Cell Tiss. Res. t62, 13--22 (1975) 9 by Springer-Verlag 1975
The Mandibular Organ of the Lobster, H o m a r u s a m e r i c a n u s
*
E. H. B y a r d a n d R. R. Shivers Department of Zoology, University of V~restern Ontario, London, Ontario, Canada D. E. Aiken I)epartment of the Environment,, Biological Station~ St.. Andrews, New Brunswick, Canada Received May 30, 1975
Summary. The lobster mandibular organ is well vascularized and its polygonal cells are arranged loosely around blood vessels and blood sinuses. Numerous mitochondria and microbodies (peroxisomes) give the acidophilic cytoplasm a finely granular appearance, but there is no evidence of secretory granules. The abundant endoplasmic reticulum is almost entirely agranular and occurs in two morphologically distinct forms: tubular and cisternal. The tubular reticulum is randomly distributed and may represent the site of synthesis and transport of the mandibular organ product. The cisternal reticulum is frequently associated with microbodies. Both forms of endoplasmie reticulum proliferate during mid to late premolt. Mandibular organ ultrastructure closely resembles that of cells known to synthesize steroids or lipids, which suggests that this organ ma,y have a similar function. There is no ftmetional evidence of involvement in molt control in Homarus, but ultrastruetural and other evidence suggests an analogy with the insect corpus allatum. Key words: Mandibular organ - - Lobster - - Agranular endoplasmie reticulum - - Microbodies - - Corpus allatum analogue. Introduction L e R o u x (1968, 1969) described a " m a n d i b u l a r o r g a n " i n the m a n d i b u l o m a x i l l a r y region of 20 different decapod species, a n d proposed t h a t m a n d i b u l a r organs a n d Y-organs are separate structures i n all decapods. This c e r t a i n l y holds for the B r a c h y u r a where the separate locations of both are well established, b u t i n other decapods the i d e n t i t y of the organs is less certain. Sochasky et al. (1972), i n s u p p o r t of L e R o u x ' s position, p o i n t e d o u t t h a t v i r t u a l l y all so-called " Y - o r g a n " work i n the Macrura had, i n fact, been done oi1 the m a n d i b u l a r organ. T h e y also described the location of a possible homolog of the Carcinus Y - o r g a n i n the lobster Homarus, a n d this identification was confirmed b y L e R o u x (1974), t h u s establishing a g r e e m e n t o n the separate identities of the m a n d i b u l a r organ a n d the Y - o r g a n i n the Macrura.
Send o//print requests to: Dr. E. H. Byard, Department of Zoology, University of Western Ontario, London, Ontario, N6A 5B7 Canada. * Supported by a National l~esearch Council of Canada grant to 1~. I~. Shivers. E. H. Byard thanks the staff of the Biological Station, St. Andrews, New Brunswick, Canada, for their time and cooperation and D. B. MeMfllan for his encouragement during the preparation of this manuscript. J . B . Soehasky provided useful criticism and access to preliminary translations.
14
E.H. Byard et al.
Aoto et al. (1974) recently published a n u l t r a s t r u c t u r a l s t u d y of molt cycle changes i n the m a n d i b u l a r organ a n d Y-organ of Palaemon paucidens, a n a t a n t i a n decapod. No comparable studies exist for the R e p t a n t i a (lobsters, crayfish, crabs, etc.). Some useful i n f o r m a t i o n on the m a n d i b u l a r organ can be found i n the descriptions of the crayfish " Y - o r g a n " given b y C h a u d o n n c r c t (1956), D u r a n d (1960), Connell (1970), Miyawaki a n d T a k e t o m i (1970), a n d Miyawaki et al. (1971). L e R o u x (1969, 1974) gave brief descriptions of the lobster m a n d i b u l a r organ a n d Y-organ, b u t m a d e no observations on fine structure a n d no a t t e m p t to correlate changes with the molt cycle. I n the present paper we will describe the gross a n d fine s t r u c t u r e of the lobster m a n d i b u l a r organ with reference to different stages of the molt cycle. Materials and Methods Mature lobsters (Homarus americanus) were obtained irom Conley's Lobster Co., St. Andrews, New Brunswick and were held at the Biological Station, St. Andrews, in 3 foot square tanks supplied with running sea water. The animals were fed herring, cod, and shrimp ad libitum every four days. The sexes were mixed, and no more than twelve animals were held per tank. The animals used in this study were all derived from a single stock (the southern Nova Scotia fishery). Molt stages were determined for lobsters with the pleopod examination technique worked out by Aiken (1973). For light microscopy, mandibular organs were fixed in Bouin's fluid and stained with hematoxylin and eosin. For electron microscopy, mandibular organs were fixed in one of the following solutions (at 4~ (a) a mixture of 1% osmium tetroxide and 6.25% glutaraldehyde, buffered to pH 7.4 with 0.063 M s-collidine (Trump and Bulger, 1966); (b) 10% paraformaldehyde containing 0.122M sucrose, 0.005M CaC12, buffered to pH 7.4 with 0.07M s-collidine, followed by 2% buffered osmium tetroxide (pH 7.4) (Winborn and Seelig, 1970); (c) 6% glutaraldehyde buffered with 0.05M cacodylate to pH 7.4 (with 2 % sucrose). After a buffered wash, this tissue was incubated according to the method of Novikoff and Goldfischer (1969) to demonstrate catalase activity. Control media contained 0.03M 