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Cyanobacterial biodiversity of semiarid public drinking water supply reservoirs assessed via next-generation DNA sequencing technology

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  • Unidersidade Federal do Tocantins

Abstract and Figures

Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.
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*For correspondence. E-mail: mbitt@usp.br; Tel.: +55 19 3429-4128; Fax:
+55 19 3447-8649
§Supplemental material for this article may be found at
http://www.springerlink.com/content/120956.
Copyright G2019, The Microbiological Society of Korea
Adriana Sturion Lorenzi1,2, Mathias Ahii Chia1,3,
Fabyano Alvares Cardoso Lopes2,4,
Genivaldo Gueiros Z. Silva2, Robert A. Edwards2,5,
and Maria do Carmo Bittencourt-Oliveir
a
1*
1Laboratory of Cyanobacteria, Department of Biological Sciences, Luiz
de Queiroz College of Agriculture, University of São Paulo (USP),
Piracicaba, SP, Brazil
2Computational Science Research Center, San Diego State University,
San Diego, California, USA
3School of Marine and Atmospheric Sciences, Stony Brook University,
Southampton Campus, New York, USA
4Laboratory of Enzymology, Department of Cell Biology, University of
Brasília (UNB), Brasília, DF, Brazil
5Department of Computer Science, San Diego State University, San
Diego, California, USA
(Received Jun 27, 2018 / Revised Nov 13, 2018 / Accepted Dec 13, 2018)
Journal of Microbiology (2019) Vol. 57
DOI 10.1007/s12275-019-8349-7
eISSN 1976-3794
pISSN 1225-8873
Cyanobacterial biodiversity of semiarid public drinking water supply
reservoirs assessed via next-generation DNA sequencing technology§
Next-generation DNA sequencing technology was applied
to generate molecular data from semiarid reservoirs during
well-defined seasons. Target sequences of 16S-23S rRNA ITS
and cpcBA-IGS were used to reveal the taxonomic groups of
cyanobacteria present in the samples, and genes coding for
cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA),
and cylindrospermopsins (cyrJ) were investigated. The pre-
sence of saxitoxins in the environmental samples was eval-
uated using ELISA kit. Taxonomic analyses of high-through-
put DNA sequencing data showed the dominance of the ge-
nus Microcystis in Mundaú reservoir. Furthermore, it was the
most abundant genus in the dry season in Ingazeira reservoir.
In the rainy season, 16S-23S rRNA ITS analysis revealed that
Cylindrospermopsis raciborskii comprised 46.8% of the cyano-
bacterial community in Ingazeira reservoir, while the cpcBA-
IGS region revealed that C. raciborskii (31.8%) was the most
abundant taxon followed by Sphaerospermopsis aphanizo-
menoides (17.3%) and Planktothrix zahidii (16.6%). Despite
the presence of other potential toxin-producing genera, the
detected sxtA gene belonged to C. raciborskii, while the mcyE
gene belonged to Microcystis in both reservoirs. The detected
mcyE gene had good correlation with MC content, while the
amplification of the sxtA gene was related to the presence of
STX. The cyrJ gene was not detected in these samples. Using
DNA analyses, our results showed that the cyanobacterial
composition of Mundaú reservoir was similar in successive
dry seasons, and it varied between seasons in Ingazeira re-
servoir. In addition, our data suggest that some biases of an-
alysis influenced the cyanobacterial communities seen in
the NGS output of Ingazeira reservoir.
Keywords:cyanotoxins, genotypic composition, NGS, pub-
lic water supply, water quality
Introduction
Northeastern Brazil has well defined rainy and dry seasons
(Almeida et al., 2009), and water scarcity has worsened in
this semiarid region due to increased frequency and duration
of drought caused by changing global climatic conditions
(World Bank, 2013). The construction of reservoirs has re-
solved this problem by ensuring water is available during the
dry season and prolonged drought. However, the discharge
of domestic and industrial sewage eutrophies these water
bodies due to the nutrient-enriched conditions of the sewage.
Eutrophic conditions coupled with high water temperatures
and long water residence times have led to the excessive pro-
liferation of cyanobacteria in semiarid reservoirs over the
years (Chellappa and Costa, 2003; Costa et al., 2006; Bitten-
court-Oliveira et al., 2014). In Pernambuco (PE) State, 90%
of the reservoirs are eutrophic (Bouvy et al., 2000), and per-
ennially have blooms of Cylindrospermopsis raciborskii (Wol-
oszynska) Seenayya and Subba Raju, Microcystis aeruginosa
(Kützing) Kützing, Microcystis panniformis Komárek et al.,
Sphaerospermopsis torques-reginae (Komárek) Werner, Laug-
hinghouse IV, Fiore and Sant’Anna Klebahn (formerly de-
scribed as Anabaena spiroides), Sphaerospermopsis aphani-
zomenoides (Forti) Zapomělová, Jezberová, Hrouzek, Hizem,
Reháková and Komárková, and Planktothrix agardhii (Go-
mont) Anagnostidis and Komárek (Bouvy et al., 2001; Molica
et al., 2005; Bittencourt-Oliveira et al., 2014).
Among the secondary metabolites produced by cyanobac-
teria, cyanotoxins such as microcystins (MCs), cylindrosper-
mopsins (CYNs), and saxitoxins (STXs) have been detected
in Brazilian semiarid water supply reservoirs (Bittencourt-
Oliveira et al., 2011a, 2014) or in cyanobacterial strains iso-
lated from these aquatic systems (Borges et al., 2015). MCs
are assembled by multifunctional enzyme complexes con-
sisting of nonribosomal peptide synthetases (NRPSs) and
polyketide synthases (PKSs) (Christiansen et al., 2003; Rou-
hiainen et al., 2004). The MC gene cluster in the model cy-
anobacterium Microcystis aeruginosa PCC7806 spans 55 kb
of DNA composed of 10 (mcyABCDEFGHIJ) bidirectionally
transcribed open reading frames (ORFs) arranged in two pu-
tative operons, mcyA-C and mcyD-J (Tillet et al., 2000; Ras-
togi et al., 2015). Similarly, STXs and CYNs are synthesized
2Lorenzi et al.
by modular NRPSs and PKSs enzyme complexes (Dittmann
et al., 2013). In cyanobacteria such as Anabaena circinalis,
Aphanizomenon, Cylindrospermopsis raciborskii, Lyngbya
wollei, and Raphidiopsis brookii the STX gene cluster (25.7–
36.0 kb) consists of up to 33 genes encoding biosynthetic en-
zymes, transporters and regulatory proteins (Neilan et al.,
2013). The CYN gene cluster (cyr) spans 43 kb of DNA and
is comprised of 15 open reading frames containing genes re-
quired for the biosynthesis, regulation, and export of the
toxin (Mihali et al., 2008).
In response to the tragic incident in Caruaru (PE) – Brazil,
in 1996, where the death of several dialysis patients occurred
after exposure to MCs contaminated water (Jochimsen et al.,
1998; Azevedo et al., 2002), the Brazilian government pro-
posed a specific regulation (Brazil, 2011) that stipulates man-
datory monitoring of water reservoirs used for public supply.
This resolution establishes maximum permissible limits of
1 μg/L for MCs and 3 μg/L for SXTs, and weekly monitoring
when the cyanobacterial cell density is higher than 10,000
cells/ml (Brazil, 2011).
The objective of several studies has been to determine the
composition and ecology of cyanobacteria in the Brazilian
semiarid water bodies (Bittencourt-Oliveira et al., 2011b,
2012a; Lira et al., 2011; Dantas et al., 2012; Moura et al., 2012;
Fonseca et al., 2015). However, the molecular ecology of
toxin-producing cyanobacteria has been poorly addressed
(Bittencourt-Oliveira, 2003; Bittencourt-Oliverira et al., 2010,
2011a, 2012b; Borges et al., 2015; Lorenzi et al., 2015).
