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Microcystin analysis in single filaments of Planktothrix spp. in laboratory cultures and environmental blooms
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Microcystin analysis in single filaments of Planktothrix spp. in laboratory cultures and environmental blooms Reyhan Akcaalana,, Fiona M. Youngb, James S. Metcalfb, Louise F. Morrisonb, Meric Albaya, Geoffrey A. Coddb a
Istanbul University, Fisheries Faculty, Ordu Cad. No:200 34470 Laleli, Istanbul, Turkey Division of Environmental and Applied Biology, School of Life Sciences, University of Dundee, Dundee DD1 4HN, Scotland, UK
b
art i cle info
A B S T R A C T
Article history:
Single filaments of Planktothrix spp. were isolated from laboratory cultures of P. agardhii
Received 15 June 2005
(NIES 595) and P. rubescens (SL 03) and from four freshwater lakes in England and Turkey.
Received in revised form
Filament lengths were measured and microcystins were extracted by freeze-thawing and
20 February 2006
boiling. Microcystin analysis of the isolated single filaments was performed by ELISA using
Accepted 20 February 2006
antibodies raised against microcystin-LR with a minimum detection limit (MDL) of
Available online 4 April 2006
11 pg filament1. In some cases a high percentage of the filaments from the environmental
Keywords:
samples and laboratory cultures were below the MDL of the assay. Based on the filaments
Planktothrix rubescens
with detectable microcystin contents, P. agardhii from Bassenthwaite Lake (England) had
Planktothrix agardhii
the lowest mean microcystin concentration (0.7 fg mm3), and the highest microcystin
Microcystin immunoassay
concentration (2.9 fg mm3) was measured in P. rubescens from Iznik Lake (Turkey). We
Single filaments
investigated the relationship for filaments with microcystin contents above MDL and their
Cyanobacteria
biovolume. Relationships varied widely although P. agardhii from Bassenthwaite showed a better (positive) relationship between filament biovolume and microcystin content than P. rubescens from environmental samples. Under culture conditions, P. rubescens showed a good relationship between filament biovolume and toxin content. & 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Cyanobacteria are well-known components of the phytoplankton communities of lakes with varying nutrient status. However, in waterbodies around the world, lakes that are classified as eutrophic often contain mass blooms of cyanobacteria. Together with the nuisance effects of cyanobacterial blooms, cyanobacteria can also produce secondary metabolites which have been shown to be toxic to animals and humans (Codd et al., 1999). Reports concerning toxic cyanobacterial blooms have been produced in many countries (e.g. Park et al., 1998; Eynard et al., 2000; Frank, 2002; Ballott et al., 2003), as a result of increasing scientific interest and public awareness, and associated health problems. Corresponding author. Tel.:+90 212 4555700; fax: +90 212 5140379.
E-mail address: [email protected] (R. Akcaalan). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.02.020
One of the most important groups of cyanobacterial toxins, due to their widespread occurrence and high toxicity, are the microcystins, mainly produced by Microcystis spp. Members of additional cyanobacterial genera e.g. Planktothrix, Anabaena, Anabaenopsis and Nostoc also produce these toxins and Nodularia spumigena has been shown to produce the related pentapeptides, the nodularins (Skulberg et al., 1993; Sivonen and Jones, 1999). In addition to Microcystis, Planktothrix spp. are very well distributed in the northern hemisphere, especially in Europe. There are several reports of mass occurrences of Planktothrix from European countries from the North to the South: Norway (Skulberg and Skulberg, 1985), Switzerland (Micheletti et al., 1998), France (Feuillade et al., 1996), Poland (Krupa and
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Czernas, 2003), Germany (Fastner et al., 1999), Austria (Dokulil and Jagsch, 1992), Italy (Loizzio et al., 1988) and Turkey (Albay et al., 2003). Bloom-forming Planktothrix species can be distinguished according to their pigment composition and habitat. While P. agardhii (green variety) prefer shallow, nutrient-rich, wellmixed waterbodies, mass occurrences of Planktothrix rubescens (red variety) have generally been recorded in the metalimnion of oligo-mesotrophic lakes that are often used as drinking water supplies (Loizzio et al., 1988; Barco et al., 2004). A survey of 55 German freshwaters showed that P. rubescens had higher microcystin concentrations than other toxic cyanobacteria, namely Microcystis spp. and P. agardhii (Fastner et al., 1999). Furthermore, the occurrence of subsurface maxima of P. rubescens makes depth-profiling in monitoring programmes more important in waterbodies needed for drinking water supply where recurrent blooms have been observed. In addition, countries are increasingly conducting monitoring programmes on their waterbodies for cyanobacteria and cyanotoxins due in part to a significant increase in public awareness. Therefore, early warning and easily applicable monitoring systems and subsequent risk assessment are becoming more important. The World Health Organisation (WHO, 1998) has derived a provisional Guideline Value (GV) for microcystin-LR, one of the over-70 known microcystin variants. This GV is 1 mg l1 microcystin-LR for drinking water (Falconer et al., 1999). In addition to direct analysis for microcystins, surrogate procedures are recommended to indicate whether microcystin concentrations are likely to be approaching concentrations that present health risks via drinking and recreational exposure. These surrogates include the determination of cyanobacterial cell concentrations in water and of chlorophyll a concentrations, when cyanobacteria are dominant (Falconer et al., 1999). The estimations of (putative) microcystin concentrations from cyanobacterial cell numbers and chlorophyll a concentrations are based on comparative cell counts, pigment analyses and microcystin analyses of environmental samples (Falconer et al., 1999) and, specifically for Microcystis, on the determination of cell number and microcystin concentration in laboratory cultures (Long et al., 2001; Lyck, 2004). Microcystin-producing blooms of Planktothrix (Oscillatoria) spp. are common among toxic mass populations of cyanobacteria (Sivonen and Jones, 1999), although little information is available on relations between microcystin concentrations and conveniently measured indices in these filamentous cyanobacteria. In this study, we have directly determined microcystin quotas in single filaments of Planktothix by immunoassay. Considerable current interest is centred on why cyanobacteria produce toxins and on mechanisms of cyanotoxin production in colonies and filaments. For example, do all of the filaments in a Planktothrix sp. population contain the toxins, and are microcystin quotas per filament proportional to filament length or biovolume? In order to help answer these questions, filaments were isolated from laboratory strains and environmental samples from British and Turkish lakes for two bloom-forming Planktothrix species, P. rubescens and P. agardhii, and analysed for microcystins by immunoassay.
2.
Materials and methods
2.1.
Environmental samples
P. rubescens filaments were collected from two lakes in western Turkey: Sapanca Lake and Iznik Lake (on 28.11.03 and 21.12.03, respectively). Sapanca Lake is an oligo-mesotrophic lake and a drinking water reservoir. Successive Planktothrix blooms have been observed in the metalimnion of the lake since the 1980s. Sapanca has a maximum depth of 55 m and a surface area of 46.8 km2. Iznik Lake is the fifth biggest lake in Turkey with a maximum depth of 70 m and a surface area of 308 km2. The lake is mesotrophic and toxic blooms of Anabaena sp. were observed in the surface water in the summer months of 2001 with 7.2 mg l1 MC-LR equivalents (R. Akcaalan, unpublished data). P. rubescens samples were collected from both lakes with a plankton net (55 mm) throughout the water column. P. agardhii filaments were isolated from two lakes in England. One P. agardhii environmental cyanobacterial bloom sample was obtained on 07.07.2003 from Elmhirst Fishing Lake, Lincolnshire, a small, shallow waterbody with a surface area of 0.014 km2. The other cyanobacterial bloom sample was taken on 03.07.2003 from Bassenthwaite Lake, a mesotrophic lake in the Lake District, Cumbria, with a maximum depth of 21 m and a surface area of 7.8 km2.
