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Microcystins and anatoxin-a in Arctic biocrust cyanobacterial communities
Toxicon 101 (2015) 35e40
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Microcystins and anatoxin-a in Arctic biocrust cyanobacterial communities Ewelina Chrapusta a, b, *, Michał We˛grzyn b, Kornelia Zabaglo a, Ariel Kaminski a, Michal Adamski a, Paulina Wietrzyk b, Jan Bialczyk a a Department of Plant Physiology and Development, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland b Department of Polar Research and Documentation, Institute of Botany, Faculty of Biology and Earth Sciences, Jagiellonian University, Kopernika 27, 31-501 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 1 April 2015 Accepted 29 April 2015 Available online 30 April 2015
In the polar regions cyanobacteria are an important element of plant communities and represent the dominant group of primary producers. They commonly form thick highly diverse biological soil crusts that provide microhabitats for other organisms. Cyanobacteria are also producers of toxic secondary metabolites. In the present study we demonstrated that biocrust-forming cyanobacteria inhabiting the Kaffiøyra Plain, the north-west coast of Spitsbergen, are able to synthesize toxins, especially microcystins (MCs, from 0.123 to 11.058 mg MC-LR per g dry weight, DW) and anatoxin-a (ANTX-a, from 0.322 to 0.633 mg ANTX-a per g DW). To the best of our knowledge, this is the first report on the presence of ANTX-a in the entire polar region. The occurrence of cyanotoxins can exert a long-term impact on organisms co-existing in biocrust communities and can have far-reaching consequences for the entire polar ecosystem. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Svalbard Kaffiøyra Polar terrestrial ecosystem Biological soil crusts Cyanotoxins
1. Introduction Cyanobacteria as cosmopolitan organisms are present in all latitudes from the polar regions to the tropical deserts (Vincent, 2000; Wynn-Williams, 2000). Their global distribution is a result of specific adaptive strategies that have developed during evolution, such as the ability to undergo oxygenic photosynthesis, to grow in a wide temperature range, to produce UV-absorbing compounds, to form colonies and spores, to fix nitrogen and to adapt to freezeethaw cycles (Vincent, 2000; Zakhia et al., 2008). In polar ecosystems cyanobacteria are the most important element of microbial and plant communities. They represent the dominant group of primary producers inhabiting extreme aquatic and terrestrial environments (Vincent, 2000; Quesada et al., 2008;
* Corresponding author. Department of Plant Physiology and Development, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. E-mail addresses: [email protected] (E. Chrapusta), michal.wegrzyn@ uj.edu.pl (M. We˛grzyn), [email protected] (K. Zabaglo), ariel.kaminski@uj. edu.pl (A. Kaminski), [email protected] (M. Adamski), paulina.wietrzyk@ doctoral.uj.edu.pl (P. Wietrzyk), [email protected] (J. Bialczyk). http://dx.doi.org/10.1016/j.toxicon.2015.04.016 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
Zakhia et al., 2008; Vincent and Quesada, 2012). Cyanobacteria first colonize the nutrient-poor habitats, thus they play a large role in the primary succession on glacial moraines (Vincent, 2000; Zakhia et al., 2008). Typically, they form cohesive, highly diverse biocrust microbial communities, with a thickness of a few centimeters, on moist soils and in freshwater reservoirs (Vincent, 2000; de los Rios et al., 2004; Quesada et al., 2008; Zakhia et al., 2008; Vincent and Quesada, 2012). These structured communities provide shelter and microhabitat for many different organisms, such as: rotifers, flagellates, nematodes, tardigrades, green algae, fungi, diatoms, archaea, heterotrophic bacteria and viruses (Quesada et al., 2008; Cary et al., 2010). Cyanobacteria in temperate and tropical areas are well known for the synthesis of toxic secondary metabolites that can be harmful for animals and humans (Chorus and Bartram, 1999; Hitzfeld et al., 2000; Carmichael, 2001; Metcalf et al., 2012; Richer et al., 2015; Metcalf et al., 2015). The production of toxins in high latitudes is largely unexplored (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2012, 2013, 2014). Several species of Antarctic cyanobacteria can produce microcystins, nodularin and cylindrospermopsin (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2012, 2014), while Arctic
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material may contain saxitoxins and microcystins (Kleinteich et al., 2012, 2013). The physiological and ecological role of cyanotoxins in cold ecosystems is poorly understood (Babica et al., 2006). Their biosynthesis is energetically expensive, thus it is suggested that they may constitute a cyanobacterial defense mechanism against grazing by herbivores and/or stimulate the formation of changes within and among populations by allelopathic interactions, which help cyanobacteria to gain an ecological advantage in these environments (Carmichael, 1997; Hitzfeld et al., 2000; Lürling, 2003; Babica et al., 2006; Schatz et al., 2007; Wood et al., 2011; Kaplan et al., 2012). Toxins are also considered as an intracellular storage of nitrogen, carbon and other elements (Carmichael, 1997). Another hypothesis says that microcystins are quorum-sensing