Introduction

With the rapid development of global aquaculture, achieving sustainable development and environmental protection while pursuing high yields has become a critical issue that needs urgent attention. Traditional monoculture systems, characterized by high stocking densities, have led to increased economic benefits but often come with a series of challenges, including water quality deterioration, frequent disease outbreaks, and environmental pollution. In particular, the accumulation and decomposition of uneaten feed and fecal matter in the water can elevate levels of harmful substances such as ammonia nitrogen and nitrite nitrogen, thereby threatening the health of cultured species and compromising food safety.1,2 To address these challenges, polyculture systems have emerged as an eco-friendly aquaculture method and have garnered widespread attention and application.

Polyculture systems involve the culture of different aquatic species within the same water body, utilizing the ecological complementarity among them. This approach can effectively enhance biodiversity within the farming system, promote the recycling of 、 nutrients, and reduce the accumulation of harmful substances and eutrophication in the water, thereby improving water quality and mitigating environmental pollution risks.3,4 Polyculture enhances ecosystem stability and sustainability by optimizing the spatial structure and hierarchy of the biological community through the occupation of various ecological and trophic niches with suitable species. Integrating finfish into shrimp ponds can influence the structure of phytoplankton communities and the composition of particulate matter, thereby impacting the system’s material cycling.5 Research by Nahon et al.6 demonstrated that polyculture can ensure that various ecological and trophic niches in shrimp ponds are occupied by suitable species, enhancing the spatial structure and hierarchy of the biological community, optimizing the ecological structure, and promoting biodiversity within the pond. Xiong et al.7 showed that Penaeus monodon (P. monodon, black tiger shrimp) can be polycultured with fish or crabs, primarily herbivorous fish. The polyculture environment was found to be significantly superior to monoculture in terms of pond ecology, facilitating healthier growth of both fish and shrimp. Zhao et al.8 reported that incorporating an appropriate density of Sinonovacula constricta (razor clam) into a polyculture system of Scylla paramamosain (mud crab) and P. monodon notably enhanced both farming efficiency and the utilization of nitrogen and phosphorus. Additionally, Xiaoqi et al.9 found that a polyculture system of P. monodon, Acanthopagrus latus (yellowfin seabream), and Siganus fuscescens (rabbitfish) effectively reduced the accumulation of ammonia nitrogen and nitrite nitrogen in shrimp ponds, promoting material cycling and yielding higher economic benefits.

In high-density aquaculture systems such as the high-level pond systems widely used in Hainan province of China and other P. monodon farming regions, the practice involves high stocking densities, high feed input, and high economic returns. However, these systems also face significant challenges, including excessive pond loading, deterioration of water quality during the middle and late stages of farming, and severe ecological damage to surrounding water bodies due to the discharge of effluent. The accumulation and decomposition of uneaten feed and aquaculture waste lead to elevated levels of harmful nitrogen compounds such as ammonia nitrogen and nitrite nitrogen, as well as outbreaks of harmful cyanobacteria like Microcystis and Oscillatoria. These factors pose a threat to the health of cultured species and food safety, representing a major challenge in the aquaculture of various species, including P. monodon.

Given the pressing need for sustainable solutions that support both environmental health and efficient production in high-density aquaculture systems, attention has increasingly focused on microbial preparations as effective tools. These biological agents offer the potential to remediate water quality problems while simultaneously promoting animal growth, thereby providing a synergistic approach to addressing the limitations of intensive aquaculture practices.10,11 For instance, the use of Bacillus preparations in water bodies has been shown to optimize the bacterial community composition in pond sediments and increase the number of aerobic bacteria, thereby enhancing the ecological functions of indigenous microbial communities and significantly improving the survival rate, body weight, and yield of cultured shrimp.12 Furthermore, studies by Xin et al.13 and Yang et al.14 indicated that Rhodopseudomonas palustris strain PS1 effectively reduced chemical oxygen demand (COD), ammonia nitrogen, nitrate nitrogen, and phosphate in intensive shrimp farming effluent, and suggested that different strains of Rhodopseudomonas palustris might exhibit varying efficacies in water purification.

