Comprehensive Investigation of a Toxic Microcystis sp. (Cyanobacteria) Collected from Snow Lake (New Mexico, USA).

The toxin producing cyanobacterium Microcystis sp. was collected in the mid October 2020 from the shallow waters of Snow Lake (New Mexico, USA). This species caused a visible bloom consisting of the pale green irregular macro colonies. Mass spectral analysis of the biomass revealed the presence of 4 derivatives of microcystin in that bloom: MC-LR (in the water and biomass), MC-RR (in biomass), MC-LY (in biomass), and MC-YR (in biomass).Next-generation sequencing allowed the retrieval of two Microcystis sequences in the bloom; which are molecular benchmarks for toxic Microcystis that may be used in future monitoring studies. Light microscopy provided evidence for the taxonomic aliation of the found morphotype as Microcystis os-aquae (Wittrock) Kirchner. However, molecular sequencing and the present situation in cyanobacterial taxonomy prevented aliation of our morphotype to Microcystis os-aquae, justifying following name – Microcystis sp. Confocal microscopy was used to determine the distribution of the cell content utilizing 3D stereo imaging. Emission spectra analysis identied the pigment composition and pigment distribution within the cells. SEM revealed 3D arrangement of the cells in the colonies, texture of the surface of the cells (perhaps dehydrated collapsed polysaccharides), F-layer and pili-like structures. Additionally, SEM/EDS analysis conrmed the F-layer using elemental composition analysis, which showed sulfur in the F-layer – typical element for that structure. Through the use of AFM, we analyzed the texture of the cell's surface and conrmed pili-like structures.


Introduction
Freshwater resources are rapidly declining due to pollution from wastewater, climate change, and harmful algal blooms (HABs; Anderson 2009; Adams et al. 2018; Alvarez et al. 2019). It is more of a concern in arid areas, especially those with extensive agriculture and industry (Anderson et al. 2017). In particular, HABs may cause substantial economic loss due to water anoxia, noxious water odor from geosmin, and the accumulation of toxins in food webs that can affect aquaculture (Izaguirre and Taylor 2007;Smith et al. 2010; Molot et al. 2014). Certain HABs that result from cyanobacteria are called cyanobacterial Harmful Algal Blooms (cHABs), and may occur in water bodies around the world (Carmichael 1994;Carmichael 2008). One of the major contributors to the cHABs are cyanobacterial taxa of the genus Microcystis. Worldwide, there are 13,731 occurrence records of the genus Microcystis in the GBIF (GBIF.org, 2021). According to this dataset, the rst record of Microcystis is from the year 1831 and, up until 1982, records of this taxa were rather scarce (up to 20 records/year worldwide). Over the last 40 years occurrences of Microcystis spp. increased to around 500 records/year, indicating either progression of the Microcystis dominated cHABs or an increase of studies dealing with the cHABs. Most of the records were strictly botanical and were coming from temperate zones, without any analytical analysis. Samples of cHABs are complex matrices with numerous biotic and abiotic variables. Many investigations target one or two variables of a cHAB, neglecting others and the relationships between each component.
