Differential Fatty Acids and Stress Biomarkers Responses in Native (Carcinus Aestuarii) and Invasive (Portunus Segnis) Crabs Exposed to Environmental Pollution

The present work aims to determine and compare the tolerance to different stress environmental conditions in two crabs’ species the native Carcinus aestuarii and the invasive Portunus segnis. In this study, C.aestuarii appeared more intolerant to environmental stressor as shown by the important changes occurred on the proximate, fatty acids and their essential compounds comparing to P.segnis. Our results revealed accumulation of metallic trace elements, macromolecular damages and antioxidant defense systems perturbations mostly in C.aesuarii gills and muscles than P.segnis, thus highlighting the importance of a multi-markers approach to assess the urban pollution in coastal ecosystems. According to this study, fatty acid proles and oxidative stress biomarker examination of both crabs indicate that the different environmental stresses signicantly inuence the tolerance of C.aestuarii compared to the invasive P.segnis which may provide physiological advantages for the achievement of their invasion in novel ecosystems. C22:5n-3, ranged between 1.28% and 6.52%), eicosatetraenoic acid (C20:4n-3, comprised between 0.05% and 6.77%), and linoleic acid (C18:2n-6, presented between 1.44% and 9.87%).Our results demonstrated that P.segnis gills contained signicant high levels of n-3 PUFA and low levels of total n-6 PUFA as compared to C.aestuarii in both studied seasons (p<0.001, One-way ANOVA). This lower level of n-3PUFA in C.aestuarii gills was proved by the decreases levels of EPA, DHA, C18:3n-3, and C20:4n-3during spring and winter seasons (p< 0.05 One-way ANOVA). Opposite variation was recorded for n-6 PUFA as evidence by statistical enhancement of the arachidonic acid (ARA; C20:4n-6) and it principal precursors (e.g. C18:2n-6 and C20:3n-6) in C.aestuarii gills among the two studied seasons when comparing with P. segnis (p< 0.05 One-way ANOVA). Similar trend was documented when compared the two seasons as evidence by an increase of SFA levels, principally C16:0, during winter in both P. segnis (+15% and +24%, respectively) and C. aestuarii (+27% and +46%, respectively) gills as compared to spring seasons. Concerning MUFA, it level remained stable between crabs gills during the spring as well as winter seasons. Nevertheless, several variation were recorded in the main PUFA composition of gills, showing decreases of n-3 PUFA and DHA and increases of n-6 PUFA and ARA in both P.segnis and C.aestuarii during winter when compared to spring (p<0.05, One-way ANOVA). lipids. native Anodontites trapesialis and exotic Limnoperna fortunei species Hence, they partly concluded that gills could reect greatly the concentration of TEs in surrounding habitat. Our results revealed that all invasive crabs behaved much better in favor of trace element build-up. The higher tolerance of P. segnis was evident from the signicant decrease of metal pollution and bioaccumulation indices, indicating more ecient resistance mechanism than C. aestuarii. A higher tolerance to the accumulation of TEs was translated by the low index of bioaccumulation which can bring physiological advantages for the success of the invasion of P. segnis over C. aestuarii populations. Bioconcentration of TEs in aquatic species by and salinity) and intrinsic (reproductive status …etc) Thus, signicant differences were expected to be observed in crabs among seasons. It has been noted that the accumulation of trace elements in both crabs’ organs seems to be more accentuated during winter season which coincides and correlate with the lower values of temperature and salinity (r ≥ 0.310;p<0.05). Apparently, this high accumulation could be related to the spawning process which occurred during the cold season as reported Glamuzina et al showed that TEs uptake signicantly enhanced the peroxidation products in mostly during the cold season. The excessive generation of ROS products may in turn maintain the overproduction of hydroxyl radical (.OH) through Fenton reaction, which cause unsaturated fatty acids oxidation, and alter membrane integrity (Ayala et al. 2014; Krumova and Cosa 2016). Our work is the rst attempt to compare FA composition of invasive and native crabs’ organs that revealing changes in both P.segnis and C.aestuarii proles in relation to environmental stressors. These changes were more marked in C.aestuarii gills tissues and elucidated by increases of SFA, mainly revealed by the signicant enhancement of palmitic (16:0), and stearic (C18:0) acids as compared to P.segnis. These results seems to be (i) an evident outcome of the lipid production occurrence (ii) an adaptive response to ensure the high energy demand needed for TEs detoxication in order to promote the membrane stability and (iii) probably ascribed to TEs toxicity. However, C.aestuarii muscles showed a decrease of SFA that can reect the damage causes in lipid metabolism as a shifty reaction beside the induced of ROS production. This supposition is conrmed by the signicant negative correlations between the lower SFA levels and TEs accumulation in muscles tissues. Additionally, it is well known that PUFA play a key role in cell development and function (Stillwell and Wassall 2003, Ruxton et al. 2005, Liu et al. 2015). The current results evidently exhibited that PUFA, n-3 PUFA levels mainly DHA and EPA and their respective precursors (ALA, C18:3n-3 and ETA, C20:4n-3) decreased signicantly in C.aestuarii tissues than P.segnis. Such variations were related to TEs accumulation and associated with


