Physiological and molecular responses of the copepods Acartia clausi and Acartia tonsa to nickel nanoparticles and nickel chloride.

Nickel compounds in dissolved form or as nanoparticles may affect planktonic invertebrates in marine ecosystems. Here, we assessed the physiological (naupliar mortality, egg production, egg hatching success) and molecular (quantitative gene expression) responses of the crustacean copepods Acartia clausi (indigenous Mediterranean species) and Acartia tonsa (model organism in ecotoxicology), to nickel nanoparticles (NiNPs) and nickel chloride (NiCl2), over time. We also measured NPs size and the temporal release of Ni ions in aqueous solution, through dynamic light scattering (DLS) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively. Nauplii of A. clausi were highly vulnerable to NiCl2 in the 48 h acute test, with an EC50 in the range of Ni concentrations measured in polluted waters. Females of both species exhibited a decreased egg production and hatching success after the 4-day exposure to NiNPs. Molecular responses in A. clausi incubated in NiNPs and NiCl2 showed a stronger up- or down-regulation, compared to A. tonsa, of genes associated with detoxification (phospholipid-hydroperoxide glutathione peroxidase, glutathione-S-transferase sigma), oxidative stress (superoxide dismutase), nervous system functioning (acetylcholinesterase), and oogenesis (vitellogenin). In conclusion, new information was here obtained on the effects of different forms of nickel on physiological and molecular responses of A. clausi, that could help to identify biomarker genes of exposure to be used as early-warning indicators. Our results also highlighted the need of employing indigenous copepod species to better evaluate the ecotoxicological impact of pollutants in different geographical area.

Prostaglandin pathway activation in the diatom Skeletonema marinoi under grazer pressure.

Prostaglandins (Pgs) are eicosanoid lipid mediators detected in all vertebrates, in some marine invertebrates, macroalgae and in diatoms, a class of eukaryotic microalgae composing the phytoplankton. The enzymes involved in the Pgs pathway were found to be differentially expressed in two strains of the diatom Skeletonema marinoi, named FE7 and FE60, already known to produce different levels of oxylipins, a class of secondary metabolites involved in the defence of diatoms against copepod predation, with FE7 being higher producer than FE60. In the present study we investigated the response of genes involved in the production of oxylipins and Pgs, evaluating their expression after the exposure to the copepod Temora stylifera. Our results highlighted a grazer feeding preference for FE60, the strain having low oxylipins content and reduced expression of Pgs enzymes, and an impact on the gene expression of the enzymes involved in oxylipins (i.e. lipoxygenase) and Pgs (i.e. cyclooxygenase) biosynthesis, especially in FE7. A time course evaluation of the gene expression over 24 h showed an upregulation of the essential enzyme in the Pgs pathway, the cyclooxygenase, in FE60 after 6 h of exposure to the grazer, differently from FE7 where no upregulation of gene expression in the presence of copepods was revealed. These results provide preliminary indications regarding the existence of a complex involvement of the Pgs pathway in the prey-predator interaction that requires further investigations.

Transcriptomic analysis reveals responses to a polluted sediment in the Mediterranean copepod Acartia clausi.

Marine sediments are regarded as sinks for several classes of contaminants. Characterization and effects of sediments on marine biota now require a multidisciplinary approach, which includes chemical and ecotoxicological analyses and molecular biomarkers. Here, a gene expression study was performed to measure the response of adult females of the Mediterranean copepod Acartia clausi to elutriates of polluted sediments (containing high concentrations of polycyclic aromatic hydrocarbons, PAHs, and heavy metals) from an industrial area in the Southern Tyrrhenian Sea (Bagnoli-Coroglio). Functional annotation of the A. clausi transcriptome generated as reference here, showed a good quality of the assembly and great homology with other copepod and crustacean sequences in public databases. This is one of the few available transcriptomic resources for this widespread copepod species of great ecological relevance in temperate coastal areas. Differential expression analysis between females exposed to the elutriate and those in control seawater identified 1000 differentially expressed genes, of which 743 up- and 257 down-regulated. Within the up-regulated genes, the most represented functions were related to proteolysis (lysosomal protease, peptidase, cathepsin), response to stress and detoxification (heat-shock protein, superoxide dismutase, glutathione-S-transferase, cytochrome P450), and cytoskeleton structure (α- and ß-tubulin). Down-regulated genes were mostly involved with ribosome structure (ribosomal proteins) and DNA binding (histone proteins, transcription factors). Overall, these results suggest that processes such as transcription, translation, protein degradation, metabolism of biomolecules, reproduction, and xenobiotic detoxification were altered in the copepod in response to polluted elutriates. In conclusion, our results contribute to gaining information on the transcriptomic responses of copepods to polluted sediments. They will also prompt the selection of genes of interest to be used as biomarkers of exposure to PAHs and heavy metals in molecular toxicology studies on copepods, and in general, in comparative functional genomic studies on marine zooplankton.

