Many cyanobacteria are capable of producing compounds that are toxic to other organisms. The cyanotoxins have been classified into four main groups according to their mode of action. In addition to the hepatoxins, cytotoxins, and gastrointestinal toxins, there are several types of neurotoxins (Codd et al., 2005). A potent neurotoxin synthesized by freshwater cyanobacteria is anatoxin-a (ANTX), likely one of the smallest toxic alkaloids described to date (LD50 of 0.2 mg/kg IP in mice, Carmichael et al., 1979). Prior to its chemical identification, ANTX was labeled the Very Fast Death Factor due to its ability to induce death within 4 min following intraperitonial injection in mice. ANTX has now been detected in lakes and rivers throughout the world (e.g., Codd et al., 2005; Cadel-Six et al., 2007; Wood et al., 2007; Aráoz et al., 2008), and, in some cases, at concentrations leading to lethal poisonings of livestock, domestic animals, and wildlife.

The factors regulating ANTX production, whether in the field or in the laboratory, are not well understood (Osswald et al., 2007). The toxin has been studied less that the more commonly encountered microcystins associated with freshwater cyanobacterial blooms (a Web of Science search since 1995 yields about a quarter of the number of papers on ANTX relative to the number of papers on microcystins). ANTX production is strain specific with cellular contents ranging up to 13,000 μg/g dry weight in Anabaena (reviewed by Osswald et al., 2007). The optimal temperature for production appears to lie between 19.8 and 22.0°C and is maximal during the exponential phase of growth in batch cultures.

Several hypotheses have been proposed to explain why toxins more generally are produced by cyanobacteria. They may be produced as a defense mechanism against grazers or other organisms in order to gain ecological advantage. Or they may be involved in cell signaling processes (Wiegand and Pflugmacher, 2005). Cyanotoxins including ANTX have been labeled as secondary metabolites as they do not appear to be involved in primary metabolism (Carmichael, 1992). In this regard, an increase in demand for resources at the cellular level would in theory lead to a decrease in ANTX production.

The availability of nutrients may play a role in the production of secondary metabolites, including nitrogen containing alkaloids such as ANTX, as suggested by the carbon-nutrient balance hypothesis (Hamilton et al., 2001). This hypothesis links the amount of secondary metabolites produced by plants to the relative abundance of nutrients. Recently, the production and composition of microcystins (MC) was investigated in relation to nitrogen to carbon supply ratios (N:C) in the freshwater cyanobacterium Microcystis aeruginosa (van de Waal et al., 2009). Consistent with the carbon-nutrient balance hypothesis, a high N:C ratio in the external media and within the cells led to higher concentrations of microcystins (and of the nitrogen-rich MC variant MC-RR) in comparison to lower N:C ratios. Despite the fact that less than 1% of the total cellular nitrogen was invested in microcystins, the availability of inorganic nitrogen appeared to influence secondary metabolite concentrations and composition. Nitrogen availability may also play an analogous role in the production of other nitrogen containing secondary metabolites such as ANTX.

In the following study, the relationship between nitrogen availability and ANTX production was investigated in Aphanizomenon issatschenkoi (Ussaczew) Proschkina-Lavrenko using an ANTX producing strain isolated from a hypereutrophic New Zealand lake (CAWBG02, Wood et al., 2007). A. issatschenkoi is a colonial planktonic species, which can produce heterocysts (Ballot et al., 2010), the primary site for atmospheric nitrogen (N₂) fixation in the Nostocales in the absence of available inorganic nitrogen. The process of nitrogen fixation being energy demanding, this study hypothesized that nitrogen fixation might lead to further reductions in the production of secondary metabolites containing nitrogen when the supply of inorganic N was low. Both intracellular and extracellular production was estimated: given its small molecular weight ANTX can be found in significant but variable amounts in solution (Rapala et al., 1993).