Introduction
Tunisian coastal ecosystems are threatened by increasing contamination due to the occurrence of chemical compounds, like polycyclic aromatic hydrocarbons (PAHs) (Barhoumi et al. 2016), pesticides (Mhadhbi et al. 2019) and metallic trace metals (TEs) ). These pollutants are released into coastal aquatic ecosystems as a result of human activities such as urbanization, industrialization, and agricultural activities. These xenobiotics affect aquatic organisms causing signi cant negative health issues and mortality . Along the Tunisian coast, the Gulf of Gabes represents high value eco-socio-system; it has been recognized through decades as a biodiversity "Hot Spot". This area has been a reservoir of marine organisms, high phytoplankton bloom and a nursery for several sh species (Ben Brahim et al. 2010;Enajjar et al. 2015). However, this shallow coastal highly productive ecosystem have been impacted by tides and submitted to urban/industrial e uents for years (Chi et et al. 2019). Previous reports demonstrated that the main cause of the disequilibrium of this ecosystem and the degradation of its water quality is the phosphogypsum discharge (Amor et al. 2018). Living marine organisms in the Gulf of Gabes have developed over time physiological adaptations physiological functions that allowed their survival and growth in such highly impacted ecosystem (Hattab et al. 2013). Despite the Gulf of Gabes is exposed to anthropogenic factors altering its natural features, it has been submerged by invasive species that represented an added pressure on its functioning and sheries. Several exotic invasive species have been recorded in Tunisian coastal ecosystems and speci cally in the Gulf of Gabes .
Biological invasions are a widespread and signi cant component of human-caused global environmental change where alien species invasion has been favored by new climatic conditions at the expense of native species (Vitousek et al. 1997;Perings et al. 2002). Climate change in uences invasive species distribution and behavior by affecting their entry pathways, establishment, spread and colonization of new habitats (Hobbs and Mooney 2005). It has been previously reported that global warming and climate changes affected native communities, altered ecosystem functions and increased the risk of biological invasion. Consequently, invasive species have been considered as the main drivers of global biodiversity loss and ecosystem degradation (Richardson et al. 2010) as they showed a strong tolerance to pollution and environmental stressors (Olden et al. 2006). Observable claims that invasive species are normally more tolerant to unfavorable conditions is still limited (Lenz and al. 2011), and therefore, further investigation is strongly recommended.
Recently, the popular invasion of the ecosystem of the Gulf of Gabes was characterized by the introduction of the blue crab (Portunus segnis), which has been introduced to the Mediterranean Sea after the opening of the Suez Canal and quickly became widespread on its coasts (Bejaoui et Bejaoui et al. 2021). In Tunisia, its invasive range covers the entire country coasts. It was recorded for the rst time in the Gulf of Gabès in 2015, but it has been assumed that its invasion occurred in Tunisian waters for at least two years before (Rabaoui et al. 2015). Like many invasive species, P. segnis invasion affected negatively native species, such as Carcinus aestuarii (C. aestuarii), through direct competition for food and habitat since it displayed faster growth rate and reproductive potential than the native species (Katsanevakis et al. 2014). This native crab (C. aestuarii) lives in shallow water and is able to withstand large variations in salinity and temperature, probably due to its tolerance and high adaptability (Yamada and Hauck 2001). C. aestuarii inhabited the Mediterranean Sea (estuarine and lagoon ecosystems) and having colonized the Adreatic Sea (Qyli et al. 2020).
In order to examine whether invasive species are more tolerant to stress compared with their native ones, we investigated the physiological responses of two marine crab species (C. aestuarii and P. segnis) against the environmental changes and trace metal pollution, that have been recently adopted in Tunisian food sector as an important and economically valuable marine products (Mili et al. 2020). Although, several studies investigated the effect of environmental pollution on native crabs, little attention has been paid to assess the tolerance of invasive crabs towards xenobiotics and their possible use as bio-indicators of pollution. Predicting alien invasive species adaptation in response to new dynamic environments, in particularly, environmental pollution presents a further serious challenge.
Up to date, there is no data available on antioxidant defense systems and their response induced by the accumulation of trace elements by comparing invasive and native crab species. For this, this study aimed to perform a comparative analysis of C. aestuarii and P.segnis to assess whether different realistic stressors can affect their metabolisms where the gills and muscles have different concentrations of trace elements. Therefore, the present study was planned to assess antioxidant networks and biochemical composition by means of established fatty acid pro les, proteins, lipids and carbohydrates contents.