or -signaling molecules that indicate changing growth conditions (Schatz et al., 2007; Wood et al., 2011; Kleinteich et al., 2013). Microcystins (MCs) are the most common and ubiquitous toxins detected in the cyanobacterial cells around the world. They belong to the group of cyclic heptapeptides recognized as hepatotoxins. To date, more than 60 variants of MCs have been isolated and characterized (Carmichael, 1997; Metcalf and Codd, 2012). MCs are potent inhibitors of protein phosphatases PP1 and PP2A in eukaryotic organisms (mammals and higher plants) (Carmichael, 1997; Chorus and Bartram, 1999). They are biosynthesized by many genera of cyanobacteria including Microcystis, Anabaena, Nostoc, Oscillatoria, Planktothrix, Chroococcus, Anabaenopsis, Phormidium, Arthrospira, Haphalosiphon and others (Metcalf and Codd, 2012). Anatoxin-a (ANTX-a) is a neurotoxic alkaloid acting as a powerful agonist at neuronal nicotinic acetylcholine receptors (Carmichael, 1997; Chorus and Bartram, 1999). ANTX-a is found mainly in the genera Anabaena, Aphanizomenon, Oscillatoria, Planktothrix, Cylindrospermum, Microcystis, Arthrospira and Phormidium (Metcalf and Codd, 2012). The basic aim of this study was the qualitative and quantitative identification of cyanotoxins in cyanobacterial biocrusts collected from the Kaffiøyra Plain, on the north-west coast of Spitsbergen. As previously postulated (Kleinteich et al., 2012, 2013), we confirmed the presence of MCs in the cells of Arctic cyanobacteria and identified an earlier undetected toxin e ANTX-a. 2. Materials and methods 2.1. Sampling and material conservation Samples of cyanobacterial biocrusts were collected from eight sampling sites during an expedition to the Kaffiøyra Plain, NW Spitsbergen (11500 000 E12 400 000 E, 78 390 000 N-78 410000 N) (Fig. 1) in July 2012. Samples were probed using sterile spatulas, air-dried, sealed in paper bags and kept in the dark at 4 C for taxonomic identification or stored frozen in 20 C until toxin analysis. 2.2. Taxonomic analysis The samples of cyanobacterial biocrusts were prepared using a Nikon SMZ1500 binocular microscope, and then identified under a Nikon H600L ECLIPSE light microscope, by standard algological techniques. For species identifications, references were made to rek, 1998; Koma rek and Anagnostidis, 2007; published keys (Koma rek et al., 2013). Koma
on a laboratory scale and then quantitatively transferred to handoperated ground-glass homogenizers. MCs and ANTX-a were extracted and purified using the methods described by Meriluoto and Codd (2005). Briefly, homogenized samples were flooded with pure methanol and shaken for 30 min at 150 rpm. The supernatants were transferred into separate test-tubes and evaporated to dryness under a nitrogen stream. The dry residues were dissolved in Milli-Q water and then were subjected to further purification and concentration by solid phase extraction (SPE). Asprepared samples were filtered through syringe filters Durapore (PVDF, Millex) and analyzed with high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). 2.3.2. Identification of cyanotoxins Toxins analysis was carried out by HPLC (Waters, Milford, MA, USA) consisting of a 600E gradient pump, 717 plus autosampler, 996 photodiode array (PDA) detector, Jetstream 2 plus column thermostat and Millenium32 SS software. The gradient mobile phase composed of water and acetonitrile (ACN), both acidified with 0.05% trifluoroacetic acid (TFA). The flow rate was 1 mL min1. Chromatograms were monitored at 200e300 nm. MCs content was measured following the procedure described by Bialczyk et al. (2014). Briefly, the samples were separated on a Purospher STAR RP-18 endcapped column (55 mm 4 mm, 3 mm) maintained at 40 C. The gradient mobile phase rose from 25% to 70% of ACN in 9 min. MCs content (mg g1 dry weight, DW) was calculated using the standard curve for pure MC-LR and expressed as its equivalent. MC-LR was identified by absorbance at 239.4 nm. ANTX-a content was determined using the method of Kaminski et al. (2013). Briefly, the mobile phase was changed from 100% to 80% of water in 18 min. The extracts were loaded into an Atlantis C18 reverse-phase cartridge column (4.6 mm 250 mm; 5 mm) protected by a guard column (C18 cartridge), thermostated at 23 C. ANTX-a was identified by comparing the UV-spectra determined for its commercial standard and quantified by absorbance at 228.8 nm. Its content was expresses as mg g1 DW. Next, the samples that had been previously tested positively on HPLC for the presence of MCs and ANTX-a were analyzed with a MicrOTOF-Q mass spectrometer (Bruker-Daltonics, Bremen, Germany) using an electrospray ion source (ESI-MS) to confirm these results. The extracts were diluted (1:1, v/v) with an aqueous mixture of 50% methanol and 1% formic acid. The positiveion mode was applied and the scan range was m/z 30e1000 in the MS and MS/MS modes. 2.4. Chemicals All reagents were analytical or HPLC grade and were purchased from SigmaeAldrich Company (St. Louis, MO, USA). The ultrapure grade water (Milli-Q water) was from Millipore Corporate (Bedford, MA, USA). The standards, (±)ANTX-a fumarate and MC-LR, were €ln, Germany) and SigmaeAldrich obtained from BioTrend (Ko Company (St. Louis, MO, USA), respectively. 2.5. Statistical analysis All data was expressed as means ± standard deviation (SD) of five independent replicates.
