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 (Mannino et al. 2017; Chan et al. 2019). The entry of invasive species such as P. segnis into the gulf of Gabes may possibly affect the regular functioning of C. aestuarii as a native species as they may compete for substrate, space and light. Despite the fact that several recent studies have raised concerns about the hazardous effects of TEs accumulation on marine species, no reports are yet available when comparing invasive and native crabs and in particularly from the Tunisian coast. Yet, this study was therefore planned to evaluate and compare the possible toxic effects of TEs bioaccumulation on the both invasive (P.segnis) and native (C.aestuarii) crabs using a multiple approach (biological, biochemical and cellular markers).
Crabs are intertidal and deposit feeders’ species (Iribarne et al. 1997), they have the capacity to bioaccumulate TEs, which are present in both phases (water and sediment) (Karar et al. 2019). 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 findings 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, sediment…etc) (Chaâbane et al. 2020). Additionally, our results demonstrated that essential elements such as Cu and Fe were detectable during the entire sampling period and were greater than the standard guidelines set by EU (2006), WHO (1993b) and USA (2006). Considering that Cd, Pb and Ni are nonessential toxic TEs, it may be assumed that there is positive bioaccumulation in both crabs’ gills since the values were below the standard guideline as recommended by EU (2006) and USA (2006). In line with this, gills commonly tend to bioaccumulate TEs more efficiently than other organs (e.g., muscles, foot and mantle), and this has been well demonstrated in several works carried out on aquatic species, such as the native Anodonta anatina and the invasive Sinanodonta woodiana (Bielen et al. 2016) and freshwater native Anodontites trapesialis and exotic Limnoperna fortunei species (Haj et al. 2019). Hence, they partly concluded that gills could reflect 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 significant decrease of metal pollution and bioaccumulation indices, indicating more efficient 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 may be influenced by physicochemical extrinsic (e.g., water temperature and salinity) and intrinsic factors (reproductive status …etc) (Pinheiro et al. 2012; Siddiqui and Saher 2021). Thus, significant 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 by Glamuzina et al (2017).
It has been reported previously that TEs accumulation in aquatic organisms may alter their metabolic functions and the macromolecular structures inducing reactive oxidative stress (ROS) production (Perrat et al. 2013; Hussain et al. 2018; Bejaoui et al. 2019). In this sense, the levels of MDA and LOOH of C. aestuarii organs, mostly in the gills, showed significant increases regardless to the high accumulation of TEs than in P.segnis. These inductions in the gills of C. aestuarii could be due to the structural and functional characteristics of this organ because it is located in direct contact with the external environment and has a small epithelium characterized by poor protection (Compere et al. 1989; Henry et al. 2012).This pattern was previously demonstrated in invasive and native bivalves related to TEs uptake (Rabeh et al. 2019). 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 significantly 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 first attempt to compare FA composition of invasive and native crabs’ organs that revealing changes in both P.segnis and C.aestuarii profiles 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 significant 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 detoxification 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 reflect the damage causes in lipid metabolism as a shifty reaction beside the induced of ROS production. This supposition is confirmed by the significant 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 significantly in C.aestuarii tissues than P.segnis. Such variations were related to TEs accumulation and associated with peroxidation process as consequence of membrane permeability depletion (Filimonova et al. 2016). This supposition is highlighted by the high correlation of PUFA, n-3 PUFA, DHA, and EPA with trace elements uptake (r≥ -0.409; p<0.05). Also, this could be attributed to its important role in mediating immunological and inflammatory responses (Thyrring et al. 2015; Chetoui et al. 2019). Consistent with this, our findings suggest that TEs uptake could be responsible for the decrease of these processes and may provoke simultaneous modifications of gills and muscles structure and function via the disruption of their physiological processes. Conversely, n-6 PUFA and ARA, as precursors of eicosanoids cascades; were significantly higher in C.aestuarii gills and muscles as compared to P.segnis which was highlighted by a positive correlation with TEs uptake. Thus, this enhancement of ARA prove the improvement in the adaptation of C.aestuarii to environmental stress mainly the invasion process of P.segnis, since it have an important physiological response (Vance and Vance 2002). Our findings were in agreement with previous reports pointed out on in situ and in vivo works indicating the changes of FA composition of aquatic species concomitant with trace elements uptake or/and exposure (Bejaoui et al. 2019; Fouzai et al. 2020).Yet, this alteration of FA composition during winter season may be attributed to the decrease of temperature and salinity which are known to affect significantly the membrane fluidity (Laurel et al. 2012; Malekar et al. 2018; Guo et al. 2019).
