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Amer. Zool., 24:893-909 (1984)

The Structure and of the Cuticle1

Robert Roer and Richard Dillaman Institute of Marine Biomedical Research, Universityof North Carolina at Wilmington, Wilmington,North Carolina 28403

Synopsis. The integument of decapod consists of an outer epicuticle, an exocuticle, an endocuticle and an inner membranous layer underlain by the hypodermis. The outer three layers of the are calcified. The is in the form of and amorphous carbonate. In the epicuticle, mineral is in the form of spherulitic calcite islands surrounded by the lipid- matrix. In the exo- and endo- the calcite aggregates are interspersed with -protein fibers which are organized in lamellae. In some species, the organization of the mineral mirrors that of the organic fibers, but such is not the case in certain cuticular regions in the xanthid . Thus, control of crystal organization is a complex phenomenon unrelated to the gross morphology of the matrix. Since the cuticle is periodically molted to allow for growth, this necessitates a bidirec- tional movement of calcium into the cuticle during postmolt and out during premolt resorption of the cuticle. In two species of crabs studied to date, these movements are accomplished by active transport effected by a Ca-ATPase and Na/Ca exchange mech? anism. The epi- and exocuticular layers of the new cuticle are elaborated during premolt but do not calcify until the old cuticle is shed. This phenomenon also occurs in vitro in cuticle devoid of living tissue and implies an alteration of the nucleating sites of the cuticle in the course of the molt.

Introduction within the matrix and mechanisms for con? While the crustacean cuticle has been trol or cessation of crystal growth. The crustacean cuticle has a rich the subject of study for over 250 years provided source of information with to each (Reaumur, 1712, in Drach, 1939), the focus respect of these central In addition, the of those investigations has generally been questions. Crustacea offer some for concerned with the process of molting. Our unique problems those interested in . approach will be slightly different; we will deal with the of the Crustacea Since the mineralized exoskeleton ofthe Crustacea is to as a mineralized tissue that is made partic? subjected periodic molting, bidirectional net movement of mineral ularly interesting by the fact that its struc? different of the molt is ture is affected by a cyclic molting process. during stages cycle This situation is in con? When investigating any mineralized tis? necessary. sharp trast to other in which sue, one must address some basic problems: calcifying systems net or 1) the chemical and crystalline form accretionary growth patterns slightly are the rule. Further? of the mineral; 2) the nature and form of shifting equilibria more, crustaceans demonstrate drastic the organic matrix; 3) the relationship differences within the same tissue between the organic and inorganic com? temporal with to the extent of and ponents of the tissue and the influence of regard capacity for mineralization. Such differ? the matrix on crystal morphology; 4) the temporal ences, marked and discrete tran? sources of mineral for deposition; 5) the by rapid sitions, allow one to ask and pathways for mineral movement into or very specific answerable about the control of out from the mineralized structures; 6) questions nucleation and mineralization. rates of mineral deposition; and 7) the nature and location of nucleation sites The subject of crustacean cuticles is as diverse as is the taxon and, hence, an ency- clopedic overview is impossible. We will restrict our review, in to the 1 From the on Mechanisms large part, Symposium of Calcification crustaceans in Biological Systemspresented at the Annual Meeting decapod although examples from other orders will serve to remind us ofthe American Society of Zoologists, 27-30 Decem? ber 1983, at Philadelphia, Pennsylvania. ofthe hazards of sweeping generalizations. 893 894 R. Roer and R. Dillaman

Structure of the Decapod im ] Ep 5, Integument

Basic cuticular structure

The integument of the decapod Crus? tacea is, in general, comprised of a rigid exoskeleton or cuticle underlain by the cel? Ex 65pm lular hypodermis (Richards, 1951). The cuticle is not homogeneous, but contains 2.2Hm/l four discrete layers. Despite the fact that this observation was made more than a cen? tury ago (Williamson, 1860) and has been the subject of numerous subsequent stud? ies, the nomenclature of the four layers of the cuticle varies from author to author. The terminology of Travis (1963), how? ever, has come to be widely accepted and will be used exclusively in the present work. These layers from the most external to the 'V-> most internal are: the epicuticle, the exo- cuticle, the endocuticle and the membra- nous layer (Fig. 1). The epicuticle is the outermost and thin- nest ofthe cuticle. It consists of tanned En 195pm layer lipoprotein impregnated with calcium salts It is bilaminar, with the 8.1 Hm/I (Travis, 1955a). basal layer pervaded by mineral-filled canals normal to the surface (Hegdahl et al, 1977c). The exocuticle immediately underlies the epicuticle. It is composed of chitin-protein fibers stacked in layers of continuously changing orientation (Travis, 1955a; Green and Neff, 1972). In 34% by weight of the organic component is chitin (Welinder, 19756). The exocuticle is hard- ened by quinone tanning and calcification (Travis, 1955a), with the mineral crystals situated between the fibers (Bouligand, 1970; Hegdahl etal, 19776). The endocuticle is the thickest and the most heavily calcified layer of the cuticle (Travis, 1955a, 1965). The endocuticle, like the is of hori? Fig. 1. Montage ofthe mineralized cuticular layers exocuticle, composed = of Carcinus maenas carapace (from Roer 1979). En zontal lamellae of chitin-protein fibers with Ex = = Num? endocuticle, exocuticle, Ep epicuticle. continuously changing orientation (Green bers the total thickness of the individual represent and Neff, 1972; etal, the and the thickness of endo- and exocuticular Hegdahl 1977a), layers material 73% chitin lamellae (1). organic being by weight in Cancer pagurus (Welinder, 19756). The endocuticle is apparently not tanned, but hardened solely by Ca salts (Travis, 1955?). Calcification in Crustacean Cuticle 895