2.3. Toxin analysis in cyanobacterial biocrusts
3. Results and discussion
2.3.1. Isolation of cyanotoxins Cyanobacterial biocrusts were very carefully separated from the substrate using sterile tweezers and scalpels. 0.1 g of experimental material derived from each of the eight sampling sites was weighed
Toxins production by cyanobacteria is common, yet most studies have focused on species inhabiting the temperate and tropical zones (Carmichael, 1997). In contrast, there is still relatively little information showing the ability of polar cyanobacteria to
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Fig. 1. Kaffiøyra Plain, NW Spitsbergen. The numbers in spots represent the sampling sites of cyanobacterial biocrusts. Map of sampling points was developed using ArcGIS 10 program. © Norwegian Polar Institute (2014a, b and c).
synthesize these bioactive compounds (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2012, 2013, 2014). HPLC analysis revealed and ESIeMS spectra confirmed the presence of two cyanotoxins, hepatotoxic MC-LR and neurotoxic ANTX-a, in biocrust communities growing on the Kaffiøyra Plain (Fig. 2).
Only one MC isoform, MC-LR, was detected. MC-LR was identified by comparing the absorption and MS/MS spectra (Fig. 3) with its standard. As seen in Table 1 the occurrence of MCs was demonstrated in all eight sampling sites. The MC-LR equivalent content ranged from 0.123 ± 0.004 mg g1 DW (sample no. 2) to 11.058 ± 0.572 mg g1 DW (sample no. 7). This study confirmed
Fig. 2. Ion spectra from the ESIeMS analysis of biocrust cyanobacterial community collected from sampling site no. 7 on the Kaffiøyra Plain, NW Spitsbergen. Product ion spectra from m/z 166.13 [M þ Hþ] is ANTX-a, and from m/z 995.55 [M þ Hþ] is MC-LR.
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Fig. 3. The presence of MC-LR in biocrust cyanobacterial community collected from sampling site no. 7 on the Kaffiøyra Plain, NW Spitsbergen. The absorption maximum of MC-LR (239.4 nm) (A), MS/MS spectrum of MC-LR (m/z 995.55 [M þ Hþ]) with its chemical structure (B).
Table 1 MCs and ANTX-a content in Arctic biocrust cyanobacterial communities collected from eight sampling sites on the Kaffiøyra Plain, NW Spitsbergen. MCs total content was expressed as an equivalent of MC-LR. Values are means ± SD of five replicates. Sampling sites
MC-LR equivalent content [mg/g DW] ANTX-a content [mg/g DW]
1
2
3
4
5
6
7
8
1.121 ± 0.082
0.123 ± 0.004
0.948 ± 0.087
0.389 ± 0.051
2.145 ± 0.181
1.051 ± 0.081
11.058 ± 0.572
0.941 ± 0.082
e
e
e
e
0.323 ± 0.018
e
0.633 ± 0.032
e
previous scientific reports on the presence of MCs in the polar habitats, especially in Arctic (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2012, 2013, 2014). The values obtained here were higher than those of Kleinteich et al. (2013) for samples from the northern Baffin Island, Northern Canada (0.106 mg g1 DW), but were similar to levels observed in the cells of Antarctic cyanobacteria from McMurdo Ice Shelf, Bratina Island and Dry Valleys (1e16 mg g1 DW) (Jungblut et al., 2006; Wood et al., 2008). ANTX-a was identified by comparison with absorption and MS/ MS spectra of standard (Fig. 4). This toxin was detected only in two of the eight sampling sites, no. 5 (0.322 ± 0.018 mg g1 DW) and no. 7 (0.633 ± 0.032 mg g1 DW) (Table 1). To the best of our knowledge, this is the first report on the presence of ANTX-a in the entire polar region. The contents of MCs and ANTX-a in cyanobacterial biocrusts of Spitsbergen were significantly lower compared to those monitored in temperate and tropical areas (up to 25 103 mg MCs g1
DW and up to 4.4 103 mg ANTX-a g1 DW) (Chorus and Bartram, 1999). Jungblut et al. (2006) hypothesized that it may be a result of low cyanobacterial abundance or low rates of their biosynthesis. Additionally, presented data confirmed that MCs in Arctic, as in other climatic zones, are the most commonly-found cyanobacterial toxins (Carmichael, 1997; Chorus and Bartram, 1999; Metcalf and Codd, 2012). Initially, it was assumed that MCs and ANTX-a content in tested biocrust communities would change with increasing distance of the sampling site from the glacier front and the weakening of environmental stress conditions. However, our results did not confirm the existence of such dependence. The occurrence and content of both toxins was related only with the species composition of particular communities. The biocrusts from the Kaffiøyra Plain showed a high biodiversity and were mostly dominated by filamentous and coccoid cyanobacteria belonging to Nostocales, Oscillatoriales and Chroococcales. Based on microscope analyses in
Fig. 4. The presence of ANTX-a in biocrust cyanobacterial community collected from sampling site no. 7 on the Kaffiøyra Plain, NW Spitsbergen. The absorption maximum of ANTXa (228.8 nm) (A), MS/MS spectrum of ANTX-a (m/z ¼ 166.13 [M þ Hþ]) with its chemical structure (B).
