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Microalgae are currently considered one of the most promising resources for biofuel production, aquaculture feedstock and new pharmaceuticals. Among them, green algae of the genus Tetraselmis are extensively studied for their lipid accumulation in nutrient-starvation conditions. In this paper, we present the full-transcriptome of Tetraselmis suecica and differential expression analysis between nitrogen-starved and -repleted conditions (at stationary phase) focusing not only on lipid metabolism but giving new insights on nutrient starvation responses. Transcripts involved in signal transduction pathways, stress and antioxidant responses and solute transport were strongly up-regulated when T. suecica was cultured under nitrogen starvation. On the contrary, transcripts involved in amino acid synthesis, degradation of sugars, secondary metabolite synthesis, as well as photosynthetic activity were down-regulated under the same conditions. Among differentially expressed transcripts, a polyketide synthase and three lipoxygenases (involved in the synthesis of secondary metabolites with antipredator, anticancer and anti-infective activities) were identified, suggesting the potential synthesis of bioactive compounds by this microalga. In addition, the transcript for a putative nitrilase, enzyme used in nitrile bioremediation, is here reported for the first time for T. suecica. These findings give new insights on T. suecica responses to nutrient starvation and on possible biotechnological applications for green algae.

Dendritic Cells (DCs) recognize infectious non-self molecules and engage the adaptive immune system thereby initiating long lasting, antigen-specific responses. As such, the ability to activate DCs is considered a key tool to enhance the efficacy and quality of vaccination. Here we report a novel immunomodulatory sulfolipid named β-SQDG18 that prototypes a class of natural-derived glycolipids able to prime human DCs by a TLR2/TLR4-independent mechanism and trigger an efficient immune response in vivo. β-SQDG18 induces maturation of DC with the upregulation of MHC II molecules and co-stimulatory proteins (CD83, CD86), as well as pro-inflammatory cytokines (IL-12 and INF-γ). Mice immunized with OVA associated to β-SQDG18 (1:500) produced a titer of anti-OVA Ig comparable to traditional adjuvants. In an experimental model of melanoma, vaccination of C57BL/6 mice with β-SQDG18-adjuvanted hgp10 peptide elicited a protective response with a reduction in tumour growth and increase in survival.

Diatoms, ubiquitous primary producers in marine systems, synthesize oxylipins, which impair copepod fitness through a maternal effect. While oxylipins do not directly affect the adults, these chemicals alter basic cellular and developmental processes of copepod embryos, thereby negatively affecting egg hatching and naupliar survival. Inspecting the effects of oxylipins on copepod reproductive success is extremely challenging, because wide variations in their synthesis potentials among diatom genera, species, populations and strains have been detected. In parallel, distinct copepod species and populations can be differently sensitive to oxylipin-producing diatoms. Lately, application of chemical and molecular methods allowed to finely characterize the oxylipin profiles of single diatom strains and to highlight detoxification responses of copepods through the relative expression of selected genes of interest. Thus far, integrative experimental approaches encompassing physiological, chemical and molecular data were mostly applied to laboratory setups, whereas few in situ experiments were presented in this perspective. Moreover, only a restricted number of papers tested the effects of oxylipins on the plankton community as a whole. This review synthesizes latest advances in conceptual and methodological approaches in diatom–copepod interactions and aims at discussing possible re-shaping of the plankton community in response to oxylipin-producing diatom occurrence.

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Diatoms are unicellular algae playing a key role as photosynthetic organisms in the world’s ocean food webs. The chemical ecology of planktonic diatoms is well documented, but few studies have reported on the effects of benthic diatoms on their consumers, also due to difficulties in the collection, quantification and massive culturing of benthic species. Here for the first time we investigate the effects of feeding on two abundantly occurring benthic diatoms, Nanofrustulum shiloi and Cylindrotheca closterium, isolated from the leaves of the seagrass Posidonia oceanica, on the sea urchin Paracentrotus lividus. Adult P. lividus were fed for one month on diets of either one of the two diatoms and on the green alga Ulva rigida, used as a feeding control. By combining morphological, metabolomic and de novo transcriptomic approaches, we demonstrate toxigenic effect on embryos generated by females fed with these benthic diatoms. Furthermore, chemical analysis reveal the presence of polyunsaturated aldehydes only for N. shiloi, and a high production of other oxylipins (cytotoxic compounds on their grazers and on cancer cell lines) for both diatoms, including some additional peaks not correlated to the canonic oxylipins commonly observed in planktonic diatoms. These findings open new perspectives in the study of diatom secondary metabolites influencing their grazers.

