Biochemical and physiological adaptations in the estuarine crab Neohelice granulata during salinity...

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Comparative Biochemistry and Physiology, Part A 151 (2008) 423–436www.elsevier.com/locate/cbpa

Review

Biochemical and physiological adaptations in the estuarine crabNeohelice granulata during salinity acclimation☆

Adalto Bianchini a,⁎, Mariana Machado Lauer b, Luiz Eduardo Maia Nery a, Elton Pinto Colares a,José María Monserrat a, Euclydes Antônio dos Santos Filho a

a Departamento de Ciências Fisiológicas, Fundação Universidade Federal do Rio Grande, Campus Carreiros, Av. Itália km 8, 96201-900, Rio Grande, RS, Brazilb Programa de Pós-Graduação em Ciências Fisiológicas – Fisiologia Animal Comparada, Fundação Universidade Federal do Rio Grande,

Campus Carreiros, Av. Itália km 8, 96201-900, Rio Grande, RS, Brazil

Received 3 May 2006; received in revised form 21 November 2007; accepted 3 December 2007Available online 8 December 2007

Abstract

Neohelice granulata (Chasmagnathus granulatus) is an intertidal crab species living in salt marshes from estuaries and lagoons along the Atlanticcoast of South America. It is a key species in these environments because it is responsible for energy transfer from producers to consumers. In order todeal with the extremely marked environmental salinity changes occurring in salt marshes, N. granulata shows important and interesting structural,biochemical, and physiological adaptations at the gills level. These adaptations characterize this crab as a euryhaline species, tolerating environmentalsalinities ranging from very diluted media to concentrated seawater. These characteristics had led to its use as an animal model to study estuarineadaptations in crustaceans. Therefore, the present review focuses on the influence of environmental salinity onN. granulata responses at the ecological,organismic and molecular levels. Aspects covered include salinity tolerance, osmo- and ionoregulatory patterns, morphological and structuraladaptations at the gills, and mechanisms of ion transport and their regulation at the gills level during environmental salinity acclimation. Finally, thisreview compiles information on the effects of some environmental pollutants on iono- and osmoregulatory adaptations showed by N. granulata.© 2007 Elsevier Inc. All rights reserved.

Keywords: Neohelice granulate; Chasmagnathus granulatus; Crab; Gills; Ionoregulation; Osmoregulation; Salinity; Pollution

Contents

1. General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4242. Environmental salinity effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4243. Tolerance to salinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4254. Hemolymph osmoregulation patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4255. Hemolymph ionoregulation pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4266. Gill morphology and structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4277. Enzymes associated with hemolymph iono- and osmoregulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4288. Mechanisms of gill NaCl transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429

☆ This paper is part of the 5th special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by MarceloHermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Carolina A. Freire (Brazil), Tania Zenteno-Savín (Mexico) and the editors ofCBP. This issue is dedicated to the memory of Knut Schmidt-Nielsen (1915–2007), a great mind in comparative physiology, with books translated into 16 languages,including Spanish and Portuguese, and Cicero Lima (1934–2006), journalist, science lover and Hermes-Lima's dad.⁎ Corresponding author. Fundação Universidade Federal do Rio Grande, Departamento de Ciências Fisiológicas, Av. Itália km 8 – Campus Carreiros, 96.201-900,

Rio Grande, RS, Brazil. Tel.: +55 53 3233 6853; fax: +55 53 3233 6848.E-mail addresses: [email protected], [email protected] (A. Bianchini).

1095-6433/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpa.2007.12.001

mailto:[email protected]

mailto:[email protected]

http://dx.doi.org/10.1016/j.cbpa.2007.12.001

424 A. Bianchini et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 423–436

9. Mechanism of ion transport regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43210. Aquatic pollutants and osmoregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43211. Conclusions and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

1. General aspects

The main goal of the present review is to discuss thebiochemical and physiological responses of the estuarine crabNeohelice granulata (Crustacea, Decapoda, Brachyura) toenvironmental saline stress. Aspects covered will includesalinity effects on N. granulata responses at the ecological,organismic and molecular levels. Crab salinity tolerance and theionic and osmotic regulation patterns associated with environ-mental salinity changes will be discussed. Morphological andstructural adaptations, as well as the mechanisms of iontransport and their regulation at the gills level during salinityacclimation will also be analyzed and modeled. Finally, thisreview will compile information on the biochemical andphysiological effects of some environmental pollutants on theiono- and osmoregulatory adaptations showed by N. granulata.

The burrowing crab N. granulata, formerly known as Chas-magnathus granulata or C. granulatus (Sakai et al., 2006), is themost abundant macroinvertebrate on intertidal mudflats and saltmarshes of the South Western Atlantic (Iribarne et al., 2000). Itlives in both vegetated and non-vegetated areas (Bass et al., 2005)along the South Atlantic coast from Rio de Janeiro (Brazil) toPatagonia (Argentina) (Boschi, 1964). Density and sex ratio varyamong seasonal periods and areas. Spatial and temporal variationsin sex ratio and size frequency distributions among microhabitatssuggest that the structure ofN. granulata populations is dynamic,with a clear spatial segregation of sizes and sexes. Interpopula-tional differences in density, size and reproductive period are notsimply attributed to changes associated with a latitudinal cline.Other factors, such as food availability and thermal stress seem tobe implicated (Bass et al., 2005).

N. granulata has important ecological roles in intertidalmudflats and salt marshes, controlling or affecting differentphysico-chemical and biological aspects in these environments.For example, it excavates and maintains large semi-permanentopen burrows that work as passive traps for debris, increasingthe retention of this material in mudflats (Iribarne et al., 2000).N. granulata is also considered as a key species in the energytransfer in mudflats and salt marshes, grazing on the cordgrassSpartina densiflora (D' Incao et al., 1990). Evidences suggestthat N. granulata affects cordgrass production by herbivory onnew shoots (Bortolus and Iribarne, 1999). This crab also playsan important role on the structure of the meiofauna communityin estuarine intertidal habitats (Rosa and Bemvenuti, 2005). Forexample, polychaete distribution and availability to shorebirdsare affected by N. granulata activity, establishing an importantshorebird–burrowing organism interaction (Botto et al., 2000;Palomo et al., 2003; Iribarne et al., 2005). Another example ofthe ecosystem engineering ability of N. granulata is related to

the fact that the environmental heterogeneity produced by itsactivity has an effect on the infaunal behavior, risk of mortality,and the zonation pattern (Escapa et al., 2004).

2. Environmental salinity effects

As for other estuarine animals, environmental salinity drivesseveral aspects of N. granulata biology. It is a key parameter inthe larval biology (Anger, 2003). For example, pre-hatchingsalinity is very important for larval survival, growth, andduration of development under normal condition or food stress(Gimenez and Anger, 2001; Gimenez, 2002; Gimenez andTorres, 2002; Gimenez and Anger, 2003). Also, interpopula-tional differences in low salinity tolerance of embryos and larvaeare related to environmental differences between habitats (Bassand Spivak, 2003). However, first larval stages and juveniles ofN. granulata have a better survival and development in highsalinities (Boschi et al., 1967; Rieger and Nakagawa, 1995).