The total amount of ANTX produced by A. issatchenkoi CAWBG02 was lower in this study than that reported for this strain when first isolated. For a 23 day culture Selwood et al. (2007) reported a maximum concentration of 1105 μg L⁻¹, whereas a maximum of 146 μg L⁻¹was estimated here. This could stem in part from differences in growing conditions related to differences in the composition of media, temperature, and light as well as aeration since the present cultures were not bubble aerated which Selwood et al. (2007) mentioned could double the toxin production. Furthermore differences in both extraction and analytical procedures for ANTX could also lead to differences: there are at present no common standard methods for most algal toxins or certified reference materials in use. The cellular content reported is within the range of that reported for a recently isolated strain of A. issatschenkoi from northeastern Germany (Ballot et al., 2010). Collectively the species appears less toxic than the benthic cyanobacterium Phormidium favosum which produces 8000 μg g⁻¹ per dry weight (Gugger et al., 2005) or previous reports for Anabaena strains (summarized in Osswald et al., 2007), although the problem of analytical interference from phenylalanine may have inflated these earlier estimates of ANTX which did not clearly separate these isobaric compounds.

In this study, nitrogen limited the biomass yield of A. issatschenkoi in the low N cultures as evidenced by lower dry weights and overall final yields under the 1 and 5% relative to 100% N (Table 3). The growth rate was also reduced in the 1% cultures and, to a lesser extent, in 5% cultures. Nitrogen deficiency was also reflected in cellular constituents: in particular lower chlorophyll a concentrations and overall lower ratios of phycocyanin to chlorophyll a (Figure 2). The phycobiliproteins, being nitrogen-rich accessory pigments, are typically reduced relative to chlorophyll a under nitrogen stress in cyanobacteria (Turpin, 1991).

When soluble inorganic nitrogen becomes limiting in the environment, many cyanobacteria belonging to the Nostocales, such as Aphanizomenon, are capable of fixing atmospheric nitrogen to compensate for their needs for that specific nutrient. Hence, the particulate organic nitrogen content should have been more or less constant throughout the 40 days in all three treatments if N₂ fixation had occurred to compensate for nitrogen limitation. On days 20 and 30, the C:N ratios were significantly higher for cultures grown in 1 and 5% N compared to 100% N treatment. This further confirms that nitrogen was limiting and that N₂ fixation did not compensate for the N-deficiency, particularly in the 1% N treatment. In fact, heterocysts were not evident in the cultures and were either non-existent or too small to differentiate from vegetative cells. This may be consistent for CAWBG02 as it did not produce heterocysts in culture with or without nitrogen supplementation in the original report (Wood et al., 2007). With respect to another non-fixing cyanobacterium, van de Waal et al. (2009) reported molar C:N ratios ranging from 6.25 to 7.14 for M. aeruginosa when neither N nor C was limiting. A. issatschenkoi in the present study had lower C:N ratios overall. Nevertheless, significantly higher ratios of C:N were reached in the cultures grown under low nitrogen concentrations (Table 3).

According to the carbon-nutrient balance hypothesis, nutrients should be allocated to the production of secondary metabolites once growth is assured (Hamilton et al., 2001). Because cyanotoxins are generally considered secondary metabolites (Carmichael, 1992), their production should increase whenever the availability of essential nutrients increases. A higher production of anatoxin-a, an alkaloid that contains a nitrogen atom, was expected under higher nitrogen availability. In this study, the highest nitrogen supply corresponded to the highest growth rate and yield (Table 3) of CAWBG02 but not necessarily to higher total ANTX concentrations (Figure 3). In fact, the highest concentrations of total ANTX were produced by cultures grown in 5% N while the lowest mean concentrations of total ANTX were found in the 100% N treatment although the differences were not highly significant. Highly significant differences were observed in ANTX cellular contents (Figure 4): the 5% N cultures produced the highest cellular content and the 100% N treatment the lowest. The 100% N cultures had the highest growth rate, the highest biomass yield, the highest levels of particulate organic nitrogen and the highest phycocyanin to chlorophyll a ratios (Table 3; Figure 2), yet they produced the lowest ANTX cellular content. Additional concentrations of nitrogen should be tested to confirm these results based on only three contrasting treatments. If ANTX is indeed a type of defense compound then, its production might be a mechanism that confers ecological advantage particularly under low nutrient conditions that are common in aquatic systems during summer stratification when cyanobacteria tend to thrive and zooplankton grazing is typically high (Sommer et al., 1986).