Apoptosis or programmed cell death is the result of complex biochemical and genetic signals triggered by normal physiological events or by exposure to toxic compounds. Apoptosis can be revealed early in cells and tissues due to morphological and biochemical alterations of the cell. Here, we review recent progress in the study of the induction and detection of apoptosis in zooplankton copepods after their exposure to different apoptotic-inducer compounds, and we propose methods to predict copepod mortality even when animals appear normal. We also discuss the use of higher harmonic generation microscopy and multiphoton fluorescence microscopy techniques as possible tools to visualize apoptosis in vivo copepods without using any fluorescent probes.

The copepod Temora stylifera was fed with the pennate diatom Pseudo-nitzschia delicatissima for 15 days. This diatom does not produce polyunsaturated aldehydes (PUAs) but only synthesizes other oxylipins such as the hydroxyacid (5Z,8Z,11Z,13E,15S,17Z)-15-hydroxy-5,8,11,13,17-eicosapentaenoic acid (15S-HEPE). Effects of this monoalgal diet were compared to copepods fed with the PUA-producing diatom Skeletonema marinoi and the control dinoflagellate Prorocentrum minimum which does not produce any oxylipins. Both egg production and hatching success were negatively affected by the two diatoms compared to the control diet. Both diatoms also induced malformations in the offspring, with the number of malformed nauplii increasing dramatically with time. Malformed nauplii were apoptotic (TUNEL-positive) indicating imminent death. In terms of recruitment, only 4 healthy nauplii female−1 day−1 with S. marinoi and 9 healthy nauplii female−1 day−1 with P. delicatissima were produced during the 15-day experimental period. The results indicate that P. delicatissima induced negative effects comparable to those of S. marinoi even though it did not produce PUAs. This is the first study that tests the biological activity of oxylipins other than PUAs on copepod reproduction.

Traditionally, diatoms have been regarded as providing the bulk of the food that sustains the marine food chain and important fisheries. However, this view was challenged almost two decades ago on the basis of laboratory and field studies showing that when copepods, the principal predators of diatoms, feed on certain diatom diets, they produce abnormal eggs that either fail to develop to hatching or hatch into malformed (i.e. teratogenic) nauplii that die soon afterwards. Over the years, many explanations have been advanced to explain the causes for reproductive failure in copepods and other marine and freshwater invertebrates including diatom toxicity, or nutritional deficiency and poor assimilation of essential compounds in the animal gut. Here we review the literature concerning the first possibility, that diatoms produce cytotoxic compounds responsible for growth inhibition and teratogenic activity, potentially sabotaging future generations of grazers by inducing poor recruitment. The cytotoxic compounds responsible for these effects are short chain polyunsaturated aldehydes (PUAs) and other oxygenated fatty acid degradation products such as hydroxides, oxo-acids, and epoxyalcohols (collectively termed oxylipins) that are cleaved from fatty acid precursors by enzymes activated within seconds after crushing of cells. Such toxins are suggested to have multiple simultaneous functions in that they not only deter herbivore feeding but some also act as allelopathic agents against other phytoplankton cells, thereby affecting the growth of competitors, and also signalling population-level cell death and termination of blooms, with possible consequences for food web structure and community composition. Some oxylipins also play a role in driving marine bacterial community diversity, with neutral, positive or negative interactions depending on the species, thereby shaping the structure of bacterial communities during diatom blooms. Several reviews have already been published on diatom-grazer interactions so this paper does not attempt to provide a comprehensive overview, but rather to consider some of the more recent findings in this field. We also consider the role of diatom oxylipins in mediating physiological and ecological processes in the plankton and the multiple simultaneous functions of these secondary metabolites.