For example, many researchers interested in molecular analysis of the members of the cHABs ignore morphology completely (Lyra et al. 2001;Ouellette et al. 2006). In contrast to that, botanical ( oristic) investigations focused on morphology exclusively (Komárek and Komárková 2002). Historically, all the taxonomy of Microcystis were based on colony shape and cell morphology observed in the light microscope (Komárek and Anagnostidis 1995; Komárek and Anagnostidis 1998), and if a classical oristic paradigm is applied, all other lines of evidence are missing. We brie y discuss different techniques for the study of cHABs below. Physiological and biochemical studies usually deal with the Flayer (AKA, EPS-extracellular polysaccharides (Hoiczyk and Baumeister 1995)) or particular aspects of photosynthesis or cell structure (Kruip et al. 1994 There are many "grey areas" in this topic and perhaps many undescribed cyanophages are waiting for scienti c recognition and might be utilized in order to control cHABs. Pili-like structures as well as F-layer might play an important role in the cyanobacterial protection mechanism against cyanophages. The taxonomy of Microcystis is quite problematic. There is a massive amount of molecular information from 16S rRNA sequencing as well as whole genome sequencing (Herdman and Rippka 2017). However, the type specimen of the type species for this genus is not molecularly established which makes the true phylogenetic position of Microcystis ambiguous. The best solution for this problem would be epitypi cation or neotypi cation of the type species Microcystis aeruginosa. Additionally, the threshold for the species demarcation in the phylogenetic clade of Microcystis is perhaps smaller than in other more well-established cyanobacterial taxa (Mai et al. 2018 This research aimed to comprehensively characterize toxic bloom forming cyanobacterium -Microcystis sp. collected from the alpine lake: Snow Lake (New Mexico, USA). Utilizing advanced microscopy and spectroscopy we thoroughly studied many aspects of these natural populations, such as toxin analysis, pigment characterization, elemental composition of the F-layer etc. Graphical and factoid information (16S rRNA sequences) obtained in this research might be useful in further development of the cHAB monitoring systems.

Sample collection and storage
Samples of cHABs were collected from the shallow waters of Snow Lake (New Mexico, USA) on 10/8/2020. More detailed information is available on the web via CRIS (Melekhin et al. 2013)  Bacterial speci c primers: 27F/1492R with attached barcodes were used for ampli cation. Analysis utilized 10 thousand sequences per assay. The 16S rRNA gene ampli cation was done using a 35-cycle PCR (5 cycle used on PCR products). It used the HotStarTaq Plus Master Mix Kit (QIAGEN, USA) utilizing following steps: 94°C for 3 min., followed by 35 cycles of 94°C for 30 sec, 53°C for 40 sec. and 72°C for 90 sec., after which a nal elongation step at 72°C for 5 min. The PCR products were observed in the 2% agarose gel, following ampli cation. The PCR pool was puri ed using Ampure PB beads (Paci c Biosciences). The SMRTbell libraries (Paci c Biosciences) were prepared following the manufacturer's guide and sequencing done at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on the PacBio Sequel following the manufacturer's guidelines. Sequences were processed using the MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA). All sequences were denoised, and chimeras removed (Edgar 2016). Two Microcystis sequences were deposited into NCBI GenBank with following accession numbers: OK160994, OK160995. Megaphylogeny containing 9700 (including 2 sequences of Microcystis) sequences was performed in ARB software package (Ludwig 2004

Microscopy and Spectroscopy
Fresh bloom material was observed on the same day as a collection using a Zeiss Axioscope light microscope with DIC optics (Zeiss, Oberkochen, Germany). Morphometric features were measured using Zeiss AxioVision LE (Zeiss, Oberkochen, Germany).
Leica TCS SP5 II confocal microscope (Leica Microsystems, Germany) was utilized for the living cells observations. Argon laser (488 nm) at 16% intensity was used for the excitation (for the determination of the special pigment distribution), uorescence emission spectra for carotenoids ranged from 510 to 605 nm, uorescence emission spectra for chlorophyll a ranged from 680 to 690 nm. Sequential scan of these two spectra was implemented using HyD 4 channel in order to reduce photobleaching. Emission spectra were determined based on xyλ scan and (Vermaas et al. 2008). Pictures showing pigment distribution were taken with following lens: 63x/1.4 Oil. For the aerotopes observation different setting were used, they were: 1) re ection signal were collected at 484-493 nm using PMT2 channel, 2)PMT3 was used to capture all photosynthetic pigments at 582-717 nm 3) HC PL FLUOTAR 20x 0.5 DRY lens was utilized. Same laser was used for this analysis with different intensity, which was 45%. Lastly, for the pigment pro ling, xyλ scan was utilized starting from 490 nm and ending with 700 nm. Pigments were named according to (Vermaas et al. 2008).
