Molecular response to toxic diatom-derived aldehydes in the sea - PowerPoint PPT Presentation

Molecular response to toxic diatom-derived aldehydes in the sea urchin Paracentrotus lividus Stefano Varrella e-mail: stefano.varrella@szn.it Stazione Zoologica Anton Dohrn, Naples, Italy

Model organism: Paracentrotus lividus • Relevant on structure of benthic marine community CLASSIFICATION • Domain: Eukaryota Mediterranean sea and Atlantic ocean Kingdom: Animalia • Extraction and manteinance of gametes are easy Phylum: Echinodermata • Trasparent embryos Class: Echinoidea • Embryos grow rapidly (pluteus stage at 48hpf) Order: Echinoida • Long reproductive period Family: Echinidae Species: Paracentrotus lividus • Good model for ecotoxicological studies on response of marine invertebrate to environmental pollutants:  physical and chemical xenobiotics  antifoulings/pesticides  low pH  heavy metals  X-rays  endocrine disrupters compounds  oxylipins derived from diatoms  UVs

Diatoms  There are more than 200 genera of living diatoms, and approximately 100,000 species.  Diatoms live in the oceans and in freshwater.  Most live in open water, although some live as surface films at the water-sediment interface (benthic), or even under damp atmospheric conditions.  They are very important in oceans, where they are estimated to contribute up to 45% of the total oceanic primary production. http://deepbluehome.blogspot.it/search?q=diatom

Marine food web Beneficial role in supporting planctonic food web. Diatoms are good food for the primary consumers of plankton.

Diatoms species produce secondary metabolities with cytotoxic activity Echinoderms (Miralto et al. 1999) Polychaetes • Oocyte viability • Sperm motility inhibition Crustaceans • Fertilization success • Cleavage inhibition • Hatching reduction • Larval toxicity (Ianora and Miralto 2010)

Oxidative metabolism of fatty acids in diatoms Hydroperoxide lyase OXYLIPINS (Ianora and Miralto 2010)

The effects of decadienal on sea urchin embryos Decadienal Decadienal induces teratogenesis and apoptosis Control 1.32 µM 2.63 µM 3.95 µM 5.26 µM Control 1.32 µM 2.63 µM 3.95 µM (Romano et al., 2003) (Romano et al., 2010) Increasing concentrations of decadienal First molecular studies (Romano et al., 2011) (Marrone et al., 2012)

….. The other PUAs? Octadie diena nal Heptadie ienal  Abundant compounds among PUAs  No molecular evidences

Stress responses of sea urchin after exposure to heptadienal and octadienal Molecular Morphologic  Do  Do PUAs affect the PUAs have molecular targets? embryogenesis?  Could  When do PUAs act on these targets be considered as possible embryo development? biomarkers for environmental stress response?

Experimental procedures Sea urchin eggs Decadienal Incubation for 10 min with Octadienal aldehydes Heptadienal Fertilization http://www.alnmag.com/news/2012/02/sperm-pathways-sea-urchins Morphological observations Control embryos at 48 hpf at 48 hpf

Do PUAs affect the embryogenesis? PUAs treatments induced Dose-dependent increase of abnormal plutei TERATOGENESIS Control ( embryos in sea water without aldehydes) (Varrella et al., 2014)

Experimental procedures Sea urchin eggs Decadienal Incubation for 10 min with Octadienal aldehydes Heptadienal Fertilization http://www.alnmag.com/news/2012/02/sperm-pathways-sea-urchins Morphological observations at Control embryos 72, 96 hpf at one week hpf until one-week

Do PUAs affect the embryogenesis? Abnormal embryos (%) Heptadienal Octadienal Decadienal 72, 96 hpf Developmental times (hpf) Embryos number (%) Control One week Controllo Decadienal 40 ’ df Heptadienal Octadienal Normal Delayed Abnormal

Experimental procedures Sea urchin eggs Incubation for 10 min with aldehydes Fertilization http://www.alnmag.com/news/2012/02/sperm-pathways-sea-urchins PUAs additions 10 min pf 40 min pf 2 hpf 3 hpf 5 hpf 8 hpf 2 blastomeres 8 blastomeres 32 blastomeres Early blastula Late blastula Morphological Control embryos observations at 48 hpf at 48 hpf

