Immune response to abamectin-induced oxidative stress in Chinese mitten crab, Eriocheir sinensis

Immune response to abamectin-induced oxidative stress in Chinese mitten crab, Eriocheir sinensis

Ecotoxicology and Environmental Safety 188 (2020) 109889 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal ho...

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Ecotoxicology and Environmental Safety 188 (2020) 109889

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Immune response to abamectin-induced oxidative stress in Chinese mitten crab, Eriocheir sinensis

T

Yuhang Honga,∗,1, Hongmei Yina,1, Yi Huanga, Qiang Huanga, Xiaozhen Yangb a

Key Laboratory of Application of Ecology and Environmental Protection in Plateau Wetland of Sichuan, Xichang University, Xichang, 415000, Sichuan Province, China Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Engineering Research Center of Agriculture, Shanghai Ocean University, 999 Huchenghuan Road, Lingang New District, Shanghai, 201306, China

b

A R T I C LE I N FO

A B S T R A C T

Keywords: Abamectin Eriocheir sinensis ROS Immune response

It is known that abamectin (ABM) inflicts oxidative damage on aquatic animals; however, knowledge about the immune response under pesticide-induced oxidative stress is incomplete. In the present study, several cellular and humoral immune parameters, including total haemocyte counts (THC), lysosomal membrane stability (LMS), activities of acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LZM) were investigated to reveal the effects of ABM exposure on the immune defence mechanisms of the important freshwater crab, Erocheir sinensis. According to the results, a significant increase of THC was found in low concentration groups (0.03 and 0.06 mg/L), while dramatic decreases occurred in high concentration groups (0.12 and 0.24 mg/L) after 96 h of exposure. We also detected significant increases of reactive oxygen species (ROS) in haemocytes at 0.12 and 0.24 mg/L, and there was a dose- and time-dependent decrease of lysosomal membrane stability. These results suggest that the excessive generation of ROS induced by ABM may be leading the massive collapse of lysosomal membrane, which in turn may be causing the sharp drop of haemocyte counts in E. sinensis. The increase of hydrolytic enzymes ACP and AKP at low concentrations and the decrease at high concentrations also indicate an immune response associated with haemocytes status under stress. However, activities of LZM decreased significantly. After injection of Aeromonas hydrophil, mortalities increased under exposure to ABM and were positively related to ABM concentration. These results confirm that ABM exposure has the ability to impair immune defence and result in the host's susceptibility to pathogens.

1. Introduction Agrochemicals are developed to eliminate pests and increase crop productivity in the agricultural industry. However, the extensive use of these chemical products on a worldwide scale has entailed risks not only to human health but also to the non-target organisms and, more seriously, to the ecological environment. Among all agrochemicals, abamectin (ABM) is the most frequently used compound in recent decades for its efficiency in both crop protection and for pharmaceutical purposes (Bai and Ogbourne, 2016). As a member of the avermectin family of 16-membered macrocyclic lactones derived from the actinomycete Streptomyces avermitilis, abamectin has drawn a lot of attention in ecotoxicological studies (Jansson and Dybas, 1998). ABM is a mixture of avermectins containing about 80% avermectin B1a and 20% B1b, which have similar biological and toxicological properties (Johnson, 1991). The main action of ABM is its effect on glutamate and

γ-amino butyric acid (GABA)-gated chloride channel, which is located in brain cells and secured by the blood-brain barrier in mammals (Ian R. Duce et al., 1995; Omura, 2008). But nowadays, more and more studies demonstrate its high toxicity to mammals (Bai and Ogbourne, 2016). Moreover, its persistent use in environmental settings poses potential ecological risks in different ecosystems, particularly in aquatic environments. As both an antiparasitic agent and an efficient insecticide, ABM is widely used in agriculture, namely for the protection of plants and for the treatment of diseases in livestock (Campbell, 1989). In comparison to pyrethroids or organophosphates, ABM is known as being safer for the environment because of its low toxicity and capacity for degradation. Additionally, the strong bind of ABM to soil and sediment results in low accumulation in water and aquatic organisms (Davies and K Rodger, 2001). As a result, ABM is approved for use as a treatment in aquaculture to cope with aquatic parasites, and it is known to be used without manufacturers’ recommendation in some countries,