Based on the above background, this study aims to address two critical issues in current aquaculture research. First, while polyculture systems have been proven to improve water quality and enhance production efficiency, there is limited data on the optimal species ratios in specific polyculture systems, such as the combination of Mugil cephalus (sea mullet), Scylla serrata (mangrove crab), and P. monodon. Determining the optimal ratios is key to maximizing both ecological and economic benefits. Second, although microbial preparations have shown promising potential in alleviating water quality issues and improving animal health in high-density aquaculture, their potential synergistic effects with polyculture systems remain underexplored. To fill these gaps, this study is designed to evaluate three different species ratio combinations of sea mullet, mangrove crab, and shrimp, along with a control group, to compare key water quality indicators and production variables. Furthermore, by applying different micro-ecological preparation strategies to both the optimal ratio group and the control group, this study aims to develop a more scientific and sustainable polyculture strategy. The results will provide critical data support for optimizing species ratios in polyculture systems and offer insights into integrating microbial preparations to enhance aquaculture sustainability and productivity.

Materials and Methods

Ethics statement

The P. monodon is not classified as an endangered or protected species. Therefore, in China, no specific permissions are required for conducting experiments involving this species.

Experimental setup and management for mixed culture study

The experiment was conducted at the Hainan Xinbang Seed Industry Co., Ltd. experimental base using 12 concrete ponds, which included 1 control group and 3 experimental groups, with each group consisting of 3 experimental ponds. Each pond had an area of 24 m² and a water depth of 1.2 m, with the experimental period lasting 60 days. During the experiment, fresh seawater was intermittently added to compensate for water level decreases due to evaporation and other factors. Oxygen was supplied to the ponds using a Roots blower connected to a nano-tube microporous aeration disc, ensuring that the dissolved oxygen level in the water did not fall below 5.0 mg/L. Throughout the experiment, appropriate feed for the shrimp was administered according to normal farming practices, while no additional feed was provided for the Sea mullet and mangrove crab. The specifications and treatments for the mixed culture of shrimp with Sea mullet and mangrove crab are shown in Table 1.

Table 1.Specifications and treatments for the mixed culture of Shrimp with Sea mullet and mangrove crab (Mean ± SD)
Experimental Number Cultured Species Specification
(g/tails, g/individuals)		 Stocking Density
(tails/m² or individuals/m²)		 Stocking Quantity (tails, individuals)
Control Group shrimp 0.51±0.02 g 75.00 1800
Group 1 shrimp 0.51±0.02 g 75.00 1800
Sea mullet 102.47±4.31 g 0.45 10
mangrove crab 5.08±0.19 g 0.45 10
Group 2 shrimp 0.51±0.02 g 75.00 1800
Sea mullet 102.47±4.31 g 0.45 10
mangrove crab 5.08±0.19 g 0.90 20
Group 3 shrimp 0.51±0.02 g 75.00 1800
Sea mullet 102.47±4.31 g 0.90 20
mangrove crab 5.08±0.19 g 0.45 10

Water quality monitoring and yield assessment

During the experiment, water samples were taken every two weeks, with one sample collected from each experimental pond. Water temperature, dissolved oxygen, salinity, and pH were measured on-site at 10 AM on the sampling day using a multiparameter water quality analyzer. The analysis of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, soluble reactive phosphorus, total nitrogen, total phosphorus, and COD in the water samples was conducted according to the Chinese national standard GB/T 12763.4-2007.

At the end of the experiment, the yield and survival rates of shrimp, Sea mullet, and mangrove crab were assessed. The survival rate of shrimp was calculated using the following formula:

Survival Rate (%) = W1/（W2/50）/1800×100

where W1 is the total weight of shrimp at the end of the experiment, and W2 is the total weight of a random sample of 50 shrimp.

Optimization of microecological regulation in multi-trophic level aquaculture ponds

The experiment was conducted in three concrete ponds at the experimental base of Hainan Xinbang Seed Industry Co., Ltd., with each pond having the same specifications. The setup included one control pond and two experimental ponds. Based on the optimal co-culture configuration established from previous experiments, fish, crabs, and shrimp were stocked at densities of 0.90 individuals/m², 0.90 crabs/m², and 75 shrimp/m², respectively. Prior to the start of the experiment, the water in the ponds was disinfected using bleaching powder at a concentration of 5 ppm, soaked for 24 hours, and then aerated thoroughly for an additional 24 hours to remove any residual disinfectant. During the experiment, no microecological agents were added to the control pond, while specific microbial agents were administered to experimental ponds 1 and 2 as detailed in Table 2. The experiment lasted for a total of 30 days. At the end of the experiment, water samples were collected from each pond to measure key water quality indicators.

