Molecular identification of Planktothrix rubescens as the cause of a
potentially toxic intense algal bloom for a first time in Greece (Lake Ziros).
Katerina Vareli, 1 Evangelos Briasoulis, 2 George Pilidis, 1 Ioannis Sainis1 *
1 Department of Biological Applications and Technologies, University of Ioannina,
Ioannina 45110, Greece. 2 Medical School, University of Ioannina, Ioannina 45110, Greece.
*Corresponding author:
Keywords:
molecular characterization; Lake Ziros.
attributed to Microcystis, Anabaena, and Aphanizomenon genera while
Planktotrix is the most common bloom forming cyanobacterium in deep Northern
and prealpine European oligotrophic to mesotrophic lakes. In the framework of
systemic study of the cyanobacterial communities of the Lakes of Northwestern
Greece we studied the cynobacterial dynamics in Lake Ziros throughout a 15
month’s period, from January of 2006 to March of 2007. Surprisingly a severe
cyanobacterial bloom occured during the period, which upon microscopic
examination and detailed molecular cha racterization found to be caused by
Planktothrix rubescens species. The appearance of Plankothrix rubescens from
November 2006 coincided with poor cyanobacterial diversity observed and
resulted in a thick epilimnetic bloom. Genotype composition of the tota l
cyanobacterial community of the lake was analyzed by using denaturing gradient
gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region
of the rnn operon (rRNA-ITS). A Planktothrix rubescens strain closely related to
Kpr strain from Lake Klinckenberg,The Netherlands dominated. This is the first
report of Planktothrix rubescens identified as a bloom forming species in Greece.
The importance of this observation is expanded by the fact that microcystin
concentrations recorded in Lake Ziros were the highest measured ever in Greek
aquatic ecosystems examined so far. Microcystin’s levels in cyanobacterial
pellets were measured and found amongst the highest recorded worldwide.
3
Introduction
In recent years publications that deal with p roblems related to rapid increases of
cyanobacteria populations in aquatic ecosystems are increasingly appearing.
Cyanobacterial blooms are thought to have negative biological impacts on
ecosystem and wildlife and also raise concerns for potential release of biotoxins
by some cyanobacteria species. ( Sivonen and Jones, 1999 ; Kurmayer et al., 1999; Oliver et al., 2000) . The best studied cyanobacterial biotoxins are
microcystins, a family of about 60 cyclic heptapeptide hepatotoxins which share
the amino acid ADDA (3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4,6-
dienoic acid) combined with other six amino acids (Dawson, 1998; McElhiney and Lawton, 2005) Chronic exposure to microcystin LR has shown to cause
liver damage and also tumors in wild and domestic animals (Li et al., 2001; Nishiwaki-Matsushima et al., 1992) . Recently it was shown that microcystins
exert their tumor-promoting effects through inhibition of the serine/threonine
phosphatase protein phosphatase 2A (PP2A) which is a known tumor
suppressor (Xing et al., 2006). These health hazards led World Health
Organization (WHO) to set alarm levels for microcystin LR: 1μg/l as an upper
safe limit for microcystin LR concentrations in drinking water and 20 μg/l as a
guideline value for moderate health risk in recreational waters (Falconer and Humpage, 2005).
It is noteworthy, that in Mediterranean aquatic ecosystems algal blooms
are a consequence of eutrophication processes and usually consist of
Microcystis, Anabaena, and Aphanizomenon genera (Abdel-Rahman et al., 1993; Banker et al., 1997; Vezie et al., 1997; Vezie et al., 1998., Giovannardi et al., 1999; Oudra et al., 2001; Vasconcelos et al., 2001; Briand et al., 2002; Cook et al., 2004) In contrast, many freshwater lakes at Northern latitudes are
dominated by filamentous cyanobacteria of the genus Planktothrix which does
not always associated with eutrophication . Two well studied Planktothrix species
proved to have a preference for low light and are also tolerant to lower
temperatures. Planktothrix agardhii , a green pigmented cyanobacterium
predominates in hypertrophic shallow lakes in Northern Europe (Mur et al., 1999; Briand et al., 2002). Planktotrix rubescens is a red pigmented filamentous
species, and represent the most common bloom forming cyanobacterium in deep
4
Northern and prealpine European oligotrophic to mesotrophic lakes (Sivonen and Jones., 1999). Planktothrix species are potential microcystin producers and
they can also produce aplysiatoxins, neurotoxic anatoxin -a, paralytic shellfish
poisons and planktocyclin, a cyclooctapeptide protease inhibitor (Sivonen and Jones, 1999; Pommati et al., 2000 ; Baumann et al 2007). Planktotrix
rubescens are usually confined to the metalimnion of deep lakes as light
penetrates to this depth. But during periods of low insolation such as winter,
when the filaments receive low light doses, they float up to the surface, forming
visible red colored water blooms (Walsby et al 2004; Walsby et al 2005).
