Effect of dietary Bacillus subtilis supplement on Cd toxicokinetics and Cd-induced immune and antioxidant impairment of Procambarus clarkii

Cadmium (Cd), a non-biodegradable contaminant in freshwater ecosystems, can pose a serious threat to aquatic animals at high levels. In this study, the Cd toxicokinetics and the immune and antioxidant defense were explored in Procambarus clarkii exposed to different levels of Cd (0, 0.1, 1.0 mg Cd/L) or treated with 1.0 mg Cd/L and dietary Bacillus subtilis supplementation (1 × 107 cfu/g). Results from the 21-day uptake and depuration experiment revealed that Cd exposure elicited a dose- and time-dependent uptake in all crayfish tissues, and the rank order of Cd concentration was gill > hepatopancreas > exoskeleton > muscle. The one-compartment model demonstrated that gills had the highest uptake rate (ku) value after Cd aqueous exposure and the ku and elimination rate (kd) values in gill, hepatopancreas, and exoskeleton of the group with 1.0 mg Cd/L were higher than those of the group at alow Cd concentration (0.1 mg Cd/L). However, B. subtilis could decrease Cd ku and increase Cd kd in hepatopancreas, resulting in the reduction of bioconcentration factors (BCF), steady-state concentrations (Css), and biological half-life (Tb1/2). A positive correlation was found between aqueous Cd concentration and the severity of hepatopancreas histopathological injury, while B. subtilis could ameliorate the pathological damage in the high Cd group. Similarly, aqueous exposure to Cd elevated malonaldehyde (MDA) content and suppressed the activities of lysozyme (LZM), acid phosphatase (ACP) in hepatopancreas and alkaline phosphatase (AKP) in hemolymph. The activities of superoxide dismutase (SOD) and catalase (CAT) in hepatopancreas were also inhibited. Nevertheless, they were all recovered with the dietary addition of B. subtilis. In conclusion, our results indicated that exposure to Cd significantly increased Cd accumulation and toxic damages in crayfish hepatopancreas, while dietary administration of B. subtilis to crayfish significantly decreased Cd accumulation and improved the immune and antioxidant defense, leading to the prevention in toxic effects of Cd.


