Saccharomyces cerevisiae additions normalized hemocyte differential genes expression and regulated crayfish (Procambarus clarkii) oxidative damage under cadmium stress

Because China produces the most crayfish in the world, safe solutions must be improved to mitigate the risks of ongoing heavy metal stressors accumulation. This study aimed to use Saccharomyces cerevisiae as a bioremediation agent to counteract the harmful effect of cadmium (Cd) on crayfish (Procambarus clarkia). Our study used three concentrations of S. cerevisiae on crayfish feed to assess their Cd toxicity remediation effect by measuring total antioxidant capacity (TAC) and the biomarkers related to oxidative stress like malondialdehyde (MDA), protein carbonyl derivates (PCO), and DNA–protein crosslink (DPC). A graphite furnace atomic absorption spectroscopy device was used to determine Cd contents in crayfish. Furthermore, the mRNA expression levels of lysozyme (LSZ), metallothionein (MT), and prophenoloxidase (proPO) were evaluated before and following the addition of S. cerevisiae. The results indicated that S. cerevisae at 5% supplemented in fundamental feed exhibited the best removal effect, and Cd removal rates at days 4th, 8th, 12th, and 21st were 12, 19, 29.7, and 66.45%, respectively, which were significantly higher than the basal diet of crayfish. The addition of S. cerevisiae increased TAC levels. On the other hand, it decreased MDA, PCO, and DPC, which had risen due to Cd exposure. Furthermore, it increased the expression of proPO, which was reduced by Cd exposure, and decreased the expression of LSZ and MT, acting in the opposite direction of Cd exposure alone. These findings demonstrated that feeding S. cerevisiae effectively reduces the Cd from crayfish and could be used to develop Cd-free crayfish-based foods.


Feeding frequency and cadmium exposure
The experimental crayfish were divided into six boxes, each holding 80 crayfish, after seven days of acclimatization.The boxes were allocated into three groups at random: two treatment groups (TGs) and one control group (CG).For 28 days, the treatment group was fed S. cerevisiae feed (1% and 5%), whereas the control group was fed the basal commercial feed 68 .To clearly examine the removal effect of S. cerevisiae on crayfish, sampling analysis was performed on the first, fourth, eighth, twelfth, and twenty-first days of acculturation.
For 10 days, all crayfish were kept in plastic aquaria (90 cm60 cm25 cm) of aerated tap water containing Cd (1.450 mg L −1 ) and 12 days without Cd.This experiment was performed at Yibin University in the Chinese province of Sichuan.The Cd concentrations were 20 times higher than the national standard for fishery water quality.Crayfish were fed a base diet for the first ten days and then switched to a different diet for the next twelve days as shown in Table 1.
Basic feed without S. cerevisae (Group 1), S. cerevisae at 1%, and 5% supplemented in basic feed (Groups 2 and 3) were the three food groups.(2nd and 3rd groups).Each group had three replicates, each with 80 animals.Six parallel samples from each group were collected every four days for a total of four times in the removal experiment.

Graphite furnace atomic absorption spectroscopy (GF-AAS)
Cd measurements in crayfish tissue were performed using a Perkin-Elmer Analyst 800 Atomic Absorption Spectrometer outfitted with a Zeeman background correction device and an electrothermal atomizer transversely heated graphite tube (THGA) 69 .Electrodeless discarge lamps (EDLs, Perkin-Elmer) at 282.3 nm (slit width 0.7 nm) were used as radiation sources for the Cd.At room temperature, twenty-microliter aliquots of the material were injected into a graphite tube, followed by a two-step drying, pyrolysis, and atomization (one step each).The graphite tube was finally cleaned.Dilution of a certified 1000 mg L −1 Cd monoelement standard solution (Trace Cert Fluka) was used to generate standard solutions for calibration curves.Reagent blank was used to dilute all solutions.The method's detection limit, as determined by the Cd calibration curve, was Cd, 0.19 mg kg −1 .

Total antioxidant capacity (TAC) determination
The TAC was measured using the TAC Assay Kit according to the manufacturer's instructions.A Fluko Superfine Homogenizer at 1000 rpm for about 30 s was used to homogenize 300 µl of each sample with 106 hemocytes/mL.The samples were centrifuged at 12,000 g for 4 min at 4 °C.Then, working solutions containing 20 l of catalase and 170 l of 2,′-Azinobis-(3-ethylbenzthiazo-line-6-sulfonate) (ABTS) were added to 10 l sample solutions and kept at room temperature for 10 min.A microplate reader was used to measure the TAC at 414 nm (Spectramax M5 multimode microplate reader, San Francisco, CA, USA).We used soluble Trolox as a reference.The corresponding Trolox/mg protein concentration in individual samples was used to calculate the results 70 .

