Introduction

Diatom-derived α,β,γ,δ-unsaturated aldehydes (polyunsaturated aldehydes, PUA) can interfere with the hatching success and larval development of copepods as well as with the embryonic development of invertebrates (see Ianora et al., 2003 and Pohnert, 2005 for reviews). Diatoms are unicellular algae that contribute substantially to the phytoplankton and are thus an important base of the marine food web. A generally negative impact of diatoms on herbivores would have major implications for the classical concept of plankton ecosystem functioning. Therefore, numerous studies, mainly based on laboratory experiments, have been carried out to clarify this predator-prey relationship. While some authors have proposed a deleterious effect of diatoms (Ianora et al., 2003), others have not found any adverse effects (e.g., Jonasdottir et al., 1998). Moreover, no general negative relationship between copepod egg hatching and diatom biomass was detected in the global field survey by Irigoien et al. (2002). Most of these studies correlated hatching parameters to the presence or absence of diatoms rather than monitoring PUA production of the diet, even if this is the postulated determining factor in these interactions. This is surprising, since only a few marine and fresh water diatoms have been investigated for PUA-production (Pohnert, 2005). Indeed, some of the controversially discussed results on reproductive parameters (Paffenhöfer et al., 2005) might be explained by variability of PUA production in different diatom species. To overcome this limitation and to provide a basis for further ecological and modelling studies, we conducted a survey of volatile PUA-production from representative diatoms of the major classes by using a protocol based on in situ trapping of the reactive metabolites (Wichard et al., 2005).

Methods and Materials

Cultivation

Seventy one diatom-isolates from different sources were investigated (Table 1). All isolates were grown in stationary cultures using 250 ml jars containing 100 ml artificial medium (Table 1). Sylvania (Germany) “Cool white deluxe (F36W 840, 4000K)” tubes provided illumination. A light regime of 16:8 (light/dark) with 30–40 μE/m2/sec light intensity was used. Generally, the growth temperature was 15.5°C, except for Thalassiosira aestivalis and Skeletonema costatum (CCMP 784), which were grown at 13°C and 23°C, respectively. Cultures reached the stationary phase after 2–3 wk, when 60-90 ml (104−106 cells/ml culture medium, depending on species) were harvested. The cell morphology of each culture was checked prior to harvest with light microscopy. Cells were counted with a Neubauer-improved haemocytometer in four replicates. The counting variance ranged from 10–35%.

Table 1 Overview of the Isolates Investigated by Solid Pahse Microextraction and PFBHA-Derivatisation
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Quantitative PUA analysis

The cultures were harvested by filtration as described in Wichard et al. (2005). For a first rapid screening of the cultivated isolates, solid phase microextraction was performed with a polydimethylsiloxan fiber after wounding by sonication as described in Pohnert et al. (2002). To quantify PUA release upon cell damage, a protocol based on derivatization of PUA with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamin hydrochloride (PFBHA·HCl) and subsequent GC/MS (EI) analysis was applied (Wichard et al., 2005). The limit of quantification for PUA in concentrated diatom cultures was 5 ng/ml. Each analysis was performed in triplicate.

Quantitative Chlorophyll a + c analysis

In cases where PUA were determined, the chlorophyll content of the diatom isolates was quantified as well. The extraction in 90% acetone and quantification was performed in triplicate according to the standardized method No 446.0 (U.S. Environmental protection agency: Microbiological and Chemical Exposure Research and references herein). Jeffrey and Humphrey’s trichromatic equations were applied: Chlorophyll a (mg/l) = 11.85 E664 − 1.54 E647 − 0.08 E630 and Chlorophylls c1 + c2 (mg/l) = −1.67 E664 − 7.60 E647 + 24.52 E630. Each value was corrected by the absorbance at 750 nm. The ratio of PUA to Chl a + c of identical culture batches was calculated as PUA/Chl (ppm) = PUA (μg/ml)/Chl (μg/ml) × 1,000,000.

Determination of cell volume

Living cells were measured microscopically in planar view (minimum: 20 cells). Linear measurements were converted to cell volume using different geometric approximations: a cylinder for Chaetoceros compressus, Guinardia deliculata, Melosira spp., Skeletonema subsalatum, and Thalassiosira spp.; a cylinder + 2 half spheres for Skeletonema costatum and Skeletonema pseudocostatum, and a cone for Asterionellopsis glacialis. The carbon content was determined by the carbon to volume relationship based on the equation C (pg/cell) = 0.288 × volume0.811 (Menden-Deuer and Lessard, 2000). The PUA to carbon ratio was calculated as PUA/C (ppm) = PUA (fg/cell)/C (fg/cell) × 1,000,000.