2.2.
Culture of Planktothrix spp.
P. rubescens (SL 03) filaments were isolated from Sapanca Lake and grown in BG11 medium containing 17.6 mM nitrate (Rippka et al., 1979). P. agardhii strain NIES 595 was grown in the same medium. Batch cultures of both monocyanobacterial, non-axenic Planktothrix strains were grown at 20 1C and 25 mE m2 s1 under cool white fluorescent light.
2.3.
Microcystin analysis
Filaments were isolated from environmental samples and from laboratory cultures under a dissecting microscope. After isolation, each filament was washed by serial transfer through distiled water three times. The length and width of each filament were measured under a binocular microscope with a calibrated graticule and the filaments transferred to 250 ml tubes containing distiled water. Microcystin extraction of filaments in the tubes was achieved by two cycles of freeze-thawing in liquid nitrogen and then boiling the tubes for 1 min in a waterbath. The extracted filament, in distiled water, was analysed directly as methanol is not a suitable solvent for immunoassay. Metcalf and Codd (2000) found that equivalent concentrations could be obtained between boiling waterbath and methanol extraction of microcystins from cyanobacterial cells. The extracts were analysed for microcystins by immunoassay (ELISA) with reference to a gravimetric microcystin-LR standard (Metcalf et al., 2000). To determine whether any difference existed between the relationship of biovolume and microcystin quotas in single and pooled Planktothrix filaments, subsamples were taken
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from both Planktothrix cultures and filaments were isolated as above. After measurement of filament length and width, the biovolume of each cylindrical filament was calculated. Filaments were put in the same tube until total biovolumes of 5, 10, 20, 30, 40 and 50 104 mm3 were reached. Microcystins were extracted and analysed by ELISA as before.
2.4.
Statistics analysis
For filaments with microcystin quotas above the minimum detection limit (MDL, 11 pg filament1), the relationship with filament biovolume was evaluated with Pearson correlation and sample means were compared by one-way analysis of variance (ANOVA) using SPSS 11.0 for Windows (Chicago, IL, USA).
3.
Results
Six Planktothrix samples were investigated, including two laboratory cultures and four environmental samples, consisting of P. agardhii or P. rubescens. In some cases a proportion of the single filaments in investigated populations did not show the presence of microcystin above the MDL. Calculations of microcystin quota filament1, cell1 or mm3 cell volume were only performed using cell parameters of filaments above this MDL, The lowest microcystin quota per cell was found for P. agardhii (NIES 595) and the highest microcystin quota per cell was determined in P. rubescens from Iznik Lake (Table 1). On a cell quota basis, P. rubescens had a higher microcystin quota than P. agardhii, however, as a result of differences in cell dimensions, P. agardhii (NIES 595) had the higher microcystin quota per biovolume in comparison to the two red Planktothrix (SL 03 and those from Sapanca Lake), whilst P. rubescens from Iznik Lake had the highest microcystin content per cell, as well as by biovolume (Table 1).
Table 1 – Microcystin quota per cell and per unit biovolume in Planktothrix spp. from environmental samples and from laboratory cultures fg cell1
fg mm3
P. agardhii (NIES 595) P. agardhii (BL)a
75.6 91.2
1.9 0.7
P. rubescens (SL 03) P. rubescens (IL)b P. rubescens (SL)c
103.9 235.6 108.2
1.7 2.9 1.4
Mean values summarize data for filaments containing MC contents above the immunoassay MDL of 11 pg filament1. a BL, Bassenthwaite Lake. b IL, Iznik Lake. c SL, Sapanca Lake.
3.1.
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Environmental samples
The P. agardhii filaments isolated from the bloom at Elmhirst Fishing Lake were too small for the detection of microcystins in individual filaments. However, microcystin analysis of the bloom by ELISA showed that it contained 3.2 mg l1 microcystin-LR equivalents. Four single filaments were isolated from the sample and none were above the MDL of 11 pg filament1. However, when four filaments from this bloom were placed in the same tube, amounting to a total filament length of 875 mm, approximately 18 pg microcystinLR equivalents were detected. A total of 29 P. agardhii filaments were isolated from Bassenthwaite Lake and 23 filaments (79%) were above the MDL of the ELISA method. P. agardhii filaments had larger cell dimensions than the other Planktothrix spp. Thus the filament biovolumes were much higher and ranged from 1.1 to 22 104 mm3. The range of toxin quotas per filament was quite narrow and varied between 27 and 41 pg filament1 (Fig. 1A). Although the relationship between filament biovolume and toxin content was positive ðr2 ¼ 0:55; po0:01Þ, the mean values of MC quota were very similar in the three filament size classes (Table 2). P. rubescens filaments were collected from two Turkish lakes. This is the first report of P. rubescens in Iznik Lake. A total of 24 filaments were isolated from this waterbody and microcystins were detected in all of them. The length of filaments was smaller compared to P. agardhii, thus their biovolumes varied between 0.8 and 3 104 mm3. All filaments were under 2 mm in length and only two size classes were obtained with smaller filaments (o1 mm) having a slightly higher microcystin quota. Microcystin concentrations of environmental samples of Iznik lake varied between 29.2 and 114.4 pg filament1 (Fig. 1C). The highest toxin quota among all Planktothrix species investigated in this study was detected in filaments of P. rubescens from Iznik Lake (Table 1). Sapanca Lake is an oligo-mesotrophic lake and recurrent P. rubescens blooms in the metalimnion have been observed over the past two decades. Twenty-four filaments were isolated and with the exception of two, microcystins were detected in all filaments with a range of 18.4–66.1 pg filament1 and filament biovolumes varied between 0.75 and 4.6 104 mm3 (Fig. 1B). No positive correlation was found between filament biovolume and filament microcystin concentration ðp40:05Þ, although microcystin content increased slightly for each size class (Table 2).
3.2.
Laboratory cultures
A total of 72 P. agardhii filaments were isolated from the exponential growth phase of the cyanobacterial culture and 41 filaments (57%) gave positive results by ELISA. Biovolumes of filaments with detectable microcystin varied between 0.75 and 5.8 104 mm3 and toxin concentrations were very variable, ranging from 11 to 63.4 pg filament1. The hypothesis that smaller filaments would contain lower microcystin amounts, and longer filaments greater microcystin amounts, was not found here. The highest microcystin content was found in a filament with a biovolume of 2.7 104 mm3 (Fig. 2A). There was
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Fig. 1 – Relationships between biovolume and microcystin quota of P. agardhii filaments from Bassenthwaite Lake (A), and P. rubescens from Sapanca Lake (B) and Iznik Lake (C).
