Dynamics of Phytoplankton Communities and Environmental Drivers in Chinese Mitten Crab Aquaculture Ponds: Highlighting the Need for Cyanobacteria Control

: Pond culture is the primary method for cultivating Chinese mitten crab ( Eriocheir sinensis ), with phytoplankton significantly influencing their growth. Green algae benefit crab growth by serving as supplementary food, while cyanobacteria, particularly during blooms, hinder it and pose health risks. Environmental changes in nutrient levels, temperature, and light significantly affect phytoplankton communities in ponds, impacting both ecosystem stability and crab growth. However, there is a limited understanding regarding the patterns of phytoplankton changes within adult Chinese mitten crab culture ponds. This study conducted monthly collection and analysis of phytoplankton throughout the culture cycle in typical adult Chinese mitten crab culture ponds, concurrently measuring physical and chemical water parameters to explore the correlation between phytoplankton changes and environmental factors. The results revealed distinct seasonal variations in phytoplankton composition. Chlorophyta and Bacillariophyta, such as Chlorella , Pediastrum , and Cocconeis , predominated in spring, while Chlorophyta and cyanobacteria, such as Volvox , Anabaena , and Microcystis , dominated in summer, and cyanobacteria and Bacillariophyta, such as Microcystis , Dolichospermum , and Cocconeis , prevailed in autumn. Total phytoplankton density consistently increased throughout the culture period. Microcystis constituted the majority of cyanobacteria biomass throughout most months. Although the total phytoplankton biomass fluctuated, cyanobacteria biomass consistently rose each month, reaching a peak of 61.7 mg/L in October. Water temperature and pH emerged as the primary environmental drivers influencing changes in phytoplankton community structure. Cyanobacteria density reached its peak between 18 and 26 ◦ C and at a pH range of 7.5–8.5. These findings highlight the need for environmental regulation and cyanobacteria control in Chinese mitten crab culture ponds, thus promoting the health and sustainability of the Chinese mitten crab culture.


Introduction
The Chinese mitten crab (Eriocheir sinensis) is one of the most economically significant crab species in Chinese aquaculture.Due to the increasing demand for this crab, intensive pond farming has become the predominant farming practice in recent years.According to the data from the 2022 China Fishery Products Annual [1], freshwater aquaculture production of the Chinese mitten crab surged from 232,400 tons in 2000 to 808,300 tons Water 2024, 16, 1688 2 of 15 in 2021.Adult Chinese mitten crabs are primarily fed formulated diets and fresh fish kept on ice.However, only 20-40% of this food is digested and absorbed by the crabs, while the remainder enters the water and sediments in the ponds.During aquaculture, the accumulation of food residues and waste from crab metabolism in pond water leads to eutrophication, thereby increasing the frequency and duration of algal blooms [2][3][4].
Phytoplankton is an essential component of pond ecosystems and serves as the primary producer, significantly influencing the productivity of aquatic environments [5].Certain phytoplankton species, such as green algae, contribute to the growth of Chinese mitten crab by providing additional food.Conversely, other algae, such as cyanobacteria, impede the growth of Chinese mitten crabs, especially during cyanobacterial blooms, which pose a severe health threat to the cultured animals.Studies indicate that in aquaculture ponds, the extensive proliferation of cyanobacteria can lead to alternations in phytoplankton communities' structure and the destabilization of aquaculture ecosystems [6][7][8].Furthermore, extensive cyanobacteria blooms near the water surface can reduce water transparency and lower dissolved oxygen levels [9][10][11][12][13][14], resulting in significant fluctuations in pH and the deterioration of water quality, potentially causing mortality among aquaculture species.Additionally, the decomposition of dead cyanobacteria consumes a significant amount of dissolved oxygen, potentially inducing hypoxia in Chinese mitten crabs for an extended period, thus resulting in widespread mortality and economic losses [14].Moreover, prolonged algal blooms may produce and release algal toxins [15][16][17][18][19][20][21][22], posing a threat not only to the growth and quality of Chinese mitten crabs but also potentially endangering human health through the food chain.
Seasonal fluctuations in phytoplankton communities within lakes exhibit discernable patterns influenced by factors such as light availability, water temperature, nutrient concentrations, and hydrodynamics.Typically, the onset of spring initiates a period of rapid proliferation, driven by escalating temperatures, augmented sunlight, and heightened nutrient inputs.Subsequently, summer marks the peak of biomass accumulation, characterized by warm temperatures and ample nutrient availability, fostering widespread blooms.As autumn ensues, diminishing sunlight and declining water temperatures impede growth, precipitating a reduction in biomass.Winter ensues with stability, as reduced temperatures and diminished light intensities induce dormancy or minimal growth.However, it is crucial to acknowledge that variations in these patterns may arise due to localized climatic nuances, hydrological dynamics, and anthropogenic influences [23].
While most of the aforementioned studies have primarily focused on lakes, fewer have examined phytoplankton in aquaculture ponds.Ponds, being relatively small water bodies, are vulnerable to anthropogenic disturbances, which may result in distinct phytoplankton dynamics compared to lakes.This study aims to systematically monitor phytoplankton Water 2024, 16, 1688 3 of 15 variations in typical Chinese mitten crab aquaculture ponds over a cultivation cycle, while concurrently analyzing associated environmental factors.We hypothesize that, during the cultivation cycle, the continuous accumulation of residual feed and crab excrement lead to an increase in nutrient concentration in the nearly enclosed pond water, resulting in a continuous rise in algal biomass.The objective is to elucidate the species composition and dynamic patterns of the phytoplankton community, along with analyzing the driving factors, to establish a foundational understanding of regulating the aquatic environment of Chinese mitten crab aquaculture.