The membranous layer is the innermost there are an estimated 150,000-220,000 layer of the cuticle and is in contact with pore canals per mm2 (Hegdahl et al, 1977a); the hypodermis. It consists of chitin and in Carcinus maenas there are about protein, the chitin representing 74% ofthe 950,000/mm2 (Roer, 1980); and Orconectes organic material in Cancer pagurus (Wel? virilis there are 50-90 pore canals ema- inder, 19756), but contains no mineral nating from each epithelial cell or about (Travis, 1955a). 4,000,000/mm2 (Travis, 1963). Thus, the The mineral in the outer three layers of cuticle is in close contact with the hypo? the cuticle is CaC03 in the form of calcite at all levels and should be consid? or poorly crystalline, amorphous calcium ered living tissue. carbonate (Travis, 1963). The molt The hypodermis is heterogeneous, hav? cycle ing numerous cell types arranged in three Rather detailed accounts ofthe phenom? principal layers (Travis, 1955a, b, 1957, ena related to the molt cycle of the Crus? 1965). The layer in contact with the cuticle tacea come from the last century (Vitzou, and apparently responsible for its forma? 1882), but the first method for describing tion is the outer epithelial layer or the cuti? the molt cycle that could be applied to the cle secreting cells of Green and Neff (1972). Crustacea in general did not appear until The outer epithelial layer is one cell thick much later (Drach, 1939). According to and may range from squamous to colum? this method, the molt cycle is divided into nar as will be discussed below. Beneath the five stages (A-E), with further subdivisions outer epithelium is the sub-epithelial con? within each stage. Thus the postmolt period nective tissue layer (Travis, 1955a, 6) which corresponds to stages Al9 A2, Bb B2, Cl9 consists of oval reserve cells, blood sinuses C2, and C3; intermolt is stage C4; premolt with hemocytes, lipoprotein cells (Sewell, is comprised of stages D0, D/, D/', D/", 1955) and pigment cells (Green and Neff, D2, D3, and D4; and the act of or 1972). Proximal to the connective tissue molting is stage E (Drach and Tchernigov- layer is the inner epithelium which is sim? tzeff, 1967). ilar to the outer epithelium but reduced in The intermolt size. The inner epithelium is bounded on integument its inner margin by a Most adult decapods spend the majority with the exception of those regions cov? ofthe molt cycle in the intermolt condition ering the branchial chamber, where the (stage C4), molting only once or twice yearly inner epithelium elaborates a thin, non- (Passano, 1960). The cuticle has its four calcified cuticle resembling the outer epi? layers complete and is fully calcified (Fig. cuticle in other respects (Skinner, 1962). 2). The hypodermis is in its most reduced The cuticle is pervaded by vertically run? state at this time. The epithelial cells in ning pore canals first described by Valentin contact with the cuticle are extremely (1837, in Richards, 1951). Further exam? squamous (Green and Neff, 1972) having ination has revealed these to be cytoplas? a height of only 9 pm in Orconectes virilis mic extensions of the outer epithelial cells (Travis, 1965). The secretory activity of which emanate from the apical cell bor- the Golgi of the epithelial cells is at a min? ders, extend through the membranous imum (Chassard-Bouchaud and Hubert, layer, endocuticle and exocuticle, and ter? 1973; Hubert and Chassard-Bouchaud, minate at (Travis, 1963; Green and Neff, 1978) and the endoplasmic reticulum is 1972) or in (Hegdahl et al, 1977'c) the epi? reduced (Green and Neff, 1972). The pore cuticle. The pore canals may have a typical canals are still structurally intact and con? helical or twisted ribbon morphology tain cytoplasm (Green and Neff, 1972) or (Drach, 1939; Neville etal, 1969) with the may be partially filled with calcite crystals pitch of the being equal to the lamel- (Travis, 1963; Travis and Friberg, 1963; lar period (Drach, 1939). These pore canals Hegdahl et al, 1977a, 6, c). are extremely numerous. In Cancer pagurus The hypodermis is not completely inac- 896 R. Roer and R. Dillaman

EARLY PREMOLT Do-D1

LATE PREMOLT D2-D4 INTERMOLT C4 Apolysis Solation of memb. layer

POSTMOLT Membranous layer A,-C3 present Mineral resorption Cuticle complete Formation of new epi- & exocuticle

Calcification of pre-exuvial cuticle Deposition of endocuticle

Fig. 2. Schematic representation of the cuticular events associated with progression through the molt cycle. = = = = Ep epicuticle, Ex exocuticle, En endocuticle, Mb membranous layer.

