Male and female GT and PC (the same number for each sex) were collected in April 2019 and March 2020, respectively, in the mangroves of Europa and Mayotte (Malamani area) (see Figure 2 for the sampling site). All animals were hand collected at low tide when crabs are active and out of their burrows. They were placed into individual bags or boxes in order to minimize stress and fights during transport to the laboratory (either the field laboratory on Europa Island, or the CUFR marine laboratory at the University Center of Mayotte). Animals (∼130 for PC, ∼100 for GT) were placed for 3 days for acclimation in individual boxes containing natural filtered seawater (∼33‰ salinity; 1,050 mOsm.kg^–1), under a natural photoperiod (12 h light: 12 h dark) and without feeding prior to experimentation.

For the osmoregulation experiment, ∼100 PC were studied (N = 10 per salinity) in Mayotte and ∼70 GT were studied (N = 4–8 per salinity) in Europa Island. Sampling size was limited here due to the pristine nature of the ecosystem and legal environmental protection requirements. For the respirometry and osmoregulatory capacity, 24 crabs were studied for each species. No mortality was observed during acclimation and experimental processes for PC (Mayotte). No mortality was observed during acclimation for GT (Europa), but one female died after 72 h of ammonia-N exposure.

Three different experimental conditions were performed: some crabs were maintained in seawater (∼33 ppt), others were transferred to diluted seawater (∼5 ppt) or to 10 mg.l^–1 ammonia-N solution (hereafter noted, respectively, SW, DSW and N exposure). This solution was prepared by adding 1 mMol of ammonium chloride (Sigma, United States) to diluted seawater (∼5 ppt). Ammonia-N was chosen as experimental treatment because the ionized form of ammonia-N (NH4⁺) is the major component of domestic WW (Capdeville et al., 2018; Mégevand et al., 2021, submitted). This condition refers to total ammonia (ammonia-N). It represents the sum of unionized ammonia NH3 and ionized ammonia NH4⁺ (Haywood, 1983; Lemarié et al., 2004; Bermudes and Ritar, 2008). The ammonia-N concentration was chosen on the basis of concentrations that may be released by treatment plants in Mayotte (Herteman, 2010). It corresponds to a sub-lethal concentration for a fiddler crab species such as Uromastyx princeps, adults sharing similar size and morphology with P. chlorophthalmus (Azpeitia et al., 2013). Ammonia-N exposure solutions were replaced every day. Salinity of SW and diluted seawater (DSW) were tested every day and readjusted if needed.

To determine osmolality curves, crabs were exposed at different salinities with 4–8 crabs per salinity for GT, and 8–10 crabs per salinity for PC. Salinity ranged from DSW (∼4.5 ppt, ∼135 mOsm.kg^–1) to concentrated SW (∼51 ppt, ∼1,510 mOsm.kg^–1). Animals were maintained in isolated boxes for a minimum of 72 h, which is sufficient time for osmoregulation to be achieved in such estuarine crustacean species (Lovett et al., 2001; Rivera-Ingraham et al., 2016). After this period, a hemolymph sample was taken using a 0.1–0.5 ml hypodermic syringe with the needle inserted between the cephalothorax and the third and fourth periopod. Osmotic pressure (OP) of the hemolymph samples was immediately quantified in duplicate by freezing point depression osmometry (Model 3320, Advanced Instruments, Inc., Norwood, MA, United States).

At the end of this experiment, animals were put back in individual boxes in aerated SW before being released into the mangrove.

The experimental design consisted in a static, intermittent flow-through respirometry system based on Clark et al. (2013). Crabs were individually placed into 125 ml chambers allowing them to make sporadic movements considering their size, while ensuring accurate measurements of O₂ consumption. Following procedures described in Killen (2014), O₂ measurements were performed using a 4-channel fiber-optic system with contactless O₂ sensor spots (FireSting O₂, PyroScience, GmbH, Aachen, Germany) where water oxygen content was quantified once every 5 s.

Water-mixing within the chambers was achieved with magnetic stirrers located under 1 mm² mesh grid to avoid too much disturbance (Rivera-Ingraham et al., 2016). Sensors were calibrated to 100 and 0% air saturation using air-saturated water and 80 mM Na₂SO₃ solution, respectively. Chambers were filled with a tubing system providing control SW, DSW or contaminated water from aerated, filtered and temperature-controlled tanks (∼25°C). Crabs were gently introduced in the chambers and left to acclimate for 1 h in aerated seawater prior experiment.