3-amino-l,2,4-triazole (to inhibit catalase) or 5 • 10 4 M KCN to inhibit mitochondrial cytochrome oxidase. Tissue was postfixed in 2% buffered osmium tetroxide. All tissue was embedded in Epon 812 (Luft, 1961). Silver to gold sections were collected on uncoated copper grids and examined unstained, stained for 30 minutes with saturated aqueous uranyl acetate, or double stained with uranyl acetate and 0.5% lead citrate (Venable and Coggeshall, 1965). Results Lobster m a n d i b u l a r organs are a t t a c h e d to the posterior face of the m a n d i b l e near the bases of the p r o m i n e n t m a n d i b u l a r a d d u c t o r muscles (Sochasky et al. 1972). T h e y are large ( ~ t w o grains i n large males), multilobate, pale to dark green structures which have a compact appearance in situ b u t a flattened, foliaceous s t r u c t u r e when freshly dissected. I n m a t u r e males the m a n d i b u l a r organ is larger a n d more resistant to mechanical damage t h a n it is in m a t u r e females. A b r a n c h of the v e n t r a l thoracic artery enters the p a r e n c h y m a on the v e n t r a l surface a n d ramifications provide a rich blood supply (Fig. 1). The cell p o p u l a t i o n of the m a n d i b u l a r organ varies depending on location in the organ (Fig. 2). Along the zone of a t t a c h m e n t of the organ to the posterior m a n d i b u l a r a d d u c t o r muscle, the cells are elongate (longitudinal axis a b o u t 10 [zm) a n d aligned i n rows or cords (Fig. 2a). T h e y have a p r o m i n e n t nucleus a n d lightly s t a i n i n g cytoplasm. Mitotic figures are c o m m o n a n d have been observed at all
Lobster Mandibular Organ
15
Fig. 1. Light micrograph of mandibular organ of male, stage D~. Note branching blood vessel
(BV) with rapid reduction in diameter (*). Small branches seem continuous with tissue spaces admitting entry of hemocytes (H) into parenchyma, x 450
molt stages and in both sexes. Toward the central region of the mandibular organ the cells gradually enlarge (Fig. 2b). Nuclei measure up to 10 ~m and the amount of acidophilic, granular cytoplasm increases, resulting in spherical and polygonal cells up to 30 ~m in diameter. Usually part of each cell surface abuts on a blood sinus, and hemocytes are common in the spaces between the cells (Figs. 1, 2). No ducts are present in this organ. At its distal edge the cells show extensive vaeuolization (Fig. 2 c). The cell boundaries are indistinct and some nuclei are pyknotic. Although the mandibular organ cells vary in size and degree of vacuolization, their complement of cytoplasmic organelles is the same in both sexes. The fine structure of a typical mandibular organ cell is shown in Figure 3. The granular appearance of the cytoplasm of these cells under the light microscope is due to the presence of numerous mitoehondria, microbodics, and less frequent autophagie vacuoles. I n the perinuclear region vesicular Golgi complexes and lipid droplets are common and the latter sometimes occur in clusters (Fig. 4). The agranular endoplasmic reticulum of these cells has two morphologically distinct forms: tubular (TER) and cisternal (CER)(Figs. 3, 6, 7). The tubular form is the most common, occurring randomly throughout the cells (Figs. 3, 6). Occasionally, short segments have ribosomes attached to them. The granular reticulum appears to proliferate in both sexes during premolt, especially in late premolt (el. Fig. 6), resulting in large areas filled with agranular reticulum to the exclusion of other organelles. The eisternal retieulum is frequently associated with mitochondria or microbodies, and it is rare to find a microbody that does not have at least one closely
Lobster Mandibular Organ
17
apposed segment of CER. In some cases, the CER forms extensive layers around a microbody (Fig. 7). These "whorls" are often large and probably correspond to the round pale areas of cytoplasm with acidophilic centers which are readily seen with the light microscope (W, Fig. 2b). These pale areas occur at all molt stages, but are more common in stages D 1 to D 4 of premolt in both sexes. I n addition to the well developed endoplasmic reticulum, the lobster mandibular organ cell often has a convoluted plasma membrane (Fig. 5). Its invaginations extend deep into the cell, giving rise to tortuous extracellular channels which appear to be continuous with larger extracellular spaces. The plasma membrane convolutions occur at the surface of cells facing blood sinuses as well as those directly apposed to other cells. Each cell is covered by an external lamina which does not follow the invaginations of the plasma membrane (EL, Fig. 5), and separates the mandibular organ cells from adjacent blood sinuses. When mandibular organ cells are incubated with diaminobenzidine in the presence of hydrogen peroxide at pH 9.0 and then post-fixed with OsO4, a dense reaction product is found over the microbodies in the cytoplasm (Fig. 8), indicating the presence of catalase (Novikoff and Goldfischer, 1968, 1969; Locke, 1971, Beard, 1972). Microbodies have a pale, finely granular matrix, and microbody-like structures, with a more condensed matrix, are often seen (Fig. 9). Similar structures contain myelin figures and other debris embedded in their matrix (Fig. 9); they do not react with diaminobenzidine and m a y be intermediate between microbodies and autophagic vacuoles (cf. Fig. 3).
