Abstract: Cyanobacterial blooms can cause severe ecological and health problems in drinking water reservoirs. To alleviate this problem, allelopathically active submerged macrophytes can be used to reduce cyanobacterial growth. Accordingly, this study aimed to evaluate the sensitivity of strains of the Microcystis aeruginosa complex isolated from reservoirs with the presence and absence of submerged macrophytes to the allelochemicals of Ceratophyllum demersum. A coexistence experiment was carried out between the submerged macrophyte C. demersum and four Microcystis strains, with two treatments for each strain, one in coexistence with the submerged macrophyte (7 g L-1) and control (in the absence of the macrophyte). Two strains of M. aeruginosa (BMIUFRPE-06 and BMIUFRPE-07) and two of M. panniformis (BMIUFRPE-08 and BMIUFRPE-09) were used, which were isolated from Cajueiro (with submerged macrophytes) and Tapacurá (without submerged macrophytes) reservoirs, respectively. The biomass of Microcystis strains from the reservoir without macrophytes (BMIUFRPE-08 and BMIUFRPE-09) was significantly inhibited in 96% (T-test: p < 0.01) and 74% (T-test: p< 0.05), when compared to the control, respectively, with lower values of growth rates (ANOVA: p < 0.05). The strains isolated from the reservoir with macrophytes (BMIUFRPE-06 and BMIUFRPE-07) showed a growth delay (biomass reductions of 44 and 58%, respectively) in the coexistence treatment but without significant difference from the control on the sixth day of the experiment (biomass - T-test: p > 0.05; growth rate - ANOVA: p > 0.05). These results suggest that strains isolated from environments with submerged macrophytes are less sensitive to allelochemicals of these plants, as these strains may be adapted to the coexistence with submerged macrophytes.
Keywords: adaptation, allelopathy, aquatic plants, cyanobacterial blooms, Microcystis aeruginosa complex.
Ecologia
Growth of Microcystis strains isolated from environments with the presence and absence of submerged macrophytes in coexistence with Ceratophyllum demersum
Cihelio Alves Amorim alvescihelio@gmail.com
Recepción: 08 Febrero 2021
Aprobación: 27 Julio 2021
Many lakes and reservoirs may exhibit two main alternative states, one with clear water and high biomass of aquatic macrophytes, and one with turbid waters, distinguished by the high phytoplankton biomass and reduced macrophyte coverage (Scheffer, Carpenter, Foley, Folke, & Walker, 2001). In tropical waters, cyanobacterial blooms are frequent, usually due to warm climates and artificial eutrophication, which can alter the trophic interactions in the aquatic systems (Bittencourt-Oliveira, Piccin-Santos, Moura, & Aragão-Tavares, 2014; Moura, Aragão-Tavares, & Amorim, 2018; Amorim, Dantas, & Moura, 2020).
In recent years, the increase in temperature and eutrophication of water bodies has led to an increase in cyanobacterial blooms (Paerl & Otten, 2013; Burford et al., 2020), mainly those composed of the Microcystis genus, with records in 108 countries (all continents, except Antarctica) (Harke et al., 2016). These blooms negatively affect public supply reservoirs through the release of cyanotoxins, which compromise water quality, and reduce recreational and fishing activities (Davis & Koop, 2006). Among toxins, microcystins are the most studied and frequent in aquatic environments, which have been reported in 79 countries (Harke et al., 2016). Furthermore, there are more than 240 variants of microcystins, with different levels of toxicity (Svirčev et al., 2017), which can cause damage to aquatic macrophytes (Amorim, Ulisses, & Moura, 2017), zooplankton (Liang, Ouyang, Chen, Su, & Yang, 2017), fish (Boaru, Dragos, & Schirmer, 2006), and even humans (Carmichael et al., 2001).
As a control alternative, the introduction of submerged aquatic macrophytes has generated great results in reducing blooms in temperate and tropical regions (e.g., Dong, Yang, Li, Li, & Song, 2014; Amorim & Moura, 2020). Submerged macrophytes can act as a refuge for zooplankton, as they compete for light and nutrients and exudate allelochemicals; moreover, such mechanisms are the main action modes of these plants (Gross, 2003). Among the macrophytes, C. demersum L. is one of the most allelopathically active, which can release a greater amount of allelochemicals and strongly inhibit phytoplankton growth (Hilt & Gross, 2008).
