The Structure and Calcification of the Crustacean ...

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organic material in Cancer pagurus (Wel? inder, 19756), but contains no mineral. (Travis, 1955a). The mineral in the outer three layers of the cuticle is CaC03 inĀ ...
The Structure and Calcification of the Crustacean Cuticle Author(s): Robert Roer and Richard Dillaman Source: American Zoologist, Vol. 24, No. 4 (1984), pp. 893-909 Published by: Oxford University Press Stable URL: http://www.jstor.org/stable/3882781 . Accessed: 01/10/2013 08:52 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

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Amer. Zool., 24:893-909 The

(1984)

Structure

and Robert

of the

Calcification Roer

and

Richard

Crustacean

Cuticle1

Dillaman

Institute of Marine BiomedicalResearch, Universityof North Carolina at Wilmington, Wilmington,North Carolina 28403 Synopsis. The integument of decapod crustaceans consists of an outer epicuticle, an exocuticle, an endocuticle and an inner membranous layer underlain by the hypodermis. The outer three layers of the cuticle are calcified. The mineral is in the form of calcite crystals and amorphous calcium carbonate. In the epicuticle, mineral is in the form of spherulitic calcite islands surrounded by the lipid-protein matrix. In the exo- and endocuticles the calcite crystal aggregates are interspersed with chitin-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 crabs. 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 bidirectional 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 has been cuticle While the crustacean of study for over 250 years the subject 1712, in Drach, 1939), the focus (Reaumur, been has generally of those investigations with the process of molting. Our concerned we will will be slightly different; approach of the Crustacea deal with the exoskeleton as a mineralized tissue that is made partic? ularly interesting by the fact that its struc? ture is affected by a cyclic molting process. tis? When investigating any mineralized sue, one must address some basic problems: form 1) the chemical nature and crystalline of the mineral; 2) the nature and form of the organic matrix; 3) the relationship and inorganic com? between the organic of of tissue and influence the the ponents the matrix on crystal morphology; 4) the sources of mineral for deposition; 5) the for mineral or movement into pathways out from the mineralized structures; 6) and 7) the rates of mineral deposition; of nucleation nature and location sites

1 From the Symposium on Mechanismsof Calcification in Biological Systemspresented at the Annual Meeting ofthe American Society of Zoologists, 27-30 Decem? ber 1983, at Philadelphia, Pennsylvania.

for con? within the matrix and mechanisms of crystal growth. The trol or cessation has provided a rich crustacean cuticle with respect to each source of information In addition, the of these central questions. for offer some unique problems Crustacea in biomineralization. those interested exoskeleton ofthe Since the mineralized is subjected to periodic molting, Crustacea of mineral net movement bidirectional stages of the molt cycle is during different is in sharp con? This situation necessary. trast to other calcifying systems in which net accretionary growth patterns or slightly are the rule. Further? shifting equilibria drastic demonstrate crustaceans more, within the same tissue temporal differences with regard to the extent of and capacity Such temporal differ? for mineralization. tran? ences, marked by rapid and discrete sitions, allow one to ask very specific and about the control of answerable questions and mineralization. nucleation is as cuticles The subject of crustacean diverse as is the taxon and, hence, an encyis impossible. overview We will clopedic in large part, to the our review, restrict crustaceans although examples decapod from other orders will serve to remind us ofthe hazards of sweeping generalizations.

893

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R. Roer

894

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Ep

and

5, im

R. Dillaman Structure

Basic cuticular

Ex

65pm 2.2Hm/l

'V->

En

195pm 8.1 Hm/I

of the Integument

Decapod

structure

of the decapod The integument Crus? a tacea is, in general, of comprised rigid or cuticle underlain exoskeleton by the cel? lular hypodermis The (Richards, 1951). but contains cuticle is not homogeneous, four discrete the fact that layers. Despite was made more than a cen? this observation 1860) and has been tury ago (Williamson, the subject of numerous stud? subsequent of the four layers of ies, the nomenclature to author. the cuticle varies from author of Travis (1963), how? The terminology and ever, has come to be widely accepted will be used exclusively in the present work. These layers from the most external to the are: the epicuticle, the exomost internal and the membrathe endocuticle cuticle, nous layer (Fig. 1). is the outermost and thinThe epicuticle nest layer ofthe cuticle. It consists of tanned with calcium salts lipoprotein impregnated with the It is bilaminar, 1955a). (Travis, canals basal layer pervaded by mineral-filled et al, normal to the surface (Hegdahl 1977c). The exocuticle underlies the immediately of chitin-protein It is composed epicuticle. in layers of continuously fibers stacked orientation (Travis, 1955a; Green changing and Neff, 1972). In Cancer pagurus 34% by is chitin weight of the organic component is hard19756). The exocuticle (Welinder, and ened by quinone calcification tanning with the mineral 1955a), (Travis, crystals situated between the fibers (Bouligand,