Sampling Location
The ponds under investigation are located within the demonstration aquaculture farm dedicated to Chinese mitten crabs in Changzhou City, Jiangsu Province, China (31 • 35 ′ N, 119 • 28 ′ E), covering a total area of approximately 24,300 hectares.Six ponds for the cultivation of adult Chinese mitten crab were chosen for investigation and sampling.Each of these ponds occupies an area of approximately 9000 square meters.The stocking density was approximately 13,500 crabs per hectare (Figure S1).

Sampling
Monthly surveys were conducted from May to October 2022.Water samples were collected from five evenly distributed points within each pond using a 2.5 L sampler and were mixed in a bucket.Phytoplankton samples were prepared by transferring one liter of the mixed water into a sample bottle, followed by adding 15 mL of Lugol's solution and 25 mL of formaldehyde for fixation.The samples were then transported to the laboratory, concentrated through sedimentation, and enumerated under an optical microscope using a 0.1 mL counting chamber [56,57].The identification of algae was performed with reference to the literature on freshwater algae from China [58,59].A minimum of 400 cells were enumerated in each sample to achieve an accuracy exceeding 90%.Algal biovolume was estimated by considering algal cells as geometric shapes of equivalent volume, and biomass was obtained by converting the biovolume from 10 9 µm 3 to approximately 1 mg of biomass [60].
An additional 1 L of the mixed water sample was transferred into a sample bottle and stored in a low-temperature storage unit during transport to the laboratory for nutrient analysis.Total nitrogen (TN) was determined utilizing the persulfate digestion and ultraviolet spectrophotometry method [56,61].Total phosphorus (TP) was analyzed via persulfate digestion followed by the ascorbic acid method [56,61].Chlorophyll-a (chl-a) was measured using a spectrophotometric method after filtration on Whatman GF/C glass filters and extraction with cold 90% acetone [56,61].Water temperature (WT), dissolved oxygen (DO), and pH were measured in situ with a YSI multi-parameter water quality meter.

Data Analysis
Phytoplankton species dominance was calculated using the following formula: where Y is dominance; n i is the biomass of the i-th phytoplankton species; N is the total biomass of all phytoplankton; and f i is the frequency of occurrence of the i-th of phytoplankton species.Dominant species (genera) were the top ten species (genera) exhibiting the highest dominance for each month in this study.The Shannon-Wiener index (H), Pielou's evenness index (H ′ ), and Margalef's index (D) were calculated using the following formulas: where P i represents the proportion of individuals belonging to the i-th species to the total number of phytoplankton, S represents the total number of species in the sample, and N represents the total density of phytoplankton in the sample.Non-metric multidimensional scaling (NMDS) was employed to elucidate the monthly dynamics pattern of phytoplankton communities using R4.3.1 (R Foundation for Statistical Computing, Vienna, Austria).Canonical correspondence analysis (CCA) was conducted using Canoco 5.15 (Microcomputer power, Ithaca, NY, USA) to explore the relationship between phytoplankton and physicochemical factors, as the Detrended Correspondence Analysis (DCA) indicated a gradient length exceeding 4.