Several studies have demonstrated the inhibition of cyanobacteria by the allelochemicals of aquatic macrophytes. For example, Mohamed (2017) reviewed studies showing that more than 40 aquatic macrophytes can inhibit phytoplankton through allelopathy. Most of these studies were performed with Microcystis spp. (e.g., Chang, Eigemann, & Hilt, 2012; Švanys, Eigemann, Grossart, & Hilt, 2016; Amorim, Moura-Falcão, Valença, Souza, & Moura, 2019). However, this genus has a wide variability in response to macrophyte allelochemicals; for example, non-microcystin-producing strains are more sensitive to the chemical compounds of these plants (Švanys et al., 2016).
Algal populations can present a wide genetic variability, which allows their adaptation to diverse environmental conditions, such as resistance to pollutants (Behra, Genoni, & Joseph, 1999) or zooplankton predation (Vanormelingen, Vyverman, De Bock, Van der Gucht, & De Meester, 2009). For Microcystis aeruginosa (Kützing) Kützing, some studies showed that the adaptation to different environmental pressures can be related to strain genetic variability, however, this has only been tested for microcystin producing or non-producing strains. For example, non-toxic strains are better competitors for light (Kardinäal et al., 2007), while toxic strains are favored under conditions of high temperatures and nutrients (Davis, Berry, Boyer, & Gobler, 2009).
Another factor that can influence the growth performance of organisms is their place of origin, as described by the ‘new weapons hypothesis’, which predicts that non-native species are efficient in controlling populations of native species when introduced into new environments (Callaway & Ridenour, 2004). However, over time, native species can adapt to coexist with exotic species as they become established in new locations (Reigosa, Sanchez-Moreiras, & Gonzalez, 1999). Accordingly, some studies have addressed the tolerance of cyanobacteria (Hilt, 2006) and planktonic green algae, Scenedesmus obliquus (Turpin) Kützing (Al-Shehri, 2010), to allelochemicals of submerged macrophytes over time.
Biomanipulation studies using submerged aquatic macrophytes have become frequent and have shown good results in cyanobacterial bloom control (e.g., Liu et al., 2018; Amorim & Moura, 2020). However, the previous exposure of cyanobacteria to these plants in their natural environment should be considered, as they can become adapted to the allelochemicals. Accordingly, we evaluated the sensitivity of four strains of the M. aeruginosa complex isolated from reservoirs with the presence and absence of submerged macrophytes to the C. demersumL. allelochemicals. We tested the hypothesis that strains isolated from environments without submerged plants are more sensitive to allelochemicals than those with a history of macrophyte coexistence.
In the experiments, four strains of Microcystis were used: two M. aeruginosa (BMIUFRPE-06 and BMIUFRPE-07) and two M. panniformis Komárek et al. (BMIUFRPE-08 and BMIUFRPE-09). The strains were isolated from field samples and identified based on morphological characteristics (Komárek & Anagnostidis, 1986; Nguyen, Tanabe, Matsuura, Kaya, & Watanabe, 2012). Both species are included in the M. aeruginosa complex (MAC) due to their high genetic similarity (Harke et al., 2016). Although they presented different morphologies in the field, all studied strains presented the same unicellular form, with few small colonies under laboratory conditions.
The strains of M. panniformis were isolated from the Tapacurá reservoir, located at the municipality of São Lourenço da Mata - PE, in October 2017, and characterized by the absence of submerged macrophytes (Moura Júnior, Pott, Severi, & Zickel, 2019). The strains of M. aeruginosa were isolated from the Cajueiro reservoir, in the municipality of Garanhuns - PE, which has extensive stands of the submerged macrophytes Egeria densa Planch., Chara spp. and C. demersum (based on field observations during sampling dates), in November 2017. Other studies have shown that these macrophytes have the potential to inhibit M. aeruginosa through allelopathy (e.g., Van Donk & Van de Bund, 2002;Vanderstukken, Mazzeo, Van Colen, Declerck, & Muylaert, 2011; Dong et al., 2014). Both reservoirs have similar limnological and climatic characteristics (climate type As in the Köppen classification) and annual Microcystis blooms (Amorim et al., 2020). Details of the limnological conditions during the sampling dates are described in Table 1 (see Amorim & Moura, 2021).
The strains were isolated using the micro-pipetting technique and kept under acclimation under the respective laboratory conditions: temperature (25°C ± 1.5), light intensity (30 μmol m-2 s-1), pH (7.5), photoperiod (12 hours), they were also homogenized three times a day. The strains were deposited at the Microalgae Culture Collection of the Universidade Federal Rural de Pernambuco - BMIUFRPE. Cultures of isolated cyanobacteria were maintained in flasks containing ASM1 nutrient medium (Gorham, Mclachlan, Hammer, & Kim, 1964), in a climatic chamber under controlled conditions.