Glutaraldehyde xed cells were placed into agarose matrix inside the Eppendorf 2 ml tubes (Mozaffari et al. 2019). Subsequently agarose blocks with the cell pellets were washed with OsO 4 (Electron Microscopy Sciences, Hat eld, PA) at 2% dilution with 0.1 M imidazole-HCl at pH 7.2 inside the tubes. After that, agarose blocks were removed from the tubes and cut in the thin slices with visible osmium stained regions of the cells. Thin slices were placed in to sterile Petri dish with diH 2 O and were put on the shaker for 20 mins. Rinsed agarose slices were placed into 10 ml glass vial with plastic caps for the EtOH dehydration series. Series were as follows: 50% EtOH -20 mins, 80% EtOH -20 mins, 95% EtOH -20 mins, absolute EtOH -20 mins (x2). While dehydration, glass vials with the samples were places onto rotary mixer. Prior to the resin embedding, samples were rinsed with propylene oxide for 20 mins twice on the rotary mixer. Resin embedding utilized Spurr's epoxy resin (Low viscosity embedding kit, Electron Microscopy Sciences, Hat eld, PA). The rest of the procedure was followed as Robert Marc's Lab protocols (The University of UTAH). Resin blocks were cut on the Ultramicrotome in order to obtain thin sections for the TEM analysis. For the on grid negative staining (pili and cyanophages) observation, fresh grid were placed on the ltered colony and rinsed with 1% aqueous phosphotungstic acid, pH 6.5 (Vaara et al. 1984). TEM H-7650 (Hitachi High-Technologies, Tokyo, Japan) was used to observe both negative staining samples and thin section at 80 kV (high-resolution mode). Digital camera XR 60 (AMT Corp., Woburn, MA) was utilized to capture the images.
For the SEM, dehydrated cells (as it was describe in TEM section) that were not agarose embedded were observed on Tabletop Scanning Electron Microscope, Model TM-1000 (Hitachi High-Technologies, Tokyo, Japan) without sputter coating. In this analysis, accelerating voltage was 15 kV. The rest of the SEM pictures were taken on sputter-coated material utilizing Hitachi S-3400N II (Hitachi High-Technologies, Tokyo, Japan) with the following acceleration voltage -10 kV. Secondary electron detector was used.
Sputter coating was done using Pt/Au source. Energy-dispersive X-ray spectroscopy -EDS (Noran System Six 300, Thermo Electron Corp., Madison, WI) was used to determine elemental composition of the surface of the colony (F-layer). Acceleration voltage also was 10 kV. In order to compare cyanobacterial EPS with chondroitin sulfate, small portion of a Turkey's ligament was dehydrated with absolute EtOH analyzed using EDS.
Prior AFM scanning, glutaraldehyde xed cells were dried out on the cover slip in the room temperature. To acquire AFM images of the pili and cell surfaces, Bruker Dimension Fast Scan (Germany) was utilized with RTESPA probe (part #: MPP-11120-10) on the tapping mode. Amplitude error and Height sensors were implemented. Bruker NanoScope Analysis software v1.51 was used for the editing of the obtained images Toxin's analysis Lake water (600 ml) was ltered through Extract-Clean TM SPE C18-HC cartridge (part no. Modi ed gradient steps are given in Table S1. Detailed LC-PDA-MS 2 parameters are presented in Table   S2. Experimental data was acquired with MassLynx software version 4.0 (Waters TM corporation, Milford, MA, USA). Following masses: 382.2 and 742.4 were excluded from the analysis as a carryover artefacts.