Temora stylifera adult copepods were fed with four different monoalgal diets and six combinations of the same cultures for 15 days. Fecundity, hatching success, number of cannibalized embryos, fecal pellet production, adult mortality and naupliar recruitment were compared, in order to find the best diet for this species. Phytoplankton species tested were Prorocentrum minimum (PRO); Isochrysis galbana (ISO); Tetraselmis suecica (TETRA) and Rhodomonas baltica (RHO) which were supplied alone or in different combinations and at various concentrations ranging from a minimum of 1 mg C L−1 day−1 to a maximum of 66 mg C L−1 day−1. Of the ten diets tested, ISO was the worst and was unable to sustain egg production and adult survival possibly because adults were unable to ingest this alga due to its small size. TETRA was also a poor food since it negatively impacted egg production and adult survival, as well as egg hatching success, possibly due to the lack of essential compounds necessary for optimal embryogenesis. RHO and PRO were the best foods inducing highest egg production, hatching success and naupliar recruitment. Even if mean egg production rates were similar to those obtained with some mixed diets, carbon intake concentrations with mixed diets were from 3 to 33 and from 6.6 to 66 times higher than with RHO and PRO given alone, respectively. Mixed diets of ISO and PRO, especially when supplied at higher concentrations (66 mg C L−1 day−1), had a negative effect on egg hatching success and adult survival, with a corresponding reduction in naupliar recruitment. On the other hand, mixed diets of TETRA and PRO promoted high naupliar recruitment but values were similar to PRO offered alone. Our results indicate that a good monoalgal diet such as RHO and PRO can be as effective as a mixed diet to sustain the mass cultivation of T. stylifera.

The shallow-living, benthopelagic copepod species Pseudocyclops xiphophorus Wells (R Soc Edimburg 67:1967), collected over a yearly cycle from the fouling material in the brackish water Lake Faro (North-eastern Sicily), showed marked seasonal fluctuations in population abundances, with maximum numbers recorded in autumn. Highest in situ egg production rates coincided with periods of low adult and juvenile densities and vice versa, except in autumn when peaks in egg production and adult population densities were coincident. In this period, mean daily egg production rates reached a maximum of 4–5 eggs per female, when surface water temperature was 17–18°C. Egg production rates declined drastically in winter and were completely arrested when surface temperatures dropped to 10–12°C. In March, daily egg production rates began to increase again with an increase in ambient temperatures, reaching a maximum at the end of August. In the laboratory, as in the field, mean daily egg production rates were positively correlated with temperature, with values ranging from 2.2 ± 0.3 (16°C) to 8.9 ± 2.6 (30°C) (mean ± S.D.) eggs per female per day. At 32°C, P. xiphophorus females survived but did not reproduce. At 34°C, all specimens died after a few days. In terms of total egg production for the entire female lifespan, maximum values occurred at 16°C and minimum at 24°C. Temperature also dramatically affected female life span, which was shorter at higher temperatures. Development time of eggs decreased with increasing temperature, as also development time from egg to adulthood. Remating was necessary for the continued production of fertile eggs at 16°C because female life span was longer. The unique egg-laying behaviour in this species may ensure higher survival rates of egg stages compared to free-spawning and egg-carrying calanoid species. After releasing the egg pair, the female swims over the eggs with a rotatory motion, secreting a substance which facilitates the adhesion of the eggs to the bottom; she then continues to swim over the eggs until they are attached. Although egg production rates in this species are low compared to other pelagic copepods, they are within the range of values reported for egg-carrying species. The greater fecundity at higher temperatures compared to other subtemperate species indicates that the species is well adapted to the higher temperatures of coastal lagoons and brackish water lakes where it contributes to the biofouling community.

Background The dense phytoplankton blooms that characterize productive regions and seasons in the oceans are dominated, from high to low latitudes and from coast line to open ocean, by comparatively few, often cosmopolitan species of diatoms. These key dominant species may undergo dramatic changes due to global climate change. Results In order to identify molecular stress-indicators for the ubiquitous diatom species Skeletonema marinoi, we tested stress-related genes in different environmental conditions (i.e. nutrient starvation/depletion, CO2-enrichment and combined effects of these stressors) using RT-qPCR. The data show that these stressors impact algal growth rate, inducing early aging and profound changes in expression levels of the genes of interest. Conclusions Most analyzed genes (e.g. antioxidant-related and aldehyde dehydrogenases) were strongly down-regulated which may indicate a strategy to avoid unnecessary over-investment in their respective proteins. By contrast, key genes were activated (e.g. HSPs, GOX) which may allow the diatom species to better cope with adverse conditions. We propose the use of this panel of genes as early bio-indicators of environmental stress factors in a changing ocean. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1574-5) contains supplementary material, which is available to authorized users.