Corresponding author. E-mail address: [email protected] (Y. Hong). 1 These authors contributed equal to this work. https://doi.org/10.1016/j.ecoenv.2019.109889 Received 23 July 2019; Received in revised form 24 October 2019; Accepted 27 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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2. Methods and materials

such as Brazil and China (Bai and Ogbourne, 2016; Omura, 2008; Starling et al., 2019). As one of the top producers of ABM, China has scarce information about the residual effects of ABM on the environment (Chen et al., 2016). Hence, research regarding the ecotoxicological effects of ABM on aquatic organisms is urgent. Several studies have demonstrated that ABM is highly toxic to aquatic species (Novelli et al., 2012a, 2016; Tišler and Eržen, 2006; Wislocki et al., 1989). However, most studies are focused on the acute toxicity of ABM on fish, while very little research associating ABM's effects with crustacea has been reported (Bai and Ogbourne, 2016; Novelli et al., 2012b). Crustacea are an important part of the food chain in any aquatic ecosystem and demonstrate powerful sensitivity to the exposure of contaminants. The Chinese mitten crab, Eriocheir sinensis, is one of the most important freshwater species, incredibly prevalent in a variety of aquatic environments, including rivers, lakes and estuaries. It is also widely bred as a commercial species with an annual output of more than 850,000 tons in China (BFMA, 2017). Considering the possible water contamination through run-off or accidental introduction, also the direct use of ABM as a treatment for parasite control in farming ponds, the investigation of the toxic effects of ABM on E. sinensis and safety margins in practice is necessary for the aquaculture industry today (Novelli et al., 2012a). On the other hand, the widespread distribution of this species makes it possible to function as a key bioindicator for aquatic ABM contamination in China. Contaminants including pesticides are considered to induce oxidative stress in a biological system by the overproduction of reactive oxygen species (ROS). Subsequently, the oxidative damage from environmental stress is generally believed to suppress the immune system and leads to enhanced susceptibility to infectious diseases among crustaceans (Zhou et al., 2016). However, study on the interplay between oxidative stress and immune response in crustaceans is still limited. During the farming of E. sinensis, the use of ABM for parasite control will possibly induce oxidative stress in animals and have a negative impact on production. On the other hand, the fact that E. sinensis is widely spread in rivers and freshwater lakes in China, makes this species a good indicator in the assessment of pollution in China's aquatic ecosystem. Therefore, we have chosen this important species as the experimental animal and focus of our study. A previous study has demonstrated that ABM could lead to the damage of antioxidation systems through the overproduction of ROS (Zhang et al., 2017). Likewise, other studies have also observed immunodepression during exposure to humans (Corsini et al., 2008), birds (Liu et al., 2014), and fish (Wang et al., 2011), by decreasing immune enzyme activity. As an invertebrate, E. sinensis lacks adaptive immunity and has to rely on an efficient non-specific immune system to defend against pathogen invasion. The circulating haemocytes play a major role in both cellular and humoral immunity (Johansson et al., 2000). Several studies revealed that death of animals caused by the impairment of immune defence after contaminant exposure contributes to a decrease of total haemocyte counts (Cheng and Chen, 2001; Hong et al., 2017; Yeh et al., 2004). On the other hand, the phagocytic defence, which is highly dependent on ROS generation, turns out to be weakened under contaminant-induced oxidative stress. In this process, the excessive ROS are toxic to haemocytes and may disturb some crucial pathways in immune response (Zhou et al., 2016). We hypothesise that ABM exposure may induce ROS generation and oxidative stress, leading to immunological damage in crabs. For a better understanding of the relationship between oxidative stress induced by ABM and immune defence in host, an investigation of cellular and humoral immune response in E. sinensis was conducted in this study. The intracellular generation of ROS, total haemocyte counts (THC), lysosomal membrane stability (LMS) in haemocytes, and lysosomal proteolytic enzymes such as acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM) were investigated after sublethal exposure of ABM. This study will provide new insights into the impairment of physiological homeostasis in immune systems and responses under oxidative stress.

