Isabella Buttino

The insidious effect of diatoms on copepod reproduction

The productive regions of the ocean are characterized by seasonal blooms of phytoplankton which are generally dominated by diatoms. This algal class has, therefore, traditionally been regarded as providing the bulk of the food that sustains the marine food chain to top consumers and important fisheries. However, this beneficial role has recently been questioned on the basis of laboratory studies showing that although dominant zooplankton grazers such as copepods feed extensively on diatoms, the hatching success of eggs thus produced is seriously impaired. Here we present evidence from the field showing that the hatching success of wild copepods feeding on a diatom-dominated bloom is also heavily compromised, with only 12% of the eggs hatching compared with 90% in post-bloom conditions. We report on the structure of the three aldehydes isolated from diatoms that are responsible for this biological activity, and show that these compounds arrest embryonic development in copepod and sea urchin bioassays and have antiproliferative and apoptotic effects on human carcinoma cells.

Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom

The growth cycle in nutrient-rich, aquatic environments starts with a diatom bloom that ends in mass sinking of ungrazed cells and phytodetritus. The low grazing pressure on these blooms has been attributed to the inability of overwintering copepod populations to track them temporally. We tested an alternative explanation: that dominant diatom species impair the reproductive success of their grazers. We compared larval development of a common overwintering copepod fed on a ubiquitous, early-blooming diatom species with its development when fed on a typical post-bloom dinoflagellate. Development was arrested in all larvae in which both mothers and their larvae were fed the diatom diet. Mortality remained high even if larvae were switched to the dinoflagellate diet. Aldehydes, cleaved from a fatty acid precursor by enzymes activated within seconds after crushing of the cell, elicit the teratogenic effect. This insidious mechanism, which does not deter the herbivore from feeding but impairs its recruitment, will restrain the cohort size of the next generation of early-rising overwinterers. Such a transgenerational plant–herbivore interaction could explain the recurringly inefficient use of a predictable, potentially valuable food resource—the spring diatom bloom—by marine zooplankton.

A marine diatom-derived aldehyde induces apoptosis in copepod and sea urchin embryos

SUMMARY The diatom-derived aldehyde 2-trans-4-trans-decadienal (DD) was tested as an apoptogenic inducer in both copepod and sea urchin embryos, using terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labelling (TUNEL), DNA fragmentation profiling (laddering) and an assay for caspase-3 activity. DD induced TUNEL positivity and DNA laddering, but not caspase-like activation, in copepod embryos spawned by females fed for 10-15 days the diatom diet Thalassiosira rotula Meunier (in vivo), or when newly spawned eggs were exposed for 1 h to 5 μg ml-1 DD (in vitro). To our knowledge, this is the first time that evidence for an apoptotic process in copepods has been obtained by cytochemical (TUNEL) and biochemical (DNA fragmentation) approaches. The absence of caspase-like activity in copepod embryos suggests that caspase-independent programmed cell death occurs in these organisms. In sea urchin embryos, DD induced apoptosis and also activated a caspase-3-like protease. The saturated aldehyde decanal induced apoptosis at higher concentrations and after a longer incubation period than DD, indicating that α,β-unsaturation of the molecule, coupled with the aldehyde group, is responsible for the greater biological activity of DD. Since diatoms are an important food source for marine herbivores such as copepods and sea urchins, these findings may help explain why unsaturated aldehydes often induce reproductive failure, with important ecological consequences at the population level.

