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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.

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 &micro, M, 15 &micro, M, and 20 &micro, M. 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.

Oxygenated derivatives of fatty acids, collectively called oxylipins, are a highly diverse family of lipoxygenase (LOX) products well described in planktonic diatoms. Here we report the first investigation of these molecules in four benthic diatoms, Cylindrotheca closterium, Nanofrustulum shiloi, Cocconeis scutellum, and Diploneis sp. isolated from the leaves of the seagrass Posidonia oceanica from the Gulf of Naples. Analysis by hyphenated MS techniques revealed that C. closterium, N. shiloi, and C. scutellum produce several polyunsaturated aldehydes (PUAs) and linear oxygenated fatty acids (LOFAs) related to the products of LOX pathways in planktonic species. Diploneis sp. also produced other unidentified fatty acid derivatives that are not related to LOX metabolism. The levels and composition of oxylipins in the benthic species match their negative effects on the reproductive success in the sea urchin Paracentrotus lividus. In agreement with this correlation, the most toxic species N. shiloi revealed the same LOX pathways of Skeletonema marinoi and Thalassiosira rotula, two bloom-forming planktonic diatoms that affect copepod reproduction. Overall, our data highlight for the first time a major role of oxylipins, namely LOFAs, as info-chemicals for benthic diatoms, and open new perspectives in the study of the structuring of benthic communities.

Diatoms are key phytoplankton species in the world oceans and essential in the transfer of energy through marine food chains. Their beneficial role has been questioned after the discovery that some diatom species produce teratogenic compounds such as polyunsaturated aldehydes and other oxylipins that induce abortions, poor larval development and high offspring mortality in many invertebrates. To test whether maternal and post-embryonic diatom diets affected development in the copepod Paracartia latisetosa , we carried out experiments in which the PUA-producing diatom Skeletonema marinoi (SKE) was fed to this copepod species. Controls were run with the dinoflagellate, Prorocentrum minimum (PRO), which does not produce any of these compounds. Four treatments were tested: both mothers and progeny always fed on either SKE or PRO, and those in which mothers received either PRO or SKE, and neonates were switched to the other diet. Our results show that a SKE/SKE diet induced lower egg production and egg viability, as well as slower embryonic development than the control diet. Naupliar development was negatively affected with the diatom diet which blocked the metamorphosis of larvae at N3, 4 days after hatching, compared to the control diet that allowed for normal naupliar development in 10 days. Nauplii reared on PRO and spawned by females fed on SKE were able to complete development, but only after 11 days, whereas larvae reared on SKE spawned by females fed on PRO were arrested at the N5 developmental stage, 12 days after hatching. Apoptotic TUNEL-positive nauplii were detected with the SKE diet, indicating imminent death. Significant differences were also recorded by comparing biomass of equivalent naupliar stages obtained for each treatment, and the overall mean biomass female − 1 day − 1 for each diet. Growth inhibition due to oxylipin-producing diatoms may have important consequences on prey–predator relationships and on the biomass flux through marine food chains.

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.

The anti-proliferative effects of diatoms, described for the first time in copepods, have also been demonstrated in benthic invertebrates such as polychaetes, sea urchins and tunicates. In these organisms PUAs (polyunsaturated aldehydes) induce the disruption of gametogenesis, gamete functionality, fertilization, embryonic mitosis, and larval fitness and competence. These inhibitory effects are due to the PUAs, produced by diatoms in response to physical damage as occurs during copepod grazing. The cell targets of these compounds remain largely unknown. Here we identify some of the genes targeted by the diatom PUA 2-trans-4-trans-decadienal (DD) using the tunicate Ciona intestinalis. The tools, techniques and genomic resources available for Ciona, as well as the suitability of Ciona embryos for medium-to high-throughput strategies, are key to their employment as model organisms in different fields, including the investigation of toxic agents that could interfere with developmental processes. We demonstrate that DD can induce developmental aberrations in Ciona larvae in a dose-dependent manner. Moreover, through a preliminary analysis, DD is shown to affect the expression level of genes involved in stress response and developmental processes.