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each sample we identified from three to five dominant cyanobacterial species that may be potential MCs producers, such as Nostoc sp., Nostoc paludosum and Chroococcus sp. Several species of Nostoc from cold and warm environments can synthesize MCs, including N. paludosum (Sivonen et al., 1990, 1992; Chorus and Bartram, 1999; Figueiredo et al., 2004; Zurawell et al., 2005; Wood et al., 2006; Oudra et al., 2009). Nostoc muscorum, Nostoc linckia, Nostoc rivulare, Nostoc spongiaeforme and Nostoc zetterstedtii also produce MCs, however none of these have been found in biocrusts (Chorus and Bartram, 1999; Figueiredo et al., 2004; Zurawell et al., 2005; Oudra et al., 2009). Nostoc sp. is a major producer of MCs, particularly MC-LR, in Antarctic and Arctic ecosystems (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2013). Similarly, it is presumed that Nostoc species are the main MCs producers in biocrust communities from the Kaffiøyra Plain. Additionally, in two samples, no. 5 and 7 (Figs. 2 and 4) where the content of MCs was the largest and ANTX-a was also observed, we found other cyanobacterial species: Oscillatoria sp., and Phormidium sp. Oscillatoria and Phormidium are mainly postulated as MCs-producing genera, thus their occurrence in these two biocrust communities has probably contributed to increasing MCs content (Chorus and Bartram, 1999; Hitzfeld et al., 2000; Figueiredo et al., 2004; Zurawell et al., 2005; Jungblut et al., 2006). Oscillatoria tenuis, O. limosa, O. perornata and Phormidium formosum have been classified as MCs producers, but taxa identified in the Kaffiøyra samples were not similar to these species (Chorus and Bartram, 1999; Figueiredo et al., 2004; Zurawell et al., 2005). Both Oscillatoria and Phormidium are also likely candidates for the synthesis of ANTX-a, which was detected in samples no. 5 and 7. So far, a few strains of Oscillatoria, Phormidium as well as Phormidium favosum and P. autumnale were classified as associated with ANTX-a production (Edwards et al., 1992; Ar aoz et al., 2005; Gugger et al., 2005; Wood et al., 2007, 2012). However, none of them have been described in these cyanobacterial communities. The accurate identification of MCs and ANTX-a producers is not possible based on the available data, thus their synthesis could not yet be attributed to specific cyanobacterial species. 4. Conclusions The significance of cyanotoxins production in polar regions has not been elucidated. Their presence can exert a long-term impact on the biocrust-forming organisms and can have far-reaching consequences for the entire polar ecosystems. Thus, there is an urgent need to extend research on the accurate identification of toxic cyanobacteria strains that will contribute to better explanation the role of toxins in those environments. Conflict of interest statement The authors declare that there are no conflicts of interest. Ethical statement No ethical concerns. Acknowledgments We are grateful to Professor Zbigniew Lechowski for providing manifold help and advice in preparing and performing experiments and Associate Professor Ireneusz Sobota for the opportunity to stay at the research station on the Kaffiøyra Plain. Faculty of Biochemistry, Biophysics and Biotechnology is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education.
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Vincent, W.F., 2000. Cyanobacterial dominance in the polar regions. In: Whitton, B.A., Potts, M. (Eds.), The Ecology of Cyanobacteria, Their Diversity in Time and Space. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 321e340. Vincent, W.F., Quesada, A., 2012. Cyanobacteria in high latitude lakes, rivers and seas. In: Whitton, B.A. (Ed.), Ecology of Cyanobacteria II: Their Diversity in Space and Time. Springer, Dordrecht, The Netherlands, pp. 371e385. Wood, S.A., Mountfort, D., Selwood, A.I., Holland, P.T., Puddick, J., Cary, S.C., 2008. Widespread distribution and identification of eight novel microcystins in Antarctic cyanobacterial mats. Appl. Environ. Microbiol. 