral glands of D. obtusa does not seem to be of enzymatic nature, since the immunoenzymatic test for detecting ex-amylase was negative. The absence of ex-amylase confirms the results obtained by STERBA (1957), who observed that squashed labral glands do not hydrolyse starck and glycogen. Nevertheless, the presence of other enzymes cannot be ruled out. Since the glands open into the alimentary groove, outside the mouth, it is possible that a part of the secretion contains proenzymes. The presence of protein and carbohydrates in the cytoplasm of these glands suggests that the 2 compounds are bound to form glycoprotein. The presence of glycoproteins in the secretion of the labral glands might be interpreted in several ways. The labral glands of Daphnia may be involved in the feeding process, since the salivary glands of many other invertebrates, especially insects [i.e., Locusta, LAUVERJAT (1972, 1973)] secrete glycoproteins. Furthermore, the fact that these labral gland cells may release their products directly into the haemolymph and are innervated, and that many hormones are glycoprotein, suggests that in addition to their exocrine function, the glands may also have an endocrine function, as previously hypothesized (ZAFFAGNINI 1964). The different ultrastructural appearance of cell B of the posterior pair suggested that this cell may synthesize substances of a different chemical nature from that of the other 3 cells (ZAFFAGNINI and ZENI 1987). Since cell B's responses to the histochemical reactions did not differ in any way from other gland cells, it was impossible to establish whether this is so. The unusually large intercellular space between the 2 cells of the posterior pair, interpreted as the site of accumulation of the secretion products, remains colourless with all the staining techniques performed. It may be that the material in this space, flocculent at the ultrastructural level (ZAFFAGNINI and ZENI 1987), is not preserved by histological methods used. Alternatively, not being bound to microscopically demonstrable substrata, this material may be difficult to reveal histochemically. Daphnia mainly feeds on algae and bacteria. HASLER (1935, 1937) has isolated proteinases, amylases, Iipases, and peptidases from the guts of D. magna and D. pulex. Recently SCHOENBERG et al. (1984) have found evidence for cellulose digestion, although the possibility that this reflects the activity of symbionts cannot excluded. The presence of these enzymes has not, however, been immunohistochemically demonstrated. Consequently, ex-amylase we detected in the intestinal cells and the contents of the lumen of the gut of Daphnia represent an interesting finding.
Acknowledgements The authors wish to thank Prof. Dr. F. ZAFFAGNINI and Prof. Dr. A. M. BOLOGNANI FANTIN for the helpful suggestions and for the critical reading of the manuscript. We also thank Ms E. ZIRONI for her help in the typewriting of this MS.
Fig. 5. Electron micrograph of the cytoplasm of a gland cell of the anterior pair of the labrum of D. obtusa, showing a Golgi complex. G Golgi complex; Gl glycogen. THIERY reaction (time of TSC exposure = 72 h). Bar t!, 0.5 ftm. Fig. 6. Electron micrograph at the level of the contact zone between a gland cell of the anterior pair and the cell A (A) of the posterior pair. THIERY reaction (time of TSC exposure = 72 h). Bar t!, 1 ftm.
Histochemistry of the labral glands of Daphnia
179
I
Fig. 7. Electron micrograph of the gland cells of the posterior pair of the labrum of D. obtusa. Detail of the zone where the cell A (A) is contiguous to the cell B (B). Gl glycogen; IS intercellular space. THIERY reaction (time of TSC exposure = 72 h). Bar ~ 114m.
