Microbiome and transcriptome analyses reveal the influence of calcined dolomite application on Eriocheir sinensis in a rice–crab co-culture system

Co-culture systems of rice and aquatic animals can contribute to the ecological intensification of agriculture by reducing nutrient loss and the need for N fertilizer application and by enhancing nutrient-use efficiency. However, the input of high-protein diets into paddy fields, to facilitate the growth of aquatic animals, has been found to increase N pollution and acidification of the soil. Although soil amendments have been widely used to ameliorate acidic soils, reduce N2O emissions, and improve agronomic production, the relationship between soil amendments and aquatic animal remains unclear. Therefore, this study investigated the effects of calcined dolomite (hereafter referred to as dolomite) as an acidic soil amendment and Ca–Mg supplement in rice–crab co-culture using Eriocheir sinensis crabs (Chinese mitten crabs). High-throughput sequencing was used to examine crab bacterial community composition and crab hepatopancreas biology. Although the water pH was significantly increased in the dolomite group, the number, composition, and diversity of bacteria identified in crab gut microbiome did not vary significantly between the dolomite and control groups. In the dolomite group, the probiotic agents Candidatus Hepatoplasma and Lactobacillus were highly abundant in the crab gut, and immune- and retinol metabolism-related genes were significantly upregulated in the crab hepatopancreas. Overall, dolomite application increased crab health and water pH. Dolomite is a low-cost amendment, with better stability, compared to other soil amendments, thus making it ideal for sustainable and clean rice–aquatic animal co-culture.

dolomite has been widely used in agriculture in China in the recent years 12,13 .Calcined dolomite is better than uncalcined dolomite in improving soil acidity 14 .After dolomite is calcined, the CaCO 3 and MgCO 3 in the ore are transformed into CaO and MgO, which react with soil water to produce Mg(OH) 2 and Ca(OH) 2 .In addition, Mg(OH) 2 and Ca(OH) 2 can gradually release calcium and magnesium while neutralizing soil acidity, providing calcium and magnesium nutrients for the paddy field.
Rice-crab co-culture is a rice-based ecological aquaculture system commonly used in China 15,16 .It improves soil nutrient levels and increases soil-to-rice nutrient translocation capacity 7 .However, economically important aquatic animals, such as fish, crayfish, and frogs, require high-protein diets for gonad development and weight gain 17,18 .Inevitably, the input of high-protein diets into paddy fields results in increased N pollution and acidification of the soil.Our previously published study indicated that high-protein diets can reduce soil pH and microbial diversity in rice-crab co-culture 8 .Thus, this study aimed to investigate the effects of dolomite, a type of limestone that is common and affordable in China, on paddy field water quality, the abundance and community composition of microbes in Eriocheir sinensis crabs (Chinese mitten crabs), and the physiological status of the crabs when applied in a rice-crab co-culture environment.We hypothesized that dolomite amendment could increase Ca 2+ and Mg 2+ supply and have desirable effects on the physiological status of crabs.Thus, by determining the efficiency of soil amendments in rice-crab co-culture systems, we aim to provide key solutions for improving food production while promoting environmental safety.
Before the paddy field experiment, 5 dolomite concentrations were tested for their impact on water: 0 kg/m 2 (A), 0.5 kg/m 2 (B), 1.5 kg/m 2 (C), 2.5 kg/m 2 (D), and 3.5 kg/m 2 (E).Each concentration had 3 repeats, and each repeat used a self-made container (w × l × h = 40 × 50 × 20 cm) filled with 20 L of tap water.Water quality parameters were monitored every 3 days for 1 month at the Chemical Analysis Laboratory of the Shenyang Agriculture University.Temperature and pH values were tested using a pen-like pH meter (pH-100A; Shanghai Lichen Co., Ltd., Shanghai, China), dissolved oxygen (DO) was measured using a portable dissolved oxygen meter (PB-607A; Shanghai Leici Co., Ltd, Shanghai, China), and the salinity was determined directly with a pen-like salinity meter (AR-8012A; Smart Sensor Co., Ltd., Guangdong, China).Alkalinity, hardness, ammonia-nitrogen (NH 3 -N), and nitrate-nitrogen (NO 2 -N) were followed standard procedures described in APHA 19 .

Field description and experimental design
The experiments were performed in 6 paddy fields (each 17 × 10 m) at Panjin Guanghe Crab Industry Co., Ltd., Panjin, Liaoning Province, China, during the 2021 rice-growing season (over 5 months, from 1st, May to 10th October).In total, 12,000 late megalopa-stage Eriocheir sinensis crabs (Chinese mitten crabs; average weight, 7.34 ± 1.32 mg) were obtained from Panjin Guanghe Crab Industry and randomly distributed within the 6 fields (2000 crabs/field).The two treatments in the field experiment were set as no dolomite treatment (CK) and dolomite treatment (DOL).Based on the results of our previous experiment, the dolomite quantity in the DOL group was set at 1.5 kg/m 2 .During the 5-month experiment, crabs were fed commercial diets (Well Hope Foods Co., Ltd., China, containing 40% protein) at 3% of their body weight per day.

