Effects of copper on larvae of the marbled crab Pachygrapsus marmoratus (Decapoda, Grapsidae): Toxicity test and biochemical marker responses

The importance of trace elements in ecotoxicological investigations is a well-known issue when monitoring polluted areas such as commercial harbours. Copper represents one of the most common metal contaminants, often detected in these areas as it is widely employed in various fields and has many sources of inflow in the marine environment. Pachygrapsus marmoratus is a widespread intertidal crab species that has been extensively studied in ecology, ethology and population genetics. Ecotoxicological studies have also been performed, exclusively on the adult stage. In the present study we investigated the mortality and biochemical (oxidative stress and neurotoxicity) responses of P. marmoratus larvae exposure to environmental relevant concentration of copper. Results showed dose-dependent responses in terms of larval mortality, with a calculated LC50 value of 0.5 mg/L of Cu. The LC50 concentration was used as the starting point for subsequent biochemical response evaluation. Results also demonstrated dose-dependent activation of antioxidant systems assuming a compensatory antioxidant activity to prevent higher cellular damage when larvae were exposed to the highest concentrations of copper. Moreover, a significant enhancement of neurotransmitter activities was observed, assuming a possible direct interaction of copper with the enzymes or an increase of free copper ion aliquot into the cells.


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-Introduction
Commercial harbors are known to be highly contaminated areas due to ordinary and non-ordinary activities, usually associated with water column and bottom sediments contamination (Denton et al., 2005). There are various sources of contamination including anthropic activities -such as traffic, dredging, effluent discharge -and natural events -such as river overflows and rainwater runoff (Bourg and Bertin, 1996;Schiff, 1995; Guerra-Garcia and Garcia-Gomez, 2005, Bretzel andCalderisi, 2006;Lye, 2009). Almost all main classes of contaminants are present in these areas, from Polycyclic Aromatic Hydrocarbons (PAH) to trace elements (Boyden, 1975;Denton et al., 2005;De Luca et al., 2005;Bretzel and Calderisi, 2006;Sprovieri et al., 2007;Chen et al., 2013). Copper is found in higher concentrations in harbors -both in the sediments and watercompared with other coastal areas, due to the presence of various sources of introduction, such as antifouling paints (Turner, 2010), as well as depending on physicochemical conditions (Förstner et al., 1986) and the concentration or degradation of the organic matter (Teasdale et al., 2003;Caplat et al., 2005).
Due to the high presence of contaminants in commercial harbors, chemical and ecotoxicological monitoring of water and sediments are usually mandatory and organized by environmental protection agencies or similar, especially during particular events, such as dredging. Despite of well-described and regulated procedures for chemical monitoring, ecotoxicology often focuses on mesocosms bioassays and usually occurs in laboratories. These assays, fundamental for acute and chronic description of bioavailable contaminant effects, have the limitation to be a pinpoint vision on what is happening in a specific environment. For this reason, ecotoxicological evaluations on organisms living in polluted areas, may increase the accuracy data collected from classical bioassays.
Pachygrapsus marmoratus (Fabricius, 1787) (Decapoda; Brachyura; Thoracotremata; Grapsidae) inhabits the rocky coasts of Mediterranean Sea, Black Sea and north-eastern Atlantic Ocean -from Brittany to Morocco including the Canary Islands, the Azores, and Madeira. (Zariquiey, 1968;Ingle, 1980 Flores andPaula, 2001). The marbled crab is highly prolific, with females producing thousands of fertilized eggs each reproductive season -between March and September. Dispersal -influenced by tidal and wind-driven currents -occurs through a series of planktonic larval stages lasting about a month, prior to settlement (Aydin et al., 2014). Adults are relatively sedentary (Cannicci et al., 1999). The complete zoeal development