MDA, DPC, and PCO assay
The thiobarbituric reactive species (TBARS) assay was used to measure the production level of MDA 71 .PCO and DPC measurements were performed following standard protocols introduced by Li et al. 72 .PCO contents were quantified using 2,4-dinitrophenylhydrazine (DNPH), which reacted with protein carbonyl derivates to form 2,4-dinitrophenylhydrazone. OD values were computed at 370 nm, and their expression (nmols of carbonyl groups/mg protein) was based on a molar extinction coefficient of 22,000 M/cm for aliphatic hydrazones.The concentration of DPC was determined using KCl-SDS, which was used to precipitate the crosslink and separate free DNA from protein-bound DNA.To combine with DNA, Hoechst 33,258 was added.The fluorescence was measured at a specific wavelength (excitation: 350 nm; emission: 460 nm).The fluorescence ratio was calculated as a percentage of protein bound to total DNA.Total RNA was extracted away from hemocytes originating from animals at different treatments thanks to the Trizol Lysis Reagent (TaKaRa, Dalian, China) following the manufacturer's protocol.Then, the concentration and purity of Total RNA extracts were estimated using a BioSpectrometer fluorescence (Eppendorf, Hamburg, Germany) and 1.2% agarose gel electrophoresis, and genomic DNA was removed by DNase I (TaKaRa, Dalian, China) digestion.First-strand cDNA was synthesized from 2 µg of total RNA using a cDNA synthesis kit (TaKaRa, Dalian, China).

Real-time quantitative RT-PCR of proPO, LSZ, and MT
The designation procedure for quantitative fluorescent RT-PCR primers was as per the transcriptome sequences using Primer 5 software (Table 2).
In order to compare the relative levels of expression of proPO, LSZ, and MT in the samples, the housekeeping gene β-actin was also amplified with the same cDNA samples.The RT-qPCR was carried out in a total volume of 20 µl, containing 10 µl of 2 × SYBR Premix (TaKaRa), 0.4 µl of each primer (10 µM), 0.4 µl ROX dye П, 2 µl of the diluted cDNA and 6.8 µl ddH 2 O.The thermal profile for RT-qPCR was 30 s at 95 °C for 1 cycle, 5 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C for 40 cycles.An ABI 7500 real-time detection system (Applied Biosystems, Foster City, CA, USA) was utilized to run the RT-qPCR using SYBR Premix Ex Taq II (Takara, Dalian, China) based on the manufacturer's protocol.The proposed experiments were done in triplicates involving NTC.Fold change for the gene expression relative to controls was determined by the 2 −ΔΔCt method 73 .

Statistical analysis
The data were obtained for statistical analysis by analysis of the variance (ANOVA) to compare the statistical differences between the experimental groups at p < 0.05 statistical significance level using SPSS software.

Ethics statement
The animal study was reviewed and approved by the Administration Committee of Experimental Animals, Sichuan Province, China, and the Institutional Animal Care guidelines of Yibin University, China.

Cd concentration in crayfish
The Cd concentration in the edible parts of the crayfish feeding basal diet was 14.67-14.82mg/kg, as shown in Table 3, and there was no significant declining trend throughout the process of 0-21 d water purification (basal feed).
Simultaneously, S. cerevisiae at 1% supplemented in basal diet feeding crayfish had a removal effect, and there had fallen by 43.1% after 21 days of water purification (1% of S. cerevisiae).The clearance rates on the fourth and eighth days were 8.84% and 8.7%, respectively, with no significant difference between them but a significant change when fed 1% S. cerevisiae on day 0. The best clearance rate, however, was 66.45% when fed 5% S. cerevisiae on the 21st day.The elimination rates were significantly different at 0, 4, 8, 12, and 21 days.The Cd level of the Table 2. proPO, LSZ, MT, and β-actin primers used in Real-time PCR.

Sequences of specific primers (5′-3′) Length (nt) TM (°C)
Prophenoloxidase (proPO) F-TGC CTT AGG GGT GTT TTA 18 61 R-CAG GGT GAC TGG TCT TGG  18   lysozyme (LSZ) Table 3.Effect of S. cerevisiae feeding on Cd concentration in the edible parts of crayfish.In the same row, values with different capital letters superscripts mean significant differences (P < 0.05).Values with different lowercase letter superscripts in the same column indicate significant differences (P < 0.05).