Discussion
The general organization of the lobster mandibular organ is that of an endocrine structure. Extensive vascularization provides the parenchymal cells with access to the hemolymph, and there are no ducts for export of secretory products. At the ultrastructural level the most characteristic feature is the quantity of two distinct types of agranular endoplasmic reticulum, often so extensive that it displaces other organelles. In attempting to assign it a possible role or roles it is appropriate to consider the role of the agranular E R in other cells. Agranular T E R is a common feature of vertebrate cells involved in steroid synthesis (Long and Jones, 1967; Christensen and Gillim, 1969; Lofts, 1972). On the basis of a well developed agranular TER, Locke (1969) has suggested that insect oenocytes are involved in steroid metabolism. Agranular T E R is prominent in some insect corpora allata (Fukuda et al., 1966; King et al., 1966; Odhiambo 1966a, b; Aggarwal and King, 1969; Thomsen and Thomsen, 1970), and has been implicated as the site of synthesis of the methyl ester known as juvenile hormone (R611er and Dahm, 1968). Formed secretory product has not been found in either steroid synthesizing cells or in the cells of the corpus allatum. This observation is
Fig. 2a--c. Representative micrographs illustrating regional variability of cells of mandibular organ, a. area close to attachment to posterior mandibular adductor muscle; b. central region c. distaledge. Note mitotic figures (arrows); BV=blood vessel, H--hemocyte, P=pyknotic nucleus, V~vacuole, W=whorl. Female, stage C~. • 625 2 Cell.Tiss. Res.
Lobs~r Mandibular Organ
19
consistent with the idea that the extensive cisterns of the T E R contain the secretion product in a non-condensed form which is ultimately released at the cell surface. Thus, the mandibular organ cells, with their well developed agranular TER, are morphologically similar to cells involved in either lipid or steroid synthesis. The amount of agranular CER associated with a microbody varies from a single segment to large "whorls" involving several segments of endoplasmic reticulum. The whorls are slightly more common in premolt animals. Similar structures have been observed by Joly et al. (1968) and Fain-Maurel and Cassier (1969) in the corpora allata of locusts. Odhiambo (1966b) also observed agranular membrane whorls around what he termed a "granular tysosome" in the corpus allatum of the locust. The granular lysosome could be interpreted as a microbody. The close association between microbodies and agranular endoplasmic reticulum in mandibular organ cells further indicates that they may be lipid metabolizing cells (Beard, 1972). Comparison between the results reported here for H o m a r u s a m e r i c a n u s mandibular organs and those of Aoto et al. (1974)for P a l a e m o n p a u c i d e n s reveals similarities at the light microscopic level, but several cytological differences at the ultrastructural level (Table 1). Table 1. Comparison of the ultrastructural features of the mandibular organs of Palaemon paucidens and Homarus americanus Palaemon paucidens a
Homarus americanus
Agranular ER sparse Agranular ER shows no cyclic, molt related changes "Ring shaped" Golgi complexes No microbodies present
extensive agranular ER agranular ER proliferates in mid-to-late premolt vesicular Golgi complexes large population of microbodies
a based on Aoto et al. (1974) The function of the mandibular organ remains unresolved. I n general, we agree with Aoto et al. (1974) that there are no major ultrastructural changes during the molt cycle. Furthermore, it apparently does not contain molt-inducing substances (Connell, 1970; Carlisle and Connick, 1973b); lobsters deprived of their mandibular organs have survived and continued to molt for up to two years (Aiken, unpublished). Passano (1960) suggested that it might be fruitful to search for a crustacean analog of the insect corpus allatum. I t seems appropriate to explore the possibility that the mandibular organ represents this structure.
Fig. 3. Fine structure of typical mandibular organ cell. A V=autophagic vacuole, G:Golgi complex, L : lipid droplet, M ~ microbody, M T = mitochondrion, N = nucleus, N C =nucleolus, T E R = t u b u l a r endoplasmic reticulum. Male, stage D0. X 13,485 F!g. 4. Lipid droplets (L) typically in cluster. M--microbody. Male, stage D2. x 16,000 Fig. 5. External lamina (EL) that does not follow invaginations of plasma membranes of contiguous parenchymal cells (arrows). Female, stage D0. x 17,000
Lobster Mandibular Organ
21
Fig. 6. Two forms of agranular endoplasmic reticulum. Tubular form (TER) randomly distributed; cisternal form (CER) in parallel array. Male, stage D e. • 19,000 Fig. 7. Whorl of cisternal endoplasmic reticulum (CER) surrounding microbody (M). Male, stage D e. • 15,050 Fig. 8. Microbodies following incubation in diaminobenzidine plus 5 • 10 4 M KCN. Positive reaction suggestive of catalase activity. M : m i c r o b o d y , M T : m i t o c h o n d r i o n . Male, stage C4. • 26,500 Fig. 9. Structures (arrows) resembling microbodies (M) with dense matrix and myelin figure (m/) indicative of autophagic activity. Female, stage D 1. • 13,600
