Age and nutrient limitation enhance polyunsaturated aldehyde production in marine diatoms
Graphical abstract
Production of potentially defensive polyunsaturated aldehydes in marine diatoms is modulated by physiological state of the cells as related to age and nutrient status. This explains high variability in feeding experiments and also implies that diatoms at the end of a bloom are more chemically defended than at its onset.
Introduction
Diatoms are eukaryotic unicellular algae responsible for more than 50% of marine primary production (Nelson et al., 1995). They are major representatives of the seasonal blooms that occur in the ocean and have traditionally been considered the main source of food for predatory zooplankton, such as copepods. Recent evidence indicates that certain diatom species induce a drastic reduction in the reproductive response of copepods (Poulet et al., 1994) due to blockage of mitotic divisions during embryogenesis or induction of apoptosis in developing embryos (Miralto et al., 1999b, Ianora et al., 2004). At the metabolic level, hatching failure has been related to the presence of a family of polyunsaturated short-chain aldehydes (here abbreviated as PUAs). PUA production for chemical defense was first attributed to the marine planktonic diatom Thalassiosira rotula (Miralto et al., 1999b). Several different PUAs such as 2,4-heptadienal, 2,4-octadienal, 2,4,7-octatrienal, 2,4-decadienal and 2,4,7-decatrienal, and oxo-acids bearing a similar unsaturated aldehyde structure element have been described since in several marine and freshwater diatoms in culture (reviewed in Pohnert, 2005, Wichard and Pohnert, 2006) and in natural phytoplankton samples (Wichard et al., 2005b).
PUAs are not present in intact cells and are synthesized mainly by wounded cells through enzymatic transformation of free polyunsaturated fatty acids (here abbreviated as PUFAs) (Pohnert, 2000). This defensive reaction is under the control of a phospholipase A2-galactolipase/lipoxygenase/hydroperoxide lyase enzyme cascade (Pohnert, 2002, d’Ippolito et al., 2004) which is activated within seconds after crushing of the cell. As the transformation occurs immediately after cell disruption, the regulation through transcription and de novo protein biosynthesis of the enzymes is highly unlikely. A strict correlation has been found between the PUA and the PUFA composition of phospholipids and chloroplast-derived glycolipids in diatoms (Pohnert, 2002, d’Ippolito et al., 2004). Since virtually no free PUFAs have been found in intact cells, and addition of PUFAs to wounded diatoms leads to increased formation of PUAs, availability of free fatty acids appears to be a limiting factor for aldehyde production (Pohnert, 2002).
The question arises as to how the PUA production varies depending on the physiological conditions before cell disruption occurs.
A certain variability of the lipidic pool has been observed in response to changes in physiological conditions or environmental factors, including age of culture, nutrients or temperature (for a review, Groth-Nard and Robert, 1993). For many phytoplankton species, production of toxins and other secondary metabolites is strongly modulated by physiological conditions and age (reviewed in Legrand et al., 2003, Ianora et al., 2006). Diatom PUAs are rather dependant on the available lipid resources and a few key enzymes, in contrast to these toxins, which require elaborate enzymatic activity for their production. We show here that PUA production of diatoms is also strongly dependant on the physiological and environmental conditions during growth.
The bloom-forming diatom Skeletonema marinoi (separated from S. costatum, Sarno et al., 2005) produces the C7 aldehyde 2E-4Z-heptadienal (in the following heptadienal), as well as the C8 aldehydes 2E-4Z-octadienal (octadienal) and 2E-4Z,7Z-octatrienal (octatrienal), which are derived from eicosapentaenoic (EPA), hexadecatrienoic (HDTRI), and hexadecatetraenoic (HDTERTA) acid, respectively (Pohnert, 2005). S. marinoi forms dense, almost monospecific blooms in late-winter in the Northern Adriatic Sea (Mediterranean Sea) that strongly effect copepod reproduction and recruitment (Miralto et al., 1999a, Ianora et al., 2004). During the bloom, PUA production per cell correlates positively with S. marinoi cell numbers and, in turn, with phytoplankton lysis rates which are higher in the final stages of the bloom (Casotti, R., unpubl.), suggesting a potential release of PUA into the seawater without grazing.
The present study aimed at quantifying potential production of PUAs per cell in cultures of S. marinoi during the different growth phases, from exponential to declining. PUA production has been measured in cultures grown under nitrate and phosphate limitation. The data obtained show that the wound-activated production of PUAs per S. marinoi cell was higher when cultures reached the stationary growth phase. The same was true for N- and P-limited cultures.
Section snippets
Growth under standard conditions
The average growth rate of S. marinoi in exponential phase under standard conditions was 0.96 d−1 ± 0.05 d−1 SD. The cultures attained cell concentrations of 1.05 × 106 cell ml−1 at the stationary phase, and these remained constant for 6 days before the declining phase (Fig. 1a). We tested the hypothesis that nitrate and phosphate could be the factors limiting growth in the control cultures causing their entrance in the stationary phase of growth, by adding these nutrients separately to triplicate
Discussion
The increasing potential for PUA production with age of the culture or under nutrient conditions suggests the endogenous and/or environmental control of diatom toxicity. Culture conditions and bloom phase are thus very important when assessing the effect of a diatom diet upon copepod fitness or reproduction. PUAs have been shown to have teratogenic effects on predators by inducing abortions, malformations and reduced larval growth (Ianora et al., 2004). In a broader survey of different diatom
Conclusions
A strong dependence of PUA production on culture age and N- or P-limitation in cultures of S. marinoi has been observed, suggesting a direct link between toxin production, physiological conditions and nutrient stress. This has direct implications for natural conditions, since nutrient limitation is often, if not always, the factor triggering the end of a bloom at sea (Cullen, 1991). In many coastal areas, where most algal blooms occur, N:P ratios have changed considerably compared to the
Biological material
Axenic cultures of the marine diatom Skeletonema marinoi (Sarno and Zingone), strain CCMP 2092 (separated from S. costatum), were cultured in 2-l polycarbonate bottles with air bubbling in a growth chamber (Hereaeus). Axenicity was confirmed at the start and the end of every experiment on any replicate by inoculating 1 ml of culture in 0.1% peptone agar in medium. One-month old natural seawater amended with f/2 nutrients (Guillard, 1975) was used as medium. The cultures were maintained at 17 °C
Acknowledgements
We thank C. Brunet for chlorophyll a, F. Corato and D. Cassin for POC, PON and nutrient analyses. We deeply thank J.A. Berges for his helpful comments and suggestions. The project was carried out in the framework of the MarBEF Network of Excellence ‘Marine Biodiversity and Ecosystem Functioning’, which is funded by the Sustainable Development, Global Change and Ecosystems Programme of the European Community’s Sixth Framework Programme (contract GOCE-CT-2003-505446). The MPG and the DFG (PO
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