620 M.G.JOURNAL WHEATLY OF EXPERIMENTAL ZOOLOGY 283:620–640 (1999)

Calcium Homeostasis in Crustacea: The Evolving Role of Branchial, Renal, Digestive and Hypodermal Epithelia MICHELE G. WHEATLY* Department of Biological Sciences, Wright State University, Dayton, Ohio 45435

ABSTRACT Crustaceans serve as an ideal model for the study of calcium homeostasis due to their natural molting cycle. Demineralization and remineralization of the calcified cuticle is accompanied by bidirectional Ca transfer across the primary Ca transporting epithelia: gills, antennal gland (kidney), digestive system, and cuticular hypodermis. The review will demonstrate how a continuum of crustaceans can be used as a paradigm for the evolution of Ca transport mechanisms. Generally speaking, aquatic crustaceans rely primarily on bran- chial Ca uptake and accordingly are affected by water Ca content; terrestrial crustaceans rely on intake of dietary Ca across the digestive epithelium. Synchrony of mineralization at the cuticle vs. storage sites will be presented. Physiological and behavioral adaptations have evolved to optimize Ca balance during the molting cycle in different Ca environments. Intra- cellular Ca regulation reveals common mechanisms of apical and basolateral membrane trans- port as well as intracellular sequestration. Regulation of cell Ca concentration will be discussed in intermolt and during periods of the molting cycle when transepithelial Ca flux is signifi- cantly elevated. Molecular characterization of the sarco-/endoplasmic reticular Ca pump in aquatic species reveals the presence of two isoforms that originate from a single gene. This gene is differentially expressed during the molting cycle. Gene expression may be regulated by a suite of hormones including ecdysone, calcitonin, and vitamin D. Perspectives for future research are presented. J. Exp. Zool. 283:620–640, 1999. © 1999 Wiley-Liss, Inc.

Crustaceans have served comparative physiolo- food (including reingested exuviae) and drink, and gists as useful paradigms for studying the evolu- from Ca deposits stored in regions of the digestive tionary transition from water to land since they system; (3) the antennal gland (analog of kidney) occupy habitats ranging from salt water (SW) to may be involved in postfiltrational reabsorption of freshwater (FW) and via either origin onto land urinary Ca2+; and (4) the cuticular hypodermis can (Burggren and McMahon, ’88). Examining calcium effect demineralization or mineralization at differ- (Ca) homeostasis in a continuum of crustaceans ent stages of the molting cycle. Aquatic species rely may assist in understanding evolutionary changes primarily on their gills for Ca uptake from envi- in Ca regulation during the transition from ronmental water; in terrestrial species the diges- aquatic to terrestrial environments. In recent tive epithelium predominates as diet provides years crustaceans have emerged as an ideal external Ca. This review will discuss very recent nonmammalian model in which to study Ca ho- research that elucidates evolutionary strategies for meostasis by virtue of their natural molting cycle Ca homeostasis in crustaceans at four levels (Fig. 1): (1) whole organism strategies related to envi- that involves erosion/deposition of the CaCO3 cu- ticle (Wheatly, ’97). In premolt Ca is reabsorbed ronmental Ca; (2) extracellular regulation (hemo- from the old cuticle that is shed (ecdysis). The new lymph, shell, calcified deposits); (3) intracellular cuticle is mineralized primarily with external (en- regulation; and (4) molecular regulation. Ca homeo- vironmental) Ca2+ and with internal stores. Four stasis is of fundamental significance given the supreme biological importance of Ca in living or- crustacean epithelia are specialized for bidirec- 2+ ganisms. tional Ca exchange: (1) gills are the site of pas- sive Ca2+ diffusional loss and active uptake in 2+ aquatic species and may postrenally modify Ca Grant sponsor: NSF; Grant number: 9603723. content of voided urine in terrestrial species; (2) *Correspondence to: Michele G. Wheatly, Department of Biologi- 2+ cal Sciences, Wright State University, Dayton, OH 45435. E-mail: the digestive epithelium effects Ca uptake from [email protected] © 1999 WILEY-LISS, INC. EVOLUTION OF CRUSTACEAN CA TRANSPORT 621 mM). Aquatic strategies for Ca homeostasis have focused on adult decapods. Marine crabs maintain intermolt extracellular (EC) and urine Ca isoionic with respect to SW (Greenaway, ’85; Neufeld and Cameron, ’92); Ca is rarely limiting in marine or brackish environments. Intermolt FW crayfish hyperionically regulate EC Ca primarily through reabsorbing 95% Ca from urinary filtrate (Wheatly and Toop, ’89). In- land waters can vary in their Ca content and this, in turn, can influence whole organism acid-base and ion balance (Table 1). The minimum level for Ca homeostasis in crayfish is around 50 µM (Wheatly, de Souza and Hart, unpublished). In nature, crayfish can maintain Ca balance in Ca- poor water only if the diet is Ca rich (Hessen et al., ’91). External Ca can affect the electrical (Kirschner, ’94) as well as chemical gradient across epithelia. However it has little effect on apparent permeability of FW crustaceans (Rasmussen and Bjerregaard, ’95) since this is already minimal. Water Ca concentration can also influence physi- ological response to environmental change. For example, acid is more toxic to crayfish in high Ca water (Ellis and Morris, ’95). Fig. 1. Hierarchy for calcium homeostasis in crusta- Calcium homeostasis in larval/juvenile crusta- ceans. SR, sarcoplasmic reticulum; ER, endoplasmic ceans has not been well studied; however, due to reticulum; Ecd, ecdysone; CT, calcitonin; CGRP, calcito- nin gene-related product. their small size, early life-history stages have a larger SA: volume than adults and this will affect Ca fluxes in hyperregulating species. Early devel- ENVIRONMENTAL CALCIUM: opment of the crab Metopaulias depressus is com- EVOLUTIONARY STRATEGIES FOR pleted in small FW reservoirs of bromeliad leaf axils WHOLE ORGANISM CALCIUM that cannot supply the Ca demand for the develop- HOMEOSTASIS ing brood. Female crabs behaviorally supplement Intermolt calcium balance is a product Ca within the nursery by placing snail shells in the of the environment reservoir thus elevating the Ca concentration for Aquatic environments afford unlimited reservoirs nonfeeding stages; they also provide Ca-rich milli- of Ca be they marine (10 mM) or inland waters (<1 pede diet to young crabs (Diesel and Schuh, ’93).

TABLE 1. Effect of environmental Ca on hemolymph acid base and ion balance in freshwater crayfish

Water [Ca] Temp Hemolymph (mM) Species (µM) (°C) pH Ca Na Cl Reference

High calcium Procambarus clarkii 1100 15 8.0 9 199 196 Morgan and McMahon, ’82 Pacifastacus leniusculus 3200 15 7.9 9 200 201 Wheatly and McMahon, ’82 Intermediate calcium Procambarus clarkii 600 23 7.6 12 218 192 Wheatly et al., ’96 Cherax destructor 500 20 7.6 19 325 ND1 Ellis and Morris, ’95 Orconectes propinquus 100 10 7.9 10 193 178 Wood and Rogano, ’86 Astacus astacus 100 15 8.0 10 190 175 Jensen and Malte, ’90 Low calcium Procambarus clarkii 50 23 7.6 8.5 205 170 Wheatly, unpubl. Cherax destructor 50 20 7.6 19 161 ND Ellis and Morris, ’95 1ND, not determined. 622 M.G. WHEATLY Terrestrial strategies of Ca homeostasis have high-Ca diet had the effect of downregulation centered on land crabs (decapods) as well as am- (Bronner, ’96). In insects, Ca intake increased with phipods and isopods. Brachyuran crabs have in- dietary content and was postabsorptively regu- vaded land via SW (Ocypodidae, Gecarcinidae, lated at the Malpighian tubules (Taylor, ’85). This Grapsidae) or FW origins (Potamidae). Intermolt implicates use of more economical passive intake land crabs regulate EC Ca (15 mM) by control- mechanisms that are more conducive to rapid di- ling input via food and drink (Greenaway, ’85, ’93). etary switching. Land crabs will serve as a fu- Most land crabs are omnivores. Leaf litter pre- ture model to assess the regulation of intestinal dominates their diet but has been shown to be Ca transporters by dietary Ca intake. growth limiting in Gecarcinus lateralis (Wolcott Land crabs modify the Ca content of the excre- and Wolcott, ’84). Mineral deficiency can nutrition- tory product by postrenally reabsorbing 80% of ally modulate carnivory and cannibalism (Wolcott, the Ca in their isoionic urine after it has seeped ’88). Dietary Ca intake has recently been investi- into the branchial chamber (Wolcott and Wolcott, gated in the herbivorous land crabs Cardisoma ’82, ’85, ’91; Greenaway and Morris, ’89; Morris hirtipes (Greenaway and Raghaven, ’98) and Gecar- et al., ’91). Annual breeding migrations of land coidea natalis (Greenaway and Linton, ’95). Brown crabs Gecarcoidea natalis can present a challenge leaves contained twice the Ca content of green for Ca balance (Greenaway, ’94). Feeding is cur- leaves but assimilation coefficient for Cardisoma tailed for the seven days of the migration and con- hirtipes remained constant (20%) on either type of tinuous movement is incompatible with urinary leaf. Calcium retention was proportional to dietary reprocessing. These two factors combine to lower Ca intake. Assimilation was higher (37%) in Gecar- hemolymph Ca. Crabs rectify this upon arrival at coidea natalis; however most of the ingested Ca was the coast by “dipping” activity which restores EC lost in the feces. Table 2 compares the role of dif- Ca balance. ferent epithelia in intermolt Ca balance in an evo- Molting in different calcium environments lutionary continuum of decapods. In intermolt, crustaceans are typically in Ca balance; net fluxes In adult marine crabs, relatively little Ca (<20%) via gills or food are relatively low. is stored between molts in amorphous form in re- Optimality models propose that intestinal nu- gions of the gut. In full-strength SW, Ca enters trient transporters are inducible and regulated by postmolt Callinectes passively via the gills at rates dietary intake depending on the cost vs. benefit of 10 µmol · kg–1 · h–1 (Cameron and Wood, ’85; of absorption (Diamond, ’91). In mammals, re- Cameron, ’89; Neufeld and Cameron, ’92). In di- stricted Ca intake stimulated intestinal Ca2+ up- lute SW (2 ppm), postmolt crabs are able to cal- take (Ferraris and Diamond, ’89), whereas a cify as rapidly and accumulate as much Ca as

TABLE 2. Relative role of Ca transporting epithelia in whole organism Ca flux in intermolt crustaceans from different environments

Ca2+ flux (µmol · kg–1 · h–1)

Species Epithelium Net flux Influx Outflux Reference

Sea water Carcinus maenas Gill –161 +513 –674 Greenaway, ’76; Zanders, ’80 Antennal gland –31 +18 –48 Cancer magister Antennal gland –5 +6 –11 Wheatly, ’85 Homarus gammarus Antennal gland –43 +40 –83 Whiteley and Taylor, ’92 Fresh water Procambarus clarkii Gill –130 +12 –142 Wheatly and Gannon, ’95 Pacifastacus leniusculus Gill –40 Wheatly, ’89 Antennal gland –4 +35 –39 Wheatly and Toop, ’89 Austropotamobius pallipes Gill +3 Greenaway, ’74 Antennal gland –6 +41 –46 Tyler-Jones and Taylor, ’86 Terrestrial Birgus latro Antennal gland –1 +40 –41 Greenaway et al., ’90 Leptograpsus variegatus Gills +90 Morris and Greenaway, ’92 Cardisoma hirtipes Digestive +104 +542 –438 Greenaway and Raghaven, ’98 Antennal gland –6 +1 –7 Greenaway, ’89 Gecarcoidea natalis Digestive +58 +158 –100 Greenaway and Linton, ’95 EVOLUTION OF CRUSTACEAN CA TRANSPORT 623 crabs in SW using active uptake processes (Neu- to maintain Ca balance. Many retreat to sealed feld and Cameron, 1992). burrows (Ocypode) where access to exuvial Ca is In adult FW crayfish, Ca storage between molts assured (Greenaway, ’93). Often land crabs remain (in gastroliths) approximates that of marine spe- in their sealed burrows from three to four weeks cies (14%). Postmolt mineralization involves ac- during which time they do not feed (Birgus). They tive uptake of Ca primarily through the gills may also extract Ca from available burrow water during the initial 4–5 days postmolt (Wheatly and using branchial uptake mechanisms (e.g., Car- Ignaszewski, ’90; Wheatly and Gannon, ’95); any disoma, Wood and Boutilier, ’85; Pinder and Smits, deficit is restored by ingestion of exuviae (30%) ’93). Certain intertidal species obtain Ca from rock and other food items. A parchment shell is pro- pools, cliffs, or calcareous sand (Grapsus, Gecar- duced within hours with complete recalcification cinus). FW land crabs (Holthuisana) use the occurring over a few days. Removal of external hemolymph to increase Ca conservation to 65%

