Decapods account for approximately 8% of all terrestrial crustaceans (4,900 species) (Marin and Tiunov, 2013). Land invasion has occurred in numerous independent lineages of brachyuran crabs (Cannicci et al., 2020) as well as by their sister group, Anomura. The high morphological diversity among the families of Brachyura implies that taxonomic relationships within this group are still not fully resolved (Tsang et al., 2014). These crabs have transitioned to a semi-terrestrial or terrestrial environment following pathways from both marine and/or freshwater environments (Watson-Zink, 2021).

This transition from aquatic to terrestrial habitats requires physiological, ecological, and behavioral adaptations to allow the exploitation of these new environmental conditions (Lozano-Fernandez et al., 2016). This “continuum” of invasion from a fully aquatic to semi-terrestrial and terrestrial existence means that brachyuran crabs are one of the best models for studying the evolutionary pathways that have facilitated this. In other words, the ecological and morphological adjustments to life on land can be considered a “snapshot” in evolutionary terms as they are happening “now” (Burggren and McMahon, 1988; Lozano-Fernandez et al., 2016).

Most crustaceans live their entire life in the sea. Some decapod crabs live intertidally and spend short periods of time above water, while others live longer on land, turning occasionally to the ocean. True terrestrial crabs spend their adult life independent of the tidal rhythm, except for larval release (Burggren and McMahon, 1988; Anger, 1995; Turner, 2014). These species typically return seasonally (sometimes only once a year) to deposit their fertilized eggs into the ocean. Eggs hatch immediately on contact with sea water, enabling their early life stage (e.g., planktonic) development to take place in the ocean, just like their marine counterparts. This typically culminates with the emergence of a megalopa form, which undergoes a final metamorphosis to a first stage juvenile crab only when back on land. Crabs invade the land from the sea via the littoral zone and/or via estuarine and freshwater routes (Pearse, 1929; Edney, 1960; Gross, 1964; Powers and Bliss, 1983; Hartnoll, 1988). Some groups of terrestrial crabs have continued this association with freshwater, and instead of a marine larvae, they brood eggs that develop directly into young crabs (Burggren and McMahon, 1988; Cumberlidge, 2016). This strategy is analogous to that of the crayfish where larval forms remain in the eggs (kept ventrally by the female), and post-metamorphic juveniles are released during hatching, which are able to deal with the hypo-osmotic environment (Susanto and Charmantier, 2020). The phylogeny of the Brachyura maps to this pattern (Tsang et al., 2014; Figure 1), with some families containing marine, intertidal, and terrestrial species (e.g., Grapsidae) and others containing freshwater or terrestrial species only (e.g., Potamonautidae, Pseudothelphusidae, and Trichodactylidae); while others only contain semi-terrestrial and terrestrial species (e.g., Gecarcinidae, Dotillidae, and Sesarmidae). This invasion via various terrestrial microhabitats may have occurred due to feeding and behavioral needs. For example, ocypodid crabs directly invaded the land via the littoral zone (intertidal and supratidal zones) or freshwater environments where they carry out deposit feeding. Grapsoid crabs invaded land via estuarine and freshwater routes, where their lifecycle remains (Hartnoll, 1988; Takeda et al., 1996). Six grades of terrestriality have been identified that cover environments from the lower intertidal zone through estuaries and coastal forests, where crabs live in burrows, as well as arid zones (Atkinson and Taylor, 1988; Watson-Zink, 2021). However, despite the routes of invasion, crabs must be adapted for life in a predominantly low-salinity aquatic environment (e.g., freshwater), sometimes in only minimal amounts. The management of salt and water balance (e.g., osmoregulation) is absolutely integral for their survival in these varied environments. However, to truly exploit these areas, these crabs must also be able to undertake “activity” rather than just “survive” away from immersion in water, which is key to defining a true ‘land crab’ (Burggren and McMahon, 1988). In this review, we summarize our current understanding of the morphological and biochemical modifications that have taken place at the branchial chamber and associated excretory organs of semi-terrestrial and terrestrial crabs, which enable them to effectively counter the osmoregulatory challenges in a terrestrial existence. Using knowledge gained from electrophysiology, biochemistry, and more recent molecular biology techniques, we then present updated models for ion transport across all the major taxonomic groups of terrestrial crabs. This is an exciting and fast-moving field of research, and we conclude by highlighting a number of areas requiring further study.

