Effects of Selenium-enriched Bacillus Subtilis on Growth Inammatory and Intestinal Microbes of Common Carp Induced by Mercury

Mercury (Hg) is a global pollutant that affects the health of humans and ecosystems. Selenium (Se) is an essential trace element for many organisms including humans. Bacillus subtilis is widely distributed in nature, is one of the main probiotics used in aquaculture, and has a certain adsorption effect on heavy metals. The interaction between Hg and Se was rigorously studied, especially due to the observation of the protective effect of Se on Hg toxicity. The common carp was exposed to Hg (0.03 mg/L), and 10 5 cfu/g Se-rich B. subtilis was added to the feed. After 30 days of feeding, samples were taken to evaluate the growth performance, serological response, inammatory response, and intestinal microbial changes. In this study, when sh were exposed to Hg, the growth performance of the Se-rich B. subtilis plus 0.03 mg/L Hg sh group was lower than that of the control group and higher than 0.03 mg/L Hg; The levels of LZM and IgM decreased, but after supplementation with Se-rich B. subtilis, the levels of LZM and IgM increased; Hg treatment signicantly up-regulated the mRNA expression of IL-1β, IL-8, TNF-α and NF-κB P65, but down-regulated the mRNA expression of IL-10, TGF-β and IkBα. However, compared with the Hg group, the Se-rich B. subtilis plus Hg group can signicantly increase the mRNA expression levels of IL-1β, IL-8, TNF-α and NF-κB P65, but reduce the regulation of IL-10, TGF-β and IkBα expression. At the genus level, the abundance of Aeromonas in the intestines of common carp in the Hg treatment group increased, and Se-rich B. subtilis could reduce the abundance of Aeromonas (pathogenic bacteria). Through the analysis of the species, we found that the Hg group was mainly composed of Aeromonas sobria and Aeromonas hydrophila. However, in the Se-rich B. subtilis treatment group, we found that Aeromonas sobria was signicantly less than the Hg group. Because Aeromonas (pathogenic bacteria) is harmful to the sh, it can induce inammation in the sh and make the sh sick. Through microbiological analysis, it is found that Se-rich B. subtilis improves Hg-induced intestinal microbial changes, alleviates the abundance of Aeromonas, and alleviates the inammation of the sh.


Introduction
Heavy metals have become serious pollutants in the aquatic environment due to their persistence to the environment and the ability to be accumulated by aquatic organisms (Veena et al. 1997). Mercury (Hg) is a global pollutant that has been associated with kidney immune and genetic damage to animals and humans, as well as microbial diversity and function (Liu et al., 2018a;Liu et al., 2018b). Exposure to Hg can cause various diseases of the organ system (Rice et al., 2014). Fish are exposed to Hg due to pollution in inland waters, which will lead to deterioration of sh health, thereby reducing sh quality and sh production (Begam and Sengupta, 2015). the pro-in ammatory transcription factor NF-kB p65 is often a central mediator of the immune and in ammatory response; Studies have found that mercury can signi cantly induce the up-regulation of the pro-in ammatory transcription factor NF-kB p65 ( For a long time, it has been observed that Se protects animals from the toxicity of inorganic mercury and methylmercury. Parízek and Ostádalová reported one of the earliest studies on the protective effect of Se. This experiment Se protects rats from inorganic mercury-induced kidney poisoning (Parízek and Ostádalová, 1976). Subsequent studies found that this is the absorption and interaction of mercury and Se in Pseudomonas uorescens to achieve the detoxi cation of Se and mercury (Belzile et al., 2006).
Se is an essential micronutrient element that has a variety of complex effects on human health. Se is essential to human life and health, which is mainly due to its antioxidant, anti-in ammatory and antiviral properties (Wrobel et al., 2016). Lin et al reported that Se de ciency can reduce the growth performance of the kidney, spleen and skin of the young grass carp head and impair its immune function (Zheng et al., 2018). At the same time, Se supplementation can alleviate the upregulation of nuclear factor NF-KB induced by Microcystin-leucine arginine, and the upregulation of in ammatory cytokines IL-6, TNF-α, IL-1β, and TGF-β1 in cells (Adegoke et al., 2018).
Probiotics are living microorganisms that provide health bene ts to the host when supplied in su cient volumes (W.H. Organization, 2001). According to many recent studies, probiotics derived from the host's intestinal tract increase the growth rate of the host by hydrolyzing the complex polysaccharides in the host's nutrients. As a live microbial feed supplement, it is bene cial to the development of the host.

