The fiddler crabs Minuca burgersi is a crustacean from the intertidal region with wide distribution, occurring in the Western Atlantic of the USA (south of Florida), Mexico to Brazil, and all the Caribbean islands (Thurman et al. 2013). This species inhabits and eats organic debris associated with surrounding sediments, in addition to being considered an ecosystem engineer due to his high bioturbation skills at the sediment-water interface, which also modifies the availability of resources and pollutants such as trace elements (Pillon et al. 2019).
Due to its wide distribution, ease of sampling, high capacity for population recovery, and laboratory management, in addition to the extensive literature on its biological and ecological characteristics, it becomes an adequate bioindicator for these countries.
At the end of the experiment, we verified that there was a reduction, but not significant, in the concentrations of Cu or Pb between the exoskeletons released and renewed in Group A, without contamination, whose average concentrations of Cu and Pb whose variations were 14.72 µg/g and 0.12 µg/g, respectively (Fig. 2A, Table 2).
Table 2
Statistical tests results. Independent samples Student's t-tests were used to verify statistically significant differences in Cu and Pb concentrations before and after the ecdysis. Six analyzes were applied, one for each element (Cu and Pb) and group (A, B, and C).
| Shapiro-Wilk normality test | Levene's homogeneity of variances test | Independent samples Student t-test |
---|
| W | p | F | p | t | p |
Group A - Cu (0 mg/g) | 0.95 | 0.229 | 5.06 | 0.035 | 1.67 | 0.109 |
Group B - Cu (5 mg/g) | 0.96 | 0.361 | 1.12 | 0.301 | 3.66 | 0.001a |
Group C - Cu (10 mg/g) | 0.96 | 0.519 | 13.33 | 0.001a,c | 6.71 | 0.000b |
Group A - Pb (0 mg/g) | 0.96 | 0.430 | 0.00 | 0.977 | 0.24 | 0.809 |
Group B - Pb (5 mg/g) | 0.92 | 0.071 | 1.19 | 0.286 | 5.44 | 0.000b |
Group C - Pb (10 mg/g) | 0.96 | 0.359 | 0.00 | 0.967 | 3.60 | 0.002a |
ap ≤ 0.01; bp ≤ 0.001; cthe Welch's correction was applied to the t-test p-value result because of the homoscedasticity assumption violation. |
When we evaluated the crabs present in Group B and Group C, we found a significant reduction in the average concentrations of Cu and Pb in the released and renewed exoskeletons, whose variations were 22.05 µg/g and 3.09 µg/g, with 5 mg/g concentration metals in the food, and 80.89 µg/g and 2.12 µg/g, with 10 mg/g concentration metals in the food, respectively (Figs. 2B and 2C, Table 2).
Interestingly, in the renewed exoskeletons of Group B crabs, we found lower levels of concentration of Cu and Pb than renewed exoskeletons in Group A (Figs. 2A and 2B). This indicates that when offering foods containing intermediate concentrations of metals, complementary detoxification processes must have been triggered, such as the action of the hepatopancreas and gills, leading to a lesser migration of metals from the hemolymph to the exoskeleton, leaving a smaller portion of heavy metals available for adsorption in the exoskeleton of this group.
According to Pytharopoulou et al. (2008), Lemus et al. (2016), and Capparelli et al. (2018), metalloneins and similar proteins that bind to metals rich in low molecular weight cysteines are found in increased levels in the digestive gland, hepatopancreas, and gills of marine crustaceans exposed to environmental conditions contaminated with heavy metals, being essential for the pathways of trace elements in the metabolism and sequestration of metals. Thus, they act in the process of bioaccumulation of toxic metals, detoxification, homeostatic regulation of metals, protection against oxidative stress, among others.
It is worth mentioning that some studies have discussed the use of these proteins as specific metallic biomarkers because they are also related to other environmental variations, such as salinity, sex, stages, body size, seasonality, and tissues (Morgan et al. 2004; Ladhar-Chaabouni et al. 2011; Cenov et al. 2018). However, because it is a controlled experiment, this analysis bias can be discarded in our interpretations.
By doubling the concentration of metals in the food, doses of Cu, and Pb in Group C, with 10 mg/g, we found that the metals' migration behavior from the hemolymph released to the exoskeleton occurred differently (Fig. 2C). For Cu, the concentration in this exoskeleton more than doubled in relation to Group B, while for Pb, the concentrations were equivalent. This result may be related to the existence of greater affinity of this metal for the tissues that form the exoskeleton in the stiffening mechanisms during ecdysis, or even the greater availability of Cu in the hemolymph, despite the same concentration of Cu and Pb in the food offered. Several authors, e.g., Rainbow (2002); Kouba et al. (2010); Stanek et al. (2014), reported in their studies that Cu is a component of the respiratory system of crustaceans, forming hemocyanin, causing high concentrations of this metal to exist in their hemolymph when compared to other metals. Thus, it is assumed that there is a greater tendency for the occurrence of a simple migration/diffusion to the tissues that form the exoskeleton.
Despite the difference in the accumulation of metals in the released exoskeleton, it was noticed that the mechanisms of elimination of these toxic components were activated in a similar way to that observed for Group B, generating concentrations of these metals in the renewed exoskeletons, which are also equivalent.
In this way, it can be inferred that different Cu and Pb concentrations do not interfere with the normal clearance physiology of these animals for clearance (Figs.
2B and
2C).
These results indicate that ecdysis is one of the physiological processes by which this species of crustacean purges Cu and Pb concentrations (Fig. 2B and 2C). In agreement with our findings, other authors have described that the mobilization of Cu in the exoskeletons of Minuca rapax (other species related to the Fiddler crabs) for the hemolymph is dynamic, occurring whenever the animal needs this metal (Capparelli et al. 2017). For Pb, it was previously described that Minuca pugnax raised in environments with high Pb concentrations eliminated Pb by ecdysis, while the same species of crab raised in low concentrations of Pb absorbed it in soft tissues (Bergey & Weis 2007). Ramos et al. (2021) also characterized the process of detoxifying heavy metals in Ucides cordatus crabs in natura during the period of ecdysis, demonstrating to be an important detoxification route in these invertebrates.
The molecular mechanisms involved in these clearings are unknown, but it was observed that two intracellular enzymes, protein kinase C (PKC) and phosphodiesterase (PDE), were always activated when the crabs were exposed to high concentrations of Cu and Pb; high production of PKC and PDE, and in turn, stimulates the increase of ecdysteroids in the pre-ecdysis phase, which finally stimulates ecdysis (Spaziani et al. 2001).
This work differs from previous studies in that we conducted a controlled experiment in the laboratory, where the dosage of the metallic contaminant could be pre-established and the food removed after ecdysis (Fig. 2B and 2C). Since these animals can re-accumulate these metals due to their ethological behavior (White and Rainbow, 1986), ecdysis in the Cu and Pb purge can be more accurately quantified by controlling other external parameters. This sensitivity is necessary since the renewed exoskeletons' levels of contamination were practically the same observed in renewed exoskeletons of animals fed without contaminated food (Fig. 2B and 2C vs. 2A).