Spatio-temporal variation of toxin-producing gene abundance in Microcystis aeruginosa from Poyang Lake

<jats:title>Abstract</jats:title>         <jats:p><jats:italic>Microcystis aeruginosa</jats:italic> causes massive blooms in eutrophic freshwater and releases microcystin. Poyang Lake is the largest freshwater lake in China and has kept at a mid-nutrient level in recent years. However, there is little research on microcystins production in Poyang Lake. In this study, water and sediment samples from ten sampling sites in Poyang Lake were collected from May to December in 2020, and from January to April in 2021 respectively. <jats:italic>Microcystis</jats:italic> genes (<jats:italic>mcyA,mcyB,16s rDNA</jats:italic>) were quantified by real-time fluorescence quantitative PCR analysis,and then the spatial and temporal variation of <jats:italic>mcy</jats:italic> genes, physicochemical factors and bacterial population structure in the lake was analyzed. The relationship between the abundance of mcy genes and physicochemical factors in water column were also revealed. Results indicated that the microcystin-producing genes <jats:italic>mcyA</jats:italic> and <jats:italic>mcyB</jats:italic> showed significant differences in spatial and temporal levels as well, which is closely related to the physicochemical factors especially the water temperature(p &lt; 0.05) and the nitrogen content(p &lt; 0.05). The abundance of <jats:italic>mcy</jats:italic> genes in the sediment in December affected the abundance of <jats:italic>mcy</jats:italic> genes in the water column in the next year, while the toxic <jats:italic>Microcystis</jats:italic> would accumulate in the sediment. In addition to the toxic <jats:italic>Microcystis</jats:italic>, we also found a large number of non-toxic <jats:italic>Microcystis</jats:italic> in the water column and sediment, the ratio of toxic to non-toxic species can also affect the toxicity production of <jats:italic>Microcystis aeruginosa</jats:italic>. Overall, the results showed that <jats:italic>Microcystis aeruginosa</jats:italic> toxin-producing genes in Poyang Lake distributed spatially and temporally which related to the physicochemical factors of Poyang Lake.</jats:p>

section_content: Microcystis aeruginosa is one of the major species causing toxic cyanobacterial blooms in the world's freshwater systems [1]. Among the toxins produced by cyanobacteria, microcystins (MCs) are the most abundant, widely distributed and damaging algal toxins [2, 3]. MCs are usually present in the cell body. 

When the algal cells burst or disintegrate, MCs are continuously released into the water body, which can contaminate the sources of the drinking water [4, 5]. Microcystins not only affect the growth, development and metabolism of plants and animals, but can also be accumulated in various plants and animals through the food chain and food web, posing a serious threat to the health of animals, plants and humans in freshwater lake ecosystems [6, 7]. Therefore, it is necessary to monitor the changes of microcystins in the lake. 

All toxin-producing Microcystis contained the mcy gene, while the absence or inactivity of the mcy gene prevented the production of MCs. Moreover, Microcystin enzyme synthesis system is encoded by the mcy gene [8]. The mcy gene cluster contains 55 kb DNA encoding six big open reading frames (orf,mcyA-E and G), and four additional small orf (mcyF and H-J) located in the chromosome [9]. Deletion of the mcy gene destroys all isoforms of the microcystin toxin cyclic heptapeptide. Toxin-producing and non-toxinproducing Microcystis aeruginosa differ in mcy sequence [10, 11]. Thus, the sequence of the mcy gene can be used to determine the toxicity potential of Microcystis and non-toxic Microcystis. The mcyA gene is necessary for the biosynthesis of MCs [12], while the mcyB gene is a key element in the synthesis of polypeptide chains in the growth of MCs [13]. Therefore, we analyzed the gene abundances of mcyA and mcyB to detect the microcystins level in Poyang Lake. 

