Cyanobacterial and microalgal bioactive compounds – the role of secondary metabolites in allelopathic interactions

Organic compounds not directly involved in the normal growth, development and reproduction of living organisms are called secondary metabolites. Many of these compounds demonstrate biological activity, and potentially could affect the growth and development of biological systems. This process is observed worldwide and is known as allelopathy. This term was originally used by Molish (1937) to describe biochemical interactions between plants in terrestrial environments. In 1984, Rice defined allelopathy as any direct or indirect, negative or positive effect of chemical substances, produced and secreted by plants and microorganisms, on other plants and microorganisms. This definition was further developed by the International Allelopathy Society (IAS) in 1996. According to IAS, the term ‘allelopathy’ refers to any process induced by secondary metabolites produced by bacteria, fungi, algae and plants, which affects the growth and development of biological and agricultural systems. Due to their potential application in agriculture and forestry, biochemical interactions between terrestrial plants are well known. First observations were made in antiquity, for example the influence of Juglans nigra and Juglans regia on other species (Gniazdowska et al. 2004). Since then, many bioactive compounds produced by higher plants have been isolated and identified. Allelopathy, however, is not limited to terrestrial environments. Interactions caused by allelochemicals are widespread and common in freshwater, brackish and marine habitats, and occur among aquatic producers belonging to different taxonomic groups (Cembella 2003; Gross 2003). In comparison to the existing knowledge of allelopathy in terrestrial ecosystems, allelopathic interactions in aquatic habitats are still not well understood. In aquatic environments, allelochemicals may function as agents capable of incapacitating or killing the competitors. This is a chemical defense strategy and probably an important adaptive factor. Species which produce one or more bioactive compounds harmful to other species could compete better and achieve the dominance (Mulderij et al. 2005; Tillmann et al. 2008). It is known that production of bioactive secondary metabolites is highly species- or even strain-dependent (Leflaive & Ten-Hage 2007). Furthermore, allelopathic compounds released by donor species into water need to be properly hydrophilic and reach target species in effective concentrations (Gross 2003). According to the literature, allelochemicals can have several modes of action and can affect different physiological processes in living organisms, often simultaneously. Usually the influence is negative and the most common effect observed is the inhibition of target species growth. Liu et al. (2013) noticed the negative influence of Aegiceras corniculatum, the mangrove plant common in coastal and estuarial areas from India to Australia, on the diatom Cyclotella caspia. Gallic acid produced by A. corniculatum causes a reduction in growth and morphological changes in target species cells. Two seagrass species, Zostera marina and Zostera noltii, caused inhibition of growth of the toxic red tide dinoflagellate Alexandrium catanella (Laabir et al. 2013). Higher plants can also be affected by metabolites produced by microalgae or cyanobacteria. For example, living cells of Microcystis aeruginosa inhibited the growth of Lemna japonica (Jang et al. 2007), while crude extracts from M. aeruginosa cultures induced an oxidative stress in Lemna gibba (Saqrane et al. 2007). There are several reports of interactions between macrophytes and phytoplankton including cyanobacteria. Living tissues and extracts from three macrophytes, Ulva linza, Corallina pilurifera and Sargassum thunbergii, inhibited the growth of Prorocentrum donghaiense (Wang et al. 2007). Several phytoplankton species like Heterosigma akashiwo, Alexandrium tamarense and Skeletonema costatum were affected by the living tissue of Ulva lactuca (Nan et al. 2008). There are also reports on positive interactions between organisms from different taxonomic groups. Mulderij et al. (2007) observed that extract and cell-free filtrate obtained from the aquatic higher plant Stratiotes eloides stimulated the growth of two phytoplankton species: cyanobacteria Synechococcus elongatus and green algae Scenedesmus obliquus. One of the main reasons behind the interest in aquatic allelopathy is that bioactive secondary metabolites could be one of the key factors that promote the dominance of marine and freshwater harmful algal blooms (HABs) (Legrand et al. 2003). Moreover, phytoplankton organisms, especially dinoflagellates and cyanobacteria are considered to be a source of a wide variety of substances with bioactive potential. Allelopathic compounds isolated from cyanobacteria and other groups of phytoplankton include low-molecular-weight peptides, phenols, fatty acids, polysaccharides and alkaloids (Svircev et al. 2008). New biologically active substances could be important in many branches of industry, medicine and pharmacy. In recent years, screening for bioactive compounds produced by microorganisms has been performed extensively all over the world.