tive, however, for a peak in RNA synthesis Through the premolt period they change is seen during C4 (Skinner, 1966) and the from squamous to columnar. In Orconectes reserve cells of the highly vacuolated and they double their height between D0 and reduced connective tissue are engaged in D2, reaching a maximum of 54 ^m (six times the storage of materials for the ensuing their height in C4) by D3 (Travis, 1965); premolt period (Travis, 1957). the maximal height is about 56 jum in Car? cinus (Roer, 1980). The Golgi begin to The premolt integument secrete a proteinaceous paracrystalline The onset of premolt (stage D0) is marked substance and an increase in smooth endo? by apolysis, or the separation of the hypo? plasmic reticulum associated with mito? dermis from the cuticle through the action chondria is observed (Hubert and Chas? of secreted chitinase, chitobiase and pro? sard-Bouchaud 1978). Mitotic activity is tease (Jeuniaux, 1959a, 6; Bade and Stin- apparent in the epithelial cells during stages son, 1978) which cause the solation ofthe D0, D/ and D/'; and there are peaks in 02 membranous layer. Apolysis results in the consumption, protein synthesis and chitin severing ofthe pore canals (Green and Neff, synthesis during stage D2 (Skinner, 1962, 1972) (Fig. 2). The outer epithelial cells 1966; Stevenson, 1972). begin to increase in height and complexity. Premolt is also the period during which Calcification in Crustacean Cuticle 897

the matrix of the new epicuticle and exo? and proceeds proximally (Travis and Fri- cuticle is laid down beneath the old cuticle. berg, 1963;Bouligand, 1970). Mineral Epicuticular deposition takes place during apparently reaches the outer portions of stage D,, and exocuticular deposition the cuticle via the pore canals. Calcium is begins at stage D2. Because these matrices concentrated in the distal portions of the are deposited before the molt, they are epithelial cells and appears to be extruded referred to as the pre-exuvial layers (Drach, in vertical rows corresponding in position 1939). Although the organic matrix ofthe to the pore canals of the new cuticle (Tra? epi- and exocuticle is laid down pre-exu- vis, 1957, 1963, 1965; Travis and Friberg, vially, these layers do not calcify until after 1963; Chockalingham, 1971). Stage B is the molt (Paul and Sharpe, 1916; Travis, marked by the onset of endocuticle depo? 1963; Travis and Friberg, 1963). The epi? sition; here calcification is concomitant with cuticle is, however, tanned before the molt matrix formation, each organic lamella (Krishnan, 1951). being mineralized as it is laid down (Drach, Concomitant with pre-exuvial deposi? 1939; Travis, 1957,1963, 1965; Travis and tion is the partial resorption of both the Friberg, 1963). mineral and organic portions of the old Mineral deposition continues through cuticle (Drach, 1939; Travis, 1965; Roer, postmolt. Calcification spreads throughout 1980). In Gecarcinus lateralis, more than the exocuticle and eventually is found to 75% of the cuticle is resorbed (Skinner, pervade the walls of the pore canals (Tra? 1962); while in Panuliris argus only about vis, 1963; Travis and Friberg, 1963). Cal? 20% ofthe carapace is resorbed, the endo? cite crystals may finally be seen within the cuticle being completely resorbed in some lumina of the pore canals as the cell pro? areas and the exocuticle being partially cesses apparently recede to be replaced with resorbed in certain regions (Travis, 1955a). mineral (Travis, 1963; Travis and Friberg, In Carcinus, 15-20% of the mineral is 1963; Hegdahl etal, 1977a, 6, c). The end resorbed during premolt (Graf, 1978). of postmolt is marked by the deposition of Resorptive activity reaches a maximum the membranous layer during stage C3 and during stage D2 (Drach, 1939; Green and the cessation of net calcium deposition Neff, 1972), and results in a rise in hemo? (Passano, 1960). lymph Ca++ concentration (Robertson, The postmolt changes in the hypoder- 1960) and HC03~ concentration. This mal cells are also marked. By one day post? causes a slight alkalosis ofthe hemolymph, molt the dedifferentiation of these epithe? the pH rising from 7.9 to 8.1 in Astacus lial cells has already begun (Green and Neff, leptodactylus (Dejours and Beekenkamp, 1972). In Orconectes, the epithelial cells 1978). decrease from their maximal height of 54 /tm to 21 nm within two days of ecdysis, The postmolt integument the decrease continuing until a minimum The initial events following the molt are is reached at intermolt (Travis, 1965). The the tanning of the exocuticle and the cal? secretory activity of the Golgi and abun? cification of the pre-exuvial layers. The dance of endoplasmic reticulum also exocuticle is tanned by quinone formation decreases through postmolt to a virtual from dihydroxyphenols under the action absence in C4 (Hubert and Chassard-Bou? of a polyphenol oxidase transported to the chaud, 1978). cuticle from the cells epithelial (Krishnan, The Relationship between Mineral 1951; Travis, 1957; Vacca and Fingerman, and Organic Components 1975a, 6). The pre-exuvial layers begin to The calcify during stage Ax. The first crystals epicuticle of CaC03 are evident in Carcinus 10 hr The epicuticle, as mentioned above, dif- after the molt (Drach, 1937); and in Astacus fers from the exo- and endocuticles in its fluviatilis the rate of Ca deposition reaches lack of chitin and lamellar organization. a peak two days postmolt (Welinder, This organizational difference is mani- 1975a). Calcification ofthe epi- and exo- fested in the orientation ofthe mineral. A cuticles begins in the most external regions transmission electron microscopic and 898 R. Roer and R. Dillaman

