- Email: [email protected]
Morphometric analysis of the calcium-transporting sternal epithelial cells of the terrestrial isopods Ligia oceanica, Ligidium hypnorum, and Porcellio scaber during molt
Arthropod Structure & Development 29 (2000) 241±257
ARTHROPOD STRUCTURE & DEVELOPMENT www.elsevier.com/locate/asd
Morphometric analysis of the calcium-transporting sternal epithelial cells of the terrestrial isopods Ligia oceanica, Ligidium hypnorum, and Porcellio scaber during molt Juliane GloÈtzner, Andreas Ziegler* Z.E. Elektronenmikroskopie, University of Ulm, D 89069 Ulm, Germany Received 20 September 2000; accepted 11 December 2000
Abstract Isopods shed ®rst the posterior and then the anterior half of the body. Before molt, most terrestrial species resorb CaCO3 from the posterior mineralized cuticle. The mineral is stored in anterior sternal deposits, which are used to calcify the new posterior cuticle after molt. For Porcellio scaber it is known that the anterior sternal epithelium has speci®c structural differentiations for epithelial transport. These differentiations include the plasma membrane surface areas, and the volume fraction of the mitochondria. We analyzed the ultrastructure of the sternal epithelium and used a morphometric approach to study the variations of these parameters between species living in different terrestrial environments. In Ligidium hypnorum, which lives in moist environments, the plasma membrane surface area and volume fraction of mitochondria are much larger than in the semiterrestrial Ligia oceanica. This is in accordance with the relatively larger CaCO3 deposits and shorter time intervals for their formation and resorption in L. hypnorum. For P. scaber, which is adapted to mesic habitats, most values are between those of L. oceanica and L. hypnorum. However, P. scaber has even larger CaCO3 deposits which are formed and degraded within similar time intervals as in L. hypnorum. This unexpected result is considered from the standpoint of more effective mechanisms being present for epithelial ion transport. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Calcium storage; Crustacea; Cuticle; Biomineralization; Integument; Ultrastructure
1. Introduction Most terrestrial isopods (Oniscidea) store large amounts of cuticular calcium in the ®rst four anterior sternites (Herold, 1913; Verhoeff, 1940; Messner, 1965; Steel, 1982) before the molt, and reuse the stored calcium for calci®cation of the new cuticle after molt (Steel, 1993). Formation and resorption of these sternal deposits are linked with a unique, biphasic molt cycle (for a scheme of the molting and calcium movements in an terrestrial isopod see Ziegler and Merz (1999)). Isopods molt ®rst the posterior half of the body, which includes the ®fth thoracic segments, and then the anterior half of the body. The pathway for cuticular calcium during the molt cycle is well described for Porcellio scaber and Oniscus asellus (Messner, 1965; Steel, 1993; Ziegler, 1996; Ziegler and Scholz, 1997). About one week before the posterior molt, calcium is resorbed from the posterior cuticle into the hemolymph and transported across the anterior sternal epithelium * Corresponding author. Tel.: 1731-502-3444; fax: 1731-502-3383. E-mail address: [email protected] (A. Ziegler).
(ASE) into the ecdysial space, where it is stored as an amorphous, probably hydrated, CaCO3 compound (Ziegler, 1994). During intramolt, the short interval between the anterior and the posterior molt, the CaCO3 deposits are entirely resorbed within a short time period and used for calci®cation of the new posterior cuticle. In addition, Oniscidea ingest their old cuticle after each partial molt. Calcium ions originating from the old cuticle are most probably transported across the intestinal epithelium and are used to mineralize the new anterior cuticle (Steel, 1993; Ziegler and Scholz, 1997). Ultrastructural work has shown that the anterior sternal epithelial cells of P. scaber (but not the cells of the posterior sternal epithelium (PSE) which transports a smaller amount of calcium), are differentiated for epithelial ion transport (Ziegler, 1996; Ziegler, 1997a). During the deposition and resorption of the sternal CaCO3 deposits, an elaborate network of interstitial dilations and channels known as the interstitial network (IN), enlarges the surface area of the basolateral plasma membrane, and the volume and abundance of mitochondria increases. In addition, numerous folds increase the surface of the apical plasma membrane
1467-8039/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 1467-803 9(00)00030-X
242
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
during intramolt and electron-dense granules that contain calcium are located within the IN. Various types of calcium reservoirs have been developed in terrestrial and freshwater Crustaceans (for review see Greenaway, 1985) including gastroliths of many decapods (Travis, 1963; Radtke, 1983), calcium containing microspherules within the hemolymph as in Holthuisana transversa (Sparkes and Greenaway, 1984) and the reservoires in the posterior midgut caeca in terrestrial amphipods (Graf, 1978; Graf and Meyran, 1983). Storage of large amounts of cuticular calcium is thought to be an adaptation to the terrestrial environment. Among crustaceans, oniscidean isopods were most successful in the colonization of terrestrial habitats. They inhabit a wide variety of biotopes such as rocky seashores, moist and shady woodlands, mesic, and even arid habitats. The cuticle of terrestrial isopods is relatively permeable to water and therefore they are susceptible to desiccation (Quinlan and Hadley, 1983; Warburg, 1989). The development of thicker and more calci®ed cuticles is thought to have reduced water loss by evaporation (Lagarrigue, 1968) during the evolution of terrestrial isopods (Gruner et al., 1993). A strong calci®cation of the cuticle requires the formation of CaCO3 deposits with a larger storage capacity. A comparative ultrastructural investigation of the CaCO3 deposits within two of the ®ve main taxa of the Oniscidea showed that three types of deposits can be distinguished whose storage capacity correlates with the degree of terrestriation (Ziegler and Miller, 1997). The Ligiidae, which are bound to biotopes with high humidity, have CaCO3 deposits with rather small storage capacity. Most members of the semiterrestrial genus Ligia live immediately adjacent to oceans and thus have ready access to seawater. They have thin single-layered Type III CaCO3 deposits composed of numerous small spherules (Ziegler and Miller, 1997). The closely related but more terrestrial species Ligidium hypnorum inhabits moist and shady forest biotopes, and prefers environments, which are relatively rich in calcium (Dahl, 1917). Their Type II CaCO3 deposits are two-layered and have a higher storage capacity than the type III deposits (Ziegler and Miller, 1997). Crinochaeta (which comprise the Porcellionidae, Armadilliidae and other oniscidean families) inhabit a variety of biotopes, including very humid, mesic and even arid regions. They have generally large, three-layered type I CaCO3 deposits (Ziegler and Miller, 1997). Another interesting difference between the more ancestral Ligiidae and the later-evolved Crinochaeta relates to hemolymph-calcium homeostasis capabilities during the molt cycle. Hemolymph calcium rises much more during molt in Ligiidae (Numanoi, 1934; Ziegler et al., 2000a) than in Crinochaeta (BoÈhm and Eibisch, 1976; Ziegler and Scholz, 1997), suggesting that Crinochaeta developed more effective mechanisms for calcium homeostasis than Ligiidae (Ziegler et al., 2000a). Variations in calcium storage capacity and hemolymphcalcium homeostasis probably correlate with differences in
transport ef®ciency of the anterior sternal epithelium. In an attempt to establish ®rst evidence for interspeci®c variations in the morphological differentiation of the sternal epithelia, we analyzed the ultrastructure of the ASE and PSE of Ligia oceanica and L. hypnorum. Using a morphometric approach we compared the surface area of the apical and basolateral plasma membrane, and the volume fraction of the mitochondria with those of the more terrestrially evolved Crinochaeta P. scaber. 2. Materials and methods L. oceanica were obtained from the Biologische Anstalt Helgoland. They were kept in plastic containers with rocks partly submerged in arti®cial seawater, and fed with potatoes and commercially available ®sh food (Tetra). L. hypnorum were kept in plastic boxes ®lled with soil, moldy leaves and bark, and fed with fresh carrots and potatoes. P. scaber were maintained as described earlier (Ziegler, 1996). The animals were checked regularly for the appearance of sternal CaCO3 deposits. Animals in early premolt, late premolt and intramolt stage were used for investigation. Animals without any external signs of molting were de®ned as being in the early premolt stage. In some of them apolysis had already occurred, however, the secretion of the new cuticle had not yet started. Welldeveloped sternal deposits and a change in the color of the anterior tergites due to deposition of CaCO3 in the whole anterior cuticle de®ned animals in late premolt stage. Animals in intramolt stage (between posterior and anterior molt) were used after the CaCO3 deposits were partly degraded indicating that resorption of CaCO3 took place. The isopods were killed by injecting them with 12.5% glutaraldehyde in 0.1 mol l 21 cacodylate buffer. The anterior (1±4) and the posterior (5±7) sternal plates were removed, ®xed for 1±2 h in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mol l 21 cacodylate buffer (pH 7.3) and post®xed in 1% OsO4 and 0.8% K3Fe(CN)6 in 0.1 mol l 21 cacodylate buffer for 1 h. After dehydration in a series of isopropanols the specimens were transferred to propylene oxide and embedded in Epon. Ultrathin sagittal sections (60±80 nm) were cut with a diamond knife (Diatome) on a Leica Ultracut microtome, stained with 2% uranyl acetate in H2O and with 0.3% lead citrate and viewed in a Philips 400 electron microscope at 80 kV. For energy-®ltered transmission electron microscopy semithin sections (500 nm) were cut with a diamond knife, stretched on a hot-water surface and placed on Formvar-coated copper grids stabilized with carbon. The sections were stained with 2% uranyl acetate in H2O. An energy-®ltering electron microscope (Zeiss CEM 902 operated at 80 kV) equipped with a goniometer stage was used to take stereo micrographs at an energy loss of 38 eV and tilt angles of 0 and 1128. We followed the method of Merz (1967) to determine the
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
243
by L Lp =e mm with `L' as the standardized pro®le length and `e' as the width of the test area (Fig. 1). The areas of the basolateral and the apical plasma membranes per mm 2 area of the epithelium were determined by A L £ 1:273 mm2 Overlays with regular arrays of dots were used to determine the volume fraction of the mitochondria of the ASE and PSE of L. oceanica and L. hypnorum. For each test area the number of dots on the mitochondria (Pm) as well as the number of dots on the cytoplasm (Pc) were counted. The volume fraction Vm of the mitochondria was calculated by Vm Pm =Pc £ 100; % Weibel; 1969 Means and standard errors of the means were calculated for each group. ANOVA was used to detect signi®cant differences in the surface area of the apical and basolateral plasma membrane between molting stages, between the anterior and posterior epithelial cells, and between species. Bonferroni±Holm's test was used to assign signi®cant differences between selected pairs of means following signi®cant ANOVA results. 3. Results 3.1. General morphology
Fig. 1. Test area and test system on a micrograph of the anterior sternal epithelium of L. hypnorum at intramolt. The pro®le areas of half epithelial cells were taken as test areas. The applied test system consists of an overlay of coherent lines of semicircles. e, length of the test area; d, diameter of the semicircles. £ 10800.