Introduction
Cadmium (Cd), a non-essential and toxic element, is recognized as a prevalent environmental pollutant in the aquatic ecosystem. It is potentially toxic to aquatic biota and human health through bioaccumulation and bioamplification along the food chain (Fowler 2009;Zhang et al. 2019). Recently, high levels of Cd in sediments and surface water were reported, commonly accompanied with environmental disasters in rural regions (Foesch et al. 2020). Cd toxicity in aquatic organisms has been widely recorded by previous researches. The adverse impacts included the development impairment of embryo, growth reduction, tissue morphology changes, physiological and biochemical dysfunctions, and oxidative stress when exceeded the thresholds (Olsvik et al. 2016;Huo et al. 2017). At certain concentrations, Cd is involved in a complex set of chemical reactions, such as gluconeogenesis and lipid metabolism, to reduce the glucose uptake and fatty acid synthesis. Additionally, Cd exposure could decrease the transcript levels of pro-inflammatory cytokines in Danio rerio and Cyprinus carpio, suggesting Cd had an immunosuppressive effect . In crustaceans, Cd exposure even enhanced the secretion of gonadal inhibitory hormone (GIH), altered calcium homeostasis and caused DNA damage (Wang et al. 2009). It is well known that the toxicity of Cd is strongly related to its content in various tissues, and the accumulation and elimination rates directly determine the final accumulation level of Cd in different tissues. Thus, it is very important to investigate the bioaccumulation characteristics of Cd in living organisms.
Many mathematical models have been developed in recent years to mimic and limit the bioaccumulation of heavy metals in aquatic species. At present, the steady-state model, one-compartment model, and biodynamic model are typical in the studies on different pollutants. Among them, the one-compartment model simplifies the enrichment process of pollutants such as heavy metals by aquatic organisms (Zauke et al. 1995). Since the total amount of pollutants was assumed to be constant, the mass transfer process of heavy metals only occurs between the two phases of the organism and the water body (Clason et al. 2003). It has the advantage of precisely calculating the kinetic parameters in the theoretical equilibrium state even if the organism's heavy metal intake does not reach an equilibrium state (Janssen et al. 1991). As a result, the one-compartment model is currently the most extensively employed in the studies on heavy metal enrichment in aquatic species (Kahle and Zauke 2002;Sun et al. 2018).
Red swamp crayfish (Procambarus clarkii), originating from Northeastern Mexico, is a commercially important species for freshwater aquaculture worldwide (Wang et al. 2018a). Owing to its powerful enrichment ability of metals, higher concentrations of metal were reported in the tissues of crayfish than those of any other species living in the same aquatic surrounding (Bellante et al. 2015). Over the years, crayfish had been used as a sensitive bioindicator of various contaminants in aquatic environment because of its unique lifestyle and extreme tolerance to harsh survival conditions (Suárez-Serrano et al. 2010). Especially, they were recently applied to monitor the aquatic environments for metal pollution (Alcorlo et al. 2008;Zhao et al. 2019;Shui et al. 2020). It is reported that they can accumulate metals such as Cd, Cu, and Pb in two ways: waterborne and dietary route (Goretti et al. 2008). These heavy metals can distribute in multiple organs, especially in the hepatopancreas, and disrupt numerous physiological processes (Vogt 2002;Suárez-Serrano et al. 2010;Bellante et al. 2015). The crustacean hepatopancreas, which is similar to the liver of higher organisms, is the most susceptible to waterborne pollutants (Li et al. 2007) as it plays a vital role in the storage and detoxification process of heavy metals. Therefore, the hepatopancreas is considered to be a suitable organ model for toxicologic researches of heavy metals. As confirmed by the previous study, the remarkable features of Cd toxicities in crayfish are oxidative stress and obvious histological impairment of hepatopancreas . However, the relationship between bioaccumulation characteristics and Cd toxicities in crayfish has not been investigated thus far.
Bacillus subtilis (B. subtitlis) is a gram-positive bacterium that possesses a powerful extracellular protein secretion system. It has been frequently utilized in aquaculture to optimize the utilization of nutrients, modulate the metabolism of the host, and enhance the resistance from infections (Yang et al. 2019;Zhou et al. 2019;Karpov et al. 2020). Dietary addition with B. subtitlis was observed to improve the growth performance, antioxidant capacity, and immunity in crucian carp (Liu et al. 2022), and to enhance the immune responses and disease resistance in hybrid grouper (Liu et al. 2021). More production and increased activities of trypsin and amylase were also discovered in shrimps after the addition of B. subtitlis in their diets (Wang et al. 2018b;Kuebutornye et al. 2019;Vogeley 2019). However, until now, little is known about whether dietary B. subtitlis can affect Cd accumulation and release and prevent various types of damages caused by Cd stress in aquatic animals. Therefore, in the present study, we investigated the effect of dietary Bacillus subtilis supplement on Cd toxicokinetics and Cd-induced immune and antioxidant impairment inProcambarus clarkii. Specifically, the crayfish were exposed to different levels of Cd or co-treated with 1.0 mg Cd/L and dietary supplement of Bacillus subtilis for 2 weeks, followed by one-week depuration in clean water. The one-compartment model was applied to estimate kinetics parameters. Secondly, histopathological alterations, non-specific immunity responses and antioxidant defenses were determined after the 2 weeks treatment to analyze the toxicity of Cd in the crayfish.