Environmental salinity also influences juvenile and adultN. granulata distribution and abundance within differentpopulations. For example, highest abundances are observed inthe Patos Lagoon estuary (Southern Brazil) during periods ofmoderate increasing temperature and salinity. Furthermore, highabundance of ovigerous females is reported in saltwater during thesummer. These patterns are likely associated with the speciesreproductive and recruitment strategies (D'Incao et al., 1992). Inaddition to these ecological effects, environmental salinitychanges can also induce several responses at the organismic level.

At the behavioral level, environmental salinity is an externalcue involved in the memory control in N. granulata. Forexample, exposure to high environmental salinity (33‰), for atleast 4 days, triggers an enhanced long-term memory. Thisfacilitatory effect is induced by synthesis of angiotensin-II-likepeptides in several neural structures of N. granulata, thus beinginvolved in the association between context and iterative dangerstimulus in this crab species (Delorenzi et al., 2000).

At the metabolic level, salinity affects growth and biochemicalcomposition of larvae, influencing directly the number andquality of larvae reachingmetamorphosis. For example, exposureof Zoea I larvae to low salinity induces lower increments in dryweight, lipid and protein contents (Torres et al., 2002). In juvenileand adult N. granulata, acclimation to low salinity induces lipidmobilization in gills and muscle (Luvizotto-Santos et al., 2003).Carbohydrate metabolism is also modified during environmentalsalinity changes. Increases in hemolymph glucose levels areobserved after hypo- or hyper-osmotic stress (Santos and Nery,1987; Da Silva and Kucharski, 1992). However, glucosemetabolism is seasonal dependent. In the summer, saline stress(hypo- or hyper-osmotic shock) induces gluconeogenesis in gills,

425A. Bianchini et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 423–436

muscle, and hepatopancreas (Nery and Santos, 1993; Schein et al.,2004; Schein et al., 2005b), while in winter carbohydratemobilization is observed in gills and hepatopancreas (Nery andSantos, 1993). Regarding proteins, environmental salinity alsoaffects its metabolism in N. granulata. Crab response to salinitychange is dependent on the type of saline stress. For example,hepatopancreas andmuscle amino acid pools decreases during thehypo-osmotic stress, while an increase in these pools is observedduring the hyper-osmotic stress (Schein et al., 2005a). As a resultof all the metabolic adjustments required for salinity acclimation,higher oxygen consumption rates are observed in juveniles andadult crabs after acclimation to extreme environmental salinities(Santos et al., 1987; Bianchini and Castilho, 1999).

At the molecular level, environmental salinity changes affectenzyme gene expression and activity in N. granulata. Crabresponse is clearly dependent on the saline stress. For example,hyper-osmotic stress induces increase in phosphoenolpyruvatecarboxykinase gene expression and activity (Schein et al.,2004), as well as in alkaline phosphatase activity in muscle ofhigh salinity-acclimated crabs (Pinoni et al., 2005). In addition,environmental salinity changes also affect gene expression and/or activity of gill Na+/K+-ATPase, carbonic anhydrase, H+-ATPase, arginine kinase, and alkaline phosphatase. Theresponse of these enzymes and their involvement in the ionicand osmotic regulation at the hemolymph level in N. granulatawill be discussed below in details (see Section 7).

Regarding the interaction between anthropogenic activitiesand environmental factors, it is also important to stress thatsalinitymarkedly affects the toxicity of both inorganic and organicaquatic pollutants to N. granulata. For example, increasingsalinity decreases cadmium and silver toxicities in adult crabs(Vitale et al., 1999; Rodriguez et al., 2001; Lauer et al., 2005).This protective effect of salinity against acute metal toxicity islikely due to a lower metal bioavailability associated with metalcomplexation by the elevated concentrations of anions, especiallyCl− in seawater, as well as the competition between metal andcations present in higher concentrations (e.g., Na+, Ca2+ andMg2+)in seawater by the binding sites at the site of metal action at the gills(Bianchini et al., 2004; Lauer et al., 2005; Pedroso et al., 2007). Onthe other hand, salinity effect on organic compounds toxicity isdependent on the chemical tested. Substances normally involved inthe crab ionic and osmotic metabolism, such as ammonia (seeSection 8), have their toxicity affected by salinity changes whiletoxicity of xenobiotics, such as pesticides, is not altered byenvironmental salinity. For example, adult N. granulata is lesstolerant to ammonia at extreme (low or high) salinities (Rebeloet al., 1999), while no changes in organophosphorus pesticidestoxicity is observed (Monserrat et al., 1991; Monserrat andBianchini, 1995).

3. Tolerance to salinity

All responses showed by N. granulata to changes inenvironmental salinity reported above are certainly part of acomplex biochemical and physiological plasticity responsible forthe successful establishment of this species in salt marshes andintertidal mud flats. According to Gimenez (2003), this plasticity

could contribute to maintain a certain degree of populationconnectivity and persistence regardless of habitat heterogeneity.Among the crab responses to environmental salinity changes,those directly related to the ability ofN. granulata to maintain theadequate ionic and osmotic balances at the hemolymph level arecertainly imperative for the species tolerance to environmentalsalinity changes.

According to Charmantier et al. (2002), N. granulata followsa strategy of larval export in its reproductive cycle, i.e., somelarval stages live and develop well under different salinityconditions from that of juveniles and adults (Boschi et al., 1967;Rieger and Nakagawa, 1995; Charmantier et al., 2002; Novoet al., 2005).

Ontogenetic changes in N. granulata tolerance to salinity areclosely associated with its reproductive cycle. Zoea I hatch inbrackish estuarine waters, where juveniles and adults live, beforeits exportation to marine zones (Charmantier et al., 2002). Thus,larvae at Zoea I stage can be considered as being euryhaline, likejuveniles and adults (Bromberg, 1992;Miranda, 1994;Novo et al.,2005). In contrast, larvae at the Zoeal stages II to IV arestenohaline and develop in coastal adjacent area, where salinity isgenerally higher and more constant. Once the Megalopa stage isreached, larvae become euryhaline and re-invade the brackishadult environment, settling in semi-terrestrial habitats near theadults burrows (Charmantier et al., 2002). It is interesting to notethat the occurrence of a phenotypic plasticity in the developmentof the osmoregulatory capacity inN. granulatawas demonstrated.Preceding exposure of eggs and larvae to a reduced salinity (20‰)increased the hyper-osmoregulatory capacity to deal with lowsalinities (5–10‰) in all zoeal stages (Charmantier et al., 2002).This plasticity could have an important adaptive value for thespecies.

Physiological studies performed on adult N. granulatashowed that both males and females are euryhaline, toleratingexposure to low (2–5‰) or high salinities (40‰) for at least acouple of weeks. However, a sexual difference in tolerance toextremely low salinities in laboratory tests (0 and 1‰) isobserved. When the lethal time values for 50% of theindividuals tested are compared, tolerance to 0‰ salinity waslower in females than in males in both summer and winter. Thisdifference is attributed, at least in part, to the smaller size ofadult females (body mass), and consequently their larger mass-specific surface gill area in contact with the hypo-osmoticmedium (Novo et al., 2005).

