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Monitoring cYanobacteria and cYanotoxins in Alqueva dam
Amely Zavattieri; Susana Nunes; Alexandra Penha;
Maria Manuela Queiroz Martins Mantero Morais
Corresponding author: A. Zavattieri, Email: [email protected].
Rua da Barba Rala, n.º 1, Parque Industrial
e Tecnológico de Évora, 7005-345 Évora,
Portugal. Tel/Fax: (+ 351) 266 768 060
INTRODUCTION
Alqueva region is now recognized as one of the most productive and recreational area of Alentejo, in Portugal. For these reason ALEX project (Alqueva Hidro
Meteorological Experiment) included complementary physical and biological studies to asses the ecological situation of Alqueva dam. A good knowledge of the whole
system is mandatory to propose adequate management strategies. Several biological quality indicators of reservoirs are widely recognized as the phytoplankton
composition, specially cyanobacteria. Polymerase chain reaction (PCR) was selected to corroborate microscopic identification of cyanobacteria found during ALEX
campaign in the summer of 2014 , and also to verify the presence of toxic genes in the samples. This technique allows the identification of the major toxin
biosynthesis genes not requiring axenic cultures, since such cultures are difficult to obtain (Choit al., 2008). This molecular approach subsequently facilitates an
analysis of the distribution of genotypes based on the presence or absence of a combination of target genes in the samples. In the current study, an initial screening
of samples was completed by PCR using a combination of primer sets (Table 1).
MATERIAL AND METHODS
PRELIMINARY RESULTS
Study area and sampling
The field campaign in Alqueva reservoir took place from June to September 2014.
In situ measurements, water samples and biological elements were monthly
collected from three fixed platforms placed in the lacustrine zone and from
selected sites in the margins. An additional campaign for cyanobacteria was
performed in October due to a bloom situation. In each campaign, vertical profiles
of temperature, dissolved, pH, oxidation-reduction potential and turbidity were
taken. Samples were collected with a plankton net from platforms and margins and
separate in two sub-samples (100 ml each) one for microscopic identification and
isolation for in vitro culture and the other for molecular studies (the latest were
kept on ice until transported to the laboratory).
Microscopic identification of collected samples indicate the presence of serveral
species of cyanobacteria in Alqueva dam during the summer of 2014 (Fig. 2).
Isolation for in vitro culture was no easy particularly for the Genus Microcystis.
apparently the growth of this cyanobacterium is inhibited in agar medium (Allen and
Gorham, 1981). Filamentous cyanobacteria grew well in BG-11 medium (solid and
liquid) with or without the addition of antibiotics (quais usamos) (Fig. 3)
a
e
Fig. 1 Meteorological platform in which cynobacterial samples were taken with plankton net.
DNA extraction directly from local samples
Cells used to extract DNA for PCR analysis were collected by filtering 100 ml of
the sample collected as described above onto 0,2 µm pore size polycarbonate
membrane filters (Millipore), and immediately frozen in Eppendorfs (-20ºC) until
processing. Extraction of DNA was latter perform using a modification of the
protocol described by Rinta-Kanto et al., 2005. Conventional PCR was performed
for cyanobacterial reference strains and bloom samples to check the presence of
toxin genes; and second, to check the specificity of the PCR primers to amplify
single genes. The set of primers and primers targets used for PCR analysis are
summarized in table 1. Each single reaction, with a final volume of 25 µL,
contained 1 µL of template??, 0.2 µM of each primer (final concentration), 0.2 µM
dNTPs (final concentration, Invitrogen, California ), 5X GoTaq® Flexi buffer
(Promega, Madison, WI), 3 mM MgCl2 (final concentration) and 0.025 U/µL
GoTaq® G2 Flexi DNA Polymerase (Promega, Madison, WI). The PCR protocol
consisted of an initial denaturation step at 94 °C for 3 min, 35 cycles at 94 °C for
45 s, primer annealing at 50 °C for 30 s, primer elongation at 72 °C for 1 min, and
a final single step at 72 °C for 7 min. The PCR reactions were performed in a
thermocycler (Qual?)and the specificity of the reaction was verified by a 1%
agarose-gel electrophoresis.
b
f
c
g
d
h
Fig. 2 Microscopic images of the cyanobacteria identify in the summer of 2014 for Alqueva Dam .
Anabaena catenula (a); Cylindrospermopsis sp. (b) ; Lyngbya sp. (c); Oscillatoria sp. (d);
Microcystis aeruginosa (e); Aphanozomenon flos-aquae (f); Woronichinia naegeliana (g);
Microcystis aeruginosa (h)
Fig. 3 Different phases of in vitro culture. Isolated cyanobacteria on solid G-11 medium (a) ; liquid
medium without antibiotic (b).