Group Percent S. cerevisiae in additive
The concentration of Cd in crayfish during the removal period (mg/kg) www.nature.com/scientificreports/crayfish in the control group was significantly higher than that of the crayfish fed 1% and 5% S. cerevisiae, as indicated in Table 3.Despite this, no significant differences were observed among them after four days of feeding in water.On the 8th, 12th, and 21st days, the Cd concentration of crayfish with varied proportions of S. cerevisiae was significantly lower than the control group, and the Cd concentration of S. cerevisiae with different ratios was also statistically different.Finally, after 21 days, the 1% and 5% S. cerevisiae showed the most significant activity in eliminating Cd, with Cd concentrations of 8.29 and 5.06 mg/kg, respectively.

The TAC level of the hemocytes of crayfish
Except for the group of Cd + 5% S. cerevisiae on 0 d and Cd in 21 days, no significant changes in TAC levels were found following Cd exposure, Cd + 1% S. cerevisiae, and Cd + 5% S. cerevisiae throughout the treatment periods, as shown in Fig. 1.TAC levels for Cd, on the other hand, decreased significantly, falling by 63% and 52%, respectively, compared to controls.The TAC steadily increased with S. cerevisiae concentrations, peaking at Cd + 5% S. cerevisiae.TAC in crayfish hemocytes was reduced dose-dependently by Cd.

The MDA, PCO, and DPC levels in hemocytes of crayfish
The MDA content of hemocytes increased significantly (p 0.05) when compared to the control group (Fig. 2A).
MDA levels in crayfish exposed to Cd increased with time and were 4.71 and 16-fold higher than in controls, respectively.MDA decreased with 1 and 5% S. cerevisiae additions compared to Cd treatment free any S. cerevisiae, but it remained higher than the control.As observed in Fig. 2B, PCO levels in hemocytes increased dramatically over time as Cd concentration increased.After Cd (1.450 mg L −1 ) exposure, the PCO level increased to 2.11-fold higher than the controls, especially on the 21st day.With time increasing, the 1 and 5% S. cerevisiae additions not showed any decreases in PCO until eight days, but the PCO level decreased over an extended period ( 12 and 21 days) which was more remarkably with 5% S. cerevisiae addition.
The DPC level was considerably increased after Cd exposure compared to the control groups (Fig. 2C).DPC levels increased with time and exposure to greater Cd concentrations when compared to the control.With 1 and 5% S. cerevisiae additions and increasing time, the DPC level was significantly decreased, more notably with 5% S. cerevisiae additions.

The expression status of proPO in hemocytes of crayfish
Along with the increase in Cd content, there was a general downward trend in proPO expression levels (Fig. 3).When compared to the control group, proPO expression levels were significantly reduced, especially after 21 days (p 0.05).The expression of proPO in crayfish hemocytes containing Cd has decreased dramatically over time when compared to those that do not contain Cd.With the 1 and 5% S. cerevisiae additions, the proPO were significantly up-regulated beginning from the eighth day and with extended time compared to Cd without any of S. cerevisiae additions.The increase in proPO was remarkably in 5% more than 1% S. cerevisiae addition.Cd exposure inhibited the expression level of proPO in crayfish hemocytes.The expression levels of LSZ and MT in hemocytes of crayfish LSZ mRNA expression level demonstrated a dose-dependent response to Cd treatment (Fig. 4A).LSZ expression level increased rapidly beginning at 8 d in Cd treatment groups, which recorded a high level in 21d with 8.8-fold compared to control.Both 1 and 5% S. cerevisiae additions led to a decrease in the expression of LSZ, which was more in 5% than 1% S. cerevisiae addition.Cd treatment significantly increased LSZ mRNA expression levels, while S. cerevisiae additions decreased it in the hemocytes in a dose-oriented manner (Fig. 4A).Cd treatment resulted in a significant increase in MT mRNA expression levels in the hemocytes in a dose-oriented manner with a maximal response at 21 d at the exposure range of 6.2-fold compared to the control (Fig. 4B).Both 1 and 5% S. cerevisiae additions led to a decrease in the MT, especially at 12th and 21st d.Cd exposure significantly incremented MT mRNA expression levels, whereas S. cerevisiae additions did not decrease this expression in the hemocytes in a dose-oriented manner.