area of the apical and basolateral plasma membranes. The test®eld used covered about one half of an epithelial cell (Fig. 1). Measurements were done on a computer using overlays with a coherent semicircular test system (Fig. 1). The pro®le length `Lp' of the apical and the basolateral plasma membrane of each test area was determined by Lp Nd mm with `N' as the number of intersections between the test system and the pro®les of the plasma membranes and `d' as the diameter of the semicircles (Fig. 1). Lp was normalized to a standard length of 1 mm epithelium
The sternal integument comprises the cuticle, the epithelial cell layer that consists of more or less cubic cells, and the basal lamina (Figs. 2 and 3). In the anterior and posterior sternal integument of L. hypnorum a pigment cell layer underlies the basal lamina (Figs. 7, 8 and 15). In L. oceanica a pigment cell layer under the basal lamina was lacking in the ASE (Figs. 3 and 18). In the PSE it was found in only one of eight animals (not shown). 3.2. Ultrastructure Many features of the ultrastructure are very similar in L. oceanica and L. hypnorum. Therefore the description below applies for both species unless differences are described explicitly (The ultrastructure of P. scaber was described in detail in previous publications (Ziegler, 1996; Ziegler, 1997a; Ziegler and Merz, 1999)). The cuticle comprises the distal outer and inner epicuticle (Figs. 10, 24±26), the exocuticle in the middle (Figs. 3, 4, 20 and 22) and the proximal endocuticle (Figs. 2, 20 and 22). The thickness of the cuticle during late premolt, intramolt and early premolt are given in Table 1. During late premolt and intramolt the new cuticle is thickest in L. oceanica followed by L. hypnorum and P. scaber. Numerous cellular extensions of the epithelial cells protrude into
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
the pore channels of the new cuticle (Figs. 9, 15 and 20). During late premolt the cellular extensions of both the ASE and PSE end within the inner epicuticle (Figs. 10 and 15). During intramolt, only the cellular extensions of the ASE end within the inner epicuticle (Fig. 17) while the cellular extensions of the PSE end within the exocuticle leaving empty pore channels in the more apical regions of the cuticle (Figs. 20 and 22). In addition, the cellular extensions in the PSE are less abundant during intramolt than during late premolt, leading to a larger density of pore channels in distal layers of the cuticle (Fig. 22). In L. hypnorum peculiar new structures were found within the ecdysial space of most late premolt and intramolt animals. They are located close to the new anterior and posterior sternal cuticle (Fig. 15). In some early premolt animals we observed these tubular structures also at the outer sternal cuticle. In ultrathin sagittal sections they show round and elongated pro®les (Fig. 24). Stereopairs of semithin sections have revealed that these pro®les belong to tubular structures that are connected to the new epicuticle (Figs. 25 and 26). They have an inner diameter of about 20 nm and an outer diameter of about 30 nm. From the outer margin ®lamentous material extends to an approximate diameter of 100 nm (Fig. 24). During early premolt the amount of organelles in the cells of the ASE and the PSE is similar. There is little rough endoplasmic reticulum (RER), and mitochondria and Golgi stacks are rare (Fig. 2). A large portion of the cytoplasm is ®lled with lipid droplets and glycogen (Fig. 2), which resemble the glycogen granules described in P. scaber (Ziegler, 1997a). During late premolt and intramolt the cells of the ASE are rich in RER, mitochondria and Golgi stacks (Figs. 3, 8 and 16). Small vesicles with electron-dense content are often located in the vicinity of the apical plasma membrane (Figs. 3, 9, 16 and 18). In the PSE cells the abundance of most organelles resembles that in the ASE, however, mitochondria are less abundant in the PSE than in the ASE (Figs. 15, 20 and 22). The apical and the basolateral plasma membranes of the ASE have almost plane surfaces during early premolt (Fig. 2). In the PSE the apical plasma membranes are also plane, however, neighboring cells often interdigitate with one another (not shown). During late premolt and intramolt the morphology of the apical plasma membrane differs
245
between L. oceanica and L. hypnorum. In the ASE of L. oceanica many subcuticular microvilli-like extensions of the cytoplasm occur (Figs. 3, 9 and 18), whereas in the ASE of L. hypnorum the apical plasma membrane forms many subcuticular folds rather than microvilli-like extensions (Figs. 4, 8 and 10). In both species, folds increase the surface area of the basolateral plasma membrane. Laterally these folds surround ®nger-like cellular processes, which appear as more or less circular or elongated pro®les (Figs. 3, 8, 16 and 18). In the PSE subcuticular microvillilike extensions in the apical plasma membrane and subcuticular folds are rare during late premolt of L. oceanica (not shown) and L. hypnorum (Fig. 15), respectively. In intramolt, however, 2 of 6 L. oceanica (not shown) and 3 of 8 L. hypnorum (Fig. 23) have many subcuticular microvilli-like cellular extensions. In L. oceanica basolateral folds and lateral ®nger-like processes are rare during late premolt and intramolt. In L. hypnorum the basolateral plasma membrane compartment forms only a few folds in the basal region during late premolt (Fig. 15), and neighboring epithelial cells often interdigitate with one another (not shown). During intramolt, the basolateral plasma membrane was almost plane in six specimens (Fig. 22). However, in two L. hypnorum the basolateral plasma membranes form lateral folds containing ®nger-like cellular processes similar to those in the ASE (Fig. 23). These animals belonged to those with subcuticular microvilli-like cellular extensions in the apical plasma membrane mentioned above. During late premolt and intramolt interstitial spaces of the ASE contain electron-dense material in most L. oceanica and L. hypnorum (Figs. 3, 8 and 18; Table 2). The electrondense material appears either as elongated structures (Figs. 11 and 12) or as granules (Figs. 13 and 14). The granules have diameters between 20 and 290 nm. The electron-dense granules are associated with the extracellular side of ¯at regions (Fig.14) or cellular extensions (Fig. 13) of the basolateral plasma membrane. In most animals the electrondense material is mainly concentrated in the apical half of the interstitial space (Figs. 7, 8 and 23). In L. hypnorum and occasionally in L. oceanica electron-dense granules in the basal half of the interstitial space often had a smaller diameter than in the apical half (Figs. 4±6). Electron-dense particles ranging between 20 and 110 nm in size were observed in the basal lamina of many animals (Figs. 3, 8,
Figs. 2±6. Fig. 2 The anterior sternal epithelium of L. hypnorum during early premolt. £ 10200. bl, basal lamina, en, endocuticle; g, Golgi; gl, glycogen; ld, lipid droplets; m, mitochondria; n, nucleus; rer, rough endoplasmic reticulum. Fig. 3 The anterior sternal epithelium of L. oceanica during late premolt. Many subcuticular microvilli-like extensions of the cytoplasm (me) increase the surface area of the apical plasma membrane. The basolateral plasma membrane forms many folds, thereby increasing the interstitial space (is), which contains electron-dense material (arrows). Electron-dense particles appear in the basal lamina (arrowheads). £ 6800. bl, basal lamina; ce, cellular extensions; ep, epicuticle; ex, exocuticle; g, Golgi; m, mitochondria; rer, rough endoplasmic reticulum; v, vesicle. Fig. 4 The anterior sternal epithelium of a L. hypnorum during late premolt with large electron-dense granules (arrows) in the apical and smaller ones in the basal region of the interstitial space. Overview: the apical and the basolateral plasma membrane form many folds. bl, basallamina; ep, epicuticle; ex, exocuticle; m, mitochondria; mf, membrane folds; n, nucleus; rer, rough endoplasmic reticulum; rectangles, regions shown in the insets in higher magni®cation. £ 9100. Fig. 5 The anterior sternal epithelium of a L. hypnorum during late premolt. Details of apical region of the interstitial space with large electron-dense granules (arrow). £ 18200. Fig. 6 The anterior sternal epithelium of a L. hypnorum during late premolt. Detail of basal region of the interstitial space containing smaller electron-dense granules (arrow). £ 18200.
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
247
Table 1 Thickness of the cuticle (nm) within the anterior and posterior integument (mean ^ SEM) at three different molting stages (The number of measurements is given in brackets) Tissue Stage L. oceanica L. hypnorum P. scaber a
anterior late premolt 4102 ^ 272 (10) 1005 ^ 127 (10) 492 ^ 120 (8)
intramolt 4312 ^ 523 (9) 1310 ^ 131 (12) 999 ^ 123 (8)
posterior early premolt
late premolt a
14055 ^ 4107 (4) 4831 ^ 250 a (4) 8737 ^ 1716 a (4)
6228 ^ 973 (10) 1593 ^ 155 (10) 1019 ^ 310 (8)
intramolt
early premolt a
7483 ^ 1336 (8) 3925 ^ 587 a (10) 3197 ^ 811 a (8)
14235 ^ 1176 a (4) 5562 ^ 832 a (4) 9775 ^ 2228 a (4)
Cuticle contains endocuticular layers.
Table 2 Occurrence of electron-dense material within the lateral interstitium of the anterior (ASE) and posterior sternal epithelium (PSE)
16 and 19). In the ASE of L. hypnorum the occurrence of these electron-dense particles correlated with a very high amount of electron-dense material in the lateral interstitium (Fig. 8). In the PSE electron-dense material is lacking during late premolt in both species, and occurs only occasionally during intramolt (Figs. 21 and 23, Table 2). In L. hypnorum these few epithelia resemble those of most ASE with respect to the appearance of the interstitial space, distribution of electron-dense granules (Fig. 23), and electron-dense particles in the basal lamina (not shown). 3.3. Morphometry In order to compare the plasma membrane areas in different molting stages and species we normalized the values of the measurements to 1 mm 2 area of the epithelium as
described in the methods. Mean values for the normalized apical (Aat) and basolateral plasma membrane (Abl), the volume fraction of the mitochondria (Vm) and signi®cant differences between selected pairs of means are given in Figs. 27±29. In all three species we found a signi®cant increase of Aat in the ASE from early to late premolt and from early premolt to intramolt (Fig. 27). In P. scaber the increase from early to late premolt appears rather small in comparison with the other species due to a virtual lack of subcuticular folds. In the PSE Aat rises transiently during late premolt (however insigni®cantly in L. oceanica, P 0:24 (Fig. 27). In general total Aat may be divided into two subtypes: the subcuticular plasma membrane and the plasma membrane that belongs to cellular extensions into the cuticle. In most cases the percentile contribution of the cellular extensions
Figs. 7±14. Fig. 7 The anterior sternal epithelium of L. hypnorum during late premolt. Animal with electron-dense material (arrows) only in the apical region of the interstitial space and no electron-dense particles within the basal lamina. £ 6600. Fig. 8 The anterior sternal epithelium of L. hypnorum during late premolt. Animal with large amounts of electron-dense material (arrows) mostly in the apical region of the interstitial space and much electron-dense material within the basal lamina. £ 6900. bl, basal lamina; ce, cellular extensions; cu, cuticle; ld, lipid droplets; g, Golgi; is, interstitial space; m, mitochondria; mf, membrane folds; n, nucleus; pg, pigment granules; rer, rough endoplasmic reticulum; v, vesicle; arrowheads, electron-dense particles in the basal lamina. Fig. 9 and Fig. 10 The differentiation of the apical plasma membrane in the anterior sternal epithelium of L. oceanica and L. hypnorum during late premolt. ce, cellular extensions; ex, exocuticle; ie, inner epicuticle; m, mitochondria; me, subcuticular microvilli-like extensions of the cytoplasm; mf, membrane folds; oe, outer epicuticle; pc, pore channel; rer, rough endoplasmic reticulum; v, vesicle. L. oceanica (Fig. 9) with apical subcuticular microvilli-like cellular extensions. £ 26600. L. hypnorum (Fig. 10) with apical folds. £ 23700. Figs. 11±14 The structure of the electron-dense material in the anterior sternal epithelium of L. hypnorum. The electron-dense material often forms elongated structures (arrowheads), which ®ll parts of the intercellular space (Fig. 11, £ 35200. Fig. 12 £ 41600). Electron-dense granules on the tips of lateral cellular projections (arrowheads, Fig. 13 £ 82800). Small electron-dense granules associated with the extracellular side of ¯at regions of the basolateral plasma membrane (arrowheads, Fig. 14, £ 36400). bl, basal lamina; is, interstitial space; m, mitochondria; rer, rough endoplasmic reticulum.