Sampling area
The Gulf of Gabes is located on the eastern coast of Tunisia (34° 05′ 37″; 10° 26′ 13″), in the western Mediterranean basin (Figure 1). It covers an area of 90 km 2 which has an average depth of 40 m and a maximum width of 250 km. The Gulf of Gabes is known as the most dynamic ecosystem, devoting around 40% of the national aquatic production (DGPA 2015) and is considered as an important nursery for several species of sh (Enajjar et al. 2015).This ecosystem was recently identi ed as one of the eleven consensus eco-regions of the Mediterranean and is considered, along with the Venetian Plateau region, to be a shallow, phytoplankton blooming region known to be oligotrophic, as it is governed by the in ux of nutrients generated from Atlantic surface waters from Gibraltar (Krom et al. 2010, Ayata et al. 2017. Despite the hydrodynamics and biogeochemistry of this area, the Gulf of Gabes received important daily industrial and agricultural discharges that affect its quality ).

Sampling crabs and tissues preparations
A total of 50 sexually mature P. segnis and C. aestuarii of commercial size (Weight ≈ 180 g and length ≈10 cm) were collected from the Gulf of Gabes at about 50 m depth on spring and winter 2019. Winter season corresponded to the spawning period of both species where gamete release occurred. While, the spring period is marked by the beginning of gametogenesis process in which gametes formation and development in gonads starts (Tureli and Yesilyurt 2017). Samples were carried to the laboratory in aerated box with seawater and acclimated during 6 days. After acclimation, invasive and native crab individuals were sacri ced to remove gills and muscles tissues. Regardless of sex, for the biochemical analysis, organs were immediately homogenized with Tris-HCl buffer (20mM, pH 7.4) in cold, and then centrifuged during 25 min at 10.000 × g. The obtained supernatant extracts were stored at -80°C until proximate and oxidative stress analyses. For trace elements accumulation and fatty acids' analyses, gills and muscles were conserved in liquid nitrogen.

Metallic trace elements analysis
TEs analysis in muscles and gills tissues was made according to the Carvalho et al (2000) protocol. Each gill and muscle tissues were dried at 150°C until reaching constant weight and measured with precision scale (0.01g dray weight). Dry tissues (1g) were ground to powder using an agate mortar then dried powders were mineralized in Te on bombs in a closed microwave digestion labstation (Ethos D, Milestone Inc.) using concentrated nitric acid solution (4mL, 69%) and hydrogen peroxide (2mL, %). Digestates were diluted to an appropriate volume of 50 ml prior to being analyzed. TEs concentrations were determined using an inductively coupled plasma mass spectrometry (ICP-MS, Agilent, 7500ceModel). Results were expressed in mg/g of dry weight (DW). Analytical accuracy was checked by analyzing Certi ed Reference Materials (Charleston, SC, USA) to check the analytical precision. The results obtained for standard reference materials were within the 95% con dence interval and consistent with the certi ed values for all TEs. The obtained TEs concentrations were compared to maximum permitted limits for human consumption established by Montenegrin national legislation and other international and national guidelines ( Table 1). Table 1 Trace element concentrations (mg/g) in P. segnis and C. aestuarii muscles and gills collected from the Gulf of Gabes during spring and winter seasons.

