Effects of toxic Microcystis genotypes on natural colony formation and mechanism involved

Microcystis is a globally distributed bloom-forming genus of cyanobacteria (Haande et al. 2007; Shen & Song 2007). In natural waters, Microcystis cells are enveloped by an amorphous mucilage sheath and form colonies with different sizes and morphologies (Haider et al. 2003; Wang et al. 2015). Colony formation can protect Microcystis cells from zooplankton grazing, viral or bacterial attack, desiccation and other potential negative environmental factors, and increase floating velocity, all of which provide a competitive advantage over other phytoplankton genera (Kearns & Hunter 2000; Wu et al. 2007; Xiao et al. 2012; Yang & Kong 2012). However, the mechanisms underlying how and why Microcystis form colonies remain poorly understood.

When Microcystis colonies isolated from the natural waters are cultured in the laboratory, their colonial phenotype is hard to maintain and always disaggregates into unicellular cells (Bolch & Blackburn 1996; Yang et al. 2006). This morphological variation is apparently related to the lack of some environmental factors that stimulate colony formation in laboratory cultures (Li & Dai 2016), and these environmental factors may be contributing to colony formation in the natural conditions. Various biotic and abiotic factors, such as zooplankton grazing pressure (Yang et al. 2008), heterotrophic bacteria co-cultivation (Shen et al. 2011), fluid motion (Li et al. 2013a), ultraviolet radiation (Quesada & Vincent 1997), high calcium levels (Wang et al. 2011), elevated heavy metal concentrations (Bi et al. 2013), low temperature (Trainor 1993) and illumination (Li et al. 2013b), have been identified to be stimulators of Microcystis colony formation in laboratory experiments.

Co-culture investigations have proved that environmental factors could affect the selection of toxic and non-toxic genotypes, and toxic cells always dominated in the growth-limiting conditions (Dziallas & Grossart 2011; Leblanc Renaud et al. 2011; Lei et al. 2015). Toxic Microcystis genotypes containing microcystin synthetase (mcy) gene cluster could produce and secrete microcystins (MCs) (Tillett et al. 2000). Gan et al. (2012) found that addition of MCs led to increased Microcystis colony size, while depletion of MCs caused colony size to decrease. It was suggested that MCs released by toxic genotypes acted as an internal factor that played a pivotal role in the development of Microcystis colonies. This knowledge, however, was obtained via laboratory experiment and needed to be proved in natural conditions. Toxic genotypes always coexist with non-toxic genotypes in natural Microcystis colonies (Davis et al. 2009; Li et al. 2012). Kurmayer et al. (2003) found that the proportion of toxic Microcystis genotypes varied within colonies in Lake Wannsee (Germany). The abundance and proportion of toxic Microcystis genotypes always varies in spatial and temporal scales in different freshwater systems (Li et al. 2014). More field data from a range of freshwater ecosystems are needed to assess the effects of toxic genotypes and MCs production on wild Microcystis colony formation.

Haihe River is the largest water system in North China. Its mainstream flows through Tianjin City and empties into Bohai Bay (Wang et al. 2014). The mainstream of Haihe River is an important urban emergency reserve water source, and also acts as a flood drainage channel in Tianjin City (Dai & Yu 2014). However, it has been seriously polluted by industrial, agricultural and domestic effluents (Zhao et al. 2014), and suffers annual cyanobacterial blooms dominated by Microcystis during summer (Zhou et al. 2013).

In this study, a field investigation, including the proportion of toxic Microcystis genotypes and MC cellular production of toxic genotypes in four different colony size classes, was conducted in the mainstream of Haihe River. These findings are helpful in elucidating the relationship between the proportion of toxic Microcystis genotypes and colony formation, as well as MCs cellular production of toxic genotypes and colony formation during blooms.

1 L of integrated water sampled per sampling site was fixed with Lugol's solution. Phytoplankton species were identified according to the methods of Hu & Wei (2006) and counted using a blood counting chamber under microscope (×40). Microcystis colonies were separated before being counted according the method of Bi et al. (Bi et al. 2015). To separate super-large colonies (>90 μm), large colonies (20–90 μm), middle colonies (8–20 μm) and small colonies (0.45–8 μm), water samples were poured gently through sieves (Lingyun Rubber and Plastic Co., Ltd) with four different mesh sizes (90, 20, 8 and 0.45 μm) within minutes (Yang et al. 2009; Bi et al. 2015).