Table 2.Microbial agent application in each concrete pond
Group Bacillus subtilis Bacillus licheniformis Saccharomyces cerevisiae
Application Rate (CFU/m³) Application Frequency (days/cycle) Application Rate (CFU/m³) Application Frequency (days/cycle) Application Rate (CFU/m³) Application Frequency (days/cycle)
Control Group 0 - 0 - 0 -
Group 1 2.5×108 10 1.0×108 10 1.0×108 10
Group 2 5.0×108 10 1.0×108 10 2.0×108 10
Group 3 5.0×108 15 1.0×108 15 2.0×108 15

Data processing and analysis

For data processing and analysis, ANOVA (Analysis of Variance) was conducted using GraphPad software to evaluate the effects of different treatments on water quality indicators, survival rates and yields. Significant differences were determined at a p-value of less than 0.05. All graphs were generated using Microsoft Excel to visualize the data.

Results

Water quality indicator analysis in mixed culture experiment

The results of the water quality assessments indicated that with the increase of cultivation time, all the tested indices showed a continuous upward trend. Meanwhile, compared to the control group, the total nitrogen, total phosphorus, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, active phosphorus, and COD in the experimental groups all showed different degrees of decrease at various sampling points. In most cases, this reduction trend amplified over time (Fig. 1). Moreover, comparative analysis revealed that most water quality indices in Group 3 were significantly lower than those in the control and the other two experimental groups (Table 3). Specifically, at the final sampling point, the reductions in total nitrogen, total phosphorus, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, active phosphorus, and COD reached their maxima, at 60.09%, 38.13%, 57.14%, 55.88%, 52.47%, 43.02%, and 32.54%, respectively. Additionally, correlation analysis demonstrated a significant negative correlation between the Sea mullet stocking density and all water quality indices (P <0.05) (Table 4); whereas the association of mangrove crab was generally negative with these indices, it was not significant except for total phosphorus (P >0.05). Furthermore, Sea mullet showed a higher level of correlation with all measured water indices compared to mangrove crab (Table 4).

Figure 1
Figure 1.Trends in nitrogen, phosphorus, and COD levels in different polyculture ponds (mean ± SD). T1 represents experimental group 1, T2 represents experimental group 2, and T3 represents experimental group 3.
Table 3.Changes in nitrogen, phosphorus, and COD in different polyculture ponds (Mean ± SD)
Testing Indicators Sampling Date
Group		 2020.10.05 2020.10.20 2020.11.04 2020.11.19
Total Nitrogen (mg/L) Control Group 0.66±0.0499a 1.17±0.0408a 1.68±0.045a 4.31±0.0245a
Group 1 0.51±0.0294b 0.98±0.0287b 1.47±0.0408b 3.08±0.034b
Group 2 0.45±0.0249c 0.89±0.0205c 1.39±0.0125bc 2.71±0.045c
Group 3 0.43±0.0216c 0.79±0.0125d 1.18±0.0125c 1.72±0.0741d
Total Phosphorus (mg/L) Control Group 0.24±0.017a 0.36±0.0143a 0.57±0.0094a 0.7±0.0062a
Group 1 0.22±0.0155b 0.28±0.0103b 0.47±0.0143a 0.59±0.0147b
Group 2 0.21±0.0085c 0.27±0.0085b 0.36±0.0103b 0.51±0.0143c
Group 3 0.2±0.0062c 0.24±0.0082c 0.31±0.0082c 0.43±0.0094d
Ammonia Nitrogen (mg/L) Control Group 0.11±0.0095a 0.13±0.0087a 0.2±0.0051a 0.45±0.0129a
Group 1 0.11±0.0057ab 0.13±0.0019a 0.18±0.005b 0.3±0.0131b
Group 2 0.11±0.0012a 0.12±0.0023b 0.17±0.0074b 0.27±0.0053c
Group 3 0.1±0.0046b 0.11±0.0065c 0.14±0.003c 0.19±0.0017d
Nitrite Nitrogen (mg/L) Control Group 0.08±0.0036a 0.15±0.0034a 0.2±0.0038a 0.34±0.0096a
Group 1 0.08±0.0042b 0.13±0.0029b 0.18±0.0039b 0.27±0.0093b
Group 2 0.07±0.0009c 0.1±0.0059c 0.17±0.0017b 0.24±0.0154c
Group 3 0.07±0.0038d 0.11±0.0036c 0.13±0.003c 0.15±0.003d
Nitrate nitrogen (mg/L) Control Group 0.03±0.0028a 0.06±0.0027a 0.1±0.0035a 0.22±0.0017a
Group 1 0.02±0.0025b 0.05±0.002b 0.08±0.003b 0.17±0.0024b
Group 2 0.02±0.0017c 0.05±0.0017c 0.08±0.0005c 0.17±0.0028b
Group 3 0.02±0.0013c 0.04±0.0014d 0.06±0.0009d 0.1±0.0057c
Active Phosphorus (mg/L) Control Group 0.05±0.0046a 0.07±0.0032ab 0.1±0.0059a 0.17±0.0074a
Group 1 0.05±0.0019b 0.07±0.003a 0.09±0.0023b 0.15±0.0069b
Group 2 0.05±0.0011bc 0.06±0.0008b 0.08±0.0012c 0.14±0.0076c
Group 3 0.05±0.0031b 0.06±0.0012c 0.07±0.001d 0.1±0.0034d
COD
(mg/L)		 Control Group 2.67±0.0785a 2.75±0.0205a 4.23±0.0408a 4.61±0.1087a
Group 1 2.57±0.0694a 2.87±0.0287b 3.67±0.0556b 4.03±0.0712b
Group 2 2.49±0.0205b 2.57±0.0616c 3.58±0.0535b 3.86±0.0909c
Group 3 2.47±0.0464c 2.66±0.0386c 2.78±0.0330c 3.11±0.0510d