Preparing Se-rich B. subtilis and Preparing diet
Commercial feed as a basic diet. Se-rich B. subtilis is added to the basic feed For the detailed steps of preparing Se-enriched B. subtilis, please refer to Xinchi S et al (Shang et al., 2021). The mercury content in 20 ml water samples from different aquariums was collected. Table 1 displays the actual mercury concentration. The probiotics are diluted with sterile normal saline, fully homogenized and added to the basic feed according to the needs of the experiment ( nal dose of bacteria: 105 cfu/g feed; nal concentration of Se: 0.5 ppm) (Shang et al., 2021). The same volume of sterile saline was added to the basic diet to prepare a control. Store all feed in a refrigerator at 4℃.

Feed and experimental design
Common carp (6.2 ± 0.1 g) was purchased from an aquatic fry farm (Jilin Province, China) and transported to the laboratory. We randomly divided 360 sh into four groups and divided these groups evenly into 12 tanks (80 L; 3 replicates per group; 30 sh per tank). After the experimental sh were domesticated and reared for 2 weeks, the healthy common carps were randomly divided into 4 treatment groups (Se-rich B. subtilis, control group, Se-rich B. subtilis plus 0.03 mg/L Hg and 0.03 mg/L Hg). After the experiment started, they were fed twice a day at 8 o'clock and 18 o'clock, according to the sh body mass accounting for (1-2)% of the daily feeding amount. 80L aerated tap water in the water tank, the daily water exchange rate is 1/2 of the total.

Growth performance
Observe the development performance of common carp after one month of breeding. The calculation of coe cients was made below: Rate of survival (SR, %) = 100 × (ultimate quantity of sh/initial amount of sh), Weight gain proportion (WGR, %) = 100 × [(ultimate body weight − primary body weight)/primary body weight], given rate of increase (SGR, %/day) = 100 × [(ln ultimate body weight -ln primary body weight)/days].

Serum immunological test
Elisa kit (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, Jiangsu) is used to determine serum immunoglobulin M (IgM) levels and lysozyme (LZM) activity.

Reverse-transcriptase real-time PCR (RT-PCR)
At the end of the exposure test, the expression levels of immune-related genes in the spleen and kidney tissues were measured. The Trizol tool (Takara, Dalian, China) was used to extract total RNA from the spleen and kidney. Use RT-PCR cDNA tool (Takara, Dalian, China) to synthesize clean RNA with OD260/OD280 absorption ratio 1.8-2.0 as a template (Wang, et al., 2021). The primers were synthesized by Kumei Biotechnology Co., Ltd., Jilin. RT-PCR is used to quantify the expression levels of 7 immune response-related genes (IL-8, NF-KB P65, IkBα, IL-1β, TNF-α, IL-10, and TGF-β). Using housekeeping gene β-actin as an internal control (Yin, et al., 2018). Table 3 shows the sequence of the given primers used in this study. The RT-PCR reaction takes a total volume of 20 uL, including 1 uL cDNA, 2 uL each primer, 7 uL treated DEPC water and 10 uL SYBR Premix Ex Taq Master Mix. The thermal reaction conditions are as   follows: 95°C for 5 minutes, 95°C for 5 seconds, 60°C for 30 seconds, 72°C for 30 seconds, cycle 30 times. The RT-PCR reaction is repeated 3 times for each sample. Convert the data to Ct values after each reaction. The relative gene expression is determined by 2 −△△CT . (QIIME) tool (version 1.17) to analyze the raw readings. UPARSE is used to cluster OTU, with an analogy cutoff rate of 97%, and UCHIME is used to identify and remove chimeric sequences.Using the RDP classi er against the SILVA (SSU115) 16S rRNA database, with a con dence threshold of 70%, it is used to analyze the classi cation of each 16S rRNA gene sequence. SPSS 20.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Information was shown as mean ± standard deviation (S.D.) for every group. The whole test was made for three times. One-way exploration of variance (ANOVA) was adopted for the determination of the signi cance variations among the groups, which was followed by Tukey's various contrast experiment. The signi cance level was set at P < 0.05.