Proximate composition analysis
Carbohydrate content was measured in muscles and gills of the invasive and native crabs according to Dubois et al (1956 ethanol and 1% chloroform. MT level was measured at 412 nm after adding 0.25 M NaCl, 1 mM EDTA and 0.6 mM DTNB to the pellets and the MT concentration was estimated using GSH as a reference standard and expressed as µmol of GSH/ mg of protein. Reduced glutathione (GSH) was determined according to the method of Ellman (1959). The reaction mixture was prepared with phosphate buffer (0,1M, pH 7.5), salicylic acid (4%) and DTNB (10mM). A yellow product was formed whose absorbance was determined at 412 nm. Reduced glutathione concentration was estimated using a standard curve previously prepared, and expressed as µg/mg of protein.
Glutathion peroxidase (GPx) was measured using GSH as a conjugation substrate based on the method previously described by Flohe and Gunzler (1984).
Superoxide dismutase (SOD) was determined spectrophotometrically at 580 nm according to Beauchamp and Fridovich (1971), and based on the ability of superoxide dismutase to inhibit the reduction of nitro-blue tetrazolium (NBT) by superoxide anion. One SOD unit was de ned as the amount of enzyme required to inhibit 50% of the NBT photo-reduction in comparison with tubes lacking the extracts. Activity was expressed as U/ mg of protein.

Anthropogenic indices
Metal pollution index (MPI) was evaluated to compare the total metal content at the different sampling sites and organs (Usero et al. 1997) and was calculated as follows: Equation (1) MPI=(Cf 1 × Cf 2 × Cf 3 ×……Cf n ) 1/n Where Cf 1 , Cf 2 , Cf 3 and Cf n (mg/g) were the average concentration of TEs in each organs and each species.
Bioconcentration factor (BCF) was determined to assess the relationship of TEs levels in invasive and native crabs, and was calculated as follows (Mendoza-Carranza et al. 2016): Equation (2) BCF=C native/C invasive where C native and C invasive (mg/g) stand for the average concentration of TEs in the crabs species.
Individual Mean Bioaccumulation index (IMBI) was calculated according to the equation given by Maes et al (2005) and ranged as follows: 0 < IMBI < 1.
Equation (3) IMBI=ICi / Cimax /n where, n is the total member of metals, Ci is the individual metal concentration of the heavy metal I, Cimax is the maximum observed concentration of the heavy metal I.

Data analysis
The resulted data was expressed as mean ± standard deviation (SD. The statistical analysis was performed applying R version 4.0.4 (R Core Team, 2021) (Harrell et al. 2021). Our results were analyzed for normality and homogeneity of variance using Kolmogorov-Smirnov and Levene's tests, respectively. A oneway analysis of variance (ANOVA) was used via Tukey HSD to establish the signi cant variation between the invasive and native crabs at p< 0.05. Principal Component Analysis (PCA) and correlation matrix between tested parameters were run by the statistical software program R version 2.15.2 and applied to assess the association between both crabs' species and measurements parameters in the gills and muscles tissues. The heatmap was generated with the R package heatmaply (Galili et al. 2021).

Metallic trace elements contents in P. segnis and C. aestuarii muscles and gills
Differences in TEs concentrations between P. segnis and C. aestuarii were detected and presented in Table 1. In particular, C. aestuarii exhibited the highest concentration of TEs in both gills and muscles (p<0.05, One-way ANOVA).
Pb, Cd, Cu, Fe and Ni levels were two-fold lesser in P. segnis gills than in C. aestuarii. Similarly, muscles tissues showed signi cant lower amounts of TEs (Pb, Cd, Cu and Ni) in P. segnis as compared to C. aestuarii (p<0.05, One-way ANOVA), except Fe which remained constant for both crabs tissues. During the winter, the contents of Cu and Cd in both crabs' tissues decreased signi cantly when compared to spring (Table 1). Only, gills tissues showed a signi cant decrease of Pb and Fe during winter in both P. segnis and C. aestuarii (p<0.05, One-way ANOVA). However, Ni contents showed a similar variation in both spring and winter seasons.