Recent advances in sequencing technologies allied with bio-
informatics tools have increased the possibility of large-scale
studies of cyanobacterial communities (Steffen et al., 2012;
D’Agostino et al., 2016). Next-Generation Sequencing (NGS)
technologies employed in metagenomics, comparative ge-
nomics, and metatranscriptomics have significantly contri-
buted to the understanding of the ecology and control of
harmful blooms of cyanobacteria (Li et al., 2011; Steffen et
al., 2012, 2015; Penn et al., 2014; Voorhies et al., 2016; Wood-
house et al., 2016; Walter et al., 2018). Therefore, the objec-
tives of the present study were to: (1) to characterize the di-
versity of cyanobacteria and cyanotoxin genes in Ingazeira
and Mundaú reservoirs (Brazil) during the rainy and/or dry
seasons using NGS, and (2) determine whether the detected
cyanotoxin genes were associated with the presence of toxins
in the investigated environmental samples. The Ingazeira and
Mundaú reservoirs are very important semiarid water bodies
that provide potable water to more than 300,000 people (SRH,
2000), and are perennially plagued with noxious bloom-for-
ming and potential toxin-producing cyanobacteria (Bitten-
court-Oliveira et al., 2014). This work represents an effort to
evaluate the Brazilian semiarid cyanobacterial composition
using NGS, and the results provide valuable data on the di-
versity and abundance of cyanobacteria in public water sup-
ply reservoirs in unique ecosystems worldwide. The public
availability of these data will serve as a basis for comparison
with other distinct environments, which will aid the compre-
hensive understanding of the cyanobacterial community of
specific ecosystems.
Materials and Methods
Site description and sample collection
In addition to supplying drinking water to over 300,000 in-
habitants during periods of drought, which are characteristic
of the semiarid climate, Ingazeira and Mundaú reservoirs in
northeastern Brazil are used for irrigation, fishing, ranching
and bathing (Bouvy et al., 2000). The reservoirs are located
in the “Agreste” phytogeographic zone of Pernambuco State
(Supplementary data Fig. S1), which is characterized by re-
gular rainy and dry regimes, and an average yearly temper-
ature of 26°C. Cyanotoxins have been frequently detected in
these reservoirs (Lorenzi et al., 2018).
Surface water samples were previously collected preferen-
tially from the middle of Ingazeira (08°36󼛂41.2󼛃S; 36°54󼛂G
23.7󼛃W) and Mundaú (08°57󼛂21.1󼛃S; 36°30󼛂07.3󼛃W) reser-
voirs by Bittencourt-Oliveira et al. (2014). Water samples were
collected twice from Ingazeira (I1 - Apr 14 and I18 - Oct 13,
2009) and Mundaú (M1 - Mar 17 and M27 - Nov 09, 2009)
reservoirs using 20-μm mesh plankton net. Sample I1 was
collected in the rainy season, while samples I18, M1, and M27
were obtained in the dry season. Due to problems associated
with the sampling difficulties and sample availability, inter-
seasonal comparison was only possible for the Ingazeira re-
servoir but not the Mundaú reservoir. Further, the compari-
son was made only for samples from the same reservoir be-
cause there is a 58.57 km-straight line distance between Inga-
zeira and Mundaú reservoirs, and the NGS technology was
employed for site discovery to investigate related samples.
Metagenomic DNA extraction, PCR amplification, and next-
generation sequencing
Environmental water samples were subjected to DNA ex-
traction immediately after samplings using a cetyltrimethyl-
ammonium bromide (CTAB)-based extraction method (Ro-
gers and Bendich, 1985) as described previously (Lorenzi et
al., 2015), and at least three biological replicates were car-
ried out per sample. The samples were stored at -20°C until
analysed. In the present study, three technical replicates of
PCR reactions targeting the 16S rRNA gene with the 16S–23S
intergenetic segment (Strunecký et al., 2011) and cpcBA-IGS
(Neilan et al., 1995) region were performed on pooled DNA
samples. In order to investigate the presence of microcystins,
cylindropermopsins, and saxitoxins genes, the mcyE (Rantala
et al., 2004), cyrJ (Mihali et al., 2008), and sxtA (Smith et al.,
2011) genes were targeted, respectively. PCR reactions were
performed using 10 ng of total DNA and pureTaq Ready-
To-Go PCR Beads kit (GE Healthcare) in a GeneAmp PCR
System 9700 Thermal Cycler (Applied Biosystems). PCR ther-
mocycling conditions were in accordance with those speci-
fied by the authors indicated above. Negative controls (with-
out DNA) were prepared using the same reaction conditions
and primers. Subsequently, amplicons stained with SYBR®
Gold Nucleic Acid Gel Stain (Invitrogen) were analyzed on
1.0% agarose gels after electrophoresis in 0.5 × TBE run-
ning buffer. PCR products were subjected to shotgun sequen-
cing. Paired-end libraries were prepared with the Nextera
XT DNA Library Prep Kit part# FC-131-1096 (Illumina) ac-
cording to the manufacturer’s instructions. The libraries were
Next-generation DNA sequencing of cyanobacterial blooms 3
sequenced on a MiSeq Personal Sequencing System (Illumina)
using the 500-cycle MiSeq reagent kit v2 (2 × 250) (Illumina).
An average of 60,238.9 reads per sample was obtained from
a subset of the original 12 samples.
Data preprocessing, annotation, and analyses
Paired-end reads were merged using PEAR software (Zhang
et al., 2013) to produce consensus sequences and increase the
annotation accuracy. Low-quality bases (quality score < 20)
from merged and unmerged sequences were trimmed from
both ends using the Phred algorithm with the python script.
Merged and unmerged trimmed sequences from the same
sample were concatenated into a single file for each sequence
category (16S-23S rRNA ITS, cpcBA-IGS, mcyE, and sxtA),
and the replicates were analyzed separately. These files were
uploaded to the Metagenomics Rapid Annotation (MG-
RAST) server (Meyer et al., 2008), and made publicly acces-
sible under the project accession ‘Metacommunity of cya-
nobacterial blooms – ESALQ USP / SDSU’ and accession
numbers 4676155.3 to 4676165.3 and 4676167.3.
Unassembled DNA sequences (250-bp length) of each sam-
ple dataset were annotated with the BLASTN 2.2.32+ soft-
ware (Altschul et al., 1997) using a cut-off E-value of 1e-5 and
97% minimum sequence identity against the NCBI-NT da-
tabase for downstream analyses. Tables of frequencies of the
hits to each target sequence for each sample dataset were ge-
nerated and normalized by dividing by the total number of
hits to remove bias difference in read lengths and sequencing
efforts. Heat maps, which are graphical representations of
individual values contained in a matrix in colors, were con-
structed from the predicted relative abundance of the taxa
using a homemade python script, and Euclidean distance em-
ployed as the distance method. Only the best and relevant
number of hits for each query sequence was used in the count.
The abundances of DNA sequences from the water samples
were compared using the Statistical Analysis of Metagenomic
Profiles (STAMP) software version 2.1.3 (Parks et al., 2014),
to test the hypothesis that there were no significant differ-
ences between the samples and provide exploratory plots for
analyzing the profile of samples from the same researvoir.
P values were calculated using the Welch’s t-test (P < 0.05),
and correction was applied using Bonferroni (16S-23S rRNA
ITS and cpcBA-IGS) and Benjamini-Hochberg (mcyE and
sxtA) methods. Taxa with a small effect size were removed
by filtering (effect size < 1.0), and asymptotic confidence
intervals (95%) were calculated. Correlation matrix based
principal component analysis (PCA), a process that employs
orthogonal transformation, was performed on abundance
measures using the Factoextra 1.0.3 package with the R ver-
sion 3.2.2 software (R Development Core Team, 2015). With
an ellipse level of 0.95, the PCA was used to convert the set
of observations of possibly correlated variables into a set of
values of linearly uncorrelated variables. Cross-assembly of
cyanobacterial sequences was conducted using MIRA (Che-
vreux et al., 2004) by concatenating all the sequenced reads
from all the samples into one unique file. Individual sequ-
ences compared to the cross-assembly were visualized using
distance and cladogram from the crAss (Dutilh et al., 2012)
output.