radiographic study by Hegdahl and co- the hypodermal cells underlying the cuti? workers (1977c) on the epicuticle of Cancer cle (Drach, 1939; Travis, 1963; Hegdahl pagurus revealed that mineral was restricted et al, 19776; Giraud-Guille and Quintana, to vertical canals, 100-250 nm in diame? 1982). When calcification is complete, ter, in the proximal layer. These canals are however, the distribution of mineral within presumably the distal terminations of the these layers is relatively homogeneous pore canals. The mineral was in the form (Hegdahl et al, 1977a, 6; Giraud-Guille and of CaC03 crystals or crystal aggregates and Quintana, 1982). were distributed unevenly and surrounded Although Bouligand (1970) claims that by epicuticular tissue. there is no orientation of the calcite crys? We have also observed a non-homoge? tals with respect to the chitin-protein fibers neous and discontinuous distribution of in Carcinus, other evidence points to the mineral in the epicuticle of Carcinus maenas. contrary. The crystal aggregates clearly are While a 24 hr treatment of Carcinus cuticle aligned with the organic fibers as deter? with 5.25% sodium hypochlorite results in mined by transmission electron micros? a complete removal of the epicuticle, brief copy in Gaetice depressus (Yano, 1975) and treatment renders the tissue only partially in Cancer pagurus (Hegdahl et al, 1977a, anorganic and reveals mineral clumps 6), and by secondary ion mass spectroscopy which are spherulitic aggregates (Figs. 3 and X radiodography in Carcinus (Giraud- and 4). The loss of these aggregates fol? Guille and Quintana, 1982). Indeed, Heg? lowing the complete hypochlorite removal dahl and co-workers (1977a) have clearly of the organic material suggests that these demonstrated rod-shaped crystal aggre? are both discrete from one another and gates interspersed with the chitin-protein from the mineral components of the fibers. underlying exocuticle. We have further investigated, by scan? ning electron microscopy, the orientation The exocuticle and endocuticle of the mineral components and the rela? The exo- and endocuticles have in com? tion of this orientation to the organic fibers. mon a regular array of chitin-protein fibers The organization of the cuticle is made arranged in lamellae defined by a rotation more apparent by the comparative obser? in the orientation of parallel sheets of these vations of untreated cuticles, EDTA-decal- fibers (Mutvei, 1974 and Figs. 6 and 16). cified cuticles and those rendered anor- The two layers differ in the lamellar spac? ganic by sodium hypochlorite. In Carcinus ing with the interlamellar distance being (Figs. 5-8) the composite nature of the less in the exocuticle than the endocuticle exoskeleton is clearly seen as the inter- (approximately 2 fim and 8 nm respectively spersion ofthe organic fibers with similarly in Carcinus; Dillaman and Roer, 1980). oriented crystal aggregates. The orienta? Mineral first appears as small crystals of tion of the chitin-protein fibers demon? calcite observed around the perimeters of strates a continuous spiral rotation with the pore canals and then throughout the each lamella corresponding to a 180? chitin-protein fibrillar network (Travis, deviation (Fig. 6). Throughout the exo- and 1963; Yano, 1975). Calcification of the endocuticles of Carcinus, the mineral is exocuticle appears to be concentrated ini? found as long rod-shaped elements dis- tially in the periphery of and interstices playing the same continuously changing between prisms defined by the margins of orientation as seen in the chitin-protein

Plate 1. Scanning electron micrographs of Carcinus maenas cuticle. Fig. 3. Partially anorganic epicuticle. x 700. Fig. 4. Partially anorganic epicuticle. x 5,500. Fig. 5. Untreated endocuticle. Note pore canals (arrow). x 10,000. Fig. 6. Decalcified endocuticle. Note pore canals (arrow). x 5,000. Fig. 7. Anorganic endocuticle. Note the pore canal space (ps). x 10,000. Fig. 8. Anorganic endocuticle. Note the individual spherulitic elements comprising rod-shaped crystal aggregates (arrow) x 40,000. Calcification in Crustacean Cuticle 899 900 R. Roer and R. Dillaman fiber network (Figs. 7 and 8). Inspection of Carcinus: minute spherulites are organized the mineral rods at higher magnification in strands interspersed between the organic reveals that these are aggregates of calcite fibers (Fig. 17). In the cone-shaped regions, spherulites strung together in the orien? however, the organization of the mineral tation of the organic fibers (Fig. 8). is not as well defined by the chitin-protein While this pattern of mineralization fiber orientation (Figs. 18 and 19). The appears to obtain in all of the Cancridae crystal aggregates form spherules of much and Portunidae thus far investigated, the larger diameter (0.25 /xm as compared to situation in the Xanthidae appears to vary 0.05 /mi) and demonstrate a far less orga? somewhat. The overall appearance of the nized orientation (Fig. 20). The fusion of untreated cuticles of the xanthids is quite these aggregates with one another is also similar to that of the other families (Fig. apparently not as robust as the fiber-ori- 9) but, macroscopically, has a porcelainous ented region, as the large spherules are character and, in general, is considerably often displaced by hypochlorite treatment. thicker, particularly in the stone , Men- The lack of correlation in the xanthids ippe mercenaria. Likewise, decalcified prep? between the chitin-protein fiber orienta? arations of Menippe or Neopanope texana tion and, in certain regions, the orientation cuticle show no major differences from ofthe crystal aggregates precludes a simple those of other crabs studied (Fig. 10). Upon hypothesis for the control of mineral mor? bleach treatment, however, the anorganic phology and organization. Thus it does not cuticles demonstrate regions of mineral in appear that calcitic spherules merely which the orientation and structure differ nucleate randomly upon the organic fibers markedly from one another. These differ? and simply fill the spaces between them. It ent regions of mineral in the anorganic is clearly not solely the orientation of the Menippe cuticle are not merely surface fea? chitin-protein lamellae which governs the tures nor artefacts resulting from the orientation of the crystal aggregates. While hypochlorite treatment. The atypical the cone-shaped regions of mineral in Men? regions are cone-shaped and evident at the ippe and Neopanope do not appear to cor? inner surface, at the exocuticular/endo- respond to any morphological or anatom? cuticular and boundary throughout the ical features of the integument, clearly the intervening endocuticle (Figs. 11 and 12). nature of the matrix or of the cells under? This pattern is not evident in Carcinus (Fig. lying the matrix differs in these regions. It is 13), but likewise apparent in Neopanope, may be that while the organic fibers appear the other xanthid we have observed to date uniform, they may possess nucleation sites (Fig. 14). whose characteristics vary from place to The ultrastructure of the untreated and place. the decalcified cuticle of Menippe shows Sources of Mineral for Deposition patterns of organization very similar to that of Carcinus even as far as the interlamellar Most of the Crustacea possess some distances ofthe exo- and endocuticles (Figs. means for the retention and storage of part 15 and 16). In the regions ofthe anorganic of the calcium resorbed from the cuticle cuticle not contained within the cone- during premolt. The postmolt mobiliza? shaped structures, the mineral morphol? tion of this calcium provides an endoge? ogy and orientation is also very similar to nous source of calcium for the calcification