In Greece, nine out of thirty three lakes examined so far suffer
eutrophication (Cook et al., 2004). Thick cyanobacterial scums are yearly
observed and the dominant genera during thes e blooms found to be Microcystis,
Anabaena, Anabaenopsis and Aphanizomenon (Cook et al., 2004; Vardaka et al., 2005).
Lake Ziros is a closed hydrological system located in NW Greece in low
coastal terrains of Pindos mountainous range. This is a carstic lake that occupies
an area of 60ha and a maximum depth of 56 m used for recreational and
irrigation purposes it remains completely unstudied. Due to its deepness, clarity,
isolation and distance from any anthropogenic activities the lake is thought to be
oligotrophic. Cyanobacterial populations have never been characterized and
blooms have not been reported for this lake to date.
We studied in detail the cyanobacterial community dynamics in Lake Ziros
throughout a 15 month’s period, from January of 2006 to March of 2007 and
report herein the identification of Planktothrix rubescens as the bloom causing
cycobecterial species in that case. To our knowledge this is the first study
establishing that Planktothrix rubescens is also a bloom forming species at least
in one Greek lake. The importance of this observation is broadened by the fact
that microcystin concentrations recorded in Lake Ziros were the highest
measured ever in Greek aquatic ecosystems examined so far.
Materials and Methods
Field sampling
Lake Ziros water was sampled from January 2006 to March 2007 at monthly
intervals. Water samples were collected immediately below the surface in sterile
5
bottles. Samples were kept cool until they were processed within 1h from
collection.
The cyanobacterial cells in the lake samples were concentrated by
centrifugation. For genomic DNA extraction we used the following protocol which
is based on a number of different previously published protocols (Neilan, 1995; Wu et al., 2000; Zwart et al., 2 005). The pellet was resuspended in 10ml of
100mM Tris, 50mM EDTA, 100mM NaCl, 0.1% sarkosyl pH8 and incubated at
300 C with mild shaking for 20min. After centrifugation at 8000rpm for 10min, the
pellet was washed with 10ml TES buffer (50mM Tris, 5mM EDTA, 50mM NaCl
pH8), resuspended in 2.5ml TES, lysozyme was added to obtain a final
concentration of 5mg/ml and the solution was incubated at 37 0 C for 1hr. Then,
SDS was added to a final concentration of 2% and the solution incubated for
30min at 370 C. Proteinase K was added to a final concentration of 150µg/ml and
the mixture incubated overnight at 48 0 C with vigorous shaking. In cases with
poor solubility, 0.5g zirconium beads (0.1mm diameter) were added and the
tubes were vigorously shaken (5.000 rpm) on a Mini Bead-beater (Biospec
products, Bartlesville, OK, USA) for 2min with intermittent cooling on ice.
Samples were centrifuged for 5min at 10,000g and the supernatant extracted
once with 1V of phenol and 0.5V of chloroform:isoamyl alcohol 1:1. The aqueous
phase was transferred to a new tube. Heat treated RNAse A was added to a final
concentration of 50µg/ml and the solution incubated for 1hr at 37 0 C. Solutions
were extracted twice with 1V of phenol and once with 1V of chloroform:isoamyl
alcohol (24:1). The DNA was then precipitated by adding 1V ammonium acetate
4M and 2V ice cold isopropanol and centrifuging at 14,000xg for 30 min.
Subsequently, the DNA was dissolved in MilliQ water and purified on a Wizard
column (Promega, Madison, WI, USA) according to t he manufacturer’s
recommendations.
PCR amplification was performed in an Eppendorf Mastercycler gradient
thermocycler in a 50 µl reaction volume containing approximately 100ng of DNA.