Test chemicals and diet preparation
Cadmium chloride hydrate (CdCl 2 ·2.5 H 2 O, 99% purity) was obtained from Sinopharm Chemical Reagent (Shanghai, China). The alive B. subtilis (ANSB060) and commercial pellet feed were purchased from Tongwei Feed Co., Ltd, China, and Zhongke Biotic Co., Ltd, China, respectively. The bacteria were dissolved in sterile purified water to adjust the cell count of 1 × 10 7 cfu/g based on the findings from the previous studies (Dong et al. 2018b;Xu et al. 2021) and our preliminary experiment, and then evenly sprayed onto the feed pellets for crayfish. An adhesive (Nanhua, China) was applied to improve probiotic stability and decrease the release of probiotic from the pellets. The diets were air-dried under ventilated conditions and stored at room temperature. The rations fed every day were prepared on the same day to ensure the active concentration of bacteria.

Experimental protocol
The crayfish with average wet weight of 9.87 ± 2.1 g and average length of 5.32 ± 1.0 cm were provided by a local commercial farm (Jianli, Hubei, China). Before the experiment, they were temporarily housed for 14 days in glass aquaria (0.6 m × 0.45 m × 0.45 m) containing aerated, carbon filter, dechlorinated tap water, and a natural photoperiod. The water quality parameters included dissolved oxygen: 4.70 ± 0.56 mg/L; pH: 7.95 ± 0.11; and water temperature: 23.81 ± 0.64 ℃. Commercial pellet (Haid, China) was given once daily at 3-5% of their body weight.
Three hundred and sixty crayfish with similar sizes were randomly divided into 4 groups with three replications. These groups were designed as 0 (control), 0.1 mg/L, 1.0 mg/L, and 1.0 mg/L + B. subtilis. The control group was treated with normal filtered water but without adding any Cd. Different concentrations of exposure solutions were prepared by adding an appropriate amount of Cd 2+ stock solutions to dechlorinated tap water. The lower Cd concentration (0.1 mg/L) was selected according to the maximum permitted concentration in freshwater, and the highest Cd exposure concentration (1.0 mg/L, 1/80 LC 50 ) was determined in the previous 96-h LC 50 estimated value (80.0 mg/L) for crayfish (Tan et al. 2012). Cd concentrations in water were measured every day by Graphite Furnace Atomic Absorption Spectroscopy (GFAAS, ICE3000, Thermo, USA). The detection limits for Cd in crayfish were set according to the National Standard of China (GB 5009.15-2014). In order to assess the toxicokinetic parameters of Cd, crayfish was exposed to Cd solutions for 2 weeks, followed by 1-week depuration in clean water. The antioxidant and immune indexes were detected at day 14. To ensure the stability of Cd concentration, one-third of the water volume in the tank was renewed every day, and a suitable amount of exposure solution was re-added to the water. Crayfish were fed twice a day during the experiment. Other experimental conditions were kept the same as those during acclimation.

Sample collection
Six crayfish were randomly selected from three parallel groups at every time point and anesthetized for 10 min with ice. The hemolymph was extracted from the pericardial cavity of the crayfish with a 2.5-mL sterile syringe to examine the activities of enzymes involved in the nonspecific immunity and antioxidant. The hepatopancreas, gills, muscle, and crustal tissue were dissected from each crayfish. Approximate 100 g of tissues was freeze-dried and homogenized for the quantification of Cd. About 500 mg of hepatopancreas was washed with ice-cold phosphatebuffered saline and then homogenized (1:10 w/v) at 4 °C for the test of biochemical parameters. After being centrifuged at 12,000 g for 30 min at 4 °C, the supernatants were collected. Additionally, other parts of the hepatopancreas samples were fixed in a 4% paraformaldehyde solution for histological observation.

Cadmium quantification
According to the early reported method , the Cd concentrations in various tissues of crayfish were detected by Graphite Furnace Atomic Absorption Spectrometry.
The concentration of Cd could be obtained by the equation: where A 1 indicates the concentration of Cd in the digestion solution of the sample in mg/L, A 2 indicates the Cd concentration in the blank solution in mg/L, V indicates the volume of digestion solution in mL, and M denotes the dry weight of the sample in grams.

