Introduction

Over the past few years, heavy metal-caused pollution in aquatic systems has drawn increasing attention1. Cadmium (Cd) is a non-decomposable pollutant that has brought concerns about environmental issues and its impact on human health worldwide2,3. Cd's toxicity can be bio-amplified in the food chain4. Cd can trigger several harmful changes, including biochemical changes, morphological disruptions in cellular structures, and physio-biological dysfunctions5,6 through stimulating increased accumulation of reactive oxygen species (ROS) in living organisms7,8,9.

Oxidative stress from excess reactive oxygen species suggested that DNA damage and protein oxidation may be responsible for cadmium toxicity10,11,12,13. Excessive ROS accumulation in the antioxidant defence system was brought on by an imbalance between ROS production and removal14,15,16. In addition to impairing cellular processes, ROS also mediates the signaling pathways involved in physiological processes17,18,19. The prophenoloxidase (proPO) activating system (proPO-AS) is a special invertebrate defence mechanism that is essential for melanization in response to environmental stress20. Based on Söderhäll and Smith21 reports, the inactive proPO zymogen in granular and semi-granular hemocytes is initially transformed into active phenoloxidase (PO) before catalyzing the oxidation of phenols to quinines, which are later polymerized to melanin22. Hemocytes are the main structural source of proPO in crustaceans, according to Cerenius and Söderhäll23, Terwilliger and Ryan24, Matozzo and Marin25. Furthermore, Cd-induced damage to lysosomal membranes leads to the release of lysozyme (LSZ) based in granular hemocytes26. Additionally, lysozyme is employed as a sensitive and trustworthy biomarker to assess the toxicity of environmental contaminants27. Additionally, the main role of the metal ion detoxification processes is played by the protein metallothionein (MT), a low-molecular-weight, cysteine-rich, and metal-binding protein28,29. Importantly, it has been shown that metals play a major role in regulating MT mRNA expression at the transcription level28,30 and that inducing MT is thought to be essential for reducing Cd toxicity, such as oxidative damage31.

Red swamp crayfish (Procambarus clarkia) is a commercially significant freshwater cultured species32. The main reason for regarding this species as a typical bioindicator of toxic pollutants in aquatic environments is that they have a lengthy lifecycle, widespread distribution, and fairly simple anatomy33,34. According to an earlier study, the hepatopancreas are vital, multifunction organs for crustaceans' nutrient absorption, metabolism, and immune function35. The hepatopancreas was introduced as the core target of numerous environmental stresses36,37.

It has been reported that environmental Cd accumulates in crayfish tissues. Pollution levels in the environment have shown a positive correlation with concentrations in tissue samples38. Tissue levels were frequently found to be positively correlated with distance from the source of pollution39,40,41. The Cd is taken up and accumulated by crayfish from both the environment and food42. The Cd accumulation and detoxification are primarily carried out by the hepatopancreas in crayfish43 and other crustaceans44,45. The highest accumulation of Cd in crustaceans exposed to various Cd concentrations has been reported in the gills46.

To determine the contamination of these metal contaminants, the hepatopancreas and bioaccumulation ratio between the hepatopancreas and abdominal muscle of P. clarkii exposed to heavy metals have been used47,48,49. Bioremediation is an effective, broad, cost-effective, and environmentally acceptable cleanup technology50. Over the past several decades, there has been extensive research on cadmium removal by biosorption51,52. Microorganisms like fungi, algae, or bacteria can remove cadmium during biosorption53,54. Heavy metal (e.g., Cd2+, Pb2+, Cu2+) elimination by Saccharomyces cerevisiae has been attributed to their adsorption capacity. S. cerevisiae has previously been used to remove lead from milk, and research has shown that it is a natural biosorbent that can do so55. The CAD1 protein of S. cerevisiae contains domains bZIP and PAP1 that are closely related to Cd overexpression resistance56. S. cerevisiae has a part in heavy metal detoxification mechanisms, including sulfur refactoring pathways in yeast, glutathione production mechanisms, and reducing oxidative stress-related gene and protein expression57. Heavy metal ions with decreased toxicity of metallothionein and plant chelating peptide binding were later discovered58. Previous research has demonstrated that high heavy metal contamination in wheat can be overcome by using S. cerevisiae as a native remedy59. Amirnia et al.60 demonstrated the efficacy of removing heavy metals from industrial effluents using living yeast cells in a self-contained continuous adsorption system.

Furthermore, the use of S. cerevisiae to remove cadmium from fish such as Cyprinidae has a highly promising future61,62,63. At present, there are no reports on the possible role of S. cerevisiae in the attenuation of heavy metal concentrations in crayfish. On the other hand, several investigations in various aquaculture species have been undertaken, and the potential of S. cerevisiae as a probiotic has been investigated64,65,66. Similarly, Siddik, et al.64 shown in juvenile barramundi, Lates calcarifer, that a basal meal supplemented with 1% S. cerevisiae and 1% Lactobacillus casei alters physiological performance and promotes gut microbiome. The main objective of this study is to examine the ability of S. cerevisiae as a bioremediation method to combat Cadmium stress. The effects of a basal diet with free and varied dosages of S. cerevisiae, as well as different feeding days, on crayfish cadmium removal were examined across a 10-day exposure trial and a 12-day cadmium removal experiment. To validate our removal test, we also measured total antioxidant capacity (TAC) and oxidative stress indicators such as malondialdehyde (MDA), protein carbonyl derivates (PCO), and DNA–protein crosslink (DPC). In addition, four additional conformations were studied, as well as the mRNA expression levels of lysozyme (LSZ), metallothionein (MT), and prophenoloxidase (proPO) in crayfish. The research findings are expected to open new avenues for develo** feed additives, heavy metal removal technology, and new removal agents for crustaceans and aquatic animals.