In aquatic environments, chemical information is transmitted by diffusion and the main problem is the low concentration of extracellular compounds in the spaces between cells. Bioactive compounds with a low molecular weight are favored due to their faster diffusion (Leflaive & Ten-Hage 2007). Compared to allelochemicals identified in higher plants, little is known about metabolites produced by microalgae and cyanobacteria. Any such identified compounds, however, show much structural variety and include alkaloids, phenols, organic acids, long-chain fatty acids and cyclic peptides. Many allelochemicals have been described by their chemical characteristics, molecular weights and activity type, but the chemical structures of most of them remain unknown (Legrand et al. 2003). The effects of allelopathic compounds depend on the type of interaction between species, mode of action and activity. One of the commonly observed effects is inhibition of photosynthesis, usually by inhibition of electron transfer in Photosystem II (PSII). Fischerellin A produced by cyanobacteria Fischerella musciola is one of the identified factors affecting PSII, with up to four different targeting sites (Gross et al. 1991; Srivastava et al. 1998). Another widespread effect observed is the generation of Reactive Oxygen Species (ROS). An unidentified compound secreted by Microcystis sp. caused an oxidative stress in the target dinoflagellate species (Sukenik et al. 2002). There are reports on the formation of ROS in target cells by nostocine A, produced by cyanobacteria from Nostoc genera (Hirata et al. 2003, 2004). The generation of ROS, depending on the type and increasing concentration, may even lead to the programmed cell death or cyst formation in some species (Vardi et al. 1999; Leflaive & Ten-Hage 2007). Allelopathic interactions resulting in the cell death of target species are often observed. This is usually due to the hemolytic, lytic and membrane-disruptive properties of the secreted compounds. For example, microalgae Prymnesium parvum causes rapid damage to plasmatic membranes of Rhodomonas baltica cells. This fast process also suggests a direct impact on cell membranes instead of enzymatic processes like hydrolysis or pore formation (Schmitt et al. 1999; Skovgaard et al. 2003). Most recently, Ma et al. (2011) described the membrane-disruptive properties of compounds obtained from the dinoflagellate Alexandrium tamarense, but the exact mechanism of this process remains unknown. Some species, especially from cyanobacteria like Anabaena flos-aquae, can induce cell paralysis of target species’ cells leading to faster settling, which could lead to a ‘competitor-free zone’ (Kearns & Hunter 2001). According to the literature data, many aquatic organisms produce extracellular enzymes, a prerequisite for nutrition and the use of complex substrates. Among cyanobacteria, several isolated strains could inhibit the activity of α-glucosidase activity (Jüttner & Wu 2000). A low-molecular-weight compound, collected from A. flos-aquae, inhibited the activity of α-amylase enzyme (Winder et al. 1989). Another known enzyme inhibitor is okadaic acid produced by some dinoflagellates like Prorocentrum lima. This compound is a serine-threonine protein phosphatase inhibitor. The stronger effect of P. lima exudates compared to purified acid suggests that other allelochemicals are involved (Sugg & Van Dolah 1999). Some allelochemicals can affect nucleic acid synthesis. For example, two alkaloids isolated from Calothrix sp. and Fischerella sp. (calothrixine A and hapalindole E respectively) had a negative impact on RNA polymerase activity (Doan et al. 2000). Moreover, calothrixine A can also affect DNA synthesis. Allelopathic substances may also cause morphological and ultrastructural changes. Valdor et al. (2007) reported several changes induced by living cells, extracts and pure microcystins on target organisms, including elongated and vacuolized cells, granular cell content, fragmented trichomes, an increased number of heterocysts and, in colony-forming species, a tendency to disaggregate into isolated cells. Morphological and structural modifications, including bleaching and vacuolization, thylakoid degeneration as well as disappearance of cell structures like nuclei, were observed in Chlamydomonas sp. cells when exposed to a crude extract obtained from Fischerella sp. cultures (Gantar et