2.1. Animals and chemicals Adult crabs, Eriocheir sinensis (Crustacean: Decapoda: Grapsidae) were purchased from a commercial farm in Shanghai, China. The crabs were acclimated in a recirculating aquaculture system with constant aeration for two weeks. Animals were fed once a day at 7:00 p.m. with a commercial crab ration, and residuals and faeces were removed 2 h after feeding. During the acclimation, water temperature was maintained at 20 ± 1 °C, pH = 7.4 and dissolved oxygen at 6.5–7 mg/L. Then, crabs without any amputated limbs or other signs of external damage were chosen for the experiment. All experimental protocols were reviewed and approved by the Animal Bioethics Committee, Xichang University, China. The ABM (B1a, > 90% pure, CAS # 7175141-2) that was used in this test was purchased from Sigma-Aldrich Chemicals (Germany) and other chemical reagents were purchased from Sinochem Crop Care Co. LTD (Shanghai). 2.2. Experimental design Based on the information about 48 h- and 96 h- LC50 values of ABM on E. sinensis from our previous study and the application dosages in a crop farm (Novelli et al., 2016) and a rice farm (Pei et al., 2009), crabs were exposed to a gradient of ABM concentrations (0.03, 0.06, 0.12, and 0.24 mg/L, which represent 1/32, 1/16, 1/8 and 1/4 of 96 h LC50, respectively) in glass aquaria (75 cm × 45 cm × 60 cm) for 96 h. In addition, a control group without any ABM was also set. Individual crabs were randomly assigned to each group, with 10 crabs allocated for each treatment. Water in aquaria was aerated constantly and half renewed once a day by adding fresh water containing the same concentration of ABM to minimize the variance of the given concentration. Water conditions were monitored daily and no mortality was observed during the exposure. To avoid rapid photodegradation of ABM during the test, each aquarium was covered with a plastic plate to make the illumination less than 300 lux. 2.3. Determination of ABM in test water Before sampling, the concentrations of ABM (B1a as major component) in the test water were analysed at baseline (0 h) and 24 h after exposure (before renewal) by means of high-performance liquid chromatography coupled with mass spectrophotometry (HPLC-MS) (Agilent 6495, USA). Each water sample, with 50 mL per tank, was taken and filtered through a polytetrafluoroethylene membrane (0.22 μm). Samples were extracted by dichloromethane three times and dehydrated by anhydrous sodium sulphate. After vacuum concentration, extracts were transferred to a centrifuge tube and diluted with methanol to a volume of 2 mL. For the analysis of ABM in water samples, the following chromatographic conditions were used: a ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm, Agilent, USA); a column temperature of 40 °C; a mobile phase consisting of formic acid and acetonitrile, with gradient elution at a ratio of 95: 5 initially, 85: 15 at 2 min, 60:40 at 4 min, 20:80 at 8 min, 0:100 at 10 min and 60: 40 at 12 min; flow rate, 0.4 mL/min; injection volume, 20 μL. The mass spectrometer equipped with an electrospray ionization (ESI) source with the following conditions: capillary voltage, 3000 V; N2 was used as both dry gas (gas) and nebulizer gas (sheath gas); nebulizer voltage 500 V; gas temperatures, 250 °C; sheath gas temperature, 350 °C; gas flow rate 15 mL/min and sheath gas flow rate 12 mL/min. Procedures were run under the multiple reaction monitoring (MRM) program. Detailed information of analytical conditions of MRM is given in Table 1. Prior to sample analyses, HPLC-MS system performance and calibration were verified by an external standard method. The concentrations used to calculate linearity were between 25 ng/mL to 400 ng/mL with five points. The calibration curve is shown in 2