References Aggarwal, S. K., King, R. : A comparative study of the ring glands from wild type and 1 (2) gl m u t a n t Erosophila melanogaster. J. Morph. 129, 171-200 (1969) Aiken, D. E. : Proecdysis, setal development, a n d molt prediction in the American lobster (Ho~arus americanus). J . Fish. Res. Bd. Can. 30, 1337-1344 (1973) Aoto, T., Kamiguchi, u Hisano, S. : Histological and ultrastructural studies on the Y-organ and the mandibular organ of the freshwater prawn, Palaemon paucidens, with special reference to their relation with the molting cycle. Jour. Fac. Sci. Hokkaido Univ. Ser. VI, Zool. 19 (2), 295-306 (1974) Beard, M. E. : Identification of peroxisomes in the r a t adrenal cortex. J. Histochem. Cytochem. 20, 173-179 (1972) Carlisle, D. B., Connick, R. O. : Crustecdysone (20-hydroxyecdysone): site of storage in the crayfish Orconectes propinquus. Canad. J. Zool. 51, 417-420 (1973b) Chaudonneret, J. : Le syst~me nerveux de la r~gion gnathale de l'4cr~visse Cambarus a//ini8 (Say). Ann. Sci. Nat. Ser. 11, Zool. Biol. Anim. 18, 33-61 (1956) Christensen, A . K . , Gillim, S . W . : The Gonads, K . W . McKerns (ed.). Amsterdam: NorthHolland, 1969 Connell, P. A. M. : The hormonal control of molting in the dwarf crayfish, Cambarellusshu]eldti. P h . D . Thesis, D e p a r t m e n t of Biology, Tulane University, 156 pp. 1970 Durand, J. B. : Limb regeneration and endocrine activity in the crayfish. Biol. Bull. (Woods Hole) 118, 250-261 (1960) Fain-Maurel, M. A., Cassier, P. : ]~tude infrastructurale des corpora allata de Locusta mi!lratoria migratorioides (R and F.), phase solitaire, au cours de la m a t u r a t i o n sexuelle et des cycles ovariens. C. R. Acad. Sci. (Paris) Ser. D 268, 2721-2723 (1969) Fukuda, S., Eguchi, G., Takeuchi, S.: Histological and electron microscopical studies on sexual differences in the structure of the corpora allata of the moth of the silkworm, Bombyx mori. Embryologia 9, 123-158 (1966) Joly, L., Joly, P., Porte, A., Girardie, A. : ]~tude physiologique et ultrastructurale des corpora allata de Locusta migratoria L. (Orthoptbre) en phase gr6gaire. Arch. Zool. exp. gen. 109, 703-728 (1968) King, R., Aggarwal, S. K., Bodenstein, D. : The comparative submicroscopic cytology of the corpus allatum-corpus cardiacum complex of wild type a n d / e s adult female Drosophila melanogaster. J. exp. Zool. 161, 151-176 (1966) Le Roux, A. : Description d'organes mandibulaires noveaux chez les Crustac6s D6capodes. C . R . Acad. Sci. (Paris), S6r. D Sci. Nat. 266, 1414-1417 (1968). (Description of new mandibular organs in the decapod Crustacca. Fish. Res. Board Can, Transl. Ser. No. 2015) Le Roux, A. : Aspects histologiques et histochimiques des organes mandibulaires chez les Crustaces Decapodes. Bull. Soc. Zool. Ft. 94, 299-300 (1969). (Histological a n d histochemical appearance of the mandibular organs in dceapod crustaceans. Fish. Res. Board Can., Transl. Ser. No. 2408) Le Roux, A. : Mise au point s propos de la distinction entre l'organe Y e t l'organe mandibulaire chez les Crustac6s eucarides. C. R. Acad. Sci. (Paris), S6r. D Sci. Nat. 278, 1261-1264 (1974). [Improvement concerning the distinction between the u organ and the mandibular organ in eucarid crustaceans. Fish. Mar. Service (Can.), Transl. Ser. No. 3090]
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Locke, M. : The ultrastructure of the oenocytes in the molt/intermolt cycle of an insect. Tissue a n d Cell 1, 103-154 (1969) Locke, M. : The origin and fate of microbodies in the fat body of an insect. J. Cell Biol. 48, 61-78 (1971) Lofts, B.: The Sertoli cell. Gen. comp. Endocr., Suppl. 3, 636-648 (1972) Long, J. A., Jones, A. L. : The fine structure of the zona glomerulosa and the zona fasciculata of the adrenal cortex of the opossum. Amer. J. Anat. 120, 463-471 (1967) Luft, J. H. : I m p r o v e m e n t s in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409-414 (1961) Miyawaki, M., Taketomi, Y. : Structural changes induced in the cells of Y gland of crayfish b y an administration of ecdysterone. Zool. Mag. 79, 150-155 (1970) Miyawaki, M., Taketomi, Y., Couch, E. F. : The susceptibility of Y gland cells of the crayfish Procambaru8 clarkii, to exogenous steroids. Annot. Zool. Jap. 44, 42-46 (1971) Novikoff, A. B., Goldfischer, S. : Visualization of microbodies for light and electron microscopy. J. Histoehem. Cytochem. 16, 507 (abs.) (1968) Novikoff, A.B., Goldfischer, S. : Visualization of peroxisomes (microbodies) and mitoehondria with diaminobenzidine. J. Histoehem. Cytochem. 17, 675-680 (1969) Odhiambo, T. R. : The fine structure of the corpus allatum of the sexually mature male of the desert locust. J. Insect Physiol. 12, 819-828 (1966a) Odhiambo, T. R. : Ultrastructure of the development of the corpus allatum in the adult male of the desert locust. J. Insect Physiol. 12, 995-1002 (1966b) Passano, L. M. : Molting and its control, p. 473-536. I n : T. H. W a t e r m a n (ed.), The physiology of crustaeea. Vol. 1. New York: Academic Press (1960) RSller, H., Dahm, K. H.: The chemistry and biology of juvenile hormone. Recent Progr. Hormone Res. 24, 651-680 (1968) Sochasky, J. B., Aiken, D. E., Watson, N. H. F. : Y-organ, molting gland, and mandibular organ: a problem in~ decapod crustacea. Canad. J. Zool. 50, 993-997 (1972) Thomsen, E., Thomsen, M. : Fine structure of the corpus allatum of the female blow-fly, Calliphora erythrocephala. Z. Zellforsch. l l 0 , 40-60 (1970) Trump, B. F., Bulger, R. E. : New ultrastructural characteristics of cells fixed in a glutaraldehyde-osmium tetroxide mixture. Lab. Invest. 15, 368-379 (1966) Venable, J. H., Coggeshall, P. : A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408 (1965) Winborn, W. B., Seelig, L. L. : Paraformaldehyde and s-collidine- a fixative for preserving large blocks for electron microscopy. Texas Rep. Biol. Med. 28, 347-361 (1970)