248
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
(Ace) to Aat is zero in the sternal epithelium during early premolt (exceptions are stated explicitly, below). In the ASE of L. oceanica Ace is 34% during late premolt and 28% during intramolt, versus 54 and 7% in the PSE. In the ASE of L. hypnorum Ace is 5 and 12% during late premolt and intramolt, respectively, and 27 and 4% in the PSE. In the ASE of P. scaber Ace is 1% during early and late premolt, and 4% during intramolt. In the PSE Ace is 13% during late premolt and 38% during intramolt. In the PSE of P. scaber the signi®cant difference in Aat between early premolt and intramolt by 1.15 mm 2 is entirely due to an increase of Ace. In all three species Abl in the ASE increases from early premolt to late premolt and intramolt (insigni®cant for P. scaber during late premolt) (Fig. 28). In the PSE Abl does not change signi®cantly within species (P . 0.05), however, during early premolt Abl of L. hypnorum is considerably smaller than in L. oceanica (Fig. 28). This difference is due to the smaller height of the epithelial cells in Ligidium as compared to the other two species (data not shown). Nevertheless, we found the largest value for Abl in the ASE of L. hypnorum during intramolt. In the ASE all three species show an increase of Vm from early premolt to late premolt by a factor of at least 2.0 and a somewhat smaller increase from early premolt to intramolt (not signi®cant for L. oceanica; P 0:2 and P 0:57; respectively) (Fig. 29). The largest increases by factors of about 3 occurred in L. hypnorum during late premolt and intramolt. In the PSE Vm does not change signi®cantly within and between species (Fig. 29). 4. Discussion The present study shows that in the ligiids L. oceanica and L. hypnorum the ASE is differentiated for an increased ion transport during the formation and resorption of the sternal CaCO3 deposits. These differentiations are similar to that found previously in the ASE of the crinochete P. scaber. Interspeci®c variations in the differentiation of the sternal integument concern the relative thickness of the new incomplete cuticle, the subcuticular enlargements of the apical plasma membrane, the shape of the interstitial spaces,
249
and the temporal distribution of electron-dense granules between the epithelial cells. Quantitative differences were found for the surface area of the apical and basolateral plasma membrane and the volume density of the mitochondria. During molt the epithelium of the integument functions in resorption of the organic and mineral components of the old cuticle, and secretion and calci®cation of the new one. In addition, the ASE forms and degrades the large sternal CaCO3 deposits that serve as calcium reservoirs during the biphasic molt. For P. scaber it has been shown, that the analysis of the ASE and, as a control, the PSE permits to distinguish morphological differentiations for ion transport from those for the secretion and resorption of the organic compounds of the cuticle (Ziegler, 1996; Ziegler, 1997a). Secretion of the new anterior cuticle already begins during the formation of the sternal deposits, thereby establishing a barrier for the diffusion of ions between the CaCO3 deposit and the epithelium. Therefore, delayed secretion of the ®rst cuticular layers in the anterior sternites during late premolt and intramolt is regarded as a mechanism to speed storage and resorption of CaCO3 (Messner, 1965; Steel, 1993; Ziegler, 1997a). A comparison of the cuticle during molt within the sternal integument shows that in the anterior sternal integument the delay of growth is least in L. oceanica followed by L. hypnorum and largest in P. scaber (Table 1, (Ziegler, 1997a)). In the crinochete O. asellus the delay in cuticle growth is similar to that in P. scaber (Messner, 1965; Steel, 1982). For the ®rst time we describe tubular structures connected to the sternal epicuticle within the ecdysial gap of L. hypnorum or, in some animals, at the sternal body surface. At the current stage of investigation we cannot correlate these structures with any function. Possibly they serve as sponges to retain moisture, or they have some function during the molting process. In future experiments we plan to investigate the water-absorbing properties of the tubular structures and their distribution in molting and non-molting animals. The deposition and resorption of CaCO3 requires the 1 movement of Ca 21, HCO2 across the epithelia 3 and H (Fig. 30A, B). It is not clear whether movements of calcium ions occur by active transcellular or passive paracellular
Figs. 15±19. Fig. 15 The posterior sternal epithelium of L. hypnorum during late premolt. £ 7800. bl, basal lamina; ce, cellular extensions; ep, epicuticle; ex, exocuticle; g, Golgi; gl, glycogen; hl, hemolymph; ld, lipid droplets; m, mitochondria; n, nucleus; pg, pigment granules; rer, rough endoplasmic reticulum; v, vesicle; arrows, tubular structures. Fig. 16 The anterior sternal epithelium of L. hypnorum during intramolt. The apical and the basolateral plasma membrane form many folds. Large amounts of electron-dense material (arrows) appear in the interstitial space (is). Electron-dense particles occur in the basal lamina (arrowheads). £ 7700. Fig. 17 The anterior sternal epithelium of L. hypnorum during intramolt. Cellular extensions protruding into the inner epicuticle. £ 15300. bl, basal lamina; ce, cellular extensions; ex, exocuticle; g, Golgi; ep, epicuticle; m, mitochondria; mf, membrane folds; rer, rough endoplasmic reticulum; v, vesicle. Fig. 18 The anterior sternal epithelium of L. oceanica during intramolt. The apical plasma membrane forms small subcuticular microvilli-like extensions of the cytoplasm (me). The basolateral plasma membrane forms many folds. The interstitial space (is) contains a comparatively small amount of electron-dense material (arrows). Many electron-dense particles appear in the basal lamina (arrowheads). £ 7900. Fig. 19 The anterior sternal epithelium of L. oceanica during intramolt. Electron-dense particles (arrowheads) in the basal lamina at higher magni®cation. £ 20000. bl, basal lamina; ce, cellular extensions; ex, exocuticle; m, mitochondria; rer, rough endoplasmic reticulum; v, vesicle.
250
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
Figs. 20±23. Fig. 20 The posterior sternal epithelium of L. oceanica during intramolt. Animal with almost ¯at apical and basolateral plasma membrane. £ 6500. Fig. 21 The posterior sternal epithelium of L. oceanica during intramolt. Animal with electron-dense material within the interstitial space (arrows). £ 9400. bl, basal lamina; ce, cellular extensions; en, endocuticle; ep, epicuticle; ex, exocuticle; m, mitochondria; n, nucleus; rer, rough endoplasmic reticulum; v, vesicle. Fig. 22 The posterior sternal epithelium of L. hypnorum during intramolt. Animals with almost ¯at apical and basolateral plasma membranes. £ 8500. Fig. 23 The posterior sternal epithelium of L. hypnorum during intramolt. Animal with subcuticular microvilli-like extensions of the cytoplasm (me) and many folds of the basolateral plasma membrane and electron-dense material within the interstitial space (is). £ 10700. bl, basal lamina; ce, cellular extensions; en, endocuticle; ep, epicuticle; ex, exocuticle; gl, glycogen; hl, hemolymph; m, mitochondria; n, nucleus; pc, pore channels; pg, pigment granules; rer, rough endoplasmic reticulum; v, vesicle.
252
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
Fig. 27. The apical plasma membrane surface area of the anterior (ASE) and posterior (PSE) sternal epithelial cells of L oceanica, L. hypnorum and P. scaber at three different molting stages (means ^ SE). Asterisks on top of the columns indicate signi®cant differences from the early premolt stage. Horizontal brackets with asterisks indicate statistically signi®cant differences between the means. Signi®cant differences between the ASE and PSE are indicated below the columns. (***P , 0.001, *P , 0.05, a: P , 0.05) N, number of animals; n, number of test ®elds.
mechanisms (Neufeld and Cameron, 1993). For the transcellular pathway (Fig. 30) verapamil sensitive calcium channels were proposed for the in¯ux of calcium into the cells and a Ca 21-ATPase, Na 1/Ca 21- and Ca 21/H 1exchange mechanism for the extrusion of calcium from the cells to the other side of the epithelium (Roer, 1980; Greenaway and Dillaman et al., 1995; Neufeld and Cameron, 1993; Wheathly, 1997; Ahearn et al., 1999). For the ASE of P. scaber a high concentration of Na/K-ATPase was demonstrated in the basolateral (Ziegler, 1997b) and a V-type H 1-ATPase in the apical plasma membrane (Ziegler et al., 2000a). In general, large plasma membrane surfaces in conjunction with abundant or large mitochondria are regarded as differentiations for high ion transport activity across the
epithelial cells. The signi®cant increase in the apical and basolateral plasma membrane surface area and the increase of the volume density of the mitochondria from early premolt to late premolt and/or intramolt shows that in all three species analyzed the ASE cells become differentiated for epithelial ion transport. The differences between the plasma membrane surfaces of the ASE and PSE cells suggest that ion transport across the epithelial cells play a role in the formation and resorption of the sternal CaCO3 deposits. In L. hypnorum the increase in the apical plasma membrane surface area is caused by development of subcuticular folds similarly to P. scaber (Ziegler, 1997a). In contrast L. oceanica increases the apical plasma membrane by subcuticular microvilli-like extensions. The latter have been described for other crustaceans and insects
Figs. 24±26 (previous page). Fig. 24 Tubular structures (arrows) in the ecdysial space of L. hypnorum (intramolt). In thin sagittal sections the structures show round and elongate pro®les. £ 86500. Fig. 25 ( £ 38100) and Fig. 26 ( £ 48100) Tubular structures (arrows) in the ecdysial space of L. hypnorum (intramolt). Stereopairs of semithin (500 nm) sagittal sections taken at an energy loss of 38 eV, showing that the structures form tubules and are connected to the outer epicuticle. ie, inner epicuticle; oe, outer epicuticle.
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
253
Fig. 28. The mean basolateral plasma membrane surface area of the anterior (ASE) and posterior (PSE) sternal epithelial cells of L oceanica, L. hypnorum and P. scaber at three different molting stages (means ^ SE). Asterisks on top of the columns indicate signi®cant differences from the early premolt stage. Horizontal brackets with asterisks indicate statistically signi®cant differences between the means. Signi®cant differences between the ASE and PSE are indicated below the columns. (***P , 0.001, **P , 0.01, *P , 0.05) N, number of animals; n, number of test ®elds.
in conjunction with the secretion of the cuticle (KuÈmmel and Claasen et al., 1970; Arsenault and Castell et al., 1984; Buchholz and Buchholz, 1989; Locke, 1969). In L. oceanica the spatial and temporal distribution suggests that they may function in the mineralization of the cuticle and deposition and resorption of the CaCO3 deposits. In the ASE they appear during formation and resorption of the sternal deposits, whereas in the PSE they occur only in a fraction of the animals analyzed. In these animals they appear only in the intramolt stage, during which mineralization of the exocuticle takes place (whereas the cuticle is secreted in both molting stages). A role in mineralization of the cuticle was also attributed to intracuticular cellular extensions (Travis, 1965; Roer, 1980; Giraud-Guille, 1984; Roer and Dillaman, 1984). In addition they might be involved in the resorption and deposition of CaCO3 deposits (Ziegler, 1996). The latter would be of particular signi®cance in the anterior sternal integument of L. oceanica in which the new cuticle is comparatively thick during late premolt and intramolt. A recent freeze±etch analysis of the ASE and PSE cells of P. scaber has shown that the membrane particle density within the plasma membrane of the cellular extensions is signi®cantly larger than in the subcuticular apical plasma membrane (Ziegler and Merz, 1999). In general, the contri-
bution of cellular extensions into the cuticle to the apical membrane surface area correlates with the thickness of the exocuticle before the appearance of the ®rst endocuticular layers during intramolt (an exception is the decrease from 34 to 28% from late premolt to intramolt in L. oceanica). After the ®rst endocuticular layers are secreted shortening of the cellular extensions leads to the observed reduction in their contribution to the total apical plasma membrane surface area in the posterior integument of L. oceanica and L. hypnorum. In comparison, in P. scaber the elongation of cellular extensions in the posterior integument from late premolt to intramolt explains the large contribution of intracuticular to total apical plasma membrane. Variations in the magnitude of differentiations indicative for epithelial ion transport between species may be interpreted in conjunction with variations in the need for large storage capacity of the CaCO3 deposits and/or variations in deposition/resorption rates. Our results show that the degree of differentiation is much larger in L. hypnorum than in L. oceanica. For animals in late premolt this difference seems to be related to larger deposits rather than to differences in transport rate, since the formation of the deposits requires 5±7 days in Ligia (Nicholls, 1931; Numanoi, 1934), and 6±8 days in L. hypnorum (present observations). A shorter intramolt stage of 15±20 h in L. hypnorum versus 2±3 days
254
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
Fig. 29. The mean volume fraction of the mitochondria within the cytoplasm of the anterior (ASE) and posterior (PSE) epithelial cells of L. oceanica, L. hypnorum and P. scaber at three different molting stages (means ^ SE). Asterisks on top of the columns indicate signi®cant differences from the early premolt stage. Horizontal brackets with asterisks indicate statistically signi®cant differences between the means. Signi®cant differences between the ASE and PSE are indicated below the columns (***P , 0.001, *P , 0.05). N, number of animals; n, number of test ®elds. Data for P. scaber from (Ziegler, 1997a).
in L. oceanica seems to be responsible for the larger degree of differentiation, in addition to the relative larger storage capacity of the CaCO3 deposits. Both, the storage capacity and a quick resorption of the CaCO3 deposits are probably necessary to avoid excessive desiccation during molt in the more terrestrial environment of Ligidium as compared with that of Ligia. The differentiation to ion transport in the ASE seems to be more pronounced in P. scaber than in L. oceanica, but, with the exception of the apical plasma membrane during intramolt, is less than in L. hypnorum. This is of particular interest because the sternal deposits of P. scaber have a larger storage capacity than those of the Ligiidae (Ziegler and Miller, 1997), and the time-periods for the formation and resorption of the deposits are similar to those observed in L. hypnorum. The comparatively small increase in the volume density of the mitochondria suggests that P. scaber developed more ef®cient mechanisms for epithelial ion transport. Earlier, Ziegler et al. (2000b) proposed that a more developed mechanism would exist based on a better hemolymph calcium homeostasis during molt in crinochetes than in Ligia species. Changes in hemolymph calcium concentration during molt are explained by
imbalances between the resorption of calcium from the posterior cuticle into the hemolymph and concomitant transport of calcium from the hemolymph across the ASE to the sternal CaCO3 deposits during premolt, and vice versa during intramolt. A comparatively high increase in calcium concentration of 47% was found in the hemolymph of Ligia pallasii (Ziegler et al., 2000b) and of 50±60% in Ligia exotica (Numanoi, 1934). In the Crinochaeta storage and resorption of calcium is accompanied by rather small increases in hemolymph calcium; e.g. only 25% in Armadillidium nasatum and 19% in P. scaber (BoÈhm and Eibisch, 1976; Ziegler and Scholz, 1997). More ef®cient calcium transport probably evolved because of a greater need to conserve calcium in isopods adapted to mesic versa species that live in rather moist habitats. At the present state of knowledge we can only speculate how more ef®cient mechanisms for ion transport may have evolved. An increase in the surface area of the apical plasma membrane as in the anterior sternal epithelial cells of L. oceanica and L. hypnorum during late premolt is one way to increase the ¯ux rate for ions involved in CaCO3 deposit formation or resorption. Such an increase, however, also increases nonspeci®c back-¯ux of ions (Fig. 30, C). Possibly, in P.