Discussion
Cadmium has a strong bioenrichment effect in crayfish, and its biometabolic half-life is long.The majority of heavy metals are extremely toxic and non-biodegradable.Cu(II), Cd(II), Zn(II), and Pb(II) maximum permissible concentrations in China are 2.0, 0.1, 5.0, and 1.0 mg/L, respectively 74,75 .As a result, in order to meet increasingly stringent environmental quality standards, they must be removed from waste effluents.To treat such effluents, various methods such as chemical precipitation, ion exchange, electrodeposition, membrane separation, and adsorption have been used 76 .Traditional chemical precipitation was the most cost-effective method, but it is inefficient.Ion exchange and reverse osmosis were both effective in general but had high maintenance and operational costs 77 .Adsorption, particularly with low-cost natural sorbents, is also one of the few promising methods for removing toxic metals from aqueous environments.The previous research of Zhang et al. 78 demonstrated the efficacy of Cadmium removal using MgCl 2 -modified biochar (MgC600) derived from crayfish shell waste.
Bioremediation is a natural alternative process to incineration, catalytic degradation, adsorbent use, physical removal, and ultimately pollutant destruction 79,80 .Microorganisms are a biological tool for metal removal because they can be used to remove, concentrate, and extract heavy metals from polluted aquatic habitats 81 .The bioremediation strategy is based on biological agents' high metal-binding ability, which aids in the extraction of heavy metals from very dilute solutions.Bioremediation using microorganisms is very beneficial and can also adapt to extreme conditions in polluted areas.Because microorganisms act on pollutants even in very dilute solutions, bioremediation with microorganisms is very beneficial and can also adapt to extreme conditions 79 .
Our study found that Cd could not be eliminated quickly when crayfish were raised in pure water without S. cerevisiae, which largely agrees with the previous studies' results 82,83 .The concentration of Cd in crayfish with temporary feeding or conventional diet could not be effectively removed by natural metabolism, which is also applied to oysters 84 .There is a general agreement over the benefits of S. cerevisiae in the biosorption of limited concentrations of Cd and alleviation of contaminated foods with the employment of green technologies under the disguise of a natural, low-cost, and abundant sorbent 85 .The mechanism of Cd toxicity in S. cerevisiae has been studied 86 in conjunction with Cd-induced UPR, intracellular ROS levels, and cell death, all of which may play important roles in Cd-induced toxicity, though the mechanism by which S. cerevisiae removes cadmium from crayfish is not well studied.To the best of our knowledge, the distribution of Cd in crayfish is controlled by the p38 Mitogen-activated Protein Kinase (MAPK) by modulating the accumulation of Cd in different crayfish tissues under Cd-stressed conditions 87 .We hypothesized that S. cerevisiae and crayfish cells might have competitive adsorption for cadmium.The S. cerevisiae species was used to remove Cd from milk obtained at the highest Cd removal (70%) rate.This proportion was at 80 μg/L of Cd concentration in milk samples after the final storage time of the four-day 88 .As shown in Table 1, when S. cerevisiae was used in crayfish feed, the highest cadmium removal rate was only 29.7% at 5% of S. cerevisiae but higher than 1%.Therefore, it is assumed that the increased concentrations of S.cerevisiae biomass provided a more binding site for Cd and hence a higher capacity for Cd removal.Cd, as is well known, cannot produce free radicals directly.On the other hand, Cd can indirectly stimulate the production of ROS through the superoxide radical and the hydroxyl radical 89 .Furthermore, an intriguing mechanism was presented to explain Cd's indirect role in ROS generation, in which Cd was thought to replace Fenton-active metals such as iron and copper in cytoplasmic and membrane proteins (e.g., Ferritin), increasing the number of freely iron and copper ions that participate in oxidative stress via Fenton reactions 90 .
Importantly, Wätjen and Beyersmann 91 back up the preceding findings.Excess ROS are normally eliminated by the antioxidant system to maintain the body's redox status 92 .However, when the generation of ROS surpasses the body's antioxidant defences, lipid peroxidation, protein modification, DNA damage, and other oxidative effects are induced 93 .Membrane lipid peroxidation has been identified as one of the functional repercussions of oxidative damage 94,95 .MDA, a traditional marker of lipid peroxidation, reflects the degree of oxidative damage 96 .As a result, the severity of oxidative stress caused by Cd can be determined based on changes in MDA levels in organs or cells 97,98 .
Numerous studies have shown that Cd-induced reduction of antioxidant enzyme activities can inhibit the scavenging process of ROS, which can lead to an increase in MDA levels in cells or organisms 93,99,100 .An earlier study discovered that Cd could induce ROS generation in the hemocytes of the crab S. henanense 26 .In the present study, Cd exposure increased the level of MDA compared to Cd exposure with S. cerevisiae as feed additives, indicating the efficiency of S. cerevisiae in removing the Cd effect from crayfish cells.As we discovered in this study, Cd (21 d) significantly inhibited TAC activity in crayfish hemocytes, whereas TAC activity was recovered by using S. cerevisiae as feed additives.Previous research by Zhou et al. 28 demonstrated that Cd (2.900 mg L1, 14 and 21 days) exposure significantly inhibited TAC activity by increasing MDA concentrations in crab hemocytes.