Physical and Chemical Parameters
TN exhibited an initial increase followed by a subsequent decrease during the study period.TN reached its peak in August.TP fluctuated without clear trends.The nitrogenphosphorus ratio (TN/TP) demonstrates an initial rise followed by a subsequent decline.
Chlorophyll-a (chl-a) exhibits significant fluctuations, with a notable increase in September.The pH varies within the range of 7.05-9.6,peaking above 9.0 from June to August.Water temperature (WT) fluctuates notably across months, demonstrating an overall pattern of initial increase followed by decline.August records the highest water temperature at 33.6 • C, while October marks the lowest at 16.6 • C (Figure 1).
where Pi represents the proportion of individuals belonging to the i-th species to the total number of phytoplankton, S represents the total number of species in the sample, and N represents the total density of phytoplankton in the sample.Non-metric multidimensional scaling (NMDS) was employed to elucidate the monthly dynamics pattern of phytoplankton communities using R4.3.1 (R Foundation for Statistical Computing, Vienna, Austria).Canonical correspondence analysis (CCA) was conducted using Canoco 5.15 (Microcomputer power, Ithaca, NY, USA) to explore the relationship between phytoplankton and physicochemical factors, as the Detrended Correspondence Analysis (DCA) indicated a gradient length exceeding 4.

Physical and Chemical Parameters
TN exhibited an initial increase followed by a subsequent decrease during the study period.TN reached its peak in August.TP fluctuated without clear trends.The nitrogenphosphorus ratio (TN/TP) demonstrates an initial rise followed by a subsequent decline.
Chlorophyll-a (chl-a) exhibits significant fluctuations, with a notable increase in September.The pH varies within the range of 7.05-9.6,peaking above 9.0 from June to August.Water temperature (WT) fluctuates notably across months, demonstrating an overall pattern of initial increase followed by decline.August records the highest water temperature at 33.6 °C, while October marks the lowest at 16.6 °C (Figure 1).

Phytoplankton Species
The investigation identified a total of 184 phytoplankton species belonging to 93 genera (Figure S2).Among them, Chlorophyta exhibited the highest species richness, comprising Water 2024, 16, 1688 5 of 15 44 genera and 99 species.Next are cyanobacteria, with 26 species in 17 genera, and Bacillariophyta, consisting of 14 genera and 24 species.Other genera are from Euglenophyta, Chrysophyta, Pyrrophyta, Cryptophyceae, and Xanthophyta.Dominant species in the investigated ponds from May to October primarily belong to cyanobacteria, Bacillariophyta, Cryptophya, Euglenophyta, and Chlorophyta.Chlorophyta and cyanobacteria exhibit a higher number of dominant species that are present every month.Other algae typically have only 1-2 dominant or non-dominant species each month (Table 1).There was a distinct turnover regarding the dominant species, with an increase in cyanobacteria species and a decrease in Chlorophyta species.The number of dominant species in cyanobacteria demonstrated an overall fluctuating increasing trend, rising from only one species in May to six species in October.Conversely, the number of dominant species in Chlorophyta depicted an overall fluctuating decreasing trend, declining from seven species in May to one species in October.This suggests that the dominant species in Chinese mitten crab aquaculture ponds have shifted from Chlorophyta to cyanobacteria with seasonal changes.
The dominant genera in cyanobacteria have generally transitioned from only Microcystis to Microcystis, Anabaena, Dolichospermum, and Planktonthrix.Initially dominated solely by Microcystis from May to July, the dominant genera transitioned to a shared dominance of Microcystis and Anabaena in August, and eventually to a shared dominance among Microcystis, Anabaena, Dolichospermum, and Planktonthrix.It is noteworthy that the proportion of Microcystis and Anabaena within the dominant genera of cyanobacteria demonstrated an overall upward trend.Meanwhile, the proportion of filamentous cyanobacteria also increased and was particularly pronounced in September and October.
Several dominant genera were identified within Chlorophyta, showing a transition pattern from Chlorella, Pediastrum, and Cosmarium to Desmodesmus and Kirchneriella.In May, there was little disparity in the dominance values of Chlorella, Pediastrum, Pseudopediastrum, and Cosmarium.Chlorella emerged from May to July, exhibiting a decrease in dominance value.The dominance of Desmodesmus primarily demonstrated a fluctuating upward trend, maintaining a dominant position throughout, except for June, and emerging as the most prevalent genus within Chlorophyta for three consecutive months starting in August.