Histochemistry of the labral glands of Daphnia
181
References BANCROIT, J. D., and STEVENS, A., Theory and practice of histological techniques. Churchill-Livingstone, Edinburgh 1977. CANNON, H. G., On the labral glands of a Cladoceran (Simocephalus vetulus) with a description of its mode of feeding. Quart. J. Microsc. Sci. 66, 213-234 (1922). - Function of the labral gland in Chirocephalus. Nature 136, 758 (1935). CANTIN, M., and BENCHIMOL, S., Localization and characterization of carbohydrates in adrenal medullary cells. J. Cell BioI. 65,463-479 (1975). CUNNINGTON, W. A., Studien an einer Daphnide, Simocephalus sima. Beitriige zur Kenntnis des Centralnervensystems und der feineren Anatomie der Daphniden. Z. Naturwiss. 37,447-520 (1903). DORNESCO, G. T., et STEOPOE, J., Les glandes tegumentaires des Phyllopodes Anostraces. Ann. Sci. Nat. Zool. 20, 29-69 (1958). FRYER, G., Secretions of the labral and trunk limb glands in the c1adoceran Eurycercus lamellatus. Nature 195, 97 (1962). - Functional ontogenetic changes in Branchinecta ferox (MILNE-EDWARDS) (Crustacea: Anostraca). Phil. Trans. Roy. Soc. London 3038, 229- 343 (1983). GHARAGOZLOU-VAN GINNEKEN, I. D., Contribution 11 l'etude infrastructurale des glandes labrales de quelques harpacticoides (Crustace, Copepodes). Arch. BioI. 88, 79-100 (1977). HASLER, A. D., The physiology of digeslion of plankton Crustacea. I. Some digestive enzymes of Daphnia. BioI. Bull. Woods Hole 68,207-214 (1935). - Further studies on the digestive enzymes of (A) Daphnia and Polyphemus, (B) Diaptomus and Calanus. BioI. Bull. Woods Hole 72, 290- 298 (1937). Hsu, S. M., RAINE, L., and FANGER, H., Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparative between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577-580 (1981). LASCHI, R., and GOVONI, E., Staining methods for semithin sections. In: Electron Microscopy in Human Medicine (1. V. JOHANNESSEN, ed.). Vol. 1: Instrumentation and Techniques. McGraw Hill, New York 1978. LAUVERJAT, S., Role des cellules zymogenes dans les secretions salivaires de Locusta migratoria (Orthoptere, Acridoidea). Tissue Cell 4, 301- 310 (1972). - Ultrastructure des glandes salivaires de Locusta migratoria. (Orthoptere, Acridoidea). Arch. Zool. expo gen. 114, 129-147 (1973). NICHOLSON, K. W., and YONGE, C. M., Function of the labral glands in Chirocephalus. Nature 136, 608 (1935). PEARSE, A. G. E., Histochemistry. Theoretical and Applied, 4th ed., vol. 2: Analytical Technology, ChurchillLivingstone, Edinburgh 1985. POCHON-MASSON, J., RENAUD-MoRNANT, J., et CALS, P., Contribution ilia connaissance des glandes tegumentaires metameriques d'un Crustace Meiobenthique interstitiel (Crustacea Mystacocarida). Cytologie structurale et infrastructurale. Arch. Zool. expo gen. 116, 123-146 (1975). SCHOENBERG, S. A., MACCUBBIN, A. E., and HODSON, R. E., Cellulose digestion by freshwater microcrustacea. Limnol. Oceanogr. 29, 1132-1137 (1984). STERBA, G., Die Riesenzellen der Daphniden-Oberlippe. Z. Zellforsch. 47, 198-213 (1957). THIERY, J. P., Mise en evidence des polysaccharides sur coupes fines en microscopie electronique. J. Microscopie 6, 987-1018 (1967). ZAFFAGNINI, F., Prime indagini suI controllo endocrino della muta e della riproduzione in Daphnia magna (Crustacea: Cladocera). Arch. Zool. Ital. 49, 157-179 (1964). - and ZENI, c., Ultrastructural investigations on the labral glands of Daphnia obtusa (Crustacea, Cladocera). J. Morphol. 193, 23- 33 (1987). ZENI, c., and ZAFFAGNINI, F., Occurrence of innervation in labral glands of Daphnia obtusa (Crustacea, Cladocera). J. Morphol. 198, 43-4l! (1988). Authors' address: Dr. CRISTINA ZENI, Institute of Comparative Anatomy, University of Ferrara, Via L. Borsari 46, 1-44100 Ferrara.
Institute of Comparative Anatomyl), University of Ferrara, Ferrara, Italy, and Department of Animal Biology2), University of Modena, Modena, Italy
A preliminary histochemical study on the labral glands of Daphnia obtusa (Crustacea, Cladocera) By CRISTINA ZENI') and ANTONELLA FRANCHINI 2 ) With 7 Figures (Received November 17, 1989)
Summary The gland cells located in the upper lip of the c1adoceran Daphnia were studied by histochemical reactions to establish the chemical nature of some substances they synthesize. Neutral polysaccharides were found to be present, but acid glycosaminoglycans absent. Large amounts of proteins and ribonucleoproteins are also present, lipid substances were not detected. Immunohistochemical methods failed to reveal lX-amylase in the labral gland cells, though the enzyme was detected in the cells of the intestine. The secretion products of the labral gland cells are probably glycoproteins. The results are discussed in terms of the possible roles of these substances in the animal's physiology.
