HSP70 in Gills and Hepatopancreas of Mangrove Crabs Ucides cordatus : Comparison between Contaminated and Pristine Environments

: This study analyzed ﬁeld and acclimatized (7 days) mangrove Ucides cordatus crabs from polluted and unpolluted environments to compare their HSP70 levels. The animals were cryo-anesthetized and dissected. Gills (anterior and posterior) and hepatopancreas were collected to evaluate total proteins and HSP70 levels using ELISA (Enzyme-Linked Immunosorbent Assay) method. The acclimatized animals from polluted environments showed higher HSP70 levels in the hepatopancreas than ﬁeld animals. Results showed higher HSP70 levels in laboratory animals from the polluted environment than in ﬁeld animals in the posterior gills. The regulation to decrease the damage caused by the environment and the acclimatization process may not be sufﬁcient to stabilize physiological responses, especially in animals from polluted environments.


Introduction
The contamination of the aquatic environment has been increasing every year as a result of anthropic action, mainly due to increased urbanization, the expansion of industrial activity, and the exploitation of natural resources [1,2].Animals from estuarine and coastal regions are directly affected by local pollution through the disposal of chemicals and toxic metals [3,4].Among the most-studied estuarine animals is the Ucides cordatus ("uçá") crab.These animals feed mainly on the litter of mangrove areas, as well as the sediment itself.They promote bioturbation and incorporate organic matter into the sediment.Finally, they have slow growth and a long life cycle, and are abundant and easy to capture in the field [5].
In contact with crustaceans, metals may cause physiological and morphological damage to gills and hepatopancreas, organs that are the first entry sites of water, food, and contaminants in these organisms.Therefore, estuarine animals present strategies to signal the degree of pollution in a given area, which assist in their survival [3].One strategy expresses a family of stress proteins, the heat shock proteins (HSPs).These proteins play critical roles in metabolism, such as folding, translocation, and refolding denatured proteins under stress or non-stress conditions [6,7].
HSP70 presents various functions, especially in multiple cellular stress responses [7].This vital protein is present in bacteria to mammals and has been identified in various Crustacea [6].The increase of HSP70 in response to external stressors in crab species of Scylla paramamosain, Callinectes sapidus, and Callinectes danae, among other groups, has been amply evidenced, revealing that HSP70 is a versatile molecular indicator that can assess the presence of external stressors and environmental contamination [8,9].Studies carried out with Chironomus tentans larvae showed an increase in the expression of HSP genes, mainly HSP70, in animals present in contaminated environments [10].In addition, exposure to cadmium has increased the expression of HSP70-related genes in Scylla paramamosain hepatopancreas [11].Likewise, on exposure to copper, the marine crab Portunus trituberculatus showed increased levels of HSP genes, such as HSP90 in gills [6].
However, although important, the interactions between anthropogenic stressors and the effects on organisms in the field are poorly understood and rarely investigated [12].Crustaceans, especially crabs, have been identified as tolerant to metals [13], and it is essential to compare animals from regions with different contamination levels to assess their survival, even in the presence of contaminants [4].Previous studies showed that animals from contaminated environments had high levels of stress biomarkers when compared to animals present in uncontaminated environments.Thus, we hypothesize that HSP70 levels will increase in animals belonging to contaminated environments relative to animals present in pristine environments.Regarding acclimatization, recent studies with Callinectes danae showed that the acclimatization process could interfere with the physiological results of animals present in contaminated and uncontaminated environments, also corroborating our hypothesis [4].
Therefore, this study aimed to analyze the levels of HSP70 in the mangrove crab U. cordatus, comparing the origin of the animals (contaminated or pristine environments) and how they are maintained (field or acclimatized in the laboratory).

Studied Regions
Itanhaém is located in the state of São Paulo (Itanhaém estuary; 24 • 10 14.76 S-46 • 48 2.96 W) and has a high level of contamination by metals, mainly from landfills, and dumps, which contribute to the contamination of estuarine systems [3,14].The Itanhaém region has high levels of cadmium (0.050 mg L −1 ), lead (0.1 mg L −1 ), copper (0.050 mg L −1 ), chromium (0.1 mg L −1 ), iron (0.5 mg L −1 ), nickel (0.1 mg L −1 ), and phosphorus (0.07 mg L −1 ) dissolved in water.These values are above the levels allowed by the ISQG (Interim Sediment Quality Guidelines), determining a high degree of environmental pollution [15].The Juréia-Itatins Ecological Station is located on the southern coast of the State of São Paulo (24 • 26 7.43 S-47 • 4 46.12W); being preserved, protected, and considered a conservation unit since 2006 [3,16].Due to its small population, this region is an unpolluted place with traditional extractive activities [13,17].The Juréia-Itatins Ecological Station covers one of the largest areas of environmental protection.The area is widely used by researchers as it is considered a region without anthropic interference.Metal levels are below the detection levels and parameters established by the ISQG, so this area is used as a reference for a pristine environment [18].