When do PUAs act on embryo development? Aldehydes additions at different developmental times Decadienal Abnormal Plutei (%) Heptadienal 10 min bf 10 min pf 40 min pf Octadienal 2 hpf 3 hpf 5 hpf 8 hpf Concentrations (µM) (Varrella et al., 2014)

PUAs action time 10 minutes before fertilization 10 minutes after PUAs

Experimental procedures Sea urchin eggs Decadienal Incubation for 10 min Octadienal with aldehydes Heptadienal http://www.alnmag.com/news/2012/02/sperm-pathways-sea-urchins Fertilization Determination of teratogenic and dose-dependent concentrations of aldehydes Analysis of 31 by Real Time q-PCR

Do PUAs have molecular targets? Genes analyzed by Real-Time qPCR Development and Detoxification Skeletogenesis Stress differentiation MT hat Hsp70 SM30 sox9 MT4 Hsp60 BMP5-7 BP10 MT5 Hsp56 SM50 Blimp MT6 MT Nec MT7 Alix MTase uni MT8 Wnt5 GS p16 Wnt6 MDR1 cytb p19 Wnt8 CAT p38 MAPK 14-3-3 ε (Marrone et al., 2012; Varrella et al., 2014)

The key stages of P. lividus embryogenesis Swimming blastula Early blastula 5 hpf 9 hpf Prism Pluteus 24 hpf 48 hpf

Effects of decadienal on gene expressions at different developmental stages Hsp70 SM50 SM50 SM30 Hsp60 uni Wnt6 MT4 MT6 hat MT4 SM30 MT6 SM50 MT8 Stress Skeletogenesis Development/Differentiation Detoxification Up-regulated genes Down-regulated genes (Modified from Varrella et al., 2014)

Effects of heptadienal on gene expressions at different developmental stages Hsp70 p38 MAPK Hsp56 Wnt6 MTase p38 MAPK SM50 hat Alix sox9 hat MT4 Wnt5 SM50 Wnt6 Wnt8 Stress p19 Skeletogenesis p16 Development/Differentiation uni Detoxification SM50 Up-regulated genes MT4 Down-regulated genes SM30 MT8 SM50 (Modified from Varrella et al., 2014)

Effects of octadienal on gene expressions at different developmental stages MTase SM50 Hsp70 Wnt6 MTase MT6 SM50 SM30 hat MT7 SM50 SM30 CAT BMP5-7 SM30 SM30 p19 SM50 SM50 Stress p16 Skeletogenesis Development/Differentiation MT4 Detoxification Up-regulated genes Down-regulated genes (Modified from Varrella et al., 2014)

Summary Decadienal Heptadienal Octadienal Hsp70 Hsp70 Hsp70 Hsp60 MTase MTase hsp56 p38 MAPK SM50 SM50 SM50 SM30 uni SM30 uni p19 BMP5-7 p16 p19 hat hat Wnt5 Stress Wnt6 Wnt6 Wnt6 Skeletogenesis Wnt8 hat Development/Differentiation sox9 Detoxification Alix Up-regulated genes CAT MT4 MT4 Down-regulated genes MT4 MT6 MT6 MT6 MT8 MT8 MT8 (Modified from Varrella et al., 2014)

Morphological conclusions  Do PUAs affect the sea urchin embryogenesis? The aldehydes induced teratogenesis on sea urchin embryos in a dose-dependent manner. The teratogenic effect after a week of PUAs exposure appeared to be stronger for decadienal and heptadienal.  When do PUAs act on embryo development? The aldehydes could compromise the normal embryonic development affecting embryos before and/or soon after the fertilization

Molecular conclusions  Do PUAs have molecular targets? And could these targets be considered as possible biomarkers for environmental stress response? The aldehydes have different molecular targets, affecting the expression levels of different genes at different times of embryonic development. Stress response Biomarkers

....more in general Ecological relevance considering the importance of diatoms blooms in nutrient-rich acquatic environments … .. .....molecular evidence for the toxic effects of the diatom-derived PUAs .....novel tools for understing the cellular mechanisms of the response to aldehydes exposure to the benthic organisms. Future perspectives  Effects of many other Oxylipins (HEPEs)  Functional networks of the PUAs target genes  Oxyditave stress effects induced by Oxylipins