Table 1. PCR primers used
GENE
SIZE
(bp)
REFERENCE
780
Neilan et al. 1997; Jungblut et al. 2005
754
Neilan et al. 1997
220
Neilan et al. 1997
GGCATTCCTAGTTATATTGCCATACTA
GCCCGTTTTTGTCCCTTTGCTGC
300
Wilson et al., 2000
mcyA-Cd1F
mcyA-Cd1R
AAAATTAAAAGCCGTATCAAA
AAAAGTGTTTTATTAGCGGCTCAT
297
Hisbergues et al. 2003
mcyB2959F
mcyB3278R
TGGGAAGATGTTCTTCAGGTATCCAA
AGAGTGGAAACAATATGATAAGCTAC
350
Nonneman & Zimba 2002
PKEF1
PKER1
CGCAAACCCGATTTACAG
CCCCTACCATCTTCATCTTC
755
Ouahid et al. 2005).
HEPF
HEPR
TTTGGGGTTAACTTTTTTGGCCATAGTC
AATTCTTGAGGCTGTAAATCGGGTTT
472
Jungblut e Neilan 2006
PKSM4
PKMS5
M13
M14
GAAGCTCTGGAATCCGGTAA
AATCCTTACGGGATCCGGTGC
GGCAAATTGTGATAGCCACGAGC
GATGGAACATCGCTCACTGGTG
650
Schembri et al. 2001
597
Schembri et al. 2001
PRIMER
SEQUENCE 5’-3’
27F
809R
740F
1494R
Micr184F
Micr431R
AGAGTTTGATCCTGGCTCAG
GCTTCGGCACGGCTCGGGTCGATA
GGCYRWAWCTGACACTSAGGGA
TACGGTTACCTTGTTACGAC
GCCGCRAGGTGAAAMCTAA
AATCCAAARACCTTCCTCCC
GYL2
GYL4
Microcystin production
mcyB, gene present in
Microcystis that produce
16sRNA specific fragment for
cyanobacteria
16s specific for Microcystis
Specific for Cilindrospermopsis
raciborskii
microcistin
Gene mcyE, from Genera
Microcystis
mcyE & ndaF, microcystin and
nodularin sinthetase
Cylindrospermopsin
polyketide synthetase (pks)
Cylindrospermopsin peptide
synthetase (ps)
REFERENCES
Choi GG., Bae MS., Ahn CY, Oh HM. 2008 Induction of axenic culture of Arthrospira (Spirulina) platensis based on antibiotic sensitivity of
contaminating bacteria. Biotechnology Letters 30, 87–92.
Allen EAD., Gorham PR. 1981. Culture of planktonic cyanophytes on agar, In W. W. Carmichael (ed.), The water environment: algal toxins
and health. Plenum Publishing. Corp., New York. p. 185-192.
Hisbergues M, Christiansen G, Rouhiainen L, Sivonen K, Borner T. 2003. PCR-based identification of microcystin-producing genotypes of
different cyanobacterial genera. Archives of Microbiology 180(6):402-410.
Jungblut A-D, Neilan BA. 2006. Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin,
synthetase genes in three orders of cyanobacteria. Archives of Microbiology 185(2):107-114.
Neilan BA., Jacobs D., DelDot T., Blackall LL.,Hawkins PR., Cox PT., Goodman AE. 1997. rRNA sequences and evolutionary relationships
among toxic and nontoxic cyanobacteria of the genus Microcystis. Int.ernational Journal of Syst.ematic Bacteriol.ogy 47:693-697.
Nonneman D, Zimba PV. 2002. A PCR-based test to assess the potential for microcystin occurrence in channel catfish production ponds.
Journal of Phycology 38(1):230-233.
Ouahid Y, Perez-Silva G, del Campo F. 2005. Identification of potentially toxic environmental Microcystis by individual and multiple PCR
amplification of specific microcystin synthetase gene regions. Environmental Toxicology 20(3):235-242.
Rinta-Kanto JM, Ouellette AJA., Twiss MR., Bridgeman TB., Wilhelm SW. 2005. Quantification of toxic Microcystis spp. during the 2003 and
2004 blooms in western Lake Erie using quantitative real-time PCR. Environmental Science and Technolology 39:4198–4205.
Schembri MA, Neilan BA, Saint CP. 2001. Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis
raciborskii. Environmental Toxicology 16(5):413-421.
Wilson KM, Schembri MA, Baker PD, Saint CP. 2000. Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii
and design of a species-specific PCR. Applied and Environmental Microbiology 66(1):332-338.
Fig. 4 Electrophoretic gel with the results of the 10 primers used
PCR procedure confirmed the presence of cyanobacteria in the Alqueva by way of
the conservative region of the cyanobacterial genome in 16SrRNA with product of
780bp (1) and 754bp (2) Fig 4. The presence of Microcystis was also confirmed by
the sequences 220bp (3). It was also possible to observed specific for ........and
(Fig. 4). On the other hand, it was no possible to identify specific genes for
Cylindrospermopsis raciborskii. being necessary to identify the specie of
Cylindrospermopsis found by microscopical observation. The results associated
with the production of the toxin Cylindrospermopsin like cylindrospermpsin
polyketide synthetase (pks) and polipeptide synthetase were also no conclusive
being necessarily to adjust the PCR reaction conditions for the fragments 597bp
(9) and 650bp (10).
CONCLUSIONS
The combination of microscopical identification and PCR confirmation with specif
primers was very usefull for a . In the future we will introduce pRT-PCR for a rapid
identification of cyano and toxins as well as quantification of.........