Cyanotoxin analysis
MCs and CYNs results were retrieved from Bittencourt-
Oliveira et al. (2014), and STXs results from Lorenzi et al.
(2018). These data were used for comparisons with the de-
tection of potential toxin-producing cyanobacterial geno-
types in the samples. The ELISA method is widely used for
cyanotoxin detection and quantification in field and labo-
ratory samples, and many studies have demonstrated a strong
correlation between ELISA and LC-MS results (Babica et
al., 2006; Wood et al., 2008; Bláhová et al., 2009; Ballot et al.,
2010), and HPLC-DAD (R = 0.96, P < 1 × 10−10) (Metcalf et
al., 2000).
Results
Cyanobacterial diversity
Heat maps generated from sequencing data on the basis of
the relative abundance of cyanobacteria are shown in Fig. 1.
The cyanobacterial profiles were variable between seasons
in the Ingazeira reservoir. The 16S-23S rRNA ITS sequence
results showed that Cylindrospermopsis raciborskii (46.8%)
was dominant in the rainy season. Furthermore, C. raci-
borskii (31.8%) was the most dominant species followed by
Sphaerospermopsis aphanizomenoides (17.26%) and Plank-
tothrix zahidii (16.4%) on the basis of cpcBA-IGS sequences.
Microcystis aeruginosa was the most abundant cyanobacte-
rial species in Ingazeira reservoir in the dry season with re-
gard to 16S-23S rRNA ITS sequences (Fig. 1), while M. pan-
niformis was dominant on the basis of cpcBA-IGS sequences.
The 16S-23S rRNA ITS sequences of uncultured cyanobac-
terium were as abundant as those of M. aeruginosa in the dry
season. Moreover, the sequences of uncultured Microcystis
sp. comprised 18.18% of total cpcBA-IGS sequences in the
dry season.
Microcystis was the most abundant genus in the two con-
secutive dry seasons in Mundaú reservoir (Fig. 1). With re-
gard to the 16S-23S rRNA ITS sequences, the proportion
(57.97% for M1, and 56.10% for M27) of M. aeruginosa was
similar in both dry seasons. Despite the occurrence of M.
aeruginosa in M1, M. flos-aquae significantly contributed to
the abundance of the Microcystis genus on the basis of cpcBA-
IGS sequences. On the other hand, M. flos-aquae was domi-
nant in M27, followed by M. aeruginosa and M. panniformis
according to cpcBA-IGS sequence analysis. In addition, the
proportion of M. aeruginosa population was almost unaltered
in M1 and M27 based on cpcBA-IGS sequences. Analysis of
cpcBA-IGS sequences revealed a similar relative abundance
of Radiocystis fernandoii in M1 and M27.
Characterization of cyanotoxin genes
The detected mcyE gene belonged to Microcystis in Ingazeira
reservoir, despite the presence of other potential MC-pro-
ducing genera in samples I1 and I18 of the rainy and dry
seasons, respectively (Fig. 1). The proportion of Microcystis
sequences changed during the seasons, leading to the domin-
ance of Microcystis sp. CYN10 (accession number FJ393328)
in the dry season (Fig. 1). The detected sxtA gene in Ingazeira
reservoir was strongly associated with C. raciborskii in the
4Lorenzi et al.
Fig. 1. Relative abundance of cya-
nobacteria and cyanotoxins genes
in Ingazeira (I) and Mundaú (M)
reservoirs. Lowercase letters a, b,
and c represent triplicate samples.
White color (-) represents zero
counts.
rainy and dry seasons. The presence of potential CYN-pro-
ducing cyanobacteria was not detected by PCR amplifica-
tion of the cyrJ gene with the oligonucleotide primers cyn-
sulfF and cylnamR (Mihali et al., 2008).
In Mundaú reservoir, the detected mcyE gene belonged to
M. aeruginosa and Microcystis sp. RST 9501 (JQ771642) in
both dry seasons. However, the proportion of mcyE gene be-
longing to M. aeruginosa and Microcystis sp. RST 9501 (ac-
cession number JQ771642) varied between the two dry sea-
sons (Fig. 1). The detected sxtA gene in samples M1 and M27
belonged to C. raciborskii. The cyrJ gene was not detected in
these samples.
Seasonal comparison of cyanobacterial diversity and cyano-
toxin genes in the studied reservoirs
The profiles of cyanobacterial diversity in the samples col-
lected from the same reservoir were compared with the
STAMP software, and the detected differences are shown
in Fig. 2. Pairwise comparisons of 16S-23S rRNA ITS sequ-
ences of samples I1 (rainy) and I18 (dry) from Ingazeira
reservoir indicated that C. raciborskii population was the
highest in I1 (Fig. 2A, powder gray), which was indicative
of significant positive differences. On the other hand, the
population size of M. aeruginosa was the highest in I18 (Fig.
2A, gray), corresponding to negative differences between
Next-generation DNA sequencing of cyanobacterial blooms 5
(A)
(B)
Fig. 3. Principal component analysis of the most abundant taxa (A) and cyanotoxins genes (B) in Ingazeira (I) and Mundaú (M) reservoirs. Light gray col-
ored taxa show low contribution to the analysis. ITS – 16S-23S rRNA ITS; IGS – cpcBA-IGS; I1 – rainy season; I18, M1 and M27 – dry season samples.
Lowercase letters a, b, and c represent triplicate samples.
6Lorenzi et al.
Next-generation DNA sequencing of cyanobacterial blooms 7
the populations. As visualized by STAMP, highest abun-
dance of C. raciborskii and S. aphanizomenoides popula-
tions in I1 (Fig. 2C, powder gray) corresponded to positive
differences, while that of M. panniformis in I18 (Fig. 2C, gray)
corresponded to negative differences between proportions
on the basis of cpcBA-IGS sequences. In the case of mcyE
gene analysis, the overrepresentation of M. aeruginosa po-
pulation in I1 (Fig. 2E, powder gray) showed positive differ-
ences between the analyzed samples.
For Mundaú reservoir, pairwise comparisons of cpcBA-IGS
sequences of samples M1 and M27 (dry seasons) indicated
that the population of M. flos-aquae was higher in M1 (Fig.
2D, light gray), which corresponded to positive differences.
The population of M. panniformis was overrepresented in
M27 (Fig. 2D, dark gray), indicating negative differences be-
tween proportions of this species. In addition, positive and
negative differences between mcyE proportions were shown
for M. aeruginosa population, which was highest in M1 (Fig.
2F, light gray), and Microcystis sp. RST 9501 that was over-
represented in M27 (Fig. 2F, dark gray), respectively. No sig-
nificant differences were found with regard to the relative
abundance of sxtA gene sequences in Ingazeira and Mundaú
reservoirs.
Principal component analysis (PCA) showed a significant
contribution of the 16S-23S rRNA ITS sequences to the cor-
relation between the collected samples and reservoirs (Fig.
3A). PCA indicated a strong positive correlation between
the Mundaú samples M1 and M27 (dry seasons) and M. ae-
ruginosa based on 16S-23S rRNA ITS sequences. The rela-
tive abundance of the 16S-23S rRNA ITS sequences of Plank-
tothrix pseudagardhii and uncultured proteobacterium sig-
nificantly distinguished the Ingazeira I18 (dry season) sam-
ple from the I1 (rainy season) sample (Fig. 3A). The uncul-
tured proteobacterium sequences were generated using the
359F and 23S30R (Strunecký et al., 2011) primers, and were
also annotated with a cut-off E-value of 1e-5 and 97% mini-
mum sequence identity against the NCBI-NT database. Due
to the significant number of the hits obtained with the un-
cultured proteobacterium sequences, they were not omitted
from the results. The first 2 principal components accounted
for over 96% of the total variation (Fig. 3A). PCA results for
toxin-related genes are shown in Fig. 3B. The detected mcyE
gene was significantly associated with the different climatic
seasons and reservoirs (Fig. 3B), while the sxtA gene was sig-
nificantly correlated with C. raciborskii on the second prin-
cipal component. The cyanobacterial taxa related to mcyE se-
quences strongly separated the water samples and reservoirs
into four distinct quadrants. This ensured that even samp-
lings performed in consecutive dry seasons were not grouped
together. The first 2 principal components of the PCA were
responsible for over 74% of the total variation (Fig. 3B).
