Over the last centuries, the environmental impacts of heavy metal pollution have increased in the coastal areas, causing a major threat to the ecosystems and the biota they support (Ruiz et al., 2014). Among these xenobiotics, mercury (Hg) is listed as one of the most toxic elements due to its tendency to bioaccumulate and biomagnify through the food chain (Balshaw et al., 2007). The sources of mercury contamination in aquatic ecosystems include natural and anthropogenic emissions (Verlecar et al., 2008). The later generally come from incineration, fungicides, paints and industrial processes. One of the established mechanisms of Hg toxicity is its ability to induce cellular oxidative stress through the generation of reactive oxygen species (ROS) which react with macromolecules such as lipids, proteins and DNA (Uttara et al., 2009). As the cell membrane is tightly linked to cellular physiology, several authors have reported that the membrane is the primary detector of stress stimuli and activator of the cellular stress response (Dindia et al., 2013; Vígh et al., 2007). According to several authors, changes in the membrane biophysical properties as a primary response to stress, mainly trigger changes in lipid and fatty acid compositions (Los et al., 2004; Thyrring et al., 2015). Indeed, FAs, as the main constituent of the cell membrane, play crucial roles in several physiological functions as well as in the maintenance of membrane structures (Neves et al., 2015). It therefore appears that lipids and their constitutional components could be closely involved in cellular responses to pollutants such as Hg in aquatic organisms (Ferrain et al., 2018). In this context, it has been proven that the susceptibility of individual FA to peroxidation increases exponentially with an increasing number of double bonds on the carbon chain (Holman, 1954). Thus, polyunsaturated fatty acids (PUFAs) like eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are not only targets that are damaged by ROS, but also play a key role in enhancing an organism’s adaptation to environmental stress (Munro et al., 2016). Given this, FAs were recently argued to be promising bio-indicators to assess stress exposure and ecosystem health in a marine environment (Silva et al., 2017).

Nowadays, most of the available data regarding the use of FA as biomarkers for marine pollution monitoring refers to bivalves, micro and macroalgae and fish (Filimonova et al., 2016; Gonçalves et al., 2016); however, little is known about other marine organisms such as holothurians. Our previous study (Telahigue et al., 2019) investigated for the first time, the impact of acute mercury exposure on several biochemical parameters including fatty acid profile in the Holothuria forskali body wall. Holothurians, commonly known as sea cucumbers, represent an important marine resource due to their numerous nutraceutical and pharmaceutical proprieties (Bordbar et al., 2011). The sea cucumber fishery and aquaculture trade have been an active industry in Asia for a long time. In recent years, the sea cucumber trade has also flourished in some Mediterranean sea and NE Atlantic Ocean countries (Sicuro and Levine, 2011). The black sea cucumber or cotton-spinner Holothuria forskali, which is one of the most common sea cucumber species in the Mediterranean sea, is listed as a new target resource for future farming industries. (Sicuro and Levine, 2011). Like most of other sea cucumbers species, H. forskali is a deposit feeder and may ingest a large amount of sediment (Navarro et al., 2014) which make it particularly prone to heavy metal pollution. Several authors have reported that sea cucumbers could be considered as a potential bioindicator for heavy metal pollution (Turk Culha et al., 2016). Indeed, it is well established that mercury tends to accumulates in tissue in a specific manner depending on the organ’s physiological role and its regulatory ability. Generally, some tissues/organs such as body wall and respiratory tree have received particular attention to evaluate the impact of noxious stimuli in holothurians. Nonetheless, little information is available on the longitudinal muscles. In fact, muscle constitutes an interesting organ to analyze owing to the energy supply function and to its major role in locomotion and contraction movements in response to environmental stimuli and to its being a target tissue for bioaccumulation of merury (as confirmed by unpublished data obtained in our laboratory). The current study was mainly designed to evaluate the impact of Hg contamination on the fatty acid composition of H. forskali muscle. The results may provide valuable data concerning the underlying toxicity mechanism of Hg in lipid metabolism and to verify the validity of the FA profile as a biomarker of mercury intoxication in sea cucumber. Other parameters such as levels of H₂O₂ and LOOH were also assessed in order to verify the induction of an oxidative stress state in H. forskali muscle tissue following acute Hg exposure.

Heavy metals such as mercury are potential stressors that exert toxic effects through redox cycling which results in the uncontrolled production of ROS and a failure in antioxidant defense systems (Lushchak, 2011). In the current study, an over production of ROS was confirmed by the increase in the H₂O₂ level in all Hg-treated groups. Our results are consistent with several studies which reported a close correlation between the generation of intracellular ROS and the toxicity exerted by the inorganic Hg in several marine organisms (Verlecar et al., 2008; Wang et al., 2016). In this context, Patrick (2002) reported that exposure to Hg promotes the synthesis of H₂O_2, which could be converted into a reactive hydroxyl radical and lipid peroxidation (LPO) products in mitochondrial membranes. Furthermore, Hg ions are able to increase the action potential in the inner membrane of the mitochondria which regulates HO^-, O₂ ^- and H₂O₂, and negatively adjusts the defense enzymes SOD, CAT, GPx, as well as GSH (Flores et al., 2019).