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
255
Fig. 30. Ion movements in the hypodermis of crustaceans during deposition (A) and resorption (B) of CaCO3, and proposed para- and trans-cellular calcium movements in the ASE of L. hypnorum (C), and P. scaber (D) during CaCO3 deposit formation. Dashed arrows indicate energetically futile back ¯ux of calcium (see discussion for further explanation). ASE, anterior sternal epithelium; b, basal lamina; c, cuticle; Cc, calcium channel; Cp, Ca 21-ATPase; e, ecdysial space; Ex, Na/Ca-Exchanger; f, apical folds; g, electron-dense granules; h, hypodermis; Np, Na/K-ATPase; pc, paracellular pathway; tc, transcellular pathway.
scaber the evolution of more ef®cient transport molecules and/or higher density of these molecules rather than an increase of the plasma membrane surface area has lead to increased ion ¯ux rates. This would explain the rather small increase of the apical plasma membrane surface area and would avoid an excessive waste of energy by futile back ¯ux of ions (Fig. 30, D). The difference in the occurrence of electron-dense granules between P. scaber and the two ligiids analyzed may also be explained by more ef®cient calcium homeostasis mechanisms in the former. For P. scaber it has been shown that the electron-dense granules contain an organic
matrix and a high concentration of calcium (Ziegler, 1996) and phosphorus (Ziegler, unpublished) indicating that the granules contain calcium phosphate, which is quite insoluble. In P. scaber they appear to be secreted at the apical region of the lateral plasma membrane and migrate to the basal side until they disintegrate within the basal lamina. The present ultrastructural analysis indicates a similar fate for the electron-dense granules of L. oceanica and L. hypnorum. Since in P. scaber the electron-dense granules occur almost exclusively in the ASE during the resorption of the CaCO3 deposits they are thought to play a role in the transport of calcium ions from the apical to the basal side of the
256
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
epithelium. Another function may be to limit the calcium activity within the interstitial spaces in order to maintain a speci®c calcium gradient across the plasma membranes (Ziegler, 1996). In contrast to the situation in P. scaber, electron-dense granules occur in the ASE of L. oceanica and L. hypnorum as frequent during late premolt as during intramolt, and in a few cases even in the posterior sternal epithelium during intramolt. Because the calcium-containing granules cannot support a transport of calcium from basal to apical during late premolt, they are likely to be energetically inef®cient because calcium ions are transported back into the hemolymph producing a futile cycle (Fig. 30, C). Therefore, it appears likely that electron-dense calcium granules evolved for buffering the interstitial free calcium concentration within physiological limits. During the evolution of more ef®cient calcium homeostasis, however, they may nevertheless need to be present in the ASE during intramolt because here extracellular calcium ¯uctuations may still be large. Alternatively electrondense granules may have been maintained during the evolution of more ef®cient calcium homeostasis because they accelerate the resorption of the CaCO3 deposits by carrying calcium ions across the epithelium. This would mean that during evolution the electron-dense granules changed their main function from an extracellular calcium buffer in Ligiidae to a vehicle for interstitial calcium transport in Crinochaeta. 5. Conclusions The structural and morphometric results of the two ligiid species are consistent with a larger ion transport activity in L. hypnorum as compared with L. oceanica due to relatively thicker CaCO3 deposits and shorter time intervals for their resorption within the former. For P. scaber, the morphometric characteristics seem not to ®t to the simple correlation to the ion transport rate between species. Rather, it seems that P. scaber develops more effective mechanisms for epithelial ion transport during molt than the ligiids. The present data provide a ®rm base for comparative investigations between the different taxa of the Oniscidea. Further investigations within the Ligiidae and the Crinochaeta are necessary to establish how the degree of differentiation correlates with the degree of terrestriation. Acknowledgements We thank Dr Thomas Carefoot for critically reading the manuscript and linguistic corrections. References Ahearn, G.A., Duerr, J.M., Zhuang, Z., Brown, R.J., Aslamkhan, A., Killebrew, D.A., 1999. Ion transport processes of crustacean epithelial cells. Physiological and Biochemical Zoology. 72, 1±18.
Arsenault, A.L., Castell, J.D., Ottensmeyer, F.P., 1984. The dynamics of exoskeletal-epidermal structure during molt in juvenile lobster by electron microscopy and electron spectroscopic imaging. Tissue and Cell 16, 93±106. BoÈhm, G.-A., Eibisch, H., 1976. Untersuchungen zum Mineralstoffwechsel der Crustaceen Orconectes limosus (Decapoda) und Armadillidium nasutum (Isopoda) in der VorhaÈutungsphase. Zoologisches Jahrbuch fuÈr Physiologie 80, 175±183. Buchholz, C., Buchholz, F., 1989. Ultrastructure of the integument of a pelagic Crustacean: moult cycle related studies on the Antarctic krill, Euphausia superba. Marine Biology 101, 355±365. Dahl, F., 1917. Die Landisopoden SuÈdwestdeutschlands. Mitt Zool Mus Berlin 8, 405±423. Giraud-Guille, M.M., 1984. Calci®cation initiation sites in the crab cuticle: the interprismatic septa. Cell and Tissue Research 236, 413±420. Graf, F., 1978. Les sources de calcium pour les crustaceÂs venant de muer. Archs Zool Exp Gen 119, 143±161. Graf, F., Meyran, J.C., 1983. Premolt calcium secretion in midgut posterior caeca of the crustacean Orchestia: ultrastructure of the epithelium. Journal of Morphology 177, 1±23. Greenaway, P., 1985. Calcium balance and moulting in the crustacea. Biological Reviews 60, 425±454. Greenaway, P., Dillaman, R.M., Roer, R.D., 1995. Quercitin-dependent ATPase activity in the hypodermal tissue of Callinectes sapidus, during the moult cycle. Comparative Biochemistry and Physiology 111A, 303±312. Gruner, H.E., Moritz, M., Dunger, W., 1993. Arthropoda (ohne Insekta), 4 Teil. In: Gruner, H.E. (Ed.). Lehrbuch der speziellen Zoologie. Wirbellose Tiere, 1. Gustav Fischer, Jena Stuttgart, New York. Herold, W., 1913. BeitraÈge zur Anatomie und Physiologie einiger Landisopoden, HaÈutung-Secretion-Atmung. Zoologisches Jahrbuch fuÈr Anatomie und Ontogenie der Tiere 35, 457±526. KuÈmmel, G., Claasen, H., Keller, R., 1970. Zur Feinstruktur von Cuticula und Epidermis beim Fluûkrebs Orconectes limosus waÈhrend eines HaÈutungszyklus. Zeitschrift fuÈr Zellforschung 109, 517±551. Lagarrigue, J.-G., 1968. ReÂcherches biochimiques sur le squelette teguÂmentaire des isopodes terrestres. Vie et milieu 19, 173±187. Locke, M., 1969. The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting ¯uid in an insect. Journal of Morphology 127, 7±40. Merz, W.A., 1967. Die Streckenmessung an gerichteten Strukturen im Mikroskop und ihre Anwendung zur Bestimmung von Ober¯aÈchenVolumen-Relationen im Knochengewebe. Mikroskopie 22, 132±142. Messner, B., 1965. Ein morphologisch-histologischer Beitrag zur HaÈutung von Porcellio scaber latr. und Oniscus asellus l. (Isopoda terrestria). Crustaceana 9, 285±301. Neufeld, D.S., Cameron, J.N., 1993. Transepithelial movement of calcium in crustaceans. Journal of Experimental Biology 184, 1±16. Nicholls, A.G., 1931. Studies on Ligia oceanica. I. A. Habitat and effect of change of environment on respiration. B. Observations on moulting and breeding. Journal of Marine Biological Association of the United Kingdom 17, 655±706. Numanoi, H., 1934. Calcium in the blood of Ligia exotica during nonmolting and molting phases. Journal of the Faculty of Science, Imperial University of Tokyo 3, 351±358. Quinlan, M.C., Hadley, N.F., 1983. Water relations of the terrestrial isopods Porcellio laevis and Porcellionides pruinosus (Crustacea, Oniscoidea). Journal of Comparative Physiology 151, 155±161. Radtke, R.L., 1983. The deposition of organic and inorganic materials in the gastroliths of Homarus americanus. American Zoologist 23, 94. Roer, R.D., 1980. Mechanisms of resorption and deposition of calcium in the carapace of the crab Carcinus maenas. Journal of Experimental Biology 88, 205±218. Roer, R., Dillaman, R., 1984. The structure and calci®cation of the crustacean cuticle. American Zoologist 24, 893±909. Sparkes, S., Greenaway, P., 1984. The hemolymph as a storage site for
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257 cuticular ions during premoult in the freshwater/land crab Holthuisana transversa. Journal of Experimental Biology 113, 43±54. Steel, C.G.H., 1982. Stages of the intermoult cycle in the terrestrial isopod Oniscus asellus and their relation to biphasic cuticle secretion. Canadian Journal of Zoology 60, 429±437. Steel, C.G.H., 1993. Storage and translocation of integumentary calcium during the moult cycle of the terrestrial isopod Oniscus asellus (L.). Canadian Journal of Zoology 71, 4±10. Travis, D.F., 1963. The deposition of skeletal structures in the Crustacea. 3. The histochemical changes associated with the development of the mineralized gastroliths discs in the cray®sh, Orconectes virilis Hagen. Acta Histochemica 15, 269±284. Travis, D.F., 1965. The deposition of skeletal structures in the crustacea 5 The histomorphological and histochemical changes associated with the development and calci®cation of the branchial exoskeleton in the cray®sh, Orconectes virilis Hagen. Acta Histochemica 20, 193±222. È ber die DoppelhaÈutung der Landisopoden. ZeitVerhoeff, K.W., 1940. U È kologie der Tiere 37, 126±143. schrift fuÈr Morphologie und O Warburg, M.R., 1989. The role of water in the life of terrestrial isopods. Monitore Zoologico Italiano (N.S.) Monogra®a 4, 285±304. Wheatly, M.G., 1997. Crustacean models for studying calcium transport: the journey from whole organisms to molecular mechanisms. Journal of Marine Biological Association of the United Kingdom 77, 107±125. Weibel, E.R., 1969. Stereological principles for morphometry in electron microscopic cytology. International Review of Cytology 26, 235±302. Ziegler, A., 1994. Ultrastructure and electron spectroscopic diffraction analysis of the sternal calcium deposits of Porcellio scaber Latr. (Isopoda, Crustacea). Journal of Structural Biology 112, 110±116.
257
Ziegler, A., 1996. Ultrastructural evidence for transepithelial calcium transport in the anterior sternal epithelium of the terrestrial isopod Porcellio scaber (Crustacea) during the formation and resorption of CaCO3 deposits. Cell and Tissue Research 284, 459±466. Ziegler, A., 1997a. Ultrastructural changes of the anterior and posterior sternal integument of the terrestrial isopod Porcellio scaber Latr. (Crustacea) during the moult cycle. Tissue and Cell 29, 63±76. Ziegler, A., 1997b. Immunocytochemical localization of the Na 1 /K 1 ATPase in the calcium transporting sternal epithelium of the terrestrial isopod Porcellio scaber Latr. (Crustacea). Journal of Histochemistry and Cytochemistry 45, 47±446. Ziegler, A., Miller, B., 1997. Ultrastructure of CaCO3 deposits of terrestrial isopods (Crustacea, Oniscidea). Zoomorphology 117, 181±187. Ziegler, A., Merz, E., 1999. Membrane particle distribution in the sternal epithelia of the terrestrial isopod Porcellio scaber Latr. (Crustacea, Oniscidea) during CaCO3 deposit formation and resorption, a freeze± etch analysis. Journal of Structural Biology 127, 263±278. Ziegler, A., Scholz, F.H.E., 1997. The ionic hemolymph composition of the terrestrial isopod Porcellio scaber Latr. during molt. Journal of Comparative Physiology B 167, 536±542. Ziegler, A., Weihrauch, D., Bleher, R., 2000a. Expression and Immunolocalization of a V-Type H 1 -ATPase in the Anterior Sternal Epithelial Cells of P. scaber (Isopoda, Crustacea). Zoology 103, 97. Ziegler, A., Grospietsch, T., Carefoot, T.H., Danko, J.P., Zimmer, M., Zerbst-Boroffka, I., Pennings, S.C., 2000b. Hemolymph ion compositon and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt. Journal of Comparative Physiology B 170, 329±336.