To better evaluate whether each environmental sample har-
bored a specific cyanobacterial community per climatic sea-
son, their taxonomic and cyanotoxin genes compositions
were assessed with the crAss program. The crAss cladogram
showed that cyanobacterial community profiles were clearly
separated according to the sampling times and reservoirs
(Supplementary data Fig. S2). However, an exception was one
of the replicates of M27 (M27c) that was similar to those of
M1, showing that the technical reproducibility of the PCR
reactions should be taken into consideration when DNA
sequences are analyzed.
Cyanotoxins
MC and STX were detected in all the samples analyzed (Sup-
plementary data Table S1). We are aware that the ELISA
kit crossreacts with MC-LR, MC-RR, and MC-YR iso-
forms, which made it impossible to define the type of MC
detected in the different samples. The highest MC concen-
tration was recorded in Mundaú reservoir during the first
dry season, while the highest concentrations of STX were
found in Ingazeira reservoir in the rainy and dry seasons.
CYN was not detected in the samples analyzed.
Discussion
Cyanobacteria are an important group because of the roles
they play in the carbon and nitrogen cycling, environmental
and human health risk, and their biotechnological and phar-
macological applications. Historically, these organisms have
been studied on the basis of microscopic inspection, cell
counts, and biochemistry.
In the present study, the molecular analysis of environmental
samples using different target sequences aided the compar-
ison of taxonomically dominant groups in the Ingazeira and
Mundaú reservoirs per climatic season. Since these reservoirs
are located in the Brazilian semiarid region that is a particular
ecosystem worldwide, the public availability of these data will
facilitate comparisons with other environments, and provide
comprehensive information on the community of cyanobac-
teria of unique ecosystems. In addition, the molecular pro-
files obtained have valuable applications for future studies
on biochip constructions for the detection of potential cya-
notoxin-producing species in specific environments.
Cyanobacterial diversity
Several studies conducted on Brazilian semiarid reservoirs
have shown that these environments harbor similar cyano-
bacterial communities (Bouvy et al., 2003; Bittencourt-Oli-
veira et al., 2011a, 2011b, 2012a, 2014; Dantas et al., 2011;
Lira et al., 2011; Moura et al., 2011). According to Bittencourt-
Oliveira et al. (2014), cyanobacterial diversity was mainly
constituted by the toxin-producing species Cylindrospermopsis
raciborskii, Planktothrix agardhii, Sphaerospermopsis apha-
nizomenoides, and Geitlerinema amphibium in 10 reservoirs
analyzed. The only exception was the Venturosa reservoir,
where the dominant species was Merismopedia tenuissima.
Using a DNA sequence based-approach, the dominant ge-
nus found in Mundaú reservoir was Microcystis (consecu-
tive dry seasons), which is in accordance with the report by
Bittencourt-Oliveira et al. (2014) that Microcystis panniformis
was the dominant species in the reservoir. M. panniformis
is a very common species in Southern America, especially in
the eastern part of Brazil (Sant’Anna et al., 2004). However,
using morphological features for taxonomic identification
of different Microcystis species is limited by the quality of the
samples and microscopic equipment, and the competence
of the taxonomist. As the current morphological classifica-
8Lorenzi et al.
tion of Microcystis species is not supported by 16S rRNA gene
phylogenetic analysis (Willame et al., 2006) and the average
sequence divergence has been generally less than 1% (Rudi
et al., 1997; Otsuka et al., 1998), we used the 16S-23S rRNA
ITS and cpcBA-IGS sequences. Studies of the ITS region of
ribosomal RNA genes (16S-23S rRNA ITS) have distinguished
closely related prokaryotic species or populations (Bolch et
al., 1996), whereas the restriction enzyme digestion of 16S-23S
rRNA ITS has been used to resolve closely related cyanobac-
terial strains (Janse et al., 2003). Despite the inclusion of the
ITS region, the molecular marker did not effectively assess
the different Microcystis species when compared to the cpcBA-
IGS results (Fig. 1). In fact, the unification of some Micro-
cystis species, including M. aeruginosa, M. ichthyoblabe, M.
novackekii, M. viridis, and M. wesenbergii, into a single M.
aeruginosa species has been proposed under the Rules of the
Bacteriological Code (Otsuka et al., 2001; Harke et al., 2016).
In the present study, the best results were obtained using the
cpcBA-IGS region as the target molecule (Fig. 1). Compared
to the 16S-23S rRNA ITS sequences, Microcystis species were
better differentiated using the IGS sequence between the β-
and α-subunit ORFs (cpcBA-IGS), which encode for β-phy-
cocyanin and α-phycocyanin, respectively (Neilan et al., 1995).
The intergenic spacer (IGS) between the two bilin subunit
genes of the phycocyanin operon (PC) has been shown to
be a variable region of DNA sequence that is useful for the
identification of cyanobacteria to the strain level (Neilan et
al., 1995).
In Ingazeira reservoir, C. raciborskii was the dominant spe-
cies in the rainy season, while M. aeruginosa was dominant
in the dry season (Fig. 1). There were significant differences
(P < 0.05) between these taxonomic groups in the rainy and
dry seasons. The relative abundance of C. raciborskii was
higher in the rainy season, followed by a decrease in the dry
season (Fig. 2A and C). The opposite was observed for M.
aeruginosa in the same seasons. Recently, metagenomics de-
monstrated the presence of Microcystis and Cylindrosper-
mopsis in all ponds sampled in a severely drought-impacted
semiarid region of Paraíba State, Brazil (Walter et al., 2018).
Similar to the results obtained in Mundaú reservoir, the
16S-23S rRNA ITS sequence was not as discriminatory as
cpcBA-IGS in assessing the cyanobacterial diversity of Inga-
zeira reservoir (Fig. 1). Species such as P. zahidii and S. apha-
ninomenoides were only relevant in the rainy season when
the cpcBA-IGS sequence was applied as a molecular mar-
ker (Fig. 1). In the same way, Microcystis species were well
characterized by the cpcBA-IGS sequence in the dry season.
On the basis of the DNA sequencing approach, our data
showed that the composition of cyanobacterial communi-
ties changed during the rainy and dry seasons in Ingazeira
reservoir. However, using traditional microscopic techni-
ques, Bittencourt-Oliveira et al. (2014) reported multi-spe-
cies blooms of cyanobacteria, with Planktothrix agardhii as
the most abundant (69.70%) species in the rainy season in
Ingazeira reservoir. Furthermore, the authors reported that
Geitlerinema amphibium (52.76%) and Planktothrix agardhii
(32.67%) were dominant in the reservoir in the dry season.
Although DNA sequencingGtechniques can efficiently assess
previously undetectable organisms in an environment, some
bias introduced during the processing steps of the study such
as DNA extraction protocol, sequencing artifacts, DNA copy
numbers, and primer design may produce different results
when compared to traditional methods (Brooks et al., 2015).
Large discrepancies in the proportion of Gram-negative bac-
teria have already been observed using next-generation se-
quencing, microscopy, and culture-based methods (Lagier
et al., 2012). Despite performing at least 3 DNA extractions
and 3 PCR amplifications per sample, our results demon-
strated that some bias remained, which probably led to the
differences between the results reported by Bittencourt-Oli-
veira et al. (2014) and those of the present study. It is possible
that the primer sets we used are more specific to coccoid than
filamentous cyanobacteria. This may lead to a preferential
amplification of multi-template PCR and/or primer mis-
match, which probably caused the discrepancies observed.