74, 7243e7251. http:// dx.doi.org/10.1128/AEM.01243-08. Wood, S.A., Rueckert, A., Hamilton, D.P., Cary, S.C., Dietrich, D.R., 2011. Switching toxin production on and off: intermittent microcystin synthesis in a Microcystis bloom. Environ. Microbiol. Rep. 3, 118e124. http://dx.doi.org/10.1111/j.17582229.2010.00196.x. Wood, S.A., Selwood, A.I., Rueckert, A., Holland, P.T., Milne, J.R., Smith, K.F., Smits, B., Watts, L.F., Cary, C.S., 2007. First report of homoanatoxin-a and associated dog neurotoxicosis in New Zealand. Toxicon 50, 292e301. http://dx.doi.org/10.1016/ j.toxicon.2007.03.025. Wood, S.A., Smith, F.M.J., Heath, M.W., Palfroy, T., Gaw, S., Young, R.G., Ryan, K.G., 2012. Within-mat variability in anatoxin-a and homoanatoxin-a production among benthic Phormidium (Cyanobacteria) strains. Toxins 4, 900e912. http:// dx.doi.org/10.3390/toxins4100900. Wood, S.A., Stirling, D.J., Briggs, L.R., Sprosen, J., Holland, P.T., Ruck, J.G., Wear, R.G., 2006. Survey of cyanotoxins in New Zealand water bodies between 2001 and 2004. N. Z. J. Mar. Freshw. Res. 40, 585e595. http://dx.doi.org/10.1080/ 00288330.2006.9517447. Wynn-Williams, D.D., 2000. Cyanobacteria in deserts e life at the limit? In: Whitton, B.A., Potts, M. (Eds.), The Ecology of Cyanobacteria, Their Diversity in Time and Space. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 341e366. Zakhia, F., Jungblut, A.D., Taton, A., Vincent, W.F., Wilmotte, A., 2008. Cyanobacteria in cold ecosystems. In: Margesin, R., Schinner, F., Marx, J.C., Gerday, C. (Eds.), Psychrophiles: from Biodiversity to Biotechnology. Springer-Verlag, Berlin Heidelberg, Germany, pp. 121e135. Zurawell, R.W., Chen, H., Burke, J.M., Prepas, E.E., 2005. Hepatotoxic cyanobacteria: a review of the biological importance of microcystins in freshwater environments. J. Toxicol. Environ. Health B. Crit. Rev. 8, 1e37. http://dx.doi.org/10.1080/ 10937400590889412.
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Microcystins and anatoxin-a in Arctic biocrust cyanobacterial communities Ewelina Chrapusta a, b, *, Michał We˛grzyn b, Kornelia Zabaglo a, Ariel Kaminski a, Michal Adamski a, Paulina Wietrzyk b, Jan Bialczyk a a Department of Plant Physiology and Development, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland b Department of Polar Research and Documentation, Institute of Botany, Faculty of Biology and Earth Sciences, Jagiellonian University, Kopernika 27, 31-501 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 1 April 2015 Accepted 29 April 2015 Available online 30 April 2015
In the polar regions cyanobacteria are an important element of plant communities and represent the dominant group of primary producers. They commonly form thick highly diverse biological soil crusts that provide microhabitats for other organisms. Cyanobacteria are also producers of toxic secondary metabolites. In the present study we demonstrated that biocrust-forming cyanobacteria inhabiting the Kaffiøyra Plain, the north-west coast of Spitsbergen, are able to synthesize toxins, especially microcystins (MCs, from 0.123 to 11.058 mg MC-LR per g dry weight, DW) and anatoxin-a (ANTX-a, from 0.322 to 0.633 mg ANTX-a per g DW). To the best of our knowledge, this is the first report on the presence of ANTX-a in the entire polar region. The occurrence of cyanotoxins can exert a long-term impact on organisms co-existing in biocrust communities and can have far-reaching consequences for the entire polar ecosystem. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Svalbard Kaffiøyra Polar terrestrial ecosystem Biological soil crusts Cyanotoxins
1. Introduction Cyanobacteria as cosmopolitan organisms are present in all latitudes from the polar regions to the tropical deserts (Vincent, 2000; Wynn-Williams, 2000). Their global distribution is a result of specific adaptive strategies that have developed during evolution, such as the ability to undergo oxygenic photosynthesis, to grow in a wide temperature range, to produce UV-absorbing compounds, to form colonies and spores, to fix nitrogen and to adapt to freezeethaw cycles (Vincent, 2000; Zakhia et al., 2008). In polar ecosystems cyanobacteria are the most important element of microbial and plant communities. They represent the dominant group of primary producers inhabiting extreme aquatic and terrestrial environments (Vincent, 2000; Quesada et al., 2008;
* Corresponding author. Department of Plant Physiology and Development, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. E-mail addresses: [email protected] (E. Chrapusta), michal.wegrzyn@ uj.edu.pl (M. We˛grzyn), [email protected] (K. Zabaglo), ariel.kaminski@uj. edu.pl (A. Kaminski), [email protected] (M. Adamski), paulina.wietrzyk@ doctoral.uj.edu.pl (P. Wietrzyk), [email protected] (J. Bialczyk). http://dx.doi.org/10.1016/j.toxicon.2015.04.016 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