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, reflecting 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 al. 2018; Bejaoui et al. 2019).These results were further confirmed by a negative correlation found between the contents of proteins, lipids, glycogens and the peroxidation products (MDA, LOOH, AOPP and PCO) (r≥; -0.400; p <0.05) and demonstrated the toxic effect of the TE uptake that targeted the organ functioning and causes cellular stress.
In accordance with this, the generation of peroxidation products in response to TEs accumulation activates antioxidant machinery which includes both antioxidant enzymes and non-enzymatic compounds to neutralize the ROS produced by avoiding potential oxidative damage to cellular components (Ross et al. 2006; Messina et al. 2014). To cope with oxidative stress produced by ROS generation, SOD as the most powerful and the primary detoxification enzyme, removes the superoxide radical through the process of dismutation to oxygen and hydrogen peroxide (2O2+− + H+ →H2O2+O2) (Ighodaro et al. 2018). Additionally, GPx, a main intracellular enzyme, breakdown the hydrogen peroxides (H2O2) 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 significantly enhanced as compared to P.segnis organs. These responses could probably indicate the use of these enzymes during the removal of free radicals excess essentiallyO2 and H2O2 produced following TEs accumulation. On the other hand, GST reached higher activity in C.aestuarii than P.segnis act in the detoxification 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 exclusion of lipid hydroperoxid generated by the radical assault of lipid molecules which is demonstrated by a high positive correlation with lipid peroxidation products (r≥; 0.528; p <0.01). These results align strongly with previous works, where TEs accumulation have been shown to disrupt the redox statut balance of native bivalves Mytilus galloprovincialis than the invasive one Brachidontes pharaonis (Rabeh et al. 2019). Other findings showed that antioxidants enzymes increased mostly during the winter season that underline the activation of biotransformation and detoxification processes as an effort to minimize the oxidative damage caused by TEs bioaccumulation (Maranho et al. 2015; Uluturhan et al. 2019). Explicitly, GST is a phase II detoxification enzymes that remove the ROS generation products mainly by catalyzing GSH-dependent conjugation and redox reactions (Eroglu et al. 2015). Along with, GSH, is a tripeptide, confers cellular protection by directly reducing free radicals and conjugating endogenous and exogenous electrophiles (Lushchak 2012). This non-enzymatic antioxidant latter was found to increase in C.aestuarii gills and muscles as GSH exert its detoxifying function by acting as a direct forager of ROS and electrophilic compounds (Hellou et al. 2012). The enhancement levels of GSH were reported by Campillo et al (2013) and Capo et al (2015) in R.decussatus and P. nobilis respectively, collected from contaminated sites. Like GSH, metallothionein (MT) as major source of cellular thiols, has a high ability to bind metallic ions (Mao et al. 2012). Additionally, MTs have a key role in the bioaccumulation and detoxification of essential and non-essential TEs and participate in their homeostatic regulation to meet enzymatic and other metabolic demands (Mao et al. 2012). This investigation showed induction of MTs in C.aestuarii organs which seems to take part in the detoxification of excessive TEs uptake in both gills and muscles, since MT has the ability to limit the effect of hydroxyl (OH) and superoxide (O2−) radicals by removing them (Amiard et al. 2006). This phenomenon has been demonstrated in several aquatic species in the laboratory (Aich et al. 2017) and from contaminated natural habitats (Tremblay et al. 2020). Generally, the notable difference in the antioxidants levels in both crabs’ tissues was recorded between seasons. Accordingly, higher redox status responses found in winter in comparison to spring is affected by the implied a relation between the bilogical status and TEs metabolism. Analogous results from previous research on TEs assessment in different aquatic species revealing high antioxidants responses during the cold season (Marques et al. 2018; Uluturhan et al. 2019; Mansour et al. 2020).
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 modifications 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 works carried out on aquatic organisms (Karar et al. 2019; Ghribi et al. 2020).