Note: Different letters indicate significant differences (P < 0.05) for the same indicator between sampling times.

Table 4.Correlation analysis of water quality indicators in shrimp farms with Sea mullet and mangrove crab introduction
Water quality indicators Parameter Mugil cephalus Scylla serrata
Total nitrogen Correlation -0.31 -0.20
Coefficient 0.01 0.08
Total phosphorus Correlation -0.40 -0.30
Coefficient 0.00 0.02
Ammonia nitrogen Correlation -0.34 -0.22
Coefficient 0.01 0.07
Nitrite nitrogen Correlation -0.38 -0.22
Coefficient 0.00 0.06
Nitrate nitrogen Correlation -0.30 -0.17
Coefficient 0.02 0.13
Active phosphorus Correlation -0.27 -0.14
Coefficient 0.03 0.18
COD Correlation -0.42 -0.23
Coefficient 0.00 0.06

Comparative analysis of shrimp growth and survival rates in different experimental groups

The results of the aquaculture study are presented in Table 5. At the end of the experiment, the average body weight of shrimp in Group 1 showed an increase compared to the control group, though the difference was not statistically significant (P > 0.05). In contrast, the average body weights of shrimp in Groups 2, 3, and 4 were significantly higher than the control group (P < 0.05), with increases of 0.18 g/individual, 0.26 g/individual, and 0.47 g/individual, respectively. The total yields were 11.95 kg, 12.52 kg, 11.89 kg, and 12.69 kg for the control group and Groups 1, 2, and 3, respectively. Analysis of variance indicated significant differences among the groups (P < 0.05).

Table 5.Comparison of indicators across experimental groups in aquaculture (Mean ± SD)
Indicators
Groud		 Weight (g) Number of Survivors (tails) Survival Rate (%) Yield (kg)
Control Group 7.33±0.07a 1630.38±11.67a 90.58 11.95±0.04a
Group 1 7.51±0.05b 1666.74±8.24b 92.60 12.52±0.19b
Group 2 7.59±0.02b 1546.20±8.53c 85.90 11.89±0.29a
Group 3 7.80±0.03c 1627.20±14.96a 90.40 12.69±0.13b

Note: Different letters indicate significant differences (P < 0.05) for the same indicator between sampling times.

Survival rates varied among the groups, with Group 1 exhibiting the highest survival rate at 92.60%, which was significantly higher than the control group (P < 0.05). The increase in survival rate for Group 1 was 2.02% compared to the control group. Conversely, Group 2 showed a significant decrease in survival rate compared to the control group (P < 0.05), with a decrease of 4.68%. The survival rates for Sea mullet in Groups 1, 2, and 3 were 83.33%, 80.00%, and 85.00%, respectively, while the survival rates for mangrove crabs were 76.67%, 71.67%, and 73.33%, respectively. However, the differences in survival rates among these groups were not significant. Further analysis from Table 5 indicated that, despite the survival rate in group 3 showing no significant changes, the average body weight of shrimp in this group was significantly higher than that of all other groups (P < 0.05). Moreover, group 3 yielded the highest final production among all groups. This final yield was also significantly higher than that of the control group and group 2 (P < 0.05), with the exception of group 1.