Dynamics of Phytoplankton Cell Density and Biomass
The overall density of phytoplankton cells exhibited a continuous upward trend, rising from 1.50 × 10 7 cells/L to 46.0 × 10 7 cells/L over the study period (Figure 2).Cyanobacteria and Chlorophyta consistently maintained dominance, with cyanobacteria notably experiencing a rapid increase in cell density during September and October, peaking at 15.7 × 10 7 cells/L and 40.5 × 10 7 cells/L, respectively, representing 79.6% and 88.1% of the total monthly density.Similar to density, the overall biomass of phytoplankton also showed an increasing trend, albeit with fluctuation, rising from 8.66 mg/L to 76.8 mg/L.From May to August, the biomass of Chlorophyta exceeded that of cyanobacteria, contributing significantly to the total biomass.However, from September to October, there was a significant increase in cyanobacteria biomass, surpassing that of Chlorophyta.Particularly, in October, there was a significant surge in cyanobacteria biomass, leading to a substantial rise in total biomass.dominance value.The dominance of Desmodesmus primarily demonstrated a fluctuating upward trend, maintaining a dominant position throughout, except for June, and emerging as the most prevalent genus within Chlorophyta for three consecutive months starting in August.

Dynamics of Phytoplankton Cell Density and Biomass
The overall density of phytoplankton cells exhibited a continuous upward trend, rising from 1.50 × 10 7 cells/L to 46.0 × 10 7 cells/L over the study period (Figure 2).Cyanobacteria and Chlorophyta consistently maintained dominance, with cyanobacteria notably experiencing a rapid increase in cell density during September and October, peaking at 15.7 × 10 7 cells/L and 40.5 × 10 7 cells/L, respectively, representing 79.6% and 88.1% of the total monthly density.Similar to density, the overall biomass of phytoplankton also showed an increasing trend, albeit with fluctuation, rising from 8.66 mg/L to 76.8 mg/L.From May to August, the biomass of Chlorophyta exceeded that of cyanobacteria, contributing significantly to the total biomass.However, from September to October, there was a significant increase in cyanobacteria biomass, surpassing that of Chlorophyta.Particularly, in October, there was a significant surge in cyanobacteria biomass, leading to a substantial rise in total biomass.Concerning cyanobacteria density composition, the proportion of Microcystis increased in June and consistently remained the largest (Figure 3).Microcystis density steadily increased over time, escalating from 0.45 × 10 7 cells/L in May to 11.24 × 10 7 cells/L in September, and further to 28.74 × 10 7 cells/L in October.Planktothrix density showed an increasing trend.By October, it boasted the highest density of cyanobacteria, second only to Microcystis, with a density of 12.01 × 10 7 cells/L.The proportion of Dolichospermum (separated from the old genus Anabanae) initially increased and then decreased with fluctuations, but its density showed a continuously increasing trend, from 0.06 × 10 7 cells/L in August to 2.33 × 10 7 cells/L in October.The proportion of other cyanobacteria generally declined, while their density increased.Despite a decreasing proportion, Anabaena exhibited an overall increasing trend in density values.Since its observation in May, except for a slight dip in density in June and August, the density surged by over 50% in all other months, with October ranking as the third most abundant cyanobacteria.Concerning cyanobacteria density composition, the proportion of Microcystis increased in June and consistently remained the largest (Figure 3).Microcystis density steadily increased over time, escalating from 0.45 × 10 7 cells/L in May to 11.24 × 10 7 cells/L in September, and further to 28.74 × 10 7 cells/L in October.Planktothrix density showed an increasing trend.By October, it boasted the highest density of cyanobacteria, second only to Microcystis, with a density of 12.01 × 10 7 cells/L.The proportion of Dolichospermum (separated from the old genus Anabanae) initially increased and then decreased with fluctuations, but its density showed a continuously increasing trend, from 0.06 × 10 7 cells/L in August to 2.33 × 10 7 cells/L in October.The proportion of other cyanobacteria generally declined, while their density increased.Despite a decreasing proportion, Anabaena exhibited an overall increasing trend in density values.Since its observation in May, except for a slight dip in density in June and August, the density surged by over 50% in all other months, with October ranking as the third most abundant cyanobacteria.