al. 2008). All of the described effects could lead to changes in the growth rate. Inhibition of growth induced by identified and unknown compounds is the most common allelopathic influence observed in all phytoplankton groups. For example, there are several reports on Microcystis aeruginosa causing the growth inhibition in other cyanobacteria as well as microalgal species (Singh et al. 2001; Sukenik et al. 2002; Żak & Kosakowska 2014). Also, cyanobacteria Anabaena variabilis demonstrated a negative impact on the growth of green algae Chlorella vulgaris (Żak et al. 2012). A similar effect induced by the dinoflagellate Alexandrium tamarense (Arzul et al. 1999; Wang et al. 2006) has also been observed. Although the majority of allelopathic interactions between phytoplankton species demonstrate negative character, there are reports on positive influences of one organism on another mediated by secondary metabolites. Despite the (mostly) negative impact of Fischerella sp. on other species, the crude extract collected from cultures of this cyanobacteria stimulated the growth of another cyanobacterium, Nostoc sp. (Gantar et al. 2008). The dinoflagellate Prorocentrum minimum increased the growth of the diatom Skeletonema costatum in bi-algal cultures (Tameishi et al. 2009). Exudates obtained from Emiliania huxleyi not only stimulated the growth of the diatom Phaeodactylum tricornutum but also promoted the binding and uptake of several trace metals, including Cu, Fe, Zn and Mn (Vasconcelos & Leal 2008). More information regarding allelopathic interactions among the groups of eukaryotic microalgae and prokaryotic cyanobacteria is provided in Table 1.

According to the literature, the first observations of allelopathic interactions in aquatic environment were made in freshwater habitats and the majority of these reports concern cyanobacterial species. Little is known about the phytoplankton allelopathy in marine or, particularly, in brackish habitats. In marine environments, the majority of known interactions involve red-tide bloom-forming organisms like dinoflagellates (Cembella 2003; Gross 2003). Several ecological functions have been suggested for allelopathy. Some compounds may function as a feeding deterrent, repelling antagonistic microbes and higher order grazers. Phytoplankton species may affect microbes, zooplankton, invertebrates, fish and other vertebrates, including mammals. In particular, toxin-producing organisms with potential to form mass populations can have an impact on water resources, aquaculture, fisheries and human health (Wiegand & Pflugmacher 2004). It is known that many cyanobacterial species exhibit the optimal growth in the presence of heterotrophic bacteria. Production and excretion of extracellular metabolites may attract associated species (Kaebernick & Neilan 2001). For example, the bacterium Pseudomonas aeruginosa is attracted to the heterocysts of Anabaena sp., and these two species form a mutualistic relationship sharing the available N2. The cyanobacterium Microcystis aeruginosa, demonstrates a greater cell-specific rate of CO2 fixation in the presence of other bacteria. Furthermore, allelochemicals may also play a role in the competition between phytoplankton, compensating for competitive disadvantages like a low growth rate or low nutrient uptake. Organisms which produce and secrete bioactive compounds can also have an advantage over competitors under the same environmental conditions. Allelopathy, as a better competition strategy, could explain species succession (Wolfe 2000). The differential impact of bioactive compounds on different target species could be one of the factors causing changes in the plankton community structure (Mulderij et al. 2003). Moreover, according to Roy (2009), allelopathy acts as a strongly self-regulatory strategy of the phytoplankton community, not only as a succession regulator, but also as an effective mechanism maintaining the species diversity in the environment. The combined field observations and laboratory studies have shown that allelopathic interactions may induce bloom sequences in a eutrophic lake. Dominant cyanobacteria can inhibit other organisms, including species which were previously dominant (Keating 1977, 1978). There are other reports connecting algal succession and bloom formation with production of extracellular metabolites (Kearns & Hunter 2001; Vardi et al. 2002). For