Zakhia et al., 2008; Vincent and Quesada, 2012). Cyanobacteria first colonize the nutrient-poor habitats, thus they play a large role in the primary succession on glacial moraines (Vincent, 2000; Zakhia et al., 2008). Typically, they form cohesive, highly diverse biocrust microbial communities, with a thickness of a few centimeters, on moist soils and in freshwater reservoirs (Vincent, 2000; de los Rios et al., 2004; Quesada et al., 2008; Zakhia et al., 2008; Vincent and Quesada, 2012). These structured communities provide shelter and microhabitat for many different organisms, such as: rotifers, flagellates, nematodes, tardigrades, green algae, fungi, diatoms, archaea, heterotrophic bacteria and viruses (Quesada et al., 2008; Cary et al., 2010). Cyanobacteria in temperate and tropical areas are well known for the synthesis of toxic secondary metabolites that can be harmful for animals and humans (Chorus and Bartram, 1999; Hitzfeld et al., 2000; Carmichael, 2001; Metcalf et al., 2012; Richer et al., 2015; Metcalf et al., 2015). The production of toxins in high latitudes is largely unexplored (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2012, 2013, 2014). Several species of Antarctic cyanobacteria can produce microcystins, nodularin and cylindrospermopsin (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2012, 2014), while Arctic
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material may contain saxitoxins and microcystins (Kleinteich et al., 2012, 2013). The physiological and ecological role of cyanotoxins in cold ecosystems is poorly understood (Babica et al., 2006). Their biosynthesis is energetically expensive, thus it is suggested that they may constitute a cyanobacterial defense mechanism against grazing by herbivores and/or stimulate the formation of changes within and among populations by allelopathic interactions, which help cyanobacteria to gain an ecological advantage in these environments (Carmichael, 1997; Hitzfeld et al., 2000; Lürling, 2003; Babica et al., 2006; Schatz et al., 2007; Wood et al., 2011; Kaplan et al., 2012). Toxins are also considered as an intracellular storage of nitrogen, carbon and other elements (Carmichael, 1997). Another hypothesis says that microcystins are quorum-sensing or -signaling molecules that indicate changing growth conditions (Schatz et al., 2007; Wood et al., 2011; Kleinteich et al., 2013). Microcystins (MCs) are the most common and ubiquitous toxins detected in the cyanobacterial cells around the world. They belong to the group of cyclic heptapeptides recognized as hepatotoxins. To date, more than 60 variants of MCs have been isolated and characterized (Carmichael, 1997; Metcalf and Codd, 2012). MCs are potent inhibitors of protein phosphatases PP1 and PP2A in eukaryotic organisms (mammals and higher plants) (Carmichael, 1997; Chorus and Bartram, 1999). They are biosynthesized by many genera of cyanobacteria including Microcystis, Anabaena, Nostoc, Oscillatoria, Planktothrix, Chroococcus, Anabaenopsis, Phormidium, Arthrospira, Haphalosiphon and others (Metcalf and Codd, 2012). Anatoxin-a (ANTX-a) is a neurotoxic alkaloid acting as a powerful agonist at neuronal nicotinic acetylcholine receptors (Carmichael, 1997; Chorus and Bartram, 1999). ANTX-a is found mainly in the genera Anabaena, Aphanizomenon, Oscillatoria, Planktothrix, Cylindrospermum, Microcystis, Arthrospira and Phormidium (Metcalf and Codd, 2012). The basic aim of this study was the qualitative and quantitative identification of cyanotoxins in cyanobacterial biocrusts collected from the Kaffiøyra Plain, on the north-west coast of Spitsbergen. As previously postulated (Kleinteich et al., 2012, 2013), we confirmed the presence of MCs in the cells of Arctic cyanobacteria and identified an earlier undetected toxin e ANTX-a. 2. Materials and methods 2.1. Sampling and material conservation Samples of cyanobacterial biocrusts were collected from eight sampling sites during an expedition to the Kaffiøyra Plain, NW Spitsbergen (11500 000 E12 400 000 E, 78 390 000 N-78 410000 N) (Fig. 1) in July 2012. Samples were probed using sterile spatulas, air-dried, sealed in paper bags and kept in the dark at 4 C for taxonomic identification or stored frozen in 20 C until toxin analysis. 2.2. Taxonomic analysis The samples of cyanobacterial biocrusts were prepared using a Nikon SMZ1500 binocular microscope, and then identified under a Nikon H600L ECLIPSE light microscope, by standard algological techniques. For species identifications, references were made to rek, 1998; Koma rek and Anagnostidis, 2007; published keys (Koma rek et al., 2013). Koma