Furthermore, the dominant genus found in Mundaú reser-
voir was Microcystis (consecutive dry seasons), which is in ac-
cordance with the report by Bittencourt-Oliveira et al. (2014).
From the results obtained with environmental samples, Sipos
et al. (2007) recommended the use of a low annealing tem-
perature to reduce preferential amplification and maintain
PCR stability. In addition, the BLAST hits obtained by sear-
ching against reference databases are generally used for taxo-
nomic identification of query sequences (Kim et al., 2013),
but are limited by the sequences available in public databases.
Sequences of P. agardhii and G. amphibium isolated from
Brazilian ecosystems are extremely scarce in public data-
bases, and may contain genetic diversities that do not permit
the annealing of currently designed primers. Furthermore,
the genetic diversity of uncultured cyanobacteria should be
taken into account when designing PCR based-methods. As
previously stated above, it is important to note that the use
of morphological features for taxonomic classification of cy-
anobacteria has limitations, which also may have been re-
sponsible for the differences recorded between the results
reported in Bittencourt-Oliveira et al. (2014) and those of
the present study.
Characterization of cyanotoxin genes
The toxicity potential of water bodies from semiarid eco-
systems was recently shown by Walter et al. (2018) using
NGS. COG functional annotation of metagenomic sequences
revealed gene sequences related to toxin production in drin-
king water from a semiarid region of Paraíba State (Brazil),
including cyanopeptolin synthetase (mcn), microcystin syn-
thetase (mcy), and non-ribosomal peptide synthase genes.
The PCR-based sequencing method employed in this study
generated sequences of mcyE and sxtA genes, which are in-
volved in MC and STX biosynthesis, respectively. The mcyE
gene codes for a partial adenylation domain and a phospho-
pantetheine-binding site, the region that activates glutamic
acid in the MC biosynthesis pathway (Rantala et al., 2004).
The sxtA gene encodes a polyketide synthase (PKS)-like struc-
ture that is uncommon to STX biosynthesis in cyanobacteria
(Kellmann et al., 2008). Despite the presence of other poten-
tial toxin-producing genera, the detection of the sxtA gene
was mainly associated with C. raciborskii, and the presence
of the mcyE gene was related to Microcystis in both reser-
voirs. As previously reported, the effects of bias during sam-
ple processing, sequencing, and sequence annotation may
Next-generation DNA sequencing of cyanobacterial blooms 9
have interfered with the affiliation of the sequences. Never-
theless, by integrating molecular taxonomy and functional
features, it was possible to separate different samples from
the reservoirs analyzed (Supplementary data Fig. S2), which
demonstrated the sensitivity of DNA-based methods.
Cyanotoxins and related genes
The detected mcyE gene had good correlation with the pre-
sence of MC, while the amplification of the sxtA gene was
related to STX content (Supplementary data Table S1). The
C. raciborskii populations of Brazilian aquatic systems are
potential STX producers (Lagos et al., 1999), and the pre-
sence of the sxt biosynthetic gene cluster has been confir-
med in the T3 strain of the cyanobacterium (Kellmann et al.,
2008). Unlike the Southern American isolates, the C. raci-
borskii strains isolated from Australian and Asian aquatic
ecosystems synthesize CYN. This provided a basis for the
characterization of the cyr gene cluster in C. raciborskii AWT-
205 (Mihali et al., 2008). Although the cyrJ gene is a suitable
probe for the detection of potential CYN producers (Mihali
et al., 2008), its absence in samples collected from Ingazeira
and Mundaú reservoirs correlates with the absence of the
cyanotoxin in both reservoirs. It is important to note that CYN
was recently reported in some Brazilian semiarid water sup-
ply reservoirs containing C. raciborskii (Bittencourt-Oliveira
et al., 2011a). Despite the lack of PCR amplification of the cyrJ
gene and the absence of CYN in the present study, careful
consideration should be given to non-annealing of the oli-
gonucleotide primers and non-expression of the cyr gene
cluster.
Conclusion
The PCR-based methodology applied in this study gives an
overview of the genetic basis of the cyanobacterial profiles
present in two Brazilian semiarid public water supply reser-
voirs. Taxonomic analysis of high-throughput DNA sequen-
cing data showed that the cyanobacterial diversity did not
significantly change in Mundaú reservoir during the two con-
secutive dry seasons, and it varied over the seasons in Inga-
zeira reservoir. The detected mcyE and sxtA genes were as-
sociated with the presence of the cyanotoxins MC and STX
in both reservoirs, respectively. Using the molecular profiles
generated, biochip constructions for the detection of poten-
tial cyanotoxin-producing species in specific environments
may be obtained in future works. However, our results sug-
gested some biases of analysis on the basis of DNA sequ-
encing such as preferential amplification of multi-template
PCR, primer mismatch, and/or sequence availability in pub-
lic databases, which probably influenced the cyanobacterial
communities seen in NGS output. Based on the findings of
the present study, we recommend the integration of tradi-
tional approaches with NGS methods for the comprehensive
understanding of the community structure and function of
cyanobacterial populations in natural aquatic ecosystems.
Acknowledgements
This study was sponsored by grants from the São Paulo
Research Foundation (FAPESP – 2013/15296-2) and the Bra-
zilian National Research Council (CNPq – 442083/2014-9).
A.S.L. and M.A.C were supported by FAPESP post-doctoral
fellowships (Grant 2014/01913-2 and 2013/11306-3, respec-
tively). G.G.Z.S. was supported by NSF Grants (CNS-1305112,
MCB-1330800, and DUE-132809 to R.A.E). F.A.C.L. was
supported by CAPES graduate scholarship.
Conflict of Interest
The authors declare that there are no conflicts of interest.
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... Despite their potential importance, the structure and functioning of the microbial communities in these ecosystems have received relatively less attention compared to other freshwater bodies, such as natural lakes and rivers [13]. There are very few studies on bacterial community structures and compositions of the surface waters in Brazil [14,15]. Thus, this study aimed to (i) to explore the microbial communities in surface and bottom layer water along the Billings reservoir using the 16 S rRNA gene-based Illumina MiSeq sequencing (ii) evaluate the presence of potential pathogens in these water samples and (iii) explore the predicted functional pro les of the obtained microbial communities in the basin to determine their role in the ecosystem. ...
... Members of Cyanobacteria were detected as the most dominant genus and that all Cyanobacteria bloom tested here were toxic. These results lend further support to previous studies that demonstrated Brazilian semiarid reservoirs harbor cyanobacterial communities [15,[30][31][32][33][34]. The presence of these bacteria in high abundance in the reservoir can be linked to uploading nutrients like ammonia and to the increase in water temperature (20.7°C) at the time of sampling. ...
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Here, we describe the microbial diversity and physicochemical properties in freshwater samples from the surface and bottom layer of Billings reservoir in São Paulo state, Brazil. Twenty-two matched samples were characterized using the 16S rRNA gene Illumina MiSeq platform. Taxonomical composition revealed an abundance of Cyanobacteria phyla, followed by Proteobacteria , with 1903 and 2689 known bacterial genera in the surface and deep-water layers, respectively. Chroobacteria, Actinobacteria, Betaproteobacteria, Alphaproteobacteria, Sphingobacteriia, and Acidimicrobiia were the most dominant classes. Shannon diversity index ranging from 2.3 - 5.39 and 4.04 - 6.86 in the surface and bottom layer, respectively. Among the diverse pathogenic genera identified, Flavobacterium was the most predominant genus. Temperature and phosphorus concentration were among the most influential factors in shaping the microbial communities of both layers. Predictive functional analysis suggests that the reservoir is enriched in motility genes involved in the flagellar assembly. The overall results present new information on the significantly altered diversity composition of the bacterial community detected in Billings freshwater reservoir.