Toxicokinetic model
Uptake kinetics were expressed in terms of Cd concentration change over time. To estimate kinetics parameters, we performed non-linear regression fitting of the following one -compartment kinetic model equations simultaneously (Lister et al. 2011).
For the uptake phase (0 ≤ t ≤ t c ): And for the depuration phase (t > t c ): where C t is the total internal concentration of Cd (mg/kg w.w.) in crayfish at sampling time t (d); C 0 is the Cd concentration in crayfish measured at the beginning of the experiment (t = 0), α is uptake flux constant, which equals k u C exp , where k u is the uptake rate constant; C exp is the exposure concentration during the uptake phase; k d is the elimination rate constant; t c is the time at which crayfish were transferred to the uncontaminated (t c = 13 days) . Steady-state concentrations (C ss ) were calculated as C 0 + α/k d (Díez-Ortiz et al. 2010;Wen et al. 2011). The bioaccumulation factors based on the kinetic method (BAF) could be calculated from the kinetic parameters with the following equation: A biological half-life (the time it takes to reach half of the equilibrium value) was calculated (T b1/2 ) from the corresponding constant uptake rate constant (k u ), according to the relation T b1/2 = ln 2/k u (d). Goodness-of-fit tests to verify the applicability of the one-compartment kinetic model by F-test and t-test according to previous studies (Lin et al. 2019).

Histopathological examination
The fixed hepatopancreas were dehydrated in an ethanol series (75-95%) and embedded in paraffin. Then, 5-μm-thick sections were cut and stained with hematoxylin-eosin (HE). Finally, observation and assessment of hepatopancreas were done on a light microscope (Nikon H600L Microscope and image analysis system, Tokyo, Japan). Histological damages in the histopathological slices were quantitatively determined following the previous methods (Bernet et al. 1999;Corbett et al. 2015). A severity score value from 0 to 6 was assigned for the degree and extent of each alteration: 0-unchanged, (1 or 2)-mild, (3 or 4)-moderate, (5 or 6)-severe.

Assays of enzyme activity and MDA content
Activities of lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) in the hemolymph of crayfish were determined using the commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). The protein content, malondialdehyde (MDA) content, superoxide dismutase (SOD), and catalase (CAT) activities in the hepatopancreas homogenates were tested according to the reported method (Marklund and Marklund 1974;Satoh 1978).

Statistical analysis
The experimental data were analyzed using SPSS 22.0 (SPSS, Chicago, IL). Goodness-of-fit of the model was used to assess the quality of the method by F-test statistics and chi-square analysis. The Kolmogorov-Smirnov test and Levene's test were used for normality and consistency, and if the data did not conform to the normal distribution, a logarithmic transformation or a nonparametric method was performed. One-way ANOVA (p < 0.05) and Tukey's test were used to estimate significant differences among different groups. Three independent repetitions of the experiment were performed, and the data from all three experiments were expressed as mean ± standard error (SEM).

Cd concentrations in exposure solutions
As exhibited in Fig. 1, the mean measured Cd concentrations in the exposure medium before renewed were 0.00 ± 0.01, 0.09 ± 0.01, 0.92 ± 0.04, 0.87 ± 0.03 mg/L, respectively. The concentration of chemicals in the exposure solution must be maintained at a stable level from the reliability of toxicokinetic experiments. Low degradation efficiency (less than 20%) of Cd in the solution was confirmed at 24-h intervals; thus, Cd concentrations in exposure solutions were comparative with nominal concentrations. Dietary supplementation with B. subtilis during the 14-day exposure did not change the concentrations of Cd in solutions compared with the group exposed to the same concentration of Cd.