Statistical exploration
3 Results There was no signi cant difference between the control group and the Seenriched B. subtilis group, while the 0.03 mg/L Hg group was signi cantly reduced compared to the control group (P < 0.05). The growth performance of the se-rich B. subtilis plus Hg sh group of 0.03 mg/L was less than that of the control group and more than 0.03 mg/L Hg (P < 0.05).

Serum non-speci c immune responses
Hg is known to cause disturbances in the immune response. LZM and IgM levels for both treatment and control groups were determined (Fig. 1). When sh were exposed to Hg, LZM and IgM levels decreased. However, LZM and IgM levels increased after supplementation with Se-rich B. subtilis. The LZM and IgM extents of the Se-rich B. subtilis group grew greatly by comparing with the control group (P < 0.05; Fig. 1).

Immune-associated gene expression
Hg exposure greatly up-regulated the mRNA expression of IL-8, IL-1β, TNF-α and NF-kB P65 but downregulated the mRNA expression of IL-10, TGF-β and IkBα (Fig. 2). Nevertheless, the co-treatment with Hg and Se-enriched B. subtilis greatly increased the mRNA expression levels of IL-8, IL-1β, NF-kB P65 and TNF-α compared with the group exposed to Hg and not supplemented with dietary supplements. Downregulate the mRNA expression of IL-10, TGF-β and IkBα (P < 0.05). Compared with the control, IL-1β, TNFα, IL-8 and NF-kB P65 were up-regulated by exposure to mercury, while the consumption of Se-rich B. subtilis alleviated IL-1β, TNF-α, IL-8 and NF-kB P65 were up-regulated, and IL-10, TGF-β and IkBα were down-regulated (P < 0.05).
3.4 DNA extraction and 16S rRNA gene exploration

Statistical exploration of sequencing data
The dilution curve directly shows the rationality of the amount of sequencing data and indirectly shows the abundance of species in the sample. If the curve tends to be at, it indicates that the amount of sequencing data is gradually reasonable. In this study, after a month of feeding trials, we found that the end of the thinning curve (Fig. 3A) was attened. Therefore, we conclude that the amount of sequencing data is reasonable for our analysis.
For clarifying the effect of Hg in the intestinal ora of common carp, we performed PCoA analysis. The control group, the Se-rich B. subtilis group, the Se-rich B. subtilis plus Hg group, and the Hg group were combined and analyzed. The PCoA results showed that the microbial composition of the four groups of different diets was signi cantly different (Fig. 3B) (P < 0.05).
The chao1 index (the number of species included in the community) between the four groups found that the control group was relatively high, but the difference was not signi cant (Fig. 3C). The Shannon index (the diversity of gut microbes) found that there was no signi cant difference between different diets and groups (Fig. 3D). The results showed that the species richness and uniformity of each group of different diets did not change much.