Plate 2. Scanning electron micrographs of crab cuticle. Fig. 9. Untreated Menippe mercenariacuticle. x80. Fig. 10. Decalcified Menippe cuticle. x80. Fig. 11. Inner surface of anorganic Menippe cuticle. Note sectors of cuticle both on the surface and in fractures. x 16. Fig. 12. Outer surface of anorganic Menippe endo- (En) and exocuticle (Ex). Note rounded projections corresponding to sectors (arrow). x20. Fig. 13. Anorganic Carcinus cuticle. x 100. Fig. 14. Anorganic Neopanope texana cuticle. x70. Calcification in Crustacean Cuticle 901 902 R. Roer and R. Dillaman

4 r Calcification in Crustacean Cuticle 903

= of the new cuticle. The storage sites may ration kinetics with a Km 0.13 mM Ca be in the form of gastroliths, the calcareous (Greenaway, 1974). Active uptake of cal? concretions beneath the cuticle lining the cium may also be induced in euryhaline cardiac stomach in some freshwater crabs in dilute media. Callinectes is able to macrurans and terrestrial brachyurans; fully calcify its integument in 10 ppt sea? sternal concretions within the anterior water, although at a rate slower than that cuticle during the biphasic molt in isopods; in 30 ppt seawater (Price Sheets and Den- concretions within the posterior caeca of dinger, 1983). Both Callinectes and Carci? the midgut in amphipods; and the general nus have calcium transport mechanisms deposition of calcareous spherules in the with a low affinity, Km =10 mM Ca hepatopancreas and increase in protein- (Greenaway, 1983). bound calcium within the hemolymph (see The uptake mechanism for Ca in Aus- Graf, 1978 for review). The importance of tropotamobius is in part dependent upon the endogenous calcium for postmolt miner? presence of HC03~ in the external medium alization is dependent upon the habitat of (Greenaway, 1974), however very little is the ; Crustacea in sea water depend known regarding the sources nor uptake very little upon stores, as calcium is readily pathways for the carbonate for mineraliza? available from the medium while reserves tion. While a certain amount of carbon is are more important for freshwater and ter? likely to be absorbed from consumption of restrial forms. Thus over 92% of body cal? the exuvium and from food, other possible cium is lost upon exuviation in Carcinus sources include HC03~ from the water and while only 25% and 40% are lost in the metabolic C02. supralittoral and terrestrial isopods Ligia In preliminary experiments in which we and Oniscus respectively (Graf, 1978). injected postmolt (B^ Carcinus with 50 /.iCi of calcium from the food and each of 45CaCl and and etched Uptake NaH14CO? water is the source of exogenous calcium, the cuticle (according to Dillaman and but the relative importance of these two Ford, 1982) following a 6 hr incubation, sources varies. In general, the food accounts we found an unequal distribution of label for little of the total calcium of the new in the mineral throughout the cuticle (Fig. cuticle in the marine brachyura despite the 21). The specific activity of 14C was uni? eating ofthe exuvia in many species (Drach, form from the inner surface out to the epi? 1939; Graf, 1978). Such is not the case of cuticle while the specific activity of 45Ca was the mole crab Emerita asiatica in which there highest at the inner surface and decreased is no storage and the food accounts for monotonically toward the epicuticle. approximately 82% ofthe calcium for min? Nowhere in the cuticle was the specific eralization (Sitaramaiah, 1967). activity of 14C as high as that of 45Ca despite In freshwater and estuarine organisms the fact that the specific activity of H14C03 specific uptake mechanisms exist for the was higher in the hemolymph than was that accumulation of calcium from the dilute of45Ca. media. In fact, Gammarus pulex can attain These data imply that there is on the one complete calcification in media containing hand a much larger tissue pool for carbon? 0.1 mM calcium (Wright, 1980). The fresh? ate than for calcium and, on the other hand, water , Austropotamobius pallipes, that equilibrium of 14C in the tissue pools takes up calcium against an electrochemi- and within the cuticle is much faster than cal gradient by a system displaying satu- that of 45Ca. The location and extent of