Na or HCO3 reduced Ca uptake by 55%. In Ca- (Sparkes and Greenaway, ’84). free medium, postmolt crayfish remained in Ca In terrestrial amphipods, the best-developed balance. When Ca was reintroduced into the me- model is Orchestia cavimana which resides on the dium after four days, calcification commenced, but beach (family Talitridae, Meyran et al., ’84, ’86). at reduced rates. Thus active Ca-uptake mecha- Calcium reabsorbed from the old cuticle is tem- nisms appear to be maximally active in the ini- porarily stored in the posterior caeca (PC) which tial postmolt period. However a secondary increase are diverticula of the midgut. in Na uptake upon reintroduction of external Ca, Terrestrial isopods (suborder Oniscoidea) have confirmed that Ca uptake is partially Na-depen- emerged in recent years as an interesting crusta- dent (Wheatly and Gannon, ’95). During five days cean model for Ca homeostasis by virtue of their in acidic water (pH 5.2), Ca uptake was 60% re- biphasic molting cycle which has been modeled duced, and total body Ca was 40% lower compared on the species Oniscus asellus (Steel, ’93), and with postmolt crayfish in neutral pH (Zanotto and Porcellio scaber (Ziegler, ’96). Apolysis and epicu- Wheatly, ’95). These effects could not be repro- ticle secretion occur synchronously throughout the duced in high CO2 acid water suggesting that the animal until day 10 of premolt. At this time, Ca response was due to lowering of ambient CO2 reabsorbed from the posterior cuticle is deposited rather than a direct effect of pH. In alkaline wa- in the anterior 4 sternites in the space between ter (pH 9.2) Ca uptake was 30% elevated. the old cuticle and the sternal epithelium (20% Juvenile/larval Ca balance during the molting body Ca). Additional Ca is stored in the epimeral cycle has been studied in crayfish Procambarus plates and throughout the general cuticle of the clarkii. The calcium content of whole body, exu- anterior region with total Ca storage totaling 48%. viae and gastroliths had scaling exponents of 0.93– Ecdysis of the posterior half (Ep) occurs on day 1.27 (Wheatly and Ayers, ’95). Large crayfish 16 followed by rapid remineralization using the demineralize the cuticle more effectively in pre- transposed Ca (12 hr). Following a period called molt and store less mineral compared to smaller intramolt (24 hr), anterior ecdysis (Ea) follows on crayfish. Small crayfish remineralize more rap- day 17, followed by calcification of the new ante- idly in postmolt commensurate with increased rior cuticle with Ca from the hemolymph. Syn- molting frequency. Allometric relationships (slope chrony is regained during days 17–23 postmolt. 0.85–0.98) have recently been demonstrated on The isopods preferentially reingest their exuviae postmolt day 1 for both net Ca flux and unidirec- in order to recycle additional Ca. Complete min- tional Ca influx (Zanotto et al., unpublished). eralization requires Ca from ingestion of other Marine land crabs store Ca internally in gas- food items. troliths (5–15%) although only at the same level as their aquatic counterparts; instead they rely EXTRACELLULAR CA REGULATION on reingesting Ca in the shed exuviate. Wheatly In crustaceans, the majority of internal Ca is (’97) estimated the rate of postmolt digestive Ca2+ located extracellularly either in the acellular uptake in the land crab Gecarcoidea natalis to be hemolymph or regions of the body that are calci- around 12,000 µmol · kg–1 · h–1. Ca from storage fied/decalcified associated with the molting cycle. sites and reingested exuviae is used for immedi- ate calcification of critical regions. Once feeding Intermolt hemolymph Ca regulation resumes, dietary Ca will make up the deficiency. Circulating Ca is held at a constant level in a Behavioral adaptations enable many land crabs range of decapods (around 12 mM). In intermolt 624 M.G. WHEATLY crabs diffusible Ca is in equilibrium; unidirec- unaltered (Procambarus clarkii, Ca, 0.6 mM, tional Ca fluxes are equivalent both into and out Wheatly et al., ’96; Cambarus, Ca, 0.05 mM, of the animal (Table 2) suggesting exchange dif- Hollett et al., ’86; Astacus, Ca, 0.1 mM, Jensen fusion between internal and external pools (Green- and Malte, ’90). As in fish, the hemolymph acido- away, ’76). In intermolt crayfish, active Ca influx sis appears to be more pronounced in harder wa- mechanisms are silent in intermolt and unidi- ter resulting from a loss of cations in excess of rectional efflux results in a small negative Ca anions. The Ca ion offers a protective effect balance (Table 2, Wheatly and Gannon, ’95). In- against severe ion loss in hard water arising from vestigators have used a battery of environmen- the weak ionic interactions with surface ligands tal parameters in an attempt to alter circulating on the gill which serves to stabilize the apical Ca; however, in most cases it has been found membrane (McDonald, ’83). Aside from water that EC Ca is very tightly regulated. hardness, evolutionary history is also important µ Exposure of crayfish to low-Ca water (50 M; in the EC Ca response to acidification. Species Table 1) resulted in a reduced hemolymph Ca in (such as Cambarus) evolving under softer water Procambarus clarkii (Wheatly et al., unpublished) conditions of mountain streams evolved iono- while not in Cherax destructor (Ellis and Morris, regulatory mechanisms that preadapted them to ’95). Since the crayfish does not actively take up withstanding low-pH stress more successfully Ca in intermolt (Table 2), an increased gradient for passive diffusion could increase efflux. Low ex- than species originating from central basins of ternal Ca also produced an EC acidosis, and low- great rivers (Orconectes). While sulfuric acidifica- ered both circulating Na and Cl due to increased tion raises Ca, nitric acid does not (McMahon and permeability. In the wild, crayfish acclimated to Stewart, ’89). low Ca (75 µM) continue to take up Ca through- Ellis and Morris (’95) attempted to resolve the out intermolt (Hessen et al., ’91). In high Ca wa- debate by examining external acidification of the ter, the hemolymph Ca did not rise (Table 1); Southern Hemisphere crayfish Cherax destructor however, inspection of crayfish at the conclusion during long term (21 days) exposure to H2SO4 in of the experiment revealed well-developed gastroli- either low- (50 µM) or high-Ca water (500 µM). ths suggesting that excess Ca was stored. Acidification in low-Ca water produced a meta- In the past, whenever an elevation in EC Ca bolic acidosis compensated by a respiratory alka- has occurred in response to EC acidosis there has losis and accompanied by a 3.2 mM increase in been a tendency to associate this circumstan- hemolymph Ca. However, this Ca increase was tially with exoskeletal CaCO3 mobilization for not accompanied by elevated HCO3 or reduced + purposes of buffering H (Morris et al., ’86). The carapace Ca content. Thus it was concluded that only definitive study addressing the involve- it originated, if anything, from other storage sites ment of carapace in buffering hypercapnic aci- such as muscle or hepatopancreas. In low-Ca acid dosis was performed in Callinectes (Cameron, water, hemolymph Na was depressed suggesting ’85) and showed that its role was negligible. It that Ca has a role in mediating Na+ and H+ move- was postulated that this strategy was employed ments across the apical surface of branchial cells. preferentially in air when standard branchial The low-Ca level employed (50 µM) is at or below ion exchange mechanisms for acid-base regula- the minimum Ca for normal homeostasis. Under tion are inoperative (Wheatly and Henry, ’92). these conditions, crayfish appeared to maintain A number of recent studies have continued this acid-base balance at the expense of ion balance debate using external acidification or removal of by reducing H+ permeability, stimulating H+ ex- water breathers into air as experimental stimuli. – cretion, shutting down Cl /HCO3, and/or conserv- Calcium balance during acidification in FW + crustaceans is a complex issue that appears to be ing HCO3 to buffer entering H (Wheatly and affected by water hardness, acid type, and evolu- Henry, ’92). tionary history. Several studies have reported a Along the same lines, aerial exposure of aquatic significant rise in Ca associated with severe EC decapodans has in some cases resulted in elevated acidosis upon external acidification of crayfish EC Ca (Austropotamobius, Morris et al., ’86). This (Orconectes, Ca, 0.1 mM, Wood and Rogano, ’86; is typically observed in species that are poorly Procambarus clarkii, Ca, 1 mM, Morgan and adapted/evolved for air breathing and that incur McMahon, ’82). In other studies, where the EC a significant hemolymph acidosis. Species rou- acidosis was less pronounced, hemolymph Ca was tinely exposed to air in nature encounter mini- EVOLUTION OF CRUSTACEAN CA TRANSPORT 625 mal acid-base disturbance and correspondingly allel; they increase in premolt and decrease in maintain hemolymph Ca (Procambarus, Wheatly postmolt (Meyran et al., ’93). In the isopod et al., ’96; Carcinus, de Souza, and Taylor, ’91). Oniscus, the hemolymph becomes milky and vis- So the degree of acidosis appears to be the ma- cous around ecdysis again suggestive of stor- jor factor in influencing EC Ca homeostasis, age in the hemolymph (Steel, ’93). namely that hemolymph Ca only rises under se- vere EC acidosis. Confirming this hypothesis was Calcified deposits a recent study on exposure of Carcinus to lethal In intermolt crustaceans, most of the whole body Cu levels (Boitel and Truchot, ’89). An induced meta- Ca (80%) resides in the outer acellular layers of bolic acidosis later reinforced by hypercapnia and the cuticle in the form of crystalline CaCO3. Dur- accumulation of lactic acid resulted in restricted gas ing premolt the cuticle is weakened in place by exchange. A steep increase in hemolymph Ca con- CaCO3 reabsorption. Some Ca is stored in amor- centration occurred immediately before death. How- phous form in EC mineralized structures such as ever all studies to date are in agreement that any gastroliths in crayfish and crabs, and sternal de- EC Ca appearing in the hemolymph is translocated posits in isopods. Some is stored in amorphous from soft body tissues and not the carapace (de form in IC granules and a significant amount is Souza, ’92). excreted. At ecdysis, the exuviae still contain sig- nificant quantities of Ca (30–70%); this Ca can Hemolymph calcium regulation be reingested in postmolt. Calcified deposits are during molting cycle remobilized in postmolt to provide Ca for imme- It is fairly well established that hemolymph Ca diate cuticular mineralization. The bulk of re- is well regulated during the molting cycle of quired Ca however comes from external sources aquatic species (Greenaway, ’85; Zanotto and (water/food). This area has been reviewed else- Wheatly, ’93) even when whole-body Ca fluxes are where (Greenaway, ’85; Wheatly, ’96, ’97). Calci- profound. Total Ca typically increases by 40% in fied deposits exhibit some common characteristics. premolt associated with Ca reabsorption and de- First, their Ca flux is exactly the opposite of that creases in postmolt associated with mineraliza- at the cuticle (e.g., gastroliths are deposited while tion (Wheatly and Hart, ’95). The premolt increase the cuticle is being reabsorbed and vice versa). is primarily in the bound moiety; free Ca which is This presents some interesting questions with re- more relevant biologically, and can now be measured spect to synchrony of control mechanisms. In other with ion selective electrodes, remains constant words, reabsorption and deposition of CaCO3 are (Neufeld and Cameron, ’92). Crayfish precipitated routinely exhibited simultaneously at different into ecdysis by multiple limb autotomy (MLA) ex- epithelia. Second, while the calcified deposits are hibited an earlier premolt Ca peak and those pre- deposited over the space of several weeks, they cipitated into ecdysis by eyestalk removal (ER) did are reabsorbed within a period of hours. not exhibit a premolt Ca peak suggesting that Ca The best-studied calcified deposits are the gas- balance in artificially induced molts is not equiva- troliths which are paired CaCO3 concretions lent to those molts that occur naturally (Wheatly formed by the gastrolith disc in the cardiac stom- and Hart, ’95). ach of crayfish and some crabs. They are formed beginning at 40 days before ecdysis from hyper- Some FW land crabs (such as Holthuisana) trophied mitochondria that slough off into the lu- store Ca in the form of CaCO granules in the 3 men of the stomach. At ecdysis only 20% body Ca hemolymph during ecdysis (Sparkes and Green- remains in the soft crayfish and 17% of this is away, ’84). This strategy enables storage to ap- contained in the gastroliths (Wheatly and Ayers, proach 65% with hemolymph concentration ’95). The gastroliths are solubilized within 24 hr rising by 150-fold to 2 M. These granules are and the Ca is used to preferentially harden mouth- formed in subepidermal connective tissue cells parts, gastric ossicles and dactyls of walking legs (Greenaway and Farrelly, ’91); their small size so that motility and feeding are resumed. (0.26 µm) allows them to pass through the cir- In the terrestrial isopods Oniscus and Porcellio, culatory system. The hemolymph of Ocypode amorphous CaCO3 deposits combined with an or- also appears milky immediately after ecdysis ganic matrix are stored between the epithelium and Ca concentration reaches 70–100 mM and old cuticle of the anterior sternites (Steel, ’93; (Greenaway, ’93). In the amphipod Orchestia Ziegler, ’94). After Ep, reabsorption of the Ca in cavimana, total and ionized Ca change in par- these deposits is synchronized with calcification 626 M.G. WHEATLY of the new posterior exocuticle. In the FW land lated by solubilization of CaCO3. During cuticu- crab Holthuisana Ca reabsorbed from the cuticle lar mineralization the matrix behaves like a Ca in premolt is stored as large EC noncirculating sink; however, free diffusion of Ca would allow granules in narrow intercellular channels between for continuous uncontrolled mineralization which epidermal cells (large 0.8–4 µm, CaPO4). These does not occur. granules essentially get trapped so as to avoid ob- struction of small hemolymph lacunae and arte- INTRACELLULAR CALCIUM rial branches (Greenaway and Farrelly, ’91). In REGULATION amphipods, calcareous concretions are stored in Transcellular transport in intermolt the PC lumen (Meyran et al., ’84). Transepithelial Ca flux typically involves a Paracellular calcium pathway transcellular pathway summarized in Figure 2. Since cytosolic Ca is kept low (0.1–0.5 µM), this One model for transepithelial Ca transport in- will involve passive Ca influx across the plasma volves an extracellular or paracellular pathway membrane on one side of the cell and active ex- that consists of passive movement between cells trusion across the membrane on the opposing side. and across cell junctions along an electrochemi- Most crustacean Ca transporting epithelia exhibit cal gradient. The paracellular pathway has been bidirectional Ca transport at different stages in proposed for Ca storage in the midgut PC of the the molting cycle. Membrane transporters in amphipod Orchestia (premolt, Meyran et al., ’84) intermolt crustaceans have been identified in lob- and the anterior sternal epithelium (ASE) of ster hepatopancreas and antennal gland, crab Porcellio (during resorption of deposits in intra- gills, and in all three crayfish tissues using vesicle molt, Ziegler, ’96). In Orchestia, Ca enters the approaches. Entry into the cell across the outer basal surface of the caecal epithelium cells from apical membrane may be passive or via a Ca2+/ the hemolymph and is pumped into narrow IC Na+ (H+) antiporter. However active extrusion from channels to enter the lumen of the caeca predomi- the cell across the inner basolateral membrane is nantly via a paracellular route, although some via Ca2+ ATPase or indirectly via Ca2+/Na+ ex- may be pumped across the apical membrane di- changer. These active extrusion mechanisms can be rectly into the lumen. After exuviation of amphi- used either to fine tune IC levels (housekeeping) or pods, the Ca stored in the PC lumen concretions to accomplish Ca2+ translocation. This pathway re- is transported from the caecal lumen to the quires the presence of intercellular junctions that hemolymph within 24–48 hr and used to miner- limit paracellular movement. A major constraint on alize the new cuticle (Graf and Meyran, ’83, ’85). this mechanism is that Ca must be transported The PC epithelium has a network of EC channels through the cell without increasing IC Ca concen- like that of the ASE of Porcellio. During postmolt tration above the submicromolar range (see below). resorption of the calcareous concretions, spheri- Transmembrane Ca transfer will be governed by cal granules appear in the EC network. These both the electrical and chemical gradients. In cray- granules are secreted by epithelial cells and dis- fish transepithelial potential (TEP) is very sensi- integrate into smaller particles (still EC) and ul- tive to external Ca (Kirschner, ’94). At 0.034 mM timately into ionized Ca that enter hemolymph. Ca TEP was –18 mV; as external Ca increased, TEP There is ultrastructural similarity between the became more positive. The TEP is attributed to the ASE of Porcellio and the midgut epithelium of PC diffusive efflux of NaCl across the gills, with a Cl/ of Orchestia. Na perm ratio of 1. The Ca dependence of the TEP There is less support for involvement of the originates from the fact that active inward Ca paracellular route in reabsorption/mineralization transport is electrogenic. of the cuticle. In the premolt FW land crab Holthuisana, Greenaway and Farrelly (’91) do not Apical mechanisms support the paracellular route for Ca movement from molting fluid to hemolymph. Calcium would Apical transport at the lobster, Homarus, an- have to diffuse through the thickness of the tennal gland (Ahearn and Franco, ’93), and procuticle to reach the apical junctions and it is hepatopancreas (Zhuang and Ahearn, ’96) involves unlikely that such a concentration gradient ex- three independent processes, two of which involve ists since both solutions are saturated with re- carrier-mediated facilitated diffusion through 2+ + spect to CaCO3. Additionally the apical junctions antiporters that can exchange Ca for either Na are “tight.” Onset of reabsorption may be regu- or H+: (1) amiloride sensitive electrogenic 2Ca2+/ EVOLUTION OF CRUSTACEAN CA TRANSPORT 627 H+ (H+ preferred to Na+) antiport that is unique (Homarus gammarus), a weak hyperregulator to crustaceans—this transporter exhibits 2 exter- (Table 3). Affinity of both transporters was un- nal cation binding sites with markedly dissimilar changed upon dilution. However, the transport ca- apparent binding affinities and a single IC bind- pacity of both transporters in epipodites and –1 ing site (K0.5, 0.05 mM; Vmax, 7 nmol · mg · 2.5 branchiostegites increased significantly while cor- sec–1); (2) amiloride insensitive electroneutral 1Ca2+/ responding levels in the gills decreased. A related + + + 2Na antiport (Na preferred to H , K0.5, 0.23 mM; study (Haond et al., ’98) using confocal laser scan- –1 –1 Vmax, 2.27 nmol · mg · 2.5 sec ); (3) simple diffu- ning microscopy revealed that the epipodites sion through a verapamil-sensitive Ca channel that (lamellar organs inserted between the gills) and is dependent upon membrane potential. inner side of the branchiostegites are covered by a well-differentiated osmoregulatory epithelium Basolateral mechanisms that becomes increasingly differentiated during Basolateral membrane vesicles (BLMV) have external dilution. Basolateral infoldings develop, been employed to characterize basolateral Ca apical microvilli increase in height, and vesicles transport in a range of intermolt crustaceans proliferate. Meanwhile the trichobranchiate gills (Table 3). BLMV from gills of intermolt crab, remain poorly differentiated and their ultrastruc- Carcinus, acclimated to 50% SW (Flik et al., ’94) ture is unchanged upon dilution. The epipodites exhibited ATP-dependent uptake (Ca2+ ATPase and branchiostegites serve as ancillary ion trans- –1 –1 with K0.5, 149 nM; Vmax, 1.73 nmol · mg · min ) port epithelia to compensate for ion loss through and Na gradient-dependent uptake (Na+/Ca2+ with the permeable cuticle and the limited ionoregu- –1 –1 K0.5, 1780 nmol; Vmax, 9.88 nmol · mg · min ). latory capacity of the gills. Affinity of both mechanisms matched physiologi- My lab has recently characterized ATP-depen- cal IC Ca. At cytosolic Ca concentrations up to dent Ca2+ uptake into osmotically reactive inside- 500 nM, the Ca2+ ATPase is the primary mecha- out resealed BLMV (Wheatly et al., ’99) from nism of efflux; however, above 500 nM the ex- hepatopancreas, gill, and antennal gland of inter- changer predominates. The same two independent molt FW crayfish (Table 3). ATP-dependent 45Ca2+ processes of energized Ca transport were demon- uptake (Wheatly et al., ’99) was abolished by pre- strated in BLMV of lobster hepatopancreas (Zhuang treatment with either vanadate or the ionophore and Ahearn, ’98) namely a vanadate-sensitive high- A23187. Calcium affinity was equivalent in hepato- affinity but low-capacity calmodulin-dependent pancreas and gill (K0.5, 0.28 µM) but was higher 2+ plasma membrane Ca ATPase (PMCA; K0.5, 65 in antennal gland (0.11 µM). Maximal uptake was –1 –1 –1 –1 nM; Vmax, 1.07 pmol · µg · 8 sec ) and a low-af- 20.26 pmol · mg pr · min in hepatopancreas finity high-capacity Ca2+/3Na+ (or Li+) antiporter and was 5–6× higher in gill and antennal gland. + + (K0.5, 14.6 µM). When Li replaced Na in exchange An ATP titration curve indicated a K0.5 of 0.01 mM for Ca2+, the apparent affinity for Ca influx was in hepatopancreas and gill and 0.039 mM in an- not significantly affected (K0.5 for Na 14 µM, Kt tennal gland. EGTA treatment of hepatopancreas 2+ for Li 20 µM) but the Vmax was reduced by factor and antennal gland vesicles decreased Ca up- + –1 –1 of 3 (Vmax for Na , 2.72 pmol · µg · 8 sec ; Vmax take by 50%; uptake was restorable by calmodulin. for Li+, 1.03 pmol · µg–1 · 8 sec–1). The Ca2+ AT- However, in gill Ca uptake was unaffected by Pase kinetics approximated to those reported by EGTA treatment and calmodulin decreased up-