Table 2 – Mean length (7SD) and MC quota per filament (7SD) and per unit biovolume (7SD) of different size classes of Planktothrix spp. in environmental samples and laboratory cultures taken during growth phase Size classes (mm)
n
Mean length (mm)
Mean MC (pg filament1)
MC/Biovolume (fg mm3)
P. agardhii (BL)a
o1 1–2 42
10(3) 5(3) 8
0.670.2 1.670.4 3.170.8
29.472.3 30.370.96 34.973.7
1.170.6 0.470.1 0.270.06
P. rubescens (IL)b
o1 1–2 42
16 8
0.870.2 1.270.1
53.6720.6 51.1712.7
3.471 1.970.2
P. rubescens (SL)c
o1 1–2 42
5(1) 16(1) 1
0.870.3 1.470.2 2.03
28.277.1 39714.6 40
1.971 1.270.2 0.9
NIES 595
o1 1–2 42
7(9) 21(15) 13(7)
0.770.1 1.570.3 2.870.8
32.9714 32.6710.7 37712.3
3.770.5 1.870.4 1.170.2
SL 03
o1 1–2 42
8(9) 22(8) 8(5)
0.770.1 1.570.3 2.470.3
3376.5 39.778 49.878.6
2.770.5 1.670.3 1.270.1
n indicates the number of microcystin-detected filaments. The numbers in parentheses indicate the numbers of filaments which have undetectable MC quotas (MDL 11 pg filament1). Mean values summarize data for filaments containing MC contents above the immunoassay MDL. a BL, Bassenthwaite Lake. b IL, Iznik Lake. c SL, Sapanca Lake.
no correlation between single filament biovolume and microcystin quota ðp40:05Þ. On the other hand, microcystin quota per unit biovolume decreased with an increase in size class (Table 2). Similar results were found for filaments collected at different time intervals from the cultures. The only exception was for filaments collected 45 days after inoculation. Here, there was a better relationship between biovolume and microcystin quota, although 5 of the 20 filaments were below the MDL (Fig. 3). P. rubescens filaments were isolated from Sapanca Lake. After 6 months of batch growth (by serial subculture), a total of 60 filaments were analysed by ELISA and microcystin quotas for 38 of them (63%) were above the MDL. Filament
biovolumes ranged between 0.9 and 5.2 104 mm3 and microcystin contents varied between 29 and 68 pg filament1 in microcystin-detectable filaments (Fig. 2A). Correlations between single filament biovolumes and microcystin quotas of P. rubescens were high ðr2 ¼ 0:49; po0:01Þ. Similar results were obtained for filaments taken from cultures at days 15, 21, 30 and 45 (Fig. 4). Mean microcystin quotas per biovolume did not show a great deal of change with time. Although a significant correlation was found between filament biovolume and toxin quota for every sampling day, the correlation coefficient showed a decrease with time (Fig. 4). For both laboratory cultures, a significant correlation was found between pooled biovolume and microcystin
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Fig. 2 – Relationships between single filament biovolume (A), pooled filament biovolume (B) and microcystin content in the filaments of P. agardhii (NIES 595) and P. rubescens (SL 03).
concentration: r2 ¼ 0:73 for P. agardhii and 0.74 for P. rubescens (Fig. 2B). Interestingly, microcystin quotas of all Planktothrix filaments above MDL increased with increasing size class, with the exception of P. rubescens from Iznik Lake. However, microcystin quota per biovolume decreased when the size classes increased without exception (Table 2).
4.
Discussion
To our knowledge this is the first direct quantification of microcystins in single Planktothrix filaments. Previous estimations of microcystin cell quotas in toxic cyanobacteria, especially Microcystis (Orr and Jones, 1998; Long et al., 2001; Kurmayer et al., 2003; Lyck, 2004) were determined arithmetically by counting suspensions of multiple cells or filaments and analysing these by HPLC with a much higher MDL. Morrison et al. (2003) successfully used ELISA to analyse microcystins in single colonies of Microcystis. For Planktothrix, few studies have mentioned cyanotoxin filament or cell quotas until now. Using this method we were unable to determine whether some filaments in the population were negative, although a proportion was below the MDL with some samples. Our data also clearly show that there are some
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differences in microcystin filament quota both between and within species (po0:01, Table 1). The microcystin content of Planktothrix spp. filaments was initially determined by analysis of extracts from samples containing known numbers of multiple filaments. This permits an arithmetic estimation of the average microcystin content per filament, and if filament and cell dimensions are measured, of a mean microcystin content per unit biovolume and per cell. On the other hand, microcystin concentrations in Planktothrix spp. blooms in the literature have often been based on gravimetric methods and results have been presented mainly as mg/biomass. WHO guidelines currently use alert levels to advise on precautions to be taken when a cyanobacterial bloom occurs in a waterbody. Generally, chlorophyll a concentrations calculated from the cell or colony/filament numbers, have been used to derive these alert levels. The present work aimed to determine whether there was variability in microcystin quotas of single filaments from a population. Although there was variability with some samples, with new microscopy image analysis tools, it may be possible to reduce the error margins and possibly use biovolume or simply filaments, in addition to cell counts to estimate microcystin concentrations in a waterbody. Although significant correlations were achieved with detectable microcystin content and its relationship to filament length and biovolume, in some cases not all filaments were positive for microcystins by ELISA. However, these filaments would be expected to contain microcystins, although below the MDL, as shown with analysis of four filaments from Elmhirst Fishing Lake. Therefore for statistical analysis, only microcystin-positive filaments should be included, although as microcystin analytical methods, more sensitive than ELISA are conceived and developed, our understanding of the relationship between microcystin content and filament length and biovolume can be furthered. In comparative studies, analyses of concentrated environmental Planktothrix suspensions have been found to have a higher mean microcystin content per unit biomass than other genera, namely Microcystis and Anabaena (Henriksen and Moestrup, 1997; Fastner et al., 1999). Of these toxic Planktothrix species, P. agardhii had lower microcystin concentrations than P. rubescens (Fastner et al., 1999). These differences are thought to be due to the patchy distribution of mcy genes in P. agardhii populations, in contrast to P. rubescens, in which all filaments in a population contain these genes for microcystin synthesis (Kurmayer et al., 2004). Our results also showed that in addition to patchy gene distribution, the P. agardhii filaments contained lower microcystin contents than those of P. rubescens on a cell quota basis (e.g. 103.9 fg cell1 P. rubescens (SL 03) versus 75.6 fg cell1 P. agardhii (NIES 595)). Briand et al. (2002) detected the lowest microcystin concentration when numbers of P. agardhii filaments were high in a shallow lake in France. This situation was attributed to the co-occurrence of microcystin-producing and non-producing filaments and a seasonal shift in the dominance of different strains with different toxicities. Hayes et al. (2002) proposed that there may be a shift between Nodularia genotypes which have a better adaptation to the environmental conditions in cyanobacterial populations. As the organism needs to expend resources for survival, growth may become reduced and more
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Fig. 3 – Relationships between biovolume and microcystin quota in filaments of P. agardhii (NIES 595) on different sampling days.