Metal pollution index (MPI) in P. segnis and C. aestuarii muscles and gills
The results of MPI are presented in Table 2, showing that C.aestuarii tissues exhibited the highest values as compared to P.segnis (p<0.05, One-way ANOVA).Our results demonstrated that MPI values varied signi cantly between the studied crabs (p< 0.05, One-way ANOVA), as evidence by an increase in C. aestuarii gills (by 25% and 15% during spring and winter, respectively) and muscles (by 25% during winter) as compared to P. segnis. Results are presented as means ± SD (n=6).

Bioaccumulation index (IMBI) in P. segnis and C. aestuarii muscles and gills
The distribution of the bioaccumulation index (IMBI) results according to periods and species are given in the Table 2. According to our results data, IMBI was lower in P.segnis gills and muscles compared to C. aestuarii (p <0.05, One-way ANOVA). Distribution patterns of IMBI in the studied seasons showed signi cant increases during the spring and winter for Cd, Cu, and Pb in gills of both P. segnis and C. aestuarii as compared to Fe and Ni which remained stable (p > 0.05, One-way ANOVA). For muscles tissues, only Cd showed a signi cant enhancement by +43% during the winter season in C.aestuarii as compared to P.segnis. When compared between seasons, Only IMBI values showed statistical variations as demonstrated by a signi cant increase of Fe in crabs gills (p<0.05, One-way ANOVA) and a signi cant decrease of Ni in crabs muscles (p<0.01, One-way ANOVA).

Bioconcentration factor (BCF)in P.segnis and C. aestuarii muscles and gills
The bioconcentration factors (BCF) of TE in P.segnis and C.aestuarii tissues are presented in Table 2. Based on this table, BCF values greater than 1 was observed for all the tested TEs. Our results showed that BCF, index related to Pb and Cd, increased signi cantly in gills during winter season by 23% and 20% as compared to the spring. However, no signi cant change was recorded for BCF index in muscles tissue. Thus, the most signi cant decreases of the BCF values were observed during winter season for Pb, Cd, Cu and Ni in both gills and muscles (p<0.05, One-way ANOVA).

Proximate composition in P. segnis and C. aestuarii muscles and gills
Proximate composition results are presented in Figure 2, showed signi cant variations between P. segnis and C. aestuarii gills and muscles organs(p<0.05 One-way ANOVA). There were signi cant decreases in lipids, proteins and carbohydrates contents in muscles and gills of C. aestuarii as compared to P. segnis during the two studied period (p<0.05 One-way ANOVA). In turn, during winter, the proximate composition decreased in both P. segnis and C. aestuarii organs, and reached minimal values as compared to the spring season ( Figure 2).
3.4. Fatty acid composition in P. segnis and C. aestuarii gills and muscles 3.4.1. Fatty acid composition in P. segnis and C. aestuarii gills Fatty acid pro les of crabs' gills are summarized in Table 3. Generally, twenty-nine fatty acids were determined and constituted about 94.1-99.3% of the total fatty acids. Large variations in the fatty acid composition of gills tissues were shown among both crabs' species, characterized with a higher proportion of PUFA, SFA than that of MUFAs. These fatty acid pro les were classi ed into PUFA, which included 44.765-51.951%; SFA, which explained by 31.426-43.204%; followed by MUFA, which composed by 11.066-13.131% of total fatty acids in both P.segnis and C.aestuarii. Results are presented as means ± SD (n=6).
In general, dissimilar variations were observed between the two studied crabs' gills. As shown in Table 3, signi cant increases in SFA levels were observed in gills of C.aestuarii as compared to P. segnis during the assessed seasons (p<0.05 One-way ANOVA). This increase due mainly to the signi cant enhancement of palmitic (C16:0), the predominant 010 SFA in all the examined crabs, that contributed approximately to 50% of the total SFA. Additionally, stearic acid (C18:0) was the second abundant SFA, ranging from 9.06-14.23% and showed similar variations in both crabs' gills. Other SFA with odd carbon numbers like myristic acid (C14:0), and pentadecanoic acid (C15:0) were presented in small and similar levels in P.segnis and C.aestuarii gills. However, arachidic acid (C20:0) and docosenoic acid (C22:0) showed signi cant increases in C.aestuarii gills than in P. segnis mainly during the winter season (+29% and +21% respectively).
Similar trend was documented when compared the two seasons as evidence by an increase of SFA levels, principally C16:0, during winter in both P. segnis (+15% and +24%, respectively) and C. aestuarii (+27% and +46%, respectively) gills as compared to spring seasons. Concerning MUFA, it level remained stable between crabs gills during the spring as well as winter seasons. Nevertheless, several variation were recorded in the main PUFA composition of gills, showing decreases of n-3 PUFA and DHA and increases of n-6 PUFA and ARA in both P.segnis and C.aestuarii during winter when compared to spring (p<0.05, One-way ANOVA).
Regarding the seasons, our data demonstrated that both native and invasive crabs were characterized by lower amounts of PUFA and MUFA during winter (p<0.01, One-way ANOVA). These amounts were mostly related to the decline of C18:1, n-3 PUFA, EPA and DHA in P.segnis and C.aestuarii muscles during the cold season (p<0.01, One-way ANOVA). Conversely, remarkable increases of the SFA levels mainly due to the rise of its main FA such as C16:0 and C18:0 were noticed during winter season for both studied crabs'.