... Despite their potential importance, the structure and functioning of the microbial communities in these ecosystems have received relatively less attention compared to other freshwater bodies, such as natural lakes and rivers [13]. There are very few studies on bacterial community structures and compositions of the surface waters in Brazil [14,15]. Thus, this study aimed to (i) to explore and compare the microbial communities in surface and bottom layer water along the Billings reservoir using the 16 S rRNA gene-based Illumina MiSeq sequencing, (ii) evaluate the presence of potential pathogens in these water samples and (iii) explore the predicted functional pro les of the obtained microbial communities in the basin to determine their role in the ecosystem. ...
... Members of Cyanobacteria were detected as the most dominant genus, with 29 % and 23.7% average abundance in all samples from the surface and bottom layers, respectively, and all Cyanobacteria bloom tested here were toxic. These results lend further support to previous studies that demonstrated Brazilian semiarid reservoirs harbor cyanobacterial communities [15,[28][29][30][31][32]. The presence of these bacteria in high abundance in the reservoir can be linked to uploading nutrients like ammonia and to the increase in water temperature (20.7°C) at the time of sampling. ...
Preprint
Full-text available
Here, we describe the microbial diversity and physicochemical properties in freshwater samples from the surface and bottom layer of Billings reservoir in São Paulo state, Brazil. Twenty-two matched samples were characterized using the 16S rRNA gene Illumina MiSeq platform. Taxonomical composition revealed an abundance of Cyanobacteria phyla, followed by Proteobacteria , with 1903 and 2689 known bacterial genera in the surface and deep-water layers, respectively. Shannon diversity index ranging from 2.3 - 5.39 and 4.04 - 6.86 in the surface and bottom layer, respectively. Among the 120 pathogenic genera identified, Flavobacterium was the most predominant genus. Temperature and phosphorus concentration were the most influential factors in shaping the microbial communities of both layers. Predictive functional analysis suggests that the reservoir is enriched in motility genes involved in the flagellar assembly. The overall results present new information on the significantly altered diversity composition of the bacterial community detected in Billings freshwater reservoir.
... In the present study, the cyanobacteria composition of the aquatic ecosystems revealed the presence of potential microcystinproducing cyanobacteria such as Microcystis aeruginosa, Oscillatoria spp., and Planktothrix aghardii. These species have been implicated in the production of microcystins in both isolated and field samples obtained from different countries (Chia and Kwaghe 2015;Beaver et al. 2018;Lorenzi et al. 2018Lorenzi et al. , 2019Kim Tiam et al. 2019;Vergalli et al. 2019). The detection of microcystins correlated with the presence and abundance of potential microcystinproducing cyanobacteria in some of the investigated aquatic ecosystems, while in others, the increase in cyanobacteria biomass did not lead to the detection of higher concentrations. ...
... This may be related to very low cell densities of Microcystis at the point that the freshwater ecosystems connect with the marine ecosystems. Although the PCR method used in the present study is suitable for the detection of potential MC producers in environmental samples (Rantala et al. 2004), it was highly sensitive for detecting only Microcystis from Nigerian coastal waters probably due to preferential amplification of multi-template PCR and/or primer mismatch (Lorenzi et al., 2019). ...
Article
Full-text available
Microcystins (MCs) are the most studied toxins of cyanobacteria in freshwater bodies worldwide. However, they are poorly documented in coastal waters in several parts of the world. In this study, we investigated the composition of cyanobacteria and the presence of microcystins (MCs) in several coastal aquatic ecosystems of Nigeria. Direct morphological analysis revealed that members of the genus Oscillatoria were dominant with five species, followed by Trichodesmium with two species in Nigerian coastal waters. Oso Ibanilo had the highest cyanobacterial biomass (998 × 103 cells/L), followed by Rivers Ocean (156 × 103 cells/L). Except for the Cross River Ocean, cyanobacteria were present in all the investigated aquatic ecosystems. Ten (10) out of twenty water bodies examined had detectable levels of MCs. Furthermore, genomic DNA analysis for the mcyE gene of microcystin synthetase (mcy) cluster showed identities higher than 86% (query coverage > 96%) with toxic strains of cyanobacteria in all the samples analyzed. Also, the sequences of samples matched those of uncultured cyanobacteria from recreational lakes in Southern Germany. Our findings indicate that the presence of toxic cyanobacteria in coastal waters of Nigeria is of public and environmental health concern.
... The dynamics of bacterial communities during the decomposition of Microcystis were monitored by 16S rRNA gene Illumina MiSeq sequencing. Next-Generation Sequencing (NGS) analysis significantly promote deeper understanding of the ecology and control of cyanobacterial blooms (Lorenzi et al., 2019). NGS enables a large number of sequences to study the complex microbial community composition owing to their advantages of higher-quality coverage with greater breadth and depth than traditional sequencing methods Lorenzi et al., 2019;Chen et al., 2020;Yan et al., 2020). ...
... Next-Generation Sequencing (NGS) analysis significantly promote deeper understanding of the ecology and control of cyanobacterial blooms (Lorenzi et al., 2019). NGS enables a large number of sequences to study the complex microbial community composition owing to their advantages of higher-quality coverage with greater breadth and depth than traditional sequencing methods Lorenzi et al., 2019;Chen et al., 2020;Yan et al., 2020). Recently, Shi et al. (2017) employed MiSeq sequencing of the 16S rRNA genes to investigate the community dynamics and functional variation of bacteria during the long-term decomposition of cyanobacterial blooms and their study demonstrated that bacterial community composition and function obviously changed during the degradation of cyanobacterial blooms. ...
Article
In aquatic ecosystems, water microbial communities can trigger the outbreak or decline of cyanobacterial blooms. However, the microbiological drivers of Microcystis decomposition in reservoirs remain unclear. Here, we explored the bacterial community metabolic profile and co-occurrence dynamics during Microcystis decomposition. The results showed that the decomposition of Microcystis greatly altered the metabolic characteristics and composition of the water bacterial community. Significant variations in bacterial community composition were observed: the bacterial community was mainly dominated by Proteobacteria, Actinobacteria, Planctomycetes, and Bacteroidetes during Microcystis decomposition. Additionally, members of Exiguobacterium, Rhodobacter, and Stenotrophomonas significantly increased during the terminal stages. Dissolved organic matters (DOM) primarily composed of fulvic-like, humic acid-like, and tryptophan-like components, which varied distinctly during Microcystis decomposition. Additionally, the metabolic activity of the bacterial community showed a continuous decrease during Microcystis decomposition. Functional prediction showed a sharp increase in the cell communication and sensory systems of the bacterial communities from day 12 to day 22. Co-occurrence networks showed that bacteria responded significantly to variations in the dynamics of Microcystis decomposition through close interactions between each other. Redundancy analysis (RDA) indicated that Chlorophyll a, nitrate nitrogen (NO3⁻-N), dissolved oxygen (DO), and dissolved organic carbon (DOC) were crucial drivers for shaping the bacterial community structure. Taken together, these findings highlight the dynamics of the water bacterial community during Microcystis decomposition from the perspective of metabolism and community composition, however, further studies are needed to understand the algal degradation process associated with bacteria.
... Moreover, none of them used a model simulating an everyday exposure, i.e., oral ingestion of the toxin, sublethal doses and subacute exposure. Indeed, MC-LR is commonly found in water supplies worldwide (Lorenzi et al., 2019;Walter et al., 2018;Thuret-Benoist et al., 2019;Svirčev et al., 2019); about 80% of the exposure to MC-LR occur by the ingestion of contaminated water (Greer et al., 2018); and nearly 2.1 billion people still do not have access to safe drinking water (Grojec, 2017) associated with the fragility of the traditional water treatment system, which is inefficient for the complete removal of MCs, mainly in developing countries (Westrick et al., 2010). ...