The gills are a multifunctional organ in aquatic crabs. Generally, most brachyuran aquatic crabs have nine pairs of phyllobranchiate gills with distinct morphological differences, with each pair specialized for gas exchange, ion regulation, acid–base balance, and/or ammonia excretion (Freire et al., 2008; Charmantier et al., 2009; Henry et al., 2012). Primarily, respiratory epithelia are characterized by thin cells and ion-transporting epithelia by thick cells (Freire et al., 2008; Taylor and Taylor, 1992). Posterior gills, by comparison, show features of ion regulatory epithelia with thick cells with numerous mitochondria, apical microvilli, and basolateral foldings (Barra et al., 1983; Taylor and Taylor, 1986; Compère et al., 1989). To overcome the osmotic and respiratory challenges associated with terrestrial life, some of the most remarkable physiological, morphological, genomic, and behavioral modifications occur in the gills, with the primary role of the gills in terrestrial crabs being osmoregulation rather than respiration. In most terrestrial crabs, salts and water are recycled from the urine at the gills. Reduction in the number of gill pairs is correlated with the degree of terrestrial adaptation in brachyuran crabs (Gray, 1957; Taylor and Taylor, 1992; Takeda et al., 1996; Lin et al., 2002).

Conversely, however, it is difficult to predict the degree of adaptation to terrestrial life by the number of gills because terrestrial and semi-terrestrial species vary greatly in size as well as phylogenetically (Lin et al., 2002; Table 2). Morphological changes are correlated with the trade-off between gas exchange and ionic regulation in the gills. For example, the semi-terrestrial species Ocypode stimpsoni has a reduced number of anterior gill pairs at the expense of newly developed air-breathing evaginated lungs (Morris, 2002; Tsai and Lin, 2012; Tseng et al., 2020). Gecarcinidae, Grapsidae, and Varunidae species show expanded and smooth lungs (Wolcott and Wolcott, 1991; Farrelly and Greenaway, 2005). In terrestrial Grapsoid and Ocypodidae crabs, the number of anterior gill pairs and surface area are reduced, and the posterior surface area is increased (Marin and Tiunov, 2013; Tseng et al., 2022). The terrestrial species Gecarcinus lateralis and Orisarma dehanii (as Chiromantes dehaani) have eight or nine pairs of gills, similar to marine, intertidal, and bimodal species (Copeland and Fitzjarrell, 1968; Takeda et al., 1996; Tseng et al., 2020). Terrestrial crab species such as Geograpsus grayi, Geograpsus crinipes, Gecarcoidea natalis, and Tuerkayana celeste (as Cardisoma hirtipes) show a reduced planar gill surface as well as possessing nodules that maintain wide spacing between the lamellae (Farrelly and Greenaway, 1992). Reduction in the surface area and thickening of the gill area impair diffusive ion losses. In bimodal crustaceans, the gills partly lose their role in respiration due to the development of “lungs,” which facilitate oxygen uptake from the air, with the modified gills used mostly for osmo/ionic regulation and CO₂ excretion (Farrelly and Greenaway, 1992). Water-breathers require thin gill lamellae with a large surface area to obtain adequate oxygen, while, in air-breathers, the lamellae are stiff and well-spaced to facilitate effective ventilation and rapid draining after immersion. The lamellae of the gills of terrestrial crabs have chitinous ridges which keep them functional in air. These are examples of the additional respiratory structures developed as a consequence of the new environmental conditions encountered on land (Diaz and Rodriguez, 1977). Parallel development is seen in other air gills such as in the book lungs of Araneida (Pohunkova, 1969). In the gills and branchiostegites of the terrestrial crabs Gecarcinus and Birgus latro, O₂ diffuses directly from air, and branchiosteites are able to excrete CO₂ directly into the air via carbonic anhydrase (CA) (Henry, 1994). In this group of crustaceans, the phyllobranch gill surface area is found to be reduced, and the surface area of the branchiostegites is found to be increased to reduce the diffusion distance of branchiostegal tissue compared to that of gills (Henry, 1994; Farrelly and Greenaway, 2005). Coenobitids have developed a third respiratory organ, the abdominal lung, which is formed from highly vascularized patches of a very thin and intensely folded dorsal integument. Adult B. latro, which does not inhabit a gastropod shell, has taken this modification a step further and has developed a highly complex branchiostegal lung that is expanded laterally and evaginated to increase the surface area. The blood/gas diffusion distance is short, and oxygenated blood is returned directly to the pericardium via pulmonary veins. The presence of a protective mollusk shell in terrestrial hermit crabs has favored the evolution of an abdominal lung, which explains why in its absence a branchiostegal lung has been developed in B. latro (Farrelly and Greenaway, 2005).

Summary: The primary role of the gills in terrestrial crabs is osmoregulation rather than respiration. In most terrestrial crabs, salts and water are recycled by gills from the urine, and the surface area of gills per weight is reduced compared to that in aquatic crustaceans. The gill lamellae are stiffened and thickened.

