Teratogenic effects of diatom metabolites on sea urchin Paracentrotus lividus embryos

Abstract

The diatom-derived polyunsaturated aldehydes (PUAs), 2-trans,4-trans-decadienal, 2-trans,4-trans-octadienal, 2-trans,4-trans,7-octatrienal, 2-trans,4-trans-heptadienal, as well as tridecanal were tested on early and later larval development in the sea urchin Paracentrotus lividus. We also tested the effect of some of the more abundant diatom polyunsaturated fatty acids (PUFAs) on development, in particular 5,8,11,14,17-eicosapentaenoic acid (EPA), one of the main precursors of diatom PUAs, as well as 4,7,10,13,16,19-docosahexaenoic acid (DHA), 6,9,12,15-octadecatetraenoic acid (stearidonic acid), 6,9,12-octadecatrienoic acid (gamma-linolenic acid) and 9,12-octadecadienoic acid (linoleic acid). PUAs blocked sea urchin cell cleavage in a dose dependent manner and with increasing chain length from C7 to C10 PUAs, with arrest occurring at 27.27 microM with heptadienal, 16.13 microM with octadienal, 11.47 microM with octatrienal and 5.26 microM with decadienal. Of the PUFAs tested, only EPA and stearidonic acid blocked cleavage, but at much higher concentrations compared to PUAs (331 microM for EPA and 181 microM for stearidonic acid). Sub-lethal concentrations of decadienal (1.32-5.26 microM) delayed development of embryos and larvae which showed various degrees of malformations depending on the concentrations tested. Sub-lethal concentrations also increased the proportion of TUNEL-positive cells indicating imminent death in embryos and larvae. Using decadienal as a model PUA, we show that this aldehyde can be detected spectrophotometrically for up to 14 days in f/2 medium.

Keywords: TUNEL; apoptosis; development arrest; diatom; fatty acids; oxylipins; sea urchin; teratogens.

Figure 1

Biosynthetic sketch for the synthesis of oxylipins from fatty acids via lipoxygenase enzymes (LOX) in marine diatoms. R₁ represent methyl terminal part and R₂ the carboxylic end of C16 or C20 fatty acid precursors. R₁ and R₂ may differ in terms of length and degree of unsaturation depending on the position of O₂ addition by LOX in the fatty acid molecule (modified from Fontana et al. [6]).

Figure 2

(a) Cleavage inhibition in sea urchin embryos following PUAs treatment. (b) Percentage of hatched sea urchin larvae. Sea urchin embryos were treated at increasing concentrations of 2-trans,4-trans-decadienal (), aldehyde mix (see text) (), 2-trans,4-trans-octadienal (), 2-trans,4-trans,7-octatrienal (), 2-trans,4-trans-heptadienal () and tridecanal (▪). Values (means ± S.D.; N = 600) are the results of three different experiments.

Figure 3

(a) Cleavage inhibition in sea urchin embryos following PUFAs treatment at increasing concentrations. (b) Percentage of hatched sea urchin larvae with increasing concentrations of fatty acids. EPA (), EPA-ME (), DHA (), stearidonic (), linoleic (▾) and γ-linolenic () acids. Values (means ± S.D.; N = 600) are the results of three different experiments.

Figure 4

(a) Variation with time in decadienal concentration in f/2 medium. Each point is a mean value of three replicates calculated from a standard curve. (b) UV spectrum in f/2 medium of decadienal (red) and decadienoic acid (black).

Figure 5

Paracentrotus lividus 48 hours plutei after incubation in decadienal at 1.32 (b), 2.63 (c), 3.95 (d) and 5.26 μM (e) compared to control embryo (a).

Figure 6

Percentage of abnormal sea urchin larvae following 48 hours treatment with decadienal ranging from 1.32–6.58 μM. Control is reported as 0 μM decadienal concentration. Yellow bars = retarded larvae; blue bars = abnormal pluteus larvae; red bar = abnormal gastrulae and blastulae; green = dead pre-hatched embryos. Values (means ± S.D.; N = 600) are the results of three different experiments.

Figure 7

Tunel positive embryos incubated for 48 hours in decadienal at increasing concentrations (lower row) and corresponding images at transmitted light (upper row). Control Pluteus (a,b). Larvae after 48 hours incubation in decadienal 1.32 (c,d), 2.63 (e,f), 3.95 (g,h) μM.

Figure 8

Percentage of TUNEL-positive embryos treated with increasing concentrations of decadienal. The percentage of larvae negative to TUNEL staining is shown in yellow. Pink indicates the percentage of embryos with nuclei that were positive to TUNEL localized in the arms and intestine. Blue indicates the percentage of larvae with apoptotic nuclei in the entire body. Values are means of three different experiments for approximately 200 embryos for each data point.