Materials and methods

Crayfish sampling

In September 2020, 480 adult male crayfish with similar sizes (10.011.10 cm length; 20.300.31 g wet weight) were obtained from a crayfish company (Yibin Haide Fishery Technology Co., LTD) in Yibin, China.

Yeast strains and growth conditions

S. cerevisiae was isolated from the intestinal tract of Procambarus Krai and was preserved in the Department of Agriculture, Forestry and Food Engineering, Yibin University, Reserve number (FYBNL) M2020121. S. cerevisiae was grown in yeast peptone dextrose adenine (YPDA; Qingdao Biological Technology Co., Ltd., Qingdao, China) overnight at 30°C before being resuspended in sterile phosphate-buffered saline (PBS). The plate dilution counting method was used to determine the colony forming units (CFU) per milliliter of S. cerevisiae culture. A mixture of 1% and 5% S. cerevisiae powder was made by grinding basic feed into powder and mixing it with S. cerevisiae powder. A specified amount of water was added, mixed, and heated on the electric stove for several minutes67,68. The feed was vacuum-dried overnight at 30 °C and stored at 4 °C. Every 3 days, the feed was prepared in the same way.

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 feed68. 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.

Table 1 The basic of crayfish diet composition.
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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 results70.

MDA, DPC, and PCO assay

The thiobarbituric reactive species (TBARS) assay was used to measure the production level of MDA71. 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 extraction and cDNA synthesis

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).

Table 2 proPO, LSZ, MT, and β-actin primers used in Real-time PCR.
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In order to compare the relative levels of expression of proPO, LSZ, and MT in the samples, the housekee** 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 ddH2O. 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 method73.

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.

Results

Cd concentration in crayfish

The Cd concentration in the edible parts of the crayfish feeding basal diet was 14.67–14.82 mg/kg, as shown in Table 3, and there was no significant declining trend throughout the process of 0–21 d water purification (basal feed).

Table 3 Effect of S. cerevisiae feeding on Cd concentration in the edible parts of crayfish.
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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 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.

Figure 1
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.

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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.

Figure 2
figure 2

Effect of Cd and S. cerevisiae addition on (A) MDA, (B) PCO contents, and (C) DPC coefficient 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.

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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.

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.

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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.

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.

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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, respectively74,75. 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 used76. 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 costs77. 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 MgCl2-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 destruction79,80. Microorganisms are a biological tool for metal removal because they can be used to remove, concentrate, and extract heavy metals from polluted aquatic habitats81. 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 conditions79.

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' results82,83. 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 oysters84. 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 sorbent85. The mechanism of Cd toxicity in S. cerevisiae has been studied86 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 conditions87. 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-day88. 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 radical89. 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 reactions90.

Importantly, Wätjen and Beyersmann91 back up the preceding findings. Excess ROS are normally eliminated by the antioxidant system to maintain the body's redox status92. However, when the generation of ROS surpasses the body's antioxidant defences, lipid peroxidation, protein modification, DNA damage, and other oxidative effects are induced93. Membrane lipid peroxidation has been identified as one of the functional repercussions of oxidative damage94,95. MDA, a traditional marker of lipid peroxidation, reflects the degree of oxidative damage96. As a result, the severity of oxidative stress caused by Cd can be determined based on changes in MDA levels in organs or cells97,98.

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 organisms93,99,100. An earlier study discovered that Cd could induce ROS generation in the hemocytes of the crab S. henanense26. 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 depletion101, and the potential for genotoxic and cytotoxic effects owing to an increase in the ROS102. 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 protein72. 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 DPC103. 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 sperms97. 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 DNA72. Evidence has proven that Cd exposure engenders cell necrosis, characterized by cell membrane disintegration followed by intracellular content dissemination104,105.

ROS generation is an emerging step in Cd-induced cytotoxicity which is followed by a decrease in mitochondrial membrane potential106. Mitochondrial damage is usually accompanied by the activation of caspases and programmed cell death107,108. 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 organisms102 and suppresses immune responses110. In invertebrates, particularly crustaceans, the prophenoloxidase activating system is critical for immunity111. A serine protease cascade response would convert proPO to active PO, similar to the complement activating system in vertebrates112. Ammonia was found to have a significant impact on proPO gene expression113. The activity of proPO was significantly reduced in freshwater crayfish Procambarus clarkii after exposure to copper103. 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 harm115. LSZ is an essential lysosomal enzyme capable of lysing bacterial membranes, avoiding the risk of bacterial infections116. 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 increased115. 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 hemocytes26.

MT is a high-affinity ligand for Cd absorption, transport, and detoxification119. It has a high antioxidant capacity and protects cells from the cytotoxic effects of ROS120. Earlier studies have shown that Cd-mediated MT mRNA expression scavenges ROS production under Cd stress121, 122. Furthermore, there is evidence that MT expression levels increased significantly after 3 days of Cd challenge in the clam Mactra veneriformis115. Exposure to Cd has been associated with increased MT expression in Pacific oysters Crassostrea gigas123, hard clams Meretrix lusoria (Chang et al., 2007), and scallops Argopecten irradians124. 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 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

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