Also, the same previous study by Zhou et al. 28 found a decrease in TAC activity accompanied by an increase in MDA levels, which is consistent with the above reports.Furthermore, there is evidence that Cd exposure can cause antioxidant enzyme inhibition, GSH depletion 101 , and the potential for genotoxic and cytotoxic effects owing to an increase in the ROS 102 .Carbonyl is a typical biomarker for protein damage following exposure to the ROS, which negatively affects the amino acids in the protein side chains for producing carbonyl in the protein 72 .DNA-protein crosslinks (DPC) are thought to be constantly formed in cells during metabolism, such as via the interaction of glucose-6-phosphate with lysine amino groups, and ROS also produces a high percentage of DPC 103 .In our study, Cd exposure resulted in significant increases in DPC and PCO levels, while using S. cerevisiae as feed additives resulted in a decrease, indicating their efficiency in removing Cd toxicity in crayfish cells.
Prior research by Zhou et al. 28 revealed that the effects of Cd on the accumulation of the PCO and the emergence of DPC in crab hemocytes were quite obvious.Previous research found that acute Cd (58 and 116 mg L1, 7 d) exposure could cause a significant increase in PCO and DPC in S.henanense sperms 97 .Thus, it was reported that Cd could induce DPC generation through (1) excessive ROS-induced oxidative damage to amino acids and proteins and (2) Cd direct interference with the covalent combination of amino acids in the protein and the nucleotide of DNA 72 .Evidence has proven that Cd exposure engenders cell necrosis, characterized by cell membrane disintegration followed by intracellular content dissemination 104,105 .
ROS generation is an emerging step in Cd-induced cytotoxicity which is followed by a decrease in mitochondrial membrane potential 106 .Mitochondrial damage is usually accompanied by the activation of caspases and programmed cell death 107,108 .Overall, it has been demonstrated that excessive ROS production is associated with lipid, protein, and DNA damage, resulting in impaired cellular structure and functions.According to Qin et al. 109 , S. henanense hemocytes are classified into large granular, semi-granular, and hyaline.Acute Cd also affected hemocyte organelles.Cd causes oxidative stress in aquatic organisms 102 and suppresses immune responses 110 .In invertebrates, particularly crustaceans, the prophenoloxidase activating system is critical for immunity 111 .A serine protease cascade response would convert proPO to active PO, similar to the complement activating system in vertebrates 112 .Ammonia was found to have a significant impact on proPO gene expression 113 .The activity of proPO was significantly reduced in freshwater crayfish Procambarus clarkii after exposure to copper 103 .Furthermore, Cd exposure reduced proPO gene expression in crayfish hemocytes in the current study.The application of S. cerevisiae as feed additives has intensified the expression level of proPO.This phenomenon was consistent with the Sun et al. 114 report, which revealed that Cd exposure downregulated the expression of proPO in the hepatopancreas of the crab S.henanense.
Aquatic defence effectors such as LSZ and MT can respond well to stress factors by protecting organisms from serious harm 115 .LSZ is an essential lysosomal enzyme capable of lysing bacterial membranes, avoiding the risk of bacterial infections 116 .According to Tyagi et al. 117 , LSZ expression levels were increased in the black tiger shrimp Penaeus monodon after the bacterial pathogen challenge.Furthermore, when the clam Mactra veneriformis was exposed to Cd and Hg for 5 and 7 days, LSZ expression levels increased 115 .On the other hand, in the study of Jakiul Islam et al. 118 on the European seabass, Dicentrarchus labrax, they found an increase in LSZ expression levels during extreme cold events at various salinities.Here, we discovered that LSZ expression was up-regulated in crayfish hemocytes under Cd stress.LSZ expression levels increased rapidly beginning at 8 days in Cd treatment groups, reaching a peak in 21 days with an 8.8-fold increase compared to controls.Both 1 and 5% S. cerevisiae additions resulted in a decrease in LSZ expression, with the 5% S. cerevisiae addition being more effective than the 1% S. cerevisiae addition.In contrast, it was down-regulated when the feed additive S. cerevisiae was included in the diet.This finding suggests that S. cerevisiae effectively mitigates the negative effects of Cd exposure.LSZ expression levels were up-regulated in the hemocytes of crab S. henanense under Cd stress (1.450, and 2.900 mg L −1 ) in a previous study by Zhou et al. 28 .However, previous research found no significant changes in LSZ activity when crabs were subjected to Cd stress in S. henanense hemocytes 26 .MT is a high-affinity ligand for Cd absorption, transport, and detoxification 119 .It has a high antioxidant capacity and protects cells from the cytotoxic effects of ROS 120 .Earlier studies have shown that Cd-mediated MT mRNA expression scavenges ROS production under Cd stress 121,122 .Furthermore, there is evidence that MT expression levels increased significantly after 3 days of Cd challenge in the clam Mactra veneriformis 115 .Exposure to Cd has been associated with increased MT expression in Pacific oysters Crassostrea gigas 123 , hard clams Meretrix lusoria (Chang et al., 2007), and scallops Argopecten irradians 124 .In this experiment, we discovered that MT mRNA expression levels were induced, and more transcription of MT mRNA was activated in crayfish treated with high Cd concentrations.In contrast, it was downregulated with Cd when S. cerevisiae was used as a feed additive.Zhou et al. 28 observed that high Cd concentrations induced MT mRNA expression levels and activated more MT mRNA transcription in S. henanense than in low Cd concentrations.Fang, et al. 115 reported a similar result in the clam M. veneriformis.It is hypothesized that this is MT's defence mechanism against Cd toxicity.Thus, we identified the importance of S. cerevisiae additive in regulating MT expression and providing adequate protection from Cd toxicity in the examined crayfish.