The composition of cyanobacteria biomass exhibited different changes compared to their densities (Figure 3).While the proportion of Microcystis generally increased, the proportion of Anabaena generally decreased, despite that its biomass values continuously increased.In May, Microcystis biomass accounted for 40.7% of cyanobacteria biomass.However, it unequivocally dominated cyanobacteria in June and continued to increase.In June, Microcystis biomass increased by almost twentyfold compared to May.From June to October, Microcystis biomass continued to rise, representing 79.6% of cyanobacteria biomass in October.Meanwhile, different Microcystis species peaked in different months (Figure 4).Planktothrix and Dolichospermum biomass showed a monthly increasing trend.Planktothrix ranked the second highest among cyanobacteria in biomass in October.Dolichospermum biomass showed a monthly increasing trend, rapidly escalating from 0.08 mg/L in August to 1.16 mg/L in September, and then rapidly rising to 3.67 mg/L in October.Notably, Spirulina was observed in May.Although its density was not high, its biomass accounted for one-third of cyanobacteria biomass in that month, owing to its large cell volume (Figure 4).The composition of cyanobacteria biomass exhibited different changes compared to their densities (Figure 3).While the proportion of Microcystis generally increased, the proportion of Anabaena generally decreased, despite that its biomass values continuously increased.In May, Microcystis biomass accounted for 40.7% of cyanobacteria biomass.However, it unequivocally dominated cyanobacteria in June and continued to increase.In June, Microcystis biomass increased by almost twentyfold compared to May.From June to October, Microcystis biomass continued to rise, representing 79.6% of cyanobacteria biomass in October.Meanwhile, different Microcystis species peaked in different months (Figure 4).Planktothrix and Dolichospermum biomass showed a monthly increasing trend.Planktothrix ranked the second highest among cyanobacteria in biomass in October.Dolichospermum biomass showed a monthly increasing trend, rapidly escalating from 0.08 mg/L in August to 1.16 mg/L in September, and then rapidly rising to 3.67 mg/L in October.Notably, Spirulina was observed in May.Although its density was not high, its biomass accounted for one-third of cyanobacteria biomass in that month, owing to its large cell volume (Figure 4).The composition of cyanobacteria biomass exhibited different changes compared to their densities (Figure 3).While the proportion of Microcystis generally increased, the proportion of Anabaena generally decreased, despite that its biomass values continuously increased.In May, Microcystis biomass accounted for 40.7% of cyanobacteria biomass.However, it unequivocally dominated cyanobacteria in June and continued to increase.In June, Microcystis biomass increased by almost twentyfold compared to May.From June to October, Microcystis biomass continued to rise, representing 79.6% of cyanobacteria biomass in October.Meanwhile, different Microcystis species peaked in different months (Figure 4).Planktothrix and Dolichospermum biomass showed a monthly increasing trend.Planktothrix ranked the second highest among cyanobacteria in biomass in October.Dolichospermum biomass showed a monthly increasing trend, rapidly escalating from 0.08 mg/L in August to 1.16 mg/L in September, and then rapidly rising to 3.67 mg/L in October.Notably, Spirulina was observed in May.Although its density was not high, its biomass accounted for one-third of cyanobacteria biomass in that month, owing to its large cell volume (Figure 4).

Changed in Phytoplankton Diversity Index
Throughout the period from May to October 2022, there was a discernible downward trend in the diversity index of phytoplankton within Chinese mitten crab aquaculture ponds.This trend suggests a reduction in phytoplankton species richness and potential alterations in community composition, leading to decreased overall diversity (Figure 5).

Changed in Phytoplankton Diversity Index
Throughout the period from May to October 2022, there was a discernible downward trend in the diversity index of phytoplankton within Chinese mitten crab aquaculture ponds.This trend suggests a reduction in phytoplankton species richness and potential alterations in community composition, leading to decreased overall diversity (Figure 5).