Following acclimation, the system was filled with the exposure solution. A flush pump was switched on for 20 min to ensure proper mixing and was cut off during 40 min. During that time, the chambers were sealed. The decrease in oxygen content could be analyzed to indicate the rate of oxygen uptake. After the 40-min cycle, the pump was turned on to flush the metabolic chambers with aerated seawater (or DSW or DSW enriched with ammonia-N) during 20 min. Due to the presence of aerobic and anaerobic microorganisms capable of degrading organic compounds and consuming O₂ (Shchegolkova et al., 2016), a 20 min flushing was run to ensure 99% air saturation. The 40 min measurements allowed to make accurate O₂ measurements without falling under the threshold of 70% air saturation in order to maintain aerobic metabolism and to avoid hypoxic stress (Ombres et al., 2011; Rodgers et al., 2016). O₂ measurements were taken at T-1h (SW), T1h, T24h, and T96h after exposure (SW as control treatment, DWS or DSW with ammonia-N) for all individuals (N = 8 per condition). This 96 h exposure time was chosen in order to better compare our results with data from the literature focused on the effects of pollutants and particularly ammonia-N on crustaceans. Many studies are based on the 96h LC50 with environmental concentrations of ammonia-N and regularly include histological analyses (De Freitas Rebelo et al., 2000; Wang et al., 2003; Barbieri et al., 2016; Weihrauch and O’Donnell, 2017). Only one female GT exposed to ammonia-N died after 72 h of exposure.

At the end of the 96 h respirometry exposure, a hemolymph sample from each animal was taken (N = 7–8 per condition) and OP was assessed as described above.

Following the 96 h respirometry experiment, crabs were anesthetized and euthanized on ice for tissue sampling of the hepatopancreas, anterior and posterior gills in order to perform the histology measurements. Samples were fixed in Bouin’s fixative solution for 48 h for histological analyses. Individual molting stage was verified through examination of an epipodite under a dissecting microscope after dissection (to avoid handling-associated stress on the biological markers considered).

For histological analyses, posterior gills (pair 7 or 8) and small samples of the hepatopancreas were rinsed in 70% ethanol following immersion in Bouin’s fixative solution for 48 h. They were then dehydrated in a series of graded alcohols and embedded in paraffin. Tissue sections (4 μm) were cut with a Microtome 2125RT Leica, then placed on Glycerin albumin precoated glass slides and deparaffinated. They were stained following Masson’s Trichrome Staining Protocol (Martoja and Martoja-Pierson, 1967) using Haematoxylin Groat, Fuschine Ponceau and Aniline Blue staining solutions. Samples were examined using Leica DM6 microscope equipped with a Leica DMC 2900 and the associated software LAS X.

For the hepatopancreas analyses, pictures of the hepatopancreas for each individual were taken, each of them depicting 1–4 tubules. Images were scaled and analyzed using ImageJ software (version 1.51r). Tubules of the hepatopancreas were randomly selected for each individual. Four parameters were measured on each of those hepatopancreas tubules and, then, averaged for each individual (N = 3–8): the area of the tubules, the number of vacuoles per tubule, the area of the B-cell vacuoles, the percentage of B-cell vacuolization area per tubule. Posterior gill epithelial thicknesses were measured from areas where nuclei were visible using a 40× objective (Figure 6C). Along this single-layered epithelium, cell heights were systematically measured from the base of the cuticle to the basal side of the cells facing the hemolymph lacuna. For statistical purposes, gills were divided into three sections along longitudinal (basal, central, and apical) and transversal axes (proximal, middle, and distal). For each of these areas, photographs were taken at the same magnification (60×) using the CapturePro software. Measurements of epithelial gill thickness were then performed using Image as well.