Chemosensory-Related Genes in Marine Copepods.

Living organisms deeply rely on the acquisition of chemical signals in any aspect of their life, from searching for food, mating and defending themselves from stressors. Copepods, the most abundant and ubiquitous metazoans on Earth, possess diversified and highly specified chemoreceptive structures along their body. The detection of chemical stimuli activates specific pathways, although this process has so far been analyzed only on a relatively limited number of species. Here, in silico mining of 18 publicly available transcriptomes is performed to delve into the copepod chemosensory genes, improving current knowledge on the diversity of this multigene family and on possible physiological mechanisms involved in the detection and analysis of chemical cues. Our study identifies the presence of ionotropic receptors, chemosensory proteins and gustatory receptors in copepods belonging to the Calanoida, Cyclopoida and Harpacticoida orders. We also confirm the absence in these copepods of odorant receptors and odorant-binding proteins agreeing with their insect specificity. Copepods have evolved several mechanisms to survive in the harsh marine environment such as producing proteins to respond to external stimulii. Overall, the results of our study open new possibilities for the use of the chemosensory genes as biomarkers in chemical ecology studies on copepods and possibly also in other marine holozooplankters.

First Report of OvoA Gene in Marine Arthropods: A New Candidate Stress Biomarker in Copepods.

Ovothiol is one of the most powerful antioxidants acting in marine organisms as a defense against oxidative stress during development and in response to environmental cues. The gene involved in the ovothiol biosynthesis, OvoA, is found in almost all metazoans, but open questions existed on its presence among arthropods. Here, using an in silico workflow, we report a single OvoA gene in marine arthropods including copepods, decapods, and amphipods. Phylogenetic analyses indicated that OvoA from marine arthropods separated from the other marine phyla (e.g., Porifera, Mollusca) and divided into two separate branches, suggesting a possible divergence through evolution. In the copepod Calanus finmarchicus, we suggest that OvoA has a defense role in oxidative stress as shown by its high expression in response to a toxic diet and during the copepodite stage, a developmental stage that includes significant morphological changes. Overall, the results of our study open possibilities for the use of OvoA as a biomarker of stress in copepods and possibly also for other marine holozooplankters. The finding of OvoA in copepods is also promising for the drug discovery field, suggesting the possibility of using copepods as a new source of bioactive compounds to be tested in the marine biotechnological sector.

Historical control data in ecotoxicology: Eight years of tests with the copepod Acartia tonsa.

The calanoid copepod Acartia tonsa is one of the most frequently used organisms in acute, short-term bioassays to assess effects induced by marine matrices or chemicals on different life stages. Physiological responses in such tests can be highly variable and historical control data (HCD), values recorded from previous studies performed under similar conditions, can be useful to recognise the average responses over time. Here, we analysed egg hatching success and larval (naupliar) immobilisation/mortality of A. tonsa Mediterranean strain, cultured in laboratory conditions since 2008 and used as model organisms in ecotoxicology tests. Our aims were to evaluate the physiological response and sensitivity of A. tonsa over eight years of bioassays, and to compare our HCD with reference values, in order to assess the suitability of such a long-term culture for ecotoxicology studies. Acartia tonsa eggs were exposed for 48 h to the reference toxicant nickel chloride (NiCl2) and the % of egg hatching success and naupliar viability were compared to controls. A total of 59 acute tests, displayed in Shewhart-like control charts, showed a high mean percentage of egg hatching success (85.60% ± 5.90 SD) recorded for the whole period, and a low mean percentage of naupliar immobilisation/mortality (6.73% ± 6.38 SD) in controls. Effective concentration (EC50) for NiCl2 registered a stable mean of 0.14 mg Ni/L (± 0.047 SD) over time. Overall, our long-term dataset confirms the suitability of this copepod species for ecotoxicology studies even after years of culturing in laboratory conditions. It is advisable that other laboratories with long-term datasets made their own control charts, to allow data comparison and to improve test protocols. Considering our HCD, we suggest an EC50 of NiCl2 of 0.14 ± 0.09 mg Ni/L for acute tests with the Mediterranean strain of A. tonsa.

