Lipoxygenase Pathways in Diatoms: Occurrence and Correlation with Grazer Toxicity in Four Benthic Species

Abstract

Oxygenated derivatives of fatty acids, collectively called oxylipins, are a highly diverse family of lipoxygenase (LOX) products well described in planktonic diatoms. Here we report the first investigation of these molecules in four benthic diatoms, Cylindrotheca closterium, Nanofrustulum shiloi, Cocconeis scutellum, and Diploneis sp. isolated from the leaves of the seagrass Posidonia oceanica from the Gulf of Naples. Analysis by hyphenated MS techniques revealed that C. closterium, N. shiloi, and C. scutellum produce several polyunsaturated aldehydes (PUAs) and linear oxygenated fatty acids (LOFAs) related to the products of LOX pathways in planktonic species. Diploneis sp. also produced other unidentified fatty acid derivatives that are not related to LOX metabolism. The levels and composition of oxylipins in the benthic species match their negative effects on the reproductive success in the sea urchin Paracentrotus lividus. In agreement with this correlation, the most toxic species N. shiloi revealed the same LOX pathways of Skeletonema marinoi and Thalassiosira rotula, two bloom-forming planktonic diatoms that affect copepod reproduction. Overall, our data highlight for the first time a major role of oxylipins, namely LOFAs, as info-chemicals for benthic diatoms, and open new perspectives in the study of the structuring of benthic communities.

Keywords: biosynthesis; chemical ecology; diatoms; fatty acid derivatives; lipoxygenase; mass spectrometry; oxylipins.

Figure 1

LC-MS analysis of the four benthic diatoms. Chromatographic profiles of (a) C. closterium, (b) N. shiloi, (c) C. scutellum and (d) Diploneis sp.; Internal standard: 16-hydroxy-hexadecanoic acid (STD). Abbreviations are in agreement with d’Ippolito et al. [34]. Structures are reported in Table S4 of the Supplementary Material.

Figure 2

LOX activity in the four benthic diatoms. (a) Levels of oxylipins as μmol per carbon mg; (b) Levels of FAHs as μmol per carbon mg; (c) statistical correlation between oxylipin and FAHs levels. Data are reported as means ± SD (n = 3). Statistical differences were evaluated by One-Way ANOVA followed by Tukey’s post-hoc analysis for multiple comparisons (Supplementary Tables S1 and S2; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Identification of active lipoxygenases by MS/MS fragmentation of epoxyalcohols in C. closterium, N. shiloi and C. scutellum. For simplicity, the polyunsaturated chain of EPA is represented by the 1,3-pentadiene moiety that undergoes to enzymatic oxidation. R₁ and R₂ are variable alkyl residues to complement the structure of EPA (C20:5) or HTrA (C16:3). (a) Biosynthesis of fatty acids by LOX pathways; (b) MS parent ion and MS/MS fragmentation of epoxyalcohols analyzed as methyl esters in positive mode; (c) Diagnostic MS fragments used for the regiochemical identification of epoxyalcohols of diatoms. Assignments and abbreviations are in agreement with d’Ippolito et al. [34]. Structures are reported in Table S4 of the Supplementary Material.

Figure 4

Ternary diagram based on survival of sea urchin plutei, level of oxylipins and level of FAHs. Graphical representation derives by sum to unity (100%) of the respective variables with percentage of abnormal plutei that is strictly dependent on oxylipins and FAHs levels. Single values are reported in Table S3 of the Supplementary Information.

Figure 5

Correlation between feeding effect on the sea urchin Paracentrotus lividus and LOX products in the four benthic diatoms. (a,b) correlation of abnormal plutei with oxylipins and FAHs in the three species with active LOX pathways (C. closterium, N. shiloi and C. scutellum); (c,d) correlation of abnormal plutei with oxylipins and FAHs including Diploneis sp.

Figure 6

Lipoxygenase pathways and oxylipins of C. closterium, N. shiloi and C. scutellum (see abbreviation section for the name of the compounds). Abbreviations are in agreement with d’Ippolito et al. [34]. Structures are reported in Table S4 of the Supplementary Material.