Both GT and PC are strong osmoregulators across the tested salinities since they can maintain a gradient of greater than 600 mOsm.kg^–1 above the ambient medium when exposed to salinities close to FW (5–10 mOsm.kg^–1, Figure 3A). Both species hyper-regulated at lower salinities (hemolymph osmolality actively maintained at levels above that of DSW) and hypo-regulated at higher salinity (hemolymph osmolality maintained below that of SW). The difference between external medium and hemolymph osmolality significantly increased as salinity decreased when both species were exposed to DSW during 96 h (Figures 3B,C). GT can maintain osmoregulatory capacity values of ∼50 mOsm.kg^–1 in SW and ∼600 mOsm.kg^–1 in DSW after 96 h, and these values are significantly different [Figure 3B, one-way ANOVA F(2,20) = 89.44, p < 0.001]. The same results are observed in PC with osmoregulatory values of ∼ 100 mOsm.kg^–1 in SW and ∼ 600 mOsm.kg^–1 in DSW [Figure 3C, one-way ANOVA, F(2,20) = 1913, p < 0.001]. However, a break in the curve is detected at low salinities, when it became increasingly difficult for the animals to osmoregulate. For GT, this break occurs at a higher salinity (∼260 mOsm.kg^–1) than for PC (∼118 mOsm.kg^–1). Moreover, PC was able to maintain the same osmoregulatory capacity in DSW with ammonia-N as in DSW (Figure 3B), whereas a significant decrease in osmoregulatory capacity is observed in GT exposed to ammonia-N compared with DSW (Figure 3C): crabs cannot maintain the haemolymph-seawater interval as in DSW.

Respiration rate measurements are depicted in Figure 4A for GT and Figure 4B for PC. They include temporal responses for each treatment at T-1h (SW), T0h, T24h, and T96h. In GT, a significant effect (p < 0.001) of exposure time on O₂ consumption (μmol O₂⋅m^–1 g^–1) was observed. The interaction between treatment and exposure time was significant [χ²(6, N = 31) = 16.831, p < 0.01]. Post hoc multiple comparisons on the interaction terms showed that crabs exposed to ammonia-N significantly increased their respiration rate after 96 h of exposure compared to the other time steps and with DSW (Figure 4A). No effects of the osmotic or pollutant shock was observed immediately after exposure (T0h compared to T-1h).

In PC, no significant effect of treatment or exposure time was found, but their interaction was significant [χ²(6, N = 32) = 44.5581, p < 0.01]. Post hoc multiple comparisons on the interaction terms showed that crabs exposed to SW significantly decreased their respiration rate after 96 h exposure compared with the other time steps (Figure 4B). Crabs exposed to DSW progressively increased their respiration rate until 96 h (significant difference between T-1h and T96h and between T0h and T96h). At 96 h of exposure, a significant difference was observed between SW and DSW only.

The histology of the three main cell types of the pancreatic tubules of GT and PC is presented in Figure 5I: (1) R-cells (Restzellen, resorptive cells), the most abundant cell type in the hepatopancreas; (2) F-cells (Fibrillenzellen, fibrillar cells), which have a columnar shape and basophilic cytoplasm with an intense staining; (3) mature B-cells (Blasenzellen, blister-like cells), which are the largest cells showing large subapical vacuoles.

In GT, no effect of treatment was observed for all parameters measured in the hepatopancreas (One-way ANOVAS, Table 1, Figures 5A–D). In PC, a significant effect of treatment (p < 0.01) was reported regarding the percentage of B-cells vacuolization in hepatopancreas tubules. Tukey post hoc showed an increased vacuolization process in DSW and ammonia-N compared to SW (Figure 5H).

No effect of treatment was observed in the gills of GT across the three levels of longitudinal axis (apical, central, and basal) (one-way ANOVAs, Table 2 and Figure 6A). However, in PC, a significant effect of treatment (p < 0.0001) was reported at each level of the longitudinal axis (one-way ANOVAs, Table 2). Tukey post hoc tests revealed that the gill epithelium of crabs exposed to ammonium for 96 h was significantly thicker than those exposed to SW or DSW.