ARTHROPOD STRUCTURE & DEVELOPMENT www.elsevier.com/locate/asd
Morphometric analysis of the calcium-transporting sternal epithelial cells of the terrestrial isopods Ligia oceanica, Ligidium hypnorum, and Porcellio scaber during molt Juliane GloÈtzner, Andreas Ziegler* Z.E. Elektronenmikroskopie, University of Ulm, D 89069 Ulm, Germany Received 20 September 2000; accepted 11 December 2000
Abstract Isopods shed ®rst the posterior and then the anterior half of the body. Before molt, most terrestrial species resorb CaCO3 from the posterior mineralized cuticle. The mineral is stored in anterior sternal deposits, which are used to calcify the new posterior cuticle after molt. For Porcellio scaber it is known that the anterior sternal epithelium has speci®c structural differentiations for epithelial transport. These differentiations include the plasma membrane surface areas, and the volume fraction of the mitochondria. We analyzed the ultrastructure of the sternal epithelium and used a morphometric approach to study the variations of these parameters between species living in different terrestrial environments. In Ligidium hypnorum, which lives in moist environments, the plasma membrane surface area and volume fraction of mitochondria are much larger than in the semiterrestrial Ligia oceanica. This is in accordance with the relatively larger CaCO3 deposits and shorter time intervals for their formation and resorption in L. hypnorum. For P. scaber, which is adapted to mesic habitats, most values are between those of L. oceanica and L. hypnorum. However, P. scaber has even larger CaCO3 deposits which are formed and degraded within similar time intervals as in L. hypnorum. This unexpected result is considered from the standpoint of more effective mechanisms being present for epithelial ion transport. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Calcium storage; Crustacea; Cuticle; Biomineralization; Integument; Ultrastructure
1. Introduction Most terrestrial isopods (Oniscidea) store large amounts of cuticular calcium in the ®rst four anterior sternites (Herold, 1913; Verhoeff, 1940; Messner, 1965; Steel, 1982) before the molt, and reuse the stored calcium for calci®cation of the new cuticle after molt (Steel, 1993). Formation and resorption of these sternal deposits are linked with a unique, biphasic molt cycle (for a scheme of the molting and calcium movements in an terrestrial isopod see Ziegler and Merz (1999)). Isopods molt ®rst the posterior half of the body, which includes the ®fth thoracic segments, and then the anterior half of the body. The pathway for cuticular calcium during the molt cycle is well described for Porcellio scaber and Oniscus asellus (Messner, 1965; Steel, 1993; Ziegler, 1996; Ziegler and Scholz, 1997). About one week before the posterior molt, calcium is resorbed from the posterior cuticle into the hemolymph and transported across the anterior sternal epithelium * Corresponding author. Tel.: 1731-502-3444; fax: 1731-502-3383. E-mail address: [email protected] (A. Ziegler).
(ASE) into the ecdysial space, where it is stored as an amorphous, probably hydrated, CaCO3 compound (Ziegler, 1994). During intramolt, the short interval between the anterior and the posterior molt, the CaCO3 deposits are entirely resorbed within a short time period and used for calci®cation of the new posterior cuticle. In addition, Oniscidea ingest their old cuticle after each partial molt. Calcium ions originating from the old cuticle are most probably transported across the intestinal epithelium and are used to mineralize the new anterior cuticle (Steel, 1993; Ziegler and Scholz, 1997). Ultrastructural work has shown that the anterior sternal epithelial cells of P. scaber (but not the cells of the posterior sternal epithelium (PSE) which transports a smaller amount of calcium), are differentiated for epithelial ion transport (Ziegler, 1996; Ziegler, 1997a). During the deposition and resorption of the sternal CaCO3 deposits, an elaborate network of interstitial dilations and channels known as the interstitial network (IN), enlarges the surface area of the basolateral plasma membrane, and the volume and abundance of mitochondria increases. In addition, numerous folds increase the surface of the apical plasma membrane
1467-8039/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 1467-803 9(00)00030-X
242
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
during intramolt and electron-dense granules that contain calcium are located within the IN. Various types of calcium reservoirs have been developed in terrestrial and freshwater Crustaceans (for review see Greenaway, 1985) including gastroliths of many decapods (Travis, 1963; Radtke, 1983), calcium containing microspherules within the hemolymph as in Holthuisana transversa (Sparkes and Greenaway, 1984) and the reservoires in the posterior midgut caeca in terrestrial amphipods (Graf, 1978; Graf and Meyran, 1983). Storage of large amounts of cuticular calcium is thought to be an adaptation to the terrestrial environment. Among crustaceans, oniscidean isopods were most successful in the colonization of terrestrial habitats. They inhabit a wide variety of biotopes such as rocky seashores, moist and shady woodlands, mesic, and even arid habitats. The cuticle of terrestrial isopods is relatively permeable to water and therefore they are susceptible to desiccation (Quinlan and Hadley, 1983; Warburg, 1989). The development of thicker and more calci®ed cuticles is thought to have reduced water loss by evaporation (Lagarrigue, 1968) during the evolution of terrestrial isopods (Gruner et al., 1993). A strong calci®cation of the cuticle requires the formation of CaCO3 deposits with a larger storage capacity. A comparative ultrastructural investigation of the CaCO3 deposits within two of the ®ve main taxa of the Oniscidea showed that three types of deposits can be distinguished whose storage capacity correlates with the degree of terrestriation (Ziegler and Miller, 1997). The Ligiidae, which are bound to biotopes with high humidity, have CaCO3 deposits with rather small storage capacity. Most members of the semiterrestrial genus Ligia live immediately adjacent to oceans and thus have ready access to seawater. They have thin single-layered Type III CaCO3 deposits composed of numerous small spherules (Ziegler and Miller, 1997). The closely related but more terrestrial species Ligidium hypnorum inhabits moist and shady forest biotopes, and prefers environments, which are relatively rich in calcium (Dahl, 1917). Their Type II CaCO3 deposits are two-layered and have a higher storage capacity than the type III deposits (Ziegler and Miller, 1997). Crinochaeta (which comprise the Porcellionidae, Armadilliidae and other oniscidean families) inhabit a variety of biotopes, including very humid, mesic and even arid regions. They have generally large, three-layered type I CaCO3 deposits (Ziegler and Miller, 1997). Another interesting difference between the more ancestral Ligiidae and the later-evolved Crinochaeta relates to hemolymph-calcium homeostasis capabilities during the molt cycle. Hemolymph calcium rises much more during molt in Ligiidae (Numanoi, 1934; Ziegler et al., 2000a) than in Crinochaeta (BoÈhm and Eibisch, 1976; Ziegler and Scholz, 1997), suggesting that Crinochaeta developed more effective mechanisms for calcium homeostasis than Ligiidae (Ziegler et al., 2000a). Variations in calcium storage capacity and hemolymphcalcium homeostasis probably correlate with differences in
transport ef®ciency of the anterior sternal epithelium. In an attempt to establish ®rst evidence for interspeci®c variations in the morphological differentiation of the sternal epithelia, we analyzed the ultrastructure of the ASE and PSE of Ligia oceanica and L. hypnorum. Using a morphometric approach we compared the surface area of the apical and basolateral plasma membrane, and the volume fraction of the mitochondria with those of the more terrestrially evolved Crinochaeta P. scaber. 2. Materials and methods L. oceanica were obtained from the Biologische Anstalt Helgoland. They were kept in plastic containers with rocks partly submerged in arti®cial seawater, and fed with potatoes and commercially available ®sh food (Tetra). L. hypnorum were kept in plastic boxes ®lled with soil, moldy leaves and bark, and fed with fresh carrots and potatoes. P. scaber were maintained as described earlier (Ziegler, 1996). The animals were checked regularly for the appearance of sternal CaCO3 deposits. Animals in early premolt, late premolt and intramolt stage were used for investigation. Animals without any external signs of molting were de®ned as being in the early premolt stage. In some of them apolysis had already occurred, however, the secretion of the new cuticle had not yet started. Welldeveloped sternal deposits and a change in the color of the anterior tergites due to deposition of CaCO3 in the whole anterior cuticle de®ned animals in late premolt stage. Animals in intramolt stage (between posterior and anterior molt) were used after the CaCO3 deposits were partly degraded indicating that resorption of CaCO3 took place. The isopods were killed by injecting them with 12.5% glutaraldehyde in 0.1 mol l 21 cacodylate buffer. The anterior (1±4) and the posterior (5±7) sternal plates were removed, ®xed for 1±2 h in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mol l 21 cacodylate buffer (pH 7.3) and post®xed in 1% OsO4 and 0.8% K3Fe(CN)6 in 0.1 mol l 21 cacodylate buffer for 1 h. After dehydration in a series of isopropanols the specimens were transferred to propylene oxide and embedded in Epon. Ultrathin sagittal sections (60±80 nm) were cut with a diamond knife (Diatome) on a Leica Ultracut microtome, stained with 2% uranyl acetate in H2O and with 0.3% lead citrate and viewed in a Philips 400 electron microscope at 80 kV. For energy-®ltered transmission electron microscopy semithin sections (500 nm) were cut with a diamond knife, stretched on a hot-water surface and placed on Formvar-coated copper grids stabilized with carbon. The sections were stained with 2% uranyl acetate in H2O. An energy-®ltering electron microscope (Zeiss CEM 902 operated at 80 kV) equipped with a goniometer stage was used to take stereo micrographs at an energy loss of 38 eV and tilt angles of 0 and 1128. We followed the method of Merz (1967) to determine the
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
243
by L Lp =e mm with `L' as the standardized pro®le length and `e' as the width of the test area (Fig. 1). The areas of the basolateral and the apical plasma membranes per mm 2 area of the epithelium were determined by A L £ 1:273 mm2 Overlays with regular arrays of dots were used to determine the volume fraction of the mitochondria of the ASE and PSE of L. oceanica and L. hypnorum. For each test area the number of dots on the mitochondria (Pm) as well as the number of dots on the cytoplasm (Pc) were counted. The volume fraction Vm of the mitochondria was calculated by Vm Pm =Pc £ 100; % Weibel; 1969 Means and standard errors of the means were calculated for each group. ANOVA was used to detect signi®cant differences in the surface area of the apical and basolateral plasma membrane between molting stages, between the anterior and posterior epithelial cells, and between species. Bonferroni±Holm's test was used to assign signi®cant differences between selected pairs of means following signi®cant ANOVA results. 3. Results 3.1. General morphology
Fig. 1. Test area and test system on a micrograph of the anterior sternal epithelium of L. hypnorum at intramolt. The pro®le areas of half epithelial cells were taken as test areas. The applied test system consists of an overlay of coherent lines of semicircles. e, length of the test area; d, diameter of the semicircles. £ 10800.