Highlights: • The diatom PUA decadienal strongly impacted demographic traits of Pseudodiaptomus annandalei. • Decadienal negatively affected survival, development and reproduction in a dose-dependent manner. • Nauplii successfully molted to copepodites; however copepodites were not able to molt to the adult stage when exposed to decadienal. • Negative effects on female survival were not recorded beyond 6 days of exposure to decadienal suggesting an evolutionary adaptive response to PUA with time. • Progressive tolerance to decadienal suggest evolutionarily adaptive response to PUA with time. Abstract: The euryhaline calanoid copepod Pseudodiaptomus annandalei perennially occurs in tropical and subtropical brackish water ecosystems and harbors supporting harmful algal blooms. In the present paper we provide a quantitative assessment of the influence of the diatom-derived poly-unsaturated aldehyde (PUA), 2-trans-4-trans-decadienal on demographic traits of P. annandalei. The survival and reproduction of P. annandalei were recorded in life table experiments, using a cohort of adult males and females exposed to a range of concentrations (0.75, 1.5, 3 and 4.5 μM) of decadienal. Higher mortality of female cohorts and lower reproduction were recorded with increasing concentrations of decadienal (3 and 4.5 μM). The estimated survival time for cohort females (control: 9.7 days; 4.5 μM decadienal: 4.7 days) and developmental stages of copepods (control: 10.5 days; 4.5 μM decadienal: 1.0 days) in decadienal applied medium (Isochrysis galbana + decadienal) was significantly lower than those in control (only I. galbana). Detrimental effects further eroded recruitment by increasing naupliar mortality and impairing copepodite development to adulthood. Since P. annandalei is widely used in the aquaculture industry in Asia, the present results call for a limited use of PUA producing diatoms as algal food to avoid impairing copepod reproduction and development.

In this study, we analysed the effect of population density and food concentration on the fecundity of a Mediterranean strain of Acartia tonsa to maximize egg production. During 4-day feeding experiments, egg hatching success and faecal pellet production were also followed. The algae Rhinomonas reticulata was supplied at different concentrations corresponding to 250, 500, 1000, 1500, 2000 and 3000 μg C L−1 day−1 at the following adult copepod density: 40, 80 and 160 ind. L−1. Our results show a positive relationship between algal concentration and egg production under all experimental conditions confirming that the quantity of food strongly limits A. tonsa fecundity. Maximum egg production (57 eggs per female) was reached at the lowest density and at the maximum food concentration. Percentage of egg hatching success was not dependent on the quantity of food used. At the same food concentration, an increase in population density from 40 to 80 ind. L−1 induced an increase in faecal pellet production per couple which did not correspond to an increase in egg production, suggesting that higher energetic costs were shifted to swimming activity. Productivity of the A. tonsa Mediterranean strain is mainly limited by the quantity of food rather than by crowding conditions.

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.

Effects of the toxic Gulf of Mexico dinoflagellate Karenia brevis on its copepod grazers are equivocal, in that this dinoflagellate is ingested by various copepods, but may be toxic to, and/or nutritionally inadequate for grazers. Recent studies have shown that when the Mediterranean copepod Calanus helgolandicus fed on K. brevis, egg production rates and egg viability declined steadily over time, reaching almost zero levels after 5 days of feeding ( Turner et al., 2012 ). The K. brevis strain used in that study was chemically analyzed and shown not to contain any brevetoxins, but may have contained other harmful metabolites. Here we present gene expression analyses by reverse transcription-quantitative PCR (RT-qPCR) in C. helgolandicus after different exposure times (3, 5 and 8 days) to the same non-brevetoxin-containing K. brevis strain examined previously. The analyzed genes have a primary role in generic stress responses, defense systems (e.g. aldehyde, free fatty acid and free radical detoxification) and apoptosis regulation in other organisms. After 3 days of feeding on K. brevis, gene expression levels in copepods did not show significant changes. However, after 5 days of exposure, expression of stress-response genes was significantly reduced. After prolonged exposure for 8 days, expression of genes for 5 of 6 aldehyde dehydrogenases was strongly reduced, but expression of other genes was not coherently reduced, or increased. Microscopic observations revealed that after only a single day of feeding on K. brevis, freshly-spawned eggs were morphologically similar to those produced by copepods feeding on control diets, but after 3 days of feeding on K. brevis, eggs exhibited altered membrane cell morphology and apoptotic features such as granulation and degeneration of the cytoplasm matrix, compared to the unaltered eggs produced by copepods feeding on control diets. The changes in gene expression levels in copepods feeding on K. brevis were similar to those noted previously for copepods feeding upon diatoms that contained oxylipins, which interfered with copepod reproductive success. Changes in gene expression in copepods feeding upon toxic algal diets may offer important early warning signals of copepod stress responses to these diets.