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Table 1 Analytical conditions of multiple reaction monitoring (MRM). Compound

Precursor Ion (m/z)

Product Ion (m/z)

Cell Accelerator Voltage (V)

Fragmentor Voltage (V)

Collision Energy (eV)

Polarity

Abamectin

895.5 895.5

751.4 449.2

3 3

380

50 380

Positive Positive

Fig. 1(R2 = 0.9998) and the relative standard deviation result of accuracy is 98.93%.

2.7. Lysosomal membrane stability assay Lysosomal membrane stability (LMS) was determined by the Neutral Red Retention assay according to the protocol described by Jensen et al. (Jensen et al., 2007) and Zhou et al. (Zhou et al., 2016) with some modifications. Aliquots of haemocytes (100 μL) were pipetted to 1.5 mL centrifuge tubes and an equal volume of neutral red working solution (0.33%, Sigma) was added. The mixture was incubated for 1 h at 10 °C and then, tubes were centrifuged at 200 G for 5 min and washed twice in TBS. 100 μL of 1% acetic acid in 50% ethanol was added to the tube and covered with foil. Tubes were incubated at 20 °C for 15 min and the amount of neutral red was detected by a spectrophotometer at 550 nm. The LMS was represented as O.D. per mg protein in haemocytes. The protein content in samples was spectrophotometrically determined as previously described (Lowry, 1951), using bovine serum albumin as a standard.

2.4. Sample preparation After exposure to ABM, four individuals from each group were randomly taken at 24 and 96 h, respectively. Crabs were anaesthetized in an ice bath for 10 min and the haemolymph was drawn with a sterile 1-mL syringe from the unsclerotised membrane. Haemolymph was 1:1 and was diluted immediately with a sterile anticoagulant (30 mM of trisodium citrate, 338 mM of NaCl, 115 mM of glucose, and 10 mM of EDTA) and one portion (aliquots of 100 μL) was used for the THC assay. Then, the rest was centrifuged at 1200 rpm for 5 min at 4 °C to collect haemocytes for the ROS and LMS assays. The supernatant as cell free haemolymph (CFH) was collected for the detection of ACP, AKP and LZM activities.

2.8. Humoral immune parameters 2.5. Total haemocyte counts

The activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) in CFH were determined by a spectrophotometric method at 520 nm according to the manufacturer's protocols in detection kits (Nanjing Jiancheng Bioengineering Institute, China). In addition, the lysozyme (LZM) activity was also measured at 530 nm by a detection kit. Each enzyme activity was expressed as units of activity per mL of haemolymph (U/mL).

After the haemocyte collection, the THC assay was performed by using a haemocytometer under a Leica DM-i8 inverted phase-contrast microscope (Leica, Germany). Three replicates were made for each sample, and the THC in each group was expressed as numbers of haemocytes ml−1 haemolymph.

2.9. Effects of ABM on the antibacterial ability of E. sinensis

2.6. Intracellular ROS determination

To investigate whether ABM exposure has an impact on the antibacterial ability of E. sinensis, an additional 100 crabs were used in this experiment. Crabs were divided into five groups (as mentioned above) and each crab was injected with Aeromonas hydrophila at a density of 1 × 108 mL−1 in saline (Lv et al., 2014). After 96 h exposure to ABM, the mortality of the crabs in each group was calculated to evaluate the effect of ABM on pathogen defence in E. sinensis.