Many studies have analyzed the abundance of mcy genes to detect the microcystins levels in water bodies [14, 15]. The SYBR Green qPCR method is characterized by high speci city, reproducibility, rapidity, and has been widely used for the quantitative detection of gene expression [16, 17]. Anil Kumar et al. found the presence of the toxic cyanobacterium by employing polymerase chain reaction (PCR) ampli cation of six genes of the mcy genes cluster (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG) in seven eutrophicated ponds of Varanasi [18]. The light microscopic (LM) observation and PCR ampli cation of mcyE and mcyB genes in water samples from nine shallow waterbodies situated in Central and Eastern Bulgaria demonstrated that the MC-producers were Microcystis aeruginosa and M. wesenbergii [19]. Myriam et al. also reported the presence of microcystin-producing genes mcyA and mcyB in surface sediments to assess the potential toxicity of estuarine sediments [20]. It has been reported that environmental factors such as light intensity, pH, high nitrogen concentration, temperature, and iron starvation can affect microcystin production levels [21]. The relative abundance of these gene types was found to be correlated with pH and nitrate concentrations by analyzing the various composition of mcy genes in western Lake Erie [22]. In China, there have been many studies measuring the abundance of MCs in freshwater lakes such as Chaohu, Taihu, and Dianchi using quantitative real-time PCR [23] [24] [25]. 

However, fewer studies were carried in Poyang Lake. Moreover, no studies recorded how environmental factors affect the abundance of MCs. 

In this study, we collected water samples (0. 5m water depth from the water surface) from ten sample sites from the north to the east in Poyang Lake, and then quanti ed Microcystis aeruginosa toxicity genes (mcyA, mcyB,16s rDNA) by real-time uorescence quantitative PCR analysis. We analyzed the spatial and temporal variation of mcy genes, physicochemical factors and bacterial population structure in the lake, and then explored their relationship. The temporal and spatial differences in the abundance of mcy genes in Poyang Lake may cause by environmental factors and the overwintering characteristics of Microcystis. By analyzing the factors which affect the abundance of mcy genes, we can monitor these factors to predict the changes of mcy genes in freshwater lake ecosystems and the Microcystis aeruginosa toxins contamination.
section_name: Introduction

section_name: Material And Methods

section_content: Poyang Lake (28°4′-29°46′N, 115°49′-116°46′E) lies on the southern bank of the middle and lower reaches of the Yangtze River [26]. The lake is an essential source of drinking water, as well as water for irrigation, aquaculture and industry. The lake has all the major functions of the ecosystem, including the function of providing, supporting and regulating the environment, as well as offering cultural and recreational functions for the local population [27]. Poyang Lake has great seasonal hydrological uctuation, from April to September, the main lake and the sub-lake of Poyang Lake are connected together because of the rising water level; from October to March, the dry water period, the lake and the main lake are separated because of the falling water level [28]. 

Ten sampling stations (numbered 1-10 were established according to the geographic characteristics of Poyang Lake (Fig. 1 ). According to the variation of water level in Poyang Lake and the occurrence of cyanobacteria in this lake, eld samples were collected from May to December in 2020, and from January to April in 2021. Sites 1-5 were sampled in all the studied months, while water samples from other sites (sites 5-10) were collected from June to August during the rainy season. Each sample was a mixture collected from the top (0. 5 m surface water) and the bottom (0. 5 m over sediment) of the water column with 5L Schindler sampler. The water sample was ltered through a 0. 22 µm microporous membrane for 500 ml and stored at -20°C in the refrigerator. The samples were then used for subsequent real-time uorescence PCR.
section_name: Study sites and sample collection

section_content: Water quality parameters

In the eld, transparency was measured with Secchi discs and water depth was measured with a digital ultrasonic echosounder. Water temperature, conductivity, turbidity, and pH were determined using a vestar portable multi-parameter measuring instrument (Orion, CO, USA). Chlorophyll content was analysed by the hot ethanol method. Chemical oxygen demand (CODMn), total nitrogen (TN), and total phosphorus (TP) contents were analyzedb by APHA [29, 30].
section_name: Physicochemical parameters

section_content: One-litre integrated water sample for phytoplankton was preserved with 1% acidi ed Lugol's iodine solution and concentrated to 30 mL after sedimentation for 48 h. After mixing, 0. 1 mL concentrated samples were counted directly under ×400 magni cation.
section_name: Phytoplankton