on a laboratory scale and then quantitatively transferred to handoperated ground-glass homogenizers. MCs and ANTX-a were extracted and purified using the methods described by Meriluoto and Codd (2005). Briefly, homogenized samples were flooded with pure methanol and shaken for 30 min at 150 rpm. The supernatants were transferred into separate test-tubes and evaporated to dryness under a nitrogen stream. The dry residues were dissolved in Milli-Q water and then were subjected to further purification and concentration by solid phase extraction (SPE). Asprepared samples were filtered through syringe filters Durapore (PVDF, Millex) and analyzed with high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). 2.3.2. Identification of cyanotoxins Toxins analysis was carried out by HPLC (Waters, Milford, MA, USA) consisting of a 600E gradient pump, 717 plus autosampler, 996 photodiode array (PDA) detector, Jetstream 2 plus column thermostat and Millenium32 SS software. The gradient mobile phase composed of water and acetonitrile (ACN), both acidified with 0.05% trifluoroacetic acid (TFA). The flow rate was 1 mL min1. Chromatograms were monitored at 200e300 nm. MCs content was measured following the procedure described by Bialczyk et al. (2014). Briefly, the samples were separated on a Purospher STAR RP-18 endcapped column (55 mm 4 mm, 3 mm) maintained at 40 C. The gradient mobile phase rose from 25% to 70% of ACN in 9 min. MCs content (mg g1 dry weight, DW) was calculated using the standard curve for pure MC-LR and expressed as its equivalent. MC-LR was identified by absorbance at 239.4 nm. ANTX-a content was determined using the method of Kaminski et al. (2013). Briefly, the mobile phase was changed from 100% to 80% of water in 18 min. The extracts were loaded into an Atlantis C18 reverse-phase cartridge column (4.6 mm 250 mm; 5 mm) protected by a guard column (C18 cartridge), thermostated at 23 C. ANTX-a was identified by comparing the UV-spectra determined for its commercial standard and quantified by absorbance at 228.8 nm. Its content was expresses as mg g1 DW. Next, the samples that had been previously tested positively on HPLC for the presence of MCs and ANTX-a were analyzed with a MicrOTOF-Q mass spectrometer (Bruker-Daltonics, Bremen, Germany) using an electrospray ion source (ESI-MS) to confirm these results. The extracts were diluted (1:1, v/v) with an aqueous mixture of 50% methanol and 1% formic acid. The positiveion mode was applied and the scan range was m/z 30e1000 in the MS and MS/MS modes. 2.4. Chemicals All reagents were analytical or HPLC grade and were purchased from SigmaeAldrich Company (St. Louis, MO, USA). The ultrapure grade water (Milli-Q water) was from Millipore Corporate (Bedford, MA, USA). The standards, (±)ANTX-a fumarate and MC-LR, were €ln, Germany) and SigmaeAldrich obtained from BioTrend (Ko Company (St. Louis, MO, USA), respectively. 2.5. Statistical analysis All data was expressed as means ± standard deviation (SD) of five independent replicates.
2.3. Toxin analysis in cyanobacterial biocrusts
3. Results and discussion
2.3.1. Isolation of cyanotoxins Cyanobacterial biocrusts were very carefully separated from the substrate using sterile tweezers and scalpels. 0.1 g of experimental material derived from each of the eight sampling sites was weighed
Toxins production by cyanobacteria is common, yet most studies have focused on species inhabiting the temperate and tropical zones (Carmichael, 1997). In contrast, there is still relatively little information showing the ability of polar cyanobacteria to
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Fig. 1. Kaffiøyra Plain, NW Spitsbergen. The numbers in spots represent the sampling sites of cyanobacterial biocrusts. Map of sampling points was developed using ArcGIS 10 program. © Norwegian Polar Institute (2014a, b and c).
synthesize these bioactive compounds (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2012, 2013, 2014). HPLC analysis revealed and ESIeMS spectra confirmed the presence of two cyanotoxins, hepatotoxic MC-LR and neurotoxic ANTX-a, in biocrust communities growing on the Kaffiøyra Plain (Fig. 2).
Only one MC isoform, MC-LR, was detected. MC-LR was identified by comparing the absorption and MS/MS spectra (Fig. 3) with its standard. As seen in Table 1 the occurrence of MCs was demonstrated in all eight sampling sites. The MC-LR equivalent content ranged from 0.123 ± 0.004 mg g1 DW (sample no. 2) to 11.058 ± 0.572 mg g1 DW (sample no. 7). This study confirmed
Fig. 2. Ion spectra from the ESIeMS analysis of biocrust cyanobacterial community collected from sampling site no. 7 on the Kaffiøyra Plain, NW Spitsbergen. Product ion spectra from m/z 166.13 [M þ Hþ] is ANTX-a, and from m/z 995.55 [M þ Hþ] is MC-LR.
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Fig. 3. The presence of MC-LR in biocrust cyanobacterial community collected from sampling site no. 7 on the Kaffiøyra Plain, NW Spitsbergen. The absorption maximum of MC-LR (239.4 nm) (A), MS/MS spectrum of MC-LR (m/z 995.55 [M þ Hþ]) with its chemical structure (B).