Heatmap analysis of fatty acid composition
The hierarchical clustering based on all determined fatty acids is reported in Figure 3. These fatty acids were presented into a module demonstrating that C.aestuarii and P.segnis gills clustered together and separated from muscles organs (Figure 3). Our results revealed that gills were the most sensitive organs that show the highest responsive mainly during spring on C.aestuarii as compared to P.segnis as evidence by high amounts of SFA, n-6 PUFA and their main compounds (ARA, C18:2n-6..).

Lipid peroxidation indices in P. segnis and C. aestuarii muscles and gills
Our results showed a signi cant, increase in MDA levels (22% and 41%, respectively), and LOOH levels (40%, and 39%, respectively) in gills of C.aestuarii in comparison with P.segni samong the spring and winter seasons ( Figure 4). As regards muscles, signi cant increases were observed during spring for MDA (59%) and LOOH (71%) and during winter only for MDA (72%). However, when comparing the two seasons, both lipid peroxidation indices (MDA and LOOH) were decreased signi cantly during the winter season (p<0.05, One-way ANOVA).

Protein oxidation indices in P. segnis and C. aestuarii muscles and gills
Based on the obtained data in Figure 5, there is signi cantly increased of AOPP and PCO levels in C.aestuarii gills during spring (20% and 29% respectively) and winter (70% and 1% respectively) in comparison with P. segnis. They also showing signi cantly increases more than 50%, in C.aestarii muscles as compared to P.segnis among the spring and winter seasons.

Antioxidants responses in P. segnis and C. aestuarii muscles and gills
Antioxidants activities are provided in Table 5. Our results revealed that MTs, GSH, levels and SOD, GPx, GST activities increased in C. aestuarii gills in comparison with P. segnis (p<0.01, One-way ANOVA). Therefore, our results displayed signi cant increases of MTs (+49%), GSH (+55%), levels and SOD (+67%),GPx (+103%), GST(92%) activities in C.aestuarii muscles tissues during winter season as compared to P. segnis. Yet, during spring season, the MTs levels showed a signi cant increase by 81% in C. aestuarii muscles when compared to P. segnis. However, similar trends were recorded for GSH levels, SOD, GPx and GST activities between C. aestuarii and P. segnis muscles during spring season. The comparison between seasons showed that winter period was marked by the highest antioxidants activities in both crabs tissues as compared to the spring season (p<0.05, One-way ANOVA). Results are presented as means ± SD (n=10).