Daughter ion masses used as a criterion used to switch to MS 2 are given in Table S3. MassLynx software

Results And Discussion
Morphological description of the bloom and bloom forming cyanobacteria Clearly visible cHAB was observed in the shallow part of the Snow Lake in the fall season of 2020. In contrast to our morphological observation, CyANapp designed by EPA predicted a wider distribution of the bloom within the lake (Fig. S1). CyANapp estimation is based on calculated re ectance near 681 nm, called CI index (Schaeffer et al. 2018;Mishra et al. 2021). The bloom occurred in the form of the irregular pale green clusters of the colonies close to the shore. They seemed to be in the water column all the way to the bottom in the shallow waters (up to 1 meter deep). The bloom collected on 10/8/2020 appeared signi cantly different to the summer collection (8/1/2020), which looked healthier. Below freezing night air temperatures may have affected colony growth in the fall. Light microscope observation revealed that 3 different species of cyanobacteria co-occurred in the cHAB sample: Microcystis os-aquae (Wittrock) Kirchner (Fig. 1), Aphanizomenon os-aquae Ralfs ex Bornet et Flahault, and Dolichospermum sp. Representatives of other taxonomic groups of algae were not seen in that particular water sample. However, many amoebas, agellates, and bacteria occurred. Based on the colony shape, coccoid cyanobacterium observed in the bloom sample was identi ed as Microcystis os-aquae (Wittrock) Kirchner (Fig. 1A, B). Further microscopic and phylogenetic investigations were not able to con rm this a liation giving only genus epithet -Microcystis sp. Reason for that is the lack of the type specimen sequence of Microcystis os-aquae (Wittrock) Kirchner. DIC imaging of the colonies (Fig. 1B) showed that the majority of the cell had aerotopes (dark regions within the cells). Aerotopes appear in this con guration since gas sharply scatters the light making aerotopes rigid and dark. Some cells did not have aerotopes (upper right corner on Fig. 1B). Figure 1B depicts an individual agellate with an obviously ingested individual Microcystis cell. This suggests bioaccumulation of the toxins in the food web, which will be the subject of future research.
Toxins found in the bloom sample LC/MS MRM analysis detected the presences of 4 derivatives of microcystin in the biomass sample collected from Snow Lake (Fig. 2). Microcystin -LR, -RR, -LY, and -YR were found in the biomass extract.
For the rst 3 microcystins we had standards (Fig. 2) con rming appropriate mass transitions (995.6 → 135.1, 520 → 135.1, 1002.7 → 135.1) in appropriate retention time. Additionally, microcystin-LR was found in water samples with the following concentration 0.3 μg/L, which is substantially lower than EPA thresholds for the total microcystins. Even though the cHAB consisted of several species, we assume that Microcystis spp. mostly contributed to toxin production. Considering the oligotrophic nature of Snow Lake (Low N and P, data is not shown) it was surprising to detect such a vivid cHAB with toxins released into the water column. With climate change and extensive agricultural practice in New Mexico and the USA in general, monitoring various water bodies applying analytical methods (HPLC-MS/MS, MRMs etc.) is crucially important.

Phylogenetic analysis on the major taxa in the bloom
Phylogenetic analysis of the 16S rRNA gene ampli ed from the bloom biomass found that two found cyanobacterial sequences clustered within the conventional "Microcystis" clade (Fig. 3, Fig. S2). Even though this clade contained 752 sequences that were identi ed as Microcystis, none of these sequences were sequences of the type specimen, Microcystis aeruginosa. The type species of the genus Microcystis aeruginosa was described from Weißenfels, Germany (near Leipzig, in stagnant water). In order to determine whether or not our Microcystis os-aquae (based on morphological observation) belonged to the true Microcystis clade, epity cation of type specimen or a new type of Microcystis aeruginosa should be established. So far, there are at least two different clades on our phylogenetic tree including sequences with Microcystis a liation. Epitypi cation/neotypi cation especially for toxic and potentially toxic taxa of cyanobacteria are quite important since many cyanobacterial species express cryptic characteristics (same morphology, different genotype). Epitypi cation/neotypi cation will label the lineages with hazardous strains which are going to be highly valuable during next-generation monitoring studies. So far, only a few "old" toxic cyanobacteria have been established correctly based on the Code (Turland et al.

2018).
Advanced microscopic and spectroscopic analyses of the Microcystis sp.