Plate 3. Scanning electron micrographs of Menippe mercenariacuticle. Fig. 15. Untreated endocuticle. Note pore canals (arrow). x 5,100. Fig. 16. Decalcified endocuticle. Note pore canals (arrow). x 5,000. Fig. 17. Anorganic endocuticle from lamellar region. x 21,000. Fig. 18. Inner surface of anorganic endocuticle. Note regions of lamellate (1)and non-lamellate (n) organization. x 25. Fig. 19. Crystal organization in non-lamellate region of endocuticle. x 10,000. Fig. 20. Spherulites of non-lamellate region of endocuticle. x 20,000. 904 R. Roer and R. Dillaman

10

o + CM o .a-o?o..2-0-i2. r, i^? O ' O O o O 0 O O ? E E a o

o (0

a CO o o o o o o -cr-o-3>?ctfx?~flL 73 o o ?^nO-p0-OoO-6-O-?-~-C o oQ oo o**> ? ?? Inner Outer surface surface Cuticular Region Fig. 21. Distribution of 45Ca2+(?) and ,4COs2~(O) in two pieces of cuticle from a postmolt Carcinus maenas 6 hr after injection as determined by controlled EDTA-etching analysis.

the tissue pools of calcium and carbonate nation for normal calcification. Indeed, are as yet unknown, however. evidence points to a transepithelial trans? port of mineral to effect both deposition Pathways and Mechanisms of and resorption of minerals from calcareous Mineral Translocation tissues. Few studies have been performed in an Ultrastructural studies of the posterior effort to elucidate the mechanisms by which caecal epithelium of Orchestia (Graf and calcium and carbonate are translocated into Meyran, 1983) and of the gastrolith epi? and out from mineralized structures within thelium of Procambarus clarkii (Ueno, 1980) the Crustacea. Digby (1964, 1965) has pos- have demonstrated cells displaying the tulated that mineral deposition of crusta? properties ofa typical transport epithelium cean cuticle occurs through electrode with elaborate apical microvilli, basal action mediated by the semi-conductor infoldings and numerous mitochondria. nature of the quinone moieties of the cuti? Mizuhira and Ueno (1983) have hypothe? cle itself in response to the differing saline sized an active role of the mitochondria in concentrations within and without the ani? calcium translocation in the gastrolith epi? mal. The lack of such divergent concen? thelium. They detected large amounts of trations in most Crustacea and the rela? calcium within the mitochondrial matrices tively large potentials which must be by energy dispersive X-ray analysis and induced appear to preclude such an expla- electron energy-loss spectroscopy. Their Calcification in Crustacean Cuticle 905

proposal is that the mitochondria effect the sumably carbonate, is apparently effected transepithelial translocation of calcium by the cytoplasmic extensions of the hypo- during the elaboration of the gastroliths. dermal cells extending up into these regions In the hypodermis of the shrimp, Cran? within the pore canals. gon crangon, electron-probe X-ray micro- While we now possess some knowledge analysis has also demonstrated calcium of the mechanisms of calcium transport peaks within the epithelial cells during the associated with mineral structures in select periods of maximal resorption of the car? Crustacea, virtually nothing is known apace mineral (stage D) and while maximal regarding the supply of carbonate to these rates of deposition are occurring (stage B) tissues. It has been established by histolog? (Hubert and Chassard-Bouchaud, 1978). ical and biochemical means, however, that Epithelial involvement in the processes of carbonic anhydrase is localized within the calcium translocation during premolt epithelial cells of the hypodermis and in resorption and postmolt deposition of car? the cuticle (pore canals?) of the anomuran apace mineral was further elucidated by Clibanarius olivaceous (Chockalingham, Roer (1979, 1980) in Carcinus. In vitro uni? 1971) and of Carcinus (Giraud, 1977a, b, directional flux studies were performed on 1981). The amount of enzyme is highest premolt hypodermis removed from during postmolt calcification (stages A! to beneath the dorso-branchial carapace and C3). The role of carbonic anhydrase in the net calcium uptake experiments were con? calcification process is unclear, but inhi? ducted on pieces of postmolt integument bition of the enzyme by acetazolamide from the same region. These data dem? results in decreased rates of mineralization onstrated that both resorption and depo? in vitro (Giraud, 1977a) and in vivo (Giraud, sition of calcium were effected by active 1981) as judged by polarized light micros? transport across the hypodermis. Maximal copy. Whether the enzyme acts in a trans? x in vitro resorption rates were 22.6 10~8 port capacity or simply in the supply of mole/cm2-hr in stage D2, and deposition bicarbonate for mineralization has not been ? rates were 8 9 x 10~8 mole/cm2-hr in established. Acetazolamide has no effect stages A{ and A2. These stages correspond on in vitro calcium transport in Carcinus to those in which maximal resorption and hypodermis (Roer, unpublished data), so if deposition have been found to occur in vivo a role in carbonate transport is established (Drach, 1939; Green and Neff, 1972; Wel? it would be unlinked to the movement of inder, 1975a; Vigh and Dendinger, 1982). calcium. By employing ion substitutions and var? nucleation and the control of ious inhibitors, this calcium transport mechanism was found to involve both a Ca- Crystal Growth ATPase and Na/Ca exchange (Roer, As mentioned previously, preparation for 1980). These results have recently been the molt in the calcified crustaceans verified using another brachyuran, Calli? involves the resorption of the mineral and nectes sapidus (C. Mangum, personal com? organic components of the old cuticle and munication). the simultaneous deposition of elements of These physiological studies in combi- the new cuticle. During premolt, the entire nation with morphological investigations organic matrix ofthe two outermost layers (Travis, 1963; Bouligand, 1970; Yano, of the new cuticle (the epi- and exocuticle) 1975) point to the importance ofthe pore are elaborated beneath the old cuticle. The canals to the calcification ofthe pre-exuvial pre-exuvial layers do not calcify until after cuticle. The epi- and exocuticles are elab? the molt (Drach, 1939; Travis, 1963, 1965; orated before the molt, but only calcify Travis and Friberg, 1963). In Carcinus during postmolt. Mineral must reach dis? maenas, for example, the first crystals of tances 70 /*m or more from the apical sur? CaC03 are not observed in the epi- and faces of the epithelial cells for the rapid exocuticle until 10 hr after ecdysis (Drach, hardening of these layers following exu? 1937). Moreover, if a premolt crab is pre- viation. This transport of calcium, and pre- maturely removed from its old exoskele- 906 R. Roer and R. Dillaman

