Accumulation of Polyunsaturated Aldehydes in the Gonads of the Copepod Acartia tonsa Revealed by Tailored Fluorescent Probes

Polyunsaturated aldehydes (PUAs) are released by several diatom species during predation. Besides other attributed activities, these oxylipins can interfere with the reproduction of copepods, important predators of diatoms. While intensive research has been carried out to document the effects of PUAs on copepod reproduction, little is known about the underlying mechanistic aspects of PUA action. Especially PUA uptake and accumulation in copepods has not been addressed to date. To investigate how PUAs are taken up and interfere with the reproduction in copepods we developed a fluorescent probe containing the α,β,γ,δ-unsaturated aldehyde structure element that is essential for the activity of PUAs as well as a set of control probes. We developed incubation and monitoring procedures for adult females of the calanoid copepod Acartia tonsa and show that the PUA derived fluorescent molecular probe selectively accumulates in the gonads of this copepod. In contrast, a saturated aldehyde derived probe of an inactive parent molecule was enriched in the lipid sac. This leads to a model for PUAs'' teratogenic mode of action involving accumulation and covalent interaction with nucleophilic moieties in the copepod reproductive tissue. The teratogenic effect of PUAs can therefore be explained by a selective targeting of the molecules into the reproductive tissue of the herbivores, while more lipophilic but otherwise strongly related structures end up in lipid bodies.     

Morphological and ultrastructural characterization of sea urchin immune cells

The free circulating coelomocytes in the coelomic cavity of echinoderms are considered to be immune effectors by phagocytosis, encapsulation, cytotoxicity, and by the production of antimicrobial agents. Although echinoderms (especially sea urchin embryo) have been used as a model organisms in biology, no uniform criteria exist for classification of coelomocytes in echinoderms, and few studies have reported about the biological functions of their coelomocytes. Hence, we study the coelomocytes in the echinoid sea urchin, Paracentrotus lividus, and describe their morphological and ultrastructural features using light and transmission electron microscopes. We classify the coelomocytes of P. lividus into red spherule and colorless spherule cells, small cells, vibratile cells, and phagocytic cells; petaloid and filopodial cells. To our knowledge, this is the first report describing ultrastructural details of the coelomocytes of P. lividus. J. Morphol. 276:583–588, 2015. © 2015 Wiley Periodicals, Inc.     

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.     

Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus

We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus; this study, performed at DNA level only, lends itself as starting point to verify that these clusters could contain transcribed genes, then, to demonstrate the presence of heterogeneity at functional RNA level, also. In the present work we report in P. lividus ribosomes the existence of several transcribed variants of the 5S rRNA and we associate all transcribed variants to the cluster to which belong.     

First steps towards Echinometra lucunter embryo cryopreservation

We have studied the sensitivity to cryoprotecting agents of different embryos of the local sea urchin, Echinometra lucunter which is the species used for embryo-larval bioassays in Brazil. We have located significant differences between both species sensitivity to cryoprotecting agents; while for P. lividus propylene glycol was the less toxic compound for most development stages, whereas for E. lucunter is was the most toxic and the least toxic was Dimethyl sulfoxide. There is a significant difference between development stages as well; in the case of P. lividus, the blastula embryo was the most resistant to the cryoprotecting agents, meanwhile for E. lucunter, it was the fertilized oocyte. This is a very promising result, a really early embryo that is not extremely sensitive to Me₂SO. Our next aim is to develop a cryopreservation protocol for E. lucunter early embryos and test them in an embryo-larval bioassay.     

Defensome against toxic diatom aldehydes in the sea urchin Paracentrotus lividus

Many diatom species produce polyunsaturated aldehydes, such as decadienal, which compromise embryonic and larval development in benthic organisms. Here newly fertilized Paracentrotus lividus sea urchins were exposed to low concentration of decadienal and the expression levels of sixteen genes, implicated in a broad range of functional responses, were followed by Real Time qPCR in order to identify potential decadienal targets. We show that at low decadienal concentrations the sea urchin Paracentrotus lividus places in motion different classes of genes to defend itself against this toxic aldehyde, activating hsp60 and two proteases, hat and BP10, at the blastula stage and hsp56 and several other genes (14-3-3ε, p38 MAPK, MTase, and GS) at the prism stage. At this latter stage all genes involved in skeletogenesis (Nec, uni, SM50 and SM30) were also down-expressed, following developmental abnormalities that mainly affected skeleton morphogenesis. Moreover, sea urchin embryos treated with increasing concentrations of decadienal revealed a dose-dependent response of activated target genes. Finally, we suggest that this orchestrated defense system against decadienal represents part of the chemical defensome of P. lividus affording protection from environmental toxicants.     