Cd accumulation and depuration in different tissues of crayfish
Cd caused a dose-dependent increase in mortality of crayfish after 2 weeks of Cd exposure (Fig. 1B). The highest mortality (21.11 ± 0.64%) was found in the highest Cd group during the 21-day uptake and depuration experiment, while dietary addition of B. subtilis significantly decreased their mortality. No significant change in the mean body weight was detected among all the groups during the entire Cumulative mortality (%) ** * * B experiment period (data were not shown). The Cd concentrations in gills, exoskeleton, and muscle increased stepwise within the first 2 days in 0.1 mg Cd/L and 1.0 mg Cd/L groups, while in hepatopancreas, Cd concentrations increased rapidly after the start of the Cd exposure (Fig. 2). They achieved a steady state in all tissues within the 14-day Cd exposure. The maximum Cd concentration in gills, hepatopancreas, muscle, and exoskeleton under 1.0 mg Cd/L exposure were 31.39 ± 0.58, 24.40 ± 0.60, 1.30 ± 0.06, and 10.83 ± 0.44 mg/kg w.w., respectively. After then, their concentration decreased during the following depuration phase. 79.75 ± 0.59%, 80.31 ± 0.05%, 99.13 ± 0.44% of Cd were eliminated from gills, muscle, and exoskeleton, respectively, after the 7-day depuration. However, only 17.03 ± 0.59% of Cd was eliminated in the hepatopancreas. Diet supplemented with B. subtilis could significantly decrease the maximum level of Cd in the hepatopancreas. Additionally, there were no significant differences in the levels of Cd in the gill, exoskeleton, and muscle between the crayfish treated with Cd alone and the crayfish co-administrated with Cd and B. subtilis.
To further analyze the toxicokinetic data, a one-compartment model was employed to evaluate the Cd toxicokinetic parameters, including uptake rate (k u ), elimination rate (k d ), half-life (T b1/2 ), and bioconcentration factors (BCF) in different tissues ( Table 1). The distinct abilities to accumulate and eliminate Cd were found in the different tissues. Gills had the highest k u value, followed by hepatopancreas and exoskeleton, while the muscle showed the lowest value. The k u and k d values in the gill, hepatopancreas, and exoskeleton of the group with 1.0 mg Cd/L were lower than those of the group with 0.1 mg/L Cd concentration. When compared with 1.0 mg Cd/L exposure, co-administrated with Cd and B. subtilis depicted a lower k u value in all tissues, but a lower k d value in the gill, muscle, and exoskeleton and a higher k d value in hepatopancreas. Moreover, the crayfish co-treated with Cd and B. subtilis exhibited a higher BCF and C ss in gill, while a lower BCF and C ss in hepatopancreas when compared with the crayfish exposed to 1.0 mg Cd/L only. The rank order of BCF for Cd was gill > hepatopancreas > exoskeleton > muscle. Interestingly, the supplementation of B. subtilis weakened the BCF of Cd in hepatopancreas, while had no effect on the gills, muscle, or exoskeleton.
As shown in Table 2, the coefficients of determination (R 2 ) for Cd biokinetics in crayfish four tissues ranged from 0.9812 to 0.9945, respectively. F-test revealed that the equations were significant and the one-compartment model was suitable for obtaining the toxicokinetic parameters for the accumulation and depuration experiments. Furthermore, paired t-test indicated that there was no significant difference between the model-fitted output and the measured values.

Effects of Cd exposure and B. subtilis supplement on hepatopancreas histopathological structure
Exposure to Cd caused significant pathological damage to hepatopancreas in all experimental groups. Tubule degeneration, tubule lumen dilatation, and epithelium vacuolization frequently occurred after exposure to 0.1 and 1.0 mg Cd/L ( Fig. 3A-C), and local autolytic disintegration of tubular epithelial increased compared with the control group. A positive correlation was found between Cd exposure concentration and histopathological degree in the hepatopancreas and the R 2 was 0.9333 (Table 3). Furthermore, 1.0 mg Cd/L exposure demonstrated apparent local autolytic disintegration of tubular epithelial and epithelium vacuolization, which were remarkably recovered when co-treated with Cd and B. subtilis (Fig. 3C-E).