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A C C E P T E D M A N U S C R I P T 3 of P. marmoratus underwent six stages and the mean duration in days have already been described by Cuesta and Rodríguez (2002): Zoea I: 3.5; Zoea II: 4; Zoea III: 4.5; Zoea IV: 4.5; Zoea V: 4; Zoea VI: till the end of the larval development.
Crabs are among the most important organisms in estuarine and coastal food-webs because they connect primary producers and organic detritus to secondary consumers, promoting nutrient cycling and water quality (Madeira et al., 2014). Moreover, intertidal organisms, such as P. marmoratus, are considered model species in studies focusing on temperature (see Vinagre et al., 2012) osmotic (Jayasundara et al., 2007) and trace elements stress (Fratini et al., 2008;Tejada et al., 2015;Rainbow et al., 2000). Adults of P.
marmoratus exposed to different metal contaminates showed bioaccumulation in the hepatopancreas and gills (Fratini et al., 2008), oxidative stress responses (Tejada et al., 2015) and effect on the rate of uptake (Rainbow et al., 2000). Moreover, correlation between salinity acclimation and the effect of metal ion on different crab species has been shown. Vitale et al. (1999) found that at low salinity (2.5‰) cadmium has a median lethal effect (LC50) on Neohelice granulata (Chasmagnatus granulatus) more than 20 times lower than at high salinity (30‰). Bianchini et al. (2008) also reported numerous other implications of salinity related toxicity of copper, cadmium and other trace elements on Neohelice granulate. Concerning the larval stages, standard measures of toxicity such as mortality and LC50 after metal exposures (Ahsanullah et al., 1978;Mortimer et al., 1994;Botton, 2000;Greco et al., 2001;Ferrer et al., 2006;Neil et al., 2005) have already been investigated in other crab species. However, to our knowledge, there is no available data on biochemical responses of P. marmoratus larvae. Considering that studies using biochemical biomarkers can be helpful in establishing cause-effect relationships of specific contaminants and have been used to predict their effect on natural populations (Clements, 2000), The aim of this work is to investigate the effect of copper on larvae (zoea I) of the intertidal crab P. marmoratus after 48h acute copper exposure, evaluating survival (LC50) and biochemical responses in terms of oxidative stress and neurotoxicity.

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4 during spawning period (Mouneyrac et al., 2001) -from June to August 2018 (Note 1). The crabs were carefully transported to the laboratory and maintained in 20 L tanks (2-3 individuals per tank), filled with 5 L of filtered seawater (S=37‰, T=20±2 °C) collected from Quercianella (Livorno, Italy). Each aquarium was also provided with stones as hiding places and exposed areas where the females could aerate the eggs. Water was changed every two days. At the same time of each water renewal, fresh Ulva lactuca was added as food.
Note 1: According to the national law (Decree 26/14), that implements the Directive UE 2010/63 on the protection of animals used for scientific purposes, crabs are not under protection regimen.

-Larvae spawning, collection and maintenance
Larval spawning of P. marmoratus generally occurs synchronised with the new moon and full moon (Saigusa and Hidaka, 1978). For this reason, tanks were maintained at natural light and photoperiod. Shortly before the spawning, P. marmoratus females bearing fully mature embryos are easily recognizable, because of the size and colour of the eggs and due to their behaviour -spending more time outside the water and moving their pleiopods to aerate embryos. Females approaching spawning were isolated from the other females and transferred in 1 L beakers, with the same setup as the 20 L tanks. Spawning was checked daily.
Once released, larvae were gently removed from beakers with a sterile 3 mL plastic pipette and put in flasks with fresh filtered seawater (FSW; S=37‰, T=20±2°C) and kept in the same conditions as adults. Larvae were fed daily with algal suspension (Isochrysis galbana + Rhodomonas reticulata) (10 5 cells/mL).

-Chemicals
Copper, as copper (II) sulfate pentahydrate (CuSO4*5H2O) (Sigma-Aldrich ® ) was used as reference toxicant for both 48-h acute toxicity assay and in-vitro exposure for biomarkers evaluation. All the concentrations presented in the graphics and tables are reported as Cu 2+ . A 250 mg/L CuSO4*5H2O stock solution was prepared dissolving 50 mg of the salt directly in ultrapure water. All dilutions were prepared in FSW.
Nominal and measured concentrations for all testing dilutions are reported in Table 1.

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5 Six concentrations of copper (II) sulfate pentahydrate (CuSO4*5H2O) (0.25 -0.50 -1 -2 -4 -8 mg/L Cu 2+ ) were used for the mortality assay. Day-1 larvae were used as model organisms for a 48-h mortality assay.
Three replicates for each copper concentration and control were prepared in 100 mL glass beakers. Each beaker was filled with 50 mL of sample or control. 10 actively swimming larvae were carefully pipetted in each beaker. All containers were then covered with Parafilm ® strips to prevent water evaporation. All beakers where maintained at 20±1 °C, under a photoperiod of 12:12 light: darkness, for 48 h, without food.
Mortality in each beaker was registered at 24 and 48 h of exposure and dead organisms removed. The criterion for determining death was the absence of movement in 1-2 min of observation. 2.6 -Biochemical analysis 2.6.1 Superoxide dismutase (SOD) activity SOD activity was determined following Magnani et al. (2000) method, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol. The autoxidation of pyrogallol in the presence of EDTA at pH 8.2 is 50%. The principle of this method is based on the competition between the pyrogallol autoxidation by O2 •¯ and the dismutation of this radical by SOD. Absorption was read at the wavelength of 420 nm against Tris-EDTA buffer at time zero and after 1 minute of the addition of pyrogallol. SOD activities are expressed as units (U)/mL where one unit (U) is defined as the amount of enzyme required to cause 50% inhibition of

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A C C E P T E D M A N U S C R I P T 6 pyrogallol autoxidation.