energy may be spent to produce microcystin, which is potentially costly for the organism (Welker et al., 2004). In contrast to other cyanobacteria which may bloom during only one season, Planktothrix can dominate the phytoplankton throughout the year, which indicates the versatility of the genus (Davis et al., 2003). The occurrence of microcystin synthetase genes in different Planktothrix species is variable (Kurmayer et al., 2004). In our study, all P. rubescens filaments from Iznik Lake and 92% of the filaments from Sapanca Lake contained quantifiable microcystin. In the laboratory cultures the proportion of filaments containing microcystin above the MDL reduced to 63% for P. rubescens filaments. The percentage of P. agardhii above the MDL was 77% for environmental samples and 57% for filaments from laboratory cultures. However, this did not indicate whether filaments with no detectable microcystin possessed mcy genes or microcystins, although this was not the purpose of the study. Other studies have shown that inactive and active microcystin genotypes can co-occur in the same cyanobacterial population (Nishizawa et al., 1999; Kurmayer et al., 2004) although little is known about the regulation of the genes and their relationship to microcystin pools. The same species from different waterbodies can contain markedly different concentrations of microcystins ðpo0:01Þ, as seen with P. rubescens. Whilst one strain from Iznik Lake had a 2.9 fg mm3 mean microcystin quota, P. rubescens from
Sapanca Lake contained 1.4 fg mm3. These two lakes are located in the same region but differ in trophic status. It is known that environmental factors have an effect on microcystin production by the same species (Sivonen and Jones, 1999) although variation is generally within an order of magnitude. Similarly, the mean microcystin quota of P. rubescens filaments showed a slight difference between isolates taken from culture (1.7 fg mm3) and directly from the natural population in Sapanca Lake (1.4 fg mm3), although the cultured P. rubescens originated from the same lake and were isolated shortly before microcystin immunoassays. Nevertheless, the microcystin quotas of the P. rubescens filaments from Lake I˙znik were double that of the other two red Planktothrix isolates. When two environmental samples of P. rubescens were compared, we found a differences between microcystin quota and filament biovolume (Fig. 1). The same situation was observed with P. agardhii filaments from lakes and from laboratory cultures. On the other hand, there was no apparent relationship between single filament biovolume and microcystin quota for NIES 595, whilst pooled filament biovolumes showed a strong positive relationship ðr2 ¼ 0:7; po0:01Þ. This indicates that each filament does contribute to the total microcystin concentration. It is possible that these differences may be due to the existence of differences in the microcystin pool size in individual cells in the Planktothrix filament. Such differences could arise due to an intercellular
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Fig. 4 – Relationships between biovolume and microcystin quota in filaments of P. rubescens (SL 03) on different sampling days. division of metabolism including microcystin biosynthesis, or to the translocation of microcystins between cells in the filament, although there is no gross morphological differentiation between cells in Planktothrix filaments to support these possibilities. As Shapiro (1998) suggested, if a bacterial population is considered as a multicellular organism, it might be possible to say that some filaments produce lower amounts of microcystins than others and that this represents a differentiation between the filaments in the population. However, in all isolated specimens where microcystin content was quantifiable, microcystin quota per biovolume decreased with an increase in length, invariably (Table 2).
Acknowledgements We thank the following for support: the TUBITAK-CHEVENING Studentship programme (RA) and the Research Fund of The University of Istanbul (Project no. UDP-326/03062004, RA); the UK Natural Environment Research Council for a Ph.D. Studentship (FMY); and the European Commission (TOXIC, EVK1-CT-2002-00107: JSM, GAC; PEPCY, QLRT-2001-02634: LFM, GAC). We also thank Adnan Sumer and Ozcan Gaygusuz for assistance in field sampling.
R E F E R E N C E S
5.
Conclusion
ELISA can directly quantify microcystins in single filaments of cyanobacteria. This ability will contribute to the risk assessment of microcystin-containing cyanobacterial blooms. Single filament immunoassay for microcystins has indicated wide differences in toxin quota per unit biovolume and filament length between filaments of single laboratory clones and in environmental samples. The differences may be attributed to variations in microcystin synthetase gene expression (Kurmayer et al., 2004) and to possible changes in the biosynthesis and multiple fates of the microcystins.
Albay, M., Akc-aalan, R., Tu¨fekc-i, H., Metcalf, J.S., Beattie, K.A., Codd, G.A., 2003. Depth profiles of cyanobacterial hepatotoxins (microcystins) in three Turkish freshwater lakes. Hydrobiologia 505, 89–95. Ballott, A., Pflugmacher, S., Wiegand, C., Kotut, K., Krienitz, L., 2003. Cyanobacterial toxins in Lake Baringo, Kenya. Limnologica 33, 2–9. Barco, M., Flores, C., Rivera, J., Caixach, J., 2004. Determination of microcystin variants and related peptides present in a water bloom of Planktothrix (Oscillatoria) rubescens in a Spanish drinking water reservoir by LC/ESI-MS. Toxicon 44, 881–886. Briand, J.F., Robillot, C., Quiblier-Lloberas, C., Bernard, C., 2002. A perennial bloom of Planktothrix agardhii (Cyanobacteria) in a shallow eutrophic French lake: limnological and microcystin production studies. Arch. Hydrobiol. 153, 605–622.
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Codd, G.A., Bell, S.G., Kaya, K., Ward, C.J., Beattie, K.A., Metcalf, J.S., 1999. Cyanobacterial toxins, exposure routes and human health. Eur. J. Phycol. 34, 405–415. Davis, P.A., Dent, M., Parker, J., Reynolds, C.S., Walsby, A.E., 2003. The annual cycle of growth rate and biomass change in Planktothrix spp. in Blelham Tarn, English Lake District. Freshwater Biol. 48, 852–867. Dokulil, M.T., Jagsch, A., 1992. Dynamics of phosphorus and nitrogen loading and its effect on phytoplankton in Mondsee, Austria. Hydrobiologia 243/244 (Dev. Hydrobiol. 79), 389–394. Eynard, F., Mez, K., Walther, J., 2000. Risk of cyanobacterial toxins in Riga waters. Water Res. 34, 2979–2988. Falconer, I., Bartram, J., Chorus, I., Kuiper-Goodman, T., Utkilen, H., Burch, M., Codd, G.A., 1999. Safe levels and safe practices. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water. A Guide to Their Public Health Consequences, Monitoring and Management. E&FN Spon, London, pp. 155–178. Fastner, J., Newman, U., Wirsing, B., Weckesser, J., Wiedner, C., Nixdorf, B., Chorus, I., 1999. Microcystins (hepatotoxic heptapeptides) in German freshwater bodies. Environ. Toxicol. 14, 13–22. Feuillade, M., Jann-Para, G., Feuillade, J., 1996. Toxic compounds to Artemia from blooms and isolates of the cyanobacterium Planktothrix rubescens. Arch. Hydrobiol. 