RNA-Seq and differential gene expression analysis in Temora stylifera copepod females with contrasting non-feeding nauplii survival rates: an environmental transcriptomics study.

BACKGROUND: Copepods are fundamental components of pelagic food webs, but reports on how molecular responses link to reproductive success in natural populations are still scarce. We present a de novo transcriptome assembly and differential expression (DE) analysis in Temora stylifera females collected in the Gulf of Naples, Mediterranean Sea, where this copepod dominates the zooplankton community. High-Throughput RNA-Sequencing and DE analysis were performed from adult females collected on consecutive weeks (May 23rd and 30th 2017), because opposite naupliar survival rates were observed. We aimed at detecting key genes that may have influenced copepod reproductive potential in natural populations and whose expression was potentially affected by phytoplankton-derived oxylipins, lipoxygenase-derived products strongly impacting copepod naupliar survival. RESULTS: On the two sampling dates, temperature, salinity, pH and oxygen remained stable, while variations in phytoplankton cell concentration, oxylipin concentration and oxylipin-per-diatom-cell production were observed. T. stylifera naupliar survival was 25% on May 23rd and 93% on May 30th. De novo assembly generated 268,665 transcripts (isoforms) and 120,749 unique 'Trinity predicted genes' (unigenes), of which 50% were functionally annotated. Out of the 331 transcript isoforms differentially expressed between the two sampling dates, 119 sequences were functionally annotated (58 up- and 61 down-regulated). Among predicted genes (unigenes), 144 sequences were differentially expressed and 31 (6 up-regulated and 25 down-regulated) were functionally annotated. Most of the significantly down-regulated unigenes and isoforms were A5 Putative Odorant Binding Protein (Obp). Other differentially expressed sequences (isoforms and unigenes) related to developmental metabolic processes, protein ubiquitination, response to stress, oxidation-reduction reactions and hydrolase activities. DE analysis was validated through Real Time-quantitative PCR of 9 unigenes and 3 isoforms. CONCLUSIONS: Differential expression of sequences involved in signal detection and transduction, cell differentiation and development offered a functional interpretation to the maternally-mediated low naupliar survival rates observed in samples collected on May 23rd. Down-regulation of A5 Obp along with higher quantities of oxylipins-per-litre and oxylipins-per-diatom-cell observed on May 23rd could suggest oxylipin-mediated impairment of naupliar survival in natural populations of T. stylifera. Our results may help identify biomarker genes explaining variations in copepod reproductive responses at a molecular level.

De Novo Transcriptome Assembly and Gene Expression Profiling of the Copepod Calanus helgolandicus Feeding on the PUA-Producing Diatom Skeletonema marinoi.

Diatoms are the dominant component of the marine phytoplankton. Several diatoms produce secondary metabolites, namely oxylipins, with teratogenic effects on their main predators, crustacean copepods. Our study reports the de novo assembled transcriptome of the calanoid copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi. Differential expression analysis was also performed between copepod females exposed to the diatom and the control flagellate Prorocentrum minimum, which does not produce oxylipins. Our results showed that transcripts involved in carbohydrate, amino acid, folate and methionine metabolism, embryogenesis, and response to stimulus were differentially expressed in the two conditions. Expression of 27 selected genes belonging to these functional categories was also analyzed by RT-qPCR in C. helgolandicus females exposed to a mixed solution of the oxylipins heptadienal and octadienal at the concentration of 10 µM, 15 µM, and 20 µM. The results confirmed differential expression analysis, with up-regulation of genes involved in stress response and down-regulation of genes associated with folate and methionine metabolism, embryogenesis, and signaling. Overall, we offer new insights on the mechanism of action of oxylipins on maternally-induced embryo abnormality. Our results may also help identify biomarker genes associated with diatom-related reproductive failure in the natural copepod population at sea.