The Mandibular Organ of the Lobster, H o m a r u s a m e r i c a n u s
*
E. H. B y a r d a n d R. R. Shivers Department of Zoology, University of V~restern Ontario, London, Ontario, Canada D. E. Aiken I)epartment of the Environment,, Biological Station~ St.. Andrews, New Brunswick, Canada Received May 30, 1975
Summary. The lobster mandibular organ is well vascularized and its polygonal cells are arranged loosely around blood vessels and blood sinuses. Numerous mitochondria and microbodies (peroxisomes) give the acidophilic cytoplasm a finely granular appearance, but there is no evidence of secretory granules. The abundant endoplasmic reticulum is almost entirely agranular and occurs in two morphologically distinct forms: tubular and cisternal. The tubular reticulum is randomly distributed and may represent the site of synthesis and transport of the mandibular organ product. The cisternal reticulum is frequently associated with microbodies. Both forms of endoplasmie reticulum proliferate during mid to late premolt. Mandibular organ ultrastructure closely resembles that of cells known to synthesize steroids or lipids, which suggests that this organ ma,y have a similar function. There is no ftmetional evidence of involvement in molt control in Homarus, but ultrastruetural and other evidence suggests an analogy with the insect corpus allatum. Key words: Mandibular organ - - Lobster - - Agranular endoplasmie reticulum - - Microbodies - - Corpus allatum analogue. Introduction L e R o u x (1968, 1969) described a " m a n d i b u l a r o r g a n " i n the m a n d i b u l o m a x i l l a r y region of 20 different decapod species, a n d proposed t h a t m a n d i b u l a r organs a n d Y-organs are separate structures i n all decapods. This c e r t a i n l y holds for the B r a c h y u r a where the separate locations of both are well established, b u t i n other decapods the i d e n t i t y of the organs is less certain. Sochasky et al. (1972), i n s u p p o r t of L e R o u x ' s position, p o i n t e d o u t t h a t v i r t u a l l y all so-called " Y - o r g a n " work i n the Macrura had, i n fact, been done oi1 the m a n d i b u l a r organ. T h e y also described the location of a possible homolog of the Carcinus Y - o r g a n i n the lobster Homarus, a n d this identification was confirmed b y L e R o u x (1974), t h u s establishing a g r e e m e n t o n the separate identities of the m a n d i b u l a r organ a n d the Y - o r g a n i n the Macrura.
Send o//print requests to: Dr. E. H. Byard, Department of Zoology, University of Western Ontario, London, Ontario, N6A 5B7 Canada. * Supported by a National l~esearch Council of Canada grant to 1~. I~. Shivers. E. H. Byard thanks the staff of the Biological Station, St. Andrews, New Brunswick, Canada, for their time and cooperation and D. B. MeMfllan for his encouragement during the preparation of this manuscript. J . B . Soehasky provided useful criticism and access to preliminary translations.
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E.H. Byard et al.
Aoto et al. (1974) recently published a n u l t r a s t r u c t u r a l s t u d y of molt cycle changes i n the m a n d i b u l a r organ a n d Y-organ of Palaemon paucidens, a n a t a n t i a n decapod. No comparable studies exist for the R e p t a n t i a (lobsters, crayfish, crabs, etc.). Some useful i n f o r m a t i o n on the m a n d i b u l a r organ can be found i n the descriptions of the crayfish " Y - o r g a n " given b y C h a u d o n n c r c t (1956), D u r a n d (1960), Connell (1970), Miyawaki a n d T a k e t o m i (1970), a n d Miyawaki et al. (1971). L e R o u x (1969, 1974) gave brief descriptions of the lobster m a n d i b u l a r organ a n d Y-organ, b u t m a d e no observations on fine structure a n d no a t t e m p t to correlate changes with the molt cycle. I n the present paper we will describe the gross a n d fine s t r u c t u r e of the lobster m a n d i b u l a r organ with reference to different stages of the molt cycle. Materials and Methods Mature lobsters (Homarus americanus) were obtained irom Conley's Lobster Co., St. Andrews, New Brunswick and were held at the Biological Station, St. Andrews, in 3 foot square tanks supplied with running sea water. The animals were fed herring, cod, and shrimp ad libitum every four days. The sexes were mixed, and no more than twelve animals were held per tank. The animals used in this study were all derived from a single stock (the southern Nova Scotia fishery). Molt stages were determined for lobsters with the pleopod examination technique worked out by Aiken (1973). For light microscopy, mandibular organs were fixed in Bouin's fluid and stained with hematoxylin and eosin. For electron microscopy, mandibular organs were fixed in one of the following solutions (at 4~ (a) a mixture of 1% osmium tetroxide and 6.25% glutaraldehyde, buffered to pH 7.4 with 0.063 M s-collidine (Trump and Bulger, 1966); (b) 10% paraformaldehyde containing 0.122M sucrose, 0.005M CaC12, buffered to pH 7.4 with 0.07M s-collidine, followed by 2% buffered osmium tetroxide (pH 7.4) (Winborn and Seelig, 1970); (c) 6% glutaraldehyde buffered with 0.05M cacodylate to pH 7.4 (with 2 % sucrose). After a buffered wash, this tissue was incubated according to the method of Novikoff and Goldfischer (1969) to