Sample collection
To investigate the transcriptome and 16S rRNA changes in crabs induced by dolomite, 15 healthy crabs were randomly collected from each of the CK and DOL treatment groups on October 10, 2021.The hepatopancreas and intestines of each crab were extracted and placed in separate 2-mL RNase-free tubes.They were immediately frozen in liquid nitrogen until RNA and DNA extraction.All sampling was performed on a sterilized workbench.

DNA extraction and PCR amplification
Total genomic DNA was extracted from the crab intestine samples using an OMEGA Soil DNA kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer's instructions, and stored at − 20 °C for further analysis.The quantity and quality of the extracted DNA were evaluated using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.Briefly, the V3-V4 region of the bacterial 16S ribosomal RNA gene was amplified using PCR with the primers F 5′-ACT CCT ACG GGA GGC AGC A-3′ and R 5′-CGG ACT ACHVGGG TWT CTAAT-3′.The forward primer ITSF (5′-GGA AGT AAA AGT CGT AAC AAGG-3′) and reverse primer ITSR (5′-GCT GCG TTC TTC ATC GAT GC-3′) were used to amplify the fungal internal transcribed spacer (ITS) V1 regions 20 .Thermal cycling consisted of initial denaturation at 98 °C for 5 min, followed by 28 cycles of denaturation at 98 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, with a final extension of 5 min at 72 °C.Amplicons were extracted from 2% agarose gels and purified, using Vazyme VAHTS DNA Clean Beads (Vazyme, Nanjing, China) according to the manufacturer's instructions, and then quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA).The diversity and composition of the microbial communities in the soil and crab intestine samples were analyzed based on the raw sequencing data obtained using the Illumina MiSeq platform at Shanghai Personal Biotechnology Co., Ltd.(Shanghai, China), according to standard protocols.www.nature.com/scientificreports/

Bioinformatics and statistical analysis
After sequencing, raw FASTQ data were processed using QIIME2 and R v. 3.2.0,with slight modifications based on official tutorials (https:// docs.qiime2.org/ 2019.4/tutor ials/).According our previously published methods, amplicon sequencing variant (ASV)-level alpha diversity indexes, taxonomic composition and abundance, relative abundance, hierarchically clustered and heat map analysis was used to identify high-dimensional biomarker taxa with significantly different abundances among the DOL and CK groups 8 .Heat maps were generated using the heatmap.2function from the gplots R package, based on Spearman correlation matrices (without q-value thresholding).The raw reads have been deposited with the NCBI (BioProject numbers PRJNA788377).

Transcriptome sequencing of crab hepatopancreas
cDNA library preparation and transcriptome sequencing Total RNA was extracted from the crab hepatopancreas using Trizol Reagent (Invitrogen Life Technologies), after which, RNA concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific).mRNA was purified from total RNA that had been predigested at 37 °C for 1 h using DNase I and a Micropoly (A) PuristTM mRNA purification kit (Ambion, Austin, TX, USA).Equal amounts of mRNA samples from five crabs were pooled to generate one mixed sample, and three biological repetitions were conducted for both CK and DOL groups.A separate Illumina sequencing library was prepared using 10 µg of mRNA per sample.

De novo assembly and gene annotation
The original FASTQ data were filtered, and reads with connectors, lengths less than 50 bp, and an average sequence quality less than Q20 were removed.The high-quality sequences were spliced from scratch to obtain transcripts.These transcripts were clustered, and the longest transcripts were selected as unigenes 21

Statistical analyses
Significant differences at the 95% level were identified by one-way ANOVA followed by Tukey's multiple range test using SPSS software (v.17.0; IBM Corp., Armonk, NY, USA).To identify differentially expressed genes (DEGs) across samples or groups, edgeR (v.3.2) was used.The DEGs were then subjected to enrichment analysis of GO functions and KEGG pathways.The related analyses were performed using the "corrgram" package in the R environment (R v. 3.2.0).The data were plotted using Origin v. 9.0 (OriginLab, Northampton, MA, USA).