Assessment of the relative sensitivity of the copepods Acartia tonsa and Acartia clausi exposed to sediment-derived elutriates from the Bagnoli-Coroglio industrial area.

The sensitivity of the copepods Acartia tonsa, commonly used in standardized tests for environmental risk assessment and A. clausi, the dominant autochthonous congener species in the Mediterranean Sea, was assessed using sediment-derived elutriates from the industrial area of Bagnoli-Coroglio and nickel chloride as referent toxicant. Acute A. clausi naupliar immobilization test showed EC50 for elutriates E25, E56 and E84 of 23.3%, 80.5% and >100%, respectively, compared to 59.5%, 66.6% and >100% in A. tonsa. In the 7 day sublethal test, a reduction in A. clausi egg production rates was observed in all elutriates, but only in E56 for A. tonsa. Elutriate 56, which contained the highest amount of polycyclic aromatic hydrocarbons, also induced 70% mortality in A. clausi females. Although A. clausi was more sensitive than A. tonsa, the two species had convergent responses to the three elutriates, thus opening the venue for a potential use of A. clausi in standardized ecotoxicity tests.

Density-dependent oxylipin production in natural diatom communities: possible implications for plankton dynamics.

Oxylipins are important signal transduction lipoxygenase-derived products of fatty acids that regulate a variety of physiological and pathological processes in plants and animals. In marine diatoms, these molecules can be highly bioactive, impacting zooplankton grazers, bacteria and other phytoplankton. However, the ultimate cause for oxylipin production in diatoms is still poorly understood, from an evolutionary perspective. Here we analysed production of particulate linear oxygenated fatty acids (LOFAs, previously named non-volatile oxylipins) from natural phytoplankton collected weekly for 1 year. We demonstrate for the first time that diatoms are the main LOFA producers in natural phytoplankton assemblages. Interestingly, LOFA-per-cell production decreased with increasing diatom density and was not due to major changes in diatom community composition. An inverse relation was confirmed at a global scale by analysing diatom lipoxygenase unigenes and metagenomes from Tara Oceans datasets. A network analysis suggested that different LOFAs could contribute to modulate co-variations of different diatom taxa. Overall, we offer new insights in diatom chemical ecology, possibly explaining the evolution of oxylipin synthesis in diatoms.

UPLC⁻MS/MS Identification of Sterol Sulfates in Marine Diatoms.

Diatoms are unicellular eukaryotic organisms that play a key ecological and biogeochemical role in oceans as major primary producers. Recently, these microalgae have also attracted interest as a promising source of functional products with widespread relevance. Progress in the knowledge of cell and molecular biology of diatoms is envisaged as a key step to understanding regulation of their life cycle in marine environments as well as facilitating their full and profitable exploitation by biotechnological platforms. Recently, we identified sterol sulfates (StS) as regulatory molecules of cell death in the diatom Skeletonema marinoi. As these compounds may have a general role in diatom physiology and chemical signals in aquatic systems, we investigated a suitable tool for their analysis in laboratory and field samples. Herein, we describe a sensitive, fast, and efficient ultra performance liquid chromatography⁻mass spectrometry (UPLC⁻MS) method for qualitative and quantitative analysis of StS from crude extract of diatoms and other microalgae. The method was applied to 13 different strains of our collection of marine protists. This first study suggested a species-specific distribution of StS and identified the sulfated derivatives of 24-methylene cholesterol and 24-methyl cholesterol as the most common members in diatoms.

De novo transcriptome assembly and differential gene expression analysis of the calanoid copepod Acartia tonsa exposed to nickel nanoparticles.