Osmoregulation is one of the key regulatory functions in aquatic organisms and represents an excellent indicator of the physiological status of crustaceans (Lignot et al., 2000). Crustaceans can be classified as strong, moderate, or weak osmoregulators depending on their capacity to tolerate low salinity and the magnitude of the hemolymph-seawater osmotic difference (Henry et al., 2012). Results indicate that both PC and GT are strong osmoregulators, but the breaking point in the osmolality curve (Figure 2) occurs at a higher salinity in GT compared to PC. Therefore, the physiological osmoregulatory limit toward low salinity for GT is reached before PC. Also, they do not share the same isosmotic point. It is higher in GT (∼1,000 mOsm.kg^–1) than in PC (∼830 mOsm.kg^–1). Similar results have been found in sympatric fiddler crabs living under different salinity conditions in the Gulf of Mexico (Thurman, 2004). Hypersaline species such as Leptuca subcylindrica and Minuca rapax are isosmotic for media between 840 and 940 mOsm.kg^–1 (26–30‰) whereas, freshwater/brackish water species such as Minuca longisignalis are isosmotic at 732 mOsm.kg^–1 (23‰). The ability to maintain hemolymph osmolality at different environmental salinities is well correlated with the habitat of the species (Lin et al., 2002), but may also depends on the species physiology itself. Consequently, a species living near estuaries or river mouths must be highly tolerant to salinity variation (hypothesis 1). Ecological knowledge about habitat preferences and distribution for both studied species tend to confirm this hypothesis. GT usually inhabits exposed, lower intertidal sandy or mud flat shores fringed with mangrove forest, coral rubble or rock (Dawn and Frith, 1977; Koga et al., 2000). This species is also considered as very marine, often living on shore and never near muddy river mouths (Crane, 1975). It has the largest longitudinal range of fiddler crabs, spanning the entire breadth of the Indian Ocean and Western Pacific subrealms, as described by Rosenberg (2020). PC does not live close to the open sea but near high-tide levels on the muddy banks and flats of mangrove estuaries, close to the mouth of streams or rivulets (Crane, 1975). It is one of the dominant species living in the study site of Mayotte and is one of the three endemic species from the East Africa Province (as defined by Rosenberg, 2020). This province also comprises three other species extended into other provinces, including GT. Thus, both species can be found in Europa and Mayotte islands even if they do not share the same habitat level and salinity conditions.

Since benthic crustaceans can frequently experience elevated levels of localized ammonia-N when they bury in sediments with high organic contents (Weihrauch et al., 1999), it is believed that this necessitates an adaptive mechanism to remove excessive ammonia-N build-up within their hemolymph (Weihrauch et al., 2004; Romano and Zeng, 2012). Ammonia excretion has been widely studied in aquatic crustaceans and it is established that this function is intimately linked to the gills and their osmoregulatory function (see Weihrauch et al., 2004 for a complete description of the model).

The mechanisms for ammonia-N excretion in bimodal crabs are very diverse and complex (for a review, see Weihrauch and O’Donnell, 2017), as many membrane transporters are involved mainly in the anterior and posterior gills as well as in the antennal gland. The sodium pump Na⁺/K⁺-ATPase (NKA), K⁺-channels and Na⁺/H⁺ exchangers (NHE) are among the key membrane proteins involved although more transporters could be at play (Masui et al., 2002, 2009; Lucu and Towle, 2003; Gonçalves et al., 2006; Freire et al., 2008; Weihrauch and O’Donnell, 2017). As already described, a number of these transporters playing a crucial role in trans-epithelial ammonia excretion are also involved in the ionoregulatory machinery (Weihrauch and O’Donnell, 2015, 2017). The best example for this is NKA, one of the key transporters responsible for energizing active NaCl transport processes in invertebrates and vertebrates alike (Larsen et al., 2014). The NH₄⁺ pump (often referred to NH₄⁺/K⁺-ATPase) is equally important. However, this process reaches its limits when crustaceans are exposed to excessively high amounts of ammonia-N, generally resulting in reduced hemolymph Na⁺ levels (Romano and Zeng, 2012). This phenomenon has been observed in several species living under various salinity conditions: the American clawed lobster, Homarus americanus (Young-Lai et al., 1991), the Kuruma shrimp, Marsupenaeus japonicus (Chen and Chen, 1996), the burrowing crab Neohelice granulata (Rebelo et al., 1999), the freshwater crayfish Pacifastacus leniusculus (Harris et al., 2001) and the mud crab Scylla serrata (Romano and Zeng, 2007a), amongst others. We can relate these data to our results on osmoregulatory capacity: increased ammonia-N excretion could induce a leak in Na⁺ ions followed by a decrease in OC for GT, but not for PC. This may suggest that PC could set-up physiological mechanisms that allow a tolerance capacity to high ammonia-N levels in DSW. However, GT could not (rapidly) mobilize such tolerance. In most Crustaceans (i.e., H. americanus and P. japonicus), a decrease in hemolymph Na⁺ levels is directly linked with reduced hemolymph osmolality and/or OC, but this is not observed in the euryhaline mud crab S. serrata (Romano and Zeng, 2007a). These authors propose that this crab species relies on other mechanisms to maintain osmotic pressure even in case of Na⁺ reduced levels, as for example, by increasing the organic osmolytes such as hemolymph free amino acids (FAA). One can hypothesis that the difference observed in OC between GT and PC may result in different abilities to excrete ammonia-N (through NKA, NH₄⁺/Na⁺ exchanger, and/or other transporters). With ammonia-N in DSW, GT may exhibit lower NKA activity, as reported in the blue swimmer crab Portunus pelagicus exposed to ammonia-N at low Na⁺/K⁺ ratio (Romano and Zeng, 2011), leading to lower Na⁺ levels in the hemolymph compared to PC. A possible explanation could be that PC may better cope with high levels of ammonia-N in its hemolymph than GT, by maintaining the activity level of its membrane transporters. Measuring ammonia-N, ion concentration and pH levels in the hemolymph would help to confirm this hypothesis.