For internal transcribed spacer sequences (ITS) amp lification we used a
6
combination of two cyanobacteria - specific primers: 16S GC-CSIF primer and
23S ULR primer. These amplicons, spanning the entire rRNA -ITS and referred
as ITSc amplicons. Primer sequences and PCR conditions were as described
earlier by Janse (Janse et al., 2003). For PCR amplification of mcyE gene
fragment we used four primer sets as described earlier (Rantala et al., 2006) . All
potential microcystin producing genera were targeted with the use of a general
reverse primer (mcy-R4) and a general forward primer (mcyE-F2). Microcystin
producing Anabaena, Microcystis and Planktothrix spp. were targeted with the
same general forward primer (mcyE-F2) and one of the following genus specific
reverse primers mcyE-12R, mcyE-R8 and mcyE-plaR3 respectively (Rantala et al., 2006). For the semi nested PCR procedure, 1 -2μl of the first PCR reaction
was used as template for the second PCR reaction. PCRs were also performed
as previously described (Rantala et al., 2006) . In all cases, a proofreading DNA
polymerase was used (Expand High Fidelity DNA polymerase, Roche).
Denaturing Gradient Gel Electrophoresis (DGGE), Cloning and sequencing
DGGE was performed essentially as described earlier by Muyzer (Muyzer et al., 1993) with minor modifications as describe d by Janse et al. 2003 (Janse et al., 2003).
A small piece of the gel from the middle of all bands detected after ethidium
bromide staining was excised and incubated in 50µl sterile MilliQ water O/N at 4 0
C. The eluent was reamplified by using the origina l primer set and run on a
DDGE gel to confirm its identity. The new PCR products were purified using a
Macherey-Nagel DNA clean-up kit (Nucleospin Extract), and subsequently they
were cloned using a TOPO TA cloning Kit (Invitrogen) according to the
manufacturer’s instructions. Inserts were fully determined by sequencing both
strands. Sequencing was performed by Macrogen [Macrogen Inc, Seoul, Korea].
Microcystin extraction and ELISA measurement.
Cyanobacterial pellets lyophilized within 1hr of collection. Microcystin extraction
was performed as previously described (Karlsson et al., 2005) with minor
modifications as follows. Two to 15mg of lyophilized cyanobacterial pellets were
homogenized with 10ml of 75% methanol in water for 3min through grinding.
Samples were sonicated for 15 min, and then incubated at room temperature
7
(RT) for 20 min with vigorous shaking. The above step repeated three times. At
the end of each incubation, the samples centrifuged at 8500rpm for 10min.
Supernatants were transferred to new tubes and stored at 4 0 C. Pellets were
resuspended in 10ml 75% methanol and incubated overnight (O/N) with vigorous
shaking. The entire process was repeated twice the following day and all
supernatants were pooled.
We used commercially available ELISA kits (Abraxis, Warminster, PA) to
measure microcystin and nodularin loads according to the manufacturer’s
instructions. For ELISA measurement, appropriate volumes of each sample were
evaporated to dryness by speed vac at low temperatures and the residues were
then dissolved in 50µl of water.
Nucleotide sequences and accession numbers
The sequences were deposited at GenBank and were assigned accession
numbers EU233391 through EU233412.
Microscopic examination of the cyanobacterial sampl e collected during
the algal bloom (4/3/07) revealed that a well defined genus was present and that
was Planktothrix (Figure 1, lower right panel). Moreover due to the pinkish
appearance of the bloom, we concluded that the red -pigmented Planktothrix
rubescens was the prevailed species in the lake Ziros. The same picture was
obtained from samples corresponding to March of 2006 and from November,
December, January and February of 2007 (data not shown).
To characterize the Planktothrix species and/or Plaktothrix st rains which
were responsible for the bloom and to achieve high resolution analysis of the
total cyanobacterial community, we used denaturing gradient gel electrophoresis
profile of amplified ITSc fragments, spanning the entire rRNA -ITS sequences of
all the cyanobacteria (Janse et al., 2003) . Figure 2 shows the cyanobacterial
community profiles during the entire study period from January of 2006 to March
of 2007. Figure 2 reveals that the cyanobacterial diversity was low and more or
less constant. It is characteristic that from December to March we observed
exactly the same pattern consisting of only two bands (26 and 27, 28 and 29, 30
8
and 31, 32 and 33 respectively for each month). These two bands were also
present in March (band numbers 8 and 9) and Novembe r (band numbers 23 and
24) of 2006. To investigate the cyanobacterial community dynamics, both
prominent and faint bands were excised, reamplified, cloned and sequenced.