Conclusion
Despite the fact that the third biggest known freshwater crayfish species has been shown to have high levels of harmful metals, our understanding of the potential dangers of consuming this species lags well behind that of finfish.Because China produces the most crayfish in the world, safe solutions to counteract the hazards of ongoing heavy metal stressors as heavy metals build must be improved.The goal of this study was to use S. cerevisiae as a bioremediation agent to mitigate the negative effects of Cd on crayfish (P.clarkia).The results showed that S. cerevisiae at 5% supplemented in fundamental feed had the best removal effect, with Cd removal rates at days 4, 8, 12, and 21 being 12%, 19%, 29.7%, and 66.45%, which were significantly higher than the crayfish's basal diet.TAC levels were increased by the addition of S. cerevisiae.On the other hand, it reduced MDA, PCO, and DPC levels, which had risen as a result of Cd exposure.Furthermore, it increased proPO expression, which was decreased by Cd exposure, and decreased LSZ and MT expression, acting in the opposite direction as Cd exposure alone as shown in Fig. 5.These findings show that feeding S. cerevisiae effectively reduces crayfish Cd levels and could be used to develop Cd-free crayfish-based foods.

Figure 1 .Figure 2 .
Figure 1.Total antioxidant capacity (TAC) in crayfish hemocytes at different treatments.Data are means ± SD, n = 6 crayfish per treatment at each time point.Compared to the control group, significances are indicated by *p < 0.05 and **p < 0.01.

Figure 3 .
Figure 3.The expression levels of proPO in the hemocytes of crayfish.Data are means ± SD, n = 6 crayfish per treatment at each time point.Compared to the control group, significances are indicated by *p < 0.05 and **p < 0.01.

Figure 4 .
Figure 4.The expression levels of LSZ and MT in crayfish hemocytes.Data are means ± SD, n = 6 crayfish per treatment at each time point.Compared to the control group, significances are indicated by *p < 0.05 and **p < 0.01.

Figure 5 .
Figure 5.The effect of S. cerevisiae additions on the differential expression of oxidative-related genes in P. clarkii in the presence of Cd toxicity

Table 1 .
The basic of crayfish diet composition.The nutritional levels of basal diet were 28.5% crude protein, 6.2% crude fat, 41.9% carbohydrate and 8.1% crude ash.