Thirty days before the experiments, young and apical branches of C. demersum were collected in the Carpina reservoir, in the municipality of Lagoa do Carro - PE (Table 1) and transported to the laboratory. After sampling, the plants were washed several times, with a soft bristle brush and jets of distilled water to remove sediments and periphyton. The cultures were carried out in aquariums containing tap water (which remained resting for five days for chloride removal), with dimensions of 20 cm3 and capacity of 8 L, under the same above-mentioned conditions, and with constant aeration.
The experiments were carried out in an air-conditioned room with the same conditions described for the culture of the strains. Erlenmeyer flasks with a capacity for 1000 mL containing 500 mL of ASM1 culture medium were inoculated with the cyanobacterial strains in the exponential growth phase from the stock cultures (mean biomass of 23.5 mg L-1).
Three days before starting the experiments, the plants were washed several times with distilled water, after that, they were transferred to Erlenmeyer flasks with ASM1 culture medium for acclimatization. Two treatments (control and coexistence with macrophyte) were established for each Microcystis strain, with four replicates for each condition. The control treatment consisted of a culture of strains in the absence of the plant, while in the coexistence treatment a branch of macrophyte (7 g L-1 of fresh weight) was added to the cyanobacterial cultures. This amount of plant was chosen because biomasses of C. demersum higher than 5 g L-1 present greater allelopathic effects on cyanobacteria (Gross, Erhard, & Iványi, 2003).
Negative controls, using plastic plants, were not employed because some studies have shown that macrophytes cause no physical interference on phytoplankton and cyanobacteria (e.g., Vanderstukken et al. 2011, Vanderstukken, Declerck, Decaestecker, & Muylaert, 2014). The higher nutrient concentration in the ASM1 media excluded the possibility of nutrient limitation, while the regular homogenization of the cultures provided similar light availability to all Microcystis cells, preventing light limitation.
Aliquots of 1 mL of all experimental units were collected on days 0, 2, 4, and 6 to determine cyanobacterial cell densities, by counting the cells in a Fuchs-Rosenthal chamber (hemocytometer). The biovolume was determined according to the calculation of the geometric forms proposed by Sun and Liu (2003). The biomass of the strains was estimated by multiplying the density by the biovolume. The growth rates (μ) of the cyanobacterial strains were calculated according to Wood, Everroad, & Wingard (2005): μ (d-1) = (ln(Nt) - ln(Nt0))/t - t0, where N represents the biomass values after six days of the experiment (t) and at the initial day (t0).
The growth of branches of C. demersum in coexistence with Microcystis was analyzed. For that, branches were weighed on an analytical balance, at the beginning (day 0) and the end (day 6) of the experiment. Both were evaluated based on the fresh weight of the macrophyte.
Significant differences between the treatments for each strain during each sampling day, and in the growth of C. demersum (coexistence treatments) were verified through the Student's T-test (normal data) and the Mann-Whitney U test (non-normal data). A two-way ANOVA was performed to compare the growth rates (between treatments and strains) after six days of the experiment. Before the analyses of variances, data normality and homoscedasticity were verified by the Kolmogorov-Smirnov and Bartlett's tests, respectively. Statistical analyses were performed in the R program and the level of significance was adjusted to p < 0.05 (R Core Team, 2019).
The aquatic macrophyte C. demersum affected the growth of Microcystis strains differently and depending on the origin and history of the strains. Strains isolated from the reservoir with submerged macrophytes (BMIUFRPE-06 and BMIUFRPE-07) showed a decrease in growth, beginning on the fourth day in the coexistence treatment compared to control. On the 6th day, BMIUFRPE-06 and BMIUFRPE-07 decreased biomass by 44 and 58%, respectively; however, without significant differences between the treatment with macrophytes and the control (Figure 1 A and B). In contrast, the growth of strains isolated from the reservoir without submerged macrophytes (BMIUFRPE-08 and BMIUFRPE-09) was significantly inhibited in the coexistence treatment from the fourth day until the end of the experiment, when the strains biomass was reduced by 96 and 74%, respectively (Figure 1 C and D). Both strains isolated from the reservoir without submerged plants reached lower biomasses on the sixth day than at the beginning of the experiment (Figure 1 C and D).
The strains isolated from the reservoir with macrophytes showed no differences in growth rates between the control and coexistence treatments. However, in the coexistence treatment, the BMIUFRPE-06 strain showed a lower growth rate (0.14 d-1) compared to the BMIUFRPE-07 strain (0.40 d-1). On the other hand, the strains isolated from the reservoir without macrophytes presented significant reductions in the growth rates when in coexistence with C. demersum. Lower values of growth rates in the coexistence treatment were observed for the strain BMIUFRPE-08 (-0.05 d-1), followed by BMIUFRPE-09 (0.01 d-1). These two strains differed in growth rates in control conditions, but not in the coexistence treatment due to the intense inhibition of growth (Figure 2).