Fig. 1. Montage ofthe mineralized cuticular layers of Carcinus maenas carapace (from Roer 1979). En = endocuticle, Ex = exocuticle, Ep = epicuticle. Num? bers represent the total thickness of the individual layers and the thickness of endo- and exocuticular lamellae (1).

etal, 1970; Hegdahl 19776). is the thickest and the The endocuticle most heavily calcified layer of the cuticle The endocuticle, 1955a, 1965). (Travis, is composed of hori? like the exocuticle, fibers with zontal lamellae of chitin-protein orientation (Green changing continuously and Neff, 1972; Hegdahl etal, 1977a), the material 73% chitin being by organic in Cancer 19756). (Welinder, pagurus weight is apparently The endocuticle not tanned, but hardened solely by Ca salts (Travis, 1955?).

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Calcification

in Crustacean

The

membranous layer is the innermost of the and is in contact cuticle with layer the hypodermis. It consists of chitin and 74% ofthe protein, the chitin representing in material Cancer organic pagurus (Wel? but contains no mineral inder, 19756), (Travis, 1955a). The mineral in the outer three layers of the cuticle is CaC03 in the form of calcite or poorly crystalline, calcium amorphous carbonate (Travis, 1963). The hypodermis is heterogeneous, hav? cell types arranged in three ing numerous 1955a, b, 1957, principal layers (Travis, 1965). The layer in contact with the cuticle and apparently for its forma? responsible tion is the outer epithelial layer or the cuti? cle secreting cells of Green and Neff (1972). The outer epithelial layer is one cell thick and may range from squamous to colum? nar as will be discussed below. Beneath the outer epithelium is the sub-epithelial con? nective tissue layer (Travis, 1955a, 6) which consists of oval reserve cells, blood sinuses with hemocytes, cells (Sewell, lipoprotein cells (Green and Neff, 1955) and pigment to the connective tissue 1972). Proximal is the inner which is sim? layer epithelium ilar to the outer epithelium but reduced in size. The inner epithelium is bounded on its inner margin by a basement membrane with the exception of those regions cov? where the chamber, ering the branchial inner epithelium elaborates a thin, noncalcified cuticle resembling the outer epi? cuticle in other respects (Skinner, 1962). The cuticle is pervaded by vertically run? ning pore canals first described by Valentin exam? (1837, in Richards, 1951). Further ination has revealed these to be cytoplas? mic extensions of the outer epithelial cells which emanate from the apical cell borextend the membranous ders, through endocuticle and exocuticle, and ter? layer, minate at (Travis, 1963; Green and Neff, et al, 1977'c) the epi? 1972) or in (Hegdahl cuticle. The pore canals may have a typical helical or twisted ribbon morphology Neville 1939; etal, (Drach, 1969) with the pitch of the helix being equal to the lamellar period (Drach, 1939). These pore canals are extremely numerous. In Cancer pagurus

Cuticle

895

are an estimated 150,000-220,000 mm2 canals per (Hegdahl et al, 1977a); pore are in Carcinus maenas about there 950,000/mm2 (Roer, 1980); and Orconectes virilis there are 50-90 pore canals emafrom each cell or about epithelial nating 4,000,000/mm2 (Travis, 1963). Thus, the cuticle is in close contact with the hypo? dermis at all levels and should be consid? ered living tissue. there

The molt cycle Rather detailed accounts ofthe phenom? ena related to the molt cycle of the Crus? tacea come from the last century (Vitzou, for describing 1882), but the first method the molt cycle that could be applied to the in general did not appear until Crustacea much later (Drach, to 1939). According this method, the molt cycle is divided into five stages (A-E), with further subdivisions within each stage. Thus the postmolt period to stages Al9 A2, Bb B2, Cl9 corresponds is stage C4; premolt C2, and C3; intermolt is comprised of stages D0, D/, D/', D/", D2, D3, and D4; and the act of ecdysis or molting is stage E (Drach and Tchernigovtzeff,

1967).