section_content: The DNA in water was extracted using the E. Z. N. A Water DNA Extraction Kit (OMEGA, USA) and the DNA in sediment was extracted using The FastDNA® SPIN Kit for Soil (MP Biomedicals, USA) from the algal cells and the lter membranes according to the kit. The NanoDrop 2000 UV spectrophotometer (Thermo, USA) was used to determine the concentration and purity of DNA.
section_name: DNA extraction

section_content: With reference to the microcystin synthesis genes (mcyA, mcyB) and the gene sequence of Microcystisspeci c 16S rDNA (M16S) in the NCBI. The primers were designed using Primer 5 software and the primer information is shown in Table 1 [31]. 

PCR reaction system: 2. 5µL of DNA10µmol/L upstream primer 1µL, 10µmol/L downstream primer 1µL, 10µmol/L downstream primer 1µL, SYBR®Green Realtime PCR Master Mix (OSAKA, JAPAN) 12. 5µL, ddH2O water 8µL, the total volume was 25µL. The PCR program was initiated with a 1-min hold at 95℃, followed by 40 cycles each consisting of a denaturing step at 95℃ for 15 s, an annealing step at 60℃ for 15s and an elongation step at 72℃ for 45s. Three negative controls without DNA were included in each PCR run.
section_name: Primer design and PCR ampli cation

section_content: Microcystis aeruginosa PCC7806 was used as the standard strain for the quanti cation of both total Microcystis and MC-producing Microcystis. The standard curve was established by correlating known DNA concentrations (in cell equivalents) with the threshold cycle (CT) values of diluted samples [32, 33]. 

Ten millilitres of culture of strain PCC7806 containing 3. 4×10 7 cells/mL (determined by a direct microscopic count) were ltered through a 0. 22-µm nominal pore-size Nuclepore Track-Etch Membrane. A series of 10-fold dilutions (ranging from 3. 4×10 6 cells to 3. 4 cells) of the PCC7806 DNA template standard solution was used as the external standard for the qPCR. CT calculations were completed automatically for each qPCR assay using iQ5 software (version 3. 0; Bio-Rad, Hercules, CA) and the maximum correlation coe cient approach. In this approach, the threshold was automatically determined to obtain the highest possible correlation coe cient (R 2 ) for the standard curve.
section_name: Standard curve preparation

section_content: Statistical analyses were performed using the SPSS v. 22. 0 software (SPSS, Chicago, USA) unless otherwise speci ed. One-way analysis of variance (ANOVA) was performed to compare differences among physicochemical variables and major bacterioplankton taxa among samples. Spearman correlation analysis was performed between all possible pairs of environmental variables and bacterioplankton diversity. To evaluate the variations among physicochemical factors and the effects of physicochemical factors on bacterial communities, principal component analysis (PCA) was done using CANOCO v. 5. 0. 4 software (Microcomputer Power, CA, USA).
section_name: Statistical analysis

section_name: Results

section_content: By measuring the physicochemical parameters of the water column and plotting the correlation heat map, we found that the physicochemical parameters showed signi cant temporal and spatial differences (Table 1, Fig. 3a-c ). Transparency (50. 00-150. 00m), pH ( Furthermore, according to the criteria for determining eutrophication in freshwater lakes, it was found that the concentrations of TP, TN and Chl-a exceeded the threshold values during the sampling period (TP > 0. 03 mg/L; TN > 0. 65 mg/L; Chl-a > 9 µg/L), indicating that all ten sampling sites were under eutrophication during the sampling process. Analysis of bacterial communities in the water column and sediment (Fig. 3d, e, g, h) revealed signi cant temporal and spatial differences in the diversity indices of the bacterial communities. The correlation heat map (Fig. 3f, I ) was obtained by co-analyzing them with the environmental physicochemical parameters of the water column. The analysis revealed that OTUs, Chao1, and Shannon were signi cantly and positively correlated with conductivity in the sediment, where OTUs were also signi cantly and positively correlated with SS (p < 0. 05). In the water column, OTUs, Chao1, Shannon were signi cantly and positively correlated with DO, Chl-b, Chl-a, and negatively correlated with water temperature (p < 0. 05).
section_name: Physicochemical factors and bacterial communities in Poyang Lake

section_content: To investigate the spatial and temporal distribution of the mcyA and mcyB in water bodies, we extracted DNA from 500 ml of water samples and analyzed them via real-time uorescence quantitative PCR, and calculated the corresponding gene copy numbers using standard curves. 

