**6. Microviridin Ecology**

Microviridins play a significant ecological function as antifeeding agents against cyanobacterial natural predators. This activity is correlated with their ability to inhibit proteolytic enzymes (Figure 9). The first study to explain this mechanism was performed by Rohrlack et al. (2014) [48]. A previous work, however, had already indicated microviridins as an agent capable of causing the interrupting the feeding of *Daphnia* microcrustacean via enzymatic inhibition. This ability can partly explain the dominance of these microorganisms in some habitats, including those with a high population density of *Daphnia* [49]. In a similar way, protease inhibitors are produced by terrestrial plants to protect against herbivores. Metatranscriptomic analyses of the Kranji Eutrophic Reservoir, located in Singapore, revealed important information on the functional dynamics between different bacterial phyla, including cyanobacteria, which were dominant microorganisms, especially those belonging to the *Microcystis* genus. The microviridin transcripts were found in high quantities, along with those involved in the buoyancy and photosynthetic operation. The highest peak of the gene expression related to microviridin biosynthesis was observed when the population of *Daphnia* moved from the mesopelagic zone to the epipelagic zone, corroborating its antipredator activity [49].

**Figure 9.** Ecological role of microviridins as antifeedant against the microcrustacean *Daphnia.*

Kaebernick et al. (2001) [50] compared the feeding inhibition of *Daphnia galeata* and *D. pulicaria* by a microcystin-producing *Microcystis* (MRD) and a microcystin-deficient *Microcystis* (MRC), and it has been realized that this hepatotoxin is not associated with the ingestion rate reduction in both planktonic grazers. However, this metabolite was responsible for causing both species to decrease their survival rates. Before the death provoked by this hepatotoxin, these microcrustaceans remained immobile in the bottom of the vial and shifted only when the surrounding area suffered disturbances. In addition, the filter legs and antenna were momentarily paused, and the midgut was disrupted.

The same authors [50] described further effects of *Microcystis* strain UWOCC MRC ingestion by *D. galeata* and *D. pulicaria*. These microcrustaceans had a dysfunction in the peritrophic membrane. This membrane acts as a barrier formed by the chitin–protein complex created by the midgut cells. The consumption of *Microcystis* made this organ more enervated, as a result of which, food transport was impaired, resulting in particle aggregation in this region and in the digestive diverticula. The ingestion of these cells also disturbed the molting process. The old integument was not entirely separate from the *Daphnia* body, attached to the legs and filter antennas, and strongly hindered the ability of these species to swim and feed themselves. Individuals subjected to these conditions were more likely to die of malnutrition within two days. It was also confirmed that the freshly developed integument remained soft both in the presence of the old integument and after its mechanical removal. Under field conditions, these affected species would become easy prey to predators, since they would not be able to flee to any shelter.

The typical chitin–protein complex occurs in both structures (peritrophic membrane and integument), indicating that the reported effects were probably caused by the same bioactive compound in which the microviridin variant was cogitated. By its ability to inhibit the serine protease, this oligopeptide could be preventing the tyrosine conversion into dihydroxyphenylalanine (DOPA) and its subsequent transformation into dopamine by the enzyme DOPA decarboxylase [51]. Dopamine is involved in the cross-linkage of orthoquinones, which results in the cuticle sclerotization [52]. A complementary process was proposed by Rohrlack et al. (2003) [48]. According to this mechanism, *Daphnia*'s death was associated with incomplete protein digestion, which resulted in an important amino acid deficiency for tegument development and other structures. Ingestion of the strain of *Microcystis* UWOCC CBS, a producer of microviridin J, causes the same activity in the molting process of *D. pulicaria*. However, several additional findings have been made. Particles derived from food suspensions were found on the entire surface of the *Daphnia* body. This dysfunction was possibly due to the secretion of body fluids. Deformation on the freshly generated tegument has become more intense as the effort to eradicate it by these animals has increased. The same phenomenon was visualized when only the purified microviridin J was added.

Czarnecki et al. (2006) [53] detected eight microviridins distributed in three *Microcystis* strains (HUB08B03, HUB11G02 and HUB19B05) with ability to inhibit trypsin-like activity in the planktonic crustacean *Daphnia*. In addition to microviridins, other classes of protease inhibitors, such as some cyanopeptolins, were found in the extract obtained from these cyanobacteria. This ability of unique cyanobacterium or different cyanobacteria from the same genus can generate a variety of combinations of different oligopeptides with distinct proteolytic targets and inhibitory activity. This feature acts as an evolutionary barrier, preventing the adaptation process among zooplankton population.