Shifts in Phytoplankton Community Dynamics
Non-metric multidimensional scaling (NMDS) was used to explore the transitions in phytoplankton communities across different sampling times (Figure 6).The species composition of phytoplankton communities demonstrates a shift along the first axis (NMDS1), with May and June exhibiting similarity in species composition, and July and August showing a similar pattern.In contrast, the species composition of phytoplankton in September resembles that of July and August, whereas samples from October are distinctly positioned to the right, indicating divergence from the other samples.

Shifts in Phytoplankton Community Dynamics
Non-metric multidimensional scaling (NMDS) was used to explore the transitions in phytoplankton communities across different sampling times (Figure 6).The species composition of phytoplankton communities demonstrates a shift along the first axis (NMDS1), with May and June exhibiting similarity in species composition, and July and August showing a similar pattern.In contrast, the species composition of phytoplankton in September resembles that of July and August, whereas samples from October are distinctly positioned to the right, indicating divergence from the other samples.

Relationship between Phytoplankton and Environmental Factors
Phytoplankton composition in Chinese mitten crab aquaculture ponds is predominantly influenced by TP, WT, and DO, with TP exhibiting the highest explanatory power, as revealed by CCA (Figure 7).CCA1 and CCA2 contribute 14.7% and 6.6%, respectively, totaling 21.3% in explanatory power.TN and TP exhibited a weaker influence on dominant species.

Relationship between Phytoplankton and Environmental Factors
Phytoplankton composition in Chinese mitten crab aquaculture ponds is predominantly influenced by TP, WT, and DO, with TP exhibiting the highest explanatory power, as revealed by CCA (Figure 7).CCA1 and CCA2 contribute 14.7% and 6.6%, respectively, totaling 21.3% in explanatory power.TN and TP exhibited a weaker influence on dominant species.

Relationship between Phytoplankton and Environmental Factors
Phytoplankton composition in Chinese mitten crab aquaculture ponds is predominantly influenced by TP, WT, and DO, with TP exhibiting the highest explanatory power, as revealed by CCA (Figure 7).CCA1 and CCA2 contribute 14.7% and 6.6%, respectively, totaling 21.3% in explanatory power.TN and TP exhibited a weaker influence on dominant species.The primary environmental factors driving changes in phytoplankton community structure are WT and pH (p < 0.01).Notably, Chlorophyta exhibits a significant positive correlation with WT and pH, whereas cyanobacteria show the contrary, displaying a negative correlation with WT and pH.Chlorophyta's higher values tended to be presented at elevated temperatures or pH levels, whereas cyanobacteria's higher density was primarily presented within the temperature range of 18-26 • C and pH range of 7.5-8.5.