demonstrate catalase activity. Control media contained 0.03M 3-amino-l,2,4-triazole (to inhibit catalase) or 5 • 10 4 M KCN to inhibit mitochondrial cytochrome oxidase. Tissue was postfixed in 2% buffered osmium tetroxide. All tissue was embedded in Epon 812 (Luft, 1961). Silver to gold sections were collected on uncoated copper grids and examined unstained, stained for 30 minutes with saturated aqueous uranyl acetate, or double stained with uranyl acetate and 0.5% lead citrate (Venable and Coggeshall, 1965). Results Lobster m a n d i b u l a r organs are a t t a c h e d to the posterior face of the m a n d i b l e near the bases of the p r o m i n e n t m a n d i b u l a r a d d u c t o r muscles (Sochasky et al. 1972). T h e y are large ( ~ t w o grains i n large males), multilobate, pale to dark green structures which have a compact appearance in situ b u t a flattened, foliaceous s t r u c t u r e when freshly dissected. I n m a t u r e males the m a n d i b u l a r organ is larger a n d more resistant to mechanical damage t h a n it is in m a t u r e females. A b r a n c h of the v e n t r a l thoracic artery enters the p a r e n c h y m a on the v e n t r a l surface a n d ramifications provide a rich blood supply (Fig. 1). The cell p o p u l a t i o n of the m a n d i b u l a r organ varies depending on location in the organ (Fig. 2). Along the zone of a t t a c h m e n t of the organ to the posterior m a n d i b u l a r a d d u c t o r muscle, the cells are elongate (longitudinal axis a b o u t 10 [zm) a n d aligned i n rows or cords (Fig. 2a). T h e y have a p r o m i n e n t nucleus a n d lightly s t a i n i n g cytoplasm. Mitotic figures are c o m m o n a n d have been observed at all
Lobster Mandibular Organ
15
Fig. 1. Light micrograph of mandibular organ of male, stage D~. Note branching blood vessel
(BV) with rapid reduction in diameter (*). Small branches seem continuous with tissue spaces admitting entry of hemocytes (H) into parenchyma, x 450
molt stages and in both sexes. Toward the central region of the mandibular organ the cells gradually enlarge (Fig. 2b). Nuclei measure up to 10 ~m and the amount of acidophilic, granular cytoplasm increases, resulting in spherical and polygonal cells up to 30 ~m in diameter. Usually part of each cell surface abuts on a blood sinus, and hemocytes are common in the spaces between the cells (Figs. 1, 2). No ducts are present in this organ. At its distal edge the cells show extensive vaeuolization (Fig. 2 c). The cell boundaries are indistinct and some nuclei are pyknotic. Although the mandibular organ cells vary in size and degree of vacuolization, their complement of cytoplasmic organelles is the same in both sexes. The fine structure of a typical mandibular organ cell is shown in Figure 3. The granular appearance of the cytoplasm of these cells under the light microscope is due to the presence of numerous mitoehondria, microbodics, and less frequent autophagie vacuoles. I n the perinuclear region vesicular Golgi complexes and lipid droplets are common and the latter sometimes occur in clusters (Fig. 4). The agranular endoplasmic reticulum of these cells has two morphologically distinct forms: tubular (TER) and cisternal (CER)(Figs. 3, 6, 7). The tubular form is the most common, occurring randomly throughout the cells (Figs. 3, 6). Occasionally, short segments have ribosomes attached to them. The granular reticulum appears to proliferate in both sexes during premolt, especially in late premolt (el. Fig. 6), resulting in large areas filled with agranular reticulum to the exclusion of other organelles. The eisternal retieulum is frequently associated with mitochondria or microbodies, and it is rare to find a microbody that does not have at least one closely
Lobster Mandibular Organ
17
apposed segment of CER. In some cases, the CER forms extensive layers around a microbody (Fig. 7). These "whorls" are often large and probably correspond to the round pale areas of cytoplasm with acidophilic centers which are readily seen with the light microscope (W, Fig. 2b). These pale areas occur at all molt stages, but are more common in stages D 1 to D 4 of premolt in both sexes. I n addition to the well developed endoplasmic reticulum, the lobster mandibular organ cell often has a convoluted plasma membrane (Fig. 5). Its invaginations extend deep into the cell, giving rise to tortuous extracellular channels which appear to be continuous with larger extracellular spaces. The plasma membrane convolutions occur at the surface of cells facing blood sinuses as well as those directly apposed to other cells. Each cell is covered by an external lamina which does not follow the invaginations of the plasma membrane (EL, Fig. 5), and separates the mandibular organ cells from adjacent blood sinuses. When mandibular organ cells are incubated with diaminobenzidine in the presence of hydrogen peroxide at pH 9.0 and then post-fixed with OsO4, a dense reaction product is found over the microbodies in the cytoplasm (Fig. 8), indicating the presence of catalase (Novikoff and Goldfischer, 1968, 1969; Locke, 1971, Beard, 1972). Microbodies have a pale, finely granular matrix, and microbody-like structures, with a more condensed matrix, are often seen (Fig. 9). Similar structures contain myelin figures and other debris embedded in their matrix (Fig. 9); they do not react with diaminobenzidine and m a y be intermediate between microbodies and autophagic vacuoles (cf. Fig. 3).