Comparison at the genus levels
All sequences were identi ed at the genus level. We selected thirty data from the genus for analysis. The ve main genera in the control group were Verrucomicrobiaceae, Cetobacterium, Pseudorhodobacter. Gemmobacter and Aeromonas.The most common genera in the Hg group include Verrucomicrobiaceae, Gemmobacter, Cetobacterium, Aeromonas and Pseudomonas. After Hg exposure, the abundance of Aeromonas and Roseomonas increased signi cantly (P < 0.05). At the same time, after Hg exposure, the abundance of Pseudorhodobacter and Verrucomicrobiaceae was signi cantly reduced (P < 0.05). However, in the Hg treatment group, we found that the increase of Aeromonas and Rosemonas was reduced. The decrease of Pseudomonas and Verrucomicrobiaceae was suppressed (P < 0.05). In addition, we also found that Verrucomicrobiaceae in the Se-rich B. subtilis group also signi cantly decreased (Fig. 4A, B) (P < 0.05).

Comparison at the Species level
Similarly, we selected 30 data from the species for analysis. The most important species of intestinal microbes in the Hg group are Verrucomicrobiaceae_unclassi e, Aeromonas_sobria, Aeromonas hydrophila and Aeromonas spp. The most microbes in the control group were Cetobacterium_somerae, Gemmobacter_sp._yp3 and Pseudomonas_poae. Compared with the control group, after Hg exposure, the abundance of Cetobacterium_somerae, Pseudomonas_poae, Verrucomicrobiaceae_unclassi ed and Gemmobacter_sp._yp3 were signi cantly reduced, and Aeromonas sobria, Aeromonas hydrophila and Aeromonas hydrophila were signi cantly increased (P < 0.05). At the same time, in the Hg treatment group, it was found that the increase of Aeromonas sobria, Aeromonas hydrophila and Aeromonas spp was suppressed, while the decrease of Verrucomicrobiaceae_unclassi e was suppressed. Pseudomonas_poae and Cetobacterium_somerae increased signi cantly (Fig. 4C, D) (P < 0.05).