During migration of crabs to the land, both in evolutionary terms and through ontogeny, the most crucial challenge is prevention of the dehydration of extracellular and intracellular compartments. In arthropods, water is contained in the three main body compartments, namely, the head, thorax, and abdomen; the hemolymph and other body fluids, the cuticle of the exoskeleton, and various other tissues (muscle, alimentary canal, fats, etc.). Free or bulk water is lost by heating. In comparison, second, structured-bound water is closely associated with membranes, proteins, and nucleic acids, and visceral water, which includes all structured water layers, is only transiently bound to molecular structures. The intracellular compartment is roughly twice as large as the extracellular compartment. Intracellular fluids represent approximately two-thirds of the total body weight. Volume depletion (extracellular fluid volume deficit) is induced by loss of sodium and water, whereas water loss can result in hypertonicity (Drost- Hansen, 1971; Drost- Hansen, 1976).

In terrestrial brachyurans, water is retained within the branchial chambers, which allows ion uptake in the gills. When terrestrial crabs do not drink water, hemolymph volume decreases and plasma osmolality increases, resulting in dehydration of extracellular and transcellular compartments. In terrestrial crabs, the dominant route of desiccation is across the cuticle and gills. In most insects and crabs, the body surface is relatively leaky with transcellular water loss. When crustaceans are exposed to desiccation, gas exchange decreases due to gill lamella collapse, reducing the diffusional surface area (Morris and Oliver, 1999). Invasion of terrestrial environments from aquatic environments necessitates the need for optimal regulation of extracellular and intracellular compartments. Active ion transport systems are often associated with maximizing ion uptake at these surfaces and generation of electrical potential gradients. However, extracellular and intracellular osmoconcentrations must be aligned to avoid cellular disruption. The gill lamellae of terrestrial crabs are modified owing to the reduction in the gill area, which in part explains the decreased in the respiratory function of the gills in terrestrial crabs (Farrelly and Greenaway, 1992). The decrease in the branchial area of terrestrial and semi-terrestrial crabs is thus one significant morphological adaptation to avoid desiccation (Pearse, 1929; Rabalais and Cameron, 1985). In turn, tolerance of desiccation is paralleled by the reduced gill surface area (Rabalais and Cameron, 1985). It is likely that the body surface permeability of terrestrial crabs is also reduced; however, this requires further study. However, the evolution of osmoregulation in Uca spp. is constrained by the tolerance to desiccation and the reduction in osmoregulatory capability. The air survival of desiccation and body and water weight loss in terrestrial crabs are shown in Table 1. Semi-terrestrial brachyurans can absorb water from moist substrates to compensate water loss due to desiccation and urinary excretion (Hartnoll, 1988). Seasonal differences have also been recorded. Extracellular fluid volume in the terrestrial crab G. natalis was 27.9% body mass during the wet season but only 22.7% in the dry season (Morris and Ahern, 2003).

Urine production decreases during desiccation, i.e., inulin clearance (urine/hemolymph) decreases in the crabs Cardisoma carnifex and Gecarcoidea lalandii, indicating decrease in loss of water (Harris and Kormanik, 1981). When T. celeste (as C. hirtipes) is exposed to air for 9 days and only given fresh water to drink, the osmotic pressure of both the hemolymph and urine remained constant, although the ionic composition of urine differed from that of the hemolymph. The antennal gland reabsorbed Ca²⁺ and Mg²⁺ from the urine. Reduction in the flow of urine minimized water loss from the crab. The final excretory product was diluted, containing only 10% of the concentration of ions measured in the hemolymph. Water loss to the air was minimized by reduced final excretory product flow, but drinking was insufficient to prevent dehydration. This indicates that T. celeste may require periodic immersion to maintain long-term water balance (Dela Cruz and Morris, 1998). In a separate experiment, when T. celeste (as C. hirtipes) was maintained in fresh water, it was shown to have a urine:hemolymph ratio (for ⁵¹Cr EDTA) close to 1, showing that no water was added or removed from the primary urine and demonstrating the adaptation of this species to fresh water (Greenaway, 1989). Later work on T. celeste (as Discoplax hirtipes) and G. natalis demonstrated the hormonal control (crustacean hyperglycemic hormone (CHH) of mechanisms utilized to prevent dehydration (Turner et al., 2013). Although not testing the effect of dehydration explicitly, this work showed that CHH had species-specific and seasonally variable actions on Na⁺ and urine production, including at the driest times of the year and during the migration process, when these crabs are particularly at risk of dehydration.