example, toxic cyanobacteria Planktothrix agardhii caused a rapid decrease in Trachelomonas (Euglenophyta) biomass during blooms in a highly eutrophic dam reservoir. Although the total number of taxa observed dropped to the minimum when the concentration of a microcystin was at the maximum, the negative impact of MC-LR on Trachelomonas requires further study (Grabowska & Wołowski 2014). Thus, allelopathy may play a significant role in the induction, maintenance and termination of blooms in aquatic habitats. According to the available data, allelopathic influence could increase during a stress, for example, under nutrient-deficient conditions (N or P). Prymnesium parvum increased the production of allelopathic compounds, negatively affecting other phytoplankton species, when the N:P ratio was altered. The addition of filtrates from cultures of P. parvum grown under nutrient-deficiency conditions had a stronger negative effect on target cells, compared to filtrates obtained from non-deficient cultures. This negative impact was more pronounced when target organisms were also grown under nutrient deficiency conditions (Fistarol et al. 20003; Granéli and Johansson 2003; Fistarol et al. 2005). In addition, cell-free filtrates collected from Chrysomchromulina growing on the P-deficient medium, strongly inhibited the growth of the diatom Skeletonema costatum (Mykelstad et al. 1995). Granéli and Flynn (2006) observed the increase in Karenia mikimotoi toxicity under N-deficiency. Stressed conditions promote the production of allelochemicals, resulting in an advantage over potential competitors for the limited nutrient. Moreover, lysis of the co-occurring phytoplankton species releases organic N and P to the environment, which can support organisms with allelopathic potential. According to Li et al. (2012), in addition to the competition for nutrients, mixotrophy and allelopathy were the factors determining the dominant dinoflagellates species. The importance of allelopathy is probably enhanced not only in the case of abiotic stress, but also during introduction of new species into the environment. Production of an allelochemical and its continuous release, as well as delayed adaptation of target species, could give an advantage to the allelopathic species (Reigosa et al. 1999). There are suggestions that terrestrial plants and some aquatic plants use allelopathy as a spreading mechanism (Macias et al. 2008). It is possible that phytoplankton organisms may use a similar strategy during invasion of new habitats.

Phytoplankton species from all aquatic habitats are considered to be a great source of novel secondary metabolites. In particular, dinoflagellates and cyanobacteria are known to produce a wide variety of bioactive compounds. Even a single species could produce many secondary metabolites of various chemical groups (Tan et al. 2007). Screening for new biologically active substances is performed extensively all over the world, followed by identification and development of phytoplankton culture techniques. Microalgal and cyanobacterial metabolites demonstrate an interesting range of biological activities, including antimicrobial and antitumoral properties (Svircev et al. 2008). Examples of bioactive properties of compounds derived from phytoplankton organisms are presented in Table 2. Allelopathic phytoplankton species in freshwater and marine environments could affect the equilibrium and limit the biodiversity of other autotrophs, invertebrates and vertebrates (Rohrlack et al. 2001; Liu et al. 2002; Pflugmacher 2002). Therefore, the use of water containing allelopathic agents including toxins for irrigation could potentially affect terrestrial plants. Allelochemicals could thus potentially be used as biodegradable, environment-friendly herbicides or biocontrol agents, as a result of their natural origin and short half lives in comparison with traditional, synthetic pesticides (Gantar et al. 2008; Qian et al. 2009). Moreover, many allelopathic compounds exert their influence through other mechanisms than commercially available herbicides and exhibit bioactivity at low concentrations, making them ideal compounds for the development of new herbicides and agrochemicals (Vyvyan 2002). In addition, some studies describe the stimulatory influence of microalgal species on seed germination (Zhongqiang et al. 2005) and there are reports on the possible use of cyanobacterial