Catalase (CAT) activity
CAT activity was determined by measuring the decrease of hydrogen peroxide in absorbance at 240 nm (Aebi, 1983). The mixture was composed of 200 μL of supernatant and 500 μL of 50 mM phosphate buffer (pH 7) and the reaction was started by the addition of 300 μL of hydrogen peroxide (30mM). The decrease in absorbance was recorded every 15 seconds up to 3 min. Catalase activity was expressed as μmol H2O2/min/mg protein.

Glutathione reductase (GR) activity
GR activity was determined using the method described in Wheeler et al. (1990). GR catalyses the reduction of glutathione disulfide (GSSG) to the sulfhydryl form glutathione (GSH), functioning as dimeric disulfide oxidoreductase and utilizes a FAD prosthetic group and NADPH to reduce one molar equivalent of GSSG to two molar equivalents of GSH. The absorbance was measured at 340 nm, the enzymatic activity was determined using ε=6.22 mM -1 cm −1 and the results expressed in nmol/min/mg protein.

Cholinesterases (ChEs) activities
ChEs activity was measured according to Ellman et al. (1961) method in 100 mM phosphate buffer (pH 8.0), 10 mM DTNB, and different concentrations of substrates (AChE; BChE; PChE). Enzyme activity was recorded continuously for 5 min at 412 nm and the specific activity was corrected for the spontaneous hydrolysis of the substrate and expressed as nmol/min/mg protein, using a molar extinction coefficient of 13.6x10 3 M -1 cm -1 (the yellow dianion of 5-thio-2-nitrobenzoic acid, TNB).

-Data analysis
All data were represented as mean ± standard deviation. One-way ANOVA were performed for data analyses (PRISM software, Graphpad Software). Magnitude values with p<0.05 were considered statistically significant. The mortality assay result, as LC50 value, was calculated using the PROBITS method (Finney, 1971).

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Mortality assay
Results of 24-h and 48-h readings of larval mortality assay are reported in Table 2. No mortality was registered in controls after 24 h and 6.66% after 48 h. These results were used for a LC50 calculation using the method of PROBITS (Finney, 1971). LC50 values were calculated both for 24-h and 48-h of exposure, both as copper sulphate pentahydrate and as copper ion, and are reported in Table 3.

Superoxide dismutase (SOD) activity
SOD results for 1-h exposure of P. marmoratus to CuSO4*5H2O (0.125; 0.25 and 0.5 mg/L Cu 2+ ) are showed in Fig. 1A. All the treatment groups, except the 0.125 mg/L group, were significantly different (p < 0.05) from control, while SOD activity was significantly higher in the specimens exposed to the two highest concentrations (0.25 and 0.50 mg/L Cu 2+ ) in comparison to larvae exposed to 0.125 mg/L Cu 2+ (p<0.05).

Catalase (CAT) activity
A significant dose-dependent increase of CAT activity was detected in larvae exposed to all the contaminant concentrations (0.125; 0.25; 0.5 mg/L Cu 2+ ) in comparison to controls (p < 0.05). No significant differences were observed among exposure concentrations (Fig. 1B).

Glutathione reductase (GR) activity
Larvae exposed to 0.25 mg/L Cu 2+ showed a GR activity significantly higher than the control group (p < 0.05) while no differences were observed when comparing the larvae exposed to 0.125 mg/L Cu 2+ with the non-exposed larvae (Fig. 1C). The specimens exposed to the highest exposure concentration (0.50 mg/L Cu2 + ) exhibited significant lower inhibition of the activity in comparison to control. Significant differences were observed between 0.125 and 0.5 mg/L Cu 2+ (p<0.05), showing higher enzyme activity in larvae exposed to the lowest contaminant concentration.

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8 In AChE activity results, no significant differences were observed among exposed and non-exposed larvae (data not shown).
BChE activity results -reported in Figure 2B -showed that 0.25 and 0.50 mg/L Cu 2+ treated groups were significantly different from control and 0.125 mg/L Cu 2+ (p < 0.05) showing higher BChE activity. No significant differences were observed among specimens contaminated with these two higher exposure concentrations. Regarding the PChE results, the group exposed at 0.50 mg/L Cu 2+ was significantly different (p < 0.05) from the control (12.04 ± 9.06 nmol/min/mg protein) showing higher enzyme activity (50.15 ± 16.01 nmol/min/mg protein) compared to all the remaining concentrations ( Fig. 2A).