Confocal Microscopy (CM) was used in order to determine pigment distribution within the living cells of Microcystis sp. and to observe the shape of the cells (Fig. 4, Fig. S3,4). Confocal analysis of the emission spectrum showed that carotenoids were localized in the same place as chlorophylls, which is logical based on the con gurations of the photosynthesis apparatus (Vermaas et al. 2008). In general, pigments that were associated with thylakoids were distributed irregularly within the cells. Basically, Microcystis sp. cells were mostly packed with two different types of matter: thylakoids (yellow color on Fig. 4) and aerotopes (concave areas/black in Fig. 4, and Fig. S4). Re ecting channel imaging used in CM (both stereo 3D and regular) con rmed that (Fig. S5A,B). Green channel (emission at 500-700 nm) showed pigments within the cells, and red re ecting channel (excitation/re ection close to 490 nm) showed aerotopes (SFig. 5A,B). Aerotopes containing gas were not emitting any light, they re ected the laser. Additionally, we obtained spectrometric data showing different pigments (Fig. 4). There were chlorophyll a, phycocyanin (PC), allophycocyanin (APC), and allophycocyanin B (APC-B). Moreover, HPLC-MS/MS (plus UV-vis) analysis have detected several lipophilic pigments in the bloom sample: chlorophyll a in a radical form, zeaxanthin, fucoxanthin, and dinoxanthin. Partly Chlorophyll a and zeaxanthin came mostly from cyanobacteria. Fucoxanthin and dinoxanthin are more typical for other taxonomic groups of algae such as dinophyceae indicating heterogeneity of the algal species composition, which was not detected in taxonomic analysis. Lastly, "true" shapes of the cells and distribution of the cell content was determined using stereo z-stalk image (Fig. S4). Cells were spherical or hemispherical after division.
These features are quite important in systematics and will be used if these Microcystis populations are a new taxon.
TEM was used to investigate thylakoid arrangement. As before, thylakoids were found to be irregular (Fig.  5A). Thylakoid membranes were entangled together with aerotopes as was observed in CM. In some cases, thylakoids surrounded groups of the aerotopes, sometimes membranes were around a single aerotope. Our thin section TEM imaging was able to resolve the structure of a cell wall (Fig. 5B). Cell membrane, peptidoglycan cell wall, outer membrane, and F-layer (aka extracellular polysaccharides -EPS) were clearly recognizable. Negative staining TEM showed presence of pili-like structures (Fig. 5C) (Bhaya et al. 1999). We observed pili as a "fence" that prevents cyanophages from attacking the cells. Further experiments with wild type and pililess mutants of Microcystis together with cyanophages may shed light on this unanswered question. FFT on the pili images was used in the attempt to detect a structure of a helical protein (Fig. S6D). There were only typical Airy disk patterns without signs of a protein subunit. Additionally, we linearly and radially spread (in Photoshop) 1 pixel wide portion of edge of the individual pilus in order to enhance protein structure on FFT. None of these modi cations revealed protein structure. Most likely, the resolution of the microscope was not great enough to allow observation of that structure. Colonies observed on the grids (negative staining, no thin section) were surrounded by many different cyanophages with the capsid size between 125 and 190 nm (Fig. 5D). Taxonomically they belonged to 2 different families: Myoviridae and Podoviridae (Martin and Kokjohn 1999), the most abundant was the latter. Capsid measurement between our morphotype and the newly observed representative of Podoviridae with capsid diameter of 120 nm were the closest (Watkins et al. 2014). Most of the cyanophages were pushed away from the cells by pili and F-layer (Fig. S6A,B,C). Sometimes narrow spaces between the cells contained cyanophages in close contact (Fig. S6B). It seems that the pili and F-layer effectively protected cells against cyanophages. This was corroborated by the fact that none of the cells of Microcystis sp. were infected with cyanophages or caption of images was before regeneration and assembly of the cyanophages within the cells. Literature on cyanophages is rather scarce (Sullivan et  , and we hope that our observation will encourage microbiologists to pay more attention to this aspect of the cyanobacterial life cycle. Additionally, we hope that microbiologists and virologists will collaborate more often in order to elucidate questions related to cyanobacteria-cyanophage