The calanoid copepod Acartia tonsa is a reference species in standardized ecotoxicology bioassay. Despite this interest, there is a lack of knowledge on molecular responses of A. tonsa to contaminants. We generated a de novo assembled transcriptome of A. tonsa exposed 4 days to 8.5 and 17 mg/L nickel nanoparticles (NiNPs), which have been shown to reduce egg hatching success and larval survival but had no effects on the adults. Aims of our study were to 1) improve the knowledge on the molecular responses of A. tonsa copepod and 2) increase the genomic resources of this copepod for further identification of potential biomarkers of NP exposure. The de novo assembled transcriptome of A. tonsa consisted of 53,619 unigenes, which were further annotated to nr, GO, KOG and KEGG databases. In particular, most unigenes were assigned to Metabolic and Cellular processes (34-45%) GO terms, and to Human disease (28%) and Organismal systems (23%) KEGG categories. Comparison among treatments showed that 373 unigenes were differentially expressed in A. tonsa exposed to NiNPs at 8.5 and 17 mg/L, with respect to control. Most of these genes were downregulated and took part in ribosome biogenesis, translation and protein turnover, thus suggesting that NiNPs could affect the copepod ribosome synthesis machinery and functioning. Overall, our study highlights the potential of toxicogenomic approach in gaining more mechanistic and functional information about the mode of action of emerging compounds on marine organisms, for biomarker discovering in crustaceans.

New oxylipins produced at the end of a diatom bloom and their effects on copepod reproductive success and gene expression levels.

Diatoms are dominant photosynthetic organisms in the world's oceans and are considered essential in the transfer of energy to higher trophic levels. However, these unicellular organisms produce secondary metabolites deriving from the oxidation of fatty acids, collectively termed oxylipins, with negative effects on predators, such as copepods, that feed on them (e.g. reduction in survival, egg production and hatching success) and, indirectly, on higher trophic levels. Here, a multidisciplinary study (oxylipin measurements, copepod fitness, gene expression analyses, chlorophyll distribution, phytoplankton composition, physico-chemical characteristics) was carried out at the end of the spring diatom bloom in April 2011 in the Northern Adriatic Sea (Mediterranean Sea) in order to deeply investigate copepod-diatom interactions, chemical communication and response pathways. The results show that the transect with the lowest phytoplankton abundance had the lowest copepod egg production and hatching success, but the highest oxylipin concentrations. In addition, copepods in both the analyzed transects showed increased expression levels of key stress-related genes (e.g. heat-shock proteins, catalase, glutathione S-transferase, aldehyde dehydrogenase) compared to control laboratory conditions where copepods were fed with the dinoflagellate Prorocentrum minimum which does not produce any oxylipins. New oxylipins that have never been reported before for microalgae are described for the first time, giving new insights into the complex nature of plant-animal signaling and communication pathways at sea. This is also the first study providing insights on the copepod response during a diatom bloom at the molecular level.

Effects of the oxylipin-producing diatom Skeletonema marinoi on gene expression levels of the calanoid copepod Calanus sinicus.

Diatoms are eukaryotic unicellular plants that constitute one of the major components of marine phytoplankton, comprising up to 40% of annual productivity at sea and representing 25% of global carbon-fixation. Diatoms have traditionally been considered a preferential food for zooplankton grazers such as copepods, but, in the last two decades, this beneficial role has been challenged after the discovery that many species of diatoms produce toxic metabolites, collectively termed oxylipins, that induce reproductive failure in zooplankton grazers. Diatoms are the dominant natural diet of Calanus sinicus, a cold-temperate calanoid copepod that supports secondary production of important fisheries in the shelf ecosystems of the Northwest Pacific Ocean, Yellow Sea, Sea of Japan and South China Sea. In this study, the effect of the oxylipin-producing diatom Skeletonema marinoi on C. sinicus has been evaluated by analyzing expression level changes of genes involved in defense and detoxification systems. Results show that C. sinicus is more resistant to a diet of this diatom species in terms of gene expression patterns, compared to the congeneric species Calanus helgolandicus which is an important constituent of the temperate waters of the Atlantic Ocean and northern Mediterranean Sea. These findings contribute to the better understanding of genetic and/or phenotypic flexibility of copepod species and their capabilities to cope with stress by identifying molecular markers (such as stress and detoxification genes) as biosensors for environmental perturbations (e.g. toxins and contaminants) affecting marine copepods.