Article
Microcystin-LR (MC-LR) is a potent cyanotoxin that can reach several organs. However subacute exposure to sublethal doses of MC-LR has not yet well been studied. Herein, we evaluated the outcomes of subacute and sublethal MC-LR exposure on lungs. Male BALB/c mice were exposed to MC-LR by gavage (30 µg/kg) for 20 consecutive days, whereas CTRL mice received filtered water. Respiratory mechanics was not altered in MC-LR group, but histopathology disclosed increased collagen deposition, immunological cell infiltration, and higher percentage of collapsed alveoli. Mitochondrial function was extensively affected in MC-LR animals. Additionally, a direct in vitro titration of MC-LR revealed impaired mitochondrial function. In conclusion, MC-LR presented an intense deleterious effect on lung mitochondrial function and histology. Furthermore, MC-LR seems to exert an oligomycin-like effect in lung mitochondria. This study opens new perspectives for the understanding of the putative pulmonary initial mechanisms of damage resulting from oral MC-LR intoxication.
... While the richness and function of physiologically distinct and functionally complementary microorganisms are being described with increasing prevalence in planktonic blooms (Louati et al., 2015;Steffen et al., 2012), diversity in many benthic cyanobacterial blooms, which represent an important dimension of this global cyanobacteria phenomena, remains almost wholly unexplored (Bouma-Gregson et al., 2019. Often, targeted amplicon sequencing approaches focused solely on Cyanobacteria are utilized when characterizing benthic cyanobacterial blooms, limiting the taxonomic and functional scope of detection, and biasing perceptions of community composition (Jovel et al., 2016;Lorenzi et al., 2019). The apparent disparity in sequencing effort between planktonic and benthic blooms leaves the influence of community coordination and intra-mat trophic interactions on benthic cyanobacterial bloom ecology largely unknown. ...
Article
Anthropogenic forcing is spurring cyanobacterial proliferation in aquatic ecosystems worldwide. While planktonic cyanobacterial blooms have received substantial research attention, benthic blooms of mat-forming cyanobacteria have received considerably less attention, especially benthic mat blooms on coral reefs. Resultingly, numerous aspects of coral reef benthic cyanobacterial bloom ecology remain unknown, including underlying biodiversity in the mat communities. Most previous characterizations of coral reef cyanobacterial mat composition have only considered the cyanobacterial component. Without an unbiased characterization of full community diversity, we cannot predict whole-community response to anthropogenic inputs or effectively determine appropriate mitigation strategies. Here, we advocate for the implementation of shotgun sequencing techniques to study coral reef cyanobacterial mats worldwide, utilizing a case study of a coral reef benthic cyanobacterial mat sampled from the island of Bonaire, Caribbean Netherlands. Read-based taxonomic profiling revealed that Cyanobacteria was present at only 47.57% relative abundance in a coral reef cyanobacterial mat, with non-cyanobacterial members of the sampled mat community, including diatoms (0.78%), fungi (0.25%), Archaea (0.34%), viruses (0.08%), and other bacteria (45.78%), co-dominating the community. We found numerous gene families for regulatory systems and for functional pathways (both aerobic and anaerobic). These gene families were involved in community coordination; photosynthesis; nutrient scavenging; and the cycling of sulfur, nitrogen, phosphorous, and iron. We also report bacteriophage (including prophage) sequences associated with this subtidal coral reef cyanobacterial mat, which could contribute to intra-mat nutrient cycling and bloom dynamics. Overall, our results suggest that Cyanobacteria-focused analysis of coral reef cyanobacterial mats underestimates mat diversity and fails to capture community members possessing broad metabolic potential for intra-mat nutrient scavenging, recycling, and retention that likely contribute to the contemporary success of cyanobacterial mats on reefs. We advocate for increased collaboration between microbiologists and coral reef ecologists to unite insights from each discipline and improve efforts to understand mat ecology.
... STX belongs to the large family of guanidinium-containing marine natural products due to the presence of two guanidino groups, which are responsible for its high polarity (Berlinck, 2002;Sapse et al., 2006). Nearly 60 natural analogues of STX have been identified, which are produced by several cyanobacteria, such as Raphidiopsis raciborskii and Clyndrospermopsis raciborskii (Lorenzi et al., 2019;Wiese et al., 2010). ...
Article
As a type of cyanobacterial toxins, saxitoxin (STX) is receiving great interest due to its increasing presence in waterbodies. However, the underlying mechanism of STX-induced adverse effect is poorly understood. Here, we examined the developmental toxicity and molecular mechanism induced by STX using zebrafish embryos as an animal model. The embryonic toxicity induced by STX was demonstrated by inhibition of embryo hatching, increase in mortality rate, abnormal heart rate, abnormalities in embryo morphology as well as defects in angiogenesis and common cardinal vein remodeling. STX induced embryonic DNA damage and cell apoptosis, which would be alleviated by antioxidant N-acetyl-L-cysteine. Additionally, STX significantly increased reactive oxygen species level, catalase activity and malondialdehyde content and decreased the activity of superoxide dismutase and glutathione content. STX also promoted the expression of vascular development-related genes DLL4 and VEGFC, and inhibited VEGFA expression. Furthermore, STX altered the transcriptional regulation of apoptosis-related genes (BAX, BCL-2, P53 and CASPASE 3). Taken together, STX induced adverse effect on development of zebrafish embryos, which might be associated with oxidative stress-induced apoptosis.
Chapter
Prokaryotes were the earliest life forms on Earth but, although they originated an estimated 2 billion years before eukaryotic cells, both prokaryotes and eukaryotes share the same basic molecular mechanisms, indicating that both stem from a primordial ancestor. Bacteria, including cyanobacteria, produce a vast variety of secondary metabolites capable of controlling multiple eukaryotic cell functions. Some metabolites direct morphogenetic processes (in both cyanobacteria and higher algal taxons), while others are toxic to eukaryotes, helping prokaryotes colonize a wider variety of ecological niches. In addition, bacteria often use secondary metabolites to control other bacterial groups. On the other hand, eukaryotic cells can also synthesize secondary metabolites with either bacteriostatic or bactericidal capabilities, to counteract either cyanobacterial or eubacterial organisms. Cyanobacteria are an ancient lineage of photosynthetic microorganisms, but their study was neglected for many years. Recent publications demonstrate that cyanobacterial genomes encode a large variety of natural products, with broad mechanisms of action, many of which are probably yet unknown. Current advances in genome sequencing, making it faster and cheaper, should see a great increase in the number of completed cyanobacterial genomes, which promises many interesting discoveries in the near future.
Article
DNA-based analyses of bacterial communities were performed to identify the bacteria co-occurring with cyanobacterial blooms in samples collected at a single site over 2 years. Microcystis aeruginosa was the most predominant species (81% in 2018, and 94% in 2019) within the phylum Cyanobacteria, and microcystins were detected during all cyanobacterial blooms. The stereo microscope and scanning electron microscope observations showed bacterial associations on and around the aggregated M. aeruginosa cells. Culture-independent analyses of filtered bacterial communities showed that the Flavobacterium species in phylum Bacteroidetes (19%) was dominant in the cyanobacterial phycosphere, followed by the Limnohabitans species in Betaproteobacteria (11%). Using principal component analysis, major bacterial genus, including Microcystis and Flavobacterium species, were clustered during cyanobacterial blooms in both years. To identify key bacterial species that develop long-term symbiosis with M. aeruginosa, another culture-independent analysis was performed after the environmental sample had been serially subcultured for 1 year. Interestingly, Brevundimonas (14%) was the most dominant species, followed by Porphyrobacter (7%) and Rhodobacter (3.5%) within the Alphaproteobacteria. Screening of 100 colonies from cyanobacterial bloom samples revealed that the majority of culturable bacteria belonged to Gammaproteobacteria (28%) and Betaproteobacteria (57%), including Pseudomonas, Curvibacter, and Paucibacter species. Several isolates of Brevundimonas, Curvibacter, and Pseudomonas species could promote the growth of axenic M. aeruginosa PCC7806. The sensitivity of M. aeruginosa PCC7806 cells to different environmental conditions was monitored in bacteria-free pristine freshwater, indicating that nitrogen addition promotes the growth of M. aeruginosa.
