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Toxicol Sci
2016 Jun 01;1512:419-33. doi: 10.1093/toxsci/kfw053.
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First Morphological and Molecular Evidence of the Negative Impact of Diatom-Derived Hydroxyacids on the Sea Urchin Paracentrotus lividus.
Varrella S
,
Romano G
,
Ruocco N
,
Ianora A
,
Bentley MG
,
Costantini M
.
Abstract
Oxylipins (including polyunsaturated aldehydes [PUAs], hydoxyacids, and epoxyalcohols) are the end-products of a lipoxygenase/hydroperoxide lyase metabolic pathway in diatoms. To date, very little information is available on oxylipins other than PUAs, even though they represent the most common oxylipins produced by diatoms. Here, we report, for the first time, on the effects of 2 hydroxyacids, 5- and 15-HEPE, which have never been tested before, using the sea urchin Paracentrotus lividus as a model organism. We show that HEPEs do induce developmental malformations but at concentrations higher when compared with PUAs. Interestingly, HEPEs also induced a marked developmental delay in sea urchin embryos, which has not hitherto been reported for PUAs. Recovery experiments revealed that embryos do not recover following treatment with HEPEs. Finally, we report the expression levels of 35 genes (involved in stress, development, differentiation, skeletogenesis, and detoxification processes) to identify the molecular targets affected by HEPEs. We show that the 2 HEPEs have very few common molecular targets, specifically affecting different classes of genes and at different times of development. In particular, 15-HEPE switched on fewer genes than 5-HEPE, upregulating mainly stress-related genes at a later pluteus stage of development. 5-HEPE was stronger than 15-HEPE, targeting 24 genes, mainly at the earliest stages of embryo development (at the blastula and swimming blastula stages). These findings highlight the differences between HEPEs and PUAs and also have important ecological implications because many diatom species do not produce PUAs, but rather these other chemicals are derived from the oxidation of fatty acids.
FIG. 1. Cleavage inhibition in sea urchin embryos following decadienal (at the concentrations of 1.3, 1.6, 2.6, 3.3, 5.3, 6.6, 8.2, 9.9, 11.8, 13.1, 14.5, 15.8, 17.1, 18.4, and 19.7âμM; black line; data from Romano et al., 2010) and 5- and 15-HEPE (at the concnetrations of 6, 7, 8, 9, 10, 15, 30, 50, 70 and 90âμM; blue line) treatments. Control is reported as 0âμM concentration. Abbreviation: HEPE, hydroxyacids.
FIG. 2. Abnormal and delayed embryos after HEPEs treatments. Top panel: percentage (%) of abnormal and delayed embryos when Paracentrotus lividus newly fertilized eggs were exposed to different concentrations of the polyunsaturated aldehydes decadienal (1.32, 2.63, 3.95, 5.26, and 6.58âμM; Romano et al., 2010) and 5- and 15-HEPEs (6, 7, 8, 9, 10, 15, and 30âμM) at 48âh post fertilization (hpf). Significant differences (meanâ±âSD) compared with the control (4.3â±â0.8 abnormal embryos; 3.3â±â1.0 delayed embryos, data not shown): *Pâ<â.05, **Pâ<â.01, ***Pâ<â.001. One-way ANOVA (Pâ<â.05) with Tukeyâs multiple comparison test. Experiments were conducted in triplicate using 3 egg groups collected from 3 different females. Bottom panel (A) control embryos (in filtered sea water) without HEPEs) at 48 hpf; (B) delayed embryos observed in samples treated with HEPEs at the concentrations from 6 to 10 µM and (C) delayed embryos observed in samples treated with HEPEs at 15 and 30 µM at 48 hpf. HEPE, hydroxyacids.
FIG. 3. Real-time quantitative PCR (qPCR) at blastula stage. Histograms show the differences in expression levels of analyzed genes belonging to different functional classes (stress, detoxification, development and differentiation and skeletogenesis), followed by real time qPCR. Paracentrotus lividus embryos were grown in the presence of 15-HEPE and 5-HEPE at 7.0 µM and collected at 5âh post fertilization. Data are reported as a fold difference compared with control (meanâ±âSD) embryos in sea water without HEPEs. Fold differences greater thanâ±2 (see dotted horizontal guidelines at values of 2 and â2) were considered significant. Abbreviation: HEPE, hydroxyacids.