Ethical approval
Our study did not involve endangered or protected species.In China, the breeding and capture of Chinese mitten crabs (Eriocheir sinensis) and collecting rice plant in rice fields do not require specific permits.All efforts were made to minimize animal suffering and discomfort.The experimental protocol was approved by the Animal Ethics Committee of Shenyang Agriculture University (permit number 2021041601).This study is reported in accordance to ARRIVE guidelines (https:// arriv eguid elines.org/ arrive-guide lines).All animal and plant surveys were carried out in accordance with the approved guidelines of Shenyang Agriculture University Experimental Animal Management Committee.

Impact of dolomite on water quality
Figure S1 presents the water temperature, DO, pH, salinity, alkalinity, hardness, NH 3 -N content, and NO 2 -N content of water in the laboratory and field simulations of the rice field system treated with different quantities of dolomite.During the 30-day experiments, no significant differences in temperature, salinity, NH 3 -N, or NO 2 -N were found among the 5 groups (P > 0.05).The pH of dolomite-treated water in all groups was significantly higher than that of the control group (P < 0.05), increasing gradually with increased amounts of dolomite.DO was not conspicuously altered in the high-dolomite groups (2.5 kg/m 2 and 3.5 kg/m 2 ).However, with 0.5 kg/m 2 of dolomite, DO was significantly reduced compared to the other groups (P < 0.05).Alkalinity in the 3.5 kg/m 2 group increased significantly after 3 days compared to the other groups.

Impact of dolomite on crab intestine bacterial diversity
A total of 868,803 raw sequence reads were generated from the gut samples.After quality filtering and denoising, 786,111 clean reads were retained, with an average of 63,959 non-singleton reads per sample (Table 1).We used alpha diversity indices to estimate bacterial richness and diversity in the DOL and CK groups.Chao1, Shannon, and Simpson indices did not differ significantly between the DOL and CK groups (Fig. 1).This finding was supported by the rarefaction and rank abundance curves, which illustrate the richness and diversity of each sample, respectively (Figs.S2, S3).Beta diversity was evaluated using principal coordinates analysis and non-metric multidimensional scaling (Figs.S4, S5).The five most abundant gut bacterial phyla were Tenericutes, Proteobacteria, Firmicutes, Bacteroidetes, and TM7 (Fig. 2A).Dolomite had no effect on taxonomic composition at the phylum level but caused significant differences at the genus level.As shown in Fig. 2B The heat map showing correlations of phylum-level abundance within different time points revealed that TM7, Firmicutes, and Bacteroidetes were highly abundant in the control group (Fig. 3A).However, at the genus level, Lactobacillus, C. Hepatoplasma, Shigella, Paracoccus, and Rhodobacter were more abundant in the dolomite group than in controls (Fig. 3B).These patterns further demonstrate that probiotic bacteria populations were significantly higher in the dolomite group than in the control group.

Crab hepatopancreas MiSeq gene expression analyses
A summary of the transcriptomic sequences is presented in Table 2.In the NR database, 90.92% of unigenes were annotated (Table 3).Compared with the control group, the dolomite group showed 376 DEGs (P < 0.05), with 224 upregulated genes and 152 downregulated genes (Table S1).Heat map clustering results also indicated that most DEGs could be divided into two classes (Fig. 4).
The results of GO analysis showed that the top three pathways under the "cellular component" category were "metal ion binding", "cation binding", and "metallopeptidase activity" (Figure S4).A directed acyclic graph of the "cellular component" category was constructed (Fig. 5), which indicated that metal binding-related pathways were enriched.Using KEGG enrichment analysis, the most significantly enriched metabolism-related pathway was found to be "fructose and mannose metabolism" followed by "retinol metabolism", which was predominantly enriched in the upregulated DEGs (Figure S6).
After the uncharacterized genes among the 376 DEGs were eliminated between the control and dolomite groups, we obtained 157 upregulated genes (Table S2) and 118 downregulated genes (Table S3).The DEGs related to immune function, including glutathione peroxidase, catalase, cytochrome P450, cryptocyanin, heat shock protein (HSP) 70, and HSP 90, were significantly upregulated in the dolomite group.Moreover, four retinol dehydrogenase (RDH) genes and three ecdysteroid-related genes were also upregulated.