The influence of six monoalgal diets was tested on the reproductive success of the copepod Acartia tonsa over a 15-day period in order to define the most favorable diet for the optimization of this copepod species to be used in aquaculture and in ecotoxicology applications. The cryptophytes Rhinomonas reticulata and Rhodomonas baltica induced highest egg production rates (mean = 24.4 eggs female− 1 day− 1and 21.9 eggs female− 1 day− 1) and hatching success (mean = 76% and 86.1%) over the 15-day period, respectively. Lowest egg production rates were recorded with both diatoms Phaeodactylum tricornutum (mean = 8.8 eggs female− 1 day− 1) and Skeletonema marinoi (mean = 8.4 eggs female− 1 day− 1). These two diatoms also had detrimental effects on hatching success (mean = 44.1% and 46.5%, respectively) and adult survival. No adults survived for longer than 13 days with a diet of P. tricornutum. Moreover, nauplii produced by females that had fed on diatoms for > 10 d, were positively stained for the apoptotic fluorescent marker TUNEL, indicating imminent death. The prasinophyta Tetraselmis suecica induced low fecundity (mean = 13.4 eggs female− 1 day− 1) and hatching success (mean = 62%), and after 15 days only 37.5% of the adults survived. Isochrysis galbana induced low egg production per female (

There may be biogeographic aspects of interactions between toxic phytoplankton and their zooplankton grazers. Grazers that naturally co-occur with a given species of toxic phytoplankton may have evolved mechanisms to counteract adverse effects of phytoplankton toxins. Conversely, grazers that have no co-evolutionary experience with a toxic phytoplankter may be more susceptible to its effects. This may also be true for toxic metabolites other than neurotoxins that adversely affect copepod reproduction and development such as the well-described diatom oxylipins. In order to test this hypothesis, we investigated interactions in terms of feeding, reproductive success and survival of a copepod feeding on a potentially toxic dinoflagellate species with which the copepod does not naturally co-occur: the red tide dinoflagellate Karenia brevis from the Gulf of Mexico and the copepod Calanus helgolandicus from the Mediterranean. We additionally compared interactions of these two allopatric plankters with various co-occurring sympatric ones, including the copepod Temora stylifera and a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense, both from the Mediterranean, and as a control, the non-toxic dinoflagellate Prorocentrum minimum. Both Mediterranean copepods fed, reproduced and survived on diets of K. brevis from the Gulf of Mexico, but neither copepod did as well on this diet as on diets of A. tamarense from the Mediterranean or the control diet of P. minimum. Of the two copepods, T. stylifera fared worse on a diet of K. brevis than did C. helgolandicus. Egg hatching success for both copepods feeding on K. brevis was sharply diminished over the seven days of the experiments. This was surprising, since we subsequently discovered that our culture of K. brevis contained no brevetoxins. However, our culture appeared to contain another as yet unidentified compound. Similarly, we have previously demonstrated that our culture of A. tamarense, which contained no saxitoxins or aldehydes, did contain another unidentified compound that reduced egg hatching success by inhibiting sperm from fertilizing eggs. Thus, although diets of the dinoflagellate from the Gulf of Mexico (K. brevis) caused adverse effects on both of the Mediterranean copepods, T. stylifera was also adversely affected by the Mediterranean dinoflagellate (A. tamarense). The copepod T. stylifera which co-occurs in the Gulf of Mexico with the dinoflagellate K. brevis was far more adversely affected by feeding on a sole diet of this dinoflagellate than was the Mediterranean copepod (C. helgolandicus) which never naturally co-occurs with this dinoflagellate. Thus, any putative biogeographic effects of copepods feeding on allopatric versus sympatric dinoflagellates did not clearly emerge from our data. However, since both species of dinoflagellates appear to contain additional undescribed chemicals other than the neurotoxins for which they are best known, and these compounds are potentially related to the adverse effects we recorded, it appears that understanding of the biogeography must await further defining the biochemistry of some harmful algae:grazer interactions.

In this study, we applied different fluorescent techniques to analyse the induction of apoptosis in the copepod Acartia tonsa nauplii exposed to low concentrations of nickel chloride (NiCl2). Newly hatched and later naupliar stages were exposed to increasing concentrations of NiCl2 (0.016, 0.025 and 0.063 mg·L−1) for 7 days and then stained with annexin V–FITC, a vital fluorescent probe commonly used to visualise apoptotic cells in live samples. Nauplii were also stained with TUNEL, a non-vital fluorescent probe used to detect apoptosis in fixed copepods. Moreover, we used for the first time, two-photon fluorescence (2PF) microscopy and higher harmonic generation microscopy (second SHG and third THG harmonic generation) to study apoptosis induction in A. tonsa nauplii without the use of fluorescent probes. 2PF and THG intensity increased in samples exposed to higher Ni concentrations, with respect to the control, whereas SHG signals were similar in all treated samples and visualised muscles. Future perspe...