The mean extracellular to total toxin concentration ratios were higher in 1% N treatment than in the other two treatments, the extracellular fraction almost exceeding the particulate fraction in stationary phase (Figure 3). Stress conditions in cyanobacteria can lead to permeability changes of the cell membrane that result in leakage (Whitton and Potts, 2000). However, it should be noted that the extracellular ANTX concentrations presented in this study were likely underestimated, as is likely the case for previous studies (Rapala et al., 1993). ANTX is quite labile under light and may degrade once released into the medium (Stevens and Krieger, 1988). Short-term radio-labeling experiments would be required to estimate more accurately the extracellular release. Furthermore, although the cultures were initially bacteria free, prepared and sub-sampled under sterile conditions, it is possible that some biodegradation arose.

This study suggests that toxin production may be higher when cyanobacterial cells are under moderate nutrient stress. Recently, Kurmayer (2011) reported that another nitrogen-fixing cyanobacterium Nostoc produced higher levels of microcystin cell content under stress conditions, particularly under low phosphorous (P-PO₄) and low light irradiance, even though growth rates were reduced up to 100-fold compared to controls. As a result, microcystin content per cell was negatively correlated to P-PO₄ and irradiance. Interestingly nitrogen reduction did not have the same effect, but, in this case, the number of heterocysts increased suggesting some alleviation of N-deficiency. Even if Nostoc produced higher levels of microcystin per cell when grown under stress conditions, the total toxin production was still lower than when grown under optimal conditions. In contrast, in the present study, both cellular and total ANTX concentrations in A. issatschenkoi were higher in cultures grown at intermediate N concentrations (Figures 3 and 4). Similarly, Rapala et al. (1993) observed that ANTX cellular contents were higher in both A. flos-aquae and Anabaena flos-aquae when grown in nitrogen-free media as opposed to nitrogen-rich control media. In this study, mean ANTX cell contents of CAWBG02 were both significantly lower in 100 and 1% N media than in 5% N. Stress conditions may increase ANTX cellular production to a certain point where cyanobacterial cells are still capable of producing the toxin without compromising primary metabolism. Mean cellular ANTX concentrations for the 1% N were slightly higher than those of the 100% N, but the difference was not significant. Based on cellular composition, the 1% N cultures were more severely stressed and the N concentration may have been too low to sustain much toxin production.

The results presented in this study suggest that ANTX production may increase in A. issatschenkoi under moderate nitrogen limitation. According to the carbon-nutrient balance hypothesis and our current understanding of secondary metabolism, the opposite was expected. One possible ecological advantage of such a strategy could be that when nitrogen is limiting and population density declines, additional population losses through grazing would be minimized via higher cell specific toxin concentrations. In the present experiments batch cultures were used as they best represent algal bloom dynamics typical of nuisance cyanobacterial growth in nature. However, it is possible that factors that are not well controlled in batch cultures may have influenced the results. Growth rates were slightly different among the treatments and might have had an effect on ANTX concentrations. Although it has not been specifically demonstrated for anatoxin-a, in the case of microcystins the cellular growth rate is the primary factor regulating production (Orr and Jones, 1998). However, in this case the higher growth rate observed in the 100% N culture did not lead to the higher anatoxin concentration or cellular content. Further studies encompassing a wider range of nutrient concentrations and using other toxin producing cyanobacterial taxa are necessary to confirm these results and to determine if physiological stress maximizes toxin production more generally.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.