Discussion
The general organization of the lobster mandibular organ is that of an endocrine structure. Extensive vascularization provides the parenchymal cells with access to the hemolymph, and there are no ducts for export of secretory products. At the ultrastructural level the most characteristic feature is the quantity of two distinct types of agranular endoplasmic reticulum, often so extensive that it displaces other organelles. In attempting to assign it a possible role or roles it is appropriate to consider the role of the agranular E R in other cells. Agranular T E R is a common feature of vertebrate cells involved in steroid synthesis (Long and Jones, 1967; Christensen and Gillim, 1969; Lofts, 1972). On the basis of a well developed agranular TER, Locke (1969) has suggested that insect oenocytes are involved in steroid metabolism. Agranular T E R is prominent in some insect corpora allata (Fukuda et al., 1966; King et al., 1966; Odhiambo 1966a, b; Aggarwal and King, 1969; Thomsen and Thomsen, 1970), and has been implicated as the site of synthesis of the methyl ester known as juvenile hormone (R611er and Dahm, 1968). Formed secretory product has not been found in either steroid synthesizing cells or in the cells of the corpus allatum. This observation is
Fig. 2a--c. Representative micrographs illustrating regional variability of cells of mandibular organ, a. area close to attachment to posterior mandibular adductor muscle; b. central region c. distaledge. Note mitotic figures (arrows); BV=blood vessel, H--hemocyte, P=pyknotic nucleus, V~vacuole, W=whorl. Female, stage C~. • 625 2 Cell.Tiss. Res.
Lobs~r Mandibular Organ
19
consistent with the idea that the extensive cisterns of the T E R contain the secretion product in a non-condensed form which is ultimately released at the cell surface. Thus, the mandibular organ cells, with their well developed agranular TER, are morphologically similar to cells involved in either lipid or steroid synthesis. The amount of agranular CER associated with a microbody varies from a single segment to large "whorls" involving several segments of endoplasmic reticulum. The whorls are slightly more common in premolt animals. Similar structures have been observed by Joly et al. (1968) and Fain-Maurel and Cassier (1969) in the corpora allata of locusts. Odhiambo (1966b) also observed agranular membrane whorls around what he termed a "granular tysosome" in the corpus allatum of the locust. The granular lysosome could be interpreted as a microbody. The close association between microbodies and agranular endoplasmic reticulum in mandibular organ cells further indicates that they may be lipid metabolizing cells (Beard, 1972). Comparison between the results reported here for H o m a r u s a m e r i c a n u s mandibular organs and those of Aoto et al. (1974)for P a l a e m o n p a u c i d e n s reveals similarities at the light microscopic level, but several cytological differences at the ultrastructural level (Table 1). Table 1. Comparison of the ultrastructural features of the mandibular organs of Palaemon paucidens and Homarus americanus Palaemon paucidens a
Homarus americanus
Agranular ER sparse Agranular ER shows no cyclic, molt related changes "Ring shaped" Golgi complexes No microbodies present
extensive agranular ER agranular ER proliferates in mid-to-late premolt vesicular Golgi complexes large population of microbodies
a based on Aoto et al. (1974) The function of the mandibular organ remains unresolved. I n general, we agree with Aoto et al. (1974) that there are no major ultrastructural changes during the molt cycle. Furthermore, it apparently does not contain molt-inducing substances (Connell, 1970; Carlisle and Connick, 1973b); lobsters deprived of their mandibular organs have survived and continued to molt for up to two years (Aiken, unpublished). Passano (1960) suggested that it might be fruitful to search for a crustacean analog of the insect corpus allatum. I t seems appropriate to explore the possibility that the mandibular organ represents this structure.
Fig. 3. Fine structure of typical mandibular organ cell. A V=autophagic vacuole, G:Golgi complex, L : lipid droplet, M ~ microbody, M T = mitochondrion, N = nucleus, N C =nucleolus, T E R = t u b u l a r endoplasmic reticulum. Male, stage D0. X 13,485 F!g. 4. Lipid droplets (L) typically in cluster. M--microbody. Male, stage D2. x 16,000 Fig. 5. External lamina (EL) that does not follow invaginations of plasma membranes of contiguous parenchymal cells (arrows). Female, stage D0. x 17,000
Lobster Mandibular Organ
21
Fig. 6. Two forms of agranular endoplasmic reticulum. Tubular form (TER) randomly distributed; cisternal form (CER) in parallel array. Male, stage D e. • 19,000 Fig. 7. Whorl of cisternal endoplasmic reticulum (CER) surrounding microbody (M). Male, stage D e. • 15,050 Fig. 8. Microbodies following incubation in diaminobenzidine plus 5 • 10 4 M KCN. Positive reaction suggestive of catalase activity. M : m i c r o b o d y , M T : m i t o c h o n d r i o n . Male, stage C4. • 26,500 Fig. 9. Structures (arrows) resembling microbodies (M) with dense matrix and myelin figure (m/) indicative of autophagic activity. Female, stage D 1. • 13,600