Flik et al. (’94) in crab gill. At IC Ca activities take. Q10 ranged from 1.43 to 2.06. Addition of Na that might occur in a typical cell (100–500 nM) (5 mM) caused a 60% increase in Ca uptake that greater than 90% of Ca efflux takes place via the was inhibitable by preincubation with ouabain in- Ca ATPase. At IC Ca of 1,000–10,000 nM, the dicating that the Na pump generates a Na+ gra- Ca2+/3Na+ assumes a greater role in Ca efflux dient favorable for Ca accumulation via the Na+/ from the cell. It has been postulated that such Ca2+ exchanger. Capacity for 45Ca2+ uptake was an concentrations may be attained during elevated order of magnitude greater in gill than in either transcellular Ca flux associated with storage and hepatopancreas or antennal gland. In vitro bran- remobilization of Ca deposits in hepatopancreas. chial 45Ca2+ uptake capacity was 6× greater than in Flik and Haond (unpublished) have compared vivo unidirectional influx; however, in antennal kinetics of both Ca2+ ATPase and Na+/Ca2+ ex- gland Ca pumps operate at capacity. Sodium gradi- changer in full-strength SW and after 2-week ac- ent-dependent uptake into vesicles is being exam- climation to 70% SW in branchial tissues (gills, ined using standard techniques and flow cytometry. epipodites, and branchiostegites) of the lobster Comparison of kinetics across the evolutionary 628 M.G. WHEATLY ) Reference –1 1 · mg –1 max V exchanger + /Na 2+ 0.5 M) (nmol · min µ K 14.60 339Ahearn, ’98 Zhuang and )( –1 · mg 2 –1 max V ATPase Ca 2+ Ca 0.5 M) (nmol · min µ C( ° Temp K GillsEpipoditesBranchiostegites 37GillsGills 37 0.21Epipodites 37Branchiostegites 0.24 0.15 37GillsHepatopancreas 37 37 0.23 8Antennal gland 37 37 0.21 3 0.15 20 37 0.15 0.27 4.34 0.11 13 37 4.26 8 2.74 0.28 8 0.14 6 332 0.58 3.95 256 248 4.02 0.63 1.78 ND 3.24 Flik and Haond, unpubl. 391 ND Flik and Haond, unpubl. 310 Flik and Haond, unpubl. 45 ND ND 130 ND Flik and Haond, unpubl. Flik and Haond, unpubl. ND Flik et al., ’94 Wheatly et al., ’99 Flik and Haond, unpubl. Wheatly et al., ’99 Wheatly et al., ’99 Hepatopancreas 20 0.07 133 TABLE 3.TABLE Kinetics of Ca pump and exchanger in intermolt crustacean basolateral membrane vesicles is corrected for membrane configuration. Homarus gammarus Carcinus maenas Homarus gammarus Procambarus clarkii Homarus americanus max ND, not determined. V Species Tissue 1 2 Dilute SW (60%) Fresh water Sea water EVOLUTION OF CRUSTACEAN CA TRANSPORT 629 continuum (Table 3) reveals that affinities of both lateral (postmolt branchial uptake, gastrolith Ca pump and exchanger (except for lobster hepato- remobilization, premolt cuticular reabsorption, di- pancreas) were comparable in all species. How- gestive absorption) requires that Ca enters the ever Vmax decreased with evolution into fresh apical membrane via channels and exchangers water, probably associated with reduced perme- outlined above. Inside the cell, Ca is released from ability and passive fluxes. IC stores. Transport across the basolateral mem- In intermolt aquatic crustaceans, active Ca in- brane is via Ca2+ ATPase and Ca2+/3Na+. flux at the gills is minimal (Table 2). Ca influx If Ca2+ ATPase is functional during bidirectional across isolated perfused gills of intermolt shore transcellular Ca transport then one would expect crab (Pedersen and Bjerregaard, ’95) occurred its activity to switch location between basolateral mainly by passive mechanisms. The electrochemi- and apical at different stages of the molting cycle. cal gradient is directed into marine crabs at 30 For example, in hypodermis one would expect it and 20 ppt and out of the animal below 6 ppt to be located basally during premolt and apically (Neufeld and Cameron, ’92, ’93). Similarly, in FW during postmolt. In Callinectes hypodermal quer- crayfish there is virtually negligible active Ca in- cetin-sensitive Ca2+ ATPase (Greenaway et al., ’95) flux at the gills in intermolt; however, production was localized along basolateral membranes below of a dilute urine necessitates continuous reabsorp- the apical junction in premolt (stage D2). By tion of Ca from urinary filtrate at the nephridial postmolt stage, A2 Ca2+ ATPase was localized canal cells of the antennal gland. along apical membranes and microvilli. At B2, In land crabs, Ca2+ ATPase activity has been similar activity was seen and the apical cytoplasm reported in whole gill homogenates associated contained small vacuoles with activity. Minimal with Ca reabsorption from voided urine (Birgus activity was observed in intermolt. Similarly, dur- latro, Morris et al., ’91; Leptograpsus variegatus, ing basal to apical transport Ca2+ ATPase activity Morris and Greenaway, ’92). In the terrestrial was evident in apical microvilli of the storage epi- anomuran Birgus latro, the specific activity (10 thelium in Orchestia (Meyran and Graf, ’86). Thus –1 –1 nmol · min · mg pr ) and affinity (K0.5 < 9 µM; the Ca pump can be located on either membrane Morris et al., ’91) were typical of those reported depending upon the directionality of transport. in aquatic species (Table 3). In the supratidal The Na+/Ca2+ exchangers derive their energy from brachyuran Leptograpsus variegatus the affinity a Na+ gradient established by the Na+ pump. The of Ca2+ ATPase was 6–34 nM and specific activity Na+ pump was localized in basolateral membrane –1 –1 was 8 nmol · min · mg pr (Morris and Green- of the ASE and PSE of the isopod Porcellio scaber away, ’92). Digestive epithelia would be continu- (Ziegler, ’97a). It remained localized exclusively ously involved in uptake of Ca from food in in basolateral membranes irrespective of direction- intermolt, the primary time for feeding. ality of the Ca transfer and was present during Bidirectional transcellular transport formation and resorption of CaCO3 deposits. The during molting cycle ASE exhibited greater Na pump activity than the PSE. Activity was maximal during premolt and The models for apical to basolateral Ca move- intramolt when basolateral membrane area in- ment or vice versa (basolateral to apical) have creased. been explored in Wheatly (’97) for both mineral- Typically, Ca2+ ATPase levels are elevated at izing (hypodermis, gastrolith disc) and non- 2+ mineralizing epithelia (gill, antennal gland, and mineralizing sites. Ca ATPase activity was el- hepatopancreas). Basolateral to apical transport evated three- to fourfold in cuticular hypodermis (postmolt cuticular mineralization or premolt gas- during postmolt of both blue crab (Cameron, ’89) trolith formation) requires that Ca flows from and crayfish (Wheatly, ’97). One caveat is that the hemolymph into cells through channels in the data from Callinectes may represent nonspecific basolateral membrane (Fig. 2). Ca is sequestered alkaline phosphatase (Flik et al., ’94). In a cy- 2+ in mitochondria/organelles and the rest is trans- tochemical study, Ca ATPase increased in cray- ported out of the apical membrane by the amil- fish gastrolith disc both in premolt calcification oride-insensitive carrier system or Ca ATPase for and postmolt reabsorption (Ueno and Mizuhira, delivery to the mineralizing matrix of cuticle/gas- ’84). Specific activity of Ca2+ ATPase did not alter troliths. Transcellular Ca2+ transport was proposed during the molting cycle at the gill in either study for Carcinus (Roer, ’80) and Callinectes (Cameron, or in the supratidal crab Leptograpsus (Morris and ’89) during cuticle calcification. Apical to baso- Greenaway, ’92). 630 M.G. WHEATLY