The main ontogenetic changes in N. granulata tolerance tosalinity are schematically summarized in Fig. 1. Note that thesechanges are closely related to the crab reproductive cycle, aswell as to the ontogenetic changes in its osmoregulatory pattern(see Section 4).

4. Hemolymph osmoregulation patterns

Among estuarine animals, different osmoregulatory patternsare observed. For example, molluscs are osmoconformers whileteleost fish are osmoregulators (Barnes et al., 1988; Schmidt-Nielsen, 1996). In turn, crustaceans are generally osmoregula-tors in low salinities and osmoconformers in high salinities (for

Fig. 1. Ontogenetic changes in the osmoregulatory patterns associated with the ontogenetic development of the estuarine crab Neohelice granulata along itsreproductive cycle. Inserted graphs represent the osmoregulatory patterns for the different developmental stages. Graph axes represent the environmental (x axis) andthe hemolymph (y axis) osmotic concentrations (mOsm/kg H2O). The isosmotic line is represented by the dotted line. Figure was drawn based on information reportedby Boschi et al. (1967), Mañe-Garzon et al. (1974), Bromberg (1992), Miranda (1994), Bromberg et al. (1995), Rieger and Nakagawa (1995), Santos (1999),Charmantier et al. (2002), and Novo et al. (2005).


review: Péqueux, 1995; Kirschner, 2004). However, adultN. granulata is clearly characterized as a hyper–hypo-osmor-egulating crab (Bromberg, 1992; Miranda, 1994; Novo et al.,2005). As described for the salinity tolerance, ontogeneticchanges in the osmoregulatory pattern of N. granulata are alsoobserved.

Osmoregulatory patterns showed by N. granulata strictlymatch with the ontogenetic changes occurring in the crabecology. Zoea I larvae hatching in brackish estuarine waters areslight hyper-regulators at intermediate salinities (10–17‰) andhyper-osmoconformers at higher salinities, while Zoeae II to IVdeveloping in coastal adjacent area are generally hyper-osmoconformers. After metamorphosis to Megalopa, theosmoregulation pattern changes to hyper–hypo-regulation, andthe osmoregulatory capacities under both hypo- and hypersalineconditions strongly increase in Crab I and throughout the laterjuvenile development. Crab ability to re-invade the brackishadult environment only occurs with the first ontogeneticappearance of the hyper–hypo-regulation in the Megalopastage (Charmantier et al., 2002). At this time, larvae must in factto cope with important changes in environmental salinity, whichare typical in mudflats and salt marshes. Studies performed onadult N. granulata showed that both males and females arecapable of hyper-osmoregulation in low salinities (2–5‰) and

hypo-osmoregulation in high salinity (40‰), with an isosmoticpoint close to 29‰ salinity (Bromberg, 1992; Miranda, 1994;Novo et al., 2005).

The main ontogenetic changes occurring in the osmoregu-latory pattern of N. granulata are schematically summarized inthe Fig. 1. Note that these changes are closely related to the crabreproductive cycle and its tolerance to salinity.

5. Hemolymph ionoregulation pattern

In N. granulata, studies on hemolymph ionoregulation wereonly performed on adults. As observed in other euryhalinecrustaceans like Carcinus maenas, Eriocheir sinensis and Cal-linectes sapidus (for review: Péqueux, 1995; Kirschner, 2004),hemolymph hyper-osmotic regulation in males and femalesN. granulata is achieved by hyper-regulation of both Na+ andCl− concentration. In turn, hemolymph hypo-osmotic regulationis achieved by hypo-regulation of bothNa+ and Cl− concentration(Mañe-Garzon et al., 1974; Bromberg, 1992; Miranda, 1994;Bromberg et al., 1995; Santos, 1999; Novo et al., 2005).

As for salinity tolerance, N. granulata ability to regulatehemolymph osmotic, Na+ and Cl− concentrations is dependenton season and sex. In summer, adult males show a better osmoticand ionoregulatory capacity in low salinity (Mañe-Garzon et al.,


1974; Bromberg, 1992; Miranda, 1994; Bromberg et al., 1995;Santos, 1999). Furthermore, they have a better capacity to hyper-regulate both hemolymph osmolality and Na+ concentrationthan females, especially in winter. However, adult females showa better capacity to hypo-regulate both hemolymph osmolalityand Na+ concentration than males in both summer and winter(Novo et al., 2005). Also, the dynamics of the osmotic and ionicregulation is seasonally variable. Females show larger and fastervariations in osmolality and ion (Na+ and Cl−) concentrationsafter 2 days of hypo-osmotic stress (20→5‰) in winter than insummer, while males have an opposite response. Furthermore,males achieve faster the hemolymph osmotic and ionic steadystate after the hyper-osmotic stress (20→40‰) in winter than insummer (Novo et al., 2005). These seasonal differences in crabresponse to environmental salinity changes are explainedconsidering changes ion permeability and active transportassociated with seasonal changes in lipid composition andphysiology at the gills level (Santos, 1999).

Adult N. granulata hyper-regulates hemolymph K+ concen-tration in salinities 5 and 20‰ and conforms it in salinity 40‰(Miranda, 1994; Novo et al., 2005). Only few studies have dealtwith hemolymph K+ regulation in euryhaline crustaceans.However, the K+ hyper-regulation observed in N. granulata atsalinities up to 20‰ is similar to that shown by freshwaterorganisms. On the other hand, a no clear pattern of regulation ofhemolymph K+ concentration in seawater can be observed incrustaceans, even among crabs. Ionoconformation, as observedin N. granulata, was also reported in the grapsoid crab Pa-chygrapsus marmoratus. On the other hand, hyper-regulationwas observed in the stenohaline crab Maia squinado and theeuryhaline crabs C. maenas and Dromia vulgaris, while hypo-regulation was reported in the lobster Nephrops norvegicus(Barnes et al., 1988; Schmidt-Nielsen, 1996). Therefore, furtherstudies are needed to check if a pattern of hemolymph K+

regulation is present or not in euryhaline crabs, and if exists,biochemical and physiological studies would be necessary toidentify and characterize the mechanism(s) involved.

As described for other euryhaline crustaceans likeC. maenas,E. sinensis, and C. sapidus (for review: Péqueux, 1995;Kirschner, 2004), the posterior gills of N. granulata are themain sites for uptake of Na+ and Cl− in diluted media. Evidencesfor that come from data of structural, enzymatic, iontransport, eletrophysiological, and molecular biology studies(Luquet et al., 2000; Schleich et al., 2001; Luquet et al., 2002a;Luquet et al., 2002b; Tresguerres et al., 2003; Kirschner, 2004;Genovese et al., 2004; Luquet et al., 2005). The active uptakeof Na+ and Cl− serves to counteract the diffusive losses of Na+

and Cl− occurring under hypo-osmotic condition, thuscontributing to maintain the ionic and osmotic balances atthe hemolymph level. Regarding the hypo-osmoregulatoryability of N. granulata, it has been shown by structural,enzymatic, ion transport, eletrophysiological, and molecularbiology studies that gills are also involved in the acclimation tohigh salinity in this crab species (Luquet et al.; 2002b;Genovese et al., 2004; Luquet et al., 2005). However, datagenerated are less conclusive and we cannot at present have aclear picture of the mechanisms involved in the hypo-

regulation in N. granulata. Most of these studies will bepresented and discussed in detail in the following sections.