By using water from the shell, Coenobita crabs can survive longer under desiccation than Gecarcinidae and B. latro crabs (Burggren and McMahon, 1981). Osmoregulation in B. latro differs from the patterns seen in other coenobitids as, in the absence of a mollusk shell, the body fluids are regulated: directly against the environment. In Gecarcinids and Coenobita, water stores are maintained near the branchial cavity, possibly assisted by the excretion of HCO₃ across the gills in correlation with the movement of Cl⁻ (Burggren and McMahon, 1981). In common with other coenobitids, B. latro is remarkably tolerant of hemoconcentration. In most natural field conditions, only fresh water is available for drinking, and B. latro maintains its blood concentration in the range 650–750 mOsmol.kg^-1, with the lower hemolymph concentrations preferred in wet conditions and maintained even if the salt intake and excretion are high (Greenaway, 2001). Body water loss by desiccation induces an increase in hemolymph osmoconcentration. Where only saline water is available (e.g., atolls), the osmotic concentration (osmolality) of the body fluids increases; the animals have been shown to tolerate concentrations in excess of 1,100 mOsmol.kg^-1 for long periods (Gross, 1964; Taylor et al., 1993). When B. latro is dehydrated, hemolymph osmolarity increased up to 1,050 mOsmol.kg^-1. Under these conditions, crabs showed a preference for drinking fresh water to restore original hemolymph osmolarity (Combs et al., 1988). Under dehydration conditions caused by a few days without water, B. latro tolerates a 20% loss in body water. However, the overall result of dehydration was an increase in hemolymph osmolality from 20% to 30%, resulting in increased concentrations of Ca²⁺, Mg²⁺, and K⁺. Hadley (1994) classified adaptive strategies for water balance in terrestrial arthropods as behavioral avoidance, enhanced water conservation, dehydration tolerance, or hydration (the net absorption of water vapor from unsaturated air). The crabs Scylla paramamosain and Eriocheir sinensis exposed to air are able to absorb water from moist substrates to obtain oxygen, but eventually their gills dry and they cannot survive (Niu et al., 2020). Terrestrial crabs avoid heat by finding shelter in burrows and/or under rainforest tree cover, and they are often crepuscular or nocturnal, which helps them in further reducing dehydration. For example, G. lateralis burrows in dry ground above the littoral zone on sandy beaches where the only real moisture source is dew (Diaz and Rodriguez, 1977). Cardisoma guanhumi in Venezuela is found in low-lying areas up to 8 km away from sea, inhabiting mangrove swamps, where it digs down to the ground water level (Herreid and Gifford, 1963). Ocypode quadrata lives in the supralittoral zone on sandy beaches, where it digs deep holes above the high tide mark, approximately 500 m into the dunes. However, all three of these species are rapidly rehydrated with soil moisture. O. quadrata has the lowest threshold (<5% water) for bulk uptake from damp sand. Water is collected by capillary tufts of setae and drains into the branchial chamber (Wolcott, 1984).

Desiccation negatively affects not only many fundamental homeostatic mechanisms, such as acid–base regulation and oxidative stress but also secondary processes such as those linked to reproduction, e.g., embryo viability and hatching ability (Marochi et al., 2021; Table 1). Effects of water deprivation for a period of 18 h on hemolymph osmolality, eggs, and larval survival of female semi-terrestrial crabs Aratus pisonii were studied (Marochi et al., 2021). It was found that embryonic development is severely impacted after 18 h of water deprivation, and larval survival was negatively affected by the accumulation of metabolic waste products, which cannot be eliminated without water. Respiratory responses to activity in T. celeste (as C. hirtipes) were assessed with regard to humidity and dry season. The wet-season crabs were more active and exhibited respiratory acidosis, while the quiescent dry-season crabs showed a decrease in their locomotory activities (Weinstein et al., 1994). Metabolic activity was close to the limit of the aerobic scope, oxygen diffusion was limited, and oxygen partial pressure in branchiostegal lungs was depressed. Under experimental conditions, 90% humidity induced increased metabolic acidosis, while re-oxidation of L-lactate was very slow. The increased humidity in the air enabled greater activity in T. celeste, and together with increased rainfall, it instigated breeding migration in the wet season (Morris, 2005; Turner et al., 2011). Desiccation can also affect the Coenobite olfactory system (Krang et al., 2012) and can induce an oxidative stress response (Capparelli et al., 2021). Dehydrated C. carnifex experienced acute metabolic alkalosis as the hemolymph HCO₃ concentration was 70% above the control value, which was probably caused by the blockage of base excretion and delayed respiratory acidosis in the absence of water (Wood et al., 1986). The increase in hemolymph osmolytes was variable, and the total O₂ consumption and CO₂ excretion from the hemolymph decreased by approximately 55%, suggesting low gas exchange and metabolic depression. The oxygenation of the postbranchial hemolymph of Coenobita was not affected during dehydration since O₂ pressure and hemolymph O₂ capacity increased due to hemoconcentration. The associated metabolic acidosis and decrease in HCO₃ ⁻ during dehydration recover after 24 h of rehydration, and restoration of levels of hemolymph ions and osmolality occurs, with a nearly complete return to normal acid–base status (Burggren and McMahon, 1981). The acid–base response was progressive metabolic alkalosis, but this was only partially compensated by the increase in hemolymph CO₂ levels (Wood et al., 1986). Quickly after rehydration, this base load was removed from the hemolymph, and the values returned to those close to the control (Wood et al., 1986). In the semi-terrestrial crab H. formosensis, desiccation induces an increase in hemolymph CO₂, i.e., respiratory acidosis. This is reduced by bicarbonates and ammonia accumulation in hemolymph within 24 h of emersion to restore the hemolymph pH to the control value. The mangrove crabs Helice formosensis are regularly exposed to air, and the mean percentage of water content in these terrestrial arthropods demonstrates their ability to compensate acid–base disturbance (Allen and Weihrauch, 2021).