Animals
Male and adult specimens of the "uçá" crab (U. cordatus) from polluted and pristine regions (Table 1) (n = 20 animals/studied region) were collected.Adult male Ucides cordatus crabs were used during the intermolt period to minimize possible physiological effects of the reproductive cycle and molting periods [3].Then, the animals were transported to the IB-USP laboratory and were washed to remove the sludge.n = 10 animals/region were cryo-anesthetized and dissected at this moment (field animals) to remove the gills (anterior and posterior) and hepatopancreas.Each organ removed from each animal was separated into 2.5 mL plastic tubes and frozen at −80 • C until analysis of HSP70.
The remaining animals (acclimated animals, n = 10 animals/region) were placed in different tanks with artificial seawater (Red Sea Salt) with 20‰ salinity.During the period in the laboratory, the animals were fed with Rhizophora mangle leaves ad libitum, collected from the unpolluted sampling site.Under these conditions, the animals were kept for 7 days (3 days before the dissection, the animals were non-fed).At the end of the acclimation period, the animals were cryo-anesthetized and dissected, with the removal of anterior and posterior gills and hepatopancreas.The organs were removed and separated in 2.5 mL plastic tubes, frozen at −80 • C until the analysis of HSP70.The separation procedures between field and acclimatized animals, the maintenance of the animals, and the cryoanesthesia process described above were carried out for the animals from the region considered polluted and the animals from the region considered unpolluted (pristine).
Table 1.Weight (mean ± standard deviation) and width of the carapace (mean ± standard deviation) of animals from unpolluted and polluted areas.The asterisk represents a significant difference between animals from both groups.

Studies Area
Average

Total Protein Analysis
Initially, samples were extracted to quantify the total proteins of each removed organ for posterior HSP70 analyses.A small fraction of the sample (approximately 1 cm from each organ studied) was cut and placed in a 1.5 mL plastic tube with 540 µL extraction buffer (TBS 1× and EDTA 1 mmol L −1 ) and 60 µL of protease inhibitor cocktail (Roche, 10×).The samples were crushed and centrifuged at 4 • C for 10 min at 13,000× g.Then, the supernatant was removed and transferred to another 1.5 mL plastic tube, centrifuged again under the same conditions described above.This procedure was repeated until the samples became clear.Protein quantification occurred through the method of Bradford (Bradford reagent for 0.1-1.4mg/mL of protein, Sigma Aldrich, Saint Louis, MO, USA) [19] in triplicate, and the concentrations were calculated in µg/mL.After protein quantification, the extracted samples were stored in a freezer at -20 • C for further analysis of HSP70.

HSP70 Analysis
For the analysis of HSP 70 levels, ELISA assays were performed in triplicate using an adapted immunoassay protocol [20,21].The extracts (100 µL, containing 50 µg of total proteins) were added to 96-well plates (Corning Costar, 96-well EIA/RIA Assay Microplate) and incubated for 24 h, 1 h in an orbital shaker, and the remaining time in a refrigerator at 4 • C.After this period, all the solution on the plate was discarded, and three washes with PBS solution containing 0.05% Tween-20 (PBS-T) were performed for 1 min each.Then, to block unspecific binding, each plate was incubated for 24 h with 3% BSA, 1 h in an orbital shaker, and the remaining time in the refrigerator at 4 • C.After blocking, the solution was discarded, and the plates were washed with PBS-T buffer three times for 1 min.After washing, the plates were incubated with a specific primary antibody (anti-HSP 70 rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:500 in PBS-T with 0.1% BSA, for 2 h 30 min, in an incubator at 29 • C.After incubation, the solution was discarded, and the plate was washed three times for 1 min with PBS-T buffer.Subsequently, the plate was incubated with the secondary antibody peroxidase-conjugated and specific (goat anti-rabbit Igg-Hrp, Santa Cruz Biotechnology, Santa Cruz, CA, USA), in the dilution of 1:1000, for 2 h 30 min, in an incubator at 29 • C.After the second incubation, the solution was discarded, and the plates were washed with PBS-T buffer three times for 1 min.The development process used 3,3,3,5,5-tetramethylbenzidine (TMB, commercial KPL development kit) as the substrate for the peroxidase, following the manufacturer's recommendations.In each well of the microplate 80 µL of development solution were added and incubated in the dark for 10 min.After this period, the development reaction was stopped with 50 µL of phosphoric acid 2 mmol L −1 and read in a microplate reader (SpectraMax M5, Molecular Devices, LLC., San Jose, CA, USA) at 405 nm (25 • C).The reaction blank was done with PBS 1×.HSP70 concentrations were calculated as µg/µg of total protein.