138, 175–186. Frank, C.A.P., 2002. Microcystin-producing cyanobacteria in recreational waters in south western Germany. Environ. Toxicol. 17, 361–366. Hayes, P.K., Barker, G.L.A., Batley, J., Beard, S.J., Handley, B.A., Vacharapiyasophon, P., Walsby, A.E., 2002. Genetic diversity within populations of cyanobacteria assessed by analysis of single filaments. Antonie van Leeuwenhoek 81, 197–202. Henriksen, P., Moestrup, O., 1997. Seasonal variations in microcystin contents of Danish cyanobacteria. Nat. Toxins 5, 99–106. Krupa, D., Czernas, K., 2003. Mass appearance of cyanobacterium Planktothrix rubescens in Lake Piaseczno, Poland. Water Qual. Res. J. Canada 38, 141–152. Kurmayer, R., Christiansen, G., Chorus, I., 2003. The abundance of microcystin-producing genotypes correlates positively with colony size in Microcystis sp. and determines its microcystin net production in Lake Wannsee. Appl. Environ. Microbiol. 69, 787–795. Kurmayer, R., Christansen, G., Fastner, J., Bo¨rner, T., 2004. Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp. Environ. Microbiol. 6, 831–841. Loizzio, A., Sechi, N., Volterra, L., Contu, A., 1988. Some features of a bloom of Oscillatoria rubescens D.C. registered in two Italian reservoirs. Water Air Soil Pollut. 38, 263–271. Long, B.M., Jones, G.J., Orr, P.T., 2001. Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate. Appl. Environ. Microbiol. 67, 278–283. Lyck, S., 2004. Simultaneous changes in cell quotas of microcystin, chlorophyll-a, protein and carbonate during different
growth phases of a batch culture experiment with Microcystis aeruginosa. J. Plankton Res. 26, 727–736. Metcalf, J.S., Codd, G.A., 2000. Microwave oven and boiling waterbath extraction of hepatotoxins from cyanobacterial cells. FEMS Microbiol. Lett. 184, 241–246. Metcalf, J.S., Bell, S., Codd, G.A., 2000. Production of novel polyclonal antibodies against the cyanobacterial toxin microcystin-LR and their application for the detection and quantification of microcystins and nodularin. Water Res. 34, 2761–2769. Micheletti, S., Schanz, F., Walsby, A.E., 1998. The daily integral of photosynthesis by Planktothrix rubescens during summer stratification and autumnal mixing in Lake Zu¨rich. New Phytol. 139, 233–246. Morrison, L.F., Metcalf, J.S., Codd, G.A., 2003. Analysis of microcystins by ELISA in single colonies of Microcystis sp. from UK waters. Abstracts of the Third Symposium for European Freshwater Sciences, Edinburgh, 13–18 July, 2003, p. 97. Nishizawa, T., Asayama, M., Fujii, K., Harada, K., Shirai, M., 1999. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. J. Biochem. 126, 520–529. Orr, P.T., Jones, G.J., 1998. Relationship between microcystin production and cell division rates in nitrogen limited Microcystis aeruginosa cultures. Limnol. Oceanogr. 43, 1604–1614. Park, H., Kim, B., Kim, E., Okino, T., 1998. Hepatotoxic microcystins and neurotoxic anatoxin-a in cyanobacterial blooms from Korean lakes. Environ. Toxicol. Water Qual. 13, 225–234. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1–61. Shapiro, J.A., 1998. Thinking about bacterial populations as multicellular organisms. Annu. Rev. Microbiol. 52, 81–104. Sivonen, K., Jones, G., 1999. Cyanobacterial toxins. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water. A Guide to Their Public Health Consequences, Monitoring and Management. E&FN Spon, London, pp. 1–14. Skulberg, O.M., Skulberg, R., 1985. Planktic species of Oscillatoria (Cyanophyceae) from Norway. Characterisation and classification. Arch. Hydrobiol. 38/39 (Suppl. 71), 157–174. Skulberg, O.M., Carmichael, W.W., Codd, G.A., Skulberg, R., 1993. Taxonomy of toxic cyanophyceae (Cyanobacteria). In: Falconer, I.R. (Ed.), Algal Toxins in Seafood and Drinking Water. Academic Press, London, pp. 187–209. Welker, M., Christiansen, G., von Do¨hren, H., 2004. Diversity of coexisting Planktothrix (Cyanobacteria) chemotypes deduced by mass spectral analysis of microcystins and oligopeptides. Arch. Microbiol. 182, 288–298. WHO, 1998. Guidelines for Drinking Water Quality, second ed. Addendum to vol. 2, Health Criteria and other Supporting Information. World Health Organisation, Geneva.
40 (2006) 1583 – 1590
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Microcystin analysis in single filaments of Planktothrix spp. in laboratory cultures and environmental blooms Reyhan Akcaalana,, Fiona M. Youngb, James S. Metcalfb, Louise F. Morrisonb, Meric Albaya, Geoffrey A. Coddb a
Istanbul University, Fisheries Faculty, Ordu Cad. No:200 34470 Laleli, Istanbul, Turkey Division of Environmental and Applied Biology, School of Life Sciences, University of Dundee, Dundee DD1 4HN, Scotland, UK
b
art i cle info
A B S T R A C T
Article history:
Single filaments of Planktothrix spp. were isolated from laboratory cultures of P. agardhii
Received 15 June 2005
(NIES 595) and P. rubescens (SL 03) and from four freshwater lakes in England and Turkey.
Received in revised form
Filament lengths were measured and microcystins were extracted by freeze-thawing and
20 February 2006
boiling. Microcystin analysis of the isolated single filaments was performed by ELISA using
Accepted 20 February 2006
antibodies raised against microcystin-LR with a minimum detection limit (MDL) of
Available online 4 April 2006
11 pg filament1. In some cases a high percentage of the filaments from the environmental
Keywords:
samples and laboratory cultures were below the MDL of the assay. Based on the filaments
Planktothrix rubescens
with detectable microcystin contents, P. agardhii from Bassenthwaite Lake (England) had
Planktothrix agardhii
the lowest mean microcystin concentration (0.7 fg mm3), and the highest microcystin
Microcystin immunoassay
concentration (2.9 fg mm3) was measured in P. rubescens from Iznik Lake (Turkey). We
Single filaments
investigated the relationship for filaments with microcystin contents above MDL and their
Cyanobacteria
biovolume. Relationships varied widely although P. agardhii from Bassenthwaite showed a better (positive) relationship between filament biovolume and microcystin content than P. rubescens from environmental samples. Under culture conditions, P. rubescens showed a good relationship between filament biovolume and toxin content. & 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Cyanobacteria are well-known components of the phytoplankton communities of lakes with varying nutrient status. However, in waterbodies around the world, lakes that are classified as eutrophic often contain mass blooms of cyanobacteria. Together with the nuisance effects of cyanobacterial blooms, cyanobacteria can also produce secondary metabolites which have been shown to be toxic to animals and humans (Codd et al., 1999). Reports concerning toxic cyanobacterial blooms have been produced in many countries (e.g. Park et al., 1998; Eynard et al., 2000; Frank, 2002; Ballott et al., 2003), as a result of increasing scientific interest and public awareness, and associated health problems. Corresponding author. Tel.:+90 212 4555700; fax: +90 212 5140379.