BLAST searches revealed that 22 out of 33 sequences have nearly completely
homologous counterparts in the GenBank/EMBL/DDBJ databases (Table 1).
Table 1 shows also that DGGE band numbers 8, 9, 23, 24 and 26 -33 are highly
homologous to Planktothrix rubescens sequences already deposited to
GenBank/EMBL/DDBJ databases. ITS sequences correspond ing to Planktothrix
rubenscens from Lake Ziros have two different sizes of 493bp and 657 -663bp.
Small size bands ( 9, 24, 27, 29, 31, 33) are 99% to100% homologous to
Planktothrix rubescens KPr-o DGGE band from Lake Klinckenberg (AY827784).
Bands with the larger size (8, 23, 26, 28, 30, 32) are 98% homologous to
Planktothrix rubescens KPr-b DGGE band (AY827783) from Lake Klinckenberg
also. The two ITSc PCR products correspond to two different 16S -23S operons
of the same species, Planktothrix rubescens (Janse et al., 2005). Planktothrix
rubescens sequences are the only sequences detected during the bloom.
Other bands highly homologous to database entries were also found
[Table 1]. Bands numbered 7, 11, and 14 are identical and their sequences are
99% homologous to an already deposited sequence (EF150979) corresponding
to an uncultured Nostocaceae cyanobacterium from the neighboring Lake, lake
Pamvotis. Two other bands, 10 and 12 are also identical and 98% homologous
to the uncultured Microcystis DGGE band 72 (EF151001), while band 15 is 98%
homologous to uncultured Microcystis DGGE band 69 (EF150998) from Lake
Pamvotis also. Band numbers 19, 20 and 22 are nearly identical with the major
part of them being 98% homologous to Synechococcus sp. LBP1 (AF330247)
and to the Uncultured cyanobacterium TH320 -3-10 (EF513333). The remaining
DGGE bands found to have lower homologies to already deposited sequences
(Table 1)
In an effort to test the possibility of the existence of potential toxic strains
in our samples we studied the composition of potential microcystin producers
with general and genus-specific microcystin synthetase gene E (mcyE) PCR
(Rantala et al., 2006) . When using general mcyE primers, potential MC
producers found to be present in the following samples co llected: from January
9
of 2006 to April of 2006 and from September of 2006 to March of 2007.
Amplification products from samples collected during January, February, April,
September, and October of 2006 are just detectable (Figure 3, A1). PCR
products corresponding to Planktothrix sp. mcyE gene fragment detected in
March of 2006 and from October of 2006 until March of 2007 (Figure 3, A2). The
samples were analyzed also with two other genus specific mcyE primers suitable
for specific amplification of Microcystis and Anabaena mcyE gene fragments. No
other product was amplified when using primers specific for Anabaena or
Microcystis mcyE gene fragments (data not shown). However, when used as
template the general amplification product, we detected PCR products s pecific
for Microcystis sp. or Anabaena sp. in samples from months 4, 5, 6 (Fig. 3, B2)
and 1, 2, 5, 7 (Fig.3, B3) respectively. In this semi nested PCR procedure,
Planktothrix specific mcyE gene products are detected throughout the study with
the exception of samples collected during months 5, 6, 7, 8 and 9 of 2006 ( Fig.1,
B1).
Finally we measured microcystin levels in our samples, since it is well
documented that this is the toxin expected to be produced from the genera found
to prevail in Lake Ziros. Microcystin concentrations in lake water samples were
measured by using ELISA and were expressed as microcystin –LR equivalents.
Table 2 shows that microcystin concentrations ranged from 0 to 6725 μg/ g
freeze-dried material. In the end of winter until early spring of 2006 we measure
low levels of microcystins. The highest concentration during this period was
measured in March and it was 355 μg/gr. Afterwards, microcystin ’s
concentrations decline and found to be around zero from May to September. In
October sample, microcystin’s levels are detectable again but too low (49μg/gr).