Effects of Cd exposure and B. subtilis supplement on non-specific immunity response and oxidative stress
The activities of LZM, ACP, and AKP were determined to reflect non-specific immunity response of crayfish, while the content of MDA and activities of SOD and CAT were chosen to represent the situation of oxidative stress. As shown in Fig. 4, they were apparently inhibited after exposure to 0.1 and 1.0 mg Cd/L for 2 W. Specifically, the AKP activities of the crayfish exposed to 0.1 mg Cd/L and 1.0 mg Cd/L decreased by 21.05 ± 0.05% and 73.32 ± 0.45%, respectively (Fig. 4C). The decrease in the activities of LZM, ACP, and AKP were blocked by the addition of B. subtilis (Fig. 4A-C) and the activities of SOD and CAT depicted a similar trend ( Fig. 4A and E). The content of MDA was significantly increased in the 1.0 mg Cd/L group, but the increase was effectively inhibited by the supplement of B. subtilis (Fig. 4F). Spearman's correlation analysis demonstrated that the activities of LZM, ACP, AKP, SOD, and CAT were significantly negatively correlated with the levels of Cd concentration, and the correlation coefficients (R 2 ) ranged from 0.8500 to 0.9667. In addition, a positive correlation between MDA content and Cd concentration was found (Table. 3).