The results in Fig. 4 showed that the toxin-producing genes of Microcystis aeruginosa in the water column varied signi cantly with time. It showed an increasing trend from April to September when Poyang Lake is at its water abundance and a peak in the toxin-producing genes of Microcystis aeruginosa in September. We also found that the average copy number of 16s rDNA in water was higher than the average copy number of mcyA and mcyB, which indicated that, there were also non-toxinproducing Microcystis aeruginosa in the Poyang Lake besides the toxin-producing ones(Fig. 4a ). 

Comparing the annual variability of microcystin at sampling sites S1-S5, a relative quantitative analysis using January as a reference yielded similar results. Microcystis aeruginosa toxin-producing genes varied considerably with the seasons, with mcyA being higher in summer and autumn, and mcyB being higher in June, July and August (Fig. 4d, e ). The results in Fig. 5 showed that Microcystis aeruginosa toxinproducing genes in sediment were more abundant in spring, with an increasing trend from January to April when Poyang Lake was in the dry season. The peak of Microcystis aeruginosa toxin-producing genes was reached in April. We found that the average copy number of 16s rDNA in the sediment was higher than the average copy number of mcyA and mcyB, indicating that the sediment also contained non-toxic producing Microcystis aeruginosa (Fig. 5a ). Combining the temporal and spatial distributions of the Microcystis toxicogenic genes mcyA and mcyB at S1-S10, we can conclude that the accumulation of Microcystis toxins in the sediment was higher at sampling sites near the edge of the lake and more in spring(Fig. 5b, c ). Similar results were obtained when comparing the annual variation of microcystin at sampling sites S1-S5, with January as a reference for relative quanti cation, where mcyA and mcyB showed an increasing trend from January to April (Fig. 5d-e ).
section_name: Temporal variation in the abundance of mcy genes

section_content: The spatial distribution of the microcystin-producing genes mcyA and mcyB from S1 to S10 showed that the sampling sites near Duchang had the highest abundance of microcystin-producing genes during the high water period, while the microcystin -producing genes mcyA and mcyB showed a more obvious spatial and temporal distribution trend (Fig. 4b, c ). The mcyA was accumulating higher in the central sampling sites (S4, S5) in summer and autumn; while mcyB was accumulating higher at S4 in summer (Fig. 4d, e ). In sediment, there was more mcyA accumulated at sampling sites at the lake inlet (S1, S3) in spring; while mcyB was higher at S3 (Fig. 5d-e ).
section_name: Spatial variation in the abundance of mcy genes

section_content: In the sediment, we found that both mcyA and mcyB were signi cantly positively correlated with WT NO 3 -N Chl-a, and negatively correlated with DO. mcyA was also signi cantly negatively correlated with OTUs, while mcyB was also signi cantly negatively correlated with NO 2 -N (p < 0. 05) (Fig. 6a ). In the water column, both mcyA and mcyB were signi cantly positively correlated with Chl-a and STP, and negatively correlated with OTUs, CO and NH 3 -N; while mcyA was signi cantly positively correlated with TP and DTP, and negatively correlated with DTN; and mcyB was signi cantly negatively correlated with DO, positively with NO3-N (p < 0. 05) (Fig. 6b ). In Fig. 7, it showed that NO 3 -N and Chl-a change signi cantly from June to October at ten stations. In order to elaborate the classi cation of physicochemical parameters, a principal component analysis (PCA analysis) was performed on all physicochemical parameters and bacterial population pro les. It was found that only in the water column the mcyA and mcyB differed signi cantly in time. It suggested that the variation in the toxin-producing genes of Microcystis aeruginosa in the water column is strongly related to the season (Fig. 6c-d ).
section_name: Analysis of the relationships between mcy genes with Physicochemical factors and key control factors