Increased energy expenditure for osmoregulation and ammonia-N excretion may be reflected by increased metabolic indicators such as oxygen consumption (Gomez-Jimenez et al., 2004).

Our results showed no effects of DSW in respiration rate of GT across the 96 h of exposition compared to the controls in DSW. In PC, respiration rate progressively increases in DSW, whereas O₂ consumption in SW progressively decreased until 96 h of exposure. Euryhaline crabs are able to compensate for diffusion loss of ions in dilute media by active uptake of gill epithelium (Genovese et al., 2004). The most energy-demanding ion transport mechanism is the active transport driven by the NKA (Lucu and Towle, 2003), with active ion pumping in specialized epithelial cells that are also mitochondria rich (Freire et al., 2008; McNamara and Faria, 2012). Ammonia-N excretion and hyper-osmoregulation mainly rely on these mechanisms (Weihrauch et al., 2004) which generally increases when estuarine/marine crustaceans are exposed to diluted seawater (reviewed in Romano and Zeng, 2012). Based on the large loss of salt in diluted media, these crustaceans must invest considerable amounts of energy in active transport/uptake of salt in order to compensate for the large rates of diffusive salt loss (Henry et al., 2012). When osmotic stress is coupled with ammonia-N stress, an increase in O₂ consumption is observed in GT after 96 h exposure, but this was not observed in PC. This could be directly linked to the OC decrease observed in this species. An increased energy demand is likely to occur in case of ammonia-N prolonged exposure. For instance, GT could rely on different ionic transport mechanisms without necessarily modifying its gill epithelial thickness (as discussed in Section “Histological Changes to Explain Paraleptuca chlorophthalmus and Gelasimus tetragonon Differential Ammonia-N Tolerance”). Even with an increased uptake of O₂, it is possible that the maintenance of GT hydromineral balance and ammonia-N excretion function is less efficient when facing these stressors than in PC. This could highlight a physiological trade-off because of the lack of other adaptive mechanisms, but also due to any physiological differences between the two species. In PC maintained in DSW, the increase in O₂ uptake could be due to an increased energy demand for osmoregulation (among others). However, a lower oxygen consumption in DSW with ammonia-N could suggest an increased anaerobic metabolism or a different compromise between gas exchange and osmoregulation at the gill surface (aka the osmo-respiratory compromise) (Robertson et al., 2015). A clear functional specialization has been reported for several crab species, posterior gills being the main effectors of the “compensatory” process, while anterior gills are mostly involved in gas exchange (Taylor and Taylor, 1992; Pequeux, 1995; Genovese et al., 2004). Anterior gills are also known to be actively involved in ammonia excretion (Weihrauch et al., 1998, 1999) and in some hyper-regulating species such as green shore crab, active ammonia excretion rates being lower in posterior osmoregulatory gills compared to anterior gills (Weihrauch et al., 1998). These results indicate that osmoregulatory processes are not directly linked to ammonia excretion and that excretion of toxic ammonia could proceed independently of other physiological processes (Weihrauch and O’Donnell, 2017). Large areas in the lamellae of the posterior gill epithelium reveals thick ion-transporting cells with well-developed basolateral membranes associated with numerous mitochondria in several species of euryhaline crabs such as Neohelice granulata (previously Chasmagnathus granulatus) (Genovese et al., 2004). Most of the NKA activity is generally restricted to specific areas of posterior gill lamellae lined by this kind of epithelium (Towle and Kays, 1986).