Statistical Analysis
HSP70 levels were expressed as mean ± standard deviation.Normality and homogeneity were verified by the Kolmogorov-Smirnov and Levene tests, respectively.A t-test analysis was used to compare animals' weight and carapace width from polluted environments with those from pristine environments.The same analysis was also used to compare animals from the different sampling sites (polluted or unpolluted) in the field or under laboratory conditions.Finally, a two-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to describe the influence of the environment (polluted or unpolluted) and the animal maintenance conditions (field or acclimatized) on the HSP70 levels.The program used was Origin 2018 for graphic design and Sigma Stat for two-way ANOVA and t-test.The level of significance adopted was 5%.

Results
Table 1 shows the data referring to the weight and width of the carapace of animals from uncontaminated and contaminated environments.Animals from the pristine area present greater weight and mean carapace width than animals from the polluted area (p = 0.05).
Figure 1 shows the levels of HSP70 present in the three organs studied: anterior gills, posterior gills, and hepatopancreas.The results are a comparison between animals from regions with different levels of pollution (polluted and unpolluted) analyzed after collection (field) or after the acclimatization period in the laboratory (7 days).According to the results, the hepatopancreas had the highest levels of HSP70, followed by posterior gills and anterior gills.The different levels of environmental pollution (polluted and unpolluted environment) and the maintenance conditions of the animals (field or acclimatized) were factors that influenced the results.The anterior gills (Figure 1A) did not show significant differences in HSP70 levels from different environments (contaminated or pristine) or the maintenance conditions of the animals (field or acclimated).However, field animals tended to have higher HSP70 levels compared to acclimatized animals.

Discussion
The crab U. cordatus has been widely used in population and ecological studies due to its use as a fishing resource and its economic importance for populations in coastal regions [3].Comparing the two areas (polluted and unpolluted), the animals present in the unpolluted area have greater weight and mean carapace width than animals in the polluted area.The presence of individuals with larger carapaces in the unpolluted area is evidence of the low fishing activity [22][23][24][25] and the environmental quality of the region, Regarding the posterior gills (Figure 1B), significant HSP70 levels were found in acclimatized animals from polluted environments compared to acclimatized animals from unpolluted environments (p = 0.049).In addition, an increase in HSP70 levels was also observed in acclimatized animals from polluted environments compared to field animals (p = 0.049).
The hepatopancreas (Figure 1C) showed that field animals showed higher HSP70 levels than acclimatized animals from unpolluted regions (p = 0.049).Similarly, field animals from polluted environments showed increased HSP70 levels than field animals from unpolluted environments and acclimatized animals.After the acclimatization process, animals from polluted environments showed higher HSP70 levels compared to animals from unpolluted environments (p = 0.030).