Analysis of water quality parameters in the multi-trophic aquaculture experiment

After the multi-trophic aquaculture experiment concluded, the water quality parameters of each pond were analyzed (Fig. 2). The average total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (COD) in the control pond were 6.3023 mg/L, 0.3456 mg/L, and 6.98 mg/L, respectively. In contrast, the TN, TP, and COD concentrations in Experimental Pond 1 were 4.1323 mg/L, 0.1467 mg/L, and 5.73 mg/L; Experimental Pond 2 showed significant reductions to 1.1423 mg/L, 0.0738 mg/L, and 3.87 mg/L; and Experimental Pond 3 had values of 1.3010 mg/L, 0.0784 mg/L, and 4.64 mg/L.

Figure 2
Figure 2.Effect of different micro-ecological preparations on major water quality indicators (mean ± SD). Different letters indicate significant differences (P < 0.05) in the same indicator among different experimental ponds.

In terms of water quality improvement, Experimental Pond 2 exhibited the most remarkable performance, with TP reduced by 79% and COD by 44%, significantly surpassing the other experimental ponds. Compared to Experimental Pond 1, TN in Experimental Pond 2 decreased by 72.34%, TP by 49.68%, and COD by 32.45%. Compared to Experimental Pond 3, TN, TP, and COD in Experimental Pond 2 were reduced by 12.20%, 5.87%, and 16.59%, respectively.

Discussion

In recent years, extensive research has been conducted on water quality regulation in shrimp farming and the polyculture of shrimp with various fish species.15,16 Studies have shown that filter-feeding and omnivorous fish species can enhance the utilization of water space and feed resources, as well as improve the aquatic environment.17 However, previous studies have primarily focused on Litopenaeus vannamei (Pacific white shrimp) and often involved single-species co-cultures, leaving a gap in understanding the dynamics of multi-species systems involving P. monodon. This study aims to address this gap by exploring the complementary ecological niches among species through the co-culture of Sea mullet and mangrove crabs, while also investigating the regulatory strategies of different microecological agent formulations in the fish-shrimp-crab polyculture system.

The Sea mullet is a euryhaline, omnivorous fish species that primarily feeds on diatoms and organic detritus. Its feeding habits help reduce sediment accumulation at the pond bottom and inhibit the growth of pathogenic bacteria such as Vibrio.18 Mangrove crabs, as benthic omnivores, play a role in controlling fouling organisms like sea snails and utilize dead shrimp as feed, contributing to pond cleanliness.19 These ecological roles highlight their potential to improve water quality and maintain ecological balance in shrimp farming systems. The water quality assessment results from the co-culture experiment revealed that all measured parameters exhibited an increasing trend over time, aligning with the natural nutrient accumulation process during the aquaculture cycle. Compared to the control group, the co-culture of Sea mullet and mangrove crabs significantly reduced the levels of total nitrogen (TN), total phosphorus (TP), ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, active phosphorus, and chemical oxygen demand (COD). Among the experimental groups, Group 3 (0.90 Sea mullet individuals/m², 0.45 mangrove crabs/m², and 75 shrimp/m²) demonstrated the most substantial reductions in these parameters, ranging from 32.54% to 60.09%. These findings suggest that the co-culture of Sea mullet and mangrove crabs effectively improved water quality. Furthermore, correlation analysis highlighted a significant negative relationship between Sea mullet stocking density and most water quality parameters, underscoring its pivotal role in water quality regulation within the system. In the co-culture system of Litopenaeus vannamei and Sea mullet, water quality parameters such as nitrite nitrogen, nitrate nitrogen, ammonia nitrogen, chemical oxygen demand, total phosphorus, and total nitrogen were significantly lower compared to the monoculture ponds of shrimp. These findings are similar to the results observed in this experiment.17

The analysis of shrimp growth and survival rates indicated that, although the survival rate of shrimp in Group 3 was slightly lower than that in Group 1, this group achieved the highest weight gain and final yield of shrimp. This suggests that the combination of Sea mullet and mangrove crabs in Group 3 was particularly advantageous for shrimp growth. In contrast, Group 2 showed a significant decline in shrimp survival, which may be associated with the highest stocking density of mangrove crabs. This implies that, under conditions of insufficient feed supply, mangrove crabs may prey more frequently on the shrimp. Therefore, in practical co-culture systems, it is crucial to strictly regulate the stocking density and size of mangrove crabs to minimize their negative impact on the primary cultured species.