1. Introduction Far fewer histochemical, biological, and ultrastructural studies have been made on the buccal gland system of crustaceans than of that of other invertebrates, and only limited information is available on the labral glands of non-malacostracans crustaceans, i.e., branchiopods. Most histomorphological and histochemical studies date back 30 a or more (CUNNINGTON 1903; CANNON 1922, 1935; NICHOLSON and YONGE 1935; STERBA 1957; DORNESCO and STEOPOE 1958; FRYER 1962). Some more recent reports, however, have supplied ultrastructural details on the labral glands of harpacticoid copepods (GHARAGOZLOU-VAN GINNEKEN 1977) and daphnid cladocerans (ZAFFAGNINI and ZENI 1987). The upper lip of adult daphnids has 4 large cells on each side, arranged in pairs (CANNON 1922; STERBA 1957). The ultrastructural appearance of the cytoplasm is similar in 3 of the 4 cells (the anterior pair and cell A of the posterior pair) and differs in cell B of the posterior pair (ZAFFAGNINI and ZENI1987). The cells are the site of intense synthetic activity, and are in contact with a duct cell, whose narrow, cuticularized lumen appears to contain no secretion (ZAFFAGNINI and ZENI 1987). The presence of nerve fibres in contact with these gland cells has recently been demonstrated (ZENI and ZAFFAGNINI 1988). The probable function of the labral glands have been discussed, and some authors have hypothesized that in anostracans they may be responsible for the production of a sticky substance that might facilitate swallowing when brushed onto food particles (CANNON 1935; DORNESCO and STEOPOE 1958; FRYER 1983). Other authors have denied that there is any relationship between these gland cells and feeding, asserting instead that the glands have the same function as decapod tegumental glands, namely, the formation of the external cuticle (NICHOLSON and YONGE 1935). As for the labral glands of cladocerans, it has been hypothesized that their secretion is utilized for the food ingestion, as claimed for anostracans (CANNON 1922; STERBA 1957; FRYER 1962). 13'
176
C. ZENI and A. FRANCHINI
They have also been hypothesized to have an endocrine function (ZAFFAGNINI 1964). The information currently available on tlIe chemical nature of their secretion is insufficient to identify tlIem as salivary glands, as suggested by GHARAGOZLOU-VAN GINNEKEN (1977) for the labral glands of harpacticoid copepods. This work represents a first attempt to histochemically characterize the material produced by gland cells of the labrum of Daphnia, in order to obtain information about their function.
2. Material and methods Adult females of Daphnia obtusa, collected in a pond at the Zoological Institute of Bologna University, were fixed in Bouin's fluid and 10% buffered formol and embedded in paraffin. Histomorphological stains (haematoxylineosin, Azan-Mallory) and histochemical reactions were applied to 5 to 7 flm sections to detect proteins and polysaccharides. Other animals were frozen with CO2 and sliced with a Cryocut. These sections were fixed in Baker's formol-calcium and tested for lipids. The following histochemical methods were employed. Proteins: The reaction with mercury-bromophenol blue after MAZIA, BREWER and ALFERT (1953), the Ninhydrin-Schiff reaction after YASUMA and IcHIKAWA (1953); MOREL and SISLEY'S reaction after GLENNER and LILLIE (1959); the reaction with DOD after BARNETT and SELIGMAN (1952); the performic acid-Schiff reaction after PEARSE (1951); the ferric ferricyanide method after CHtvREMONT and FREDERIC (1943), see (PEARSE 1985). Ribonucleoproteins: Azur A; gallocyanin chromalum; pyronine (PEARSE 1985). These reactions were also carried out against digestion with RNase (I mglml distilled water, I h at 37 QC). Polysaccharides: The PAS reaction; Alcian blue 8GX pH = 2.5 (PEARSE 1985). Lipids: The Sudan black B method after BAYLISS and ADAMS (1972); Oil Red 0 after LILLIE and ASHBURN (1943); Nile blue sulphate and BAKER'S (1946) acid haematein (see BANCROFT and STEVENS 1977). Immunocytochemical method for detection of ex-amylase, according to Hsu et aI. (1981), was done on sections Q obtained from animals fixed in GPA. Antihuman ex-amylase Sigma (1:250) was applied for 24 h at 4 C. Negative controls of the reaction omitted the primary antibody; positive controls utilized human submandibular gland sections. A few animals were fixed in 2.5 % glutaraldehyde in 0.1 molll cacodylate buffer at pH = 7.4 and postfixed in OS04 in the same buffer. After dehydration in alcohols and propylene oxide, the animals were embedded in toto in Araldite. Semithin sections, obtained with a Reichert OM U3 ultramicrotome, were stained with toluidine blue and PAS technique (LASCHI and GOVONI 1978). Ultrathin sections obtained using a diamond knife, placed on 100 mesh Formvar-coated aurum grids, were stained according to Thiery's (1967) method. The exposure to thiosemicarbazide (TSC) varied from I h to 3 d. The grids were observed with a Hitachi H-800 electron microscope at the Electron Microscopic Centre of Ferrara University.