section_content: Comparing the abundance of mcyA and mcyB in sediment and water of the same stations in Fig. 4 and Fig. 5, it was obviously that the areas with higher abundance of mcy genes in the water column from June to August would have higher abundance of mcy genes in the sediment from next January to April. It was more obvious in S3 where the water ow was smoother, and mcyA was more easily measured in sediment than mcyB. Comparing the abundance of mcy genes in the sediment in different months (Fig. 5d, e), we found that microcystin levels in sediments accumulated with time. The following year, the areas with higher levels of microcystins in the sediment would detect an earlier increase in the abundance of mcy genes in the water column.
section_name: The relationship between sediment and water columns of variation in mcy genes

section_content: In the global warming scenario, the eutrophication of freshwater lakes has increased. To a certain extent, it leads to the massive growth and reproduction of cyanobacteria [34]. Excessive cyanobacteria eventually leads to an increase in the concentration of microcystins and has a serious impact on the ecosystem function. It is necessary to study the variation of toxin-producing genes of Microcystis aeruginosa in lake water. Poyang Lake is the largest freshwater lake in China and a globally important wetland with multiple functions [35]. As the Yangtze River pours and tops up the North Lake, the sediment carried by the Ganjiang River accumulates in the North Lake, which leads to the accumulation of sediment in the North Lake [36, 37]. According to the geographical location, S1 is near the Yangtze River inlet, S1-S3 are all near Xingzi County, S4 and S5 are near Duchang County, S1-S5 are located in the north lake area of Poyang Lake, and S6-S10 are in the south lake area of Poyang Lake. Since the intake of the Yangtze River is in the North Lake and S3-S5 are close to the urban area, we mainly focus on the sampling points in the North Lake area for the analysis. 

In this study, the microcystin-producing genes mcyA and mcyB showed signi cant spatial and temporal differences. It is closely related to the hydrological conditions and environmental factors of Poyang Lake. 

The spatial variation in the abundance of the toxin-producing gene in Microcystis aeruginosa is mainly caused by the changes in the nitrogen content of the water column and the water velocity in Poyang Lake; the temporal variation is mainly caused by the temperature and hydrological seasonal changes (Fig. 8 ). 

Microcystins in the water column increased from April to September in high water period, while in the sediment they increased from January to April in periods of low water abundance. On one hand, it is related to the physiological properties of Microcystis aeruginosa, especially its over-wintering characteristics. Water temperature is crucial for the growth of cyanobacteria. It is known that cyanobacteria can grow and develop over a wide range of temperatures. The most suitable temperature range is 20-35℃ and the higher temperatures are bene cial for their growth development and outbreaks [38]. Kong et al. [39] proposed a "four-stage theory" for the formation of cyanobacterial blooms, which suggested that cyanobacterial growth and bloom formation go through four stages: overwintering dormancy, spring recovery, growth and aggregation, and formation of blooms. Exploring survival, recovery and microcystin release of Microcystis aeruginosa in cold or dark condition, Yi Ding et al. [40] found that Microcystis aeruginosa has a higher winter survival vigor in dark, low-temperature conditions on the surface of lake substrates in winter. It was also found that Microcystis aeruginosa had a stronger overwintering survival rate when domesticated at 15℃. According to the over-wintering characteristics of Microcystis aeruginosa, Microcystis aeruginosa in the sediment started to recover with the increasing water temperatures in April. As the result, the toxin levels in the sediment began to rise in April. After April, Microcystis aeruginosa in the sediment accumulated and oated into the water column. As the result, the levels of the toxin-producing gene in the sediment decreased. For another hand, it was related to the hydrologic characteristics of Poyang Lake. The high water period of Poyang Lake was from April to September when temperature increased [41]. During high water period, the water levels rose and the nutrients in the water column increased, which is bene cial to the growth and reproduction of Microcystis aeruginosa; while during the dry water period, the density of Microcystis became higher and the nutrients were less conducive to the growth and reproduction of Microcystis. Before high water period, Microcystis aeruginosa started to rise and proliferated as water temperatures rose. The high water period began, and then the mcy genes in the water column increased from April to September. At the same time, Microcystis aeruginosa in the sediment was re-enriched into the water column in the following summer. The microcystins in the sediment accumulated annually, and most of Microcystis aeruginosa recovered as temperatures rise, rising to the surface, growing and reproducing; but there was also a proportion of them dormant in the sediment and accumulated annually [42, 43]. Although the biomass of cyanobacteria in the water column gradually decreased in autumn, the biomass on the substrate surface increased, suggesting that cyanobacteria settle in large numbers in sediment towards the end of the bloom [44]. This also resulted in the accumulation of large amounts of microcystins in organisms that feed on sediment in the water column. Microcystis sinking is widespread during water blooms, with studies showing that algal sinking uxes can exceed 10 9 cell/m 2 /d, with a maximum of 3. 7x10 10 cell/m 2 /d which has a higher proportion of algae sink in the water column in September and October [45, 46]. This is also consistent with the trend we found in the growth of toxin-producing genes in Microcystis aeruginosa in the water column. Consequently, the management of cyanobacterial blooms during high water period is essential to reduce microcystin levels in the water column. 