area of the apical and basolateral plasma membranes. The test®eld used covered about one half of an epithelial cell (Fig. 1). Measurements were done on a computer using overlays with a coherent semicircular test system (Fig. 1). The pro®le length `Lp' of the apical and the basolateral plasma membrane of each test area was determined by Lp Nd mm with `N' as the number of intersections between the test system and the pro®les of the plasma membranes and `d' as the diameter of the semicircles (Fig. 1). Lp was normalized to a standard length of 1 mm epithelium
The sternal integument comprises the cuticle, the epithelial cell layer that consists of more or less cubic cells, and the basal lamina (Figs. 2 and 3). In the anterior and posterior sternal integument of L. hypnorum a pigment cell layer underlies the basal lamina (Figs. 7, 8 and 15). In L. oceanica a pigment cell layer under the basal lamina was lacking in the ASE (Figs. 3 and 18). In the PSE it was found in only one of eight animals (not shown). 3.2. Ultrastructure Many features of the ultrastructure are very similar in L. oceanica and L. hypnorum. Therefore the description below applies for both species unless differences are described explicitly (The ultrastructure of P. scaber was described in detail in previous publications (Ziegler, 1996; Ziegler, 1997a; Ziegler and Merz, 1999)). The cuticle comprises the distal outer and inner epicuticle (Figs. 10, 24±26), the exocuticle in the middle (Figs. 3, 4, 20 and 22) and the proximal endocuticle (Figs. 2, 20 and 22). The thickness of the cuticle during late premolt, intramolt and early premolt are given in Table 1. During late premolt and intramolt the new cuticle is thickest in L. oceanica followed by L. hypnorum and P. scaber. Numerous cellular extensions of the epithelial cells protrude into
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
the pore channels of the new cuticle (Figs. 9, 15 and 20). During late premolt the cellular extensions of both the ASE and PSE end within the inner epicuticle (Figs. 10 and 15). During intramolt, only the cellular extensions of the ASE end within the inner epicuticle (Fig. 17) while the cellular extensions of the PSE end within the exocuticle leaving empty pore channels in the more apical regions of the cuticle (Figs. 20 and 22). In addition, the cellular extensions in the PSE are less abundant during intramolt than during late premolt, leading to a larger density of pore channels in distal layers of the cuticle (Fig. 22). In L. hypnorum peculiar new structures were found within the ecdysial space of most late premolt and intramolt animals. They are located close to the new anterior and posterior sternal cuticle (Fig. 15). In some early premolt animals we observed these tubular structures also at the outer sternal cuticle. In ultrathin sagittal sections they show round and elongated pro®les (Fig. 24). Stereopairs of semithin sections have revealed that these pro®les belong to tubular structures that are connected to the new epicuticle (Figs. 25 and 26). They have an inner diameter of about 20 nm and an outer diameter of about 30 nm. From the outer margin ®lamentous material extends to an approximate diameter of 100 nm (Fig. 24). During early premolt the amount of organelles in the cells of the ASE and the PSE is similar. There is little rough endoplasmic reticulum (RER), and mitochondria and Golgi stacks are rare (Fig. 2). A large portion of the cytoplasm is ®lled with lipid droplets and glycogen (Fig. 2), which resemble the glycogen granules described in P. scaber (Ziegler, 1997a). During late premolt and intramolt the cells of the ASE are rich in RER, mitochondria and Golgi stacks (Figs. 3, 8 and 16). Small vesicles with electron-dense content are often located in the vicinity of the apical plasma membrane (Figs. 3, 9, 16 and 18). In the PSE cells the abundance of most organelles resembles that in the ASE, however, mitochondria are less abundant in the PSE than in the ASE (Figs. 15, 20 and 22). The apical and the basolateral plasma membranes of the ASE have almost plane surfaces during early premolt (Fig. 2). In the PSE the apical plasma membranes are also plane, however, neighboring cells often interdigitate with one another (not shown). During late premolt and intramolt the morphology of the apical plasma membrane differs
245
between L. oceanica and L. hypnorum. In the ASE of L. oceanica many subcuticular microvilli-like extensions of the cytoplasm occur (Figs. 3, 9 and 18), whereas in the ASE of L. hypnorum the apical plasma membrane forms many subcuticular folds rather than microvilli-like extensions (Figs. 4, 8 and 10). In both species, folds increase the surface area of the basolateral plasma membrane. Laterally these folds surround ®nger-like cellular processes, which appear as more or less circular or elongated pro®les (Figs. 3, 8, 16 and 18). In the PSE subcuticular microvillilike extensions in the apical plasma membrane and subcuticular folds are rare during late premolt of L. oceanica (not shown) and L. hypnorum (Fig. 15), respectively. In intramolt, however, 2 of 6 L. oceanica (not shown) and 3 of 8 L. hypnorum (Fig. 23) have many subcuticular microvilli-like cellular extensions. In L. oceanica basolateral folds and lateral ®nger-like processes are rare during late premolt and intramolt. In L. hypnorum the basolateral plasma membrane compartment forms only a few folds in the basal region during late premolt (Fig. 15), and neighboring epithelial cells often interdigitate with one another (not shown). During intramolt, the basolateral plasma membrane was almost plane in six specimens (Fig. 22). However, in two L. hypnorum the basolateral plasma membranes form lateral folds containing ®nger-like cellular processes similar to those in the ASE (Fig. 23). These animals belonged to those with subcuticular microvilli-like cellular extensions in the apical plasma membrane mentioned above. During late premolt and intramolt interstitial spaces of the ASE contain electron-dense material in most L. oceanica and L. hypnorum (Figs. 3, 8 and 18; Table 2). The electrondense material appears either as elongated structures (Figs. 11 and 12) or as granules (Figs. 13 and 14). The granules have diameters between 20 and 290 nm. The electron-dense granules are associated with the extracellular side of ¯at regions (Fig.14) or cellular extensions (Fig. 13) of the basolateral plasma membrane. In most animals the electrondense material is mainly concentrated in the apical half of the interstitial space (Figs. 7, 8 and 23). In L. hypnorum and occasionally in L. oceanica electron-dense granules in the basal half of the interstitial space often had a smaller diameter than in the apical half (Figs. 4±6). Electron-dense particles ranging between 20 and 110 nm in size were observed in the basal lamina of many animals (Figs. 3, 8,
Figs. 2±6. Fig. 2 The anterior sternal epithelium of L. hypnorum during early premolt. £ 10200. bl, basal lamina, en, endocuticle; g, Golgi; gl, glycogen; ld, lipid droplets; m, mitochondria; n, nucleus; rer, rough endoplasmic reticulum. Fig. 3 The anterior sternal epithelium of L. oceanica during late premolt. Many subcuticular microvilli-like extensions of the cytoplasm (me) increase the surface area of the apical plasma membrane. The basolateral plasma membrane forms many folds, thereby increasing the interstitial space (is), which contains electron-dense material (arrows). Electron-dense particles appear in the basal lamina (arrowheads). £ 6800. bl, basal lamina; ce, cellular extensions; ep, epicuticle; ex, exocuticle; g, Golgi; m, mitochondria; rer, rough endoplasmic reticulum; v, vesicle. Fig. 4 The anterior sternal epithelium of a L. hypnorum during late premolt with large electron-dense granules (arrows) in the apical and smaller ones in the basal region of the interstitial space. Overview: the apical and the basolateral plasma membrane form many folds. bl, basallamina; ep, epicuticle; ex, exocuticle; m, mitochondria; mf, membrane folds; n, nucleus; rer, rough endoplasmic reticulum; rectangles, regions shown in the insets in higher magni®cation. £ 9100. Fig. 5 The anterior sternal epithelium of a L. hypnorum during late premolt. Details of apical region of the interstitial space with large electron-dense granules (arrow). £ 18200. Fig. 6 The anterior sternal epithelium of a L. hypnorum during late premolt. Detail of basal region of the interstitial space containing smaller electron-dense granules (arrow). £ 18200.
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
247
Table 1 Thickness of the cuticle (nm) within the anterior and posterior integument (mean ^ SEM) at three different molting stages (The number of measurements is given in brackets) Tissue Stage L. oceanica L. hypnorum P. scaber a
anterior late premolt 4102 ^ 272 (10) 1005 ^ 127 (10) 492 ^ 120 (8)
intramolt 4312 ^ 523 (9) 1310 ^ 131 (12) 999 ^ 123 (8)
posterior early premolt
late premolt a
14055 ^ 4107 (4) 4831 ^ 250 a (4) 8737 ^ 1716 a (4)
6228 ^ 973 (10) 1593 ^ 155 (10) 1019 ^ 310 (8)
intramolt
early premolt a
7483 ^ 1336 (8) 3925 ^ 587 a (10) 3197 ^ 811 a (8)
14235 ^ 1176 a (4) 5562 ^ 832 a (4) 9775 ^ 2228 a (4)
Cuticle contains endocuticular layers.
Table 2 Occurrence of electron-dense material within the lateral interstitium of the anterior (ASE) and posterior sternal epithelium (PSE)
16 and 19). In the ASE of L. hypnorum the occurrence of these electron-dense particles correlated with a very high amount of electron-dense material in the lateral interstitium (Fig. 8). In the PSE electron-dense material is lacking during late premolt in both species, and occurs only occasionally during intramolt (Figs. 21 and 23, Table 2). In L. hypnorum these few epithelia resemble those of most ASE with respect to the appearance of the interstitial space, distribution of electron-dense granules (Fig. 23), and electron-dense particles in the basal lamina (not shown). 3.3. Morphometry In order to compare the plasma membrane areas in different molting stages and species we normalized the values of the measurements to 1 mm 2 area of the epithelium as
described in the methods. Mean values for the normalized apical (Aat) and basolateral plasma membrane (Abl), the volume fraction of the mitochondria (Vm) and signi®cant differences between selected pairs of means are given in Figs. 27±29. In all three species we found a signi®cant increase of Aat in the ASE from early to late premolt and from early premolt to intramolt (Fig. 27). In P. scaber the increase from early to late premolt appears rather small in comparison with the other species due to a virtual lack of subcuticular folds. In the PSE Aat rises transiently during late premolt (however insigni®cantly in L. oceanica, P 0:24 (Fig. 27). In general total Aat may be divided into two subtypes: the subcuticular plasma membrane and the plasma membrane that belongs to cellular extensions into the cuticle. In most cases the percentile contribution of the cellular extensions
Figs. 7±14. Fig. 7 The anterior sternal epithelium of L. hypnorum during late premolt. Animal with electron-dense material (arrows) only in the apical region of the interstitial space and no electron-dense particles within the basal lamina. £ 6600. Fig. 8 The anterior sternal epithelium of L. hypnorum during late premolt. Animal with large amounts of electron-dense material (arrows) mostly in the apical region of the interstitial space and much electron-dense material within the basal lamina. £ 6900. bl, basal lamina; ce, cellular extensions; cu, cuticle; ld, lipid droplets; g, Golgi; is, interstitial space; m, mitochondria; mf, membrane folds; n, nucleus; pg, pigment granules; rer, rough endoplasmic reticulum; v, vesicle; arrowheads, electron-dense particles in the basal lamina. Fig. 9 and Fig. 10 The differentiation of the apical plasma membrane in the anterior sternal epithelium of L. oceanica and L. hypnorum during late premolt. ce, cellular extensions; ex, exocuticle; ie, inner epicuticle; m, mitochondria; me, subcuticular microvilli-like extensions of the cytoplasm; mf, membrane folds; oe, outer epicuticle; pc, pore channel; rer, rough endoplasmic reticulum; v, vesicle. L. oceanica (Fig. 9) with apical subcuticular microvilli-like cellular extensions. £ 26600. L. hypnorum (Fig. 10) with apical folds. £ 23700. Figs. 11±14 The structure of the electron-dense material in the anterior sternal epithelium of L. hypnorum. The electron-dense material often forms elongated structures (arrowheads), which ®ll parts of the intercellular space (Fig. 11, £ 35200. Fig. 12 £ 41600). Electron-dense granules on the tips of lateral cellular projections (arrowheads, Fig. 13 £ 82800). Small electron-dense granules associated with the extracellular side of ¯at regions of the basolateral plasma membrane (arrowheads, Fig. 14, £ 36400). bl, basal lamina; is, interstitial space; m, mitochondria; rer, rough endoplasmic reticulum.