The intermolt

integument Most adult decapods spend the majority ofthe molt cycle in the intermolt condition once or twice C4), (stage molting only yearly (Passano, 1960). The cuticle has its four and is fully calcified layers complete (Fig. The is in its most reduced 2). hypodermis state at this time. The epithelial cells in with the cuticle are extremely contact (Green and Neff, 1972) having squamous a height of only 9 pm in Orconectes virilis (Travis, 1965). The secretory activity of cells is at a min? the Golgi of the epithelial imum and Hubert, (Chassard-Bouchaud and Chassard-Bouchaud, Hubert 1973; reticulum is 1978) and the endoplasmic reduced (Green and Neff, 1972). The pore canals are still structurally intact and con? tain cytoplasm (Green and Neff, 1972) or may be partially filled with calcite crystals 1963; Travis and Friberg, 1963; (Travis, et al, 1977a, 6, c). Hegdahl is not completely The hypodermis inac-

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R. Roer

896

and

R. Dillaman

EARLY PREMOLT Do-D1

LATE PREMOLT D2-D4

INTERMOLT C4 Apolysis Solation layer

of

memb.

POSTMOLT A,-C3

Membranous layer present Cuticle complete

Calcification pre-exuvial Deposition endocuticle

Mineral resorption of new Formation & exocuticle epi-

of cuticle of

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

for a peak in RNA synthesis tive, however, is seen during C4 (Skinner, 1966) and the reserve cells of the highly vacuolated and in reduced connective tissue are engaged of materials for the ensuing the storage premolt

period

(Travis,

1957).

The premolt integument The onset of premolt (stage D0) is marked of the hypo? by apolysis, or the separation dermis from the cuticle through the action and pro? of secreted chitobiase chitinase, tease (Jeuniaux, 1959a, 6; Bade and Stinson, 1978) which cause the solation ofthe membranous layer. Apolysis results in the severing ofthe pore canals (Green and Neff, cells 1972) (Fig. 2). The outer epithelial in increase and to complexity. begin height

the premolt period they change Through In Orconectes from squamous to columnar. D0 and they double their height between of 54 ^m (six times D2, reaching a maximum in C4) by D3 (Travis, their height 1965); the maximal height is about 56 jum in Car? The Golgi begin to cinus (Roer, 1980). a proteinaceous secrete paracrystalline and an increase in smooth endo? substance with mito? reticulum associated plasmic is observed and Chas? chondria (Hubert is Mitotic sard-Bouchaud 1978). activity apparent in the epithelial cells during stages D0, D/ and D/'; and there are peaks in 02 and chitin consumption, protein synthesis during stage D2 (Skinner, synthesis 1966; Stevenson, 1972). Premolt is also the period during

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1962, which

Calcification

in Crustacean

and exo? the matrix of the new epicuticle cuticle is laid down beneath the old cuticle. takes place during Epicuticular deposition D,, and exocuticular deposition stage begins at stage D2. Because these matrices are deposited before the molt, they are referred to as the pre-exuvial layers (Drach, the organic matrix ofthe 1939). Although is laid down pre-exuepi- and exocuticle these do not calcify until after vially, layers the molt (Paul and Sharpe, 1916; Travis, 1963; Travis and Friberg, 1963). The epi? tanned before the molt cuticle is, however, (Krishnan, 1951). with pre-exuvial Concomitant deposi? tion is the partial resorption of both the mineral and organic of the old portions cuticle (Drach, 1939; Travis, 1965; Roer, In Gecarcinus lateralis, more than 1980). is resorbed 75% of the cuticle (Skinner, 1962); while in Panuliris argus only about the endo? 20% ofthe carapace is resorbed, cuticle being completely in some resorbed areas and the exocuticle being partially resorbed in certain regions (Travis, 1955a). In Carcinus, of the mineral is 15-20% resorbed (Graf, 1978). premolt during reaches a maximum Resorptive activity 1939; Green and during stage D2 (Drach, Neff, 1972), and results in a rise in hemo? Ca++ concentration (Robertson, lymph and HC03~ concentration. This 1960) causes a slight alkalosis ofthe hemolymph, the pH rising from 7.9 to 8.1 in Astacus and Beekenkamp, leptodactylus (Dejours 1978). The postmolt integument The initial events following the molt are the tanning of the exocuticle and the cal? cification of the pre-exuvial layers. The is tanned by quinone exocuticle formation from dihydroxyphenols under the action of a polyphenol oxidase transported to the cuticle from the epithelial cells (Krishnan, 1951; Travis, 1957; Vacca and Fingerman, 1975a, 6). The pre-exuvial layers begin to calcify during stage Ax. The first crystals of CaC03 are evident in Carcinus 10 hr after the molt (Drach, 1937); and in Astacus the rate of Ca deposition reaches fluviatilis a peak two days postmolt (Welinder, Calcification ofthe 1975a). epi- and exocuticles begins in the most external regions