In DSW, no changes were observed in the thickness of the gill epithelium for the two species (Figures 6A,B). However, when exposed to ammonia-N for 96 h, branchial epithelium thickness of PC significantly increases along the longitudinal axis, whereas this does not occur for GT. An increase in osmoregulatory epithelial patches surface of posterior gills in crabs exposed to either dilute or hyper-saline water (i.e., when crabs osmoregulate) has already been observed (Genovese et al., 2000; Lovett et al., 2006). The authors suggest that these gills could be involved both in active ion uptake and active excretion of ions depending on the medium salinity, especially with the progressive increase of NKA activity during 6 days observed in crabs exposed to dilute seawater (Lovett et al., 2006). In PC, this response may be directly related to a strategy allowing increased ammonia-N excretion and osmoregulation may be through NKA increased activity, as discussed above. By increasing the thickness of the basolateral epithelium of posterior gill cells, PC may increase the number of ion transporters (mainly NKA) and mitochondria allowing, therefore, an increase in its osmoregulatory and ammonia-N excretion capacities. The increase in epithelial thickness does not occur in DSW although a higher metabolic rate is observed in this condition. Therefore, PC could rely on two different strategies depending on the nature and level of stress (increased metabolic rate in DSW and/or enhanced ion transport mechanisms when exposed to ammonium by the lengthening of the basolateral epithelium), whereas GT cannot exhibit such plasticity regarding ammonia-N exposure and increases its metabolic rate.

However, though not quantified, no major disruption of cellular structure has been observed in either species exposed to DSW or Ammonia-N. In Romano and Zeng (2007b), Ammonia-N exposure for 96 h lead to different dose-dependent disruptions of gills in P. pelagicus. At 10 mg.l^–1 ammonia-N (a similar concentration as in the present study), gill lamellae showed local infiltrations of hemocytes with normal lamellae structures whereas 60 mg.l^–1 ammonia-N generated extensive hemocyte infiltrations, disrupted pillar cells and lamellae collapses.

In Crustaceans, the hepatopancreas is a key metabolic organ, expressing a variety of transporters (Ahearn et al., 1992). Its numerous tubules are involved in food digestion, detoxification of xenobiotics and nutrient absorption (Ceccaldi, 1998; Zilli et al., 2003; Ortega et al., 2014). Histological analyses of these tubules revealed few changes for the B-cells in both GT and PC (Figures 5A–G). Even if their role is still discussed, B-cells can be considered to have two functions: elimination of waste material and intracellular digestion of nutrients (Vogt, 1994). The supranuclear vacuoles resulting from the coalescence of subapical vacuoles take the form of huge lysosomes (Vogt, 1993) and their content can be discharged into the lumen by holocrine secretions (Nott et al., 1985). Our results showed a higher vacuolization in PC exposed to Ammonia-N that could be linked to an increased intracellular digestion process through B-cells vacuoles, as it has been found in a Sesarmid crab species exposed to domestic wastewater (Mégevand et al., 2021). This increase in activity would potentially lead to an increased energy expenditure but in this case, our hypothesis is that ammonia-N could have a limited effect on the hepatopancreas functioning. As mentioned by Chu (1987), the hepatopancreas overall contribution to osmoregulatory ion uptake is believed to be minimal, whereas gills are the main site for respiration, osmoregulation, ammonia-N excretion, these gills being the main organs involved for the overall organismal adaptation to changing conditions. In Ocypodids such as PC and GT, the study of the antennal gland would also be of interest in order to understand ammonia excretion mechanisms. As shown for several fiddler crabs such as Minuca pugnax or Leptuca pugilator, ammonia is known to be actively transported across the duct and excreted in the primary urine which contains a high concentration of ammonium (Green et al., 1959; De Vries et al., 1994). Other ecomorphological markers in addition to those used on the hepatopancreas and gills would be interesting to study as indicators of adaptation to changing environments. For example, Lim and Goh (2021) demonstrated that the setae present on the maxillipeds are great indicators of adaptation, especially for efficient food extraction regarding sediment quality. Fiddler crabs with greater diversity and density in setae could be able to adapt to more variable habitats since their ability to extract food from different sediment types would be better.