Based on the fresh weight of the branches of the aquatic macrophyte in the initial and final days of the experiment, we verified that C. demersum increased its biomass in coexistence with all Microcystis strains (p < 0.05), compared to the first day of the experiment (Figure 3). The branches showed similar biomasses in coexistence with all strains, varying from 8.2 to 9.2 g L-1.
In this study, we evaluated the growth of Microcystis strains isolated from reservoirs, which differed in the presence or absence of the allelopathically active submerged macrophytes. Thus, we observed that Microcystis strains isolated from environments with submerged macrophytes, including C. demersum, were less affected by the coexistence with this plant. In contrast, Microcystis strains isolated from environments without submerged macrophytes were more sensitive to the allelochemicals and their growth was inhibited at the end of the experiment.
The ‘new weapons hypothesis’ predicts that plants are efficient at controlling populations of native species when introduced into new environments (Callaway & Ridenour, 2004). In this sense, the introduction of aquatic species in a water body promotes ecological changes through the stabilization of new species (Kumar, 2000). Thus, their development affects native communities (Vitousek, D’antonio, Loope, & Westbrooks, 1996). Therefore, the introduction of a submerged macrophyte into an environment without previous records of these plants, for example, the Tapacurá reservoir (Moura Júnior et al., 2019), can inhibit the cyanobacterial strains that are not adapted to the presence of allelochemicals. However, native species can adapt to the presence of these new individuals over time (Reigosa et al., 1999). In this case, cyanobacteria, when in coexistence with submerged macrophytes for long periods, can tolerate the presence of plant allelochemicals and thus not be strongly affected, as observed in the Cajueiro reservoir.
Studies aiming to evaluate the adaptation of phytoplankton strains to allelochemicals of aquatic macrophytes are scarce. Among them, Al-Shehri (2010) showed that a strain of Scenedesmusobliquus, isolated from a lake with extensive stands of Stratiotes aloides L., was tolerant to macrophyte allelochemicals, while a strain isolated from a lake without S. aloides showed reduced growth and higher oxidative stress. However, in another study, the sensitivity of Pediastrumduplex Meyen strains to the presence of macrophytes found in their place of origin was not confirmed. The strains isolated from environments with or without Myriophyllum spicatum L. presented the same sensitivity to plant allelochemicals (Eigemann, Vanormelingen, & Hilt, 2013). These contrasting results are due to the fact that green algae are less sensitive to macrophyte allelochemicals, when compared to cyanobacteria and diatoms (Körner & Nicklisch, 2002; Hilt, Ghobrial, & Gross, 2006; Hilt & Gross, 2008; Zhu, Liu, Wang, Gao, & Wu, 2010; Chang et al., 2012; Amorim et al., 2019), or because of the variable sensitivity among strains or species (Mulderij, Van Donk, & Roelofs, 2003; Al-Shehri, 2010; Amorim et al., 2019).
In our study, the presence of submerged macrophytes in the reservoirs where Microcystis strains were collected played an important role in their sensitivity. This result may indicate that the previous coexistence of Microcystis strains with submerged macrophytes during many years may provide resistance to the allelochemicals for cyanobacteria. Moreover, native species can coevolve with other coexisting species when the introduced species stabilize in the new ecosystem (Reigosa et al., 1999). However, little is known about the adaptation of Microcystis spp. in the presence or absence of allelopathically active macrophyte species, and whether this characteristic contributes to make it a widespread cyanobacterium in water bodies worldwide, as already reported (Harke et al., 2016).
Our results showed that the presence of allelopathically active submerged macrophytes (e.g., Egeria densa, Chara spp., and C. demersum) could be an important factor in the adaptation and selection of resistant strains of Microcystis capable of growing in the presence of the allelochemicals in the Cajueiro reservoir. The construction of this reservoir was finished in 2010 (APAC, 2020), which allowed the coexistence of strains with the submerged macrophytes for more than eight years. Considering that Microcystis spp. can rapidly adapt to environmental stressors through the selection of resistant cells, they can maintain their growth even under unfavorable conditions. For example, López-Rodas et al. (2007) demonstrated that sensitive strains of M. aeruginosa became resistant to the glyphosate in less than 60 days of exposure. Also, pre-selective mutant cells, that were occasionally found among the sensitive cultures, ensured the adaptation of the strains due to the increased growth of tolerant cells under stress conditions.