Is postembryonic development in the copepod Temora stylifera negatively affected by diatom diets

Diatoms are major components of the marine microalgae and are generally considered to be the principal food source for small pelagic crustaceans such as copepods. Recently, some species of this algal class have been shown to produce abortifacient compounds (aldehydes) that block copepod embryogenesis, thereby acting as a form of birth control for predatory copepods. To test if diatoms also have deleterious effects on postembryonic development, several diatom species were used to rear larval stages of the calanoid copepod Temora stylifera to adulthood. Our results show that T. stylifera was only able to complete development from hatching to adulthood when reared with the flagellates Isochrysis galbana and the dinoflagellates Prorocentrum minimum and Oxyrrhis marina. The daily development and mortality rates observed were in the range of those reported from previous studies on T. stylifera (0.68–0.82 stage/day and 9.9–12.4%/day, respectively). In contrast, larvae reared on the diatoms Thalassiosira rotula, Skeletonema costatum and Phaeodactylum tricornutum were unable to complete development to adulthood and died without passing the naupliar phase or during the early copepodite stages. Daily mortality rates were higher than for nondiatom species (20.3–35.5%/day). Inhibitory effects on growth were not correlated to cell size of the algae. Final survivorship of larvae fed P. minimum and I. galbana significantly improved (70–80%) when larvae were generated from females preconditioned with P. minimum for 24 h. The same treatment had no beneficial effect on larvae fed with T. rotula or S. costatum, which died again before the adult stage. Although larvae completed development in one replicate with T. rotula and final survivorship improved to 34% (compared to 7% in nauplii from nonconditioned females), this value was, in any case, lower than with nondiatom diets. No morphological aberrations were found in larvae fed on diatoms, even though they were unable to complete their life cycle and died for unknown reasons. By contrast, nauplii produced by females fed the diatom T. rotula for 7 days showed strong congenital defects such as asymmetrical bodies and reduced number of feeding appendages. Our results suggest that diatoms, which have already been shown to have deleterious effects on copepod embryonic development, may also have insidious effects on larval growth of this copepod species.

Nitric Oxide Mediates the Stress Response Induced by Diatom Aldehydes in the Sea Urchin Paracentrotus lividus

Diatoms are ubiquitous and abundant primary producers that have been traditionally considered as a beneficial food source for grazers and for the transfer of carbon through marine food webs. However, many diatom species produce polyunsaturated aldehydes that disrupt development in the offspring of grazers that feed on these unicellular algae. Here we provide evidence that production of the physiological messenger nitric oxide increases after treatment with the polyunsaturated aldehyde decadienal in embryos of the sea urchin Paracentrotus lividus. At high decadienal concentrations, nitric oxide mediates initial apoptotic events leading to loss of mitochondrial functionality through the generation of peroxynitrite. At low decadienal concentrations, nitric oxide contributes to the activation of hsp70 gene expression thereby protecting embryos against the toxic effects of this aldehyde. When nitric oxide levels were lowered by inhibiting nitric oxide synthase activity, the expression of hsp70 in swimming blastula decreased and the proportion of abnormal plutei increased. However, in later pluteus stages nitric oxide was no longer able to exert this protective function: hsp70 and nitric oxide synthase expression decreased with a consequent increase in the expression of caspase-8. Our findings that nitric oxide production increases rapidly in response to a toxic exogenous stimulus opens new perspectives on the possible role of this gas as an important messenger to environmental stress in sea urchins and for understanding the cellular mechanisms underlying toxicity during diatom blooms.

Aldehyde-encapsulating liposomes impair marine grazer survivorship

SUMMARY 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 (∼7 μm) 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.

Spermiotoxicity of nickel nanoparticles in the marine invertebrate Ciona intestinalis (ascidians)

Abstract Nickel nanoparticles (Ni NPs) are increasingly used in modern industries as catalysts, sensors, and in electronic applications. Due to this large use, their inputs into marine environment have significantly increased; however, the potential ecotoxicological effects in marine environment have so far received little attention. In particular, little is known on the impact of NPs on gamete quality of marine organisms and on the consequences on fertility potential. The present study examines, for the first time, the impact of Ni NPs exposure on sperm quality of the marine invertebrate Ciona intestinalis (ascidian). Several parameters related with sperm status such as plasma membrane lipid peroxidation, mitochondrial membrane potential (MMP), intracellular pH, DNA integrity, and fertilizing ability were assessed as toxicity end points after exposure to different Ni NPs concentrations. Ni NPs generate oxidative stress that in turn induces lipid peroxidation and DNA fragmentation, and alters MMP and sperm morphology. Furthermore, sperm exposure to Ni NPs affects their fertilizing ability and causes developmental anomalies in the offspring. All together, these results reveal a spermiotoxicity of Ni NPs in ascidians suggesting that the application of these NPs should be carefully assessed as to their potential toxic effects on the health of marine organisms that, in turn, may influence the ecological system. This study shows that ascidian sperm represent a suitable and sensitive tool for the investigation of the toxicity of NPs entered into marine environment, for defining the mechanisms of toxic action and for the environmental monitoring purpose.