References Aggarwal, S. K., King, R. : A comparative study of the ring glands from wild type and 1 (2) gl m u t a n t Erosophila melanogaster. J. Morph. 129, 171-200 (1969) Aiken, D. E. : Proecdysis, setal development, a n d molt prediction in the American lobster (Ho~arus americanus). J . Fish. Res. Bd. Can. 30, 1337-1344 (1973) Aoto, T., Kamiguchi, u Hisano, S. : Histological and ultrastructural studies on the Y-organ and the mandibular organ of the freshwater prawn, Palaemon paucidens, with special reference to their relation with the molting cycle. Jour. Fac. Sci. Hokkaido Univ. Ser. VI, Zool. 19 (2), 295-306 (1974) Beard, M. E. : Identification of peroxisomes in the r a t adrenal cortex. J. Histochem. Cytochem. 20, 173-179 (1972) Carlisle, D. B., Connick, R. O. : Crustecdysone (20-hydroxyecdysone): site of storage in the crayfish Orconectes propinquus. Canad. J. Zool. 51, 417-420 (1973b) Chaudonneret, J. : Le syst~me nerveux de la r~gion gnathale de l'4cr~visse Cambarus a//ini8 (Say). Ann. Sci. Nat. Ser. 11, Zool. Biol. Anim. 18, 33-61 (1956) Christensen, A . K . , Gillim, S . W . : The Gonads, K . W . McKerns (ed.). Amsterdam: NorthHolland, 1969 Connell, P. A. M. : The hormonal control of molting in the dwarf crayfish, Cambarellusshu]eldti. P h . D . Thesis, D e p a r t m e n t of Biology, Tulane University, 156 pp. 1970 Durand, J. B. : Limb regeneration and endocrine activity in the crayfish. Biol. Bull. (Woods Hole) 118, 250-261 (1960) Fain-Maurel, M. A., Cassier, P. : ]~tude infrastructurale des corpora allata de Locusta mi!lratoria migratorioides (R and F.), phase solitaire, au cours de la m a t u r a t i o n sexuelle et des cycles ovariens. C. R. Acad. Sci. (Paris) Ser. D 268, 2721-2723 (1969) Fukuda, S., Eguchi, G., Takeuchi, S.: Histological and electron microscopical studies on sexual differences in the structure of the corpora allata of the moth of the silkworm, Bombyx mori. Embryologia 9, 123-158 (1966) Joly, L., Joly, P., Porte, A., Girardie, A. : ]~tude physiologique et ultrastructurale des corpora allata de Locusta migratoria L. (Orthoptbre) en phase gr6gaire. Arch. Zool. exp. gen. 109, 703-728 (1968) King, R., Aggarwal, S. K., Bodenstein, D. : The comparative submicroscopic cytology of the corpus allatum-corpus cardiacum complex of wild type a n d / e s adult female Drosophila melanogaster. J. exp. Zool. 161, 151-176 (1966) Le Roux, A. : Description d'organes mandibulaires noveaux chez les Crustac6s D6capodes. C . R . Acad. Sci. (Paris), S6r. D Sci. Nat. 266, 1414-1417 (1968). (Description of new mandibular organs in the decapod Crustacca. Fish. Res. Board Can, Transl. Ser. No. 2015) Le Roux, A. : Aspects histologiques et histochimiques des organes mandibulaires chez les Crustaces Decapodes. Bull. Soc. Zool. Ft. 94, 299-300 (1969). (Histological a n d histochemical appearance of the mandibular organs in dceapod crustaceans. Fish. Res. Board Can., Transl. Ser. No. 2408) Le Roux, A. : Mise au point s propos de la distinction entre l'organe Y e t l'organe mandibulaire chez les Crustac6s eucarides. C. R. Acad. Sci. (Paris), S6r. D Sci. Nat. 278, 1261-1264 (1974). [Improvement concerning the distinction between the u organ and the mandibular organ in eucarid crustaceans. Fish. Mar. Service (Can.), Transl. Ser. No. 3090]
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Locke, M. : The ultrastructure of the oenocytes in the molt/intermolt cycle of an insect. Tissue a n d Cell 1, 103-154 (1969) Locke, M. : The origin and fate of microbodies in the fat body of an insect. J. Cell Biol. 48, 61-78 (1971) Lofts, B.: The Sertoli cell. Gen. comp. Endocr., Suppl. 3, 636-648 (1972) Long, J. A., Jones, A. L. : The fine structure of the zona glomerulosa and the zona fasciculata of the adrenal cortex of the opossum. Amer. J. Anat. 120, 463-471 (1967) Luft, J. H. : I m p r o v e m e n t s in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409-414 (1961) Miyawaki, M., Taketomi, Y. : Structural changes induced in the cells of Y gland of crayfish b y an administration of ecdysterone. Zool. Mag. 79, 150-155 (1970) Miyawaki, M., Taketomi, Y., Couch, E. F. : The susceptibility of Y gland cells of the crayfish Procambaru8 clarkii, to exogenous steroids. Annot. Zool. Jap. 44, 42-46 (1971) Novikoff, A. B., Goldfischer, S. : Visualization of microbodies for light and electron microscopy. J. Histoehem. Cytochem. 16, 507 (abs.) (1968) Novikoff, A.B., Goldfischer, S. : Visualization of peroxisomes (microbodies) and mitoehondria with diaminobenzidine. J. Histoehem. Cytochem. 17, 675-680 (1969) Odhiambo, T. R. : The fine structure of the corpus allatum of the sexually mature male of the desert locust. J. Insect Physiol. 12, 819-828 (1966a) Odhiambo, T. R. : Ultrastructure of the development of the corpus allatum in the adult male of the desert locust. J. Insect Physiol. 12, 995-1002 (1966b) Passano, L. M. : Molting and its control, p. 473-536. I n : T. H. W a t e r m a n (ed.), The physiology of crustaeea. Vol. 1. New York: Academic Press (1960) RSller, H., Dahm, K. H.: The chemistry and biology of juvenile hormone. Recent Progr. Hormone Res. 24, 651-680 (1968) Sochasky, J. B., Aiken, D. E., Watson, N. H. F. : Y-organ, molting gland, and mandibular organ: a problem in~ decapod crustacea. Canad. J. Zool. 50, 993-997 (1972) Thomsen, E., Thomsen, M. : Fine structure of the corpus allatum of the female blow-fly, Calliphora erythrocephala. Z. Zellforsch. l l 0 , 40-60 (1970) Trump, B. F., Bulger, R. E. : New ultrastructural characteristics of cells fixed in a glutaraldehyde-osmium tetroxide mixture. Lab. Invest. 15, 368-379 (1966) Venable, J. H., Coggeshall, P. : A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408 (1965) Winborn, W. B., Seelig, L. L. : Paraformaldehyde and s-collidine- a fixative for preserving large blocks for electron microscopy. Texas Rep. Biol. Med. 28, 347-361 (1970)