High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study.

Despite the ecological importance of copepods, few Next Generation Sequencing studies (NGS) have been performed on small crustaceans, and a standard method for RNA extraction is lacking. In this study, we compared three commonly-used methods: TRIzol®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and quantity of RNA from copepods for NGS. Total RNA was extracted from the copepods Calanus helgolandicus, Centropages typicus and Temora stylifera and its quantity and quality were evaluated using NanoDrop, agarose gel electrophoresis and Agilent Bioanalyzer. Our results demonstrate that preservation of copepods in RNAlater® and extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus. Intriguingly, C. helgolandicus 28S rRNA is formed by two subunits that separate after heat-denaturation and migrate along with 18S rRNA. This unique property of protostome RNA has never been reported in copepods. Overall, our comparative study on RNA extraction protocols will help increase gene expression studies on copepods using high-throughput applications, such as RNA-Seq and microarrays.

Impact of the diatom-derived polyunsaturated aldehyde 2-trans,4-trans decadienal on the feeding, survivorship and reproductive success of the calanoid copepod Temora stylifera.

Many diatoms, a major class of unicellular algae contributing to over 45% of oceanic primary production, are known to induce deleterious effects on reproductive processes in crustacean copepods. This is mainly due to the production of teratogenic oxylipins, including polyunsaturated aldehydes (PUAs). Here we tested the direct effect of the PUA 2E,4E-decadienal (DD) on feeding activity, survivorship and reproductive success of the calanoid copepod Temora stylifera. DD-inoculated cultures induced high mortality at concentrations above 3 µg mL(-1) compared to controls in both males and females, with males having a higher mortality. Low DD concentrations triggered an increase in female filtration and ingestion rates. Egg production rates and hatching times were also higher in the presence of DD, whereas egg hatching success decreased with increasing DD concentration. Our study shows, for the first time, that the presence of diatom PUAs may increase feeding rates in copepods.

Insights into the transcriptome of the marine copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi.

Diatoms dominate productive regions in the oceans and have traditionally been regarded as sustaining the marine food chain to top consumers and fisheries. However, many of these unicellular algae produce cytotoxic oxylipins that impair reproductive and developmental processes in their main grazers, crustacean copepods. The molecular mode of action of diatoms and diatom oxylipins on copepods is still unclear. In the present study we generated two Expressed Sequence Tags (ESTs) libraries of the copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi and the cryptophyte Rhodomonas baltica as a control, using suppression subtractive hybridization (SSH). Our aim was to investigate differences in the transcriptome between females fed toxic and non-toxic food and identify differentially expressed genes and biological processes targeted by this diatom. We produced 947 high quality ESTs from both libraries, 475 of which were functionally annotated and deposited in GenBank. Clustering and assembling of ESTs resulted in 376 unique transcripts, 200 of which were functionally annotated. Functional enirchment analysis between the two SSH libraries showed that ESTs belonging to biological processes such as response to stimuli, signal transduction, and protein folding were significantly over-expressed in the S. marinoi-fed C. helgolandicus compared to R. baltica-fed C. helgolandicus library. These findings were confirmed by RT-qPCR analysis. In summary, 2 days of feeding on S. marinoi activated a generalized Cellular Stress Response (CSR) in C. helgolandicus, by over-expressing genes of molecular chaperones and signal transduction pathways that protect the copepod from the immediate effects of the diatom diet. Our results provide insights into the response of copepods to a harmful diatom diet at the transcriptome level, supporting the hypothesis that diatom oxylipins elicit a stress response in the receiving organism. They also increase the genomic resources for this copepod species, whose importance could become ever more relevant for pelagic ecosystem functioning in European waters due to global warming.

Copepod population-specific response to a toxic diatom diet.