Microviridin toxicity was also accessed in the fairy shrimp *Thamnocephalus platyurus*, which belonged to order Anostraca. In the course of searching for natural products with cytotoxicity property, Sieber et al. (2019) [32] detected in the extract of *M. aeruginosa* strain EAWAG 127 deleterious activity against this microcrustacean (LD50 = 0.43 mg.mL−1). A metabologenomic approach revealed the presence of two novel microviridins: microviridin 1777 and microviridin O. The former showed a LD50 value of 95 μM for *Thamnocephalus platyurus*. This activity was ascribed to the strong capacity of this peptide in inhibiting elastase and chymotrypsin activity with an IC50 of 160 nM and 100 nM, respectively.

In addition to Cyanobacteria having a low susceptibility to a zooplankton attack, these microorganisms are also the target of various pathogenic bacteria and fungi that play an important role in controlling their growth [54,55]. True zoosporic fungi, commonly known as chytrids, are among the most pathogenic organisms capable of causing a significant number of deaths in the cyanobacterial community [56]. The success of this pathogen in infecting these photosynthetic microorganisms can be attributed to the development of chemotactic zoospores and the presence of rhizoids, which are capable of locating the target and used to extract the nutritional contents, respectively. Oligopeptides produced by cyanobacteria with an inhibitory activity against a predator's key enzymes is a great defense mechanism. The comparison between the cyanobacterial strain *P. agardhii CYA126/8* with its mutants, each one with a type of disability in producing microcystins, anabaenopeptins or/and microviridins, was conclusive to defining the protective role of these metabolites. The wild strain when incubated with the chytrid strain was unaffected, while all mutant strains were infected, including those non-microviridin-producing strains [57].

Chytrides are a rich source of protease used as a mechanism to digest their hosts. Microviridins and anabaenopeptins can target these enzymes, reducing the virulence of these fungi. The vast variety of microviridins, as well as other oligopeptides, is a major obstacle in the process of the adaptation of these parasites. On the basis of the literature, the protection mechanism referred to above appears to be constitutive, since these substances typically form an oversaturated or saturated solution in the cytoplasm [57]. Microviridin was also found in bacteria belonging to the microbiome of the plant *Populus*. Unlike the lanthipeptides that are widely distributed among the member of this community, microviridins were restricted only to the genus *Chryseobacterium*, being present in 16 out of the 18 sequenced bacteria. Its role in this microbiome is not clear. A gene cluster for microviridin in this genus showed from one to four precursor peptides belonging to class I [20]. Different from cyanobacterial microviridin, the core peptide was composed of 18 amino acid residues. Only half of the microviridin clusters analyzed had a Nacetyltransferase gene. A resistant gene presence in the majority of the microviridin clusters suggested that this oligopeptide could have antibacterial properties, conferring a protection to plants against pathogenic microorganisms [58].

Other features given to microviridins are related to their allelochemical properties. Cyanobacteria produce a variety of proteases that are essential to different processes, including nutrient absorption, protein activation, unfolded or aggregated protein removal, photoacclimation and stress response [59]. Ghosh et al. (2008) [60] demonstrated that a cyanobacterial oligopeptide with the partial structure of a microviridin affected the proteolysis in *M. aeruginosa* PCC 7806, strongly inhibiting its capacity to degrade N-alphabenzoyl-DL-Arg-p-nitroanilide (BApNA). The authors' hypothesis was that microviridinproducing *Microcystis* colonies could form an aggregate that could eventually develop as a bloom and suppress the growth of competing organisms by targeting critical functions that rely on protease activity. Another possibility is that microviridins will have a significant role to play in stress conditions by self-regulating the protease activity among cyanobacterial cells and thus enhancing their survival rates [60].

Some microviridins are not easily detected in environmental samples, since they may be rapidly degraded by other bacteria. *M. viridis* has its microviridin A content totally consumed when transferred to a nonaxenic medium [21]. The aquatic bacterium *Sphingomonas* sp. B-9, firstly isolated for its microcystin-degradation ability, has hydrolytic enzymes capable of degrading different cyclopeptides, including microviridin I. The degradation of this peptide by this bacterium is very slow, lasting around 48 h to reduce 50% of its initial content. This process occurs in two steps. Initially, the residue at the C-terminal region is removed, and, subsequently, the molecule undergoes a linearization step [61].

#### **7. Regulation**

Environmental factors play an important role in the regulation of the synthesis of oligopeptides, as they can increase the growth rate and, consequently, the production of these metabolites. In certain cases, however, the best conditions for growth did not lead to the most desirable conditions for their production [62]. Microcystin was the key subject of these studies [63]. Stress situations can alter the cell's physiological state and act as trigger for increasing the construction of these molecules. Nitrogen and phosphorus bioavailability are among the most nutritional factors investigated in cyanobacterial behaviors [64,65]. Both are involved in protein synthesis and in the energy dynamic. Due to anthropic actions, these elements have become more abundant in the aquatic environment [66].

