Effect of Ultraviolet-C Radiation on the Morphology of Cyanobacteria Nostoc sp. LBALBR-2 Isolated from Supply Reservoir (Belém, Pará, Brazil)

Cyanobacteria are photosynthetic prokaryotic organisms that in aquatic environments can compose the phytoplankton and phytobenthos, being important in the primary production of these ecosystems. The objective of this work was to identify the main morphological variations of the cells, filaments and thallus of the cyanobacteria Nostoc sp. LBALBR-2 submitted to ultraviolet radiation (UV-C) in microcosm systems. The cyanobacteria Nostoc sp. was subjected to four treatments: 1- control treatment in nutrient medium; 2- treatment in nutrient medium with exposure to UV-C radiation; 3- control treatment in medium without nutrients and 4- treatment in medium without nutrients with exposure to UV-C radiation. Optical density and chlorophyll-a analyses were performed to determine the growth of Nostoc populations and microscopic analyses to characterize the morphological development of the species . Lack of nutrients generated short trichomes with terminal heterocytes, scattered solitary akinetes and appearance of hormogonia. On nutrient medium the trichomes of Nostoc sp . showed predominantly the vegetative serial form. Cultures exposed to UV-C produced anomalous cells, thick mucilage, fragmented trichomes and hormogonia. It is concluded that Nostoc sp . LBALBR-2 grew well under eutrophication conditions and although it showed cell deformation it was resistant to UV-C radiation. cianobacteria Nostoc sp. se sometió a cuatro tratamientos: 1- tratamiento testigo en medio nutritivo; 2- tratamiento en medio nutritivo con exposición a radiación UV-C; 3- tratamiento control en medio sin nutrientes y 4- tratamiento en medio sin nutrientes con exposición a radiación UV-C. Se realizaron análisis de densidad óptica y clorofila-a para determinar el crecimiento de las poblaciones de Nostoc y análisis microscópicos para caracterizar el desarrollo morfológico de la especie. La falta de nutrientes generó tricomas cortos con heterocitos terminales, acinetas solitarias dispersas y aparición de hormonas. En medio nutritivo, los tricomas de Nostoc presentaron predominantemente la forma seriada vegetativa. Los cultivos expuestos a UV-C produjeron células anómalas, mucílago espeso, tricomas fragmentados y hormonas. Se concluye que Nostoc sp. LBALBR-2 se desarrolló bien en condiciones de eutrofización y, a pesar de mostrar deformaciones celulares, fue resistente a la radiación UV-C.


Introduction
Cyanobacteria are oxygenic photosynthetic Gram negative prokaryotes that in the aquatic environment can compose the phytoplankton and phytobenthos, being important in the primary production of this ecosystem (Komárek, 2013;Silva et al., 2020).
In addition, Nostocaceae fix atmospheric nitrogen and contribute to the fertility of agricultural soils worldwide, and some species behave as harmful microorganisms in aquatic ecosystems due to toxic bloom events (Huisman et al. 2018).
Nostoc Vaucher ex Bornet et Flahault is a genus of Nostocaceae well distributed worldwide and has more than 300 recognized species, however, the genus is heterogeneous and certainly divided into several generic clusters and most species need revision at the morphological and molecular level (Komárek, 2013). In Brazil, 12 species are recognized (Werner et al., 2015) and only one species is cited for the state of Pará, Brazilian Amazon (Costa et al., 2014).
In recent decades, increasing research on cyanobacteria in simulated environments (mesocosm and microcosm) is observed in an attempt to evaluate their behavior in relation to different environmental factors, such as exposure to heavy metals (Mota et al., 2015), nitrogen and phosphorus concentrations (Hughes & Marion, 2021), stimulation to different conditions of temperatures, light, photoperiod (Huisman et al. 2018) and ultraviolet-UV light (Rastogi et al., 2014).
Regarding UV, studies address the behavior of cyanobacteria in the face of light as a stressor of biochemical processes (Jantaro et al., 2011). Cyanobacterial cells react and produce some photoprotective and antioxidant compounds such as mycosporine-like amino acids-MAAs that have been researched for application in sunscreens and anti-aging products (Soule et al., 2016;Latifi et al., 2009;Geraldes, 2019).
On the other hand, there are studies on the behavior, morphogenesis and toxin production by cyanobacteria when exposed to UV projecting a future climate change scenario due to the sharp increase in solar ultraviolet radiation (UVR) caused by the continuous depletion of the ozone layer, fueling serious concerns about the ecological consequences for all living organisms, including cyanobacteria (Rastogi et al., 2014). UV-C used for germicidal purposes is artificial and is an alternative to prevent cyanobacterial blooms in lakes and reservoirs (Tao et al., 2010;Skai et al., 2009;Usepa, 2006;Jiang et al., 2020), as shortwave UV-C radiation has shown effect on cyanobacterial cells by inhibiting their growth and reducing cyanotoxin concentration through repression of photosynthetic activity and DNA damage (Ou et al., 2012), but no effect on green algae (Tao et al., 2010).
Few studies show the changes that occur in the morphology of cyanobacteria under the effects of UV-C radiation, especially cyanobacteria from Amazonian environments. The objective of this study was to identify the main morphological variations of the cells, filaments and thallus of the cyanobacteria Nostoc sp. LBALBR-2 submitted to ultraviolet radiation (UV-C) in a simulated environmental system.