Diatom oxylipins have been observed to deleteriously impact copepod reproductive success. However, field studies have revealed very variable and case-dependent results. Therefore, the plasticity of diatom oxylipin metabolism was studied among four clones of the marine diatom Skeletonema marinoi Sarno et Zingone. Diatom oxylipin metabolism was studied by two lipoxygenase (LOX) activity assays carried out at different pH values and by oxylipin quantification. The four clones showed no major metabolic differences in terms of protein content or growth rate. However, two of the clones produced significantly higher levels of oxylipins than the other two. LOX activity measurements also indicated clonal variability in fatty acid oxidative metabolism. The presence of clone-specific differences in oxylipin metabolism may play a role in shaping diatom population dynamics by conferring selective advantages to certain clones.

In this study we develop gene expression tools in Calanus helgolandicus to study the effects of toxic diatom diets on copepod fitness and survival. We demonstrate that when adult females are fed on the control dinoflagellate Prorocentrum minimum and the flagellate Rhodomonas baltica, which are not known to produce toxic oxylipins, there are no significant changes in alpha- and beta-tubulin (microtubule subunits) gene expression levels. By contrast, the oxylipin-producing diatom Skeletonema marinoi influences tubulin expression levels which were markedly down-regulated. We scored a panel of putative reference genes (ACT, EFA, GAPDH, 18S, S7, S20, ATPs, UBI and IST) and found that two (S20 and S7) were highly stable in the tested conditions and can be used for further experiments. To normalize reverse transcription-quantitative real time polymerase chain reaction (RT-qPCR) data we also used the third best reference gene, GAPDH, considering the possibility that the two ribosomal proteins could be co-regulated. This pilot study will pave the way for further investigations on which genes are affected by diatom diets and clarify when and if a stress response or a detoxification mechanism becomes visible in C. helgolandicus. Moreover, the RT-qPCR analysis presented here may also be useful to study the effects of other diets and/or environmental factors such as salinity, temperature, pollution and other toxic compounds, on gene expression levels in this copepod species.

A pilot re-circulating system was used for the cultivation of two Mediterranean calanoid copepods: Temora stylifera and Centropages typicus. The system automatically concentrated the naupliar and copepodite stages. Temora stylifera was fed the flagellate Rhodomonas baltica or Prorocentrum minimum, whereas C. typicus was fed with a mixture of R. baltica or P. minimum and Tetraselmis suecica. Both copepods also received Isochrysis galbana. After 21 days, the T. stylifera population increased 26-fold, reaching a density of 38 000 individuals, mostly represented by nauplii (88%). The maximum density recorded was 380 ind. L−1, with a production of 370 nauplii L−1. On average, the egg hatching success for this copepod during the rearing period was 54%, with the highest viability in April and May (>75%). The C. typicus population increased more than 10-fold after 7 weeks of rearing, reaching a density of 123 000 individuals, mainly represented by nauplii (>90%). The highest naupliar production was 100 ind. L−1, with a mean egg hatching success of 68%. This system may be useful to produce nauplii and copepodite stages to be used as live, alternative or complementary food for fish larvae or to provide a ready source of organisms for physiological and bioassay studies.

Development and sex differentiation in the copepod Temora stylifera was studied in the presence of maternal and larval diets of the diatoms Thalassiosira rotula and Skeletonema marinoi, either provided alone or supplemented with the control dinoflagellate Prorocentrum minimum. Both diatoms had deleterious effects on growth compared to the control when used as pure diets, inducing very low or even zero survival from hatching to adulthood. This effect was deleted when the diet was supplemented with a good food (P. minimum) only in the case of T. rotula. By contrast, with a maternal or larval diet of S. marinoi, nauplii did not pass metamorphosis even when this alga was mixed with P. minimum. Arrested development was not due to lack of feeding since early and late nauplii (NII and NV) ingested all three algae at similar rates when used as single diets, and did not show any preference when the algae were offered as mixtures. Since mortality rates with a mixed diet of S. marinoi + P. minimum were similar or even higher than those obtained with a single diet of S. marinoi, we suggest that this diatom is more toxic than nutritionally deficient for T. stlyfera development. No males were produced in cohorts reared on pure diatom diets or with a mixture of S. marinoi + P. minimum, and intermediate male:female sex ratios were obtained with the mixed T. rotula + P. minimum diet. Possibly some diatoms produce compounds such as oxylipins or new molecules that alter sex differentiation in T. stylifera.

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.

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.