E-mail address: [email protected] (R. Akcaalan). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.02.020
One of the most important groups of cyanobacterial toxins, due to their widespread occurrence and high toxicity, are the microcystins, mainly produced by Microcystis spp. Members of additional cyanobacterial genera e.g. Planktothrix, Anabaena, Anabaenopsis and Nostoc also produce these toxins and Nodularia spumigena has been shown to produce the related pentapeptides, the nodularins (Skulberg et al., 1993; Sivonen and Jones, 1999). In addition to Microcystis, Planktothrix spp. are very well distributed in the northern hemisphere, especially in Europe. There are several reports of mass occurrences of Planktothrix from European countries from the North to the South: Norway (Skulberg and Skulberg, 1985), Switzerland (Micheletti et al., 1998), France (Feuillade et al., 1996), Poland (Krupa and
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Czernas, 2003), Germany (Fastner et al., 1999), Austria (Dokulil and Jagsch, 1992), Italy (Loizzio et al., 1988) and Turkey (Albay et al., 2003). Bloom-forming Planktothrix species can be distinguished according to their pigment composition and habitat. While P. agardhii (green variety) prefer shallow, nutrient-rich, wellmixed waterbodies, mass occurrences of Planktothrix rubescens (red variety) have generally been recorded in the metalimnion of oligo-mesotrophic lakes that are often used as drinking water supplies (Loizzio et al., 1988; Barco et al., 2004). A survey of 55 German freshwaters showed that P. rubescens had higher microcystin concentrations than other toxic cyanobacteria, namely Microcystis spp. and P. agardhii (Fastner et al., 1999). Furthermore, the occurrence of subsurface maxima of P. rubescens makes depth-profiling in monitoring programmes more important in waterbodies needed for drinking water supply where recurrent blooms have been observed. In addition, countries are increasingly conducting monitoring programmes on their waterbodies for cyanobacteria and cyanotoxins due in part to a significant increase in public awareness. Therefore, early warning and easily applicable monitoring systems and subsequent risk assessment are becoming more important. The World Health Organisation (WHO, 1998) has derived a provisional Guideline Value (GV) for microcystin-LR, one of the over-70 known microcystin variants. This GV is 1 mg l1 microcystin-LR for drinking water (Falconer et al., 1999). In addition to direct analysis for microcystins, surrogate procedures are recommended to indicate whether microcystin concentrations are likely to be approaching concentrations that present health risks via drinking and recreational exposure. These surrogates include the determination of cyanobacterial cell concentrations in water and of chlorophyll a concentrations, when cyanobacteria are dominant (Falconer et al., 1999). The estimations of (putative) microcystin concentrations from cyanobacterial cell numbers and chlorophyll a concentrations are based on comparative cell counts, pigment analyses and microcystin analyses of environmental samples (Falconer et al., 1999) and, specifically for Microcystis, on the determination of cell number and microcystin concentration in laboratory cultures (Long et al., 2001; Lyck, 2004). Microcystin-producing blooms of Planktothrix (Oscillatoria) spp. are common among toxic mass populations of cyanobacteria (Sivonen and Jones, 1999), although little information is available on relations between microcystin concentrations and conveniently measured indices in these filamentous cyanobacteria. In this study, we have directly determined microcystin quotas in single filaments of Planktothix by immunoassay. Considerable current interest is centred on why cyanobacteria produce toxins and on mechanisms of cyanotoxin production in colonies and filaments. For example, do all of the filaments in a Planktothrix sp. population contain the toxins, and are microcystin quotas per filament proportional to filament length or biovolume? In order to help answer these questions, filaments were isolated from laboratory strains and environmental samples from British and Turkish lakes for two bloom-forming Planktothrix species, P. rubescens and P. agardhii, and analysed for microcystins by immunoassay.
2.
Materials and methods
2.1.
Environmental samples
P. rubescens filaments were collected from two lakes in western Turkey: Sapanca Lake and Iznik Lake (on 28.11.03 and 21.12.03, respectively). Sapanca Lake is an oligo-mesotrophic lake and a drinking water reservoir. Successive Planktothrix blooms have been observed in the metalimnion of the lake since the 1980s. Sapanca has a maximum depth of 55 m and a surface area of 46.8 km2. Iznik Lake is the fifth biggest lake in Turkey with a maximum depth of 70 m and a surface area of 308 km2. The lake is mesotrophic and toxic blooms of Anabaena sp. were observed in the surface water in the summer months of 2001 with 7.2 mg l1 MC-LR equivalents (R. Akcaalan, unpublished data). P. rubescens samples were collected from both lakes with a plankton net (55 mm) throughout the water column. P. agardhii filaments were isolated from two lakes in England. One P. agardhii environmental cyanobacterial bloom sample was obtained on 07.07.2003 from Elmhirst Fishing Lake, Lincolnshire, a small, shallow waterbody with a surface area of 0.014 km2. The other cyanobacterial bloom sample was taken on 03.07.2003 from Bassenthwaite Lake, a mesotrophic lake in the Lake District, Cumbria, with a maximum depth of 21 m and a surface area of 7.8 km2.
2.2.
Culture of Planktothrix spp.
P. rubescens (SL 03) filaments were isolated from Sapanca Lake and grown in BG11 medium containing 17.6 mM nitrate (Rippka et al., 1979). P. agardhii strain NIES 595 was grown in the same medium. Batch cultures of both monocyanobacterial, non-axenic Planktothrix strains were grown at 20 1C and 25 mE m2 s1 under cool white fluorescent light.
2.3.
Microcystin analysis
Filaments were isolated from environmental samples and from laboratory cultures under a dissecting microscope. After isolation, each filament was washed by serial transfer through distiled water three times. The length and width of each filament were measured under a binocular microscope with a calibrated graticule and the filaments transferred to 250 ml tubes containing distiled water. Microcystin extraction of filaments in the tubes was achieved by two cycles of freeze-thawing in liquid nitrogen and then boiling the tubes for 1 min in a waterbath. The extracted filament, in distiled water, was analysed directly as methanol is not a suitable solvent for immunoassay. Metcalf and Codd (2000) found that equivalent concentrations could be obtained between boiling waterbath and methanol extraction of microcystins from cyanobacterial cells. The extracts were analysed for microcystins by immunoassay (ELISA) with reference to a gravimetric microcystin-LR standard (Metcalf et al., 2000). To determine whether any difference existed between the relationship of biovolume and microcystin quotas in single and pooled Planktothrix filaments, subsamples were taken
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from both Planktothrix cultures and filaments were isolated as above. After measurement of filament length and width, the biovolume of each cylindrical filament was calculated. Filaments were put in the same tube until total biovolumes of 5, 10, 20, 30, 40 and 50 104 mm3 were reached. Microcystins were extracted and analysed by ELISA as before.
2.4.
Statistics analysis
For filaments with microcystin quotas above the minimum detection limit (MDL, 11 pg filament1), the relationship with filament biovolume was evaluated with Pearson correlation and sample means were compared by one-way analysis of variance (ANOVA) using SPSS 11.0 for Windows (Chicago, IL, USA).
3.
Results
Six Planktothrix samples were investigated, including two laboratory cultures and four environmental samples, consisting of P. agardhii or P. rubescens. In some cases a proportion of the single filaments in investigated populations did not show the presence of microcystin above the MDL. Calculations of microcystin quota filament1, cell1 or mm3 cell volume were only performed using cell parameters of filaments above this MDL, The lowest microcystin quota per cell was found for P. agardhii (NIES 595) and the highest microcystin quota per cell was determined in P. rubescens from Iznik Lake (Table 1). On a cell quota basis, P. rubescens had a higher microcystin quota than P. agardhii, however, as a result of differences in cell dimensions, P. agardhii (NIES 595) had the higher microcystin quota per biovolume in comparison to the two red Planktothrix (SL 03 and those from Sapanca Lake), whilst P. rubescens from Iznik Lake had the highest microcystin content per cell, as well as by biovolume (Table 1).