3. Results On tlIe basis of histochemical reactions, tlIe gland cells of the labrum of D. obtusa reveal to contain proteins and ribonucleoproteins (Fig. 1). The histochemical procedures for detecting tlIe -NH z, -SH, and -S-S- groups or the aminoacid tyrosine in tlIe proteins, which might have supplied information on protein types, all produced similar positive results. It was therefore impossible to demonstrate whether any radical or aminoacid predominated. RNase digestion shows that much of tlIe cytoplasmic contents of the labral gland cells are ribonucleoprotein materials (Fig. 2). The cytoplasm of tlIe labral glands positively reacts to the PAS technique indicating tlIe presence of polysaccharides (Figs. 3, 4). On the contrary, tlIe Alcian blue technique at pH = 2.5 gave negative results. At the ultrastructural level, the Thiery's reaction showed tlIe presence of much glycogen in tlIe cytoplasm of tlIe anterior gland cells, in clothing of
Histochemistry of the labral glands of Daphnia
177
a
2 Fig. 1. Micrograph of the labral glands of Daphnia obtusa. a anterior pair; p posterior pair. Azur A. X 160. Fig. 2. Micrograph of the labral glands of D. obtusa. a anterior pair; p posterior pair. RNase digestion/Azur A. x 160. Fig. 3. Micrograph of the labral glands of D. obtusa. a anterior pair; p posterior pair. PAS. x 160. Fig. 4. Micrograph of the labral gland of D. obtusa. a portion of cytoplasm of an anterior gland cell; p posterior pair. It is visible the large cell A and a little part of the cell B (at the right bottom). PAS on semithin section. x 160.
probably be related to the greater compactness of the RER and to the major amount of glycogen observed by the electron microscope. All the histochemical reactions carried out to detect lipid substances (neutral and acid lipids, phospholipids) were negative. All the results of the histochemical tests are reported in Table I. The procedure for the immunocytochemical detection of
Table I. Histochemical reactions on the labral glands of D. obtusa. Bromophenol blue Ninhydrin-Schiff Morel-Sisley DDD Performic acid-Schiff Chevremont-Frederic AzurA RNase/ Azur A Gallocyanin chromalum RNase/Gallocyanin Pyronine RNase/Pyronine PAS Alcian blue, pH = 2.5 Sudan black B Oil redO Nile blue sulphate Acid haematein Key:
+ positive; ++
very positive;
+
+ + + ±
+
++
++ ±
++ ± ++
± weakly positive; - negative reaction.