6. Gill morphology and structure

As reported for other euryhaline crabs like C. maenas andE. sinensis (Taylor and Taylor, 1992; Péqueux, 1995; Kirschner,2004; Freire et al., 2008), the mechanism of ion transport in N.granulata is in the three posterior pairs of gills, while the threeanterior ones are specialized for both respiration and activeammonia excretion (Genovese et al., 2000; Luquet et al., 2000,2002a).

In brachyuran crabs, the epithelium of posterior gills iscomposed by amonolayer of cells sat on the cuticle in each side ofthe ion-transporting gill lamella, where aggregates of ionocytescan be found. These cells are directly responsible for the iontransport across the gills. On the other hand, the pillar cells arepresent in the gill epithelium forming columns regularly disposedin the hemolymphatic space. This feature creates a series oflacunas that allows the hemolymph flow through the lamella.Generally, the apical surface of the ionocytes is enlarged by thepresence of multiple evaginations, while its basolateral surface ischaracterized by marked membrane invaginations. Numerousmitochondria are logged in both membrane systems (Copelandand Fitzjarrell, 1968; Freire et al., 2008). The ion-transportingproteins, like the sodium pump (Na+/K+-ATPase), are closelyassociated with the basolateral invaginations facing the hemo-lymphatic space (Copeland and Fitzjarrell, 1968; Compère et al.,1989; Towle and Kays, 1986; McNamara and Torres, 1999; Freireet al., 2008). These evidences clearly indicate that the ability ofbrachyuran crabs to cope with environmental salinity changes isdirectly associated with the activity of the ionocytes. Therefore,significant changes in gill morphology and structure, similar tothose described above, also should be observed in posterior gills ofN. granulata following acclimation to the environmental salinity.

In N. granulata, three different gill epithelia are recognized(Luquet et al., 2000): (1) a thin epithelium (2.42 µm thick)lining the whole lamellae in the three anterior gills; (2) a thickcuboidal epithelium (6–12.5 µm thick) covering part of gills 4and 5 and most of the three most posterior gills; and (3) anattenuated epithelium (0.5–1.5 µm thick) bordering themarginal channels of all the gills.

As observed for other euryhaline crustaceans (for review:Péqueux, 1995), epithelium thickness in anterior gills (1–3) ofN. granulata does not significantly change during salinityacclimation. In crabs acclimated to 12, 34 and 44‰ salinity, itranges from 1.27 to 2.46 µm. On the other hand, epithelialthickness slightly increases in medial gills (4–5) and signifi-cantly increases up to 8.10 µm in posterior gills (6–8) afterexposure to hypo-osmotic (12‰ salinity or hyper-osmotic(44‰ salinity) media, when compared to the epithelialthickness observed in crabs acclimated to seawater (34‰salinity). These gill morphometric changes are associated to anaugmented cell size rather than to an increased cell proliferation.Furthermore, putative ion-transporting epithelia are associatedwith epithelial regions with higher thickness; especially in crabsacclimated to hypo- (12‰ salinity) or hyper-osmotic (44‰


salinity) media. Therefore, posterior gills have been suggestedto be involved in both hyper- (salt uptake) and hypo-regulation(salt secretion) in N. granulata, since they are mostly lined witha tick tissue, showing the basic characteristics of a salt-transporting epithelium (Genovese et al., 2000).

In gill 5, three cellular types are described: (1) the pillar cells,which possess mitochondria packed between membrane foldsonly in their interdigitations with neighboring cells; (2) type-Icells (8.0 µ thick) with low density of mitochondria; and (3)type-II cells (9.9 µm thick) rich in mitochondria, but lackingbasolateral interdigitations. The significant increase in epitheliumthickness of the dorsal portion of gill 6, observed as a function ofan environmental salinity increase, is paralleled by the appearanceof a high density of mitochondria, which are packed betweenextensive basolateral membrane interdigitations in ionocytes andpillar cells (Genovese et al., 2004). In crabs acclimated to highsalinity (45‰), the subcuticular space delimited by the infoldingof the apical membrane increases and septate junctions areshorter, suggesting that the salt secretion could be take place viathe paracellular pathway (Luquet et al., 2002a).

In summary, the development of an ion-transportingepithelia is observed in the posterior gill epithelium of N.granulata under both hyper- and hypo-osmotic conditions. Thisdevelopment is reflected by an increase in the epithelium thick-ness due to an increase in cell size. Ultrastructural changesassociated with acclimation to hypo- or hyper-osmotic condi-tions include the development of basolateral membrane interdi-gitations rich in mitochondria and conspicuous apical membraneinfoldings, as well as increase of the subcuticular space. Thesechanges are schematically depicted in Fig. 2 and are quite similarto those reported for other euryhaline crustaceans as C. maenasand E. sinensis (for review: Péqueux, 1995).

7. Enzymes associated with hemolymph iono- andosmoregulation

The structural adjustments occurring in posterior gills of N.granulata following environmental salinity changes are paral-leled by biochemical adjustments. Marked changes in the

Fig. 2. Scheme of main structural features of the posterior gills of Neohelice granula(B). C = cuticle; SS = subcuticular space; AM= apical membrane; AI = apical infoldingsintracellular fluid. Figure was drawn based on information reported by Genovese et al.

specific activity of several enzymes related to hemolymph iono-and osmoregulation are observed in N. granulata in response toosmotic stress.

Biochemical and molecular evidences clearly indicate theinvolvement of the gill Na+/K+-ATPase (GenBank accessionnumber AF548369) in the active Na+ and Cl− uptake andsecretion taking place at the posterior gills of hyper- and hypo-osmoregulating N. granulata. Despite that all gills (anterior andposterior gills) exhibit a salinity-dependent Na+/K+-ATPaseactivity, the pattern of response to environmental salinity isdifferent among them. In fact, the highest enzyme activity is foundin the posterior gills (6 to 8) (Schleich et al., 2001), which exhibitan increased activity after crab acclimation to reduced salinity(Castilho et al., 2001; Schleich et al., 2001;Genovese et al., 2004).Posterior gills of low salinity-acclimated crabs also have a higheraffinity for Na+ than anterior ones (Castilho et al., 2001). Asignificant increase in the Na+/K+-ATPase activity can even alsobe observed in vitro after perfusion of isolated posterior gills ofcrabs acclimated to high salinity with a hypo-osmotic saline(Tresguerres et al., 2003). In addition, expression of Na+/K+-ATPase α-subunit mRNA in posterior gills of crabs acclimatedto an isosmotic condition is strongly responsive to externalsalinity. During acclimation to diluted seawater, a 35 to 55-foldincrease in Na+/K+-ATPase α-subunit mRNA is observed,while acclimation to concentrated water induces a lower res-ponse (25-fold increase). In fact, a complex pattern oftranscriptional regulation is working in N. granulata. It isdependent upon the direction of salinity change and thedevelopmental background of the gills (Luquet et al., 2005).