Discussion
Phytoplankton density in Chinese mitten crab culture ponds exhibited a monthly increasing trend, primarily determined by the continuous increase in cyanobacteria density.Although Chlorophyta decreased significantly in September and October, overall, phytoplankton continued to rise due to Chlorophyta density doubling, and there was a notable increase in October.This contrasts with findings in research on phytoplankton density in natural freshwater lakes [62][63][64].After a three-year study on phytoplankton community changes in Poyang Lake, Yuan found that phytoplankton density and biomass were consistently higher in summer compared to other seasons.Similarly, in a two-year study on eutrophic lake phytoplankton community structure changes, phytoplankton density in summer, particularly in July, consistently exceeded that of other seasons.Zhu et al. monitored Taihu Lake blooms utilizing Ocean and Land Color Instrument data (OLCI) and observed that Microcystis proliferated more rapidly during high water temperatures in July and August, with other types of blooms appearing more prominently during severe Microcystis bloom outbreaks, leading to increased bloom area expansion.
Typically, phytoplankton, especially cyanobacteria, peaks in lakes during July and August.However, in this study, phytoplankton reached its maximum in October, despite nitrogen and phosphorus concentrations being lower than those in July and August.We consider that this is due to the submerged plants' growth and decline.Submerged plants, such as Hydrilla verticillata, Vallisneria natans, and Elodea nuttallii, are planted in Chinese aquaculture ponds to provide a sheltered environment and food for crabs.In summer, they grow vigorously and release more allelopathic substances, inhibiting phytoplankton growth.As autumn approaches, submerged plants decline, weakening their inhibitory effect on algae once again [65], ultimately resulting in an increase in phytoplankton density.
Studies on still-water ecosystems suggested that due to the relatively poor water fluidity, nutrients are more readily enriched, potentially causing rapid growth of phytoplankton in the short term [66][67][68].However, the nutrient concentration in the water column was not always increased in the investigated pond.The planted submerged plants may also be responsible for the nutrient concentration variation.Between May and July, the feeding quantity for the crabs gradually increased, resulting in elevated releases of nitrogen and phosphorus into the pond.Simultaneously, submerged plants underwent rapid growth, absorbing a portion of nitrogen and phosphorus.However, due to the higher nitrogen-to-phosphorus ratio of residual feed discharged into the pond (approximately 6:1) compared to the absorption ratio of plants (approximately 5:1), phosphorus absorption was more pronounced.Additionally, ponds are typically cleansed before aquaculture, and the cleansed sediment may also adsorb part of the phosphorus.Thus, prior to July, there was a gradual rise in the nitrogen concentration alongside a decline in phosphorus concentration.Following August, the growth rate of submerged plants decelerated, leading to diminished nutrient absorption.Nevertheless, the biomass of submerged plants persisted at a high level, providing an optimal substrate for attached microorganisms.Owing to the vigorous nitrification and denitrification activities of microorganisms, nitrogen concentration gradually declined, while water phosphorus concentration might rebound due to phosphorus adsorption saturation in sediment and potential resuspension triggered by crab activity.
The prolonged extreme high temperature in 2022 may also be another factor contributing to the rapid increase in cyanobacteria, especially in September and October (Figure S3).The average temperature in 2022 was 1.0 • C higher than normal.There is growing evidence indicating a correlation between elevated temperatures and the frequency of cyanobacterial bloom outbreaks.Climate warming fosters elevated water temperatures, creating favorable conditions for cyanobacterial growth and reproduction.When water temperatures reach optimal levels for growth, cyanobacteria exhibit accelerated growth rates, facilitating rapid bloom development.However, this temperature surge may also adversely affect other aquatic organisms, further exacerbating cyanobacterial bloom formation and expansion [69,70].O'Neil et al. (2012) investigated the relationship between climate change and harmful cyanobacterial blooms, highlighting that rising temperatures associated with climate change promote environments conducive to cyanobacterial proliferation.As water temperatures rise, cyanobacteria experience heightened growth rates, resulting in more frequent and severe bloom events [71].
Several studies [25,47,48] suggest that the growth rate of algae exhibited a unimodal relationship with water temperature; that is, when the water temperature is below the optimal level, the algae growth rate shows exponential growth, whereas it decreases when the temperature surpasses the optimal range, and the growth rate of algae decreases.The density of cyanobacteria increased with the increase in water temperature from May to August.Despite the decrease in water temperature in September, the density of cyanobacteria continued to rise.This could be attributed to the onset of cyanobacteria blooms in aquaculture ponds, beginning in mid-August and persisting until mid-September.These blooms proliferate extensively, dominate the ecosystem, and inhibit the growth and reproduction of other algae.Additionally, they may be influenced by zooplankton [72][73][74][75].During bloom events, zooplankton may indirectly facilitate the growth of cyanobacteria by consuming non-cyanobacteria planktonic algae, which could explain the significant decrease in green algae density observed in September.Although the water temperature in October falls below the optimal range for cyanobacteria growth [22], their density still increases significantly, possibly due to the increase in the water pH level.
Furthermore, research has shown that pH may impact the normal growth of phytoplankton by influencing cell membrane transport processes, metabolic functions related to pH regulation within cells, or the content and relative composition of amino acids within cells [44,[76][77][78].Studies conducted in various environments, such as aquaculture ponds, natural lakes, and estuaries, have indicated that pH has either a promoting or inhibiting effect on the growth and reproduction of phytoplankton.Within a certain range, phytoplankton density tends to increase with rising pH.However, in environments where pH exceeds 9, the normal growth of most phytoplankton is hindered [26, [42][43][44][45][46].The findings of this study also support the conclusion that when pH surpasses 9, algal density significantly decreases or is predominantly dominated by colony species, such as Microcystis and Volvox.
This study revealed that cyanobacteria, particularly Microcystis, dominated in aquaculture ponds, posing a threat to the industry.Firstly, the high density of cyanobacteria diminished biodiversity, resulting in ecosystem instability.Secondly, they may produce toxins that can be absorbed by the cultured Chinese mitten crabs.Previous studies showed that after exposure to MCs, several enzymes of the crab's antioxidant defense system are activated, a response that helps maintain lipid peroxidation levels but is insufficient to completely prevent tissue damage [79].Third, cyanobacterial toxins may be transferred to higher-level consumers, including humans, by accumulating in the tissues of crabs and thus affecting food safety [80].To confirm this, we determined microcystins (MCs), including MC-LR, MC-RR, and MC-YR, in both Chinese mitten crabs and their culture ponds using the UPLC-MS/MS method.The concentrations of MCs in the pond water ranged from 0.10 µg/L to 0.15 µg/L, while in the crabs' tissues, the highest recorded value was 36.48 µg/kg (unpublished data).Therefore, implementing measures is imperative to mitigate cyanobacteria proliferation, thereby fostering environmentally friendly and sustainable development of the Chinese mitten crab industry.
First and foremost, reducing nutrient concentrations in water bodies is paramount.This can be achieved by reasonable design of stocking density to diminish organic loads, enhancing feed utilization rates, minimizing the use of fresh fish as feed, and developing environmentally friendly feeds.Secondly, optimizing aquatic plant management in crab ponds is also an effective measure.This involves strategically combining different species of submerged plants and careful management to prevent the plants from an early decline.Moreover, enhancing artificial aeration and utilizing innovative materials and technologies can effectively control the growth of cyanobacteria or mitigate their harmful effects [81].