Discussion
Rice-fishery co-culture can reduce the need for chemical fertilizers, enhance land productivity, and maintain soil fertility 16,22,23 .Although the excreta of aquatic animals provides sufficient nutrients for crop growth, our previous research showed that the high-protein diets of these animals negatively affect paddy soil and cause acidification 8 .Moreover, the main cause of soil acidification in China is the accumulation of exogenous protons and the loss of basic ions such as Ca 2+ and Mg 2+ caused by the excessive application of N fertilizer 24 .
Lime is commonly applied to soil to correct soil acidity 25 .Available liming materials include limestone (CaCO 3 ), quick lime (CaO), slaked lime [Ca(OH) 2 ], and dolomite [CaMg(CO 3 ) 2 ], which vary in their ability for neutralizing acidified soil.Among these, dolomite, which is rich in Ca and Mg, has been demonstrated to be a better acidic soil conditioner than is lime; moreover, it is cost effective compared to lime 26,27 .After dolomite is calcined, its Ca 2+ and Mg 2+ content is gradually released, and the release rate steadily increases over time, which is in line with the growth rhythm 28 .In the present study, calcined dolomite addition increased water pH slowly but steadily, maintaining a consistent pH of 8.0-8.3 for 30 days; it was not affected by other water chemical indices.With the same application time and amount, quick lime and hydrated lime treatments are more efficient than is dolomite in increasing pH 29 .Nevertheless, based on the results of soil and water pH indices, the application of calcined dolomite tends to be more reliable and economical in terms of effectiveness and stability in minimizing the soil acidity problem in rice-crab co-culture.Moreover, dolomite contains Mg, which is essential for crustacean growth and development 30 .
Chinese mitten crabs are highly valued and are thus an important part of the rice-crab co-culture system.Therefore, it is necessary to understand their physiological status within this system, especially under dolomite application.The number of bacteria identified in the gut microbiome barely varied between the dolomite and www.nature.com/scientificreports/control groups, with the results of alpha and beta diversity analysis showing insignificant differences in the composition of the gut bacterial communities.The probiotic bacterium C. Hepatoplasma was more abundant in the dolomite group than in the control group, but the converse was true for the acidophilic bacteria Lactococcus and Bacteroides.C. Hepatoplasma in the intestines of juvenile Panulirus ornatus and mitten crabs may play a symbiotic role in nutrient absorption 31,32 .Moreover, Lactobacillus, which has potential probiotic properties in crab 33 , was also more abundant in the dolomite group than in the control group.These findings suggest that the microbial communities in the intestines of crabs have an overall high average similarity among the dolomite and control groups, as reflected by the biodiversity and richness of intestinal microbiota and the most dominant community members at the phylum level.Notably, Notably, in addition to the findings mentioned earlier, we observed significant upregulation of immune function-related genes, such as glutathione peroxidase, catalase, cytochrome P450, cryptocyanin, HSP 70, and HSP 90, in the hepatopancreas of crabs in the DOL groups compared to the control group.This finding aligns with previous studies in crustaceans, where immune genes are known to be stimulated in response to various stimuli.Conversely, under acidic conditions, these immune genes are typically downregulated 34 .This suggests that maintaining an adequate level of magnesium (Mg) in the environment may play a crucial role in preventing rapid bacterial proliferation and reaching infectious levels in the giant freshwater prawn (Macrobrachium rosenbergii) 35 .Over 500 mg/kg MgO supplementation resulted in the best survival and growth of M. rosenbergii post-larvae 36 .In the present study, calcined dolomite had an MgO content of 35.70%, which may also promote growth in mitten crabs.
Based on KEGG pathway analysis, the retinol metabolic pathway and related genes were significantly upregulated in dolomite crabs compared to controls.Retinol affects development through a series of enzymes that control a two-step metabolic pathway.In this metabolic pathway, retinol is first oxidized to retinaldehyde, and retinaldehyde is then oxidized to retinoic acid.Retinoic acid acts as a ligand for retinoic acid signaling activities to directly regulate gene expression.RDH is an important enzyme in retinol metabolism and can protect cells from non-aldehyde-related toxicity.In the present study, the expression of RDH-related genes was significantly upregulated, indicating that dolomite induced retinol metabolism.Lower pH and acidified environments can have a deleterious effect on the overall homeostasis of retinoic acid in cells 37 .Therefore, dolomite application helps to increase water and soil pH and eventually has a positive impact on crab health in rice-crab co-culture systems. https://doi.org/10.1038/s41598-023-39099-1

Figure 1 .
Figure 1.Alpha diversity of bacterial groups in crab gut from the dolomite and control groups in a rice-crab co-culture system.

Figure 2 .
Figure 2. Relative abundance of (A) and genus (B) in crab gut from the dolomite and control groups in a rice-crab co-culture system.

Figure 3 .
Figure 3. Hierarchical clustering analysis of phyla (A) and genus (B) in crab gut from the dolomite (L) and control groups (K) in a rice-crab co-culture system.

Figure 5 .
Figure 5. GO annotation of assembled genes.

Table 1 .
Sequences data for bacterial of crab guts in crab rice coculture system collected in July 2021 and October 2021.

Table 2 .
Summary of the transcriptomic sequences in control group and dolomites group.
SampleClean Reads No Clean Data (bp) Clean Reads % Clean Data % NCBI Accession Number

Table 3 .
Annotation in database.