As opposite to the posterior gills, data on enzyme activityadaptations for anterior gills are scarce and quite controversial.Schleich et al. (2001) and Castilho et al. (2001) reported ahigher Na+/K+-ATPase activity in anterior gills (1 to 5) duringacclimation to low salinity. On the other hand, Genovese et al.(2004) showed an opposite response, i.e., a reduced enzymeactivity at the gill 5 in crabs acclimated to a low salinity (10‰).Therefore, further research is needed to characterize a pattern ofNa+/K+-ATPase response during acclimation to changes inenvironmental salinity in anterior gills, if it exists.

ta acclimated to isosmotic condition (A) or after hyper- or hypo-osmotic stress; BM= basolateral membrane; BI = basolateral infoldings; M =mitochondria; IF =(2000), Luquet et al. (2000, 2002a), and Genovese et al. (2004).


In addition to the role of the Na+/K+-ATPase, other enzymeslike carbonic anhydrase, V-H+-ATPase (GenBank accessionnumber AF189783), arginine kinase, and alkaline phosphataseare involved in the ionic and osmotic regulation in N. granulata.Regarding the carbonic anhydrase, it is present in bothmicrosomal and cytosolic fractions of gills (Mañanes et al.,2000). However, the highest specific activity is found in themicrosomal fraction of anterior gills and in the cytosolic fractionof posterior gills (Genovese et al., 2005a). As for Na+/K+-ATPase, carbonic anhydrase activity of all gills (anterior andposterior gills) is also dependent on the environmental salinity.The pattern of carbonic anhydrase distribution among gillschanges from a more uniform distribution when crabs are osmo-and iono-conforming their body fluids to a highest enzymeactivity in posterior gills (6–8) when crabs are hyper-regulatingthese fluids (Mañanes et al., 2000; Genovese et al., 2005a).Therefore, a differential involvement of the carbonic anhydrasepresent in anterior and posterior gills in the ionoregulatorymechanisms is suggested to occur in N. granulata.

Regarding other enzymes, it seems that a vacuolar-type H+-ATPase (V-H+-ATPase) could be also involved in the iontransport across the posterior gills of N. granulata. The firstevidence of this involvement was only recently reported. It wasshown that bafilomycin A1, a specific inhibitor of the V-H+-ATPase, induced a significant decrease (77%) in the transe-pithelial potential difference (Vte) of isolated and perfusedposterior gills (Genovese et al., 2005a). A high expression of themRNA encoding for the V-H+-ATPase B-subunit was alsoreported in posterior gills of N. granulata using the quantitativereal-time PCR technique (Luquet et al., 2005). This highexpression was paralleled by a high expression of the mRNAencoding for the housekeeping protein arginine kinase, anenzyme implicated in the energy cellular flow. It is interesting tonote that a high expression of both V-H+-ATPase and argininekinase in hypo-regulating gills seems to be of commonoccurrence in euryhaline crustaceans. In fact, similar resultswere also reported for the V-H+-ATPase in the Chinese mittencrab E. sinensis (Weihrauch et al., 2001) and the arginine kinasein the shore crab C. maenas and the blue crab C. sapidus(Kotlyar et al., 2000). However, the role of these enzymes in theionic regulation in N. granulata is still not completely under-stood. Although high levels of mRNAs expressions wereobserved, only modest changes in these expressions wereobserved after transfer of crabs from isosmotic conditions (30‰salinity) to either diluted (2‰ salinity) or concentrated (45‰salinity) seawater. Therefore, the up-regulation of both argininekinase and V-H+-ATPase observed under both hyper- and hypo-regulatory conditions does not seem to be related to ionic andosmotic regulation in N. granulata. It would be more likelyassociated with a higher demand of H+ extrusion from the cell, asa consequence of a possible increased metabolic rate induced bythe salinity changes, in order to maintain the hemolymph acid-base regulation. This statement is based on the following facts:(1) the involvement of the H+-ATPase/Na+ channel system in theNa+ uptake at the apical membrane of the gill cells has beenmoreaccepted to occur in crabs living in freshwater or adapted to lowsalinity (Onken and Putzenlechner, 1995; Weihrauch et al.,

2004); (2) in euryhaline crabs where H+-ATPase activity wasreported to occur in posterior gills, the amount of enzymemeasured was very low when compared to that found infreshwater crabs (Kirschner, 2004); (3) adult N. granulatatolerates very low salinities (2–5‰) for at least a couple ofweeks in the laboratory, however survival is hardly maintainedin freshwater and only for a couple of days (Novo et al., 2005);(4) up to now, there are no reports in the literature on the naturaloccurrence of N. granulata in freshwater; (5) significantchanges in lipid (Luvizotto-Santos et al., 2003), carbohydrate(Santos and Nery, 1987; Da Silva and Kucharski, 1992; Neryand Santos, 1993; Schein et al., 2004; Schein et al., 2005b), andprotein metabolism (Schein et al., 2005a) have been reported tooccur in N. granulata as a function of environmental salinitychanges; (6) as a result of the metabolic adjustments required forsalinity acclimation, changes in oxygen consumption rates havebeen reported in juveniles and adult N. granulata (Santos et al.,1987; Bianchini and Castilho, 1999). Therefore, the observeddecrease in Vte after inhibition of the H

+-ATPase activity inducedby bafilomycin A1 (Genovese et al., 2005a) would be explainedby changes in the activity of the mechanisms of ion transportassociated with the consequent changes in intracellular pH.

8. Mechanisms of gill NaCl transport

N. granulata is a semi-terrestrial crab showing bimodalventilation. This physiological feature allows this euryhaline crabto expend long periods on land (Halperin et al., 2000). Because ofthis ecological feature,N. granulata is subjected to important andsudden changes in the environmental salinity. For example,rainfall or evaporation can generate supratidal pools containingvery diluted or hyper-concentrated water. Also, crab gills can beexposed to water at high salinities, as a result of the waterevaporation and salt concentration in the gill chamber duringemersion (Schmitt and Santos, 1993). In fact, an increasedconcentration of Na+ is observed in the hemolymph, while adecreased concentration is observed in the water chamberduring crab emersion (Luquet and Ansaldo, 1997). Therefore,N. granulata gills must be able to deal with such changes andeven to quickly switch from a salt absorptive pattern to a saltsecretive pattern or vice-versa.

Previous studies with euryhaline crabs demonstrated thepresence of ionic transport pathways from ambient medium tothe gill epithelial cells (Péqueux, 1995; Henry, 2001; Kirschner,2004). There is conflicting evidence concerning the exactmechanism of branchial ion transport, but the current ideas canbe summarized as follows.

Na+ uptake across the apical membrane of the gill cellsoccurs via a combination of Na+/H+ and Na+/NH4

+ exchanger(Henry, 2001; Kirschner, 2004), and possibly also via Na+

uptake linked to H+ extrusion by an apical H+-ATPase, as it isbelieved to occur in fish gills (Henry, 2001; Hwang and Lee,2007). As discussed above, the involvement of the H+-ATPase/Na+ channel system in the Na+ uptake at the apical membraneof the gill cells has been more accepted to occur in crabs livingin freshwater or adapted to low salinity, such as Dilocarcinuspagei and E. sinensis (Onken and Putzenlechner, 1995;


Weihrauch et al., 2004). However, previous studies also showedthe presence of H+-ATPase activity in posterior gill of theeuryhaline crabs C. maenas and Uca tangeri, yet that theamount of enzyme in both species was quite low, being only 5 to10% of that found in E. sinensis (Kirschner, 2004). In fact, theNa+/H+ exchanger has been reported to play a major role in theNa+ exchange by H+ at the apical membrane of the gill cells inmoderate strong regulator crabs like C. sapidus and C. maenas(Cameron, 1978; Towle et al., 1997; Henry, 2001). Regardinganion transport, data available clearly indicate that the apicaluptake of Cl− from the external medium to the gill cells takesplace, at least in part, via the Cl−/HCO3

− exchanger (Péqueux,1995). In both cases, carbonic anhydrase activity is providingH+ and HCO3

− as counterions for Na+ and Cl− uptake, res-pectively (Henry, 2001).