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Changing global climatic conditions and the continuous eutrophication of aquatic ecosystems have led to increased frequency, duration and toxicity of cyanobacterial blooms worldwide. This makes the provision of time series information on cyanotoxins extremely crucial for effective monitoring and management of water resources. The objective of the present study was to investigate seasonal and annual changes in microcystins (MCs), cylindrospermopsins (CYNs), saxitoxins (STXs), neo-saxitoxin (neo-STX) and anatoxin-a (ATX-a) concentrations in 11 public water supply reservoirs in the semiarid region of Brazil, from 2004 to 2011. Per time, at least one cyanotoxin was present in all the investigated reservoirs. High levels of MCs, CYNs, STXs and Neo-STX were detected simultaneously in Carpina, Duas Unas, Ipojuca, and Jucazinho reservoirs. All the investigated reservoirs had significant concentrations of MCs. The highest levels of MCs were found in Carpina (303,300.0 ng g−1) and Duas Unas (122,053.9 ng g−1) reservoirs, while the lowest concentration of the hepatotoxins was recorded in Ipojuca (10.3 ng g−1) reservoir. On the other hand, CYN was detected in four reservoirs, STXs and Neo-STX in ten reservoirs and ATX-a in two reservoirs. The first record of CYN in Carpina reservoir was obtained in 2006. In addition, Carpina reservoir had the highest concentration of STXs and Neo-STX in the dry and rainy seasons, respectively. The high concentration of cyanotoxins observed in most samples obtained from semiarid reservoirs in Brazil demonstrates the need for regular monitoring and updated management programs.
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Harmful cyanobacterial blooms have become increasingly common in freshwater ecosystems in recent decades, mainly due to eutrophication and climate change. Water becomes unreliable for human consumption. Here we report a comprehensive study carried out to investigate the water quality of several Campina Grande reservoirs. Our approach included metagenomics, microbial abundance quantification, ELISA test for three cyanotoxins (microcystin, nodularins and cylindrospermosin), and in vivo ecotoxicological tests with zebrafish embryos. Cytometry analysis showed high cyanobacterial abundance, while metagenomics identified an average of 10.6% of cyanobacterial sequences, and demonstrated the presence of Microcystis, Cylindrospermopsis and toxin coding genes in all ponds. Zebrafish embryos reared with pond water had high mortality and diverse malformations. Among the ponds analyzed, Araçagi showed the highest lethality (an average of 62.9% ± 0.8), followed by Boqueirão (lethality average of 62.5% ± 0.8). Here, we demonstrate that water from ponds undergoing extremely drought conditions have an abundance of potentially harmful cyanobacteria and their toxins. Our findings are consistent with a scenario in which polluted drinking water poses a great risk to human health.
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Cyanobacteria are ecologically one of the most prolific groups of phototrophic prokaryotes in both marine and freshwater habitats. Both the beneficial and detrimental aspects of cyanobacteria are of considerable significance. They are important primary producers as well as an immense source of several secondary products, including an array of toxic compounds known as cyanotoxins. Abundant growth of cyanobacteria in freshwater, estuarine, and coastal ecosystems due to increased anthropogenic eutrophication and global climate change has created serious concern toward harmful bloom formation and surface water contamination all over the world. Cyanobacterial blooms and the accumulation of several cyanotoxins in water bodies pose severe ecological consequences with high risk to aquatic organisms and global public health. The proper management for mitigating the worldwide incidence of toxic cyanobacterial blooms is crucial for maintenance and sustainable development of functional ecosystems. Here, we emphasize the emerging information on the cyanobacterial bloom dynamics, toxicology of major groups of cyanotoxins, as well as a perspective and integrative approach to their management.
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Objetivo: A proliferação acelerada de cianobactérias em mananciais e reservatórios tem causado sérios danos ecológicos e à saúde pública, e é um problema que desafia as instituições responsáveis pelo fornecimento de água para a população. Nesse trabalho, foi realizada a quantificação dos níveis de microcistinas, saxitoxinas e cianobactérias ao longo de 3 anos em reservatórios do semiárido do Rio Grande do Norte (Brasil). Além disso, foi avaliada a distribuição sazonal das cianotoxinas e a porcentagem de cianobactérias e cianotoxinas que estavam acima do valor permitido de acordo com a legislação brasileira.
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This review summarizes the present state of knowledge regarding the toxic, bloom-forming cyanobacterium, Microcystis, with a specific focus on its geographic distribution, toxins, genomics, phylogeny, and ecology. A global analysis found documentation suggesting geographic expansion of Microcystis, with recorded blooms in at least 108 countries, 79 of which have also reported the hepatatoxin microcystin. The production of microcystins (originally “Fast-Death Factor”) by Microcystis and factors that control synthesis of this toxin are reviewed, as well as the putative ecophysiological roles of this metabolite. Molecular biological analyses have provided significant insight into the ecology and physiology of Microcystis, as well as revealed the highly dynamic, and potentially unstable, nature of its genome. A genetic sequence analysis of 27 Microcystis species, including 15 complete/draft genomes are presented. Using the strictest biological definition of what constitutes a bacterial species, these analyses indicate that all Microcystis species warrant placement into the same species complex since the average nucleotide identity values were above 95%, 16S rRNA nucleotide identity scores exceeded 99%, and DNA–DNA hybridization was consistently greater than 70%. The review further provides evidence from around the globe for the key role that both nitrogen and phosphorus play in controlling Microcystis bloom dynamics, and the effect of elevated temperature on bloom intensification. Finally, highlighted is the ability of Microcystis assemblages to minimize their mortality losses by resisting grazing by zooplankton and bivalves, as well as viral lysis, and discuss factors facilitating assemblage resilience.
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A common misconception persists that the genomes of toxic and non-toxic cyanobacterial strains are largely conserved with the exception of the presence/absence of the genes responsible for toxin production. Implementation of -omics era technologies has challenged this paradigm, with comparative analyses providing increased insight into the differences between strains of the same species. Implementation of genomic, transcriptomic and proteomic approaches has revealed distinct profiles between toxin-producing and non-toxic strains. Further, metagenomics and metaproteomics highlight the genomic potential and functional state of toxic bloom events over time. In this review, we highlight how these technologies have shaped our understanding of the complex relationship between these molecules, their producers and the environment at large within which they persist.
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The frequency of freshwater cyanobacterial blooms is at risk of increasing as a consequence of climate change and eutrophication of waterways. It is increasingly apparent that abiotic data are insufficient to explain variability within the cyanobacterial community, with biotic factors such as heterotrophic bacterioplankton, viruses and protists emerging as critical drivers. During the Australian summer of 2012-2013, a bloom that occurred in a shallow ephemeral lake over a 6-month period was comprised of 22 distinct cyanobacteria, including Microcystis, Dolichospermum, Oscillatoria and Sphaerospermopsis. Cyanobacterial cell densities, bacterial community composition and abiotic parameters were assessed over this period. Alpha-diversity indices and multivariate analysis were successful at differentiating three distinct bloom phases and the contribution of abiotic parameters to each. Network analysis, assessing correlations between biotic and abiotic variables, reproduced these phases and assessed the relative importance of both abiotic and biotic factors. Variables possessing elevated betweeness centrality included temperature, sodium and operational taxonomic units belonging to the phyla Verrucomicrobia, Planctomyces, Bacteroidetes and Actinobacteria. Species-specific associations between cyanobacteria and bacterioplankton, including the free-living Actinobacteria acI, Bacteroidetes, Betaproteobacteria and Verrucomicrobia, were also identified. We concluded that changes in the abundance and nature of freshwater cyanobacteria are associated with changes in the diversity and composition of lake bacterioplankton. Given this, an increase in the frequency of cyanobacteria blooms has the potential to alter nutrient cycling and contribute to long-term functional perturbation of freshwater systems.The ISME Journal advance online publication, 4 December 2015; doi:10.1038/ismej.2015.218.