Summary: The vast numbers of both morphological and physiological modifications at the branchial chambers allow terrestrial and semi-terrestrial crabs to overcome the challenges posed by dehydration.

Along with the risk of dehydration, time spent away from the ocean either in their daily or seasonal activities or through their evolution poses the additional challenge of maintaining osmoregulation for semi-terrestrial and terrestrial crabs. One of the most fundamental characteristics of these species, irrespective of their marine or freshwater origin, is their ability to osmoregulate in a wide range of osmoconcentrations. Marine-to-freshwater and terrestrial colonization is a dramatic transition in the course of evolutionary history (Wolcott, 1992; Anger, 2001; Morris, 2001). Under extreme environmental conditions, terrestrial brachyurans not only have to adapt to ion absorption from almost freshwater conditions but also regulate ion absorption from brackish water conditions (recycling ions from the urine) as well as to regulate hyperosmotic challenge induced by desiccation or external sources by ion excretion. The functioning of active transport mechanisms, ion transporters, ion channels, and their interaction with gill ionocyte cells at the apical and basolateral membranes has played a crucially important role in enabling terrestrial colonization.

Freshwater and estuarine crustaceans, whose origin is a marine ancestor and who migrated multiple times between sea water, intertidal regions, and terrestrial habitats (Giomi et al., 2014) have been shown to possess a diversity of ion transport mechanisms. Some terrestrial crabs are restricted to oceanic islands, where some of these species have made evolutionary transition from aquatic to terrestrial habitats (Paulay and Starmer, 2011). On islands, as elsewhere, they have occupied and/or created burrows in a diverse range of habitats, including above the high tide line and into coastal forests, as well as in muddy areas such as mud flats, estuaries, mangroves, and freshwater streams. Encountering such extreme environmental differences has arguably greatly accelerated acclimation in these species (Prosser, 1973; Lande, 2009; Fusco and Minelli, 2010). This terrestrial existence affects the osmolality and ionic composition of the hemolymph and the expression of specific genes in the gills required for Na⁺ and Cl⁻ uptake (Yamaguchi and Soga, 2020). Molecular techniques focusing on the active transporters Na⁺/K⁺-ATPase (NKA) and V-type H⁺-ATPase (VHA) and secondary active transporters including the Na⁺/H⁺ exchanger, Na⁺/K⁺/2Cl^- (NKCC) co-transporter, and Cl⁻/HCO₃ ⁻ exchanger have become a standard approach to study the phenotypic plasticity of the physiological trait expression of osmoregulating candidate genes in terrestrial crabs in response to environmental changes (Tsai and Lin, 2007; 2012; Lee et al., 2011).

Ion transport across gill epithelia of crabs has been studied by biochemical, electrophysiological, and molecular biology methods. Experiments have been performed where the branchial chambers have been infused with saline in vivo (Wolcott and Wolcott, 1985; Wood et al., 1986; Greenaway and Nakamura, 1991; Varley and Greenaway, 1994; Morris, 2002), and isolated gills (see Allen and Weihrauch, 2021) and split gill lamella in Ussing’s type chambers have been perfused (see Henry et al., 2012), including with pharmacological inhibitors to evaluate the impact on ion flux, transbranchial potential, and/or short-circuit current to characterize transport mechanisms in the gill epithelium of crabs.

Transcriptomic data provide more evidence for the mechanisms of ion transport and specifically about the signal transduction genes responsible for salinity adaptation in this species of crab, while the proteomic analysis data show an increase in the expression of genes involved in energy and amino acid metabolism.