Vital fluorescent probes have routinely been used to distinguish viable from non-viable embryos in various veterinary and aquaculture studies. Here, we present new protocols to rapidly detect embryo viability in the copepod Calanus helgolandicus using three of these probes, fluorescein diacetate (FDA), SYTOX green and 7-aminoactinomycin D (7-AAD), and the confocal laser scanning microscope. The percentage of fluorescent-FDA embryos and non-fluorescent SYTOX green and 7-AAD embryos were compared with the percentage of hatched unstained embryos and with the percentage of embryos that had been stained, washed, and allowed to hatch. Results showed that all three dyes accurately predicted embryo viability and could be used to rapidly calculate C. helgolandicus egg-hatching success. We also tested the possible applications of SYTOX green in egg-production/egg-hatching assays in which the dinoflagellate Prorocentrum minimum or the diatom Skeletonema costatum are used to investigate for the possible negative impact of diatoms on embryo viability. Other possible applications for fluorescence methods in studies on the reproductive biology of zooplankton, and in particular of copepods, are discussed.

Eggs and embryos of the sea urchin Paracentrotus lividus were used as a model to study the effect at the cellular level of potential anti-mitotic compounds extracted from the diatom Thalassiosira rotula. Eggs and embryos incubated in a water-soluble diatom extract, corresponding to 5 × 106 and 107 cells ml−1, were totally blocked (i.e. cell division was blocked) at the one-cell stage. At lower concentrations (2.5 and 1.25 × 106 cells ml−1), the first mitotic division was inhibited in 32 ± 26% and 25 ± 3.5% of the zygotes, respectively, demonstrating the dose-dependent effect of diatom extracts on sea urchin development. Immunofluorescence dyes, specific for DNA and α-tubulin subunits, were used to stain nuclei and microtubules in sea urchin embryos during various phases of development. Images with the confocal laser scanning microscope showed that tubulin was not organised in filaments at the sperm aster and cortex levels, and that the pronuclei were not fused in embryos incubated soon after fertilisation with water-soluble diatom extracts corresponding to 107 cells ml−1. At lower diatom-extract concentrations (4 × 106 cells ml −1), fusion of the pronuclei occurred but the mitotic spindle was not formed. Microtubules were clearly de-polymerised and the chromatin appeared globular and compacted at the centre of the cell. A similar structure was observed for sea urchin embryos incubated with 0.1 mM colchicine, a potent anti-mitotic compound. When sea urchin embryos were incubated in water-soluble diatom extracts at different times prior to the first mitotic division, microtubules appeared de-polymerised at each step, from pronuclear fusion to telophase, and cell division was blocked. At the histological level, embryos incubated with 4 × 106 cells ml−1 diatom extract showed nuclear fragmentation without cytokinesis. The possible use of sea urchin embryos as a bioassay to test for other unknown compounds with cytotoxic activity in phytoplankton species is discussed.

The respiratory physiology of summer diapausing eggs of the neustonic copepodAnomalocera patersoni, maintained under constant temperature (13 °C) and light (12 h light:12 h dark) conditions, was characterized by a bell-shaped curve, with low O2 uptake levels at the beginning of dormancy. This was followed by a steady rise in O2 consumption with maximum levels of 0.002 μl O2 embryo−1 h−1 70 d after spawning. A slow diminution in O2 uptake then occurred until Day 150 when minimum values of 0.0003 μl O2 embryo−1 h−1 were recorded, coinciding with the hatching of the first embryos. Embryos continued to hatch asynchronously up to 360 d from the moment of egg laying. When eggs were subjected to 20 °C, the respiratory activity was almost three times higher than at 13 °C, even though both respiratory curves were similar. The elevated metabolism in eggs kept at 20 °C led to death of the embryos possibly due to a total depletion of metabolic reserves. ATP content also differed at the two temperatures. Diapause eggs kept at 20 °C showed no rapid rise in ATP content as opposed to those kept at 13 °C. The results of temperature shock experiments, in which eggs were first kept at winter temperatures for several weeks, after which the temperature was raised to 20 °C for another number of weeks prior to a second period of chilling at 13 °C, showed that as long as embryos were kept at 20 °C no hatching occurred. By contrast, hatching was observed after 10 d following the resumption of winter temperatures, suggesting that low environmental temperatures are an essential prerequisite for hatching of these eggs. The type of diapause inA. patersoni differs considerably from the one described in insects and in another neustonic copepod,Pontella mediterrana. In this case, there is a U-shaped respiratory curve with greatest O2 consumption prior to the onset or upon breaking of diapause. Differences in the two types of diapause seem to involve not only differences in O2 consumption levels but also in the sequence of metabolic changes with time and the metabolic requirements during sommer and winter dormancy.