Besides biomarker analysis, the determination of the fatty acid composition has proven to be a good indicator of stress and ecosystem health, due to their high sensitivity to stress factors and environmental changes (Gonçalves et al., 2016). Indeed, changes in lipid metabolism are known to be a common biochemical response to pollutant exposure and accumulation in marine organisms (Dailianis et al., 2011). Our results showed significant changes in the FA composition of treated sea cucumbers indicating an evident harmful impact of Hg on lipid metabolism. Thereby, after exposure to Hg, the FA profile of sea cucumber showed directional tendency anchored by an increase in SFAs, and the decrease in MUFAs and PUFAs levels. The observed changes are thought to be a compensatory process to defend the integrity of biological membranes and reduce cytotoxicity (Signa et al., 2015). It should also be noted that the most prominent changes in the FA composition of the Hg-treated H. forskali were mostly recorded at the lowest tested dose as confirmed further by the PCA analysis. Such findings can be attributed to a hormeosis response and suggest that low Hg dose response may be considered as a manifestation of the plasticity of biological systems. In fact, hormeosis, which is a dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition, was observed for Algae exposed to silver ions and nanoparticles (Tayemeh et al., 2020). The increased proportions of SFA mostly resulted from the increment in palmitic acid (C16:0) and may reflect an inflammation response to Hg intoxication. Indeed, the last cited FA is particularly known as a potent agent involved in the activation of proinflammatory signaling pathways (Ma et al., 2018). Another interesting finding recorded in the current study is the decrease in the MUFA group mainly due to the diminution of the oleic acid (C18:1n-9, OA) level. The latter is a nonessential FA which derives from the desaturation of stearic acid (C18:0) by the stearoyl-CoA desaturase (Scd). The recorded decline in the OA level is probably due to the inhibition of the Δ9-desaturase activity by Hg ions as reflected by the product-to-precursor ratio used in the current study. Furthermore, OA was probably used for energy production through β-oxidation and/or in modulation of membrane fluidity (Funari et al., 2003) and/or in detoxification related mechanisms as suggested by Ruiz-Gutiérrez et al., (1999). Also worthy of note, a significant depletion of the n-6 group and particularly ARA (C20:4n-6) and its precursor LA (C18:2n-6), which are presumably required for activation of eicosanoid synthesis through the arachidonic cascade. In fact, ARA is largely reported in the literature to be an important mediator involved in inflammation, immune response, and adaptation to stress through eicosanoid pathways (Calder, 2015). Likewise, we note the expenditure of EPA, another precursor to eicosanoids, which may reflect a compensatory mechanism to maintain membrane fluidity and to promote the adaptive response of the organism to stress (Delaporte et al., 2006). Another PUFA, docosahexaenoic acid (DHA), which is a main component of membrane phospholipids, was found to increase in Hg-treated animals. Such findings might reflect the ability of H. forskali to cope with Hg injuries through the induction of Δ6-desaturase activity as reflected by the enhanced 22:6n-3/22:5n-3 ratio. Indeed, it has been proven that the DHA may act as an antioxidant to reduce lipid peroxidation (Mayurasakorn et al., 2016). Furthermore, Vanse et al. (2002) reported that DHA could be involved in modulating the inflammatory response and regulating membrane homeoviscous to protect the cell from oxidative disruption caused by Hg intrusion. Similar pictures were observed for other marine organisms following exposure to metals and organic pollutants (Telahigue et al., 2019; Silva et al., 2017).

To further explore the impact of Hg on lipid metabolism, LOOH levels, primary breakdown products of long-chain fatty acid peroxidation, were determined. The increased LOOH levels recorded in all Hg-treated groups clearly reflected the dysfunction of the mitochondrial respiratory chain. Indeed, being rich in polyunsaturated fatty acids (PUFA) as other marine organisms (Liu et al., 2017), sea cucumber seemed to be vulnerable to lipid peroxidation. The continued oxidation of fatty acid, and the fragmentation of peroxides to produce aldehydes may eventually lead to losses in membrane integrity by alteration of its fluidity.

In the current study, the acute exposure to Hg promoted a state of oxidative stress as denoted by increased hydrogen peroxide and lipid hydroperoxide levels. The changes in the FA profiles of treated sea cucumber clearly indicated an evident toxic effect of Hg on the longitudinal muscle. The palette of acids, including SFA, MUFA and PUFA, determined in the muscle tissue, can be used in ecotoxicological research as an efficient test of the effect of mercury exposure. Furthermore, the prominent effects observed at the lowest dose, which is probably due to a hormeosis response, must be interpreted with caution since no information is available concerning the mechanistic difference between the toxicity and inhibitory effects of mercury to fatty acid pathways in holothurians. Lastly, our findings extend the understanding of the adaptive mechanisms of sea cucumbers and the usefulness of the FA analysis as a biomarker for Hg contamination.