From November of 2006 to March of 2007 we measured extremely high levels
for the toxin. (Table 2).
Discussion
The reddish color of Planktothrix rubescens blooms , “the Burgundy-blood
phenomenon” (Walsby et al 2005) is characteristic and frequently alerts the
population. This phenomenon viewed in Figure 1 in Lake Ziros led us to
undertake a detailed characterization of the cyanobacterial community in this
Lake which is thought to be an ol igotrophic lake and in which cyanobacterial
10
blooms have never been reported earlier. In our effort to characterize the
cyanobacterial community of the lake, surface water samples were tested
monthly.
Microscopic examination of our samples revealed Planktothrix rubescens
in samples collected in March of 2006 and from November of 2006 unt ill the
March of 2007. The identification of the genus Planktothrix in samples without
cyanobacterial bloom was not a surprising observation because Oscilatoria sp.
(Planktothrix sp) were already identified in Greek lakes. In contrast, the
domination of Planktothrix rubescens and the appearance of a dense 3cm thick
cyanobacterial bloom in surface water of Lake Ziros was an unexpected
observation. Planctothrix rubescens reported as dominant species in lakes
located in central and Northern Europe, including lakes Zurich (Walsby and Schanz 2002), Garda (Salmaso 2000), Mondsee (Kurmayer et al., 2004),
Geneva (Annevile et al., 2002), Nantua (Feuillade 1994), Steinsfjorden
(Blikstad Halstvedt et al., 2007) and Bourget (Humbert and Le Berre 2001) . In
Lac du Bourget, the proliferation of P.rubescens has been associated with the
process of restoration this ecosystem (Jacquet et al., 2005). They typically,
occur in deep stratified waters in which they can form metalimnetic layers.
Recently, during winter 2005-2006 intense blooms of the cyanobacterium
Planktothrix rubescens occurred in four freshwater reservoirs in Sicily (Naselli- Flores 2007). In Greek water bodies, Planktothrix rubescens, had never been
identified as causative agent of cyanobacterial blooms.
In our effort to fully characterize the cyanobacterial community of the lake,
DGGE profile of amplified ITSc fragments spanning the entire rRNA -ITS, was
used. We used ITSc primer amp licons for two reasons: first, because these
amplicons contain more sequence information than ITSa and ITSb amplicons
and second because they yielded sharp bands on DGGE for the most tested
genera (Janse et al., 2003). BLAST searches have been shown that Planktothrix
rubescens sequences from Lake Ziros are highly homologous to sequences of a
Planktothrix rubescens isolate (KPr) from Lake Klinckenberg – The Netherlands
(Janse et al., 2005) . ITSc DGGE profile of KPr isolate revealed that there are
two rRNA operons with different length and sequences. A short sequence named
KPr-o and a larger sequence named KPr -b. Therefore, the two identified
Planktothrix rubescens sequences from Lake Klinckenberg corresponding to two
11
different rRNA operons of the same species , a common feature of many
filamentous cyanobacteria (Janse et al., 2005) . In the case of Planktothrix
rubescens from Lake Ziros two different ITSc sequences were cloned and
sequenced, a short sequence with a 99 -100% homology to KPr-o sequence, and
a larger sequence with 98% homology to KPr -b.
Planktothrix rubescens DGGE bands were predominant in samples
collected from November of 2006 through March 2007 and they are the only
bands detected during the bloom. The cyanobacterial community in January
2006 sample consists of six predominant bands which upon cloning and
sequencing found to be more or less different from any other sequence already
deposited to databases. Sequences from band numbers 1, 5, 6, and 21 are 85% -
98% homologous to a number of sequences already deposited to databases but
the homology is restricted only in a small part of the total ITSc sequences. We
consider that these sequences represent new strains or even new species.
Some of the above mentioned sequences were found later during our st udy, but
they disappeared upon establishment of Planktothrix rubescens bloom. It is
possible that the huge amounts of Planktothrix rubescens cells during the bloom
did not allow other sequences to be obtained from DGGE fingerprints, as
suggested earlier by Casamayor et al., 2000. Two sequences 19, 20 and 22 are
highly homologous to Synechococcus sp., a major picoplankton genus in many
freshwater and marine environments (Stockner et al., 2000, Ernst et al., 2003).