Oxygen consumption and ATP content are reported for the planktonic marine copepod Pontella mediterranea during normal and diapause embryonic development. In subitaneous embryos that hatched without delay within 48 h, O2 uptake increased linearly after spawning to reach maximum levels about 25 h later. By contrast, ATP levels were initially very high but decreased rapidly within the next 5 h to reach stable values thereafter. In diapause embryos, O2 consumption followed the typical U-shaped curve described for insect diapause. An initial period of prediapause, which lasted for about 25 days, was characterized by elevated O2 uptake. This was followed by a period of diapause in which O2 consumption dropped to 25% of the values recorded during prediapause. This protracted period of dormancy, which lasted about 4 to 5 months, was followed by a period of high O2 consumption possibly due to the breaking of diapause and resumption in development. ATP content during the pre-diapause period showed a similar trend as in subitaneous embryos with high initial levels that decreased with time for the first 20 days and remained stable afterwards.

Fecundity, egg viability and fecal pellet production are reported for Acartia clausi females collected in the Bay of Naples, Italy, from April to October 1992 and fed either with a diatom (Thalassiosira rotula) or dinoflagellate (Prorocentrum minimum) diet, at food saturated conditions. The diatom diet significantly reduced both egg and fecal pellet production as well as hatching success. Blockage of egg development occurred with both axenic and non-axenic cultures of T. rotula, suggesting that inhibitors were provided by the diatoms and not by the bacteria associated with diatom cultures. Low hatching success was also artificially induced by exposing newly spawned A. clausi eggs to high concentrations of diatom extracts, indicating the presence of deleterious, inhibitory compounds blocking copepod embryogenesis. Fecundity and hatching success diminished significantly with female age. In contrast, female longevity was not significantly modified by food type. The presence of males did not significantly alter fecundity or egg viability. Females continued to produce viable eggs throughout the period of incubation, with and without males, in both food conditions, indicating that remating is infrequent and not necessary to sustain viable egg production in this species. The succession in low and high population densities may therefore be the outcome of variations in survival rates of eggs, rather than reproductive protential perse; such variations may strongly depend on the adult copepod diet.

Egg production and viability in the copepod Temora stylifera (collected in the Bay of Naples, Italy in 1992) were strongly dependent on food type. A flagellate (Isochrysis galbana) diet induced the production of good quality eggs that developed to hatching. By contrast, two diatoms (Chaetoceros curvisetum, Phaeodactylum tricornutum) resulted in poor egg quality, with hatching success as low as 20% of total egg production. With the third diatom tested, Skeletonema costatum, females produced eggs for only 3 to 4 d, after which time they either became sterile or died. These results are discussed in relation to previous findings regarding the impact of the dinoflagellate Prorocentrum minimum and the diatom Thalassiosira rotula on the hatching success of T. stylifera eggs. Low egg viability was possibly not due to an absence of remating or a deficiency of some specific essential nutrient required for egg development but to the presence of inhibitory compounds blocking cell division during early copepod embryogenesis. This questions the traditional view that diatoms are an important food item regulating copepod secondary production.