Results and discussion

Seventy one diatom-isolates were analyzed for PUA-formation upon cell damage by sonication. The diatoms were either obtained from algal collections or freshly isolated from coastal waters off Roscoff (48°45′ N and 3°58′ W, Bretagne, France) and during several cruises to Dabob Bay, Point Wells and Friday Harbour (47° 46.14′N and 122° 50.10′W/47° 44.63′ N and 122° 25.34′ W/48.535° N and 123.005° W, Washington, USA). A total of 50 different species was investigated, with an emphasis on the family Thalassiosiraceae and the species Phaeodactylum tricornutum, because these diatoms are widely used in bioassays on the reproductive success of copepods (Miralto et al., 1999; Pohnert et al., 2002; Paffenhöfer et al., 2005). Under defined culture conditions (see Method section), 27 PUA-producers were identified among the 71 isolates investigated (Table 1). Out of the PUA producers, two released the unsaturated aldehydes only in trace amounts. The PUA-production upon wounding of 20 selected isolates (18 marine and 2 freshwater) was quantified during the stationary growth phase. PUA-production ranged from 0.01 fmol PUA/cell (Thalassiosira nordenskioeldii) to 9.8 fmol PUA/cell (Thalassiosira pacifica) (Table 2). This wide range over four orders of magnitude, as well as the isolate-dependent variability of structurally different unsaturated aldehydes, reflects a high plasticity within the Bacillariophyceae.

Table 2 Quantification of PUA Per Cell, Per Chlorophyll a + c and Per Carbon Content (Stationary Phase)
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Since the calculation of PUA per cell underestimates the aldehyde contribution of species with low cell volume but probably high cell abundance in a typical herbivore diet, the PUA to carbon (PUA/C) and the PUA to chlorophyll a + c (PUA/Chl) ratio were also calculated. With respect to the PUA/Chl and PUA/C ratios, other dominant producers, such as Skeletonema pseudocostatum (PUA/Chl = 40,650 ppm) or Skeletonema costatum (SAG 19.99, PUA/C = 488 ppm), come to the fore.

Within the Bacillariophyceae, more than half of the investigated species do not produce PUA upon wounding in the stationary growth phase. In the light of the ongoing discussion about the influence of diatoms on herbivores, this result stresses that a general PUA-mediated effect can not be assumed for any given phytoplankton bloom, but that a species and strain-specific analysis is required.

Recently, the hypothesis that PUA-production could be the reason for poor copepod reproductive success during spring blooms of diatoms was proposed (Ianora et al., 2004). In this context, it is interesting to note that some of the most abundant spring-bloom forming species like Thalassiosira spp. (e.g. Th. rotula and Th. pacifica) release high amounts of PUA. These species were isolated from different habitats, such as the Adriatic Sea (Miralto et al., 1999), the coastal waters off Roscoff (NE Atlantic) and Dabob Bay (NE Pacific).

Because the ability to produce PUA is distributed heterogeneously in the major classes of Bacillariophyceae, one cannot predict the defensive potential of certain species. Moreover, PUA-production within different isolates of one species ranges widely, and thus case-specific chemical investigations accompanying bioassays are required. For example, the different Thalassiosira rotula isolates investigated release PUA in a wide range of concentrations from 0.15 to 6.34 fmol/cell. In this study, only cultures in the stationary growth phase were investigated. This culture condition was selected since it is also used in most laboratory investigations. Additional variation of PUA-production during different phases of diatom blooms or growth phases of cultures might have to be taken into account as well.

Based on this survey, we not only recommend performing future bioassays along with chemical analyses, but also urge for a reconsideration of the general conclusions drawn in the past. It is likely, that the observed reduction of hatching success in several studies/regions may not be due to the formation of deleterious PUA, but may have other causes. On the other hand, in regions where major PUA producers are the main constituents of blooms, there might be effects on the reproduction of herbivorous grazers and their population dynamics (Ianora et al. 2004; Halsband-Lenk et al. unpublished). Whether secondary production can be significantly affected by this chemically mediated interaction in such ecosystems requires further investigation.