In addition to the mechanisms of apical Na+ and Cl−

transport described above, it has also suggested a possibleinvolvement of the Na+,K+,2Cl− cotransporter in monovalention uptake by crustacean gills (Riestenpatt et al., 1996; Henry,2001). In accordance, Kirschner (2004) proposed that a Na+,K+,2Cl−-mediated Cl− uptake pathway might exist in brackishwater crabs.

Disregarding the mechanism of Na+ and Cl− operating at theapical membrane of the gill epithelium, Na+ is actively pumpedout of the gill cell into the hemolymph, in exchange of K+, bythe Na+/K+-ATPase located at the basolateral membrane of thegill cells (Péqueux, 1995; Towle et al., 2001; Lucu and Towle,2003). In turn, transport of K+ and Cl− from the gill cell to thehemolymph occurs passively down an electrochemical gradientthrough K+ and Cl− channels, respectively. Therefore, the maindriving force for the electrogenic ion uptake involving themechanisms of gill ion transport in euryhaline crabs is providedby the Na+/K+-ATPase activity (for review: Péqueux, 1995;Kirschner, 2004).

Data obtained using electrophysiological techniques showthat posterior gills of N. granulata generate a negative Vte ineither low or high salinity-acclimated crabs. In the presentreview, the Vte is reported considering the hemolymph siderespect to bath side. In low salinity-acclimated crabs, the Vte

measured in isolated and perfused posterior gills of N. granulatawas 0.4±0.7, −10.2±1.6, −10.8±1.3, and −6.7±1.3 mV forgills 5, 6, 7 and 8, respectively. The Vte of the gill 6(representative of posterior gills) was significantly inhibited byouabain (69%) and BaCl2 (60%). Furthermore, a net ouabain-sensitive influx of Na+ (1150±290 µEq/g/h) was associated withthat Vte (Luquet et al., 2002b). In turn, Onken et al. (2003)reported that the split lamellae of posterior gills of crabsacclimated to 2‰ salinity, mounted in a modified Ussingchamber and bathed on both sides with a saline containing NaCl,showed a Vte of −4.1±0.5 mV. After voltage clamping, anegative short-circuit current (Isc) was measured (−142±21 µA/cm2) at a conductance (Gte) of 44±5 mS/cm2. In high salinity-acclimated crabs, posterior gills (gill 6) isolated and perfusedwith saline solution of composition similar to the hemolymph ofcrabs acclimated to 30 or 40‰ salinity produced a Vte of −1.5±0.1 or −1.3±0.09 mV. Vte generated by posterior gills (gill 6, 7or 8) significantly increased after reducing the concentration of

both bathing and perfusion salines (from 30‰ to 20‰ or 10‰salinity) (Luquet et al., 2002b). These data clearly show that theVte generated by posterior gills is salinity-dependent, indicatingthat a net ion uptake or secretion is occurring in hyper- and hypo-osmoregulating N. granulata, respectively. Furthermore, Vte

measured in isolated and perfused posterior gills of N. granulatais similar to that reported for other estuarine crabs showingmoderate ability to regulate, like C. maenas and C. sapidus.However, it is much lower than those reported for strongregulator crabs like E. sinensis, which are adapted for living infreshwater (for review: Péqueux, 1995). These evidencessuggest that the Vte generated by euryhaline crustaceans isgenerally related to the species osmoregulatory ability.

Evidences from experiments using pharmacological, eletro-physiological and radiotracer techniques suggest that mechan-isms of ion transport involved in the net ion uptake andsecretion taking place at the posterior gills of hyper- and hypo-osmoregulating N. granulata can be fitted into the frameworkdescribed above for other euryhaline crustaceans. In fact, theelectrogenic ion uptake and secretion across the posterior gillsof N. granulata is associated with a net ouabain-sensitive Na+

influx and efflux, respectively (Luquet et al., 2002b).In posterior gills of hyper-osmoregulating N. granulata, a

high conductance epithelium is performing an active absorptionof NaCl in a coupled way by an electrogenic mechanism similarto that seen in the thick-ascending limb of Henle's loop. In thiscase, the Na+/K+-ATPase (GenBank accession numberAF548369) present in the basolateral membrane of the posteriorgill epithelium generates the major driving force for Na+ uptake(Luquet et al., 2002b; Onken et al., 2003; Luquet et al., 2005).Then, a “pump-and-leak” system would be working on thebasolateral membrane of posterior gills of N. granulata, asdescribed above for other hyper-osmoregulating crustaceans(for review: Péqueux, 1995; Kirschner, 2004). Therefore, a Na+/K+ pump is moving Na+ from the gill intracellular fluid to thehemolymph and K+ in the opposite direction, generating achemical gradient for Na+ from the hemolymph to the gillintracellular fluid and a chemical gradient for K+ in the oppositedirection. Thus, K+ leaves the gill intracellular fluid and reachesthe hemolymph through K+ channels at the basolateralmembrane, driven by its chemical gradient. There are noevidences available on the possible mechanisms involved in Cl−

transport from the intracellular fluid to the hemolymph inN. granulata. However, one can speculate that basolateralCl− channels could play this role. On the other hand,electroneutral mechanisms would be involved in Na+ and Cl−

movements from the water to the intracellular fluid through theapical membrane. Evidences from studies applying pharmaco-logical tools suggest that these movements would occurthrough Na+/H+ and Cl−/HCO3

− antiporters, respectively(Onken et al., 2003; Genovese et al., 2005a). In addition, datafrom molecular biology studies indicate that the apicalmovement of Na+ would involve the cotransporter Na+,K+,2Cl−. In fact, expression of the mRNA encoding for thiscotransporter is strongly responsive to external salinity,expression increasing by 10–20-fold in the first 24 h ofhypo-osmotic shock (30 to 2‰ salinity) (Luquet et al., 2005).


Functional hypothetical models depicting the mechanisms ofion transport involved in the active NaCl uptake in posterior gillsof hyper-osmoregulating N. granulata are shown in Fig. 3.These models consider not only the involvement of the differention transporters discussed above, but also the activity of theenzymes associated with the hemolymph iono- and osmoregula-tion in hyper-osmoregulating N. granulata, as previouslydiscussed. According to these models, in fairly high salinities(~20‰), a Na+ gradient between the environment and theionocytes generated by the activity of a basolateral Na+/K+

pump would be the driving force to operate a Na+,K+,2Cl−

cotransporter at the apical membrane of the cell. It is interestingto note that this transporter is electroneutral and thus cannot usethe apical potential difference and is an economical system, i.e.,it does not use ATP. In addition to the Na+/K+ pump, thiscotransporter also requires the operation of the Cl− channels atthe basolateral membrane of the gill cells (Fig. 3A). At salinitieslower than 10% seawater, K+ becomes limiting, and anothersystem than the Na+,K+,2Cl− cotransporter is required for hyper-regulation of NaCl in N. granulata. In the range of 5 to 20%seawater, the coupled Na+/H+ and Cl−/HCO3

− exchangers canfunction, again using the gradients as driving forces. Asdiscussed before, the Na+/K+ pump and the Cl− channels arestill necessary at the basal membrane of the gill cells, and noATPwould be used at the apical membrane of the cells (Fig. 3B).