The cyanobacterial community of the lake con tains also cyanobacteria of the
genus Nostocaceae. Most of the Nostocaceae sequences from Lake Ziros are
nearly completely homologous to ITS sequences from Lake Pamvotis, the most
proximal studied lake, but there are also sequences with lower homologies
(81%-82%). Bands corresponding to the main bloo m forming cyanobacterium in
Greek lakes were also excised and sequenced. As it was expected Microcystis
sequences from Lake Ziros were highly homologous to Microcystis sequences
from Lake Pamvotis.
Planktothrix rubescens, Microcystis sp. and many Nostocaceae species
are known as microcystin producing species (Fastner et al., 1999). In our effort
to identify potential microcystin producers in Lake Ziros, DNA extracted from
cyanobacterial pellets were examined using general and genus specific primers
for PCR amplification of the microcystin synthetase E gene, as previously
12
described (Rantala et al., 2006) . Potential microcystin producers found in
samples from 1/06, 2/06, 3/06, 4/06, 9/06, 10/06, 11/06, 12/06, 1/ 07, 2/07, 3/07.
A semi nested PCR procedure enable s us to detect potential toxic genera even
in those samples where no general product was detectable. Thus, this two step
procedure could be followed for the detection of the presence of potential
microcystin producers even in samples where producers are a minority. Based
on our data, potential toxic Planktothrix rubescens identified in all samples
except samples from 5/06 to 8/06 and it was the only potenti al toxic species
during the bloom. Potential toxic Microcystis found in samples where Microcystin
sequences were also excised (4/06, 5/06, 6/06). Potenti al toxic Anabaena sp.
found in samples from 1/06, 2/06, 5/06, 6/06 and 7/06 but the bands were faint
even after the semi nested PCR procedure. There are s amples where
microcystin producers belong to more than one genus but we did not detect a
combination of all the three genera examined in an yone of the samples studied.
This is a common feature for oligotrophic and mesotrophic lakes examined so far
(Rantala et al., 2006).
Because, it has been found that a number of naturally occurring
Planktothrix rubescens strains although containing the total microcystin
synthetase gene cluster do not produce microcystin (Kurmayer et al., 2004),
microcystin’s concentrations in concentrated and unconcentrated water samples
were also measured. In our analysis, the microcystin content in cyanobacterial
pellets was at maximum 6,725 μg/g dry weight. This value was recorded a few
days before the onset of the bloom and on the zenith of the bloom we measured
4,347 μg/g. The above observation is in agreement with previous reports
suggesting that high percentage of inactive mcy genotypes is present in lakes
with higher densities of Planktothrix rubescens (Kurmayer et al., 2004) .
Microcystin concentrations measured were the highest ever measured in Greek
lakes and amongst the highest worldwide.
Although Lake Ziros is not a drinking water supply reservoir, continuous
monitoring is suggested to maximize prevention of health hazards due to the
recreational and irrigation uses of the lake. Moreover, we consider that our
observation might sign the start of a potential chan ge in Greek aquatic
ecosystems which indicates that investigations should now be directed not only
13
to Microcystis/Anabaena dominated shallow eutrophic lakes but also to deep
oligotrophic or mesotrophic Greek lakes.
14
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Legends
Table 1: Sequence analysis of excised DGGE bands. (UFC: uncultured
freshwater cyanobacterium)
Similarity
1 475 Nostoc linckia, AF105138 177 90% 2 480 UFC, AY827734 502 75%
Aphanizomenon sp. T33, AY827797 494 74%
3 588 Phormidium sp. OL
05,AM398977 401 85% 152 82%
4 455 Nostoc sp. PCC 7120, BA000019 469 81%
Anabaena variabilis ATCC 29413, CP000117 470 80%
5 433 Phormidium uncinatum SAG 81.79,AM398952 140 95% 41 87%
6 432 Phormidium sp.AA,
cyan.DGGE band 47, EF150979
442 99%
8 657 23 Planktothrix rub.KPr DGGE band KPr-b AY827783 531 98%
9 493 Planktothrix rubescens
KPr-0, AY827784 494 99%
band 72, EF151001 524 98%
11 442 7,14 Un. Nostocaceae
cyan.DGGE band 47, EF150979
band 72, EF151001 524 98%
13 421 16,18 Un. Nostocaceae cyan.