species which are capable of fixing atmospheric nitrogen as biofertilizers in agriculture (Sinha et al. 2002; Choudhury & Kennedy 2004; Obana et al. 2007; Asari et al. 2008). Furthermore, some cyanobacterial species, due to their heavy metal uptake abilities, may be used for bioremediation (Zaccaro et al. 2001). According to the literature, crude extracts obtained from toxin-producing cyanobacteria P. agardhii and Dolichospermum lemmermannii were harmful to the larvae of invertebrate Chironomus spp. Moreover, studies revealed that pure toxins (MC-LR and ANTX) were less toxic than extracts containing 10-times less cyanotoxins (Toporowska et al. 2014). As mentioned above, allelochemicals could affect other microorganisms and invertebrates, which could be important for controlling organisms harmful to humans. It is known that many kinds of harmful insects cause various diseases, such as mosquitoes spreading malaria, dengue and yellow fevers (Harada et al. 2000). There are several reports about the larvicidal properties of some cyanobacterial species. For example, Oscillatoria sp. and Oscillatoria agardhii have been found to be harmful to larvae of Aedes aegypti (Berry et al. 2008) and Aedes albopictus (Harada et al. 2000), respectively. According to Rao et al. (1999), the cyanobacteria Westiellopsis sp. i larvicidal properties against several mosquito species, such as Aedes aegypti, Anopheles stephensi, Culex quinquefasciatusi and Culex tritaeniorhynchus. Furthermore, some organisms like Synechococcus sp. and Pseudoanabaena sp. caused 100% mortality of exposed larvae (Berry et al. 2008). Secondary metabolites could be potentially used as control agents for harmful insects and protozoa. For example, malaria is a disease caused by the malaria parasite Plasmodium falciparum. The cyclic peptides lagunamide A and B, isolated from the marine cyanobacterium Lyngbya majuscula, display significant antimalarial properties (Tripathi et al. 2010). Moreover, some compounds inhibited the growth of the chloroquine-resistant strain of P. falciparum. These metabolites, calothrixin A and B, were isolated from the cyanobacteria Calothrix sp. According to the literature, cell-free filtrates, crude cell extracts and isolated identified compounds collected from phytoplankton species demonstrated antibacterial activity against Gram-positive and Gram-negative bacteria. Antifungal activity of allelochemicals has also been reported (Tuney et al. 2006; Bhagavathy et al. 2011). Microalgae from the genus Spirogyra show antimicrobial potential. The methanolic extract of Spirogyra decimina inhibited the growth of Staphylococcus aureus and Proteus mirabilis. Methanolic extracts obtained from Spirogyra crassa and Spirogyra biformis exhibited antibacterial activity against Proteus mirabilis and Proteus vulgaris, respectively. The strongest effects were observed in the case of ethanolic extracts collected from Spirogyra grantiana, which affected the growth of three bacterial species, namely Escherichia coli, Proteus vulgaris and Proteus mirabilis (Prakash et al. 2011). According to Scholtz and Liebezeit (2012), the diatom Amphipleura pellucida showed antibacterial activity against Aeromonas fluvalis. The same authors observed a negative effect of the microalgae Leucocryptos marina and Hemidinium nasutum on the growth of Bacillus subtilis in Agar Diffusion Assays (ADA). Moreover, they also noticed a negative effect of the microalgae Crucigenia quadrata on the fungi Aspergillus niger and Wallemia sebi. However, the majority of reports on bacteriostatic, bactericidal activity and negative impact on fungi is related to cyanobacteria. In 1999, Kreitlow et al. observed a negative influence of lipophilic and hydrophilic extracts of different cyanobacterial strains on the growth of several bacterial species. Oscillatoria rubescens, Oscillatoria sp. and Limnothrix sp. inhibited the growth of Staphylococcus aureus, Bacillus subtilis and Micrococcus flavus. All of these strains are Gram-positive bacteria. The obtained cyanobacterial extracts had no influence on the growth of Gram-negative bacterial strains Escherichia coli, Proteus mirabilis and Serratia marcescens. Antibacterial and antifungal activity of Oscillatoria anguntissima and Calothrix parientina was observed by Issa (1999). Both cyanobacterial species affected the growth of four bacterial strains, namely Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus, in