Table 1 MCs and ANTX-a content in Arctic biocrust cyanobacterial communities collected from eight sampling sites on the Kaffiøyra Plain, NW Spitsbergen. MCs total content was expressed as an equivalent of MC-LR. Values are means ± SD of five replicates. Sampling sites
MC-LR equivalent content [mg/g DW] ANTX-a content [mg/g DW]
1
2
3
4
5
6
7
8
1.121 ± 0.082
0.123 ± 0.004
0.948 ± 0.087
0.389 ± 0.051
2.145 ± 0.181
1.051 ± 0.081
11.058 ± 0.572
0.941 ± 0.082
e
e
e
e
0.323 ± 0.018
e
0.633 ± 0.032
e
previous scientific reports on the presence of MCs in the polar habitats, especially in Arctic (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2012, 2013, 2014). The values obtained here were higher than those of Kleinteich et al. (2013) for samples from the northern Baffin Island, Northern Canada (0.106 mg g1 DW), but were similar to levels observed in the cells of Antarctic cyanobacteria from McMurdo Ice Shelf, Bratina Island and Dry Valleys (1e16 mg g1 DW) (Jungblut et al., 2006; Wood et al., 2008). ANTX-a was identified by comparison with absorption and MS/ MS spectra of standard (Fig. 4). This toxin was detected only in two of the eight sampling sites, no. 5 (0.322 ± 0.018 mg g1 DW) and no. 7 (0.633 ± 0.032 mg g1 DW) (Table 1). To the best of our knowledge, this is the first report on the presence of ANTX-a in the entire polar region. The contents of MCs and ANTX-a in cyanobacterial biocrusts of Spitsbergen were significantly lower compared to those monitored in temperate and tropical areas (up to 25 103 mg MCs g1
DW and up to 4.4 103 mg ANTX-a g1 DW) (Chorus and Bartram, 1999). Jungblut et al. (2006) hypothesized that it may be a result of low cyanobacterial abundance or low rates of their biosynthesis. Additionally, presented data confirmed that MCs in Arctic, as in other climatic zones, are the most commonly-found cyanobacterial toxins (Carmichael, 1997; Chorus and Bartram, 1999; Metcalf and Codd, 2012). Initially, it was assumed that MCs and ANTX-a content in tested biocrust communities would change with increasing distance of the sampling site from the glacier front and the weakening of environmental stress conditions. However, our results did not confirm the existence of such dependence. The occurrence and content of both toxins was related only with the species composition of particular communities. The biocrusts from the Kaffiøyra Plain showed a high biodiversity and were mostly dominated by filamentous and coccoid cyanobacteria belonging to Nostocales, Oscillatoriales and Chroococcales. Based on microscope analyses in
Fig. 4. The presence of ANTX-a in biocrust cyanobacterial community collected from sampling site no. 7 on the Kaffiøyra Plain, NW Spitsbergen. The absorption maximum of ANTXa (228.8 nm) (A), MS/MS spectrum of ANTX-a (m/z ¼ 166.13 [M þ Hþ]) with its chemical structure (B).
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each sample we identified from three to five dominant cyanobacterial species that may be potential MCs producers, such as Nostoc sp., Nostoc paludosum and Chroococcus sp. Several species of Nostoc from cold and warm environments can synthesize MCs, including N. paludosum (Sivonen et al., 1990, 1992; Chorus and Bartram, 1999; Figueiredo et al., 2004; Zurawell et al., 2005; Wood et al., 2006; Oudra et al., 2009). Nostoc muscorum, Nostoc linckia, Nostoc rivulare, Nostoc spongiaeforme and Nostoc zetterstedtii also produce MCs, however none of these have been found in biocrusts (Chorus and Bartram, 1999; Figueiredo et al., 2004; Zurawell et al., 2005; Oudra et al., 2009). Nostoc sp. is a major producer of MCs, particularly MC-LR, in Antarctic and Arctic ecosystems (Hitzfeld et al., 2000; Jungblut et al., 2006; Wood et al., 2008; Kleinteich et al., 2013). Similarly, it is presumed that Nostoc species are the main MCs producers in biocrust communities from the Kaffiøyra Plain. Additionally, in two samples, no. 5 and 7 (Figs. 2 and 4) where the content of MCs was the largest and ANTX-a was also observed, we found other cyanobacterial species: Oscillatoria sp., and Phormidium sp. Oscillatoria and Phormidium are mainly postulated as MCs-producing genera, thus their occurrence in these two biocrust communities has probably contributed to increasing MCs content (Chorus and Bartram, 1999; Hitzfeld et al., 2000; Figueiredo et al., 2004; Zurawell et al., 2005; Jungblut et al., 2006). Oscillatoria tenuis, O. limosa, O. perornata and Phormidium formosum have been classified as MCs producers, but taxa identified in the Kaffiøyra samples were not similar to these species (Chorus and Bartram, 1999; Figueiredo et al., 2004; Zurawell et al., 2005). Both Oscillatoria and Phormidium are also likely candidates for the synthesis of ANTX-a, which was detected in samples no. 5 and 7. So far, a few strains of Oscillatoria, Phormidium as well as Phormidium favosum and P. autumnale were classified as associated with ANTX-a production (Edwards et al., 1992; Ar aoz et al., 2005; Gugger et al., 2005; Wood et al., 2007, 2012). However, none of them have been described in these cyanobacterial communities. The accurate identification of MCs and ANTX-a producers is not possible based on the available data, thus their synthesis could not yet be attributed to specific cyanobacterial species. 4. Conclusions The significance of cyanotoxins production in polar regions has not been elucidated. Their presence can exert a long-term impact on the biocrust-forming organisms and can have far-reaching consequences for the entire polar ecosystems. Thus, there is an urgent need to extend research on the accurate identification of toxic cyanobacteria strains that will contribute to better explanation the role of toxins in those environments. Conflict of interest statement The authors declare that there are no conflicts of interest. Ethical statement No ethical concerns. Acknowledgments We are grateful to Professor Zbigniew Lechowski for providing manifold help and advice in preparing and performing experiments and Associate Professor Ireneusz Sobota for the opportunity to stay at the research station on the Kaffiøyra Plain. Faculty of Biochemistry, Biophysics and Biotechnology is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education.
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