The level of ROS in haemocytes was measured by using a ROS Assay Kit (Beyotime, China) with the DCFH-DA oxidation method (Zhang et al., 2017). Haemocytes were resuspended in a prepared culture medium according to previous report (Hong et al., 2013), with 10 μM DCFH-DA, and incubated at 28 °C for 20 min to allow the fluorescent probe to diffuse into the cells. Then, cells were washed twice in phosphate buffer saline (PBS) to remove extra DCFH-DA. The level of ROS in cells was detected using a microplate reader (SpectraMax M2, Molecular Devices, USA) with wavelengths of excitation of 488 nm and emission of 525 nm. The results were expressed as relative levels compared to control (%).

2.10. Statistical analyses The data were processed with the aid of the SPSS V21.0 software and statistically analysed by analysis of variance (ANOVA). The

Fig. 1. Calibration curve of abamectin quantification in water. 3

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Fig. 2. Abamectin concentration in test water during the exposure. Asterisks (*) over the column represent a significant difference (P < 0.05) between each group, n = 3.

Fig. 3. Effects of ABM on total haemocyte counts in E. sinensis. Asterisks (*) over the column represent the significant difference between the experimental group and the control group, one for P < 0.05, two for < 0.01 and three for < 0.001, n = 4.

normality of data was tested by the Shapiro Wilks test and a homogeneity test of variances was performed. An independent-sample t-test was used to determine any significant differences between the ABM concentration at 0 h and 24 h, and a multiple-comparison Duncan test was performed to determine any significant differences between treatment and control subjects. A P value < 0.05 was considered to represent a significant difference.

3. Results 3.1. ABM in water The results of ABM analysis in water are given in Fig. 2. The concentrations of ABM in the test water in each group at 0 h were 0.023 ± 0.004, 0.042 ± 0.004, 0.095 ± 0.010 and 0.215 ± 0.004 mg/L, respectively. The results were close to the nominal concentrations in all treatments. There was no significant variation between concentrations at 0 h and 24 h in the groups of 0.03 and 0.06 mg/L. However, a significant degradation of ABM was found at 0.12 (F = 2.417, P = 0.02) and 0.24 mg/L (F = 6.685, P = 0.007). On average, approximately 48% and 32% decreases occurred within each group at 24 h of exposure.

Fig. 4. Effects of ABM exposure on ROS level in haemocytes. Asterisks (*) over the column represent the significant difference between the experimental group and the control group, one for P < 0.05, two for < 0.01 and three for < 0.001, n = 4.

3.2. The effects of ABM on THC

3.4. Effects of ABM on lysosomal membrane stability of haemocytes in E. sinensis A negative effect of ABM on the LMS was observed in this test. After exposure to ABM, the LMS in haemocytes decreased gradually in a doseand time-dependent manner. At 96 h, the LMS was significantly reduced in all treatments compared with the control group (Fig. 5).

As shown in Fig. 3, the THC in E. sinensis increased significantly in the group of 0.06 mg/L at both 24 h and 96 h, and a significant increase in the group of 0.03 mg/L was observed after 96 h of exposure. On the contrary, the THC decreased significantly in high concentration groups (0.12 and 0.24 mg/L).

3.5. Humoral immune responses under ABM exposure 3.3. The effects of ABM on intracellular ROS generation

Fig. 6A showed that ACP activities in the groups of 0.03 and 0.06 mg/L increased significantly at 24 h, but recovered to normal level at 96 h. On the contrary, they decreased in the groups of 0.12 and 0.24 mg/L concentrations, dependently. A similar trend was found in AKP activity during the exposure. Both ACP and AKP decreased to the lowest level under 0.24 mg/L of ABM at 96 h (Fig. 6 B). In addition, LMZ activity decreased remarkably in a dose- and time-dependent manner. However, no significant change was found at 0.03 mg/L of ABM (Fig. 6 C). The data indicated that ABM at low concentrations

The ROS level in haemocytes increased significantly in all treatments, except at 0.03 mg/L. There was a dose- and time-dependent increase of ROS during the exposure. At 96 h, the group of 0.24 mg/L got the highest ROS level with about 3.5 fold in comparison with the control group (Fig. 4). The results indicated that ABM could induce the overproduction of ROS in haemocytes, which could in turn lead to oxidative damage in their physiological response. 4

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Fig. 7. Mortality of E. sinensis to injection of A. hydrophila after 96 h exposure of ABM. Asterisks (*) over the column represent the significant difference between the experimental group and the control group, one for P < 0.05, two for < 0.01 and three for < 0.001, n = 4.