Other parameters, such as temperature, pH and light intensity, have also been investigated and challenge many scientists [67,68]. An assessment of the combined effect of different environmental elements on the development of cyanopeptides can provide a link between field research and laboratory research. Any of these variables can be associated. As the culture reaches the stationary phase, the quantity of nutrients decreases, as well as the light availability among cells, thereby reducing their growth rate.

One method used by Rohrlack et al. (2007) [67] to individually determine the light impact was to track and maintain a constant nutrient load in the medium. This technique was used to analyze the production of microviridin I by *P. agardhii* strain PT2. The quantity of cell-bound microviridin I expressed in units per biovolume decreased until the eighth day. This behavior reversed when the light availability began to decline. Nitrogen and phosphate reduction also led to a decrease in the production of this microviridin. A similar trend was reported for microcystins and anabaenopeptins. Some authors strongly believe that many of these oligopeptides play the same ecological function. The loss of one can have, as a consequence, the enhanced production of another, unaffecting the cyanobacterial growth [69,70].

The influence of light intensities was also evaluated by Pereira et al. (2012) [47] on the profiles of toxic and nontoxic oligopeptides obtained from two strains of the cyanobacterium: *R. fernandoii* 28 and 86. In the course of the experiment, they employed three different irradiances, which were classified as low (25 μMol.m−2.s−1), medium (65 μMol.m−2.s−1) and high (95 μMol.m−2.s−1). Different from other oligopeptides investigated in this study, such as microcystins and cyanopeptolins, microviridins were not encountered in all growth conditions. Microviridin 1709 production reached the maximum amount when the cells of strain 28 were exposed to a medium light intensity, while microviridin 1707, identified in strain 86, was detected solely at low light conditions.

Ferreira et al. (2006) [71] evaluated the combination of different light intensities, nutritional contents, temperatures and growth phases on the oligopeptide production in distinct strains of *Microcystis* and *Aphanizomenon*, including microviridins. This protease inhibitor was detected solely in the *Microcystis* strain RST9501. In the absence of nitrate and phosphate, this peptide was produced in higher quantities. In the majority of the cases, the intracellular fraction was responsible for over 80% of the total microviridin pool. At the same nutritional conditions, an atypical behavior was found when the cells were cultivated at 20 ◦C. In this condition, the intracellular microviridin concentration diminished to 60%.

The cell-to-cell communication is also a factor to be considered in peptide production. When this mechanism is dependent on cell density, it is called quorum sensing. Nealson and Hastings (1979) [72] were pioneers in studying this phenomenon in the Gammaproteobacterium *Vibrio fischeri*. These two scientists were capable of demonstrating that the enzyme luciferase, whose role is to transform chemical energy into light energy, was expressed only at a high cell density, having its production controlled by autoinducer signaling molecules [72]. The most known autoinducers described are the acylated homoserine lactones [73]. The aquatic environment has a natural tendency to dilute the metabolites released by microorganisms. For this reason, some authors believe that oligopeptide production is regulated by quorum sensing [74]. There is little knowledge about this mechanism in cyanobacteria. During a bloom episode, the cyanobacterial population increased significantly, creating a favorable environment for quorum sensing. In this type of situation, the high cell density augments the concentration of signaling molecules in the environment [75].

To evaluate the quorum sensing effect on oligopeptide production, Pereira et al. [74] grew the cyanobacteria in a semicontinuous culture system. Hence, the biomass level and nutritional content were maintained constant. The growth rates of high and low cell density cultures were similar. Microviridin production was detected only in a low cell density culture of *R. fernandoii* (strain R28). In contrast, the microviridins N3-N9 in *N. punctiforme* PCC73102 had their synthesis optimized under high cell density conditions [19].

The cyanobacterial lifestyle may also have an effect on its oligopeptide content. Some cyanobacteria are typically located on the water and sediment surface. There are also those with a biphasic lifestyle, where they migrate to the top during the summer and to the bottom during the winter [76]. A comparative genomics of the genus *Planktothrix* with different lifestyles performed by Pancrace et al. (2016) [77] demonstrated that all planktonic strains investigated harbored the microviridin gene cluster. In contrast, in the benthic *Planktothrix*, this gene cluster was absent, with the exception of *Planktothrix* sp. PCC 11201, which is phylogenetically closer to free-living *Planktothrix*.