Fig. 2. Model of transcellular calcium pathway in crusta- Ca2+ ATPase; SR, sarcoplasmic reticulum; ER, endoplasmic ceans. CaBP, calcium binding protein; PMCA, plasma mem- reticulum. brane Ca2+ ATPase; SERCA, sarco-/endoplasmic reticulum

The postmolt cuticular hypodermis transfers Ca plasma membrane of the ASE increases in area into the outer layers via cytoplasmic extensions due to an elaborate interstitial network (IN) of (5 × 106 · mm–2) that are contained in pore canals interstitial dilations and channels. Also, mitochon- (Compère and Goffinet, ’87). An ultrastructural drial volume increases. Such ultrastructural study of the cuticle of Carcinus using K-pyroanti- changes are typically associated with elevated monate and X-ray microanalysis (Compère et al., transepithelial ion transport. After Ep, the direc- ’93) demonstrated that plasma membranes of the tion of transepithelial Ca transport across the ASE cytoplasmic extensions are densely lined by par- has to be reversed and the rate of transport has ticles of antimony precipitate indicating location to increase dramatically in order to degrade the 2+ + 2+ of transporting sites for Ca ATPase and Na /Ca CaCO3 deposits within the short intramolt period exchanger. Gills are covered in a noncalcified cu- (<24 hr). During resorption of CaCO3 deposits nu- ticle that must be shed at ecdysis. The ultrastruc- merous Ca containing osmiophilic granules are ture of the gill filament epithelium of the crayfish formed on the basolateral membrane. These de- Procambarus clarkii was examined throughout tach, move basally and disintegrate into small os- the molting cycle (Andrews and Dillaman, ’93). miophilic particles when they cross the basal Compared to the mineralizing cuticle, preexu- lamina. Their function might be to limit the Ca vial cuticle deposition is greatly delayed in the activity within the IN during resorption of the

gill filaments occurring only during the last 10% CaCO3 deposits in order to maintain a specific Ca of premolt so that increased diffusion distance gradient across the plasma membranes of the epi- is minimized. Also postexuvial cuticle is not de- thelial cells. During resorption of the CaCO de- posited at the gills, again leaving ion uptake posits, the apical plasma membrane of the ASE uninterrupted. is increased by many subcuticular folds. The PSE The anterior sternal epithelium (ASE) and pos- does not contain IN, osmiophilic granules, or api- terior sternal epithelium (PSE) of the terrestrial cal subcuticular folds. Cellular extensions of the isopod Porcellio (Ziegler, ’96, ’97b) show structural epithelial cells into the cuticle occur in both the differentiations associated with secretion and re- ASE and PSE but are more prominent in the

sorption of CaCO3. During formation of CaCO3 de- former and may play a role in transport of ions posits (late premolt and intramolt) the basolateral involved in cuticle calcification. Although the ASE EVOLUTION OF CRUSTACEAN CA TRANSPORT 631 of Porcellio is ectodermal, as is gastrolith disc, the and fatty acyl-chain composition may alter SERCA ultrastructure is actually more similar to the am- activity in different muscle types. A comparison phipod midgut PC which is of endodermal origin. with PMCA kinetics (Tables 3 and 4) reveals that SERCA activity is almost three orders of magni- Intracellular sequestration tude greater than PMCA. This difference reflects 2+ Intracellular (IC) Ca mediates signal transduc- relative abundance of pumps and has been con- tion mechanisms that modulate many physiologi- firmed by molecular studies (see below). cal functions in the cell. Therefore eukaryotic cells Using histochemical techniques we recently use active and passive mechanisms to maintain demonstrated that intramitochondrial electron- 2+ free Ca within a very narrow range (100 nM). dense Ca precipitates increased significantly in Regulation of cytosolic Ca is especially challenged crayfish nephridial-canal cells in postmolt corre- during periods of elevated transcellular Ca flux sponding with elevated transepithelial Ca flux associated with the molting cycle. Sequestration (Rogers and Wheatly, ’97). Ca reabsorbed from the mechanisms include attachment to Ca-binding FW land crab Holthuisana cuticle in premolt may proteins (calsequestrin in SR, calmodulin, cal- be stored as IC granules (0.4–2 µm, CaCO3) in reticulin), formation of CaPO4/CaSO4 granules, or epidermal cells (Greenaway and Farrelly, ’91). sequestration among many IC organelles including Membrane-bound vesicles are formed in the api- ER/SR, mitochondria, nucleus, Golgi, endosomes, or cal cytoplasm by the Golgi. Lamellae appear and lysosomes using ATP-coupled pumps. Existence of CaCO3 is deposited in the organic matrix. Gran- a Ca gradient through the cell cytosol may drive ule masses move basally and may be stored in transport through the cell. the connective tissue. The hepatopancreas can The sarco-/endoplasmic reticulum is a well-de- play a role in Ca sequestration since amorphous veloped membranous structure which controls cy- CaPO4 granules (1–5 µm) are found within the tosolic free Ca through the release of Ca from absorptive epithelial R cells in inter- and premolt channels and Ca resequestration via an ATP-de- of Callinectes and Cancer (Becker et al., ’74). These pendent Ca-uptake pump—Sarco-/Endoplasmic are then released into the hemolymph for incor- 2+ Reticulum Ca ATPase (SERCA)—that is regulated poration into the new exoskeleton during postmolt. by phospholamban (Lytton and MacLennan, ’92). Trace metals other than Ca, such as Zn and Cd, SERCA has been purified from skeletal muscle of can be incorporated into these granules, making the FW land crab Potamon (Tentes et al., ’92). The the crab hepatopancreas an environmental moni- enzyme, which has a molecular mass of 120 kDa, tor for toxicological studies (Bjerregaard and has a lower affinity for Ca than crustacean PMCA Depledge, ’94; Taylor and Simkiss, ’94; Simmons (Tables 3 and 4). It has two K0.5 for ATP (40, 330 et al., ’96; Zhuang and Ahearn, ’96). The Ca con- mM at ATP concentrations of 10–75 and >75 µM, tent of muscle increases in premolt in the white respectively). The pH optimum is 7.5 and the en- prawn (Vijayan and Diwan, 96). In postmolt this zyme is highly temperature dependent (17–40°C). Ca is remobilized for mineralization. In a separate study, SERCA activity was charac- terized in vesicles from SR of both fast (deep exten- MOLECULAR CA REGULATION sor abdominal muscle medialis) and slow (flexor Molecular work on crustacean Ca pumps has fol- muscle of carpopodite in meropodite) crayfish stri- lowed the same chronology as in vertebrates where ated muscle (Ushio and Watabe, ’93). Uptake rate SERCA pumps were initially cloned in the mid-’80s was higher in fast muscle than in slow muscle pos- from muscle, a tissue in which they are highly ex- sibly due to existence of different isoforms or differ- pressed. Within a few years this led to the cloning ential modulation via proteins such as calmodulin and sequencing of PMCA. At present the only crus- and phospholamban. Finally, different phospholipid tacean Ca transporting proteins cloned are SERCA