Water samples and seperated algal cell suspensions of each colony size class were filtered with GF/C filters, respectively. DNA extration was performed according to the protocol of Rinta-Kanto (Rinta-Kanto et al. 2005). Briefly, GF/C filters cut into pieces were subjected to lysis buffer (40 mM EDTA–400 mM NaCl–50 mM Tris–HCl [pH 9.0]. Cell lysis was achieved by the addition of lysozyme (2 mg mL⁻¹, 37 °C, 30 min) and subsequently proteinase K (100 μg mL⁻¹ in 2% sodium dodecyl sulfate, 50 °C, 2–3 h). After extraction with phenol–chloroform–isoamyl alcohol (25:24:1 [V/V/V]) once and chloroform–isoamyl alcohol (24:1 [V/V]) twice, DNA was precipitated using 100% (V/V) ethanol and 10 mol L⁻¹ ammonium acetate solution (−20 °C, overnight). The purified DNA was resuspended in 100 μL of Millipore water after washing with 70% (V/V) ethanol.

The real-time polymerase chain reaction (PCR) assay was used to quantify two genetic elements, the PC-IGS and mcyA regions. The Microcystis PC-IGS region, which is specific to the Microcystis genus, was used to quantify the abundance of the total Microcystis population (Kurmayer & Kutzenberger 2003). The mcyA gene found within the microcystin synthetase gene operon, which only appears in toxic genotypes of Microcystis, was used to quantify the toxic Microcystis population (Yoshida et al. 2007). PC-IGS was amplified using 188F (5′-GCT ACT TCG ACC GCG CC-3′) and 254R (5′-TCC TAC GGT TTA ATT GAG ACT AGC C-3′) primers (Chen et al. 2003), and the mcyA gene was amplified using M1r-F (5′-AGC GGT AGT CAT TGC ATC GG-3′) and M1r-R (5′-GCCCTT TTT CTG AAG TCG CC-3′) primers (Yoshida et al. 2007).

Standards used to determine the PC-IGS and mcyA gene copy numbers were prepared using the genomic DNA of M. aeruginosa strain PCC7806 provided by the FACHB Collection (Freshwater Algal Culture Collection of Institute of Hydrobiology, China). Algal cells from a known volume of the M. aeruginosa 7806 culture were filtered through 0.22 membranes, and the DNA was extracted as described above. The DNA concentration (ABS260 nm) and purity (ABS260 nm/ABS280 nm) were determined spectrophotometrically. The approximate genome size of M. aeruginosa 7806 is 5.2 Mb (Frangeul et al. 2008), and the average molecular weight of 1 bp was 660 g mol⁻¹ (Vaitomaa et al. 2003). Copy numbers of both genes on the basis of DNA were evaluated according to the method of Vaitomaa et al. (2003). A 10-fold dilution series of the DNAs was prepared and amplified with the PC-IGS and mcyA real-time PCR assays. Linear regression equations for obtained cycle threshold values (Ct values) were then calculated as a function of known mcyA and PC-IGS copy numbers according to Vaitomaa et al. (2003).

The real-time PCR was performed in a volume of 25 μL reaction mixture containing 12.5 μL 2 × SYBR Premix EX Taq™ (TaKaRa, Japan), 1.0 pmol of each primer, 2 μL DNA from a standard strain or 2 μL DNA from water sample, and filled up to 25 μL with sterile ultra-pure water. The PCR was carried out on the Mastercycler realplex 4 system (Eppendorf, Germany) with Mastercycler realplex software. Amplification of PC-IGS was performed as follows: initial denaturation at 95 °C for 2 min, followed by 40 cycles at 95 °C for 20 s, annealing at 56 °C for 30 s, and elongation at 72 °C for 30 s. For mcyA, the amplification included initial denaturation at 95 °C for 2 min, followed by 40 cycles: 95 °C for 20 s, 58 °C for 30 s, and 72 °C for 20 s. The melting temperature for the real-time PCR products was determined using the manufacturer's software. All of the samples were amplified in triplicate.