Coping with an aquatic environment with low salinity and elevated ammonia-N is highly species- and habitat-specific. Therefore, a fully euryhaline species such as PC, that is adapted to nutrient-rich environments (Gross, 1982; Henry et al., 2012) is likely to be less vulnerable to nutrient enrichment and may be able to develop other coping mechanisms. However, this study focuses on potential short-term acclimation capacities. It does not take into account any possible environmental determinism that could arise from micro-evolution after prolonged exposure to ammonia-N enriched conditions (in Mayotte) or differential abiotic factors such as eutrophic or oligotrophic environments.

A research program with multi-generational expositions of the two populations would be of high interest, especially for understanding ecosystem health and ecological risk implications on a longer timescale (although technically complicated to implement).

Acclimatory and adaptive capacities to different environments induce species-specific responses among Decapod crustaceans that rely on various mechanisms to maintain their metabolism (Weihrauch et al., 1999; Henry et al., 2012; Romano and Zeng, 2013).

However, brackish and intertidal environments are among the most stressful aquatic biotopes and the establishment of Crustacean communities in such habitats suppose highly adapted physiological features (Gilles and Pequeux, 1983; Charmantier et al., 2002) to cope with low and varying salinities (Henry et al., 2012), as well as constantly changing environments. For example, it has been shown by Yong and Lim (2021) that ocypodid crabs such as Ocypode gaudichaudii possess a feeding plasticity when confronted to different habitat types, which is related to their burrowing and foraging strategies. In the case of our study, salinity variations and nitrogen enrichment phenomena could also induce different levels of adaptation in feeding and foraging habits, which would be interesting to study in both species in parallel with the individual physiology.

In the present study, PC appears to have a better physiological tolerance to decreased salinity compared to GT, even if GT is a relatively strong osmoregulator (hypothesis 1). Ammonia-N triggers short-term acclimation in PC that was not observed in GT suggesting that this species could be more vulnerable to anthropogenic discharges (hypothesis 2). GT copes with 96 h ammonia-N exposure through increased metabolic rate to excrete this product, while struggling to maintain its osmoregulatory balance. However, this response does not occur in DSW condition without ammonia-N. This suggests that some cumulative deleterious effects under Ammonia-N exposure and osmotic stress occur for this species (hypothesis 3).

Ammonia-N was used in this study as a first indicator of anthropogenic effluents, but it is more likely that crab communities are confronted with different contaminants with combined effects (e.g., complex and hypoxic wastewater), often consisting of repeated exposures. Worldwide, effects of domestic effluent discharges (controlled or not) on crab community assemblage have been observed in several different mangroves with various results (Wear and Tanner, 2007; Penha-Lopes et al., 2009; Wickramasinghe et al., 2009; Capdeville, 2018). The results of the present study point (at least) to a short-term physiological acclimation to salinity variation and pollution for species such as PC, acclimated to euryhaline and anthropized conditions (already proposed in Mégevand et al., 2021, submitted), but it has been demonstrated that individual abundance for this species decreased in chronically WW impacted areas (Capdeville et al., 2018). If GT is unable to develop short-term response to salinity and ammonia-N inputs, it is likely that this species would be even more sensitive to longer exposure to pollution, or to more complex contaminants such as hypoxic WW. If the population decline of engineering species can be dreadful for the functioning of freshwater-influenced mangroves (e.g., Mayotte), what about isolated and pristine ecosystems like Europa Island? What would be the acclimation potential of representative species like GT in the event of anthropogenic effluents? In view of the results obtained in this study and those obtained during the various works mentioned above, it is possible to be pessimistic about the ecosystem risks incurred by a pristine mangrove such as that of Europa if it were to face waters discharges of anthropogenic origin.