Cuticle

897

and proceeds (Travis and Friproximally Mineral 1970). berg, 1963;Bouligand, reaches the outer portions of apparently the cuticle via the pore canals. Calcium is in the distal portions concentrated of the cells and be to extruded epithelial appears in vertical rows corresponding in position to the pore canals of the new cuticle (Tra? vis, 1957, 1963, 1965; Travis and Friberg, 1963; Chockalingham, 1971). Stage B is marked by the onset of endocuticle depo? is concomitant with sition; here calcification matrix each organic lamella formation, as it is laid down (Drach, being mineralized 1939; Travis, 1957,1963, 1965; Travis and 1963). Friberg, Mineral continues deposition through Calcification postmolt. spreads throughout the exocuticle and eventually is found to pervade the walls of the pore canals (Tra? vis, 1963; Travis and Friberg, 1963). Cal? cite crystals may finally be seen within the lumina of the pore canals as the cell pro? cesses apparently recede to be replaced with mineral (Travis, 1963; Travis and Friberg, 1963; Hegdahl etal, 1977a, 6, c). The end of postmolt is marked by the deposition of the membranous and C3 layer during stage the cessation of net calcium deposition (Passano, 1960). The postmolt in the hypoderchanges mal cells are also marked. By one day post? molt the dedifferentiation of these epithe? lial cells has already begun (Green and Neff, In Orconectes, the epithelial cells 1972). decrease from their maximal of 54 height /tm to 21 nm within two days of ecdysis, the decrease until a minimum continuing is reached at intermolt (Travis, 1965). The secretory activity of the Golgi and abun? dance of endoplasmic reticulum also decreases to a virtual through postmolt in C4 (Hubert absence and Chassard-Bou? chaud, 1978). The

Relationship and Organic

between Mineral Components

The epicuticle The epicuticle, as mentioned above, differs from the exo- and endocuticles in its lack of chitin and lamellar organization. This difference is maniorganizational fested in the orientation ofthe A mineral. transmission electron and microscopic

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R. Roer

898

and

and costudy by Hegdahl radiographic of Cancer workers (1977c) on the epicuticle pagurus revealed that mineral was restricted nm in diame? to vertical canals, 100-250 ter, in the proximal layer. These canals are of the the distal terminations presumably form was in the The mineral canals. pore and of CaC03 crystals or crystal aggregates and surrounded were distributed unevenly tissue. by epicuticular a non-homoge? We have also observed of distribution and discontinuous neous of Carcinus maenas. mineral in the epicuticle of Carcinus cuticle While a 24 hr treatment results in with 5.25% sodium hypochlorite brief removal of the epicuticle, a complete renders the tissue only partially treatment mineral and reveals clumps anorganic which are spherulitic aggregates (Figs. 3 fol? and 4). The loss of these aggregates removal the complete hypochlorite lowing of the organic material suggests that these and from one another are both discrete of the from the mineral components exocuticle. underlying The exocuticle

and endocuticle

have in com? The exo- and endocuticles fibers mon a regular array of chitin-protein a in lamellae defined rotation by arranged in the orientation of parallel sheets of these fibers (Mutvei, 1974 and Figs. 6 and 16). The two layers differ in the lamellar spac? distance being ing with the interlamellar than the endocuticle less in the exocuticle 2 fim and 8 nm respectively (approximately in Carcinus; Dillaman and Roer, 1980). Mineral first appears as small crystals of of around the perimeters calcite observed the the pore canals and then throughout fibrillar network (Travis, chitin-protein of the Calcification 1963; Yano, 1975). ini? be to concentrated exocuticle appears of and in interstices the periphery tially between prisms defined by the margins of