The semi-terrestrial crabs Leptuca panacea (as Uca panacea), (as Uca pugilator), and Minuca rapax (as Uca rapax) regulated hemolymph in brackish water up to 2,300 mOsmol.kg^-1. As a result, it has been suggested that there is a correlation between terrestrialness among crabs and their ability to regulate their hemolymph below the osmotic concentration of a hypersaline medium. Crabs, which live in environments with large fluctuations in salinity, such as estuaries, supratidal, mangroves, and terrestrial habitats, acquire hyper–hypo osmoregulatory mechanisms (Bozza et al., 2023). Most terrestrial crabs have access only to rainwater or dilute ground water and therefore have a high capacity for Na⁺ uptake obtained by drinking ground water and/or recycling ions and water from urine. Urine reprocessing takes place in the branchial chambers of semi-terrestrial and terrestrial crabs. Antennal glands open at the base of the second antennae or first maxilla within the anterior extension of the branchial chambers. Ion transport mechanisms, necessary for reclamation of salts from the urine, are present in the ventral branchiostegal region and gills, and these structures are responsible for the high rates of ion absorption (Morris et al., 1991). In all studied terrestrial crabs, except those of the Ocypodidae, the antennal gland as a site of salt reabsorption is of minor importance, and urine is isosmotic to the hemolymph. It is the gills that are adapted for the reabsorption of salts from the primary urine in most terrestrial crabs of marine origin (Wolcott, and Wolcott, 1985; Morris, 2001): O. quadrata (Wolcott and Wolcott, 1985), G. grayi (Varley and Greenaway, 1994), G. lalandii (Harris and Kormanik, 1981), C. guanhumi (Harris, 1977; Wolcott and Wolcott, 1991), C. carnifex (Harris and Kormanik, 1981; Dela Cruz and Morris, 1998), T. celeste (as Discoplax celeste) (Dela Cruz and Morris, 1998), and B. latro (Harris and Kormanik, 1981; Morris et al., 2000).

NKA and VHA are involved in Cl⁻ absorption in hyperosmoregulating brachyurans (Freire et al., 2008; Belli et al., 2009). Activation of NKA and the presence of VHA in several families of terrestrial and bimodal brachyurans are illustrated in Table 2. Recycling of ions from the urine by terrestrial crabs needs transport mechanisms similar to those of brackish water crabs (Riestenpatt et al., 1996; Towle et al., 1997; Charmantier et al., 2009; McNamara and Faria, 2012; Yang et al., 2019; Lee et al., 2022). An apically located NKCC in gills is hypothesized to drive ion reabsorption energized by an inwardly directed Na⁺ and Cl⁻ concentration gradient and maintained by basolaterally located NKA (Morris, 2001; Kirschner, 2004)). At the same time, the apical K⁺ channel should hyperpolarize the cell, enabling Cl⁻ to efflux from the cell into the hemolymph space via Cl⁻ channels (Riestenpatt et al., 1996; Onken et al., 2003; Lucu and Towle, 2010). Na uptake is increased by the Na⁺/H⁺ exchanger. The apically located Na⁺/H⁺ exchanger also participates in ion regulation and acid–base regulation in fish and crabs (Hwang and Lee, 2007; Freire et al., 2008) and ammonia excretion processes in aquatic invertebrates (Weihrauch et al., 2004; Freire et al., 2008; Tsai and Lin, 2014). The apically located Na⁺/H⁺ antiporter cannot function adequately under neutral to acidic external pH, as this exchanger is driven by the environmental and cellular concentration gradient of Na⁺ and H⁺ and not by membrane potential (Parks et al., 2008). These thermodynamic constraints that prevent this antiporter functioning under low or neutral external pH may enable the diffusion of NH₃ ⁺ out of the cell to trap H⁺ outside the cell. This exported NH₃ ⁺ will react with H⁺ and produce NH₄ ⁺.

According to the principles of thermodynamics, NKA is insufficient to drive active transport below NaCl concentrations of approximately 1.0 mmol.L^-1 (Larsen et al., 1996), and therefore, for aquatic and terrestrial animals living under conditions of low salinity, VHA function should be critical for freshwater and terrestrial adaptation. However, there are still insufficient data on the functions of these transporters relating to the transition of animals to new environments (Lee et al., 2011). Many polarized cells, by trafficking of VHA, are able to rapidly alter the density of VHA at plasma membranes by cAMP stimulated insertion and protein kinase A-dependent phosphorylation of subunit A of VHA into the apical membrane (Pastor-Soler et al., 2008; Alzamora et al., 2010; Collins and Forgac, 2020). Apically distributed membrane-bound VHA is found in Ocypodidae: Xeruca formosensis (as Uca formosensis) and O. stimpsoni and Varunidae: Chasmagnathus convexus. Orisarma dehaani (as C. dehaani) (Sesarmidae) were found to have cytoplasm distributed VHA in gill pair 6 and apically distributed VHA in gill pairs 7 and 8 (Tsai and Lin, 2007). Such ion transport mechanisms seem to be the minimum prerequisite for terrestrial life (Tsai and Lin, 2007). VHA plays a crucial role in many crustacean species living in fresh water or under extremely dilute salinity conditions (Ehrenfeld and Klein, 1997; Towle and Weihrauch, 2001; Weihrauch et al., 2001; ;Weihrauch et al., 2004 Patrick et al., 2006; Tsai and Lin, 2007). It has been suggested that for the terrestrial families of Ocypodidae, Grapsidae, Gecarcinidae, and the Coenobitid (B. latro), VHA is utilized under fresh water conditions (Figures 2–5).