Experimental Design
Four experimental treatments were used in microcosm to verify their effects on the cyanobacteria Nostoc sp. LBALBR-2 (Table 1). Experiments 1 and 2 contain nutrients (BG-11 medium) and experiments 3 and 4 contain ultrapure water type I. For each experiment, samples were taken daily for microscopic analysis, aliquots for chlorophyll-a and optical density analysis until the fourth day, then the withdrawals occurred at 3-day intervals until day 22 nd . All treatments took place in a Research, Society and Development, v. 11, n. 12, e447111234391, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i12.34391 4 climatic chamber for plant growth (Humidity, Panasonic). The radiation source was a 30W UV-C germicidal tubular lamp, Redy.

Morphological and Morphometric Characterization
The species was observed and analyzed using an inverted microscope (Axiovert A1, Zeiss Germany) at each time point of the experiments. Morphometric measurements were recorded with a camera (Axio Cam MRc, software Axio Vision, Zeiss Germany) with measurements in micrometers (µm), measuring cell diameters and lengths (width and height), length of filaments and the presence/absence of specialized cells (akinetes and heterocytes), morphological changes, presence of mucilage, among others. Staining was established according to the Faber Castell palette (1761).

Chlorophyll -a and Optical Density
Chlorophyll

Data Analysis
The cellular values, chlorophyll-a and optical density were submitted to parametric ANOVA one way (F) and nonparametric Kruskal-Wallis (H) analysis of variance to verify the differences between the times and types of experiments, considering as significant variation p< 0.05 and the post hoc tests of Tukey and Dunn, parametric and non-parametric, respectively, both performed in Past 4.03 software (Hammer et al., 2001).

Growth
We suggest that all the wavelengths analyzed express the growth of the species Nostoc sp. There was a significant difference between the times (F= 168.2; df= 9; p= 0.0005), where the curves start to differ from day 4 (T4) (Figure 1).

Thallus Morphology
The thallus of Nostoc LBALBR-2 presented as a thvick, foliaceus, dark green mass (color palette 059) in the nutrient cultures (experiments 1 and 2) ( Figure 6A). The fouling started with a thin film on the microcosm wall and a thicker layer on the surface with the appearance of gas, usually after one week of colonization ( Figures 6B and 6C).
On the other hand, cultures without nutrients (experiments 3 and 4) presented a thin, lemon yellow mass (color palette 004) (Figures 6D and 6E). It was found that after 30 days of incubation the stalk acquired a gelatinous consistency, especially in samples without nutrients ( Figure 6F).
For all experiments, starting on day 4 th of cultivation the thallus changed from amorphous colonies, with vegetative cells overlapping in irregular layers, to multiple trichomes with cells arranged in uniseriate sequences ( Figures 6G to 6I).
In cultures with nutrients (experiments 1 and 2) the trichomes were longer, on average 300 µm long ( Figures 6J to 6L).
In cultures without nutrients (experiments 3 and 4), the final cells had terminal heterocytes and the trichomes were short, on average 105 µm ( Figure 6M), and in the final times of the experiment, akinetes appeared, measuring 2.5-3.0 µm in height by 1.6-2.0 µm in diameter ( Figure 6N). In cultures exposed to UV radiation (experiments 2 and 4), the filaments were surrounded by a dense mucilaginous sheath that was dark green in nutrient-depleted cultures and yellowish in nutrient-free cultures ( Figures 6O and 6P).
Also, after 15 days of UV exposure, fragments and hormogonia appeared and increased over time. At the final times, the trichomes showed anomalous cells as a result of UV exposure (Figures 7A to 7E). Research, Society and Development, v. 11, n. 12, e447111234391, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i12.34391 Figure 7 -Photomicrograph of Nostoc sp. LBALBR-2: A and B-hormogonia in colonies grown without nutrients and exposed to UV-C; C to E-anomalous cells in trichomes exposed to UV-C.

A B C D E
Source: Authors (2022).