Table 1 – Microcystin quota per cell and per unit biovolume in Planktothrix spp. from environmental samples and from laboratory cultures fg cell1
fg mm3
P. agardhii (NIES 595) P. agardhii (BL)a
75.6 91.2
1.9 0.7
P. rubescens (SL 03) P. rubescens (IL)b P. rubescens (SL)c
103.9 235.6 108.2
1.7 2.9 1.4
Mean values summarize data for filaments containing MC contents above the immunoassay MDL of 11 pg filament1. a BL, Bassenthwaite Lake. b IL, Iznik Lake. c SL, Sapanca Lake.
3.1.
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Environmental samples
The P. agardhii filaments isolated from the bloom at Elmhirst Fishing Lake were too small for the detection of microcystins in individual filaments. However, microcystin analysis of the bloom by ELISA showed that it contained 3.2 mg l1 microcystin-LR equivalents. Four single filaments were isolated from the sample and none were above the MDL of 11 pg filament1. However, when four filaments from this bloom were placed in the same tube, amounting to a total filament length of 875 mm, approximately 18 pg microcystinLR equivalents were detected. A total of 29 P. agardhii filaments were isolated from Bassenthwaite Lake and 23 filaments (79%) were above the MDL of the ELISA method. P. agardhii filaments had larger cell dimensions than the other Planktothrix spp. Thus the filament biovolumes were much higher and ranged from 1.1 to 22 104 mm3. The range of toxin quotas per filament was quite narrow and varied between 27 and 41 pg filament1 (Fig. 1A). Although the relationship between filament biovolume and toxin content was positive ðr2 ¼ 0:55; po0:01Þ, the mean values of MC quota were very similar in the three filament size classes (Table 2). P. rubescens filaments were collected from two Turkish lakes. This is the first report of P. rubescens in Iznik Lake. A total of 24 filaments were isolated from this waterbody and microcystins were detected in all of them. The length of filaments was smaller compared to P. agardhii, thus their biovolumes varied between 0.8 and 3 104 mm3. All filaments were under 2 mm in length and only two size classes were obtained with smaller filaments (o1 mm) having a slightly higher microcystin quota. Microcystin concentrations of environmental samples of Iznik lake varied between 29.2 and 114.4 pg filament1 (Fig. 1C). The highest toxin quota among all Planktothrix species investigated in this study was detected in filaments of P. rubescens from Iznik Lake (Table 1). Sapanca Lake is an oligo-mesotrophic lake and recurrent P. rubescens blooms in the metalimnion have been observed over the past two decades. Twenty-four filaments were isolated and with the exception of two, microcystins were detected in all filaments with a range of 18.4–66.1 pg filament1 and filament biovolumes varied between 0.75 and 4.6 104 mm3 (Fig. 1B). No positive correlation was found between filament biovolume and filament microcystin concentration ðp40:05Þ, although microcystin content increased slightly for each size class (Table 2).
3.2.
Laboratory cultures
A total of 72 P. agardhii filaments were isolated from the exponential growth phase of the cyanobacterial culture and 41 filaments (57%) gave positive results by ELISA. Biovolumes of filaments with detectable microcystin varied between 0.75 and 5.8 104 mm3 and toxin concentrations were very variable, ranging from 11 to 63.4 pg filament1. The hypothesis that smaller filaments would contain lower microcystin amounts, and longer filaments greater microcystin amounts, was not found here. The highest microcystin content was found in a filament with a biovolume of 2.7 104 mm3 (Fig. 2A). There was
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Fig. 1 – Relationships between biovolume and microcystin quota of P. agardhii filaments from Bassenthwaite Lake (A), and P. rubescens from Sapanca Lake (B) and Iznik Lake (C).
Table 2 – Mean length (7SD) and MC quota per filament (7SD) and per unit biovolume (7SD) of different size classes of Planktothrix spp. in environmental samples and laboratory cultures taken during growth phase Size classes (mm)
n
Mean length (mm)
Mean MC (pg filament1)
MC/Biovolume (fg mm3)
P. agardhii (BL)a
o1 1–2 42
10(3) 5(3) 8
0.670.2 1.670.4 3.170.8
29.472.3 30.370.96 34.973.7
1.170.6 0.470.1 0.270.06
P. rubescens (IL)b
o1 1–2 42
16 8
0.870.2 1.270.1
53.6720.6 51.1712.7
3.471 1.970.2
P. rubescens (SL)c
o1 1–2 42
5(1) 16(1) 1
0.870.3 1.470.2 2.03
28.277.1 39714.6 40
1.971 1.270.2 0.9
NIES 595
o1 1–2 42
7(9) 21(15) 13(7)
0.770.1 1.570.3 2.870.8
32.9714 32.6710.7 37712.3
3.770.5 1.870.4 1.170.2
SL 03
o1 1–2 42
8(9) 22(8) 8(5)
0.770.1 1.570.3 2.470.3
3376.5 39.778 49.878.6
2.770.5 1.670.3 1.270.1
n indicates the number of microcystin-detected filaments. The numbers in parentheses indicate the numbers of filaments which have undetectable MC quotas (MDL 11 pg filament1). Mean values summarize data for filaments containing MC contents above the immunoassay MDL. a BL, Bassenthwaite Lake. b IL, Iznik Lake. c SL, Sapanca Lake.
no correlation between single filament biovolume and microcystin quota ðp40:05Þ. On the other hand, microcystin quota per unit biovolume decreased with an increase in size class (Table 2). Similar results were found for filaments collected at different time intervals from the cultures. The only exception was for filaments collected 45 days after inoculation. Here, there was a better relationship between biovolume and microcystin quota, although 5 of the 20 filaments were below the MDL (Fig. 3). P. rubescens filaments were isolated from Sapanca Lake. After 6 months of batch growth (by serial subculture), a total of 60 filaments were analysed by ELISA and microcystin quotas for 38 of them (63%) were above the MDL. Filament
biovolumes ranged between 0.9 and 5.2 104 mm3 and microcystin contents varied between 29 and 68 pg filament1 in microcystin-detectable filaments (Fig. 2A). Correlations between single filament biovolumes and microcystin quotas of P. rubescens were high ðr2 ¼ 0:49; po0:01Þ. Similar results were obtained for filaments taken from cultures at days 15, 21, 30 and 45 (Fig. 4). Mean microcystin quotas per biovolume did not show a great deal of change with time. Although a significant correlation was found between filament biovolume and toxin quota for every sampling day, the correlation coefficient showed a decrease with time (Fig. 4). For both laboratory cultures, a significant correlation was found between pooled biovolume and microcystin
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Fig. 2 – Relationships between single filament biovolume (A), pooled filament biovolume (B) and microcystin content in the filaments of P. agardhii (NIES 595) and P. rubescens (SL 03).
concentration: r2 ¼ 0:73 for P. agardhii and 0.74 for P. rubescens (Fig. 2B). Interestingly, microcystin quotas of all Planktothrix filaments above MDL increased with increasing size class, with the exception of P. rubescens from Iznik Lake. However, microcystin quota per biovolume decreased when the size classes increased without exception (Table 2).
4.