FIG. 4. Real-time quantitative PCR (qPCR) a swimming blastula stage. Histograms show the differences in expression levels of analyzed genes, followed by real-time qPCR. Paracentrotus lividus embryos were grown in the presence of 7.0 µM of 15-HEPE and 5-HEPE and collected at 9âh post fertilization (for further details see also the legend to Figure 3). Abbreviation: HEPE, hydroxyacids.
FIG. 5. Real-time quantitative PCR (qPCR) at prism stage. Histograms show the differences in expression levels of analyzed genes, followed by real-time qPCR. Paracentrotus lividus embryos were grown in the presence of 7.0 µM of 15-HEPE and 5-HEPE and collected at 24âh post fertilization (for further details see also the legend to the Figure 3). Abbreviation: HEPE, hydroxyacids.
FIG. 6. Real-time quantitative PCR (qPCR) at pluteus stage. Histograms show the differences in expression levels of analyzed genes, followed by real-time qPCR. Paracentrotus lividus embryos were grown in the presence of 7.0 µM of 15-HEPE and 5-HEPE and collected at 48âh post fertilization (for further details see also the legend to the Figure 3). Abbreviation: HEPE, hydroxyacids.
FIG. 7. Synopsis of real time qPCR experiments. Synopsis of the patterns of up- and downregulation of different classes of genes in the sea urchin, Paracentrotus lividus, in the presence of HEPEs. PUAs decadienal, heptadienal, and octadienal are reported for the sake of comparison (Varrella et al., 2014). Abbreviation: HEPE, hydroxyacids.
Andreou,
Biosynthesis of oxylipins in non-mammals.
2009, Pubmed
Andreou,
Biosynthesis of oxylipins in non-mammals.
2009,
Pubmed
Blée,
Impact of phyto-oxylipins in plant defense.
2002,
Pubmed
Blée,
Phytooxylipins and plant defense reactions.
1998,
Pubmed
Bošnjak,
Identification of ABC transporter genes in gonad tissue of two Mediterranean sea urchin species: black, Arbacia lixula L., and rocky, Paracentrotus lividus L.
2013,
Pubmed
,
Echinobase
Caldwell,
The influence of bioactive oxylipins from marine diatoms on invertebrate reproduction and development.
2009,
Pubmed
Costa,
Phylogenetic analysis and expression patterns of p16 and p19 in Paracentrotus lividus embryos.
2012,
Pubmed
,
Echinobase
Croce,
Wnt6 activates endoderm in the sea urchin gene regulatory network.
2011,
Pubmed
,
Echinobase
Cutignano,
Chloroplastic glycolipids fuel aldehyde biosynthesis in the marine diatom Thalassiosira rotula.
2006,
Pubmed
Cutignano,
LIPOXYGENASE PRODUCTS IN MARINE DIATOMS: A CONCISE ANALYTICAL METHOD TO EXPLORE THE FUNCTIONAL POTENTIAL OF OXYLIPINS(1).
2011,
Pubmed
Diller,
Stress protein expression kinetics.
2006,
Pubmed
d'Ippolito,
New C16 fatty-acid-based oxylipin pathway in the marine diatom Thalassiosira rotula.
2005,
Pubmed
D'Ippolito,
15S-lipoxygenase metabolism in the marine diatom Pseudo-nitzschia delicatissima.
2009,
Pubmed
Duboc,
Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side.
2005,
Pubmed
,
Echinobase
Fontana,
LOX-induced lipid peroxidation mechanism responsible for the detrimental effect of marine diatoms on zooplankton grazers.
2007,
Pubmed
Garg,
Divergent DNA methylation patterns associated with gene expression in rice cultivars with contrasting drought and salinity stress response.
2015,
Pubmed
Ghiglione,
Structure of the sea urchin hatching enzyme gene.
1994,
Pubmed
,
Echinobase
Goldstone,
The chemical defensome: environmental sensing and response genes in the Strongylocentrotus purpuratus genome.
2006,
Pubmed
,
Echinobase
Gudimova,
In vivo exposure to northern diatoms arrests sea urchin embryonic development.