R. Dillaman cells underlying the hypodermal the cuti? cle (Drach, 1939; Travis, 1963; Hegdahl et al, 19776; Giraud-Guille and Quintana, is complete, When calcification 1982). of mineral within the distribution however, is relatively these layers homogeneous et and al, 1977a, 6; Giraud-Guille (Hegdahl 1982). Quintana, (1970) claims that Although Bouligand of the calcite crys? there is no orientation tals with respect to the chitin-protein fibers in Carcinus, other evidence to the points The crystal aggregates contrary. clearly are with the organic fibers as deter? aligned mined electron micros? by transmission in Gaetice depressus (Yano, 1975) and copy in Cancer pagurus (Hegdahl et al, 1977a, ion mass spectroscopy 6), and by secondary in Carcinus (Giraudand X radiodography Guille and Quintana, 1982). Indeed, Heg? dahl and co-workers have clearly (1977a) demonstrated rod-shaped crystal aggre? with the chitin-protein gates interspersed fibers. We have further investigated, by scan? electron the orientation ning microscopy, of the mineral and the rela? components tion of this orientation to the organic fibers. The organization of the cuticle is made more apparent the obser? comparative by vations of untreated cuticles, EDTA-decalcified cuticles and those rendered anorIn Carcinus ganic by sodium hypochlorite. the composite of the nature (Figs. 5-8) is clearly seen as the interexoskeleton spersion ofthe organic fibers with similarly oriented The orienta? crystal aggregates. tion of the chitin-protein fibers demon? strates a continuous with spiral rotation each lamella a to 180? corresponding deviation (Fig. 6). Throughout the exo- and of Carcinus, endocuticles the mineral is as long rod-shaped found diselements the same continuously playing changing as seen in the chitin-protein orientation

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.

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Calcification

in Crustacean

Cuticle

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899

900

R. Roer

and

of fiber network (Figs. 7 and 8). Inspection the mineral rods at higher magnification reveals that these are aggregates of calcite in the orien? spherulites strung together tation of the organic fibers (Fig. 8). of mineralization While this pattern appears to obtain in all of the Cancridae and Portunidae the thus far investigated, in the Xanthidae situation to appears vary of the somewhat. The overall appearance is quite untreated cuticles of the xanthids similar to that of the other families (Fig. has a porcelainous 9) but, macroscopically, is considerably character and, in general, in the stone crab, Menthicker, particularly decalcified ippe mercenaria. Likewise, prep? of Menippe or Neopanope arations texana cuticle show no major differences from those of other crabs studied (Fig. 10). Upon bleach treatment, the anorganic however, cuticles demonstrate regions of mineral in which the orientation and structure differ from one another. These differ? markedly of mineral in the anorganic ent regions Menippe cuticle are not merely surface fea? tures nor artefacts from the resulting The treatment. hypochlorite atypical and evident at the regions are cone-shaped inner surface, at the exocuticular/endocuticular and throughout the boundary 11 endocuticle and 12). intervening (Figs. This pattern is not evident in Carcinus (Fig. in Neopanope, 13), but is likewise apparent the other xanthid we have observed to date (Fig. 14). The ultrastructure of the untreated and the decalcified cuticle of Menippe shows patterns of organization very similar to that of Carcinus even as far as the interlamellar distances ofthe exo- and endocuticles (Figs. 15 and 16). In the regions ofthe anorganic cuticle not contained within the conethe mineral structures, shaped morphol? is also very similar to ogy and orientation

R. Dillaman Carcinus: minute spherulites are organized in strands interspersed between the organic fibers (Fig. 17). In the cone-shaped regions, the organization of the mineral however, is not as well defined by the chitin-protein fiber orientation (Figs. 18 and 19). The form spherules of much crystal aggregates to (0.25 /xm as compared larger diameter 0.05 /mi) and demonstrate a far less orga? nized orientation (Fig. 20). The fusion of these aggregates with one another is also not as robust as the fiber-oriapparently ented region, as the large spherules are often displaced by hypochlorite treatment. The lack of correlation in the xanthids between the chitin-protein fiber orienta? tion and, in certain regions, the orientation ofthe crystal aggregates a simple precludes for the control of mineral mor? hypothesis and Thus it does not phology organization. that calcitic appear spherules merely nucleate randomly upon the organic fibers and simply fill the spaces between them. It is clearly not solely the orientation of the lamellae which governs the chitin-protein orientation of the crystal aggregates. While the cone-shaped regions of mineral in Men? and ippe Neopanope do not appear to cor? to any morphological or anatom? respond ical features of the integument, clearly the nature of the matrix or of the cells under? It lying the matrix differs in these regions. be that while the fibers may organic appear sites uniform, they may possess nucleation whose characteristics vary from place to place. Sources