Species which live under high salinity conditions have a hemolymph osmoconcentration maintained below that of the external medium. It has been suggested that hyporegulation could maintain a more constant osmoconcentration when in air (Jones, 1941; Gross, 1955). This ability is of great importance for the development of terrestrial life (Thurman, 2003). Under hyperosmotic challenge, the driving force for Na⁺ and Cl⁻ secretion might be the mechanisms of a basolaterally located NKCC symporter (Figure 4; Kirschner, 2004). It was hypothesized that regulation of NKCC1 was under rapid control and turned on and off via phosphorylation and dephosphorylation in response to shrinkage (hypertonic challenge) and cell swelling (hypotonic challenge) (Hoffmann et al., 2007; Marshall et al., 2009; Mantovani and McNamara, 2021). In semi-terrestrial and terrestrial crabs, regulatory effects during hyperosmotic challenge involve maintaining intracellular fluid isosmolarity with extracellular body fluid. This mechanism utilizes non-essential free amino acids together with peptides and sugars to ensure cell shrinkage, and the accompanying acute volume cell damage by osmolytes is minimized (Faria and McNamara, 2023). Hyposmoregulating crabs can mobilize free amino acids, owing to a lesser ability to secrete salts near their upper critical limits (Faria and McNamara, 2023). In the hyper/hypoosmoregulating families of Grapsidae and Ocypodidae, under hyperosmotic challenge, salt secretion could be substantiated by a basally present NKCC co-transporter and apical Cl⁻ channel (McNamara and Faria, 2012).

One of the most striking evolutionary changes during the transition from water to land is the shift in ionic transporter expression and their accompanying activity becoming dependent on the presence of salts during recycling or in these habitats. In these same groups of terrestrial crabs, high plasticity in the transport mechanisms of the gills has been developed in response to the variety of environmental salt concentrations encountered. Lipids used as a source for metabolic energy were increased during transition from fresh water to high-salinity water and vice versa (Chen et al., 2019). Transcriptomic and proteomic studies showed an upregulation of ion transport-related genes of VHA, CA, and Cl⁻ channels during low salinity acclimation of the crab S. paramamosain; however, under high salinity, they produce more free amino acids together with an upregulation in amino acid metabolism genes (Niu et al., 2020). Transcriptome analyses have further demonstrated that osmoregulatory-related genes involved in the ion transporters NKA and VHA were downregulated after rapid acclimation of the crab from fresh water to sea water (Zhang et al., 2018).

The strategy of locomotion on land is more energy-demanding than that of locomotion in air and water. During the adaptation to a terrestrial existence, a transformation in the ability to access available energy is therefore needed. The energetic demand of terrestrial adaptation of brachyurans under extreme environmental conditions must be high, and the generation of VHA and NKA that consume ATP would lead to an increase in food requirements to support energy costs (Lee, 2016). Gene regulatory mechanisms as well as biochemical and/or physiological mechanisms to reduce metabolic demands and protect tissues and organs from damage are activated (reviewed by Rivera-Ingraham and Lignot, 2017). Physiological stress occurs at the level of protein synthesis breakdown when there is a lower or higher salinity threshold and where active transport mechanisms have ceased (Henry and Weihrauch, 2024).

Iannucci et al. (2022) suggested that crab species that live in shallow intertidal waters and in terrestrial habitats exchange oxygen with air and salts at the gills and that the branchiostegal lungs (if present) are characterized by a significantly smaller genome compared to marine species. Land invasion in decapods has resulted in genome restructuring, which could in part have facilitated land adaptation and pressures on the amount of DNA. Knight et al. (2005) suggested that large genomes in plants are found in tandem with unnecessary junk DNA and that this could be a constraint on speciation. It would, therefore, follow that there could be a reduction in genome size for animals exposed to less stable environments, but this requires further investigation in terrestrial crabs.