TABLE 4. Characterization of sarco-endoplasmic reticulum Ca2+ ATPase in crustacean muscle

Temp Vmax K0.5 Species Tissue (°C) (µmol · min–1 · mg–1)(µM) Reference Procambarus clarkii Slow muscle 20 0.270 ND1 Ushio and Watabe, ’93 Fast muscle 20 0.390 ND Potamon potamios Slow muscle 37 0.450 2 Tentes et al., ’92 1ND, not determined. 632 M.G. WHEATLY pumps in Artemia and Procambarus. My lab has may contain an extra membrane spanning stretch recently obtained a partial cDNA fragment from that would put the protein’s carboxy terminus in crayfish PMCA. the lumen of the ER. This additional tail may Mammalian SERCA (MacLennan et al., ’85) modulate the intrinsic activity of the pump and pumps Ca against its electrochemical gradient us- interact with phospholamban, a well-known regu- ing the energy from hydrolysis of ATP, mediated lator of SERCA2 pumps. Mammalian SERCA genes through a covalent attachment of the γ phosphate are differentially expressed during development to the polypeptide chain and subsequent confor- (Brandl et al., ’87) and also in response to treat- mational transitions. SERCA belongs to the su- ments that alter Ca2+ pumping activity such as: perfamily of P-type ATPases which includes Na+/ changing levels of thyroid hormone (Rohrer and K+ ATPase, H+/K+ ATPase, yeast and plant H+ AT- Dillmann, ’88), chronic muscle stimulation (Briggs Pase, and bacterial K+ ATPase. A molecular evo- et al., ’90; Hu et al., ’95), or cardiac hypertrophy lution study of the entire family was published (Nagai et al., ’89). The crustacean molting cycle recently (Axelson and Palmgren, ’98). The protein would appear to be an ideal model for correlating has 1,000 amino acids forming 10 transmembrane differential expression of SERCA/PMCA with trans- domains each in a helical conformation, 4 in the epithelial Ca2+ flux.

NH2-terminal and 6 in the COOH terminal. The When my lab set out to clone SERCA pumps in first five transmembrane helices extend into the crayfish, the only existing crustacean sequences cytoplasm to form a pentahelical stalk domain. A were from muscle of brine shrimp, Artemia (Pal- cytoplasmic loop between stalk sectors 2 and 3 mero and Sastre, ’89; Escalante and Sastre, ’93, folds into a domain composed of entirely β strands, ’94). Two mRNAs (4.5 and 5.2 kb) are produced whereas the large polypeptide loop between stalk from a single gene via alternative splicing; both sectors 4 and 5 folds into two separate but com- are present in cryptobiotic embryos. Expression municating domains that bind ATP (nucleotide of the 5.2 kb decreases after naupliar hatching binding site) and accept the γ phosphate (phos- while the 4.5-kb isoform increases during embry- phorylation site). The domains involved in the en- onic development. In larval tissue the 4.5-kb ergy transduction process are highly conserved isoform is expressed in muscle fibers of append- among P-type ATPases. ages (predominantly fast-twitch). The 5.2-kb Mammalian SERCA is encoded by a family of isoform is expressed in a variety of larval tissues three homologous and alternatively spliced genes (Escalante and Sastre, ’96). The amino-acid se- encoding five isoforms (Wu et al., ’95) which have quence shares 71% homology with mammalian a distinct pattern of tissue-specific expression. SERCA2a and -b especially in the region of the SERCA1 gene codes for two isoforms: SERCA1a functional domains and putative Ca-binding site. in adult fast-twitch muscle and SERCA1b in neo- The two isoforms differ only at the C-terminal natal fast-twitch muscle. SERCA2a (shorter end of the protein (Escalante and Sastre, ’93). The isoform) is found in slow-twitch skeletal and car- last 6 amino acids of the 4.5 kb isoform are re- diac muscle (MacLennan et al., ’85; Brandl et al., placed by 30 hydrophobic amino acids in the 5.2 ’87) and SERCA2b (longer isoform) is ubiquitously kb isoform that have the potential of forming an expressed in all cell types (Gunteski-Hamblin et extra transmembrane domain. Both isoforms al., ’88; Lytton and MacLennan, ’88). SERCA3 has arise from the same gene by alternative splicing only one isoform that is expressed in nonmuscle of the last two exons. tissue. The genes for SERCA1 and SERCA2 are The Artemia SERCA gene is transcribed from 84% homologous in rabbit (MacLennan et al., ’85). two alternative promoters (Escalante and Sastre, The SERCA3 amino-acid sequence is 76% identi- ’95). These two mRNAs also differ at the initial cal with SERCA1 and SERCA2 (Burk et al., ’89). part of their 5′ untranslated region. The 5.2-kb The SERCA2 gene is closest to the invertebrate mRNA-specific 5′ untranslated region is present gene (see below). Four distinct mRNAs have been as an independent exon whose transcription is regu- revealed in a variety of tissues derived from the lated by a promoter different from the one previ- same gene transcript by alternative transcript pro- ously described that regulates the expression of the cessing (Wuytack et al., ’92). The four mRNAs are 4.5-kb mRNA. The nucleotide sequence of the 5.2 translated into only two distinct proteins that dif- kb-mRNA promoter and the transcription initiation fer only in the C terminal: a short tail of four site have been determined. Results suggest that the amino acids in SERCA2a and a more extended expression of the two protein isoforms is regulated tail of fifty residues for SERCA2b. This extension in Artemia at the transcription-initiation step in con- EVOLUTION OF CRUSTACEAN CA TRANSPORT 633 trast to the vertebrate SERCA genes 1 and 2 which on processing of the last exons of each gene which have unique promoters for transcription of the two have been shown to be tissue-specific (Gunteski- isoforms encoded by each gene. The donor-splic- Hamblin et al., ’88). In Artemia there are two dif- ing site of the penultimate exon can either be ferent tissue-specific mechanisms that regulate recognized and fused to the last exon, giving the expression of each isoform. One is the exist- rise to the mRNA coding for the shorter pro- ence of two different promoters that independently tein, or remain unrecognized, in which case a regulate the expression of each isoform. The sec- polyadenylation site is recognized before the ond mechanism would be the differential process- last exon of the gene and the mRNA coding for ing of the last two exons of the gene which is also the longer protein is originated (Escalante and tissue-specific. This second mechanism has been Sastre, ’93). conserved between Artemia and vertebrates. It is Genomic clones coding for Artemia SERCA have interesting to note that the existence of alterna- been isolated (Escalante and Sastre, ’94). Nucle- tive promoters in Artemia is possible because of otide sequence of the mRNA coding regions show the existence of one intron in the 5′ untranslated that the gene is divided into 18 exons separated region that is not present in the vertebrate genes. by 17 introns. Compared with the rabbit SERCA1 This intron might have been generated in the evo- gene, 12 of the introns are in the same position, 8 lutionary branch of the crustaceans after the di- introns in the rabbit gene are absent from Ar- vergence of protostoma from deuterostoma which temia, 4 introns present in Artemia are not found would have allowed for the development of the in rabbit and the position of 1 intron is shifted internal alternative first exon with a new pro- one base between both genes. Primer extension moter. A second possibility could be the existence and nuclease S1 protection experiments have of the first intron and the alternative promoter shown the existence of two main regions of tran- in the ancestral gene and their loss during verte- scription initiation separated by 30 nucleotides. brate evolution. The study of SERCA genes in Transcription is initiated at both regions at two crayfish will elucidate whether the existence of or three consecutive bases. A hexanucleotide that alternative promoters is specific to Artemia or a includes the initiation sites is repeated in both general feature of crustaceans. transcription-initiation regions. The nucleotide My laboratory has recently cloned and se- sequence of the promoter region shows the exist- quenced SERCA from crayfish muscle (Wheatly, ence of several putative regulatory sites includ- 1997; Zhang et al., unpublished) and heart (Chen ing some that are muscle specific such as one et al., unpublished), and we also have partial CArG box, three MEF-2, and eight putative bind- cDNA clones for SERCA in gill and antennal ing sites for muscle transcription factors of the gland. In muscle the complete 3,864-bp sequence MyoD family. includes a 145-nt noncoding sequence at the 5’ There appear to be similarities between the ver- end, an open reading frame of 3,006 nt (coding tebrate SERCA2 and the Artemia SERCA gene. for 1,002 amino acids, molecular mass of 110 kd), Both genes can be transcribed and processed to and a 705-nt untranslated 3′ region. The complete give two different mRNAs by identical mecha- heart sequence consists of 4,495 bp with a 3,060- nisms: the use of two alternative polyadenylation bp open reading frame, coding for 1,020 amino sites that involve an additional splicing event if acids. The nucleotide sequence shares 73% iden- the more distal polyadenylation site is used. In tity with Artemia and 71% with rabbit muscle. both cases the proteins encoded by the different This gene differs from the crayfish muscle SERCA mRNAs are identical except for the carboxy ter- solely in its C-terminal amino acids where the fi- minal amino acids: the shorter form codes for 6 nal 9 amino acids are replaced by 27 hydrophobic or 4 amino acids and the longer isoform codes for amino acids that potentially form an additional 30–40 amino acids. In vertebrates the shorter transmembrane domain. The deduced amino acid isoform is expressed in slow-twitch and cardiac sequence matched with more than 30 SERCAs muscle whereas the longer isoform is expressed from invertebrates and vertebrates, exhibiting in smooth muscle and nonmuscle cells. Despite 83% identity with Drosophila, 70% identity with these similarities the tissue specific expression of Artemia, and 76% identity with rabbit fast-twitch the two isoforms is regulated through different muscle. Northern analysis of tissue SERCA dis- mechanisms. In vertebrates the two isoforms are tribution revealed five isoforms (4.5, 5.8, 7.6, 8.8, transcribed from the same promoter and the gen- and 10.1 kb), some of which are tissue-specific. eration of one or the other isoform is dependent Southern analysis indicated that a single gene 634 M.G. WHEATLY codes for the five isoforms. Muscle SERCA expres- and have been able to generate muscle-specific and sion was highest in intermolt and decreased in pre/ non-muscle-specific isoforms though similar process- postmolt. Whole organism/epithelial approaches ing events. have indicated that PMCA is inactive during the Our long term objective is to characterize cray- intermolt (above). At this time SERCA would be fish PMCA. PMCA and SERCA share structural the primary mechanism available for IC Ca regu- features, the number and location of the putative lation. We expect PMCA expression to be elevated transmembrane domains, and the distribution of in postmolt when basolateral Ca efflux is elevated; the predicted secondary motifs of β sheets and the correspondingly, SERCA’s role in Ca regulation may α helices are similar; both families are inhibited diminish. SERCA expression was inversely corre- by orthovanadate and La3+. In mammals PMCA lated with PMCA expression in mammalian cells is encoded by four different genes (Strehler, ’91). (Kuo, personal communication). Work continues in Despite these similarities, there are a number of our lab to isolate genomic clones of crayfish SERCA differences between PMCA and SERCA apart from in order to understand gene regulation. their subcellular location and associated role in The relationship between these two crayfish cell Ca regulation. PMCAs have a molecular mass isoforms is similar to that observed for the two of 134 kD while the SERCAs have a molecular SERCA isoforms in Artemia (Escalante and Sastre, mass of 110 kD (Carafoli, ’94). PMCAs are regu- ’93) and mammalian SERCA2 isoforms (Lytton and lated by calmodulin, while SERCAs are regulated MacLennan, ’88). The C-terminal extensions share by phospholamban. their hydrophobic character but have no significant Two genes for PMCA in rat brain have been amino-acid homology. As mentioned earlier the ex- sequenced (Shull and Greeb, ’88); they encode tension has the potential of forming another trans- two different isoforms with 82% homology. A membrane region; in mammals the C terminus of partial sequence has recently been cloned from SERCA2a is in the cytosol whereas the C terminus PMCA in crayfish heart and muscle (Fig. 3, of SERCA2b is in the ER lumen. The conservation Zhang et al., unpublished). The crayfish nucle- of amino-acid sequence and the same alternative otide and amino-acid sequences share 65% and splicing mechanism (Shull and Greeb, ’88) between 68% identity respectively with rat PMCA. When species so distant in evolution (300 MY) suggests this partial PMCA sequence was used to probe that the two isoforms play an important physiologi- a Northern blot of mRNAs from crayfish heart, cal role. Comparison of the genomic sequences in hepatopancreas, gill, egg, muscle, and anten- Artemia and mammals (Escalante and Sastre, ’94) nal gland, it hybridized with a 7-kb band in all indicate that over 50% of the intron positions are tissues, with a prominent band in egg and an- conserved during the evolution of this gene. One tennal gland. There is an additional 5-kb band interpretation of this phenomenon is that all the in egg indicating a possible isoform or precur- introns were present in the ancestral gene and are sor. Work continues to completely sequence this being lost during evolution. The ancestral gene pump. Our long-term goal is to investigate should have had at least 26 introns, 8 were lost whether the expression of Ca pumps in cray- during evolution of Artemia, and 4 were lost in the fish is hormonally responsive. evolution of SERCA1 gene. An alternative expla- nation is that introns are added to the genes dur- Hormonal regulation: ing evolution. Sequence analysis reveals the same evolutionary strategies degree of homology of the crustacean SERCA gene In arthropods, ecdysis is coordinated by the ste- with all three vertebrate genes. Researchers have roid hormone ecdysone (Hopkins, ’92; Lachaise et hypothesized the existence of a unique (pleisomor- al., ’93). In decapods there is a transitory ecdys- phic) ancestral gene in a common protostome and one peak in the hemolymph about three weeks deuterostome ancestor from which the unique ar- before ecdysis that initiates entry into preecdysis thropod gene and three vertebrate genes were then (Ca reabsorption, gastrolith formation). This is fol- derived (apomorphs). The same alternative splic- lowed days before ecdysis by a second peak of ing was preserved in the invertebrate gene and the greater magnitude. In the isopod Oniscus asellus, vertebrate SERCA 2 gene while it was lost in the an ecdysone peak in premolt is followed by a sec- evolution of the other two vertebrate genes. The ond smaller peak 1 hr after Ep (Johnen et al., similarity between Artemia, Procambarus, and ’95). In the amphipod Orchestia, an ecdysone peak SERCA2 suggests that this common ancestral gene stimulates preexuvial Ca storage in the PC (Graf might have possessed the same genetic structure and Delbecque, ’87). The ecdysone peak reported EVOLUTION OF CRUSTACEAN CA TRANSPORT 635