Conclusions
Over the Chinese mitten crab culture period, phytoplankton exhibited a consistent increase, primarily fueled by elevated densities of cyanobacteria.Notably, environmental factors, such as water temperature and pH, emerged as key drivers shaping phytoplankton community structure, with distinct responses observed among cyanobacteria and Chlorophyta.This study identified a downward trend in the phytoplankton diversity index, suggesting potential shifts in community composition over time.Canonical correspondence analysis highlighted the influence of water temperature and pH on dominant phytoplankton genera, underscoring the importance of environmental regulation in aquaculture management.Given the dominance of cyanobacteria, particularly Microcystis, this study emphasizes the necessity of implementing targeted mitigation measures to ensure the sustainability of Chinese mitten crab aquaculture, including oxygenation, strategic planting of submerged plants, and stocking density control, especially in the context of global climate change.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/w16121688/s1, Figure S1: The location of the six investigated aquaculture ponds (A-F) for Chinese mitten crab cultivation in Changzhou City, Jiangsu Province,

Figure 2 .
Figure 2. Monthly dynamics of density and biomass of phytoplankton in Chinese mitten crab aquaculture ponds.

Figure 2 .
Figure 2. Monthly dynamics of density and biomass of phytoplankton in Chinese mitten crab aquaculture ponds.

Water 2024 , 16 Figure 3 .
Figure 3. Dynamics of cell density and biomass composition of cyanobacteria.

Figure 3 .
Figure 3. Dynamics of cell density and biomass composition of cyanobacteria.

Figure 3 .
Figure 3. Dynamics of cell density and biomass composition of cyanobacteria.

Figure 4 .
Figure 4. Monthly variation of common phytoplankton species biomass in Chinese mitten crab culture ponds.

Figure 5 .
Figure 5. Monthly dynamics of phytoplankton diversity index in Chinese mitten crab culture ponds.

Figure 5 .
Figure 5. Monthly dynamics of phytoplankton diversity index in Chinese mitten crab culture ponds.

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Figure 6 .
Figure 6.Temporal variations in phytoplankton community composition by month.

Figure 6 .
Figure 6.Temporal variations in phytoplankton community composition by month.

Figure 7 .
Figure 7. Canonical correspondence analysis (CCA) plot illustrating the relationship between phytoplankton and environmental factors.TN: total nitrogen, TP: total phosphorus, DO: dissolved oxygen, WT: water temperature.

Figure 7 .
Figure 7. Canonical correspondence analysis (CCA) plot illustrating the relationship between phytoplankton and environmental factors.TN: total nitrogen, TP: total phosphorus, DO: dissolved oxygen, WT: water temperature.

Table 1 .
Monthly top ten dominant species of phytoplankton from Chinese mitten crab culture ponds.Blue font indicates cyanobacteria, green font indicates Chlorophyta, and black font indicates other groups.