Toxicity on larval stages
The evaluation of copper toxicity, as percentage of mortality on first larval stage (Zoea I) showed a dosedependent response after both 24 and 48 hours of exposure. The calculated LC50 value decreased more than three times from 24-h to 48-h, underlining a putative time-dependent toxicity of copper, as reported in similar assays on other crab species, such as Maja squinado (Marino-Balsa et al., 2000), Cancer anthonyi (Macdonald et al., 1988), Chasmagnatus granulata (Lopez Greco et al., 2001;Ferrer et al., 2006). The authors reported a LC50 value of more than 1000 µg/L after 24 hours and 755.39 µg/L after 48 hours of exposure. Such concentrations of copper match with data obtained in the present work. P. marmoratus is generally known as a species with high resistance to different stress sources (Tejada et al., 2015), due to its peculiarity of inhabiting supralittoral crevices (Augusto and Flores, 2001). However, findings on acute copper toxicity showed that resistance present in adults is not present in larval stages. Contaminants, such as copper, can actively affect mortality percentages in the first hours of larval life, consequently affecting P. marmoratus dispersal.

Oxidative stress related enzymes
Copper can induce cellular toxicity (Gaetke and Chow, 2003) with the formation of reactive oxygen species -ROS (Bremner, 1998;Kadiiska et al., 1993). ROS differ in terms of cellular reactivity and potentially cause

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9 toxic insults to lipids, proteins and DNA (Regoli and Winston, 1999). In basal condition antioxidant systems -such as SOD, CAT, GPx, glutathione redox cycle -are involved in coping against cellular damage (Regoli and Giuliani, 2014). A concentration-dependent increase of SOD and CAT activities were observed, assuming a compensatory antioxidant activity to prevent higher cellular damage when larvae were exposed to the highest concentrations of copper. In a study conducted by Amin et al. (2010)

Neurotoxicity
Cholinesterases (ChEs) are carboxylic ester hydrolases that break down esters of choline. They include specific cholinesterase (AChE) and non-specific ones, or pseudo cholinesterase (PChE and BChE). The preferred substrate for AChE is acetylcholine, while non-specific cholinesterases prefer butyrylcholine and/or propionylcholine, depending on the species (Lionetto et al., 2011). Several studies demonstrated the

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inhibition of ChE activities in non-model species -such as crabs -after contaminants exposure. Elumalai et al. (2007) found an inhibitory effect of zinc and mercury on ChE activity of the crab Carcinus maenas.
Similar results were obtained by Narra (2015) for the freshwater crab Barytelphusa guerini after exposure to chlorpyrfos insecticide. However, in the present study, the activity trend of both examined ChEs seemed to be in contrast with results of cited literature, showing a dose-dependent increase of activity after in vitro copper exposure. Our findings can be explained with Khedher et al. (2017), which detected a significant enhancement of AChE activity in muscle tissues of the crab C. maenas after 2 days of time exposure, followed by a repression at 7 days. Similarly, Cunha et al. (2007) showed an increase of AChE activity in the gastropod Nucella lapillus exposed to 44000 µg/L Cu, which may be due to the direct interaction of this metal with the enzyme or an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis (Romani et al., 2003). Moreover, the use of ChEs to account for responses in the antioxidant enzymes and lipid peroxidation levels is suggested to be a useful application to detect the possible exposure/effect induced by contaminants on living organisms (Lionetto et al., 2011). A significant correlation between ChEs and antioxidant enzyme activities was found in several species following contaminants exposure, as reported in mussels (Lionetto et al., 2003) and fish (Lionetto et al., 2003;Kavitha and Rao, 2008). For example, in mosquito fish, Gambusia affinis, Kavitha and Rao (2008) found the inhibition of brain AChE activity directly related to the inhibition of antioxidant enzymes, while the benthic fish Mullus barbatus, AChE activity showed a significant inverse correlation with glutathione reductase activity, but not with catalase activity (Lionetto et al., 2003). The results of the present study agree with the latter ones, underlining a similar trend in P. marmoratus as Lionetto et al. (2003) found in M. barbatus.

-Conclusions
A quantifiable and dose-dependent effect of Cu in in vitro exposure on Pachygrapsus marmoratus zoea I larval stage was observed. Results showed dose-dependent responses in terms of larval mortality as well as concentration-dependent activation of antioxidant systems assuming a compensatory antioxidant activity to prevent higher cellular damage when larvae were exposed to the highest concentrations of copper. Moreover, a significant enhancement of neurotransmitter activity was observed, hypothesizing a possible direct interaction of this metal with the enzyme or an increase of free Cu aliquot into the cells.

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These results underlined the importance of investigating the effects of pollutants on different developmental stages of resistant species and to better understand their impact on dispersal behaviour of intertidal marine organisms. Further studies are necessary to consolidate these results at a different level between larval stages and adult stage to raise ecological significance from individual to population level.   activities (mean ± standard deviation), in Pachygrapusus marmoratus exposed to different concentrations (0.125; 0.25 and 0.50 mg/L Cu 2+ ) of CuSO4*5H2O. Significant differences (p ≤ 0.05) are presented with bold asterisks (*).

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