For intracellular MCs analysis, the algal cell suspensions of each colony size class were centrifuged first (10,000 rpm, 10 min) at 4 °C, and then subjected to repeated freezing (−20 °C) and thawing (4 °C). After centrifugation, the supernatant I was collected and the cell pellet was stirred with 5% (V/V) acetic acid for 20 min, and then centrifuged to collect supernatant II. The left cell pellet was stirred with 80% (V/V) aqueous methanol for 20 min, and then centrifuged to collect supernatant III. The methanol left in supernatant III was removed via rotary evaporation. The supernatant I, II and III were mixed and filtered through a 0.22 μm membrane to prepare filtration for the subsequent solid phase extraction (SPE).

MCs were extracted using an SPE C₁₈ cartridge, which yielded 78.53% and 81.49% recovery for MC-RR and MC-YR, respectively. The SPE C₁₈ cartridge activated with methanol was cleaned using distilled water, and then loaded with filtration prepared. The flow rate was 8–12 mL/min. After washed with distilled water and 20% (V/V) aqueous methanol in sequence, MCs was eluted using 60% (V/V) aqueous methanol (with 0.05% [V/V] trifluoroacetic acid). The elute collected was rotary evaporated to dryness, redissolved in 60% (V/V) aqueous methanol, and then filtrated through a 0.22 μm membrane prior to high-performance liquid chromatography (HPLC) analysis.

Extracted MCs were analyzed using HPLC System (SPD-M20A, Shimadzu, Japan) equipped with a Shim-Pack VP-ODS column (250 mm × 4.6 mm), using 60% aqueous methanol (with 0.05% (V/V) trifluoroacetic acid) at a flow rate of 1 mL/min. MCs (MC-RR and MC-YR) were identified using their characteristic absorption spectra. Certified standards of MC-RR and MC-YR (Sigma, USA) were used to create standard curves.

On the same sampling date and sampling site, relationships between MCs (MC-RR, MC-YR and sum of MC-RR and MC-YR) cellular production of toxic Microcystis genotypes and the proportion of toxic Microcystis genotypes in four colony size classes were analyzed using Pearson's correlation. All statistical tests were conducted by statistical software (SPSS Version 17.0) and P < 0.05 was considered significant.

MCs cellular production of toxic Microcystis genotypes were expressed as microgramme (μg) equivalents of MC per 10⁸ copies of the mcyA gene in different colony size classes. As shown in Table 2, MC-RR cellular production of toxic Microcystis genotypes in the mainstream of Haihe River varied with sampling dates. On each sampling date, MC-RR cellular production of toxic Microcystis genotypes decreased with increasing colony sizes at each sampling site, and MC-YR cellular production of toxic Microcysti genotypes presented the same change tendency (Table 3). Both the highest MC-RR and MC-YR cellular productions were found in colonies with a size of <8 μm, followed by colonies with sizes of 8–20 μm, 20–90 μm and >90 μm.

On each sampling date and at each sampling site, MCs (MC-RR, MC-YR and the sum of MC-RR and MC-YR) cellular production of toxic Microcystis genotypes in each colony size class was negatively correlated with the proportion of toxic Microcystis genotypes in each colony size class (Table 4). All coefficient values were below −0.57 except the one that was found at GH on August 15.

During 2015, the mainstream of Haihe River hosted cyanobacterial blooms dominated by Microcystis from July to October. Consistent with previous reports (Davis et al. 2009; Li et al. 2012), we found that toxic Microcystis genotypes coexisted with non-toxic genotypes during Microcystis blooms. Toxic Microcystis cells comprised between 0.82% and 23.98% of the total Microcystis population at five sampling sites, a range smaller than 4.4–64.3% in Lake Taihu (China) (Li et al. 2012) and 0.5–35% in Lake Mikata (Japan) (Yoshida et al. 2007). The lower proportion of toxic Microcystis (0.01–0.6%) was found in Lake Champlain, Lake Agawam, and Mill pond (USA) (Davis et al. 2009). In these freshwater ecosystems, dominance of non-toxic Microcystis was a more general phenomenon.