The use of submerged macrophytes to control eutrophication and cyanobacterial blooms has become frequent (Yu et al., 2016; Liu et al., 2018; Amorim & Moura, 2020). The main mechanism of action of submerged macrophytes in the inhibition of cyanobacteria is allelopathy (Hilt & Gross, 2008). Besides, submerged macrophytes, especially C. demersum, can control cyanobacteria even in environments with high concentrations of phosphorus (Mjelde & Faafeng, 1997), with competition for nutrients playing a lesser role compared to allelopathy (Lombardo & Cooke, 2003). This is because C. demersum is one of the submerged macrophytes with the greatest allelopathic potential (Hilt & Gross, 2008). Moreover, in our study, we prevented nutrient and light limitation for cyanobacterial growth through the use of a nutrient-rich culture medium and the regular homogenization of the cultures, which ensure adequate levels of nutrients and light to all Microcystis cells, respectively.
The high potential of C. demersum in the control of cyanobacteria was also demonstrated in a field experiment in a tropical reservoir in Northeastern Brazil, in which the introduction of this plant was efficient to reduce up to 85% of the total cyanobacterial biomass, in an environment with dense Microcystis spp. blooms and without the presence of submerged plants (Tapacurá reservoir) (Amorim & Moura, 2020). This intense reduction in the cyanobacterial bloom was attributed to allelopathy, considering that the cyanobacteria biomass was reduced by the macrophyte even after the addition of nutrients. In this case, the physicochemical parameters remained stable, there was no limitation by light or nutrients, and the zooplankton had a lesser effect on the bloom control (Amorim & Moura, 2020). This result may indicate that the introduction of a macrophyte in an environment without submerged vegetation can reduce the biomass of cyanobacteria, considering that the phytoplankton of this environment was not previously adapted to allelochemicals of this new plant, therefore corroborating the results of the present study. Nevertheless, long-term coexistence between cyanobacteria and submerged macrophytes should be considered in biomanipulation strategies with the use of submerged macrophytes since previous interactions may reduce the sensitivity of cyanobacteria to allelochemicals.
Several studies have demonstrated the adaptation of Microcystis spp. to environmental conditions, especially toxic or non-toxic strains, including light intensity, temperature, nutrient concentrations, and the presence of reactive oxygen species (e.g., Davis et al., 2009; Dziallas & Grossart, 2011). However, the adaptation to the macrophytes allelochemicals has not yet been tested for bloom-forming cyanobacterial strains. Therefore, this study indicates for the first time that strains of cyanobacteria from environments with a large abundance of submerged macrophytes may be less sensitive to allelochemicals.
Despite its potential to slow or inhibit the growth of all tested Microcystis strains, C. demersum was not affected by the coexistence with cyanobacteria, showing a significant increase in its biomass in short-term exposure to all tested strains. Amorim et al. (2017) also demonstrated that the short-term exposure (three weeks) of the submerged macrophyte Egeria densa Planch. to toxic and non-toxic strains of Microcystis did not cause morphological changes or reduction in the biomass of the plant. This result was related to the efficiency of the E. densa antioxidant system, to prevent harmful effects of cyanobacteria. However, prolonged exposures (five weeks), both to toxic and non-toxic strains, caused a reduction in length, fresh weight, and the emission of shoots and roots in E. densa (Amorim et al., 2017). Ceratophyllum demersum also has an efficient antioxidant system that reduces the harmful effects of cyanobacteria and cyanotoxins on its physiology, in addition to absorbing and transforming cyanotoxins into non-toxic molecules (Pflugmacher, 2004). Therefore, this macrophyte can be used to control cyanobacterial blooms and cyanotoxins. In addition to this macrophyte, the other species present in Cajueiro reservoir, including E. densa and Chara spp., also exhibit the potential to inhibit cyanobacteria (Mulderij et al., 2003; Vanderstukken et al., 2011).
In conclusion, Microcystis strains were inhibited by C. demersum differently, i.e., strains from environments without macrophytes were strongly inhibited, while strains from environments with submerged plants were less sensitive and showed a growth delay. Thus, we confirmed the hypothesis that strains of Microcystis isolated from reservoirs without submerged macrophytes are more sensitive to C. demersum allelochemicals; however, strains from environments with extensive submerged plant stands were less affected when grown in coexistence with the plant, which demonstrates their adaptation to allelochemicals over the years of coexistence in natural habitats.
We thank to the Conselho Nacional de Desenvolvimento Científico e Tecnológico -CNPq for a productivity grant to ANM (Process 305829/2019-0); and the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco - FACEPE for a scholarship to RHMF.
alvescihelio@gmail.com