To further optimize water quality management, this study introduced microecological agents as an effective means to regulate pond water quality and biological health. Microecological agents, as a highly effective product for regulating pond water quality and biological health, can significantly improve the water quality status of aquaculture systems and promote animal growth and development.20 For example, Bacillus subtilis enhances water quality in aquaculture by inhibiting pathogenic bacteria and decomposing organic matter, thus reducing disease risk and improving overall pond health.21 Bacillus licheniformis helps maintain clear water and reduces harmful nitrogenous compounds by breaking down leftover feed and organic waste through its enzymatic activity.22 Saccharomyces cerevisiae, through its metabolic activities and fermentation products, can improve the structure of the microbial community in the water body, thereby promoting the decomposition of harmful substances and optimizing the water quality environment.23 Based on the aforementioned optimal co-culture scheme, this study introduced Bacillus subtilis, Bacillus licheniformis, and Saccharomyces cerevisiae into the water quality regulation of P. monodon farming, and investigated the regulatory strategies of its different configurations. The water quality analysis results of the multi-trophic aquaculture experiment indicate that pond 2 demonstrated the most significant improvement in water quality. Compared to the control pond, pond 2 saw reductions of 81.88% in total nitrogen (TN), 78.65% in total phosphorus (TP), and 44.54% in chemical oxygen demand (COD). Additionally, when compared with the other ponds, pond 2 also showed a clear advantage. For instance, compared to pond 1, TN in pond 2 decreased by 72.34%, TP by 49.68%, and COD by 32.45%. Although pond 1 showed some improvement in water quality, it was still outperformed by pond 2 in all parameters. Similarly, compared to pond 3, TN in pond 2 decreased by 12.20%, TP by 5.87%, and COD by 16.59%. Despite pond 3 showing significant improvements in the parameters, it still did not match the performance of pond 2. The optimal application protocol for these agents was determined to be: Bacillus subtilis at 5.0×10^8 CFU/m³, Bacillus licheniformis at 1.0×10^8 CFU/m³, and Saccharomyces cerevisiae at 2.0×10^8 CFU/m³, applied every 10 days. These findings indicate that the water quality regulation strategies employed in pond 2 effectively reduced the accumulation of nutrients and organic matter, thereby improving the shrimp farming environment and overall pond health. This further confirms the significant role of optimized stocking densities and species combinations in water quality control, enhancing the sustainability of shrimp farming operations.

These findings collectively demonstrate that introducing sea mullet and mangrove crabs into Penaeus monodon culture ponds can create a balanced ecosystem, thereby improving water quality and enhancing shrimp productivity. The optimal co-culture scheme identified in this study (0.90 sea mullet/m², 0.45 mangrove crabs/m², and 75 shrimp/m²) demonstrated the best performance in water quality regulation and shrimp growth. Additionally, the application of microecological agents significantly improved the farming environment, supporting the sustainable development of shrimp aquaculture. This study provides valuable insights into the advantages of multi-species co-culture systems and the application of microecological agents in aquaculture. Future research could further optimize species ratios and explore the mechanisms by which these agents improve water quality and shrimp health, thereby advancing the sustainable development of aquaculture systems.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (No. 2022YFD2400503), the Central Public-interest Scientific Institution Basal Research Fund, CAFS (NO. 2024XT02), the Central Public-interest Scientific Institution Basal Research Fund, CAFS (No. 2023TD21), the Guangdong Basic and Applied Basic Research Foundation (No. 2023A1515030022).

Authors’ Contribution

Methodology: Changhong Lin (Lead). Formal Analysis: Changhong Lin (Equal), Bo Zhang (Equal), Lihua Qiu (Equal). Investigation: Changhong Lin (Equal), Bo Zhang (Equal), Lihua Qiu (Equal). Writing – original draft: Changhong Lin (Lead). Funding acquisition: Lihua Qiu (Equal), Chao Zhao (Equal). Supervision: Bo Zhang (Lead). Writing – review & editing: Lulu Yan (Equal), Chao Zhao (Equal). Conceptualization: Chao Zhao (Lead).

Competing of Interest – COPE

No competing interests were disclosed.

Ethical Conduct Approval – IACUC

Research involving animals

The P. monodon is not classified as an endangered or protected species. Therefore, in China, no specific permissions are required for conducting experiments involving this species.

All authors and institutions have confirmed this manuscript for publication.

Data Availability Statement

The data that has been used is confidential.