Fig. 5. Effects of ABM on lysosomal membrane stability in haemocytes of E. sinensis. Asterisks (*) over the column represent the significant difference between the experimental group and the control group, one for P < 0.05, two for < 0.01 and three for < 0.001, n = 4.

0.06 mg/L. A report from Novelli et al. has shown that about 40% of degradation, on average, in a 48-h our toxic test on D. magna was found, and after 96 h of exposure, the concentration of ABM in water dropped to 5% of the nominal concentration (Novelli et al., 2012a). However, no information about the illumination condition during the test was given. Therefore, there was a slow degradation of ABM in the water (no more than 50% in each group), especially in the groups of 0.03 and 0.06 mg/ L, indicating that the test concentrations are close to the nominal concentrations. Moreover, the application of ABM on a cloudy day with low illumination will present a greater risk to non-target animals and, as such, a greater potential of ecotoxicological effects of ABM on these species should be take into consideration. Haemocyte is extremely important for the innate immune response of invertebrates (Pipe and Coles, 1995). Considering the high involvement of haemocytes in both cellular and humoral immune defence in crustaceans, some physiological parameters in haemocytes, such as THC and LMS were commonly used to reflect the immune status of the host. Previous studies demonstrated that pollutants, including heavy metals and pesticides, could cause a decrease of haemocyte counts in different kinds of aquatic animals (Hong et al., 2018; Xian et al., 2010). From our results, THC in the group of 0.03 mg/L significantly increased at 96 h, which contradicts the results in the studies mentioned above. We concluded that ABM exposure at low concentrations could induce the haematopoiesis in E. sinensis. Meanwhile, only a slight increase of ROS generation was observed at 0.03 mg/L during the test. This indicates that there may be no oxidative damage in haemocytes under a low level of ABM. These results provide evidence for our hypothesis. In

could partially elevate ACP and AKP activities, but suppressed their activities at high concentrations. 3.6. Effects of ABM on antibacterial ability of E. sinensis ABM has prominent effects on the antibacterial ability of E. sinensis. After A. hydrophila injection, less than 20% of crabs died in control after 96 h of exposure. There were no significant differences in the groups of 0.03 and 0.06 mg/L compared to the control group, whereas significant increases in mortality were observed in the 0.12 mg/L and 0.24 mg/L groups (Fig. 7). The results indicate that high concentrations of ABM could impede the antibacterial ability of E. sinensis when these species are challenged with a highly pathogenic bacteria in their aquatic environment. 4. Discussion ABM could be rapidly degraded in an aquatic environment with a half life of less than 4 days, which is far below that found in sediments. Because of its hydrolytic stability, ABM was shown to have a long half life of 78.8 days in neutral water at 25 °C (Zhang, 2004). However, taking photolysis into consideration, the rapid degradation of ABM in most reports is worth noting. In the present study, a significant decrease of ABM was observed after 24 h of exposure at a relatively low illumination with less than 300 lx. However, less than 10% of the degradation occurred in the test water at concentrations of 0.03 and

Fig. 6. Effects of ABM on humoral immune response in E. sinensis. A: ACP activity; B: AKP activity; C: LZM activity. Asterisks (*) over the column represent the significant difference between the experimental group and the control group, one for P < 0.05, two for < 0.01 and three for < 0.001, n = 4. 5

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haemocyte counts and, subsequently, a decline of humoral immune enzyme activities. This could cause problems for animals, especially with regard to their resistance to pathogen invasion, and specifically at concentrations of more than 0.06 mg/L, which is still much less than the concentration that is considered to be safe for this species. Moreover, all cellular and humoral parameters in this study could be seen as sensitive biomarkers in the assessment of ABM toxicity.