248
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
(Ace) to Aat is zero in the sternal epithelium during early premolt (exceptions are stated explicitly, below). In the ASE of L. oceanica Ace is 34% during late premolt and 28% during intramolt, versus 54 and 7% in the PSE. In the ASE of L. hypnorum Ace is 5 and 12% during late premolt and intramolt, respectively, and 27 and 4% in the PSE. In the ASE of P. scaber Ace is 1% during early and late premolt, and 4% during intramolt. In the PSE Ace is 13% during late premolt and 38% during intramolt. In the PSE of P. scaber the signi®cant difference in Aat between early premolt and intramolt by 1.15 mm 2 is entirely due to an increase of Ace. In all three species Abl in the ASE increases from early premolt to late premolt and intramolt (insigni®cant for P. scaber during late premolt) (Fig. 28). In the PSE Abl does not change signi®cantly within species (P . 0.05), however, during early premolt Abl of L. hypnorum is considerably smaller than in L. oceanica (Fig. 28). This difference is due to the smaller height of the epithelial cells in Ligidium as compared to the other two species (data not shown). Nevertheless, we found the largest value for Abl in the ASE of L. hypnorum during intramolt. In the ASE all three species show an increase of Vm from early premolt to late premolt by a factor of at least 2.0 and a somewhat smaller increase from early premolt to intramolt (not signi®cant for L. oceanica; P 0:2 and P 0:57; respectively) (Fig. 29). The largest increases by factors of about 3 occurred in L. hypnorum during late premolt and intramolt. In the PSE Vm does not change signi®cantly within and between species (Fig. 29). 4. Discussion The present study shows that in the ligiids L. oceanica and L. hypnorum the ASE is differentiated for an increased ion transport during the formation and resorption of the sternal CaCO3 deposits. These differentiations are similar to that found previously in the ASE of the crinochete P. scaber. Interspeci®c variations in the differentiation of the sternal integument concern the relative thickness of the new incomplete cuticle, the subcuticular enlargements of the apical plasma membrane, the shape of the interstitial spaces,
249
and the temporal distribution of electron-dense granules between the epithelial cells. Quantitative differences were found for the surface area of the apical and basolateral plasma membrane and the volume density of the mitochondria. During molt the epithelium of the integument functions in resorption of the organic and mineral components of the old cuticle, and secretion and calci®cation of the new one. In addition, the ASE forms and degrades the large sternal CaCO3 deposits that serve as calcium reservoirs during the biphasic molt. For P. scaber it has been shown, that the analysis of the ASE and, as a control, the PSE permits to distinguish morphological differentiations for ion transport from those for the secretion and resorption of the organic compounds of the cuticle (Ziegler, 1996; Ziegler, 1997a). Secretion of the new anterior cuticle already begins during the formation of the sternal deposits, thereby establishing a barrier for the diffusion of ions between the CaCO3 deposit and the epithelium. Therefore, delayed secretion of the ®rst cuticular layers in the anterior sternites during late premolt and intramolt is regarded as a mechanism to speed storage and resorption of CaCO3 (Messner, 1965; Steel, 1993; Ziegler, 1997a). A comparison of the cuticle during molt within the sternal integument shows that in the anterior sternal integument the delay of growth is least in L. oceanica followed by L. hypnorum and largest in P. scaber (Table 1, (Ziegler, 1997a)). In the crinochete O. asellus the delay in cuticle growth is similar to that in P. scaber (Messner, 1965; Steel, 1982). For the ®rst time we describe tubular structures connected to the sternal epicuticle within the ecdysial gap of L. hypnorum or, in some animals, at the sternal body surface. At the current stage of investigation we cannot correlate these structures with any function. Possibly they serve as sponges to retain moisture, or they have some function during the molting process. In future experiments we plan to investigate the water-absorbing properties of the tubular structures and their distribution in molting and non-molting animals. The deposition and resorption of CaCO3 requires the 1 movement of Ca 21, HCO2 across the epithelia 3 and H (Fig. 30A, B). It is not clear whether movements of calcium ions occur by active transcellular or passive paracellular
Figs. 15±19. Fig. 15 The posterior sternal epithelium of L. hypnorum during late premolt. £ 7800. bl, basal lamina; ce, cellular extensions; ep, epicuticle; ex, exocuticle; g, Golgi; gl, glycogen; hl, hemolymph; ld, lipid droplets; m, mitochondria; n, nucleus; pg, pigment granules; rer, rough endoplasmic reticulum; v, vesicle; arrows, tubular structures. Fig. 16 The anterior sternal epithelium of L. hypnorum during intramolt. The apical and the basolateral plasma membrane form many folds. Large amounts of electron-dense material (arrows) appear in the interstitial space (is). Electron-dense particles occur in the basal lamina (arrowheads). £ 7700. Fig. 17 The anterior sternal epithelium of L. hypnorum during intramolt. Cellular extensions protruding into the inner epicuticle. £ 15300. bl, basal lamina; ce, cellular extensions; ex, exocuticle; g, Golgi; ep, epicuticle; m, mitochondria; mf, membrane folds; rer, rough endoplasmic reticulum; v, vesicle. Fig. 18 The anterior sternal epithelium of L. oceanica during intramolt. The apical plasma membrane forms small subcuticular microvilli-like extensions of the cytoplasm (me). The basolateral plasma membrane forms many folds. The interstitial space (is) contains a comparatively small amount of electron-dense material (arrows). Many electron-dense particles appear in the basal lamina (arrowheads). £ 7900. Fig. 19 The anterior sternal epithelium of L. oceanica during intramolt. Electron-dense particles (arrowheads) in the basal lamina at higher magni®cation. £ 20000. bl, basal lamina; ce, cellular extensions; ex, exocuticle; m, mitochondria; rer, rough endoplasmic reticulum; v, vesicle.
250
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
Figs. 20±23. Fig. 20 The posterior sternal epithelium of L. oceanica during intramolt. Animal with almost ¯at apical and basolateral plasma membrane. £ 6500. Fig. 21 The posterior sternal epithelium of L. oceanica during intramolt. Animal with electron-dense material within the interstitial space (arrows). £ 9400. bl, basal lamina; ce, cellular extensions; en, endocuticle; ep, epicuticle; ex, exocuticle; m, mitochondria; n, nucleus; rer, rough endoplasmic reticulum; v, vesicle. Fig. 22 The posterior sternal epithelium of L. hypnorum during intramolt. Animals with almost ¯at apical and basolateral plasma membranes. £ 8500. Fig. 23 The posterior sternal epithelium of L. hypnorum during intramolt. Animal with subcuticular microvilli-like extensions of the cytoplasm (me) and many folds of the basolateral plasma membrane and electron-dense material within the interstitial space (is). £ 10700. bl, basal lamina; ce, cellular extensions; en, endocuticle; ep, epicuticle; ex, exocuticle; gl, glycogen; hl, hemolymph; m, mitochondria; n, nucleus; pc, pore channels; pg, pigment granules; rer, rough endoplasmic reticulum; v, vesicle.
252
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
Fig. 27. The apical plasma membrane surface area of the anterior (ASE) and posterior (PSE) sternal epithelial cells of L oceanica, L. hypnorum and P. scaber at three different molting stages (means ^ SE). Asterisks on top of the columns indicate signi®cant differences from the early premolt stage. Horizontal brackets with asterisks indicate statistically signi®cant differences between the means. Signi®cant differences between the ASE and PSE are indicated below the columns. (***P , 0.001, *P , 0.05, a: P , 0.05) N, number of animals; n, number of test ®elds.
mechanisms (Neufeld and Cameron, 1993). For the transcellular pathway (Fig. 30) verapamil sensitive calcium channels were proposed for the in¯ux of calcium into the cells and a Ca 21-ATPase, Na 1/Ca 21- and Ca 21/H 1exchange mechanism for the extrusion of calcium from the cells to the other side of the epithelium (Roer, 1980; Greenaway and Dillaman et al., 1995; Neufeld and Cameron, 1993; Wheathly, 1997; Ahearn et al., 1999). For the ASE of P. scaber a high concentration of Na/K-ATPase was demonstrated in the basolateral (Ziegler, 1997b) and a V-type H 1-ATPase in the apical plasma membrane (Ziegler et al., 2000a). In general, large plasma membrane surfaces in conjunction with abundant or large mitochondria are regarded as differentiations for high ion transport activity across the
epithelial cells. The signi®cant increase in the apical and basolateral plasma membrane surface area and the increase of the volume density of the mitochondria from early premolt to late premolt and/or intramolt shows that in all three species analyzed the ASE cells become differentiated for epithelial ion transport. The differences between the plasma membrane surfaces of the ASE and PSE cells suggest that ion transport across the epithelial cells play a role in the formation and resorption of the sternal CaCO3 deposits. In L. hypnorum the increase in the apical plasma membrane surface area is caused by development of subcuticular folds similarly to P. scaber (Ziegler, 1997a). In contrast L. oceanica increases the apical plasma membrane by subcuticular microvilli-like extensions. The latter have been described for other crustaceans and insects
Figs. 24±26 (previous page). Fig. 24 Tubular structures (arrows) in the ecdysial space of L. hypnorum (intramolt). In thin sagittal sections the structures show round and elongate pro®les. £ 86500. Fig. 25 ( £ 38100) and Fig. 26 ( £ 48100) Tubular structures (arrows) in the ecdysial space of L. hypnorum (intramolt). Stereopairs of semithin (500 nm) sagittal sections taken at an energy loss of 38 eV, showing that the structures form tubules and are connected to the outer epicuticle. ie, inner epicuticle; oe, outer epicuticle.
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
253
Fig. 28. The mean basolateral plasma membrane surface area of the anterior (ASE) and posterior (PSE) sternal epithelial cells of L oceanica, L. hypnorum and P. scaber at three different molting stages (means ^ SE). Asterisks on top of the columns indicate signi®cant differences from the early premolt stage. Horizontal brackets with asterisks indicate statistically signi®cant differences between the means. Signi®cant differences between the ASE and PSE are indicated below the columns. (***P , 0.001, **P , 0.01, *P , 0.05) N, number of animals; n, number of test ®elds.
in conjunction with the secretion of the cuticle (KuÈmmel and Claasen et al., 1970; Arsenault and Castell et al., 1984; Buchholz and Buchholz, 1989; Locke, 1969). In L. oceanica the spatial and temporal distribution suggests that they may function in the mineralization of the cuticle and deposition and resorption of the CaCO3 deposits. In the ASE they appear during formation and resorption of the sternal deposits, whereas in the PSE they occur only in a fraction of the animals analyzed. In these animals they appear only in the intramolt stage, during which mineralization of the exocuticle takes place (whereas the cuticle is secreted in both molting stages). A role in mineralization of the cuticle was also attributed to intracuticular cellular extensions (Travis, 1965; Roer, 1980; Giraud-Guille, 1984; Roer and Dillaman, 1984). In addition they might be involved in the resorption and deposition of CaCO3 deposits (Ziegler, 1996). The latter would be of particular signi®cance in the anterior sternal integument of L. oceanica in which the new cuticle is comparatively thick during late premolt and intramolt. A recent freeze±etch analysis of the ASE and PSE cells of P. scaber has shown that the membrane particle density within the plasma membrane of the cellular extensions is signi®cantly larger than in the subcuticular apical plasma membrane (Ziegler and Merz, 1999). In general, the contri-
bution of cellular extensions into the cuticle to the apical membrane surface area correlates with the thickness of the exocuticle before the appearance of the ®rst endocuticular layers during intramolt (an exception is the decrease from 34 to 28% from late premolt to intramolt in L. oceanica). After the ®rst endocuticular layers are secreted shortening of the cellular extensions leads to the observed reduction in their contribution to the total apical plasma membrane surface area in the posterior integument of L. oceanica and L. hypnorum. In comparison, in P. scaber the elongation of cellular extensions in the posterior integument from late premolt to intramolt explains the large contribution of intracuticular to total apical plasma membrane. Variations in the magnitude of differentiations indicative for epithelial ion transport between species may be interpreted in conjunction with variations in the need for large storage capacity of the CaCO3 deposits and/or variations in deposition/resorption rates. Our results show that the degree of differentiation is much larger in L. hypnorum than in L. oceanica. For animals in late premolt this difference seems to be related to larger deposits rather than to differences in transport rate, since the formation of the deposits requires 5±7 days in Ligia (Nicholls, 1931; Numanoi, 1934), and 6±8 days in L. hypnorum (present observations). A shorter intramolt stage of 15±20 h in L. hypnorum versus 2±3 days
254
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
Fig. 29. The mean volume fraction of the mitochondria within the cytoplasm of the anterior (ASE) and posterior (PSE) epithelial cells of L. oceanica, L. hypnorum and P. scaber at three different molting stages (means ^ SE). Asterisks on top of the columns indicate signi®cant differences from the early premolt stage. Horizontal brackets with asterisks indicate statistically signi®cant differences between the means. Signi®cant differences between the ASE and PSE are indicated below the columns (***P , 0.001, *P , 0.05). N, number of animals; n, number of test ®elds. Data for P. scaber from (Ziegler, 1997a).
in L. oceanica seems to be responsible for the larger degree of differentiation, in addition to the relative larger storage capacity of the CaCO3 deposits. Both, the storage capacity and a quick resorption of the CaCO3 deposits are probably necessary to avoid excessive desiccation during molt in the more terrestrial environment of Ligidium as compared with that of Ligia. The differentiation to ion transport in the ASE seems to be more pronounced in P. scaber than in L. oceanica, but, with the exception of the apical plasma membrane during intramolt, is less than in L. hypnorum. This is of particular interest because the sternal deposits of P. scaber have a larger storage capacity than those of the Ligiidae (Ziegler and Miller, 1997), and the time-periods for the formation and resorption of the deposits are similar to those observed in L. hypnorum. The comparatively small increase in the volume density of the mitochondria suggests that P. scaber developed more ef®cient mechanisms for epithelial ion transport. Earlier, Ziegler et al. (2000b) proposed that a more developed mechanism would exist based on a better hemolymph calcium homeostasis during molt in crinochetes than in Ligia species. Changes in hemolymph calcium concentration during molt are explained by
imbalances between the resorption of calcium from the posterior cuticle into the hemolymph and concomitant transport of calcium from the hemolymph across the ASE to the sternal CaCO3 deposits during premolt, and vice versa during intramolt. A comparatively high increase in calcium concentration of 47% was found in the hemolymph of Ligia pallasii (Ziegler et al., 2000b) and of 50±60% in Ligia exotica (Numanoi, 1934). In the Crinochaeta storage and resorption of calcium is accompanied by rather small increases in hemolymph calcium; e.g. only 25% in Armadillidium nasatum and 19% in P. scaber (BoÈhm and Eibisch, 1976; Ziegler and Scholz, 1997). More ef®cient calcium transport probably evolved because of a greater need to conserve calcium in isopods adapted to mesic versa species that live in rather moist habitats. At the present state of knowledge we can only speculate how more ef®cient mechanisms for ion transport may have evolved. An increase in the surface area of the apical plasma membrane as in the anterior sternal epithelial cells of L. oceanica and L. hypnorum during late premolt is one way to increase the ¯ux rate for ions involved in CaCO3 deposit formation or resorption. Such an increase, however, also increases nonspeci®c back-¯ux of ions (Fig. 30, C). Possibly, in P.