Aquaporins of water channels are hydrophobic membrane proteins predominantly specialized for osmoregulation (Finn et al., 2014). In Carcinus maenas, the functional expression of aquaporins confirmed water transporting properties for CmAQP1 (a classical aquaporin) and CmGLP1 (an aquaglyceroporine). Higher CmAQP1 mRNA expression in some osmoconforming crabs suggests apical/subapical located channels which attenuate osmotic gradients, while its downregulation upon exposure to diluted seawater reduces water permeability (Nash et al., 2022). Genomic data from a large number of invertebrates showed that there were differences in aquaporin-coding genes between terrestrial and aquatic species. The role of aquaporins in aquatic species during terrestrialization has been examined in arthropods (Dunlop et al., 2013). A comparative phylogenetic analysis reported the potential loss of aquaporin-like coding genes in terrestrial arthropods, with lower numbers of superaquaporin genes found in this group. These results indicate that aquaporin-coding gene duplications and loss might be key mechanisms involved in the evolution of osmoregulation during terrestrial colonization (Martínez-Redondo et al., 2023). More studies are now needed on semi-terrestrial and terrestrial crabs to test this hypothesis.

One of the most fundamental adaptations facilitating the terrestrialization of crabs across phylogenetic groupings is their strong hyper-/hypo-osmoregulatory ability as a challenge to low and high external salinity. In the most terrestrial crabs, recycling of urine take place in the branchial chamber. Hyperosmotic regulatory ability is attained by active transport mechanisms (NKA and VHA), ion transporters (NKCC), ion exchangers (Na⁺/H⁺; Cl⁻/HCO₃ ⁻), and ionic channels (Na⁺, K⁺, and Cl⁻), with their interactions with gill ionocytes playing a prominent role in crab terrestrialization. During hyperosmotic challenge, basolaterally located ion transporters (e.g., NKCC) and apical excretory Cl⁻ channels prevent cell shrinkage. Minimized cell shrinkage and damage of cell volume changes is avoided by the mobilization of organic osmolytes.

However, our review has highlighted that there is still much to learn about the ionic regulatory strategies of terrestrial crabs. Further studies of the osmoregulatory roles of the gills, branchiostegal lungs, and antennal glands of semi-terrestrial and terrestrial crabs are now crucial. In particular, more work is needed to elucidate the relationship between the functions of these organs as they work together to maintain osmoregulatory homeostasis. These studies should be continued across the semi-terrestrial and terrestrial crab taxa. We advocate the continued use of a multidisciplinary approach by combining electrophysiology, biochemistry, and molecular biology to characterize the effects of the terrestrial habitat on primary and secondary active transporters at both the apical and basolateral sides of the gill epithelia. Previous work has demonstrated that in most cases, pharmacological inhibitors are nonspecific inhibitors of ion transporters. These existing studies should therefore be used as an approximation for further work on specific genes and paralogs of these transporters. In parallel, the localization of transporters will be important for understanding the combined effort and effects of different transporters in individual cells throughout the gills, branchiostegal lungs, and antennal glands. Continuing to characterize hormonal effects on transporters will also be crucial for a complete understanding of their function. For the identification of further ion transporters, transcriptome analysis should be used to identify mRNA isoforms and/or alternative splicing, i.e., of free ancestral genes during mRNA splicing. Different paralogs could be found for different ion transport functions. Whole-genome sequencing and transcriptome data that will allow for the identification of further ion transporters are still relatively rare for crustacean species. This is because the differing function of particular ion transporters might arise from the functional differences among gene paralogs and/or the functions of splice variants (isoforms; Lee et al., 2022).

The identification of specific transport mechanisms in B. latro that underlie physiological evolution during terrestrial adaptations, when compared to other semi-terrestrial and intertidal crabs, will likely enable us to elucidate more about the mechanisms that allow crabs to live on land. Transcriptomic and proteomic studies on gills, branchiostegal lungs, and antennal glands in crabs exposed to air should be conducted. More energy is needed for terrestrial adaptation, compared to life in intertidal and oceanic environments. NKA and VHA expression studies will help in identifying their role in the osmoregulation of semi-terrestrial and terrestrial crabs and any trade off with the energy needed for terrestrial life. Transcriptome studies on osmoregulation-related genes in the acclimation of crabs to the terrestrial environment will be crucial to enable us to gain new knowledge about the molecular mechanisms underpinning these. Phylogenetic studies on ion transporters will be beneficial for the determination of ion transport across families of brachyurans and anomuran terrestrial crabs when compared to other invertebrate groups. Phylogenetic studies on ion transport in families, including taxonomic units of arthropods, will help in determining the homology of ion transporters in different crab taxa.

Terrestrial adaptation by land crabs is driven by evolutionary responses to many factors, i.e., osmoregulation, respiration, reproduction, locomotion, and changes in sensory receptors (Lozano Fernandez et al., 2016). In accordance with Krogh’s principle (Krogh, 1929), land crabs retain their crown as the ideal model group for studying the evolutionary pathways and functional and morphological adjustments needed to conquer the terrestrial environment.