178
C. ZENI and A. FRANCHINI
4. Discussion Histochemical investigation of the labral glands of D. obtusa has demonstrated the presence of large amounts of protein substances in their cytoplasm. These findings are consistent with the abundance of RER observed in these cells at the ultrastructural level (ZAFFANGNINI and ZENI 1987). Much of the protein matter is associated with RNA, as previously reported by ZAFFAGNINI (1964), and confmned here by RNase digestion. The lack of cellular polarity permits no differentiation of the structural proteins from the secretory proteins. The secretion of the labral glands of non-malacostracan crustaceans (mystacocarids and copepods) is rich in polysaccharides (POCHON-MASSON et al. 1975; GHARAGOZLOU-VAN GINNEKEN 1977). In this context are included the labral glands of Daphina whose cytoplasm contains neutral polysaccharides. CANNON (1922) instead reports that the labral glands of Simocephalus lack mucins, and STERBA (1957) states that the labral glands of Daphnia are HOTCHKIss-negative. The Thiery's reaction shows that the glycogen is absent in the cell A. Therefore the PAS positivity of this cell should be due to different polysaccharides. We are not been able [also with exposure to TSC of 72 h as suggested by Thiery (1967»), to show other carbohydrates. This method, however, is considered not very sensitive for demonstration of glycoproteins (CANTIN and BENCHIMOL 1975). The secretion of the labral glands of D. obtusa does not seem to be of enzymatic nature, since the immunoenzymatic test for detecting ex-amylase was negative. The absence of ex-amylase confirms the results obtained by STERBA (1957), who observed that squashed labral glands do not hydrolyse starck and glycogen. Nevertheless, the presence of other enzymes cannot be ruled out. Since the glands open into the alimentary groove, outside the mouth, it is possible that a part of the secretion contains proenzymes. The presence of protein and carbohydrates in the cytoplasm of these glands suggests that the 2 compounds are bound to form glycoprotein. The presence of glycoproteins in the secretion of the labral glands might be interpreted in several ways. The labral glands of Daphnia may be involved in the feeding process, since the salivary glands of many other invertebrates, especially insects [i.e., Locusta, LAUVERJAT (1972, 1973)] secrete glycoproteins. Furthermore, the fact that these labral gland cells may release their products directly into the haemolymph and are innervated, and that many hormones are glycoprotein, suggests that in addition to their exocrine function, the glands may also have an endocrine function, as previously hypothesized (ZAFFAGNINI 1964). The different ultrastructural appearance of cell B of the posterior pair suggested that this cell may synthesize substances of a different chemical nature from that of the other 3 cells (ZAFFAGNINI and ZENI 1987). Since cell B's responses to the histochemical reactions did not differ in any way from other gland cells, it was impossible to establish whether this is so. The unusually large intercellular space between the 2 cells of the posterior pair, interpreted as the site of accumulation of the secretion products, remains colourless with all the staining techniques performed. It may be that the material in this space, flocculent at the ultrastructural level (ZAFFAGNINI and ZENI 1987), is not preserved by histological methods used. Alternatively, not being bound to microscopically demonstrable substrata, this material may be difficult to reveal histochemically. Daphnia mainly feeds on algae and bacteria. HASLER (1935, 1937) has isolated proteinases, amylases, Iipases, and peptidases from the guts of D. magna and D. pulex. Recently SCHOENBERG et al. (1984) have found evidence for cellulose digestion, although the possibility that this reflects the activity of symbionts cannot excluded. The presence of these enzymes has not, however, been immunohistochemically demonstrated. Consequently, ex-amylase we detected in the intestinal cells and the contents of the lumen of the gut of Daphnia represent an interesting finding.
Acknowledgements The authors wish to thank Prof. Dr. F. ZAFFAGNINI and Prof. Dr. A. M. BOLOGNANI FANTIN for the helpful suggestions and for the critical reading of the manuscript. We also thank Ms E. ZIRONI for her help in the typewriting of this MS.
Fig. 5. Electron micrograph of the cytoplasm of a gland cell of the anterior pair of the labrum of D. obtusa, showing a Golgi complex. G Golgi complex; Gl glycogen. THIERY reaction (time of TSC exposure = 72 h). Bar t!, 0.5 ftm. Fig. 6. Electron micrograph at the level of the contact zone between a gland cell of the anterior pair and the cell A (A) of the posterior pair. THIERY reaction (time of TSC exposure = 72 h). Bar t!, 1 ftm.
Histochemistry of the labral glands of Daphnia
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I
Fig. 7. Electron micrograph of the gland cells of the posterior pair of the labrum of D. obtusa. Detail of the zone where the cell A (A) is contiguous to the cell B (B). Gl glycogen; IS intercellular space. THIERY reaction (time of TSC exposure = 72 h). Bar ~ 114m.
Histochemistry of the labral glands of Daphnia
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