Diatoms are key phytoplankton organisms and one of the main primary producers in aquatic ecosystems. However, many diatom species produce a series of secondary metabolites, collectively termed oxylipins, that disrupt development in the offspring of grazers, such as copepods, that feed on these unicellular algae. We hypothesized that different populations of copepods may deal differently with the same oxylipin-producing diatom diet. Here we provide comparative studies of expression level analyses of selected genes of interest for three Calanus helgolandicus populations (North Sea, Atlantic Ocean and Mediterranean Sea) exposed to the same strain of the oxylipin-producing diatom Skeletonema marinoi using as control algae the flagellate Rhodomonas baltica. Expression levels of detoxification enzymes and stress proteins (e.g. glutathione S-transferase, glutathione synthase, superoxide dismutase, catalase, aldehyde dehydrogenases and heat shock proteins) and proteins involved in apoptosis regulation and cell cycle progression were analyzed in copepods after both 24 and 48 hours of feeding on the diatom or on a control diet. Strong differences occurred among copepod populations, with the Mediterranean population of C. helgolandicus being more susceptible to the toxic diet compared to the others. This study opens new perspectives for understanding copepod population-specific responses to diatom toxins and may help in underpinning the cellular mechanisms underlying copepod toxicity during diatom blooms.

Molecular evidence of the toxic effects of diatom diets on gene expression patterns in copepods.

BACKGROUND: Diatoms are dominant photosynthetic organisms in the world's oceans and are considered essential in the transfer of energy through marine food chains. However, these unicellular plants at times produce secondary metabolites such as polyunsaturated aldehydes and other products deriving from the oxidation of fatty acids that are collectively termed oxylipins. These cytotoxic compounds are responsible for growth inhibition and teratogenic activity, potentially sabotaging future generations of grazers by inducing poor recruitment in marine organisms such as crustacean copepods. PRINCIPAL FINDINGS: Here we show that two days of feeding on a strong oxylipin-producing diatom (Skeletonema marinoi) is sufficient to inhibit a series of genes involved in aldehyde detoxification, apoptosis, cytoskeleton structure and stress response in the copepod Calanus helgolandicus. Of the 18 transcripts analyzed by RT-qPCR at least 50% were strongly down-regulated (aldehyde dehydrogenase 9, 8 and 6, cellular apoptosis susceptibility and inhibitor of apoptosis IAP proteins, heat shock protein 40, alpha- and beta-tubulins) compared to animals fed on a weak oxylipin-producing diet (Chaetoceros socialis) which showed no changes in gene expression profiles. CONCLUSIONS: Our results provide molecular evidence of the toxic effects of strong oxylipin-producing diatoms on grazers, showing that primary defense systems that should be activated to protect copepods against toxic algae can be inhibited. On the other hand other classical detoxification genes (glutathione S-transferase, superoxide dismutase, catalase, cytochrome P450) were not affected possibly due to short exposure times. Given the importance of diatom blooms in nutrient-rich aquatic environments these results offer a plausible explanation for the inefficient use of a potentially valuable food resource, the spring diatom bloom, by some copepod species.

Aldehyde-encapsulating liposomes impair marine grazer survivorship.

In the last decade, there has been an increased awareness that secondary metabolites produced by marine diatoms negatively impact the reproductive success of their principal predators, the copepods. Several oxylipins, products of the enzymatic oxidation of fatty acids, are produced when these unicellular algae are damaged, as occurs during grazing. In the past, the dinoflagellate Prorocentrum minimum, which does not produce the oxylipin 2-trans,4-trans-decadienal (DD), has been used as a live carrier to calculate daily ingestion rates of this molecule by copepod crustaceans. However, since the interaction between oxylipins and live carriers is unknown, the question as to how much and for how long ingestion of these molecules affects copepod reproduction remains a critical point to understanding the functional role of such compounds at sea. In the investigation presented here we used giant liposomes ( approximately 7 mum) as a delivery system for the oxylipin DD, prepared in the same size range as copepod food and containing known amounts of DD. The aim of this work was to relate the ingestion of DD to the reproductive failure of the copepods Temora stylifera and Calanus helgolandicus. Liposomes were very stable over time and after 10 days of feeding, liposomes encapsulating DD reduced egg hatching success and female survival with a concomitant appearance of apoptosis in both copepod embryos and female tissues. Concentrations of DD inducing blockage were one order of magnitude lower that those used in classical feeding experiments demonstrating that liposomes are a useful tool to quantitatively analyze the impact of toxins on copepods.