the same way. Both cyanobacteria had a negative influence on several fungi: Aspergillus flavus, Aspergillus versicolor, Penicillium variabile, Trichophyton gourgii, Microsporum canis and Chrysosporium tropicum. A slightly stronger effect was observed in samples treated with compounds from Calothrix parietina. El-Sheekh et al. (2008) demonstrate that extracts from two cyanobacteria, Anabaena wisconsinense and Oscillatoria curviceps, show antibacterial and antifungal properties toward bacteria and fungi isolated from diseased fish. Both cyanobacteria affected the bacteria Lactobacillus sp., Aeromonas hydrophilia, Bacillus firmus and Pseudomonas anguilliseptica, as well as two fungi, Aspergillus niger and Saprolegnia parasitica. According to the available data, some of the isolated compounds have anti-inflammatory and antioxidant properties. Furthermore, some of the bioactive compounds isolated from microalgae and cyanobacteria may be used as antiviral agents (Tuney et al. 2006; Yasuhara-Bell & Lu 2010; Bhagavathy et al. 2011). Compounds A1 and A2 isolated from the microalgae Cochlodinium polykrikoides, display antiviral properties against influenza virus A and B and RSV (Respiratory Syncytial Virus) A and B. Metabolite A2 also affected the parainfluenza type 2 virus (Hasui et al. 1995). Cyanovirin-N collected from the cyanobacteria Nostoc ellipsosporum shows antiviral activity against SIV (Simian Immunodeficiency Virus) in monkeys and HIV-1 and HIV-2 (Human Immunodeficiency Virus) (Boyd et al. 1997). Microalgal and cyanobacterial secondary metabolites are also a promising source of antitumoral compounds. Eucapsitrione obtained from Eucapsis sp. and fischambiguine B produced by Fischerella ambigua have a cytotoxic impact on immortalized cells of the VERO line (kidney epithelial cells extracted from an African green monkey Chlorocebus sp.) (Mo et al. 2010; Sturdy et al. 2010). Aeruginazole A obtained from Microcystis sp. shows cytotoxic properties against the human leukemia cell line MOLT-4 (Raveh & Carmeli, 2010). Very interesting results were obtained with pahayokolide A isolated from Lyngbya sp. This cyclic peptide demonstrates antitumoral properties toward several human cancer cell lines, including H460 (lung cancer), SKBR3 (breast cancer), HT-29 (colon cancer) and A-498 (kidney carcinoma). Most recently, Shanab et al. (2012) noticed antioxidant and anticancer activity of aqueous extracts obtained from Nostoc muscorum and Oscillatoria sp. Cytotoxic efficiency was investigated against two cell lines: EACC (Ehrlich Ascites Carcinoma Cell) and HepG2 (Human Hepatocellular cancer). Moreover, they discovered that both antioxidant and anticancer properties increased in cultures growing under nitrogen stress conditions. According to the literature, more natural products from marine phytoplankton have been found as potential lead compounds for a drug discovery. An increasing number of lipopeptides, such as hectochlorin, lyngbyabellins, lyngbyastatin or symplostatin, could be used for the development of new anticancer drugs with specific cellular targets (Tan 2007).

Microalgae and cyanobacteria produce a wide variety of secondary metabolites. Allelopathic interactions mediated by these compounds occur in all aquatic habitats and among all taxonomic groups of phytoplankton. Allelochemicals, which usually have a negative impact, may affect photosynthesis, nucleic acid synthesis, enzymatic activity and growth. They can induce morphological and ultrastructural changes or generation of ROS, and may even cause death of target cells. Some compounds may play a role in competition, especially in nutrient deficiency conditions, and may affect species succession. More importantly, allelopathy could be one of the factors responsible for maintaining the microalgal and cyanobacterial blooms. However, apart from their role in interactions between organisms and their ecological importance, such secondary metabolites often demonstrate other bioactive properties. These compounds could be potentially used as herbicides, insecticides or other biocontrol agents. Many isolated allelopathic compounds display antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral and anticancer activity. Therefore, new biologically active metabolites may play a significant role in different branches of industry, particularly in agriculture, medicine and pharmacy.