Discussion
The harm of Cd pollution has received considerable attention in recent decades, but toxicokinetic characteristics and toxicity of Cd in crayfish have not been fully elucidated. In the present study, we investigated the Cd accumulation and depuration in crayfish at different levels of Cd exposure, and the impacts of Cd exposure on non-specific immunities and antioxidant responses. Our findings demonstrated a dose-and time-dependent uptake of Cd in all tested tissues of crayfish following aqueous exposure, and the rank order of Cd concentration in diverse tissues was gill > hepatopancreas > exoskeleton > muscle. Previous studies have also revealed this tissue specificity of heavy metal accumulation. For instance, the contents of Pb and Cd in gills and hepatopancreas of Eriocheir sinensis were significantly higher than that in muscle (Chen et al. 2010). Generally, the tissue-specific distribution of heavy metals was associated with the organ's unique roles in the metabolism, distribution, and elimination of the chemicals (Bryan 1971;Ahearn et al. 2004). A large number of studies suggested that the gill was the main entry point for metals and act as a transient storehouse for accumulated metals through water exposure in aquatic animals (Alcorlo et al. 2006;Gedik et al. 2016), while the hepatopancreas was also a crucial organ for metal storage and detoxification (Alcorlo et al. 2006;Rőszer 2014). Accordingly, in our study, gills displayed the greatest accumulation of Cd, which might be due to the high tendency of gills to absorb Cd from the exposure solution. As a vital tissue for storing and detoxifying metals, the hepatopancreas can concentrate metals from hemolymph rapidly after the gill absorption or gut uptake (Vogt 2002;Suárez-Serrano et al. 2010). However, the uptake of Cd in hepatopancreas could not be completely removed following the 7-day depuration.
Our finding showed that both k u and k d in the gill and hepatopancreas of the crayfish exposed to 0.1 mg Cd/L were higher than those of the crayfish exposed to 1.0 mg Cd/L. Many variables influence heavy metal accumulation in aquatic species, including biotic and abiotic influences. Biological factors mainly include individual size, interspecific differences, sex, age, and reproductive status. However, abiotic factors dominantly refer to various physical and chemical factors, such as salinity, temperature, pH value, content of organic matter, seasonal changes, hydrodynamic conditions, etc. (Liu et al. 2021) also observed that the k u and k d of Cd in Chlamys farreri had a negative correlation with Cd concentration in water. The calculated tissue k u and T b1/2 for Cd in hepatopancreas were significantly higher than those in the exoskeleton and muscle, while the k d value for Cd in hepatopancreas was lower than that in the other two tissues. Therefore, it was speculated that high absorption rates and low eliminate rates might be the reasons for the higher level of Cd in hepatopancreas. Interestingly, we found that the dietary addition of B. subtilis decreased Table 1 Kinetic parameters of bioconcentration of Cd in four organs of crayfish at different concentrations Here, k u represents the uptake rate; k d represents the elimination rate; brackets () show the ± SEM values. BCF represents the bioconcentration factor; C ss represents the maximum accumulation concentration in the organism when enrichment reaches theoretical equilibrium, and T b1/2 (d) represents the time required for half of the heavy metals in the organism to be released  . Local autolytic disintegration of tubular epithelial (marked by black arrow), degenerated tubules (marked by blue arrow) and vacuolization (marked by red arrow) were observed the k d in gills, muscle, and exoskeleton, but increased in hepatopancreas. As well known, the rate of Cd elimination is proportional to the level of Cd accumulation in tissues. Our previous study showed that the stable colonization of B. subtilis in crayfish gut might limit Cd absorption (Mo et al. 2022). Importantly, the dietary addition of B. subtilis decreased k u in gills, muscle, and exoskeleton in our study further verifying this hypothesis. While the reasons are still uncertain and need further investigation. However, the bioaccumulation process of heavy metals in specific tissues is temporary and may be transported to other tissues such as the liver for purification, excretion or storage (Ghosh et al. 2020). Hepatopancreas is a key organ responsible for digestion and detoxication in crayfish, and the dietary addition of B. subtilis significantly increased the activities of LZM, ACP, SOD, and CAT, enhanced the antioxidant capacity, and alleviated hepatopancreas damage in crayfish, leading to the k d in hepatopancreas being increased.
Crayfish could accumulate Cd from their surrounding environment headily, the effects of Cd exposure on the various biological processes, we therefore further investigated. A dose-dependent rise in the mortality percentage of crayfish exposed to Cd for 2 weeks was discovered in our research. These findings were consistent with earlier studies on other aquatic animals (Ma et al. 2008;Páez-Osuna and Tron-Mayen 1995). As one of the target organs, hepatopancreas toxicological research following exposure to Cd is limited. Our study demonstrated that Cd exposure caused considerable histological structure damages of hepatopancreas, including degenerated tubules, tubule lumen dilatation, and epithelium vacuolization, which was in accordance with previous findings (Martín-Díaz et al. 2006;Kaddissi et al. 2012;Stara et al. 2018;Zhang et al. 2019).