On the other hand, the picture for hypo-osmoregulatingN. granulata is quite different from that described above forthe hyper-osmoregulating crab. Only few studies have dealt withthe mechanisms involved in the NaCl secretion in N. granulataacclimated to high salinities. In fact, there is still no modelavailable integrating the mechanisms involved in NaCl secretionin hypo-regulating crustaceans. In hypo-regulating (marine) fish,

Fig. 3. Functional hypothetical models depicting the mechanisms of ion transportNeohelice granulata acclimated to intermediate (A) or low (B) salinities. H+-AThemolymph ionic and osmotic regulation. See text for further details. CA = carbonic(2002a,b), Onken et al. (2003), Genovese et al. (2005a), and Luquet et al. (2005). Gfigure are: Na+/K+-ATPase (AF548369); Na+,K+,2Cl− cotransporter (AF548368) and

a basolateral Na+,K+,2Cl− cotransporter is mediating the Cl− fluxfrom the blood to the cytosol of the ion-transporting gill cells,which is in turn driven by the Na+/K+-ATPase activity (Evans,2002). In this current model for marine fish, Cl− extrusion fromthe gill cell to the surrounding medium would occur throughapical channels, possibly the cystic fibrosis transmembraneregulator protein (Singer et al., 1998). This Cl− efflux generatesan outside negativeVte, which would drive a Na

+ efflux through aparacellular route (Evans, 2002).

As observed in marine fish, an electrogenic ion secretionacross the posterior gills of N. granulata is associated with a netouabain-sensitive Na+ efflux (Luquet et al., 2002b), indicatingthat a Na+ gradient between the hemolymph and the ionocytes isgenerated by the activity of a Na+/K+ pump (Na+/K+-ATPaseactivity). As observed in marine fish, molecular evidencesindicate that the Na+,K+,2Cl− cotransporter (GenBank accessionnumber AF548368) is also involved in the NaCl secretion in theposterior gills of N. granulata. The expression of the mRNAencoding for this cotransporter was strongly responsive to salinitychange, increasing by 60-fold after 96 h of hyper-osmotic shock(30 to 45‰) (Luquet et al., 2005). Therefore, the Na+ gradientgenerated by the Na+/K+ pump would be the driving force tooperate a Na+,K+,2Cl− cotransporter at the basolateral membraneof the cell. In addition to the Na+/K+ pump, this cotransporter alsorequires the operation of Cl− channels. However, as opposed tomarine fish, Vte measured in posterior gills of N. granulata isgenerally negative inside (hemolymph respect to seawater). Thisfact indicates that Na+ and not Cl− is actively secreted across thegill epithelium. Therefore, differently from marine fish, a higherexpression of Na+/K+ pumps (Na+/K+-ATPase activity) and Cl−

channels would be occurring at the paracellular side of theionocytes and not at their basolateral membranes (Fig. 4). In fact,

involved in the active NaCl uptake in posterior gills of hyper-osmoregulatingPase seems to be more involved in hemolymph acid-base regulation than inanhydrase. Figures were drawn based on information reported by Luquet et al.enBank accession numbers available for some of the transporters shown in theV-type ATPase (AF189783).


data from structural analysis support the idea that the salt secretionin posterior gills of N. granulata could be take place via theparacellular pathway (Luquet et al., 2002a). A functionalhypothetical model depicting the mechanisms of ion transportinvolved in the active NaCl secretion in posterior gills of hypo-osmoregulating N. granulata is shown in Fig. 4. This modelconsider not only the involvement of the different ion transportersdiscussed above, but also the activity of the enzymes associatedwith the hemolymph iono- and osmoregulation in hypo-osmoregulating N. granulata, as previously discussed. However,it is clear that further evidences confirming the presence of bothNa+/K+-ATPase activity and Cl− channels at the paracellular sideof the ionocytes, as well as the occurrence of a basolateral Na+,K+,2Cl− cotransporter in the posterior gill of N. granulata areneeded to support our hypothetical model (Fig. 4).

9. Mechanism of ion transport regulation

Data available indicate that hemolymph osmolarity would bethe primary stimulus to trigger the mechanisms involved in ionuptake regulation taking place at the posterior gills of N.granulata. It regulates the active transbranchial NaCl absorptionby modulating the activity of the basolateral Na+/K+-ATPaseand by changing a conductive pathway, probably at the apicalmembrane. These findings come from studies on the influence ofshort-term osmotic variations on some electrophysiological

Fig. 4. Functional hypothetical model depicting the mechanisms of ion transportinvolved in the active NaCl secretion in posterior gills of hypo-osmoregulatingNeohelice granulata. CA = carbonic anhydrase. Figures were drawn based oninformation reported by Luquet et al. (2002a,b), Genovese et al. (2005a), and Luquetet al. (2005). See text for further details. GenBank accession numbers available forsome of the transporters shown in the figure are: Na+/K+-ATPase (AF548369); Na+,K+,2Cl− cotransporter (AF548368) and V-type ATPase (AF189783).

properties of the posterior gills, such as Vte of isolated andperfused posterior gills, Isc and Gte measured in split gilllamellae mounted in a modified Ussing chamber, and Na+/K+-ATPase activity measurements (Tresguerres et al., 2003). Theseauthors showed that Vte significantly increased when posteriorgills were perfused with a hypo-osmotic solution (699 mOsm/L)instead of an isosmotic saline (1045mOsm/L). Also, they clearlyshowed that Vte of isolated and perfused gills was not affected bya decrease in the concentration of Na+ or Cl− in the perfusionsaline at a constant osmolarity. Furthermore, both Isc and Gte

were increased after bathing split gill lamellae with hypo-osmotic salines (Isc from −89.0 to −179.3 µA/cm2; Gte from40.5 to 47.3mS/cm2). According to Tresguerres et al. (2003), theobserved effects of reduced osmolarity were fast, reversible andgradually dependent on the magnitude of the osmotic variation.These authors also reported increased Na+/K+-ATPase activityafter isolated gill perfusion with hypo-osmotic saline (from18.73 to 41.84 µmol Pi/h/mg proteins).