Discussion
Probiotics improve animal health and nutrition by improving feed value and enzymatic effects, and play a very important role in improving animal health, nutrition and activating immune response (Dawood et   . These results may indicate that anti-in ammatory cytokines effectively suppressed the pro-in ammatory immune response, which is consistent with the up-regulation of IL-10 observed in this study. In addition, the up-regulation of IL-10 in the liver may represent an aspect of the homeostatic mechanism that controls the Hg-induced in ammatory response. Gao et al. reported that the reduction of TGF-β will aggravate the in ammatory damage of liver tissue, but the lack of Se will inhibit the expression of TGF-β and promote the production of TNF-α, IL-1β and IL-6, which may cause carp liver tissue In ammation, but Se supplementation can prevent the decrease of TGF-β .The intake of Se-rich B. subtilis will not only increase the Se content in the body, but also B. subtilis will absorb Hg and alleviate the damage of the sh (Shang et al., 2021). In this study, there may be such a mechanism. Hg intake reduced the expression of TGF-β, while the Se-rich B. subtilis plus Hg group alleviated the decrease of TGF-β. The transcription factor NF-κB controls the expression of in ammatory cytokine genes (Taro and Shizuo, 2007). It controls the expression of pro-in ammatory genes and is also a key target for regulating in ammatory diseases (Xu et al., 2005;Yang et al., 2007). Study demonstrated that by catalyzing the degradation of IkBα, NF-κB can be activated by IKK (including IKKα, IKKβ and IKKγ), which plays an important role in regulating human pro-in ammatory cytokines (Jobin and Sartor, 2000;Bollrath and Greten, 2009). In this study, we found that the expression of IkBα in the liver and spleen decreased, and the corresponding NF-κB p65 expression increased, and this phenomenon was alleviated in the Se-rich B. subtilis treatment group. So there may be such a mechanism, Se-rich B. subtilis may be involved in the regulation of the IkBα/NF-κB signaling pathway. When the body consumes too much Hg, it leads to insu cient Se content in the body and triggers the in ammatory response and activates the IkBα/NF-κB signaling pathway. After feeding Se-rich B. subtilis to supplement Se, Se inhibits the upregulation of pro-in ammatory cytokines in the cells and promotes the expression of anti-in ammatory cytokines, thereby reducing the harm of Hg to the sh The intestine is a complex ecosystem, and the intestinal ora has an important role in this ecosystem.
Intestinal ora can assist the digestion and absorption of food and promote nutrient metabolism (Sommer and Backhed, 2013). Changes in the intestinal ora can lead to disorders of the body's normal physiological functions, leading to diseases (Nicholson et al., 2012). Through previous studies, we found that Hg signi cantly reduced the activity of enzymes such as CAT and GSH-PX and triggered in ammation (Shang et al., 2021). This experiment uses Illumina high-throughput sequencing technology to explain how the composition and diversity of carp intestinal microbial communities change under Hg exposure conditions, and provide a theoretical basis for sh intestinal health and normal human growth and development. In this study, the levels of Aeromonas sobria and Aeromonas hydrophila in the intestine of common carp after Hg treatment were higher than those in the control group. Many studies have shown that changes in the diversity of intestinal ora can cause diseases such as enteritis, in ammatory diseases and obesity. (Chassaing and Gewirtz, 2017;Beaz-Hidalgo and Figueras, 2013). Therefore, Hginduced changes in intestinal ora may affect the health of common carp.
In this study, our results indicate that Verrucomicrobiaceae, Cetobacterium, Pseudorhodobacter, Gemmobacter and Aeromonas are the most important bacterial groups in common carp. The main ora in the intestines after Hg exposure are Verrucomicrobiaceae, Gemmobacter, Cetobacterium, Aeromonas and Pseudomonas. Hg exposure caused changes in the intestinal ora, and it was found that the abundance of Aeromonas in the Hg treatment group was much higher than that of the control group. Aeromonas can colonize and infect the host, and can cause diseases such as sepsis and fungal infections. The extracellular products (hemolysin, lipase and protease) produced by Aeromonas can cause soft tissue, hepatobiliary system, respiratory system and arthritis disease (Elorza et al., 2020; Lian et al., 2020). In this study, Hg exposure increased the proportion of Aeromonas in the intestines of sh. However, in the Se-rich B. subtilis plus Hg group, we found that the abundance of Aeromonas was reduced, which indicates that feeding the Se-rich B. subtilis can change the intestinal microbes of the sh and reduce the abundance of Aeromonas. Aeromonas sobria can cause oxidation in sh bodies to change superoxide dismutase, glutathione peroxidase, and up-regulate immunoglobulins IgM and TNF-α (Harikrishnan et al., 2020).Aeromonas hydrophila can cause Catla catla immune response and increase IL-1β and TNF-α (Harikrishnan et al., 2021). In this study, it was found that Aeromonas sobria and Aeromonas hydrophila were signi cantly increased, which may be another cause of the disease. Hg induction will change the Aeromonas in the common carp intestine, and increase the Aeromonas sobria and Aeromonas hydrophila in the Aeromonas, which leads to an in ammatory response in the sh. Serich B. subtilis through the action of Se and the probiotic B. subtilis, regulates the IKBα/NF-κB signaling pathway and reduces the in ammatory response. The composition of the intestinal ora was detected by 16S rRNA gene sequencing, and this phenomenon may be that the Se-rich B. subtilis improved the intestinal ora and reduced the abundance of Aeromonas, thereby reducing the in ammatory response.

Conclusions
In conclusion, our results reported the effect of Se-rich B. subtilis on common carp exposed to mercury. This provides insightful insights for the Se-rich B. subtilis to reduce mercury poisoning in common carp. In this study, Se-rich B. subtilis alleviates mercury-induced effects on common carp growth performance and in ammation by changing the changes of intestinal microbes.

Declarations
Compliance with ethics requirements All experimental and animal handling procedures were conducted according to the research protocols approved by the Institutional Animal Care and Use Committee, Jilin Agricultural University, Jilin Province, China.

Declaration of competing interest
All authors declare that they have no con ict of interest and agree to publish this article to Fish Physiol Biochem. All the data in the article is actually available.