A unique type of integumental formation is described for several members of the copepod family Pontellidae. This surface attachment structure (SAS) consists of a mass of fine setules arranged in two semicircles on a flattened area of the anterodorsal surface of the cephalosome. Using transmission electron microscopy, the SAS was shown to be continous with the cuticle and not linked to chemo- or mechanosensory cells; its function is purely mechanical. This structure is probably an energy-saving means for these large and heavy neustonts to stay attached to the surface film. The SAS is species-specific and may thus be of potential importance to the systematics and phylogeny of the Pontellidae, in the same manner as integumental pores and sensilla, which form patterns characteristic of several copepod families and genera.

The neustonic copepodAnomalocera patersoni Templeton, collected from the Gulf of Naples from January to May 1988, laid subitaneous and diapause eggs that were of equal size and dark and smooth in appearance. In January and February, most eggs were subitaneous and hatched within 2 to 3 d at room temperature. Conspicuous numbers of diapause eggs were first obtained in February when several clutches contained a second egg type which did not hatch in a subitaneous manner. By March and April, most clutches contained only diapause eggs. Transmission electron microscope studies of subitaneous and diapause eggs revealed striking morphological differences. Subitaneous eggs had a thin vitelline coat covering the plasma membrane, whereas diapause “eggs” were enveloped by a complex four-layer structure which is assembled after spawning and which probably serves as a protective shell during dormancy. No major morphological differences were discernible in the oogenic cycle of females spawning either of the two egg types. Diapause eggs were shown to be embryos in early stages of development, and segmentation of these embryos was arrested for the entire 6-mo period of investigation. Diapause eggs hatched after 7 mo of dormancy.

Transmission electron microscope studies on the morphology of subitaneous and diapause eggs in the neustonic copepodPontella mediterranea, collected in coastal waters of the Gulf of Naples from May to December 1986, revealed striking structural differences between the two egg types and the existence of a third egg type that was morphologically similar to a diapause egg but which hatched within 2 to 3 d. In smooth, subitaneous eggs the plasma membrane was surrounded by a thin outer chorion as compared to the thick and highly complex four-layer structure enveloping subitaneous eggs with short spines, and diapause eggs. This coat may be assembled just prior to or after deposition since the morphology of developing oocytes was similar in females laying either subitaneous or diapause eggs. The only other major structural difference between the two egg types was the presence of numerous, disc-like bodies in the ooplasm of diapause eggs. Development was arrested for at least 3 mo, prior to the initiation of the first cleavage, suggesting that these eggs were kept dormant by some endogenous and, possibly, structural mechanism preceeding the termination of diapause.

Phytoplankton blooms are known to induce diarrheic, neurotic, and paralytic shellfish poisoning in humans through the production of potent toxins by several dinoflagellate and flagellate species. Diatoms, another major component of the phytoplankton, have rarely been reported as toxic to marine organisms. But diatoms may be more insidious than is generally believed. High concentrations of domoic acid have been identified in Nitzschia pungens f. multiseries (Douglas and Bates, 1991), and recent studies indicate that several diatom species also contain antimitotic compounds inducing egg mortality in several invertebrates including crustaceans (several copepod species belonging to the genera Calanus, Centropages, Acartia, Eucyclops, Paracyclops

Laboratory studies on the reproductive cycle of the planktonic copepod Temora stylifera, collected from the Gulf of Naples between October 1987 and March 1988, showed that females oscillated between a light and dark gonadal condition. Histological preparations indicated that the dark condition corresponded to a ripe stage of oogenesis as compared to the light or unripe maturation state of primary oocytes. The number of eggs released within 24 h was strongly dependent on the reproductive status of the female at the time of incubation. Light unfed females rarely reverted to a dark or ripe condition and egg deposition in such cases was almost always zero. Dark females produced eggs within 24 h under all experimental food conditions, reverting from dark to light soon after egg deposition. Successively, the length of the interclutch period was strongly dependent on the type of food present. A high percentage of infertile eggs was almost always present. We suggest that short-term studies, not taking into consideration the condition of gonads at the time of incubation, will underestimate potential egg production rates. Females maintained for longer than three or four days produced mainly infertile eggs. After remating, females reinitiated the production of fertile eggs but egg viabilities never reached 100% hatching success, indicating poor efficiency in the fertilization of mature oocytes. We hypothesize a possible cause and effect relationship between egg viabilities and seasonal fluctuations in population densities.