Fig. 3. Partial nucleotide sequence and deduced amino deduced amino acid sequence with rat PMCA is illustrated acid sequence of the muscle plasma membrane Ca2+ ATPase in the third row. Amino acids that are identical are indicated (PMCA) of crayfish Procambarus clarkii. Comparison of the by a dot. in crayfish in induced molts (ER or MLA) was toplasmic receptors into the nucleus as is known larger and more protracted than observed in natu- for glucocorticoid receptors. ral molts (Wheatly and Hart, ’95). Several labs In vitro EC Ca can block MIH suppression of are attempting to establish molecular links be- ecdysteroidogenesis in crustaceans (Watson et al., tween ecdysone and expression of genes that code ’89) and increased free-IC Ca can elevate ecdy- for proteins involved in Ca homeostasis. steroid production by activating calmodulin (Matt- When the ecdysone analogue ponasterone A (25 son and Spaziani, ’86). In lobster muscle in vivo deoxy-20-hydroxyecdysone agonist of 20 hydroxyec- ecdysteroids increased actin synthesis by promot- dysone) was injected into crayfish it precipitated ing translational processing in addition to tran- premolt (Ueno et al., ’92a). Ecdysone receptors were scriptional regulation (Whiteley and El Haj, ’97). present in nuclei and cytoplasm of the crayfish hy- While we have demonstrated differential expres- podermis, Leydig, and pillar cells at premolt (small sion of SERCA during the molting cycle, we have gastrolith period) coincident with the first ecdys- not associated this with ecdysone titers. one peak and when Ca reabsorption is being in- In vertebrates, the secosteroid vitamin D works duced. This suggests that cells involved in Ca in concert with calcitonin (CT) and parathyroid hor- transport are the target of ecdysone. Nuclear mone to regulate Ca balance. Vitamin D affects cal- ecdysteroid binding is indicative of a genomic ac- cium fluxes at transporting epithelia in bone, gut, tion of ecdysone which may be related to the induc- kidney, and chick chorioallantoic membrane. It can tion of Ca transport proteins. In the gastrolith disc stimulate Ca transport by genomic action (synthe- (Ueno et al., ’92b), maximal binding to ponasterone sis of basolateral Ca pumps and Ca binding A was observed in the cytoplasm of gastrolith epi- proteins) and nongenomic action (rapid effect: thelial cells in premolt (small but not large gas- transcaltachia, liponomic effect at brush border). trolith phase) suggesting its involvement in Immunoreactivity to vitamin D-like molecules has induction of Ca secretion. The appearance of cy- been identified in whole extracts of the amphipod toplasmic receptors may reflect induction of re- Orchestia (Meyran et al., ’91a). Receptors were ceptor protein by ecdysone. In immediate postmolt found in nuclei of epithelial cells in the PC (Meyran (gastrolith reabsorption), binding was in both nu- et al., ’91b), and immunoreactivity levels were high- clei and cytoplasm implicating translocation of cy- est during postmolt/intermolt. This period coincides 636 M.G. WHEATLY with cuticular stabilization, reduced circulating Ca FUTURE PERSPECTIVES (Sellem et al., ’89), and reduced hemolymph ecdys- At the whole animal/EC level there are still one. This profile resembles the action of vitamin D questions regarding mechanisms by which envi- on the vertebrate skeleton. Application of exog- ronmental Ca (either water or food) can regulate enous vitamin D resulted in a decrease in hemo- intermolt Ca homeostasis in crustaceans. Syn- lymph Ca in pre- and intermolt and stimulated chrony of Ca-flux mechanisms at mineralizing and preexuvial Ca storage at the PC epithelium nonmineralizing epithelia is also of extreme in- (Meyran et al., ’93). terest, as are the temporal relationships between In vertebrates, calcitonin (CT) is a 32 amino- mineralization and reabsorption. Research at the acid peptide (MW 3,500 daltons) that lowers blood cellular level will comprehensively characterize Ca Ca by inhibiting release from bone. It also has a transporters in apical, basolateral, and internal variety of “neurotransmitter-like” functions. CT membranes in a range of transporting epithelia and calcitonin gene-related product (CGRP) are and as a function of molting stage. Biochemical derived from the same ancient gene by alterna- studies should be supported with ultrastructural tive splicing of a common transcript. The CT-like work. Molecular studies will continue to sequence molecules are derived from a phylogenetically old Ca-associated proteins from different crustacean peptide. While it has evolved in parallel with the tissues, and to determine their expression during need for a calcified exo- or endoskeleton, it has elevated transepithelial Ca flux occurring during no recognizable role in calcification in some or- the molting cycle. Up- and downregulation of ganisms and so its ancestral function is unknown. transporters by water Ca (aquatic species) or di- Recently CT-like molecules (27.2 kDa, larger etary Ca (terrestrial species) will also be studied. than in vertebrates) have been reported in ma- Additionally, it will be important to determine rine crustaceans (shrimp, lobster) exhibiting a whether protein expression is responsive to hor- maximal concentration in postmolt when hemo- mones. Isolation of genomic clones will clarify the lymph Ca is declining (Arlot-Bonnemains et al., complete sequences of appropriate genes and their ’86; Fouchereau-Peron et al., ’87). However, injec- intron and exon structure together with splicing tions into intermolt/premolt animals failed to pro- mechanisms. Analysis of the 5′ upstream sequences duce any effect. In the shrimp Palaemon serratus, will elucidate the existence of the putative regula- CT and CGRP activity were both found in heart tory sites and associated factors involved in gene and eyestalk; CGRP was also identified in gills regulation. (Lamharzi et al., ’92). In the blue crab Callinectes, CT-like immunoreactivity was found in hepato- LITERATURE CITED pancreas and other tissues with a small increase Ahearn GA, Franco P. 1993. Ca transport pathways in brush in premolt (2×, Cameron and Thomas, ’92). border membrane vesicles of crustacean antennal glands. Amer J Physiol 264:R1206–1213. In the amphipod Orchestia, CT-like molecules Andrews SC, Dillaman RM. 1993. Ultrastructure of the gill peak in premolt (10-fold increase) correlated with epithelia in the crayfish Procambarus clarkii at different increased hemolymph Ca (Graf et al., ’89; Meyran stages of the molt cycle. J Crust Biol 13:77–86. et al., ’93). Immunocytological localization (Graf et Arlot-Bonnemains Y, Van-Wormhoudt A, Favrel P, Fouchereau- Peron M, Milhaud G, Moukhtar MS. 1986. Calcitonin-like al., ’92) revealed two reactive organs: the PC epi- peptide in the shrimp Palaemon serratus (Crustacea, Deca- thelium, where it may be involved in Ca flux, and poda) during the intermolt cycle. Experientia 42:419–420. the CNS, where it may play a role as a neurotrans- Axelson KB, Palmgren MG. 1998. Evolution of substrate mitter. Based on temporal patterns of release, it specificities in the P-type ATPase superfamily. J Mol Evol 46:84–101. has been suggested that ecdysone regulates expres- Becker GL, Chen CH, Greenawalt JW, Lehninger AL. 1974. sion of the CT gene via an ecdysone-response el- Calcium phosphate granules in the hepatopancreas of the ement in the crayfish CT gene promoter (L. blue crab Callinectes sapidus. J Cell Biol 61:316–326. Bjerregaard P, Depledge MH. 1994. Cadmium accumulation Smith, personal communication). of Littorina littorea, Mytilis edulis and Carcinus maenas: In summary, vitamin D and CT appear to play the influence of salinity and calcium ion concentration. Mar an increased role in Ca regulation in terrestrial Biol 119:385–395. Boitel F, Truchot JP. 1989. Effects of sublethal and lethal cop- crustaceans where Ca storage is emphasized. The per levels on hemolymph acid-base balance and ion concen- fact that the hormonal regulation of Ca homeo- tration in the shore crab Carcinus maenas kept in undiluted stasis in terrestrial crustaceans appears to re- sea water. Mar Biol 103:495–502. Brandl CJ, deLeon S, Martin DR, MacLennan DH. 1987. semble that of vertebrates suggests that it may Adult forms of the Ca2+ ATPase of sarcoplasmic reticulum. be associated with preadaptation to aerial life. J Biol Chem 262:3768–3774. EVOLUTION OF CRUSTACEAN CA TRANSPORT 637