DGGE band 60, EF150990 448 82%
14 442 7,11 Un. Nostocaceae
cyan.DGGE band 47, EF150979
16 421 13,18 Un. Nostocaceae cyan.
DGGE band 60, EF150990 448 82%
17 421 Un. Nostocaceae cyan.DGGE band20,
EF150959
19 871 Un.Cyan. TH320-3-10 ,
AF330247 840 98%
21 411 Phormidium
Prochlorothrix hollandica,AM709625 132 97% 74 98%
22 869 20 Synechococcus sp.LBP1,
AF330247 840 98%
Un.Cyan. TH320-3-10 , EF513333 841 98%
23 657 8 Planktothrix rub.KPr DGGE band KPr-b AY827783 531 98%
24 493 27,31 Planktothrix rub.KPr DGGE band KPr-0 AY827784 494 100%
25 432 Phormidium sp.AA
AY827783 531 98%
AY827784 494 100%
AY827783 531 98%
AY827783 531 98%
DGGE band KPr- 0,AY827784
AY827783 531 98%
Table 2: Seasonal variation of cell -bound microcystin concentrations measured in Lake Ziros. Microcystin LR equivalents
(Microcystin-LR eq) in μg/g dry weight (DW).
Date T pH NH4 mg/l NO3 mg/l NO2 mg/l P2O5 mg/l
microcystin μg/g dw microcystin μg/l Chlα μg/l Cell number/l biovolume mm3/l
3.1.06 7 8.1 nd nd nd nd 39 0.045 0.2 4x105 0.04 7.2.06 8 8.2 bdl bdl bdl bdl 51 0.06 0.5 1x106 0.1
12.3.06 11 8 0.08 0.2 bdl bdl 355 0.32 3.5 7x106 0.8 14.4.06 15.5 8.1 0.06 0.1 bdl bdl 10 0.01 0.5 1x106 0.1 18.5.06 23.5 8.4 0.06 0.2 bdl bdl 0.005 0 0.1 2x105 0.02 12.6.06 24 8 0.09 1.1 bdl bdl 0.007 0 0.3 6x105 0.06 9.7.06 24.5 8.1 bdl 1.6 bdl bdl 0 0 0.2 4x105 0.04 7.8.06 27 8.2 bdl bdl bdl bdl 0 0 0.3 6x105 0.06
12.9.06 18 8.2 bdl bdl bdl bdl 0.05 0 0.3 6x105 0.06 8.10.06 14 8.4 bdl bdl bdl bdl 49 0.06 0.2 4x105 0.04
12.11.06 10 8 bdl 0.7 bdl bdl 3 4.79 5 1x107 1.1 9.12.06 8.5 8.1 0.13 0.5 bdl bdl 5,369 10.6 6.5 1.3x107 1.5 9.1.07 7.5 8.5 0.10 1.02 bdl bdl 5,679 13.14 7.5 1.5x107 1.7 8.2.07 9 8 0.27 3.2 bdl 0.02 6,725 35.08 18 3.6x107 4 4.3.07 14.5 7.9 0.63 7.3 bdl 0.05 4,347 199 155 3.1x108 34.9
22
Figure 1: Lake Ziros location in NW Greece (upper left panel) and a satellite
view of the lake (upper right panel). The Planktothrix rubescens bloom at lake
Ziros (lower left panel) and a microscopic observation of Planktothix rubescens
trichomes from lake Ziros (lower right panel).
23
Figure 2: Cyanobacterial community composition in Lake Ziros as revealed by
ITSc DGGE profiles. Bands indicated by numbers 1 -33 were excised, re-
amplified and sequenced.
Figure 3: Composition of potential microcystin producers in Lake Ziros.
A1: PCR products using general mcyE primers.
A2: PCR products corresponding to Planktothrix sp. mcyE gene fragment.
B1: Semi – nested PCR amplification using Planktothrix sp. specific primers and
the PCR products of A1 as templates.
B2: Semi – nested PCR amplification using Microcystis sp. specific primers and
the PCR products of A1 as templates.
B3: Semi – nested PCR amplification using Anabaena sp. specific primers and
the PCR products of A1 as templates.