addition, a study from Jia et al. indicated that ROS is an important modulator in the haematopoiesis of crabs and could promote the production of haemocytes from progenitor cells (Jia et al., 2018). Based on their findings, ROS generation induced by ABM could motivate haematopoiesis and induce a subsequent THC increase. Regarding the groups of 0.06, 0.12 and 0.24 mg/L, ROS in haemocytes increased significantly in dose- and time-dependent manners. ROS are proven to involve multiple physiological responses, and the breakdown of equilibrium of ROS production and elimination can induce cell injury, apoptosis or even necrosis (Lushchak, 2011). Therefore, intracellular ROS is often used as a sensitive biomarker in oxidative stress. Results from the present study demonstrated that high concentration exposures of ABM could induce the excessive generation of ROS, which leads to a subsequent decrease of THC due to cell damage. This could be a reason for the immune parameters inhibition in subsequent detection. Lysosomes participate in the innate immune defence by phagocytosis in cellular pathway and by releasing hydrolytic enzymes to haemolymph in humoral pathway (Liu et al., 2009). In recent years, LMS has been used as an ecotoxicological biomarker, and the destabilization of lysosomal membranes can be considered as resulting from the overproduction of ROS induced by contaminants (Itziou and Dimitriadis, 2011). In this work, the LMS decreased significantly with a dose- and time-dependent response, even at concentration of 0.03 mg/ L. Our results are in accordance with studies about scallops (Liu et al., 2009), crabs (Zhou et al., 2016) and snails (El-Gendy et al., 2019). When the lysosomal membrane collapse, the proton pump on it is damaged and the contents in lysosomes (like hydrolytic enzymes) are released into cytosol, which results in cell damage and, furthermore, in the decrease of haemocyte counts. A report from Yao et al. (Yao et al., 2008) showed that a decrease of LMS correlates well with a decrease of THC – a finding which provides strong evidence in support of our conclusion. As the main hydrolytic enzymes in lysosomes, ACP, AKP and LZM are suggested to stand as the first line of humoral defence in innate immunity and are the most sensitive to xenobiotics (Rajalakshmi and Mohandas, 2005). In the present study, significant increases of both ACP and AKP at concentrations of 0.03 and 0.06 mg/L were observed. On the contrary, the enzyme activities decreased remarkably at 0.12 and 0.24 mg/L. From Zhou's research, higher ACP and AKP activities were always associated with a decrease of LMS (Zhou et al., 2016). This finding revealed that with the damage of the lysosomal membrane in haemocytes from E. sinensis, ACP and AKP are released from cells to haemolymph, which makes them increase during the exposure to low concentrations. However, under exposure to high concentrations, their activities decreased significantly. This may be due to the sharp decrease of haemocyte counts, leading to a synthesis obstruction of immunological enzymes. Similar results were obtained from previous studies about contaminants exposure to fish and scallops, in which their immune enzyme activities were decreased in a dose-dependent manner (Dey et al., 2016; Liu et al., 2009). In addition, LMZ is regarded as another important enzyme existing in the lysosome. Our results showed a dose- and time-dependent decrease during exposure. This could also contribute to the decrease of LMS in haemocytes. To confirm whether the immune defence system is affected by exposure to ABM, crabs were challenged with the highly pathogenic bacteria A. hydrophila. The results showed that with the increase of concentration, mortalities increased significantly, particularly in high concentration groups. This result, moreover, proved that ABM could inhibit the immune defence of E. sinensis and subsequently increase the susceptibility of animals to pathogens.

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5. Conclusion Thus, the present data detected prominent toxic effects of ABM on the Chinese mitten crab, E. sinensis. The overproduction of ROS could induce a collapse of lysosomal membrane, resulting in a decrease of 6

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