Except for seasonal changes, the changes in water quality can also regulate the concentration of microcystins in the water column. In general, water quality conditions are closely related to environmental factors such as hydrological conditions and nutrient concentrations. The temporal and spatial differences in physicochemical parameters in Poyang Lake were mainly due to water temperature and different nutrient levels [47]. Warming of the lake may lead to increased seasonal uctuations in internal loads and create favorable conditions for the production of cyanobacteria during the year [48]. The temporal differences in the physicochemical parameters were mainly caused by the differences in water temperature, DO, TP, DTP, NH 3 -N, Chl-a, while the spatial differences were mainly caused by the differences in transparency, DT, and SDT. These results suggested that the dramatic changes in hydrological conditions in Poyang Lake may have some impact on the physicochemical parameters of the lake water, and then these factors indeed affected structural changes in microcystins, including amino acids in the cell, nitrogen content, light intensity, and the C-N ratio of the available nutrients [49] [50] [51]. The overwintering characteristics of cyanobacteria were also closely related to the temperature, light, DO and redox potential in the sediment. 

The spatial variation in Microcystis aeruginosa toxin-producing gene abundance was mainly caused by the changes of nitrogen content and water velocity in Poyang Lake. The level of NO 3 -N shows signi cant spatial differences. In the S3-S5 stations, the N level was higher than that in other stations, while it was observed that the abundance of toxin-producing genes in Microcystis aeruginosa was higher in areas with high N levels. So the nitrogen concentrations in water bodies can cause changes in the level of microcystins. K. Kim et al. [52] found that nitrogen stimulated Microcystis dominated blooms more than phosphorus in water column. Nitrogen limitation has been proven to inhibit Microcystis proliferation [53]. 

The signi cant decline in Microcystis blooms in late summer or autumn has been considered to be related to nitrogen depletion in water [54]. A study found that nitrogen limitation (N limitation) was more limiting to the growth of toxic Microcystis aeruginosa than non-toxic Microcystis aeruginosa, especially when incubated under high light or high temperature [55]. In Microcystis aeruginosa, the nitrogen metabolism regulator NtcA band to the promoter region of the mcyA/D and upregulated the transcription of the mcyB gene, thereby promoting the production of MCs [56]. The growth of the toxic strain required a higher concentration of nitrogen than the non-toxic strain, while the toxic strain has the highest growth rate at higher nitrogen concentrations. The non-toxic strain does not require as much phosphorus and grows better in both low and high phosphorus conditions [57]. 