The harpacticoid copepod Tisbe holothuriae is resistant to the insidious effects of polyunsaturated aldehyde-producing diatoms

Numerous species of diatoms liberate oxylipins including polyunsaturated aldehydes (PUAs) in response to cellular damage such as may occur during grazing. PUAs are cyto- and genotoxic and negatively disrupt reproductive processes in copepods, their principal grazers, although experimental evidence would suggest that the grazer response may be species specific. The reproduction of the benthic harpacticoid copepod Tisbe holothuriae was compared over two generations. Copepods were reared using four diet treatments: PUA-producing diatom strains Skeletonema marinoi (Adriatic Sea Isolate FE6) and Melosira nummuloides (CCAP 1048/6); and non-PUA-producing diatom strains Phaeodactylum tricornutum (CCAP 1052/A) and S. marinoi (Seasalter (Walney) Ltd). Life tables were generated for each treatment using measured reproductive parameters and the net reproductive rate (R₀) calculated. No significant differences were observed between the individual reproductive parameters of T. holothuriae fed PUA-producing diatoms compared to those fed non-PUA-producing diatoms although diets of P. tricornutum resulted in some decreases in individual reproductive parameters in the second generation. There were no significant differences in the R₀ values between the four tested diets. These observations indicate that T. holothuriae exhibits a tolerance of known PUA-producing diatom diets that has not been similarly demonstrated in pelagic calanoid copepods. Harpacticoid copepods may have a greater capacity to detoxify diatom oxylipins than their planktonic calanoid counterparts.     

Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)‐actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross‐reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2‐cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli‐like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.     

Hemocytes/coelomocytes DNA content in five marine invertebrates: cell cycles and genome sizes

The hemocytes/coelomocytes DNA content in five selected marine invertebrates (sea mouse Aphrodita aculeata, spiny crab Maja crispata, sea star Echinaster sepositus, sea urchin Paracentrotus lividus, and tunicate Phallusia mammillata) was investigated by flow cytometry. The cell cycle analyses identified sea mouse coelomocytes as proliferating cells and revealed that spiny crab hemocytes and sea urchin coelomocytes complete their division in the hemolymph and coelom, respectively. The genome sizes of sea mouse and spiny crab are reported for the first time. The diploid DNA content (2C) in sea mouse A. aculeate was 1.24 pg, spiny crab M. crispata 7.76 pg, red starfish E. sepositus 1.52 pg and sea urchin P. lividus 1.08 pg. The mean diploid DNA content in tunicate P. mammillata was 0.11 pg with a high interindividual variability (45%). The presented results provide a useful database for future studies in the field of invertebrate physiology, ecotoxicology, biodiversity, species conservation and phylogeny.     

Studies of Delta-Aminolevulinic Acid Dehydrase from Skeletonema costatum, a Marine Plankton Diatom

The accumulation of δ-aminolevulinic acid and activities of δ-aminolevulinic acid dehydrase were examined in the marine diatom, Skeletonema costatum, grown in the presence of levulinic acid. Levulinic acid concentrations greater than 10 mm affect growth and morphology, and inhibit chlorophyll synthesis. The algae recover from the effects of levulinic acid after 48 hours of exposure. The recovery is characterized by increased cellular cholorphyll content, decreased δ-aminolevulinic acid accumulation, decreased 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-enhanced in vivo fluorescence, and the induction of a levulinic acid-activated δ-aminolevulinic acid dehydrase which does not follow Michaelis-Menten kinetics. The data indicate that levulinic acid blocks may be ineffective in vivo, and that δ-aminolevulinic acid is metabolized to amino and dicarboxylic acids. δ-Aminolevulinic acid dehydrase activities are used to estimate the capacity for chlorophyll synthesis. Results suggest this diatom may be capable of rapid chlorophyll turnover, which would allow the plant to light-shade adapt on the time scales appropriate to vertical mixing rates in the sea.     