Correlation matrix
A Spearman correlation matrix was used to demonstrate the association between the temperature, salinity, proximate, biomarkers parameters, fatty acid groups and trace elements amounts in crabs gills and muscles (Table 6).   (Table 6). Spearman's correlation analysis showed signi cant positive relations between trace elements amounts (Pb, Cd, Cu, Fe and Ni) and lipid peroxidation (MDA and LOOH), protein oxidation (AOPP and PCO) indices and MTs levels (r ≥ 0.441; p<0.05). Nevertheless, negative correlations were observed between MDA, and AOPP with environmental parameters mostly T°C (r ≥ -0.582; p<0.01). Additionally, GSH levels showed impotents positive correlations with Pb (r=0.595; p<0.01), Fe (r=0.615; p<0.01), and Cu (r=0.862; p<0.001) and has negative and signi cant correlation with Cd (r= -0.479; p<0.05). Our results showed that SOD and GPx positively correlated with trace elements accumulation mainly Pb, Fe and Cu (r ≥ 0.654; p<0.001). These antioxidant systems have negative correlations with T°C and Spus (r ≥ -0.615; p<0.01).However, GST activity does not show any correlation with the above parameters. According to Table 6, all trace elements showed signi cant correlations with fatty acid groups except for Ni. These correlations were negative for EPA, DHA, PUFA and n3PUFA (r ≥ -0.415; p<0.01).and positive for ARA, n6PUFA and SFA (r ≥ 0.633; p<0.01).
For muscles tissues, results showed that Cu and Ni were signi cantly correlated with carbohydrates, proteins and lipids contents (r ≥ -0.423, p< 0.05) ( Table  6). Only carbohydrates have positive correlations with T° (r=0.509; p<0.01) and S psu (r = 0.476; p<0.05). Cu has signi cant positive correlations with MDA, AOPP, PCO and LOOH levels (r ≥ 0.493; p< 0.05), while MDA and AOPP showed positive correlation with Ni (r ≥ 0.543; p< 0.01). Opposite correlation was found between MDA, LOOH levels and physicochemical parameters as evidence by a negative correlation with T° (r ≥ -0.433; p< 0.05) and S psu (r ≥ -0.442; p < 0.05). According to Table 6, there were also negative correlations between GSH, MT, GPx, SOD, GST and trace elements amounts mainly Cu, Ni, Cd and Fe (r ≥ 0.465; p< 0.05). However, Pb has not any correlations with antioxidants defenses systems. With, all antioxidants biomarkers were correlated negatively with T° and S psu (r ≥ -0.495; p< 0.01) except for MT. Cu, Fe and Ni were the main trace elements that have signi cant correlations with fatty acid groups (≥ -0.442; p< 0.05). Also, there were positive correlations between abiotic parameters (T°C and Spsu) and DHA, PUFA, n3PUFA and n6PUFA (r ≥ 0.542; p< 0.01).
3.9 Principal compound analysis clusters of all redox status set in P. segnis and C. aestuarii muscles and gills In Principal component analysis (PCA), the two rst principal components explained 82.1% of total variations ( Figure 6). Our results revealed that the rst axis (modeling 64.8% of total variation) was positively associated with trace elements concentrations (Cd, Pb, Fe, Cu, and Ni), macromolecular damages parameters (MDA, LOOH, PCO and AOPP) as well as antioxidants defenses systems (GSH, MT, GPx, and GST) ( Figure 6). This axis showed negative correlations with proteins (p<0.001; r=-0.844) and lipids (p<0.001; r=-0.667) contents. The subsequent axis (modeling 17.3% of the total variation) was positively related with SOD activity and negatively with water parameters (T°C and Spsu). These axes described a physical gradient separating the studied crabs according to organs and seasons (Fig. 6). In general, PCA even allowed us to notice that the gills are the most sensitive organs compared to the muscles because they have an a nity for the accumulation of trace elements. In addition, they have important physiological responses (e.g., levels of MDA, AOPP, MT, etc.) than those observed in muscles. On the other hand, the muscles, especially during the spring season, are associated with the strong environmental variation and the levels of proteins and lipids. Plotting the rst two principal components showed that the native C.aestuarii crabs are the most sensitive compared to the P.segnis.