interactions. Microscopic colonies observed with SEM (Fig. 6A) resemble the typical morphology of Microcystis osaquae. The same identi cation was made after colonies were observed in the light microscope (Fig. 1). It is important to mention that edge of the colony (Fig. 6A, near bottom) was thinner compared with the middle part, in other words the edge of the colony had fewer layers of overlapping cells in 3 dimensional space. The low magni cation of the colony expressed a preparation artefact, in the form of random cracks within the colony. The reason for that is the dehydration procedure as well as drying. Individual cells within the colonies were less affected by SEM artefacts (Fig. 6B). Individual cells inside the colonies formed an almost regular pattern expressed as a 3D network of the cells away from each other by the same distance (Fig. 6B). All the cells were connected with each other by slimy strings with star like arrangement, they are apparently drying artefacts consisting of shranked F-layers collapsed on the pililike structures (Fig. 6C,D; Fig. S7). The trace of the F-layers was detected by EDS technique (Fig. S8) illustrating random distribution of sulfur (typical chemical element for the cyanobacterial F-layer) on the surface of the colonies. To further con rm our nding we compared the EDS spectrum of the F-layer of Microcystis sp. with chondroitin sulfate, which is similar to cyanobacterial F-layer by chemical structuresulfated glycosaminoglycan. Similar elements and relative abundance of the peaks was observed in comparative EDS analysis supporting our original assumption (Fig. S9). Research related to cyanobacterial F-layers (EPS) is of interest to many investigators and there are targeted studies trying to answer them (Li et al. 2013; Lilledahl and Stokke 2015; Tan et al. 2020). In our research, we attempted to detect the F-layer in our strain using different techniques and were successful. Figs. 6C and D shows pililike structures, the following width of 25-125 nm, this is larger than measurements based on TEM and AFM. These discrepancies might be explained by the fact that spotter coating added some width to the structure. Moreover, dehydrated F-layer collapsed on the pili could have contributed to the thicker surface of pili. Additionally, Fig. S7 con rmed the presence of many cyanophages in the sample. Lastly, SEM revealed the texture of the cell surface, which appeared to have a bumpy texture (not smooth, as it appears in the light microscope).
AFM showed a collapsed and dehydrated F-layer, which was connecting the cells (Fig. 7). Preparation of the specimen ( xation, dehydration) did not allow us to resolve individual polysaccharides chains.
However, FFT techniques showed structures similar to the polysaccharides chains (Fig. S10C, D). Fractal elements were detected by AFM: high-resolution images of the surface of the cells looked almost identical to the cell arranged in the colony expressing the same level of complexity (Fig. 11). AFM con rmed the presence of pili-like structures (Fig. 8). According to AFM, pili-like structures were in the following range (5)6-15 (17) nm. That is the smallest across the different types of microscopy we used. This may be explained by the fact that AFM measures height in contrast to TEM and SEM. Another explanation would be that pili might be submerged in debris inside the sample.
In conclusion, we establish a phylogenetic benchmark for a toxic population of Microcystis sp. from an alpine lake (Snow Lake) in New Mexico (USA). Additionally, we explored various aspects of this population utilizing different techniques. This information is important for the future monitoring of the cHABs.    One focal plane confocal image of the cells of the Microcystis sp. Chlorophylls (red channel) and carotenoids (green channel) were mostly concatenated, giving yellow color shown on the picture. Clearly visible dark concave areas on the cell surfaces are aerotopes (no emission). Various TEM images of Microcystis sp. cells and the cyanophages associated with it. A Thin section of the single cell. Note that the shape of the cell appeared to be irregular (not spherical or hemispherical), it is because of a particular plane of a thin section together with shrinkage during xation, dehydration, and embedding. B Zoomed in portion showing edge of the cell including cytoplasmic membrane etc. C On the grid, negative staining TEM image of the pili-like structures. D On the grid negative staining TEM shows cyanophages. Cyanophages were found near the colonies but not inside the cells (Fig. S6A,B,C).