Cascading Effects of Ocean Acidification in a Rocky Subtidal Community

Temperate marine rocky habitats may be alternatively characterized by well vegetated macroalgal assemblages or barren grounds, as a consequence of direct and indirect human impacts (e.g. overfishing) and grazing pressure by herbivorous organisms. In future scenarios of ocean acidification, calcifying organisms are expected to be less competitive: among these two key elements of the rocky subtidal food web, coralline algae and sea urchins. In order to highlight how the effects of increased pCO₂ on individual calcifying species will be exacerbated by interactions with other trophic levels, we performed an experiment simultaneously testing ocean acidification effects on primary producers (calcifying and non-calcifying algae) and their grazers (sea urchins). Artificial communities, composed by juveniles of the sea urchin Paracentrotus lividus and calcifying (Corallina elongata) and non-calcifying (Cystoseira amentacea var stricta, Dictyota dichotoma) macroalgae, were subjected to pCO₂ levels of 390, 550, 750 and 1000 µatm in the laboratory. Our study highlighted a direct pCO₂ effect on coralline algae and on sea urchin defense from predation (test robustness). There was no direct effect on the non-calcifying macroalgae. More interestingly, we highlighted diet-mediated effects on test robustness and on the Aristotle''s lantern size. In a future scenario of ocean acidification a decrease of sea urchins'' density is expected, due to lower defense from predation, as a direct consequence of pH decrease, and to a reduced availability of calcifying macroalgae, important component of urchins'' diet. The effects of ocean acidification may therefore be contrasting on well vegetated macroalgal assemblages and barren grounds: in the absence of other human impacts, a decrease of biodiversity can be predicted in vegetated macroalgal assemblages, whereas a lower density of sea urchin could help the recovery of shallow subtidal rocky areas affected by overfishing from barren grounds to assemblages dominated by fleshy macroalgae.     

The reproductive response of the sea urchins Paracentrotus lividus (G.) and Psammechinus miliaris (L.) to a hyperproteinated macrophytic diet

The sea urchins Paracentrotus lividus and Psammechinus miliaris are submitted to the same environmental conditions in the Bay of Brest. The relationship between seasonal changes in food source quality and their gonad production was investigated in reproducing experimentally these conditions. In a first stage two macroalgae (Palmaria palmata and Laminaria digitata) were tested. P. miliaris showed a stronger preference for P. palmata and over a year-long experiment both urchins progressively preferred P. palmata. Seasonal variations in the chemical composition of P. palmaria were observed in the Bay of Brest: total carbohydrates were important and the relative maximum (about 50%) was reached between February and August; the lipid level was low and had a relative maximum of about 1% in June and August. Total protein in P. palmaria was high compared to other seaweeds: the maximum value (25%) was observed in June, which was probably due to the maintenance of nitrogen nutrient in the bay.In the second stage of the study, seasonal changes in biochemical components of ingestion and absorption of the two sea urchins were followed in the laboratory using a monospecific diet of P. palmaria. The patterns of total carbohydrates and lipid absorption were very similar for both sea urchin species. Carbohydrates were absorbed strongly and uniformly, year round. Lipid absorption mimicked the lipid nutrient pattern in the food source. Only changes in protein absorption varied slightly between the two urchin species. Protein absorption was maximal for both species in February and June, but the quantity of absorbed protein was significantly higher in P. miliaris than in P. lividus during February. This increase was concomitant with protein storage in the sea urchin gonads, which peaked in February for P. miliaris and in June for P. lividus. P. lividus had a higher gonad production efficiency, based on gonad yield. The comparison between in situ data and the experimental results suggests that an algal diet more nitrogenous than the in situ algal food source would benefit the herbivorous P. lividus, rather than the more omnivorous species P. miliaris. Although P. milaris has been described as a species with large gonad production potential, P. lividus appears to be a more suitable species for echiniculture conditions.     

Characterization of the Impacts of Living at High Altitude in Taif: Oxidative Stress Biomarker Alterations and Immunohistochemical Changes

At high elevations, the human body experiences a number of pathological, physiological, and biochemical changes, all of which have adverse impacts on human health and organ vitality. This study aimed to investigate the alterations in the liver and kidney biomarkers, oxidative stress markers, gene expression, and cellular histology of rats maintained at high altitudes and normal sea level. A total of twenty male Wistar rats at 2 months of age were randomly assigned to two groups. The rats in group A were maintained at normal sea level in Jeddah, whereas rats in group B were maintained in an area in Taif 2600 m above sea level. After 2 months of housing, orbital blood samples were collected for the analysis of significant biochemical indicators of oxidative stress biomarkers of the liver and kidneys. Liver and kidney tissues from both groups were taken to examine the hepatorenal changes occurring at the biochemical, histological, immunohistochemical, and genetic levels. The results revealed substantial increases in the serum levels of liver and kidney biomarkers (GPT, GOT, urea, and creatinine) and decreases in the serum levels of antioxidant biomarkers (SOD, catalase, GSH, and NO). In parallel, the levels of the malondialdehyde (MDA) tissue damage marker and inflammatory cytokines (IL-1β, TNF-α, and IFN-γ) were increased in the high-altitude group compared to the normal sea level group. In addition, there were significant alterations in the oxidative and inflammatory status of rats that lived at high altitude, with considerable upregulation in the expression of hepatic VEGF, type 1 collagen, Cox-2, TNF-α, and iNOS as well as renal EPASI, CMYC, HIF-α, and EGLN-2 genes in the high-altitude group compared with controls housed at normal sea level. In conclusion, living at high altitude induces hepatorenal damage and biochemical and molecular alterations, all of which may serve as critical factors that must be taken into account for organisms living at high altitudes.