Discussion
To our knowledge this is the first direct quantification of microcystins in single Planktothrix filaments. Previous estimations of microcystin cell quotas in toxic cyanobacteria, especially Microcystis (Orr and Jones, 1998; Long et al., 2001; Kurmayer et al., 2003; Lyck, 2004) were determined arithmetically by counting suspensions of multiple cells or filaments and analysing these by HPLC with a much higher MDL. Morrison et al. (2003) successfully used ELISA to analyse microcystins in single colonies of Microcystis. For Planktothrix, few studies have mentioned cyanotoxin filament or cell quotas until now. Using this method we were unable to determine whether some filaments in the population were negative, although a proportion was below the MDL with some samples. Our data also clearly show that there are some
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differences in microcystin filament quota both between and within species (po0:01, Table 1). The microcystin content of Planktothrix spp. filaments was initially determined by analysis of extracts from samples containing known numbers of multiple filaments. This permits an arithmetic estimation of the average microcystin content per filament, and if filament and cell dimensions are measured, of a mean microcystin content per unit biovolume and per cell. On the other hand, microcystin concentrations in Planktothrix spp. blooms in the literature have often been based on gravimetric methods and results have been presented mainly as mg/biomass. WHO guidelines currently use alert levels to advise on precautions to be taken when a cyanobacterial bloom occurs in a waterbody. Generally, chlorophyll a concentrations calculated from the cell or colony/filament numbers, have been used to derive these alert levels. The present work aimed to determine whether there was variability in microcystin quotas of single filaments from a population. Although there was variability with some samples, with new microscopy image analysis tools, it may be possible to reduce the error margins and possibly use biovolume or simply filaments, in addition to cell counts to estimate microcystin concentrations in a waterbody. Although significant correlations were achieved with detectable microcystin content and its relationship to filament length and biovolume, in some cases not all filaments were positive for microcystins by ELISA. However, these filaments would be expected to contain microcystins, although below the MDL, as shown with analysis of four filaments from Elmhirst Fishing Lake. Therefore for statistical analysis, only microcystin-positive filaments should be included, although as microcystin analytical methods, more sensitive than ELISA are conceived and developed, our understanding of the relationship between microcystin content and filament length and biovolume can be furthered. In comparative studies, analyses of concentrated environmental Planktothrix suspensions have been found to have a higher mean microcystin content per unit biomass than other genera, namely Microcystis and Anabaena (Henriksen and Moestrup, 1997; Fastner et al., 1999). Of these toxic Planktothrix species, P. agardhii had lower microcystin concentrations than P. rubescens (Fastner et al., 1999). These differences are thought to be due to the patchy distribution of mcy genes in P. agardhii populations, in contrast to P. rubescens, in which all filaments in a population contain these genes for microcystin synthesis (Kurmayer et al., 2004). Our results also showed that in addition to patchy gene distribution, the P. agardhii filaments contained lower microcystin contents than those of P. rubescens on a cell quota basis (e.g. 103.9 fg cell1 P. rubescens (SL 03) versus 75.6 fg cell1 P. agardhii (NIES 595)). Briand et al. (2002) detected the lowest microcystin concentration when numbers of P. agardhii filaments were high in a shallow lake in France. This situation was attributed to the co-occurrence of microcystin-producing and non-producing filaments and a seasonal shift in the dominance of different strains with different toxicities. Hayes et al. (2002) proposed that there may be a shift between Nodularia genotypes which have a better adaptation to the environmental conditions in cyanobacterial populations. As the organism needs to expend resources for survival, growth may become reduced and more
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Fig. 3 – Relationships between biovolume and microcystin quota in filaments of P. agardhii (NIES 595) on different sampling days.
energy may be spent to produce microcystin, which is potentially costly for the organism (Welker et al., 2004). In contrast to other cyanobacteria which may bloom during only one season, Planktothrix can dominate the phytoplankton throughout the year, which indicates the versatility of the genus (Davis et al., 2003). The occurrence of microcystin synthetase genes in different Planktothrix species is variable (Kurmayer et al., 2004). In our study, all P. rubescens filaments from Iznik Lake and 92% of the filaments from Sapanca Lake contained quantifiable microcystin. In the laboratory cultures the proportion of filaments containing microcystin above the MDL reduced to 63% for P. rubescens filaments. The percentage of P. agardhii above the MDL was 77% for environmental samples and 57% for filaments from laboratory cultures. However, this did not indicate whether filaments with no detectable microcystin possessed mcy genes or microcystins, although this was not the purpose of the study. Other studies have shown that inactive and active microcystin genotypes can co-occur in the same cyanobacterial population (Nishizawa et al., 1999; Kurmayer et al., 2004) although little is known about the regulation of the genes and their relationship to microcystin pools. The same species from different waterbodies can contain markedly different concentrations of microcystins ðpo0:01Þ, as seen with P. rubescens. Whilst one strain from Iznik Lake had a 2.9 fg mm3 mean microcystin quota, P. rubescens from
Sapanca Lake contained 1.4 fg mm3. These two lakes are located in the same region but differ in trophic status. It is known that environmental factors have an effect on microcystin production by the same species (Sivonen and Jones, 1999) although variation is generally within an order of magnitude. Similarly, the mean microcystin quota of P. rubescens filaments showed a slight difference between isolates taken from culture (1.7 fg mm3) and directly from the natural population in Sapanca Lake (1.4 fg mm3), although the cultured P. rubescens originated from the same lake and were isolated shortly before microcystin immunoassays. Nevertheless, the microcystin quotas of the P. rubescens filaments from Lake I˙znik were double that of the other two red Planktothrix isolates. When two environmental samples of P. rubescens were compared, we found a differences between microcystin quota and filament biovolume (Fig. 1). The same situation was observed with P. agardhii filaments from lakes and from laboratory cultures. On the other hand, there was no apparent relationship between single filament biovolume and microcystin quota for NIES 595, whilst pooled filament biovolumes showed a strong positive relationship ðr2 ¼ 0:7; po0:01Þ. This indicates that each filament does contribute to the total microcystin concentration. It is possible that these differences may be due to the existence of differences in the microcystin pool size in individual cells in the Planktothrix filament. Such differences could arise due to an intercellular
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Fig. 4 – Relationships between biovolume and microcystin quota in filaments of P. rubescens (SL 03) on different sampling days. division of metabolism including microcystin biosynthesis, or to the translocation of microcystins between cells in the filament, although there is no gross morphological differentiation between cells in Planktothrix filaments to support these possibilities. As Shapiro (1998) suggested, if a bacterial population is considered as a multicellular organism, it might be possible to say that some filaments produce lower amounts of microcystins than others and that this represents a differentiation between the filaments in the population. However, in all isolated specimens where microcystin content was quantifiable, microcystin quota per biovolume decreased with an increase in length, invariably (Table 2).
Acknowledgements We thank the following for support: the TUBITAK-CHEVENING Studentship programme (RA) and the Research Fund of The University of Istanbul (Project no. UDP-326/03062004, RA); the UK Natural Environment Research Council for a Ph.D. Studentship (FMY); and the European Commission (TOXIC, EVK1-CT-2002-00107: JSM, GAC; PEPCY, QLRT-2001-02634: LFM, GAC). We also thank Adnan Sumer and Ozcan Gaygusuz for assistance in field sampling.
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5.
Conclusion
ELISA can directly quantify microcystins in single filaments of cyanobacteria. This ability will contribute to the risk assessment of microcystin-containing cyanobacterial blooms. Single filament immunoassay for microcystins has indicated wide differences in toxin quota per unit biovolume and filament length between filaments of single laboratory clones and in environmental samples. The differences may be attributed to variations in microcystin synthetase gene expression (Kurmayer et al., 2004) and to possible changes in the biosynthesis and multiple fates of the microcystins.
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