2016,
Pubmed
,
Echinobase
Hammond,
Early developmental gene regulation in Strongylocentrotus purpuratus embryos in response to elevated CO₂ seawater conditions.
2012,
Pubmed
,
Echinobase
Ho,
Dominant negative Bmp5 mutation reveals key role of BMPs in skeletal response to mechanical stimulation.
2008,
Pubmed
Ianora,
Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom.
2004,
Pubmed
Ianora,
Toxigenic effects of diatoms on grazers, phytoplankton and other microbes: a review.
2010,
Pubmed
Lepage,
Spatial expression of the hatching enzyme gene in the sea urchin embryo.
1992,
Pubmed
,
Echinobase
Lhomond,
Frizzled1/2/7 signaling directs β-catenin nuclearisation and initiates endoderm specification in macromeres during sea urchin embryogenesis.
2012,
Pubmed
,
Echinobase
Marrone,
Defensome against toxic diatom aldehydes in the sea urchin Paracentrotus lividus.
2012,
Pubmed
,
Echinobase
Matranga,
Embryonic development and skeletogenic gene expression affected by X-rays in the Mediterranean sea urchin Paracentrotus lividus.
2010,
Pubmed
,
Echinobase
McIntyre,
Short-range Wnt5 signaling initiates specification of sea urchin posterior ectoderm.
2013,
Pubmed
,
Echinobase
Migliaccio,
Stress response to cadmium and manganese in Paracentrotus lividus developing embryos is mediated by nitric oxide.
2014,
Pubmed
,
Echinobase
Nanjappa,
Oxylipin diversity in the diatom family Leptocylindraceae reveals DHA derivatives in marine diatoms.
2014,
Pubmed
Nemer,
Polyubiquitin RNA characteristics and conditional induction in sea urchin embryos.
1991,
Pubmed
,
Echinobase
Palotai,
Chaperones as integrators of cellular networks: changes of cellular integrity in stress and diseases.
2008,
Pubmed
Pfaffl,
Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.
2002,
Pubmed
Pfaffl,
A new mathematical model for relative quantification in real-time RT-PCR.
2001,
Pubmed
Pinsino,
Manganese interferes with calcium, perturbs ERK signaling, and produces embryos with no skeleton.
2011,
Pubmed
,
Echinobase
Pohnert,
Diatom/copepod interactions in plankton: the indirect chemical defense of unicellular algae.
2005,
Pubmed
Ragusa,
Effects of cadmium exposure on sea urchin development assessed by SSH and RT-qPCR: metallothionein genes and their differential induction.
2013,
Pubmed
,
Echinobase
Roccheri,
Cadmium induces the expression of specific stress proteins in sea urchin embryos.
2004,
Pubmed
,
Echinobase
Romancino,
Identification and characterization of PlAlix, the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus.
2013,
Pubmed
,
Echinobase
Romano,
Design and synthesis of pro-apoptotic compounds inspired by diatom oxylipins.
2013,
Pubmed
,
Echinobase
Romano,
Teratogenic effects of diatom metabolites on sea urchin Paracentrotus lividus embryos.
2010,
Pubmed
,
Echinobase
Romano,
Nitric oxide mediates the stress response induced by diatom aldehydes in the sea urchin Paracentrotus lividus.
2011,
Pubmed
,
Echinobase
Russo,
Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B.
2010,
Pubmed
,
Echinobase
Shipp,
ATP-binding cassette (ABC) transporter expression and localization in sea urchin development.
2012,
Pubmed
,
Echinobase
Smith,
Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo.
2008,
Pubmed
,
Echinobase
Todgham,
Transcriptomic response of sea urchin larvae Strongylocentrotus purpuratus to CO2-driven seawater acidification.
2009,
Pubmed
,
Echinobase
Varrella,
Molecular response to toxic diatom-derived aldehydes in the sea urchin Paracentrotus lividus.
2014,
Pubmed
,
Echinobase
Varrella,
Toxic Diatom Aldehydes Affect Defence Gene Networks in Sea Urchins.
2016,
Pubmed
,
Echinobase
Zito,
Expression of univin, a TGF-beta growth factor, requires ectoderm-ECM interaction and promotes skeletal growth in the sea urchin embryo.
2003,
Pubmed
,
Echinobase