Discussion
One of the mainly universal problems in marine ecosystems is the appearance of invasive species, resulting in a risk to the biodiversity and the ecosystem functions . The present study demonstrated that C.aestuarii is more capable to accumulate TEs and the rate of organs translocation of their measured seems to be greater, compared to P.segnis. Our ndings clearly showed that C.aestuarii has a particular capacity to accumulate Pb, Cd, Cu, Fe and Ni in higher levels in its organs, mainly in the gills. This could be explained by the direct interactions of gills with the surrounding environment (water, Additionally, these high responses were more pronounced in gills tissues since TEs were highly accumulated (r≥ 0.750; p<0.01) and with low environmental conditions (temperature and salinity) (r≥-0.462; p<0.05). Similar to our data, Mansour et al (2020) showed that TEs uptake signi cantly enhanced the lipid peroxidation products in Ruditapes decussatus mostly during the cold season.
The excessive generation of ROS products may in turn maintain the overproduction of hydroxyl radical (.OH) through Fenton reaction, which cause unsaturated fatty acids oxidation, and alter membrane integrity (Ayala et al. 2014;Krumova and Cosa 2016). Our work is the rst attempt to compare FA composition of invasive and native crabs' organs that revealing changes in both P.segnis and C.aestuarii pro les in relation to environmental stressors. These changes were more marked in C.aestuarii gills tissues and elucidated by increases of SFA, mainly revealed by the signi cant enhancement of palmitic (16:0), and stearic (C18:0) acids as compared to P.segnis. These results seems to be (i) an evident outcome of the lipid production occurrence (ii) an adaptive response to ensure the high energy demand needed for TEs detoxi cation in order to promote the membrane stability and (iii) probably ascribed to TEs toxicity. However, C.aestuarii muscles showed a decrease of SFA that can re ect the damage causes in lipid metabolism as a shifty reaction beside the induced of ROS production. This supposition is con rmed by the signi cant negative correlations between the lower SFA levels and TEs accumulation in muscles tissues. Additionally, it is well known that PUFA play a key role in cell development and function (Stillwell and  This change in fatty acid composition in organs can affect the function of proteins, mainly those of the membrane known as the vital structural constituents of several bimoleculars (Habeck et al. 2016). In this line, PCO and AOPP have been known as good markers of oxidative stress, re ecting the uncontrolled free radical generation and protein oxidation damage (Soladoye et al. 2015).The present data demonstrated that AOPP and PCO levels increased in C.aestuarii than P.segnis which might be interpreted as a defense mechanism to the occurrence of protein oxidation stress. As previously described in aquatic organisms, high levels of lipid and protein oxidation are well documented as a signal for macromolecules alteration (Hussain et  Additionally, GPx, a main intracellular enzyme, breakdown the hydrogen peroxides (H 2 O 2 ) to water; and lipid peroxides to their corresponding alcohols principally in the mitochondria (Ng et al. 2007). In our work, SOD and GPx activities in the gills and muscles of C.aestuarii were signi cantly enhanced as compared to P.segnis organs. These responses could probably indicate the use of these enzymes during the removal of free radicals excess essentiallyO 2 and H 2 O 2 produced following TEs accumulation. On the other hand, GST reached higher activity in C.aestuarii than P.segnis act in the detoxi cation of lipid hydroperoxides derived from the TEs uptake in both organs. Our results showed that the increase in GST activities in the gills and muscles promotes greater Our current work provides evidence to suggest that seasonality could affect the physiology of crabs by altering their metabolic activity and response, causing disruption of biochemical constituents (lipids, proteins, carbohydrates), and leading to the increase of FA degradation. All these modi cations seems to be closely related to the paradox of TEs accumulation, environmental conditions and also to the reproductive cycle of crabs as reported previously in several

Conclusion
According to this study, we can conclude that the accumulation of TEs induces cellular stress, stimulates the biotransformation pathway, and interferes with the antioxidant defenses of the gills and the muscles of P.segnis and C.aestuarii among seasons. The obtained results highlighted the resistance of P.segnis to environmental stressors than C. aestuarii. Mainly in winter, the native species are more sensitive, thus re ecting the high toxicity of the absorption of TEs, which reinforces the idea about a greater sensitivity of C.aestuarii coupled with the higher redox status responses and the peroxidation processes. Additionally, the evaluation of lipid composition in our present work could be considered as a new tool that helps to disclose the eventual metabolic strategy adopted by native and invasive crabs' species to ensure their resistance against pollution impacts. This knowledge seems essential for the evaluation of the impact of different pollution sources on the ecosystems status to predict the state of the organisms which live there inside the framework of biomonitoring. -Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.

Declarations
-Competing Interests: The authors have no con ict of interest to declare.
-Availability of data and materials: Not applicable The geographic position of our sampling site in the Gulf of Gabes.

Figure 2
The proximate composition in the muscles and gills of P.segnis and C.aestuarii collected from Gulf of Gabes during spring and winter seasons.

Figure 3
Malondhyaldehide (A) and lipid hydroperoxide (B) levels in the muscles and gills of P.segnis and C.aestuarii collected from Gulf of Gabes during spring and winter seasons.

Figure 4
Advanced oxidation of proteins products (A) and proteins carbonyls (B) levels in the muscles and gills of P.segnis and C.aestuarii collected from Gulf of Gabes during spring and winter seasons.