Do benthic and planktonic diatoms produce equivalent effects in crustaceans

Hippolyte inermis Leach 1814 is a benthic shrimp characterized by a peculiar mechanism of sex reversal influenced by diatom foods. In fact, the appearance of primary females in spring is due to an apoptotic early disruption of the androgenic gland and of the male gonad, triggered by still unknown compounds present in diatoms of the genus Cocconeis. The influence of diatoms on the reproductive ecology and life cycle of planktonic crustaceans has been demonstrated previously: some planktonic diatoms produce aldehydes inducing apoptosis in the embryos and in the larvae of marine copepods, reducing their viability. Both benthic and planktonic diatoms therefore produce compounds having an apoptotic effect on some tissues of target crustaceans, although the ecological significance of the two processes is different: deleterious for copepod populations, regulative for shrimps associated with Posidonia oceanica. In the present article we experimentally administered specific planktonic diatoms, their fractions and compounds known to induce apoptosis in planktonic copepods, to H. inermis postlarvae, to check whether the apoptotic effect is due to an identical family of diatom compounds, and to establish whether the processes observed in the plankton and in the benthos, respectively, are analogous or homologous, from an ecological point of view. Our results indicated that diatom compounds acting in the two systems are different, since both planktonic diatoms and their aldehydes had negligible effects on the sex ratios of cultured shrimps.

Giant liposomes as delivery system for ecophysiological studies in copepods

SUMMARY Giant liposomes are proposed as a potential delivery system in marine copepods, the dominant constituent of the zooplankton. Liposomes were prepared in the same size range as the food ingested by copepods (mean diameter of about 7 μm). The encapsulation of a hydrophilic and high molecular mass fluorescent compound, fluorescein isothiocyanate-dextran (FitcDx), within the liposomes provided a means of verifying copepod ingestion when viewed with the confocal laser-scanning microscope. Females of the calanoid copepod Temora stylifera were fed with FitcDx-encapsulated liposomes alone or mixed with the dinoflagellate alga Prorocentrum minimum. Control copepods were incubated with the P. minimum diet alone. Egg production rates, percentage egg-hatching success and number of faecal pellets produced were evaluated after 24 h and 48 h of feeding. Epifluorescence of copepod gut and faecal pellets indicated that the liposomes were actively ingested by T. stylifera in both experimental food conditions, with or without the dinoflagellate diet. Ingestion rates calculated using 3H-labelled liposomes indicated that females ingested more liposomes when P. minimum was added to the solution (16% vs 7.6% of uptake). When liposomes were supplied together with the algal diet, egg production rate, egg-hatching success and faecal pellet production were as high as those observed for the control diet. By contrary, egg production and hatching success were very low with a diet of liposomes alone and faecal pellet production was similar to that recorded in starved females. This results suggest that liposomes alone did not add any nutritive value to the diet, making them a good candidate as inert carriers to study the nutrient requirements or biological activity of different compounds. In particular, such liposomes are proposed as carriers for diatom-derived polyunsaturated aldehydes, which are known to impair copepod embryo viability. Other potential applications of liposomes as a delivery system of drugs and nutrients in copepod mass cultivation, or as carriers of pollutants to study copepod physiology in ecotoxicological experiments, are also discussed.

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.