It has been found that the proportion of toxic Microcystis varies greatly in different colony size classes. In Lake Taihu, the larger size colonies had a higher proportion of potential toxic genotype among four Microcystis colonies with size of <50 μm, 50–100 μm, 100–270 μm and >270 μm. Kurmayer & Kutzenberger (2003) estimated the proportion of toxic genotypes within Microcystis colonies to be 1.7–71% in Lake Wannsee (Germany). The larger colony size classes (>100 μm) showed the highest proportion of toxic genotypes, whereas in the smaller size classes of Microcystis colonies (<100 μm), toxic genotypes were less abundant (Kurmayer et al. 2003). This is in consistent with our results in spite of the fact that colonies were separated in different sizes in the mainstream of Haihe River. As compared to toxic genotypes, non-toxic Microcystis genotypes are better competitors for light (Kardinaal et al. 2007) and have a better fitness in low-light conditions (Sabart et al. 2013). Meanwhile, larger colonies are known to perform more pronounced vertical migration via buoyancy regulation than smaller colonies and are able to use light more efficiently than smaller colonies (Wallace et al. 2000). It could be potentially suggested that colony size and the proportion of toxic genotypes might be involved in competition for environmental resources, e.g. light. This hypothesis requires further testing.

In Lake Taihu, the MC cell quota calculated for all Microcystis cells tended to be higher during blooms in colonies with a larger size (Wang et al. 2013). Considering that non-toxic Microcystis cells have no MC-producing capacity, the MC cell quota calculated using the above method could not reflect the actual MC-producing ability of toxic cells. In the mainstream of Haihe River, MC cellular production was calculated only for the cells carrying the mcyA gene, and it tended to be higher in colonies with a smaller size. The mainstream of Haihe River, as an important urban emergency reserve water source for Tianjin City, is a semi-enclosed water body enclosed with floodgates (Dai & Yu 2014). When the mainstream of Haihe River suffered cyanobacterial blooms, regular water diversion from Luanhe River or Yuqiao Reservoir was adopted to replace cyanobacteria-polluted water (Tian 2015). Water replacement measure took away most surface water and large colonies, because large colonies have faster vertical migration (Nakamura et al. 1993) and mainly concentrate at the water surface to form blooms (Wu & Kong 2009). Microcystis cells in small-sized colonies were more susceptible to stress and grazing (Yang & Kong 2012), and less competitive for light and nutrients as compared to cells in larger-sized colonies (Wallace et al. 2000). To protect cells against competitors and/or predators, a small-sized colony needed to aggregate to a larger-sized colony. It was known that the extracellular polysaccharides (EPS) in the mucilage or sheath of Microcystis could contribute to colony formation. As demonstrated by the results of Gan et al. (2012), MCs could stimulate colony formation in Microcystis via upregulating part of the polysaccharide biosynthesis-related genes. In the present study, we found that the smallest size class of Microcystis colony (<8 μm) showed the highest MCs cellular production of toxic Microcystis, suggesting that toxic Microcystis in a small-sized colony is needed to produce more MCs to stimulate the formation of a larger-sized Microcystis colony via increasing EPS production.

In all four colony size classes, there was a negative correlation between MCs (MC-RR, MC-YR, sum of MC-RR and MC-YR) cellular production of toxic Microcystis genotypes and the proportion of toxic Microcystis genotypes. With the increasing colony sizes, MCs cellular production decreased. The less there were toxic Microcystis cells able to produce MCs, the more each toxic cell needed to produce that molecule. It has been suggested that toxic Microcystis plays an important role in the colony formation via producing the intraspecific signaling moleculars of MCs in the natural environment.

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 31640009 and 31300393), Tianjin Natural Science Fund (Grant No. 16JCYBJC29900), Project of Tianjin Science and Technology (Grant No. 15ZCZDNC00230), China Postdoctoral Science Foundation (Grant No. 2015T80212 and 2014M551013), Tianjin Innovative Team of Aquaculture Industry Technology System, and the Open Fund of Tianjin Key Laboratory of Aquatic Ecology and Aquaculture (Grant No. TJAE201501).