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
255
Fig. 30. Ion movements in the hypodermis of crustaceans during deposition (A) and resorption (B) of CaCO3, and proposed para- and trans-cellular calcium movements in the ASE of L. hypnorum (C), and P. scaber (D) during CaCO3 deposit formation. Dashed arrows indicate energetically futile back ¯ux of calcium (see discussion for further explanation). ASE, anterior sternal epithelium; b, basal lamina; c, cuticle; Cc, calcium channel; Cp, Ca 21-ATPase; e, ecdysial space; Ex, Na/Ca-Exchanger; f, apical folds; g, electron-dense granules; h, hypodermis; Np, Na/K-ATPase; pc, paracellular pathway; tc, transcellular pathway.
scaber the evolution of more ef®cient transport molecules and/or higher density of these molecules rather than an increase of the plasma membrane surface area has lead to increased ion ¯ux rates. This would explain the rather small increase of the apical plasma membrane surface area and would avoid an excessive waste of energy by futile back ¯ux of ions (Fig. 30, D). The difference in the occurrence of electron-dense granules between P. scaber and the two ligiids analyzed may also be explained by more ef®cient calcium homeostasis mechanisms in the former. For P. scaber it has been shown that the electron-dense granules contain an organic
matrix and a high concentration of calcium (Ziegler, 1996) and phosphorus (Ziegler, unpublished) indicating that the granules contain calcium phosphate, which is quite insoluble. In P. scaber they appear to be secreted at the apical region of the lateral plasma membrane and migrate to the basal side until they disintegrate within the basal lamina. The present ultrastructural analysis indicates a similar fate for the electron-dense granules of L. oceanica and L. hypnorum. Since in P. scaber the electron-dense granules occur almost exclusively in the ASE during the resorption of the CaCO3 deposits they are thought to play a role in the transport of calcium ions from the apical to the basal side of the
256
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257
epithelium. Another function may be to limit the calcium activity within the interstitial spaces in order to maintain a speci®c calcium gradient across the plasma membranes (Ziegler, 1996). In contrast to the situation in P. scaber, electron-dense granules occur in the ASE of L. oceanica and L. hypnorum as frequent during late premolt as during intramolt, and in a few cases even in the posterior sternal epithelium during intramolt. Because the calcium-containing granules cannot support a transport of calcium from basal to apical during late premolt, they are likely to be energetically inef®cient because calcium ions are transported back into the hemolymph producing a futile cycle (Fig. 30, C). Therefore, it appears likely that electron-dense calcium granules evolved for buffering the interstitial free calcium concentration within physiological limits. During the evolution of more ef®cient calcium homeostasis, however, they may nevertheless need to be present in the ASE during intramolt because here extracellular calcium ¯uctuations may still be large. Alternatively electrondense granules may have been maintained during the evolution of more ef®cient calcium homeostasis because they accelerate the resorption of the CaCO3 deposits by carrying calcium ions across the epithelium. This would mean that during evolution the electron-dense granules changed their main function from an extracellular calcium buffer in Ligiidae to a vehicle for interstitial calcium transport in Crinochaeta. 5. Conclusions The structural and morphometric results of the two ligiid species are consistent with a larger ion transport activity in L. hypnorum as compared with L. oceanica due to relatively thicker CaCO3 deposits and shorter time intervals for their resorption within the former. For P. scaber, the morphometric characteristics seem not to ®t to the simple correlation to the ion transport rate between species. Rather, it seems that P. scaber develops more effective mechanisms for epithelial ion transport during molt than the ligiids. The present data provide a ®rm base for comparative investigations between the different taxa of the Oniscidea. Further investigations within the Ligiidae and the Crinochaeta are necessary to establish how the degree of differentiation correlates with the degree of terrestriation. Acknowledgements We thank Dr Thomas Carefoot for critically reading the manuscript and linguistic corrections. References Ahearn, G.A., Duerr, J.M., Zhuang, Z., Brown, R.J., Aslamkhan, A., Killebrew, D.A., 1999. Ion transport processes of crustacean epithelial cells. Physiological and Biochemical Zoology. 72, 1±18.
Arsenault, A.L., Castell, J.D., Ottensmeyer, F.P., 1984. The dynamics of exoskeletal-epidermal structure during molt in juvenile lobster by electron microscopy and electron spectroscopic imaging. Tissue and Cell 16, 93±106. BoÈhm, G.-A., Eibisch, H., 1976. Untersuchungen zum Mineralstoffwechsel der Crustaceen Orconectes limosus (Decapoda) und Armadillidium nasutum (Isopoda) in der VorhaÈutungsphase. Zoologisches Jahrbuch fuÈr Physiologie 80, 175±183. Buchholz, C., Buchholz, F., 1989. Ultrastructure of the integument of a pelagic Crustacean: moult cycle related studies on the Antarctic krill, Euphausia superba. Marine Biology 101, 355±365. Dahl, F., 1917. Die Landisopoden SuÈdwestdeutschlands. Mitt Zool Mus Berlin 8, 405±423. Giraud-Guille, M.M., 1984. Calci®cation initiation sites in the crab cuticle: the interprismatic septa. Cell and Tissue Research 236, 413±420. Graf, F., 1978. Les sources de calcium pour les crustaceÂs venant de muer. Archs Zool Exp Gen 119, 143±161. Graf, F., Meyran, J.C., 1983. Premolt calcium secretion in midgut posterior caeca of the crustacean Orchestia: ultrastructure of the epithelium. Journal of Morphology 177, 1±23. Greenaway, P., 1985. Calcium balance and moulting in the crustacea. Biological Reviews 60, 425±454. Greenaway, P., Dillaman, R.M., Roer, R.D., 1995. Quercitin-dependent ATPase activity in the hypodermal tissue of Callinectes sapidus, during the moult cycle. Comparative Biochemistry and Physiology 111A, 303±312. Gruner, H.E., Moritz, M., Dunger, W., 1993. Arthropoda (ohne Insekta), 4 Teil. In: Gruner, H.E. (Ed.). Lehrbuch der speziellen Zoologie. Wirbellose Tiere, 1. Gustav Fischer, Jena Stuttgart, New York. Herold, W., 1913. BeitraÈge zur Anatomie und Physiologie einiger Landisopoden, HaÈutung-Secretion-Atmung. Zoologisches Jahrbuch fuÈr Anatomie und Ontogenie der Tiere 35, 457±526. KuÈmmel, G., Claasen, H., Keller, R., 1970. Zur Feinstruktur von Cuticula und Epidermis beim Fluûkrebs Orconectes limosus waÈhrend eines HaÈutungszyklus. Zeitschrift fuÈr Zellforschung 109, 517±551. Lagarrigue, J.-G., 1968. ReÂcherches biochimiques sur le squelette teguÂmentaire des isopodes terrestres. Vie et milieu 19, 173±187. Locke, M., 1969. The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting ¯uid in an insect. Journal of Morphology 127, 7±40. Merz, W.A., 1967. Die Streckenmessung an gerichteten Strukturen im Mikroskop und ihre Anwendung zur Bestimmung von Ober¯aÈchenVolumen-Relationen im Knochengewebe. Mikroskopie 22, 132±142. Messner, B., 1965. Ein morphologisch-histologischer Beitrag zur HaÈutung von Porcellio scaber latr. und Oniscus asellus l. (Isopoda terrestria). Crustaceana 9, 285±301. Neufeld, D.S., Cameron, J.N., 1993. Transepithelial movement of calcium in crustaceans. Journal of Experimental Biology 184, 1±16. Nicholls, A.G., 1931. Studies on Ligia oceanica. I. A. Habitat and effect of change of environment on respiration. B. Observations on moulting and breeding. Journal of Marine Biological Association of the United Kingdom 17, 655±706. Numanoi, H., 1934. Calcium in the blood of Ligia exotica during nonmolting and molting phases. Journal of the Faculty of Science, Imperial University of Tokyo 3, 351±358. Quinlan, M.C., Hadley, N.F., 1983. Water relations of the terrestrial isopods Porcellio laevis and Porcellionides pruinosus (Crustacea, Oniscoidea). Journal of Comparative Physiology 151, 155±161. Radtke, R.L., 1983. The deposition of organic and inorganic materials in the gastroliths of Homarus americanus. American Zoologist 23, 94. Roer, R.D., 1980. Mechanisms of resorption and deposition of calcium in the carapace of the crab Carcinus maenas. Journal of Experimental Biology 88, 205±218. Roer, R., Dillaman, R., 1984. The structure and calci®cation of the crustacean cuticle. American Zoologist 24, 893±909. Sparkes, S., Greenaway, P., 1984. The hemolymph as a storage site for
J. GloÈtzner, A. Ziegler / Arthropod Structure & Development 29 (2000) 241±257 cuticular ions during premoult in the freshwater/land crab Holthuisana transversa. Journal of Experimental Biology 113, 43±54. Steel, C.G.H., 1982. Stages of the intermoult cycle in the terrestrial isopod Oniscus asellus and their relation to biphasic cuticle secretion. Canadian Journal of Zoology 60, 429±437. Steel, C.G.H., 1993. Storage and translocation of integumentary calcium during the moult cycle of the terrestrial isopod Oniscus asellus (L.). Canadian Journal of Zoology 71, 4±10. Travis, D.F., 1963. The deposition of skeletal structures in the Crustacea. 3. The histochemical changes associated with the development of the mineralized gastroliths discs in the cray®sh, Orconectes virilis Hagen. Acta Histochemica 15, 269±284. Travis, D.F., 1965. The deposition of skeletal structures in the crustacea 5 The histomorphological and histochemical changes associated with the development and calci®cation of the branchial exoskeleton in the cray®sh, Orconectes virilis Hagen. Acta Histochemica 20, 193±222. È ber die DoppelhaÈutung der Landisopoden. ZeitVerhoeff, K.W., 1940. U È kologie der Tiere 37, 126±143. schrift fuÈr Morphologie und O Warburg, M.R., 1989. The role of water in the life of terrestrial isopods. Monitore Zoologico Italiano (N.S.) Monogra®a 4, 285±304. Wheatly, M.G., 1997. Crustacean models for studying calcium transport: the journey from whole organisms to molecular mechanisms. Journal of Marine Biological Association of the United Kingdom 77, 107±125. Weibel, E.R., 1969. Stereological principles for morphometry in electron microscopic cytology. International Review of Cytology 26, 235±302. Ziegler, A., 1994. Ultrastructure and electron spectroscopic diffraction analysis of the sternal calcium deposits of Porcellio scaber Latr. (Isopoda, Crustacea). Journal of Structural Biology 112, 110±116.
257
Ziegler, A., 1996. Ultrastructural evidence for transepithelial calcium transport in the anterior sternal epithelium of the terrestrial isopod Porcellio scaber (Crustacea) during the formation and resorption of CaCO3 deposits. Cell and Tissue Research 284, 459±466. Ziegler, A., 1997a. Ultrastructural changes of the anterior and posterior sternal integument of the terrestrial isopod Porcellio scaber Latr. (Crustacea) during the moult cycle. Tissue and Cell 29, 63±76. Ziegler, A., 1997b. Immunocytochemical localization of the Na 1 /K 1 ATPase in the calcium transporting sternal epithelium of the terrestrial isopod Porcellio scaber Latr. (Crustacea). Journal of Histochemistry and Cytochemistry 45, 47±446. Ziegler, A., Miller, B., 1997. Ultrastructure of CaCO3 deposits of terrestrial isopods (Crustacea, Oniscidea). Zoomorphology 117, 181±187. Ziegler, A., Merz, E., 1999. Membrane particle distribution in the sternal epithelia of the terrestrial isopod Porcellio scaber Latr. (Crustacea, Oniscidea) during CaCO3 deposit formation and resorption, a freeze± etch analysis. Journal of Structural Biology 127, 263±278. Ziegler, A., Scholz, F.H.E., 1997. The ionic hemolymph composition of the terrestrial isopod Porcellio scaber Latr. during molt. Journal of Comparative Physiology B 167, 536±542. Ziegler, A., Weihrauch, D., Bleher, R., 2000a. Expression and Immunolocalization of a V-Type H 1 -ATPase in the Anterior Sternal Epithelial Cells of P. scaber (Isopoda, Crustacea). Zoology 103, 97. Ziegler, A., Grospietsch, T., Carefoot, T.H., Danko, J.P., Zimmer, M., Zerbst-Boroffka, I., Pennings, S.C., 2000b. Hemolymph ion compositon and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt. Journal of Comparative Physiology B 170, 329±336.