ton, the new cuticle becomes leathery but pugilator in the control of pre-exuvial cuti? does not calcify (Paul and Sharpe, 1916). cle calcification. In these experiments, Some internal conditions must therefore pieces of pre-exuvial cuticle were removed be met in order that deposition of mineral from crabs before the molt (late stage D) may occur. and cuticle was removed from newly molted Since the mineral from the old cuticle is crabs (stage Ax and A2). Both types of cuti? resorbed through the pre-exuvial layers of cle were stripped of hypodermal tissue and the new cuticle, Travis (1963, 1965) and fixed in 4% paraformaldehyde, decalcified Travis and Friberg (1963) have suggested overnight in 0.1 M EDTA in 4% parafor? that Ca++ and C03= are present in meta- maldehyde, and the EDTA was removed stable solution in these layers before the by further rinsing in the fixative alone. The molt. As calcification does not commence cuticles were then subjected to in vitro cal? until after the molt, however, they pro? cification by successive immersions in 0.5 posed that the epithelial cells underlying M solutions of CaCl2 and NaHC03. Sub? the cuticle exert some control over its envi? sequent observation ofthe tissues by polar? ronment. They add that this control might ized light microscopy showed mineraliza? be realized via the cytoplasmic extensions tion within the cuticles from postmolt crabs, of the epithelial cells in the pore canals of but no crystal deposition within those of the pre-exuvial cuticle through the regu? premolt crabs (Roer and Dail, in prepa? lation of such factors as Ca++, C03=, phos? ration). It is apparent that some factor phate or pH. within the matrix of the cuticle is altered An alternative hypothesis has been put in the course of the molt and the ensuing forth by Yano (1972), who favors the few hours; either a nucleating agent is unmasking of nucleation sites in the pre- secreted, or an inhibitor of nucleation and/ exuvial cuticle as the means of controlling or crystal growth is removed. calcification. He suggested that an acid The control by the matrix of crystal mor? mucopolysaccharide complexed to the phology and the cessation of crystal growth protein fraction of the pre-exuvial matrix is evident in studies on carapace repair in inhibits premolt calcification. Postmolt crabs (Dillaman and Roer, 1980). Mineral enzymatic degradation of the polysaccha- deposited within the wound scab in crabs ride-protein complex would then allow was not formed in the context ofthe chitin- calcification to proceed. While there is his? protein network and appeared as arago- tochemical evidence for acid mucopolysac? nitic granules rather than the normal cal- charides in the cuticle (Yano, 1972; Giraud, citic morph. Further deposition, which 19776), no such degradative enzyme has occurred among histologically atypical been described. lamellae was amorphous CaC03. Repair A significant peak in phosphorylase and cuticle growth showed no evidence of ces? alkaline phosphatase activities is noted in sation, and hence no membranous layer the epicuticle and exocuticle of Clibanarius which normally marks the end of deposi? in stages A^ and A2 when calcification is tion. In this context, it is possible that the commencing (Chockalingham, 1971). It is secretion of the non-calcifying membra? possible that this activity could be related nous layer serves to curtail further crystal to the removal of nucleation inhibitors or growth in normal cuticle deposition. crystal poisons within the matrix. Conclusions and Future Directions The histological location of carbonic anhydrase in the interprismatic regions of Readdressing the basic questions of min? the exocuticle (Giraud, 1981) also corre? eralization in the Crustacea it seems we to the sponds initial sites of mineral depo? have a large body of data concerning the sition (Giraud-Guille and Quintana, 1982). chemical nature and distribution of min? Thus, a possible role of carbonic anhydrase eral and organic components within the in mineral nucleation must be considered. cuticle and we know, in certain species, the We are currently investigating the role sources of the calcium for mineralization of nucleating sites in the carapace of Uca and the rates at which deposition occurs. Calcification in Crustacean Cuticle 907