Briggs FN, Lee KF, Feher JJ, Wechsler AS, Ohlendieck K, moters regulate the expression of the two sarco/endoplas- Campbell K. 1990. Ca-ATPase isozyme expression in sarco- mic reticulum Ca ATPase isoforms from Artemia francis- plasmic reticulum is altered by chronic stimulation of skel- cana. DNA Cell Biol 14:893–900. etal muscle. FEBS Lett 259:269–272. Escalante R, Sastre L. 1996. Tissue-specific expression of two Bronner F. 1996. Cytoplasmic transport of calcium and other Artemia fransiscana sarco/endoplasmic reticulum Ca AT- inorganic ions. Comp Biochem Physiol 115B:313–317. Pase isoforms. J Histochem Cytochem 44:321–325. Burggren W, McMahon BR. 1988. Biology of the land crabs: Ferraris RP, Diamond J. 1989. Specific regulation of intesti- an introduction. In: Burggren WW, McMahon BR, editors. nal nutrient transporters by their dietary substrates. Ann Biology of the land crabs. New York: Cambridge University Rev Physiol 51:125–141. Press. p 1–5. Flik G, Verbost PM, Atsma W, Lucu C. 1994. Calcium trans- Burk SE, Lytton J, MacLennan DH, Shull GE. 1989. cDNA port in gill plasma membranes of the crab Carcinus maenas: cloning, functional expression and mRNA tissue distribu- evidence for carriers driven by ATP and a Na+ gradient. J tion of a third organellar pump. J Biol Chem 264:18561– Exp Biol 195:109–122. 18568. Fouchereau-Peron M, Arlot-Bonnemains Y, Milhaud G, Cameron JN. 1985. Post-moult calcification in the blue crab Moukhtar MS. 1987. Immunoreactive salmon calcitonin-like (Callinectes sapidus): relationships between apparent net H+ molecule in crustaceans: high concentrations in Nephrops excretion, calcium and bicarbonate. J Exp Biol 119:275–285. norvegicus. Gen Comp Endocrinol 65:179–183. Cameron, JN. 1989. Post-moult calcification in the blue crab Graf F, Delbecque JP. 1987. Ecdysteroid titers during the molt Callinectes sapidus: timing and mechanism. J Exp Biol cycle of Orchestia cavimana Crustacea, Amphipoda. Gen 143:285–304. Comp Endocrinol 65:23–33. Cameron JN, Thomas P. 1992. Calcitonin-like immunoreactiv- Graf F, Fouchereau-Peron M, Van-Wormhoudt A, Meyran JC. ity in the blue crab: tissue distribution variations in the molt 1989. Variations of calcitonin-like immunoreactivity in the cycle and partial characterization. J Exp Zool 262:279–286. crustacean Orchestia cavimana during a molt cycle. Gen Cameron JN, Wood CM. 1985. Apparent H+ excretion and Comp Endocrinol 73:80–84. CO2 dynamics accompanying carapace mineralization in the Graf F, Meyran JC. 1983. Premolt calcium secretion in mid- blue crab (Callinectes sapidus) following moulting. J Exp gut posterior caeca of the crustacean Orchestia: ultrastruc- Biol 114:181–196. ture of the epithelium. J Morphol 177:1–23. Carafoli E. 1994. Biogenesis: Plasma membrane calcium AT- Graf F, Meyran JC. 1985. Calcium reabsorption in the poste- Pase: 15 years of work on the purified enzyme. FASEB J rior caeca of the midgut in a terrestrial crustacean, 8:993–1002. Orchestia cavimana. Cell Tissue Res 242:83–95. Compère PH, Goffinet G. 1987. Elaboration and ultrastruc- Graf F, Morel G, Meyran JC. 1992. Immunocytological local- tural changes in the pore canal system of the mineralized ization of endogenous calcitonin-like molecules in the crus- cuticle of Carcinus maenas during the moulting cycle. Tis- tacean Orchestia. Histochemistry 97:147–154. sue and Cell 19:859–875. Greenaway P. 1974. Total body calcium and haemolymph cal- Compère P, Morgan JA , Goffinet G. 1993. Ultrastructural cium concentrations in the crayfish Austropotamobius location of calcium and magnesium during mineralisation pallipes (Lereboullet). J Exp Biol 61:19–26. of the cuticle of the shore crab, as determined by the K- Greenaway P. 1976. The regulation of haemolymph calcium pyroantimonate method and X ray microanalysis. Cell Tis- concentration of the crab Carcinus maenas (L.). J Exp Biol sue Res 274:567–577. 64:149–157. De Souza SCR. 1992. Calcium regulation during aerial expo- Greenaway P. 1985. Calcium balance and moulting in the sure in Carcinus maenas: a role for moulting hormones? J crustacea. Biol Rev 60:425–454. Physiol 446:190P. Greenaway P. 1989. Sodium balance and adaptation to fresh De Souza SCR, Taylor EW. 1991. Electrolyte and acid-base water in the amphibious crab Cardisoma hirtipes. Physiol regulation in Carcinus maenas during prolonged aerial ex- Zool 62:639–653. posure. J Physiol 435:106P. Greenaway P. 1993. Calcium and magnesium balance during Diamond J. 1991. Evolutionary design of intestinal nutri- molting in land crabs. J Crust Biol 13:191–197. ent absorption: enough but not too much. News Physiol Greenaway P. 1994. Salt and water balance in field popula- Sci 6:92–96. tions of the terrestrial crab Gecarcoidea natalis. J Crust Diesel R, Schuh M. 1993. Maternal care in the bromeliad Biol 14:438–453. crab Metopaulias depressus decapoda maintaining oxygen, Greenaway P, Dillaman RM, Roer RD. 1995. Quercetin-de- pH and calcium levels optimal for the larvae. Behav Ecol pendent ATPase activity in the hypodermal tissue of Sociobiol 32:11–15. Callinectes sapidus during the moult cycle. Comp Biochem Ellis BA, Morris S. 1995. Effects of extreme pH on the Physiol 111:303–312. physiology of the Australian “yabby” Cherax destructor: Greenaway P, Farrelly C. 1991. Trans-epidermal transport acute and chronic changes in haemolymph oxygen lev- and storage of calcium in Holthuisana tranversa (Brachyura; els, oxygen consumption and metabolite levels. J Exp Sundathelphusidae) during premolt. Acta Zool 72:29–40. Biol 198:409–418. Greenaway P, Linton SM. 1995. Dietary assimilation and food Escalante R, Sastre L. 1993. Similar alternative splicing retention time in the herbivorous terrestrial crab Gecar- events generate two sarcoplasmic or endoplasmic reticulum coidea natalis. Physiol Zool 68:1006–1028. Ca-ATPase isoforms in the crustacean Artemia franciscana Greenaway P, Morris S. 1989. Adaptations to a terrestrial and in vertebrates. J Biol Chem 268:14090–14095. existence by the robber crab, Birgus latro L.: nitrogenous Escalante R, Sastre L. 1994. Structure of Artemia franciscana excretion. J Exp Biol 143:333–346. sarco/endoplasmic reticulum Ca-ATPase gene. J Biol Chem Greenaway P, Raghaven S. 1998. Digestive strategies in two 269:13005–13012. species of leaf-eating land crabs (Brachyura: Gecarcinidae) Escalante R, Sastre L. 1995. Tissue-specific alternative pro- in a rain forest. Physiol Zool 71:36–44. 638 M.G. WHEATLY

Greenaway P, Taylor HH, Morris S. 1990. Adaptations to a DJA, Brown JA, editors. Acid toxicity and aquatic animals. terrestrial existence by the robber crab Birgus latro: VI. Cambridge: Cambridge University Press. p 171–199. The role of the excretory system in fluid balance. J Exp Meyran JC, Chapuy MC, Arnaud S, Sellem E, Graf F. 1991a. Biol 152:505–519. Variations of vitamin D-like reactivity in the crustacean Gunteski-Hamblin, A-M, Greeb J, Shull GE. 1988. A novel Orchestia cavimana during the molt cycle. Gen Comp Ca2+ pump expressed in brain, kidney and stomach is en- Endocrinol 84:115–120. coded by an alternative transcript of the slow twitch muscle Meyran JC, Graf F. 1986. Ultrahistochemical localization of sarcoplasmic reticulum Ca ATPase gene. J Biol Chem Na+-K+ATPase, Ca2+-ATPase and alkaline phosphatase ac- 263:15032–15040. tivity in a calcium-transporting epithelium of a crustacean Haond C, Flik G, Charmantier G. 1998. Confocal laser scan- during molting. Histochemistry 85:313–320. ning and electron microscopical studies on osmoregulatory Meyran JC, Graf F, Nicaise G. 1984. Calcium pathway epithelia in the branchial cavity of the lobster Homarus through a mineralizing epithelium in the crustacean gammarus. J Exp Biol 201:1817–1833. Orchestia in premolt: ultrastructural cytochemistry and X- Hessen D, Kristiansen G, Lid I. 1991. Calcium uptake from ray microanalysis. Tissue Cell 16:269–286. food and water in the crayfish Astacus astacus (L., 1758), Meyran JC, Morel G, Haussler MR, Boivin G. 1991b. Immuno- measured by radioactive 45Ca (Decapoda, Astacidea). cytological localization of 1 25 dihyroxyvitamin D3-like mol- Crustaceana 60:76–83. ecules and their receptors in a calcium-transporting Hollett L, Berrill M, Rowe L. 1986. Variation in major ion epithelium of a crustacean. Cell Tissue Res 263:345–352. concentration of Cambarus robustus and Orconectes Meyran JC, Sellem E, Graf F. 1993. Variations in haemo- rusticus following exposure to low pH. Can J Fish Aquat lymph ionized calcium concentrations in the crustacean Sci 43:2040–2044. Orchestia cavimana during a moult cycle. J Insect Physiol Hopkins PM. 1992. Hormonal control of the molt cycle in the 39:413–417. fiddler crab Uca pugilator. Am Zool 32:450–458. Morgan D, McMahon B. 1982. Acid tolerance and effects of Hu P, Yin C, Zhang KM, Wright LD, Nixon TE, Wechsler sublethal acid exposure on ionoregulation and acid-base sta- AS, Spratt JA, Briggs FN. 1995. Transcriptional regula- tus in two crayfish Procambarus clarki and Orconectes tion of phospholamban gene and translational regulation rusticus. J Exp Biol 97:241–252. of SERCA2 gene produces coordinated expression of these Morris MA, Greenaway P. 1992. High affinity, Ca2+ specific two sarcoplasmic reticulum proteins during skeletal muscle ATPase and Na+K+-ATPase in the gills of a supralittoral phenotype switching. J Biol Chem 270:11619–11622. crab Leptograpsus variegatus. Comp Biochem Physiol Jensen FB, Malte H. 1990. Acid-base and electrolyte regula- 102A:15–18. tion, and haemolymph gas transport in crayfish, Astacus Morris S, Taylor HH, Greenaway P. 1991. Adaptations to a astacus, exposed to soft, acid water with and without alu- terrestrial existence by the robber crab Birgus latro VII: minium. J Comp Physiol 160:483–490. the branchial chamber and its role in urine reprocessing. J Johnen C, Von Gliscynski U, Dircksen H. 1995. Changes in Exp Biol 161:315–331. haemolymph ecdysteroid levels and CNS contents of crusta- Morris S, Tyler-Jones R, Bridges CR, Taylor EW. 1986. The cean cardioactive peptide-immunoreactivity during the moult regulation of haemocyanin oxygen affinity during emersion cycle of the isopod Oniscus asellus. Neth J Zool 45:38–40. of the crayfish Austropotamobius pallipes: II. An investiga- Kirschner LB. 1994. Electrogenic action of calcium on cray- tion of in vivo changes in oxygen affinity. J Exp Biol fish gills. J Comp Physiol 164:215–221. 121:327–337. Lachaise F, Le Roux A, Hubert M, Lafont R. 1993. The molt- Nagai R, Zarain-Herzberg A, Brandl CJ, Fijii J, Tada M, ing gland of crustaceans: Localization, activity and endo- MacLennan DH, Alpert NR, Periasamy M. 1989. Regula- crine control (a review). J Crust Biol 13:198–234. tion of myocardial Ca2+-ATPase and phospholamban mRNA Lamharzi N, Arlot-Bonnemains Y, Milhaud G, Fouchereau- expression in response to pressure overload and thyroid hor- Peron M. 1992. Calcitonin and calcitonin gene-related pep- mone. Proc Natl Acad Sci USA 86:2966–2970. tide in the shrimp Palaemon serratus variations during the Neufeld DS, Cameron JN. 1992. Postmoult uptake of calcium molt cycle. Comp Biochem Physiol A 102:679–682. by the blue crab (Callinectes sapidus) in water of low salin- Lytton J, MacLennan DH. 1988. Molecular cloning of cDNAs ity. J Exp Biol 171:283–299. from human kidney coding for two alternatively spliced Neufeld DS, Cameron JN. 1993. Transepithelial movement products of the cardiac Ca2+ ATPase gene. J Biol Chem of calcium in crustaceans. J Exp Biol 184:1–16. 263:15024–15031. Palmero I, Sastre L. 1989. Complementary DNA cloning of a Lytton J, MacLennan DH. 1992. Sarcoplasmic reticulum. In: protein highly homologous to mammalian sarcoplasmic The heart and cardiovascular system. New York: Raven reticulum Ca-ATPase from the crustacean Artemia. J Mol Press. p 1203–1222. Biol 210:737–748. MacLennan DH, Brandl CJ, Korczak B, Green NM. 1985. Pedersen TV, Bjerregaard P. 1995. Calcium and cadmium Amino acid sequence of a Ca2+ + Mg2+-dependent ATPase fluxes across the gills of the shore crab, Carcinus maenas. from rabbit muscle sarcoplasmic reticulum, deduced from Mar Poll Bull 31:73–77. its complementary DNA sequence. Nature 316:696–700. Pinder AW, Smits AW. 1993. The burrow microhabitat of the Mattson MP, Spaziani E. 1986. Calcium antagonizes cAMP- land crab Cardisoma guanhumi: respiratory/ionic conditions mediated suppression of crab Y organ steroidogenesis in and physiological responses of crabs to hypercapnia. Physiol vitro: evidence for activation of cAMP-phosphodiesterase by Zool 66:216–236. Ca calmodulin. Mol Cell Endocrinol 48:135–151. Rasmussen AD, Bjerregaard P. 1995. The effect of salin- McDonald DG. 1983. The effects of H+ upon the gills of fresh- ity and calcium concentration on the apparent water per- water fish. Can J Zool 61:691–703. meability of Cherax destructor, Astacus and Carcinus McMahon BR, Stuart SA. 1989. The physiological problems maenas (Decapoda, Crustacea). Comp Biochem Physiol of crayfish in acid waters. In: Morris R, Taylor EW, Brown 111:171–175. EVOLUTION OF CRUSTACEAN CA TRANSPORT 639