Discussion
The crab U. cordatus has been widely used in population and ecological studies due to its use as a fishing resource and its economic importance for populations in coastal regions [3].Comparing the two areas (polluted and unpolluted), the animals present in the unpolluted area have greater weight and mean carapace width than animals in the polluted area.The presence of individuals with larger carapaces in the unpolluted area is evidence of the low fishing activity [22][23][24][25] and the environmental quality of the region, which is a conservation unit, considered pristine and free from environmental pollutants, mainly metals [3,4,13].
The analysis of HSP70 showed differences in each organ studied.The highest levels were found in the hepatopancreas, while the lowest was seen in the anterior gills.Studies with onshore crab Carcinus maenas demonstrated that HSP70 in gill tissue did not reflect the degree of metal exposure in animals collected from field sites contaminated with metals [26].In studies carried out with the mud crab estuary Scylla paramamosain, it was observed that the gills seem to be the primary and temporary target of metal accumulation, initiating the detoxification process, which will be completed in the hepatopancreas through the increased expression of HSP genes, which are related to apoptosis and the antioxidant system [11,27].In addition, crabs Scylla paramamosain exposed to cadmium showed low expression of HSP genes in gills, being significantly expressed in hepatopancreas [11], corroborating the data found in our study.Studies carried out with Callinectes danae showed that the hepatopancreas is a specifically detoxifying organ, contributing to the sequestration of metals and reducing the levels of stressful biomarkers.Thus, through the sequestration and detoxification of contaminants by the hepatopancreas, there is a reduction in the levels of circulating metals in acclimatized animals, which could influence the levels of HSP70, reducing them [4].
Field animals from polluted regions showed higher HSP70 levels when compared to animals from unpolluted regions, mainly in hepatopancreas.In posterior gills, high HSP70 levels were also observed in acclimatized animals from polluted regions compared to acclimatized animals from unpolluted areas.Studies carried out with trouts indicate an increase in HSP70 in gills and liver when present in contaminated environments [28,29].In the same way, Lithobius mutabilis accumulate high levels of HSP70 when adapted to contaminated areas [30], possibly due to a regulatory mechanism originated by the endogenous clock in the circannual rhythm [30].
The results show that the physiological responses of the animals in relation to the levels of HSP70 are closely associated with their environment of origin.Populations chronically exposed to contaminants can cope better with stress than populations present in pristine environments [31].U. cordatus crabs from contaminated environments develop greater Cd tolerance, greater metal detoxification capacity, and less genocytotoxic damage than crabs from uncontaminated regions [13].However, despite tolerance to contaminants, animals from contaminated environments do not show the same physiological response as animals from pristine regions due to exposure to various contaminants (organic or inorganic) throughout their lives [13].
Adjustments in the physiology of an organism, mainly related to biomarker proteins, can occur due to the acclimatization process [4].The laboratory's acclimatization period (7 days) did not change the HSP70 levels in the anterior gills.Studies carried out with Callinectes danae by Ortega (2022) [4] showed that 7 days of acclimatization are sufficient to minimize the effects caused by the stress of collection and transport of animals.However, according to McDonald and Wood (1993) [32], a more extended acclimatization period (about 16 days) could stabilize the physiological process.In this way, acclimatization in the laboratory for more extended periods could modify the physiological responses of the animals [33].However, in the hepatopancreas, the acclimatization period reduced the HSP70 levels in animals from the laboratory acclimatized period, compared to field animals, regardless of the environment of origin.This reduction or non-alteration of HSP70 may be associated with the stability of the ex situ environment without the alterations found in the natural environment.Asterias rubens echinoderms, for example, did not show changes in reactive oxygen species after laboratory maintenance [34].Callinectes danae from contaminated environments, when recently collected and acclimatized in the laboratory, did not show a reduction in the levels of contaminants, especially cadmium.This fact suggests the importance of the environment origin, the processes of accumulation, detoxification, and acclimatization [4].The mechanisms of acclimatization and recovery from damage caused by pollutants seem to be related to cellular repair factors.Initially, the mitotic activity and the repair of cellular components increase to later start tissue and systemic recovery.The repair and acclimatization process is estimated to be lower, around 16 days for fish gills [32].
Regarding the posterior gills, high HSP70 levels were found in acclimatized animals from polluted environments compared to field animals.Strongylocentrotus purpuratus urchins, for example, required several months in the laboratory to stabilize molecular responses related to the immune system [35].Studies carried out with Ucides cordatus showed that the gills can play a detoxifying role, sequestering contaminants through cytoplasmic granules, making them biounavailable and decreasing physiological responses to stress.However, when the contaminants are excreted, they are released from the interior of the cytoplasmic granules, remaining available in the cytoplasm, generating high physiological responses to stress [3].Thus, the responses to acclimatization can be quite varied between tissues of the same individual and should be considered with caution [36].

Conclusions
Our results suggest a physiological response in the crabs in a contaminated environment compared to animals in a pristine area.Animals sampled in the polluted region displayed higher HSP70 levels in hepatopancreas compared to animals from the unpolluted area, emphasizing the importance of this organ as a detoxifier and the importance of the HSP70 as a stress biomarker.In addition, acclimatized animals sampled in polluted locations present higher HSP70 levels in posterior gills than animals from uncontaminated areas, suggesting the influence of acclimatization on physiological processes.Therefore, our results suggest that regulation to decrease the damage caused by the environment and the acclimatization process may not be sufficient to stabilize physiological responses, especially in animals from polluted environments.

Ecologies 2022, 3 ,Figure 1 .
Figure 1.HSP70 levels (µ g/µ g of total protein) in different organs of U. cordatus from polluted and unpolluted regions analyzed in the field or after the acclimatization period.(A) Anterior gills.(B) Posterior gills.(C) Hepatopancreas.Different letters represent the significant differences (p < 0.05) between the polluted and unpolluted regions, and the asterisk represents the comparison between field and acclimatized animals.

Figure 1 .
Figure 1.HSP70 levels (µg/µg of total protein) in different organs of U. cordatus from polluted and unpolluted regions analyzed in the field or after the acclimatization period.(A) Anterior gills.(B) Posterior gills.(C) Hepatopancreas.Different letters represent the significant differences (p < 0.05) between the polluted and unpolluted regions, and the asterisk represents the comparison between field and acclimatized animals.