of Mineral

for

Deposition

Most of the Crustacea some possess means for the retention and storage of part of the calcium resorbed from the cuticle The mobiliza? during premolt. postmolt tion of this calcium an endoge? provides 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.

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Calcification

in Crustacean

Cuticle

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901

902

R. Roer

r

and

R. Dillaman

4

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Calcification

in Crustacean

of the new cuticle. The storage sites may be in the form of gastroliths, the calcareous beneath concretions the cuticle lining the in some cardiac stomach freshwater macrurans and terrestrial brachyurans; sternal concretions within the anterior cuticle during the biphasic molt in isopods; caeca of concretions within the posterior and the general the midgut in amphipods; in the of calcareous deposition spherules and in increase proteinhepatopancreas bound calcium within the hemolymph (see of Graf, 1978 for review). The importance calcium for miner? endogenous postmolt is dependent alization upon the habitat of the animal; Crustacea in sea water depend very little upon stores, as calcium is readily while reserves available from the medium are more important for freshwater and ter? restrial forms. Thus over 92% of body cal? cium is lost upon exuviation in Carcinus while only 25% and 40% are lost in the and terrestrial supralittoral isopods Ligia and Oniscus respectively (Graf, 1978). of calcium from the food and Uptake water is the source of exogenous calcium, but the relative of these two importance sources varies. In general, the food accounts for little of the total calcium of the new cuticle in the marine brachyura despite the eating ofthe exuvia in many species (Drach, 1939; Graf, 1978). Such is not the case of the mole crab Emerita asiatica in which there is no storage and the food accounts for 82% ofthe calcium for min? approximately eralization (Sitaramaiah, 1967). In freshwater and estuarine organisms mechanisms exist for the specific uptake accumulation of calcium from the dilute media. In fact, Gammarus pulex can attain in media containing calcification complete 0.1 mM calcium (Wright, 1980). The fresh? water crayfish, Austropotamobius pallipes, takes up calcium against an electrochemisatucal gradient by a system displaying

Cuticle

903

a Km = 0.13 mM Ca 1974). Active uptake of cal? (Greenaway, in euryhaline cium may also be induced crabs in dilute media. Callinectes is able to in 10 ppt sea? fully calcify its integument at a rate slower than that water, although in 30 ppt seawater (Price Sheets and Den1983). Both Callinectes and Carci? dinger, nus have calcium mechanisms transport Km =10 mM Ca with a low affinity,

ration

kinetics

with

1983). (Greenaway, The uptake mechanism for Ca in Austropotamobius is in part dependent upon the of in medium the external HC03~ presence however 1974), (Greenaway, very little is known regarding the sources nor uptake for mineraliza? pathways for the carbonate tion. While a certain amount of carbon is from consumption of likely to be absorbed the exuvium and from food, other possible sources include HC03~ from the water and metabolic C02. In preliminary in which we experiments Carcinus with 50 /.iCi (B^ injected postmolt each of 45CaCl and and etched NaH14CO? the cuticle to Dillaman and (according a hr 6 Ford, 1982) following incubation, we found an unequal of label distribution in the mineral throughout the cuticle (Fig. 21). The specific activity of 14C was uni? form from the inner surface out to the epi? cuticle while the specific activity of 45Ca was highest at the inner surface and decreased toward the epicuticle. monotonically in the cuticle Nowhere was the specific activity of 14C as high as that of 45Ca despite the fact that the specific activity of H14C03 was higher in the hemolymph than was that of45Ca. These data imply that there is on the one hand a much larger tissue pool for carbon? ate than for calcium and, on the other hand, that equilibrium of 14C in the tissue pools and within the cuticle is much faster than 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.

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R. Roer

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R. Dillaman

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