Roer RD. 1980. Mechanisms of resorption and deposition of Wheatly MG. 1985. The role of the antennal gland in ion and calcium in the carapace of the crab Carcinus maenas. J acid-base regulation during hyposaline exposure of the Exp Biol 88:205–218. Dungeness crab Cancer magister (Dana). J Comp Physiol Rogers JV, Wheatly MG. 1997. Accumulation of calcium in the B 155:445–454. antennal gland during the molting cycle of the freshwater Wheatly MG. 1989. Physiological responses of the crayfish crayfish Procambarus clarkii. Invert Biol 116:248–254. Pacifastacus leniusculus (Dana) to environmental hyperoxia: Rohrer D, Dillmann WH. 1988. Thyroid hormone markedly 2+ I. Extracellular acid-base and electrolyte status and trans increases the mRNA coding for sarcoplasmic reticulum Ca - branchial exchange. J Exp Biol 143:33–51. ATPase in the rat heart. J Biol Chem 263:6941–6944. Wheatly MG. 1996. An overview of calcium balance in crus- Sellem E, Graf F, Meyran JC. 1989. Some effects of salmon taceans. Physiol Zool 69:351–382. calcitonin on calcium metabolism in the crustacean Orches- Wheatly MG. 1997. Crustacean models for studying calcium tia during the molt cycle. J Exp Zool 249:177–181. transport: The journey from whole organisms to molecular Shull GE, Greeb J. 1988. Molecular cloning of two isoforms 2+ mechanisms. J Mar Biol Ass UK 77:107–125. of the plasma membrane Ca transporting ATPase from Wheatly MG, Ayers J. 1995. Scaling of calcium, inorganic and rat brain. J Biol Chem 263:8646–8657. organic contents to body mass during the molting cycle of Simmons J, Simkiss K, Taylor MG, Jarvis KE. 1996. Crab the freshwater crayfish Procambarus clarkii (Girard). J biominerals as environmental monitors. Bull de L’Institut Crust Biol 15:409–417. Oceanographique 14:225–231. Wheatly MG, De Souza SCR, Hart MK. 1996. Related changes Sparkes S, Greenaway P. 1984. The haemolymph as a stor- in hemolymph acid-base status, electrolytes and ecdysone age site for cuticular ions during premoult in the freshwa- in intermolt crayfish Procambarus clarkii at 23°C during ter/land crab Holthusiana transversa. J Exp Biol 113:43–54. extracellular acidosis induced by exposure to air hyperoxia Steel CGH. 1993. Storage and translocation of integumen- or acid. J Crust Biol 16:267–277. tary calcium during the moult cycle of the terrestrial iso- Wheatly MG, Gannon AT. 1995. Ion regulation in crayfish: pod Oniscus asellus L. Can J Zool 71:4–10. freshwater adaptations and the problem of molting. Am Zool Strehler EE. 1991. Recent advances in the molecular charac- 2+ 35:49–59. terization of the plasma membrane Ca ATPase. J Membr Wheatly MG, Hart MK. 1995. Hemolymph ecdysone and elec- Biol 20:1–15. trolytes during the molting cycle of crayfish: a comparison Taylor CW. 1985. Calcium regulation in vertebrates: an over- of natural molts with those induced by eyestalk removal or view. Comp Biochem Physiol 82A:249–256. multiple limb autotomy. Physiol Zool 68:583–607. Taylor MG, Simkiss K. 1994. Cation substitutions in phos- Wheatly MG, Henry RP. 1992. Extracellular and intracellu- phate biominerals. Bull de L’Institut Oceanographique lar acid-base regulation in crustaceans. J Exp Zool 14:75–79. 263:127–142. Tentes I, Pateraki L, Stratakis E. 1992. Purification and prop- Wheatly MG, Ignaszewski LA. 1990. Electrolyte and gas ex- erties of a calcium plus magnesium ATPase from Potamon change during the moulting cycle of a freshwater crayfish. potamios skeletal muscle sarcoplasmic reticulum. Comp J Exp Biol 151:469–483. Biochem Physiol B 103:875–880. Wheatly MG, McMahon BR. 1982. Responses to hypersaline Tyler-Jones R, Taylor EW. 1986. Urine flow and the role of exposure in the euryhaline crayfish Pacifastacus lenius- the antennal glands in water balance during aerial expo- culus: I. The interaction between ionic and acid-base regu- sure in the crayfish Austropotamobius pallipes (Lereboullet). lation. J Exp Biol 99:425–445. J Comp Physiol B 156:529–535. Wheatly MG, Pence RC, Weil JR. 1999. ATP-dependent cal- Ueno M, Bidmon HJ, Stumpf WE. 1992a. Ecdysteroid bind- cium uptake into basolateral vesicles from transporting epi- ing sites in gastrolith forming tissue and stomach during thelia of intermolt crayfish. Am J Physiol (in press). the molting cycle of crayfish Procambarus clarkii. His- Wheatly MG, Toop T. 1989. Physiological responses of the tochemistry 98:1–6. crayfish Pacifastacus leniusculus (Dana) to environmental Ueno M, Bidmon HJ, Stumpf WE. 1992b. Ponasterone A bind- hyperoxia II: the role of the antennal gland. J Exp Biol ing sites in hypodermis during the molting cycle of crayfish 143:53–70. Procambarus clarkii. Acta Histochem Cytochem 25:505–510. Wheatly MG, Weil JR, Douglas PB. 1998. Isolation, visual- Ueno M, Mizuhira V. 1984. Calcium transport mechanism in ization, characterization, and osmotic reactivity of crayfish crayfish gastrolith epithelium correlated with the moulting BLMV. Am J Physiol 274:R725–R734. cycle: II. Cytochemical demonstration of Ca ATPase and Mg Whiteley NM, El Haj AJ. 1997. Regulation of muscle gene ATPase. Histochem 80:213–217. expression over the moult in Crustacea. Comp Biochem Ushio H, Watabe S. 1993. Ultrastructural and biochemical Physiol 117:323–331. analysis of the sarcoplasmic reticulum from crayfish fast Whiteley NM, Taylor EW. 1992. Oxygen and acid-base dis- and slow striated muscles. J Exp Zool 267:9–18. turbances in the hemolymph of the lobster Homarus Vijayan KK, Diwan AD. 1996. Fluctuations in Ca, Mg and P gammarus during commercial transport and storage. J levels in the hemolymph, muscle, midgut gland and exosk- Crust Biol 12:19–30. eleton during the moult cycle of the Indian white prawn, Wolcott TG. 1988. Ecology. In: Burggren WW, McMahon BR, Penaeus indicus (Decapoda: Penaeidae). Comp Biochem editors. Biology of the land crabs. New York: Cambridge Physiol 114:91–97. University Press. p 55–96. Watson RD, Spaziani E, Bollenbacher WE. 1989. Regulation Wolcott TG, Wolcott DL. 1982. Urine reprocessing for salt of ecdysone biosynthesis in insects and crustaceans: a com- conservation in terrestrial crabs. Am Zool 22:897. parison. In: Koolman J, editor. Ecdysone from chemistry to Wolcott DL, Wolcott TG. 1984. Food quality and cannibalism mode of action. New York: Thieme Medical Publishers, Inc. in the red land crab Gecarcinus lateralis. Physiol Zool p 188–203. 57:318–324. 640 M.G. WHEATLY

Wolcott TG, Wolcott DL. 1985. Extrarenal modification of postmolt fluxes of calcium and associated electrolytes in the urine for ion conservation in ghost crabs, Ocypode quadrata freshwater crayfish (Procambarus clarkii). In: Romaire R, Fabricus. J Exp Mar Biol Ecol 91:93–107. editor. Freshwater crayfish. Baton Rouge: Louisiana State Wolcott TG, Wolcott DL. 1991. Ion conservation by reprocess- University. p 437–450. ing of urine in the land crab Gecarcinus lateralis (Frem- Zhuang Z, Ahearn GA. 1996. Ca2+ transport processes of lob- inville). Physiol Zool 64:344–361. ster hepatopancreas brush border membrane vesicles. J Exp Wood CM, Boutilier RG. 1985. Osmoregulation, ionic ex- Biol 199:1195–1208. change, blood chemistry, and nitrogenous waste excretion Zhuang Z, Ahearn GA. 1998. Energized Ca2+ transport by in the land crab Cardisoma carnifex: a field and laboratory hepatopancreatic basolateral plasma membranes of Hom- study. Biol Bull 169:267–290. arus americanus. J Exp Biol 201:211–220. Wood CM, Rogano MS. 1986. Physiological responses to acid Ziegler A. 1994. Ultrastructure and electron spectroscopic stress in crayfish (Orconectes): haemolymph ions, acid-base diffraction analysis of the sternal calcium deposits of status, and exchanges with the environment. Can J Fish Porcellio scaber Latr. (Isopoda, Crustacea). J Struct Biol Aquat Sci 43:1017–1026. 112:110–116. Wu K-D, Lee W-S, Wey J, Bungard D, Lytoon J. 1995. Local- Ziegler A. 1996. Ultrastructural evidence for transepithelial ization quantification of endoplasmic reticulum Ca2+-ATPase calcium transport in the anterior sternal epithelium of the isoform transcripts. Am J Physiol 269:C775–784. terrestrial isopod Porcellio scaber (Crustacea) during the Wuytack F, Raeymaekers L, De Smedt H, Eggermont JA, formation and resorption of CaCO3. Cell Tissue Res Missiaen L, Van Den Bosch L, De Jaegere S, Verboomen H, 284:459–466. Plessers L, Casteel R. 1992. Ca2+-transport ATPases and Ziegler A. 1997a. Immunocytochemical localization of Na/K their regulation in muscle. Ann NY Acad Sci 671:82–91. ATPase in the calcium transporting sternal epithelium of Zanders IP. 1980. Regulation of blood ions in Carcinus maenas the terrestrial isopod Porcellio scaber L. (Crustacea). J (L.). Comp Biochem Physiol 65A:97–108. Histochem Cytochem 45:437–446. Zanotto FP, Wheatly MG. 1993. The effect of ambient pH on Ziegler A. 1997b. Ultrastructural changes of the anterior and electrolyte regulation during the postmoult period in fresh- posterior sternal integument of the terrestrial isopod water crayfish Procambarus clarkii. J Exp Biol 178:1–19. Porcellio scaber Latr (Crustacea) during the moult cycle. Zanotto FP, Wheatly MG, 1995. The effect of water pH on Tissue Cell 29:63–76.