Non-specific immune-related enzyme activity had been considered a criterion for evaluating the immunity of an organism (Dunier et al. 1991;Wu et al. 2016). The obtained results showed that the activities of LZM, ACP, and AKP were significantly decreased compared with those of the control group after Cd exposure for 2 weeks. It is wellknown that immune-related lysozyme is mainly produced by the hepatopancreas in crayfish (Muta and Iwanaga 1996). Thus, structural injury of the hepatopancreas caused by Cd interfered with lysozyme synthesis, leading to the decreased activities of the immune-related enzyme. LZM is an alkaline protein that is capable of killing and scavenging bacteria in the blood. Consistently, ACP and AKP are important hydrolases in the immune defense system via regulating the metabolic process of the cell body (Wei and Yang 2015a;Stara et al. 2016). Oxidative stress has been considered the prime mechanism of Cd toxicity in aquatic organisms. In order to counteract elevated ROS, crustacean hemocytes could generate a great number of anti-oxidants like SOD and CAT, which compose the first defense line against excess free radicals (Nicosia et al. 2014;Stara et al. 2016). SOD can detoxify O 2− to the less-reactive H 2 O 2 , and furthermore, it is transformed into H 2 O by CAT (Oh and Lim 2006;Guo et al. 2013). Decreases in the activities of SOD and CAT were observed upon exposure to Cd in our study, which might be related to over-produced ROS due to the insufficient scavenging capability in the hepatopancreas (Qiu et al. 2011;Dong et al. 2018a). Furthermore, a significant elevated level of MDA, a biomarker of lipid peroxidation, was observed in crayfish exposed to Cd, indicating a failure of the antioxidant response (Wei and Yang 2015b).
B. subtilis, a common strain of probiotic bacteria, was used widely in animal feed, food processing, pharmaceutical production, and water stability (Olmos et al. 2011;Lopez et al. 2016;Liu et al. 2022). Although there is no significant difference in Cd concentrations in the gill, exoskeleton, and muscle between the crayfish exposed to 1.0 mg Cd/L only and co-treated with 1.0 mg Cd/L and B. subtilis, the Cd concentration in hepatopancreas of the crayfish co-treated with 1.0 mg Cd/L and B. subtilis was dramatically lower than that of the crayfish treated with 1.0 mg Cd/L only. Specifically, the obtained results showed that B. subtilis could decrease k u value and increase k d value of hepatopancreas, resulting in the reduction of BCF, C ss , and T b1/2 . This might be one of the reasons for its use in mitigating the toxicity of heavy metals; however, the underlying mechanism needs further study. Administration of B. subtilis enhanced the non-specific immunity protection and improved the antioxidant response, resulting in the decrease in mortality upon exposure to Cd. Previous studies had shown that the addition of B. subtilis significantly increased the lysozyme activity, albumin content, and leukocyte phagocytosis in Oplegnathus fasciatus, leading to enhanced antioxidant capacity and alleviated liver damage in grass carp (Liu et al. 2017;Tang et al. 2019). B. subtilis was also reported to improve the activity of the antioxidant enzyme and effectively reduce the MDA and H 2 O 2 contents (Du et al. 2006;Yuan et al. 2016). Recently, effects of B. subtilis on nonspecific immune enzyme activity, blood biochemical indexes, and related gene expression of Cynoglossus semilaevis under ammonia nitrogen stress had also been confirmed (Wang et al. 2021). Actually, the enhancement of immune parameters such as serum antioxidant and lysozyme activity, serum protein, and glucose level of Oplegnathus fasciatus by B. subtilis resulted in the increased survival of fish after infection (Liu et al. 2017). B. subtilis added to feed was confirmed to supplement the lack of endogenous enzymes in the intestine via inducing hydrolytic enzymes and improve the rate of feed utilization, thus promoting animal growth (Ochoa 2006;Olmos et al. 2011;Valdez et al. 2014;Tang et al. 2017). Therefore, dietary administration of B. subtilis to crayfish feed efficiently inhibits the accumulation of Cd and ameliorates the pathological damage in the hepatopancreas in this study by enhancing the non-specific immunity protection and improving the antioxidant response. Alternatively, B. subtilis can mediate cadmium-induced toxicity through enteral nutrition. For example, B. subtilis may weaken the Cd-induced disorder of SCFAs production in the gut (Zhang et al. 2008). Moreover, according to research by Biao Yan et al. (2022), probiotics (Bacillus coagulans and Clostridium butyricum) have been reported to alleviate contaminants-induced toxicity by regulating intestinal microbiota and metabolites in crucian carp (Carassius auratus). Also, B. subtilis was able to mineralize and F attenuate Cd accumulation in the crayfish and rice, which can be used as a cleaning probiotic applied in rice and aquatic animal coculture systems (Mo et al. 2022).

Conclusions
In this study, the exposure of crayfish to Cd in water caused a dose-and time-dependent and tissue-specific distribution of Cd in crayfish. The hepatopancreas was the main accumulating site for Cd exposure among internal tissues in crayfish, which lead to the histological structural damage in the hepatopancreas and decreased non-specific immunity response and antioxidant defense. The onecompartment model indicated that gills had the highest k u value after aqueous Cd exposure and the k u and k d values in the gill, hepatopancreas, and exoskeleton of the group with 1.0 mg Cd/L were higher than those of the group with low Cd concentration (0.1 mg Cd/L). Dietary administration of B. subtilis to crayfish significantly decreased Cd accumulation and improved the immune and antioxidant defense, leading to the prevention in toxic effects of Cd.