Discussion
The absence of nutrients and UV exposure were stressors to the Nostoc sp. LBALBR-2 population. According to Meeks et al. (2002), Nostoc cyanobacterial species mature into four forms, which are determined by environmental cues.
Multilayered vegetative cells, trichomes with vegetative cells, trichomes with akinetes and heterocytes and the hormogonia are the main differentiations observed in Nostoc (Silva et al., 2020). Thus, the population of Nostoc sp. in the present study developed better in nutrient crops generating longer trichomes. For the vegetative cycle is maintained when the amount of nutrients is sufficient for the growth of the species (Silva et al., 1989).
On the other hand, lack of nutrients generated short trichomes with terminal heterocytes and scattered solitary akinetes. On this, Dextro et al. (2018) observed that the absence of nutrients, especially nitrogen, induces heterocyte formation and low light intensity induces akinetes formation in Nostoc strains.
Akinetes are generally recognized as resistance cells, with thick walls, that accumulate protein reserves in the form of cyanophycin granules. Akinetes are produced when environmental conditions are unfavorable (Calijuri et al., 2006;Mehnert et al., 2014;Wood et al., 2021), with temperature being one of the main factors responsible for the formation of akinetes, as some cyanobacteria initiate the production of akinetes at temperatures between 20°C and 25°C, such as Aphanizomenon ovalisporum (Cirés et al, 2013), and the absence of this cell occurs at temperatures below 21°C, as in the cyanobacterium Cylindrospermopsis raciborskii (Bittencourt-Oliveira et al., 2012). However, other environmental factors should influence the formation of these cells including carbon depletion, phosphorus limitation and symbiotic interactions Allaf According to Legrand et al. (2016) akinetes are formed under various abiotic stress conditions, depending on the species and also the sampling season. Thus, the resistance of akinetes and their ability to germinate seem to follow a speciesspecific process and are so important for the maintenance of the species that they also carry the genes for toxin production. Perez et al. (2016) found that the trigger for differentiation of akinetes of the planktonic species Anabaena variabilis is low luminosity and of the terrestrial or symbiotic species Nostoc punctiforme is lack of phosphate. These authors suggest that at the time of akinetes differentiations there is a decrease in photosynthesis and respiration in both species, and this remains low in the mature akinetes, this may indicate a reduction in growth of Nostoc sp. LBALBR-2, from the present study, in the absence of nutrients, as many dispersed acini were formed.
The occurrence and quantity of heterocytes in cyanobacterial populations depend on different environmental factors, such as aerobic fixation of atmospheric nitrogen (N 2), concentration and lack of nitrate, organic components, temperatures, X-rays, irradiation, etc. Nitrogen metabolism and environmental nitrogen concentration are considered the crucial factors in their development (Rippka et al., 1979;Komarek, 2013), thus their highest abundance was in the trichomes grown in nutrientfree medium.
We suggest that hormogonia arose in response to lack of nutrients and UV exposure, although there are few studies confirming UV stress on hormogonia formation, UV is known to cause trichome fragmentation in Nostoc sp. (Singh et al., 2011). Hormogonia is a process of dispersal, phototaxis, reproduction and establishment of nitrogen-fixing symbioses and constitutes part of the life cycle of filamentous cyanobacteria, especially in Nostoc.
According to Zuniga et al. (2020) light quality and quantity, nutrient concentrations, and autogenic repressors are factors that induce hormogonia formation, which according to Gonzales et al. (2019) occurs through a regulatory network of sigmas genes that works in a cascade, coupled with specific genes that induce changes in cell architecture.
Regarding the stresses promoted by UV-C, the most significant were the production of a thick mucilaginous sheath around the Nostoc filaments and anomalous cells, which appeared at the end of UV exposure.
Studies have shown that Nostoc sp. creates enzymatic and non-enzymatic defense mechanisms to counteract the damaging effects of UV radiation, such as the production of antioxidant enzymes and mycosporin-like amino acids capable of performing UV screening, yet it dies if continuously exposed to this radiation (Richa & Sinha, 2015) or recovers when the radiation source is ceased (Phukan & Syiem, 2019). Thus, we suggest that the mucilaginous sheath that appeared on Nostoc in the present study is the production of these compounds in response to UV-C exposure.
Phukan & Syiem (2019) evaluated the oxidant and antioxidant homeostasis responses of the cyanobacteria Nostoc muscorum exposed to UV radiation and evidenced a complex defense organization developed by this species, which after being subjected to doses of UV-C radiation exhibited ultrastructural cell damage, produced reactive oxygen species (ROS) and consequently significantly induced enzymatic and non-enzymatic antioxidant production.
In the present study, we show the morphological alterations of UV-C exposure that we suppose are consequences of the biochemical and ultrastructural alterations suffered by the exposed cells. It is worth noting that not all cells showed malformations, due to the organization of the Nostoc tallus that takes place by the superposition of cells, allowing some cell layers to be more exposed than others which was also observed by Phukan & Syiem (2019).
The Amazon microbiota is poorly known when compared to its estimated high diversity. The behavior and development of the species can be studied from models made in Nostoc cultures, and their potential in biopharmaceutical production is important in contemporary scientific research. Thus, we suggest that future studies should better highlight the sequences of events identified in the present study, especially at the intracellular and biochemical level to potentiate their use as a model of cyanobacteria development.
Furthermore, the study contributes to strengthening the knowledge about the defense mechanisms of cyanobacteria and also to improving the processes of reducing blooms using UV-C radiation.

Conclusion
The effect of the radiation on the species occurred in the initial development of the thallus, the color of the colony, the shape of the cells and the length of the filaments. The nutrient cultures allowed the cyanobacteria population to develop further and the cells did not change in diameter and length, but UV-C exposure caused anomalous cells, the appearance of a thick mucilage and numerous fragmented trichomes and hormogonia, which we suggest are adaptive strategies to UV-C light stress and especially the lack of nutrients.