The abundance of Microcystis aeruginosa toxin-producing genes in the sediment was considerably higher in the central and eastern regions than in the inlet, which might attribute to the impact of water ow affecting the number of cyanobacterial cells in the sediment. Some cyanobacterial colonies containing pseudo-vacuoles, such as Microcystis aeruginosa, can form blooms on the water surface. These blooms are often larger than 1 mm in size. The formation of blooms enhances their oating ability and they can rapidly accumulate heavily at the water surface in calm situations [58]. Among the different sites, S4 and S5 near the Duchang area had smoother water ow, which favored the growth and reproduction of Microcystis aeruginosa, and therefore the abundance of mcy genes was higher in S4 and S5. Under wind-induced surface transport, large communities of Microcystis aeruginosa concentrated in the upper layers of the water might be more easily transported to downwind areas. Therefore, the toxicity of Microcystis communities might vary between upwind and downwind waters during bloom outbreaks. Microcystis aeruginosa communities were likely to be less toxic near upwind waters than near downwind waters. Poyang Lake has a southeasterly wind direction and the effect of wind speed is mainly on the smooth water surface, thus it can also explain the higher abundance of toxin-producing genes in Microcystis aeruginosa near the Duchang area. 

Additionally, we found many non-toxic Microcystis aeruginosa in Poyang Lake. Comparative photosynthesis showed that low light or nitrogen enrichment favors the competitive advantage of toxic Microcystis, while high light combined with nitrogen limitation promoted the competitive advantage of non-toxic Microcystis. Both competitive advantages can be further ampli ed by increased temperature [55]. It also reported that the ratio of toxic to non-toxic species signi cantly affectd the toxicity production of Microcystis aeruginosa [59] and that orthophosphate signi cantly affected the growth and reproduction of toxic cyanobacterial species, and ultimately affected the production of microcystins [60]. Thus, we can reduce the microcystins levels by control the levels of nitrogen in the water column. 

The abundance of mcy genes can be predicted from the variation in Chl-a as well. Chl-a level peaked in July in areas near the inlet and peaks in September in areas with smoother currents, which was similar to the variation in the abundance of the mcy genes. The concentration of chlorophyll in the water column re ects the amount of phytoplankton in the water. It suggested that the abundance of the toxin-producing gene of Microcystis aeruginosa affected the amount of chlorophyll in the water column [61, 62]. The Chl-a is a major photosynthetic pigment of the water column and thus the measurement of Chl-a can be a tracer of eutrophication in lakes [63]. Our results indicated severe eutrophication of water bodies in the northern lake area of Poyang Lake. As a result, it is possible to observe microcystins by monitoring the chlorophyll content of the water column. 

In summary, the abundance of toxin-producing genes of Microcystis aeruginosa in Poyang Lake was mainly spatially and temporally distributed. The temporal distribution was mainly in uenced by
section_name: Discussion

section_name: Acknowledgements

section_content: The authors thank the nancial support of National Natural Science Foundations of China (Grant 31960266 ; 4216010410 ) and Key Science and Technology Project of Jiangxi Province ( 20213AAG01012 )…
section_name: Acknowledgements

section_name: Funding

section_content: This study was nancially supported by National Natural Science Foundations of China (Grant 31960266 ; 4216010410 ) and Key Science and Technology Project of Jiangxi Province ( 20213AAG01012 )
section_name: Funding

section_name: Availability of data and materials

section_content: The authors con rm that the data supporting the ndings of this study are available within the article.
section_name: Availability of data and materials

section_name: Declarations

section_content: temperature and hydrological season. They in uenced the abundance of mcy genes by affecting the overwintering characteristics of Microcystis aeruginosa. While the spatial distributions of mcy genes were mainly determined by the nitrogen content and water velocity in the water column.

section_content: Ethics approval and consent to participate Not applicable.
section_name: Declarations

section_content: Not applicable.
section_name: Consent for publication

section_content: The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.
section_name: Competing interests

section_content: Lin Wu, Dawen Zhang, Tong Qiu contributed to the conception of the study; Lin Wu performed the experiment; Lin Wu, Li Zhang, Lijuan Yuan, Qiegen Liao, Jianjun Xiang contributed signi cantly to analysis and manuscript preparation; Jutao Liu, Junhui Guo performed the data analyses and wrote the manuscript. 

Spatial and temporal variation of environmental factors and bacterial communities. The microcystin-producing genes mcyA and mcyB showed signi cant spatial and temporal differences Supplementary Files This is a list of supplementary les associated with this preprint. Click to download. 

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section_name: Authors' contributions