Regarding the mechanism of cell signaling during ion uptakeregulation, Tresguerres et al. (2003) reported that part of theelevated Vte induced by gill perfusion with hypo-osmotic salinewas maintained by theophylline, suggesting that an increasedcellular level of cyclic AMP would be involved in the regulationof ion uptake triggered by the hemolymph osmolarity. In fact,Halperin et al. (2004) and Genovese et al. (2005b) reported thataddition of cAMP agonists, such as cp-cAMP, forskolin, andIBMX in the perfusion saline, induced a significant increase inthe Vte of isolated posterior gills and that dopamine (maximumstimulation at 10 µM) had a stimulatory effect on ion uptake,increasing Vte and Na+ influx. These authors also showed thatthe stimulatory effects of forskolin and dopamine on the Na+/K+-ATPase activity correlated well with those on the Vte andNa+ influx. Finally, Halperin et al. (2004) reported that thestimulatory effect of dopamine on Vte was completely abolishedby KT5720, a specific inhibitor of the cAMP-dependent proteinkinase. Taken altogether, these findings clearly indicate that themechanism of regulation triggered by changes in the hemo-lymph osmolarity in N. granulata is mediated by dopamine,while cell signaling would involve a cyclic AMP-dependentprotein kinase. On the other hand, no data is available on thestimulus triggering ion secretion in N. granulata. Furthermore,studies focused on the hypo-regulating N. granulata must beperformed to elucidate the mechanisms involved in cellsignaling during ion secretion regulation.

10. Aquatic pollutants and osmoregulation

The presence of several aquatic contaminants in estuaries canaffect iono- and osmoregulation in estuarine animals, modifyingtheir ability to cope with environmental salinity changes. In fact,it has been demonstrated that metals at sublethal concentrationsaffect mechanisms involved in hemolymph ionic and osmoticregulation in N. granulata. For example, Bianchini and Castilho(1999) showed that the gill Na+/K+-ATPase activity of crabsacclimated to 2‰ salinity was inhibited (~40%) after either invivo or in vitro exposure to sublethal concentrations of zinc(6.12 µM). Lauer et al. (2005) showed that crab exposure (96 h)


to silver (0.42 µM) induced significant inhibition of Na+/K+-ATPase activity at the posterior gills, with a consequentreduction of the hemolymph Na+ concentration. RodríguezMoreno et al. (1998) reported that cadmium inhibited in vitro(EC50=191.5 µM) the gill Na+/K+-ATPase activity in adultmale crabs during the postmolt period. However, no significantinhibition was observed in vivo. In crabs acclimated to 2.5‰salinity, Vitale et al. (1999) reported that the activity of thecarbonic anhydrase, another key enzyme involved in theosmorregulation in N. granulata, was inhibited in vivo(EC50=14.1 µM) by cadmium. These authors also showedthat cadmium (11.1 µM) induced a significantly decrease inhemolymph Na+ concentration. However, no significant effectswere observed on either the enzyme activity or the hemolymphion levels in crabs acclimated to seawater (30‰ salinity). Onthe other hand, Rodriguez et al. (2001) reported increasedhemolymph levels of Na+ and Ca2+ after both acute (96 h) andchronic (2 weeks) exposures of crabs acclimated to 12 and 30‰salinity to cadmium at concentrations ranging from 3.56 to56.04 µM. In vitro studies showed that carbonic anhydraseactivity of crabs acclimated to 2.5‰ salinity was inhibited bycadmium (IC50=21.5 µM), copper (IC50=16.2 µM), and zinc(IC50=3.75 µM) (Vitale et al., 1999).

Besides the metal effects, some organic compounds have alsobeen shown to induce iono- and osmoregulatory impairments inN. granulata. For example, exposure (96 h) to ammonia (3.5 or5.5 mM total ammonia) induced a significant decrease inhemolymph Cl− concentration in crabs acclimated to 20‰salinity. In crabs acclimated to 40‰, a significant increase in thehemolymph osmolarity was observed after exposure (96 h) to5.5 mM total ammonia (Rebelo et al., 1999). In crabs acclimatedto 2‰ salinity, Vinagre et al. (2002, 2003) showed that the Na+/K+-ATPase activity of anterior gills was significantly reducedafter crab injection with microcystin (39.2 µg/L) for 48 h.However, no significant effect was observed in the enzymeactivity at the posterior gills and a lack of effect on hemolymphNa+ and Cl− concentrations was reported.

11. Conclusions and future perspectives

Data available in the literature indicate that biochemical andphysiological adaptations showed by N. granulata to deal withthe osmotic stress imposed by salinity changes frequentlyoccurring in intertidal mud flats and salt marshes are very similarto those reported for other estuarine crabs. The ontogeny of thehemolymph iono- and osmoregulation in N. granulata is directlyrelated to its ecology. The hyper–hypo-regulatory pattern ofosmoregulation observed in adults is achieved by regulating theconcentration of major hemolymph ions (Na+ and Cl).

Posterior gills are the main organs involved in thehemolymph ion- and osmoregulation. Consequently, theyshow marked structural, biochemical and physiological changesaccording to the environmental salinity, resulting in an activeNaCl uptake in crabs acclimated to low salinity (hyper-osmoregulating crabs). On the other hand, mechanismsinvolved in crabs acclimated to high salinity (hypo-osmoregu-lating crabs) are much less well understood. Despite the fact that

data on gill morphology and structure, as well as on hemolymphionic and osmotic composition have been accumulated over thelast decade, little information is still available on the mechan-isms involved in salt secretion in N. granulata. We canspeculate that hypo-regulation would be achieved by an activesecretion of both Na+ and Cl−, which would involve a higherexpression of the Na+,K+,2Cl− cotransporter at the salt-transporting epithelium of the posterior gill. This expressionwould also be triggered by increases in hemolymph osmolarity,as observed at low salinities. However, other active substance(s)than dopamine and cAMP would mediate ion secretionregulation and cell signaling at high salinities, respectively.All these speculations need support from experimental datafrom future studies to be performed in crabs acclimated tohypersaline waters (N30‰).

In light of the above, future studies should be done toaccomplish the following goals: (1) identify the mechanismsinvolved in the hypo-osmoregulation in N. granulata; (2) under-stand the control of the ionic and osmotic regulation and cellularsignaling occurring at the posterior gills level during N. granulataacclimation to hyper-osmotic media; (3) identify the existence of apossible pattern of hemolymph K+ regulation in euryhaline crabs,including N. granulata, and if it exists, then characterize themechanism(s) involved; (4) characterize a pattern of Na+/K+-ATPase response during acclimation to changes in environmentalsalinity in anterior gills, if it exists; (5) evaluate the possible impactof other estuarine contaminants than metals, ammonia, andorganophosphorus pesticides on the ionic and osmotic regulationinN. granulata. Results from these studies would allow us to betterunderstand the patterns of ionic and osmotic regulation in N.granulata and the biochemical and physiological mechanismsinvolved in these processes, as well as to evaluate the possibleimpact of anthropogenic activities on N. granulata populationsliving in areas of risk. In this case, it would be interesting to test thecombined effects of salinity and hydrocarbons or pesticidesbecause of the increasing use and discharge of these compounds inestuaries and salt marshes, which are natural habitats of N.granulata.

Acknowledgements

A. Bianchini and J. M. Monserrat are research fellows fromthe Brazilian CNPq. Some studies reported here were supportedby Brazilian agencies (CNPq, FINEP, CAPES, and FAPERGS),Kodak Co., and International Copper Association.

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http://dx.doi.org/10.1016/j.cbpa.2007.05.008


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Biochemical and physiological adaptations in the estuarine crab Neohelice granulata during salinity...

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