controlled of We know far less about the relationship etching radioactively labeled shells. Mar. Biol. 66:133-143. between the mineral and the organic com? Dillaman, R. M. and R. D. Roer. 1980. Carapace of the cuticle, both in to ponents regard repair in the green crab, Carcinus maenas (L). J. the determination of crystal morphology Morphol. 163:135-155. and in regard to nucleation. Finally, while Drach, P. 1937. Morphogenese de la mosaique cris- talline externe dans le in the Portunidae we have some knowledge squellette tegumentaire des C. R. Hebd. Seanc. of the mechanisms and for cal? decapodes brachyoures. pathways Acad., Paris 205:1173-1176. cium we know with movement, nothing Drach, P. 1939. Mue et cycle d'intermue chez les respect to the transport of carbonate. crustacees decapodes. Ann. Inst. Oceanogr., These latter areas of investigation will Monaco 19:103-391. P. and C. 1967. Sur la me- prove fertile ground for future work; work Drach, Tchernigovtzeff. thode de determination des stades d'intermue et which will provide information not only on son application generale aux crustaces. Vie Milieu the physiology of Crustacea but also on the Ser. A: Biol. Mar. 18:595-610. basic of principles mineralization. The Giraud, M. M. 1977a. L'anhydrase carbonique et la bidirectional nature of mineral transport calcification tegumentaire chez le crabe Carcinus and the transitions in maenas Linne. C. R. Acad. Sc, Paris 284:453- sharp temporal 456. of the cuticular matrix nucleating ability Giraud, M. M. 19776. Histochimie des ideal in which to premieres provide systems study etapes de la mineralisation de la cuticle du crabe these aspects of calcification. Carcinus maenas: Tentatives de localisation de l'anhydrase carbonique. C. R. Acad. Sc, Paris ACKNOWLEDGMENTS 284:1541-1544. Giraud, M. M. 1981. Carbonic anhydrase in We wish to our thanks to the La activity express the integument of the crab Carcinus maenas dur? Que Center for Corrosion Technology of ing the intermolt cycle. Comp. Biochem. Physiol. 69A:381-387. the International Nickel Company for the Giraud-Guille, M. M. and C. 1982. Sec? generous use of their electron Quintana. scanning ion of the crab calcified facilities. Part of this work was ondary microanalysis microscope cuticle: Distribution of mineral elements and a from N.C. Sea Grant. supported by grant interactions with the cholesteric organic matrix. Biol. Cell 44:57-68. References Graf, F. 1978. Les sources de calcium pour les crus? Bade, M. L. and A. Stinson. 1978. Activation of old taces venant de muer. Arch. Zool. Exp. Gen. 119: cuticle chitin as a substrate for chitinase in the 143-161. F. C. 1983. Premolt calcium molt of Manduca. Biochem. Biophys. Res. Comm. Graf, andj. Meyran. 84:381-388. secretion in midgut posterior caeca of the crus? tacean Orchestia: Ultrastructure of the Bouligand, Y. 1970. Aspects ultrastructuraux de la epithe? lium. 177:1-23. calcification chez les crabes. 7th Congres Int. Micr. J. Morphol. Electronique, Grenoble, pp. 105-106. Green, J. P. and M. R. Neff. 1972. A survey ofthe Chassard-Bouchaud, C and M. Hubert. 1973. Etude fine structure of the integument of the fiddler ultrastructurale du tegument des Crustaces Deca- crab. Tissue Cell 4:137-171. podes en fonction du cycle d'intermue. I. Pres? Greenaway, P. 1974. Calcium balance at the post- ence de cellules secretrices a activite cyclique dans moult stage of the freshwater crayfish, Austro- l'epiderme de Palaemon serratus Pennant. J. potamobiuspallipes (Lereboullet). J. Exp. Biol. 61: Microscopie 16:75-83. 35-45. Chockalingham, S. 1971. Studies on the enzymes Greenaway, P. 1983. Uptake of calcium at the post- associated with calcification of the cuticle of the moult stage by the marine crabs Callinectessapidus Clibanarius olivaceous. Mar. Biol. 10: and Carcinus maenas. Comp. Biochem. Physiol. 169-182. 75A:181-184. Dejours, P. and H. Beekenkamp. 1978. L'equilibre Hegdahl, T.,J. Silness, and F. Gustavsen. 1977a. The acide-base de l'hemolymphe au cours de la mue structure and mineralization of the carapace of chez l'Ecrevisse. C R. Acad. Sc, Paris 286:1895- the crab (Cancer pagurus L.). 1. The endocuticle 1898. Zool. Scripta 6:89-99. Digby, P. S. B. 1964. The mechanism of calcification Hegdahl, T.,F. Gustavsen, andj. Silness. 19776. The in crustacean cuticle. J. Physiol. 173:29P-30P. structure and mineralization of the carapace of Digby, P. S. B. 1965. Semi-conduction and electrode the crab (Cancer pagurus L.). 2. The exocuticle. processes in biological material. I. Crustacea and Zool. Scripta 6:101-105. certain soft-bodied forms. Proc. Roy. Soc. B 161: Hegdahl, T.,F. Gustavsen, andj. Silness. 1977c. The 504-525. structure and mineralization of the carapace of Dillaman, R. M. and S. E. Ford. 1982. Measurement the crab (Cancer pagurus L.). 3. The epicuticle. of deposition in molluscs by Zool. Scripta 6:215-220. 908 R. Roer and R. Dillaman

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