Egg production rates and/or hatching success in the copepods Acartia clausi, Calanus helgolandicus and Temora longicornis were negatively affected by a late spring (May-June 2003) phytoplankton bloom in the North Adriatic Sea, dominated mainly by the large diatom Cerataulina pelagica. Highest total concentrations of 3.3- 104 cells-ml(-1) were located in the vicinity of the Po River, which also corresponded to the area where the highest numbers of phaeophorbides were measured (0.779, 0.528 and 0.419 mu g.l(-1), respectively, compared to an average of the remaining stations of 0.183 +/- 0.049 SD), suggesting some grazing on the bloom. Phytoplankton biomass in terms of carbon was dominated by diatoms, representing on average 42% of total phytoplankton carbon and more than 80% at several stations. Cerataulina pelagica, Cyclotella spp., Chaetoceros spp. and small unidentified centric diatoms dominated the diatom community numerically but C. pelagica was by far the dominant diatom in terms of carbon due to its large cell size. This species represented more than 60% of the diatom biomass at nine of the 14 stations sampled, and was absent only at one station, which was the most offshore station sampled during the cruise. Although polyunsaturated aldehydes (PUAs) were not detected, other oxylipins which are hydroxy and keto derivatives of eicosapentaenoic and docosahexaenoic acids that affect copepod reproduction were found in these samples. Hence, we can attribute the negative impact of diatoms not only to PUAs, as previously believed, but also to these compounds. This is the first direct evidence of the presence of oxylipins other than PUAs in marine blooms dominated by diatoms.

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. (C) 2016 Elsevier B.V. All rights reserved.

In situ seasonal oscillations in female abundances, fecundity, egg-hatching success and survivorship of the first naupliar stage (N1) of the copepods Temora stylifera and Centropages typi- cus, collected from February 2000 to February 2001, were studied at a coastal station located in the Gulf of Naples (Mediterranean Sea). Adult T. stylifera and C. typicus females reached maximum abundances in different periods of the year, even though both species had similar breeding patterns with maximum egg-production rates (120 eggs female -1 d -1 ) in spring and early summer. Hatching success was generally >80%, showing weak seasonal trends in both species. In contrast, survivorship of N1 nauplii was much lower in T. stylifera (mean = 12%), compared to C. typicus (mean = 67%); on nearly 40% of the sampling dates, not a single N1 larva survived to moult to the second larval stage. On average, only 11 T. stylifera nauplii m -3 d -1 were recruited in situ over the year studied, compared to 139 C. typicus nauplii m -3 d -1 . Only seasonal changes in egg- and pellet-production rates in both species were correlated with total integrated chlorophyll a (chl a), whereas hatching success and naupliar survival were not correlated with either chl a concentrations or with phytoplankton compo- sition. Laboratory experiments showed that maternal feeding on Isochrysis galbana (ISO) or Proro- centrum minimum (PRO) for 7 d did not enhance naupliar survival in either copepod species, indicat- ing that the negative effects of maternal diet did not disappear after feeding on a high quality food source. Naupliar survivorship was enhanced to >90% with the addition of a K algal culture medium to the natural phytoplankton assemblage, indicating that N1 nauplii were 'drinking' and absorbing dissolved material from the medium. Our results show that high hatching success in copepods can be followed by very low early larval survivorship and that the causes for low survival are not only related to maternal diet but also to the quality of the water in which the nauplii hatch. Scoring maternal and environmental effects on copepod recruitment rates should therefore also consider mortality of early naupliar stages.

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.

A unialgal diet of a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense strongly modified egg production and hatching success in the copepod Temora stylifera, even though grazing rates were relatively constant over time. Both exponential and stationary cultures of A. tamarense reduced egg production and hatching success, but the effect of stationary cultures on hatching success was dramatic, with egg viability dropping to 0% after 24 h of feeding. HPLC analy- ses revealed that the A. tamarense clone was non-neurotoxic, with a mean toxin content of about 0.005 fmol per cell, indicating that adverse effects were not due to saxitoxins or neosaxitoxins. 1 H-NMR analyses also revealed that diatom-derived PUSCAs (polyunsaturated short-chain alde- hydes) were not responsible for reduced hatching rates, since these compounds were absent in A. tamarense as compared to the diatom Skeletonema costatum. Extracts of A. tamarense and the diatom-derived PUSCA 2-trans-4-trans-decadienal were also assessed in terms of biological effects on sea urchin embryo cell divisions. A. tamarense did not show anti-mitotic properties, as extracts did not block first-cell cleavage compared to decadienal. However, A. tamarense extracts did block fer- tilization success when sea urchin oocytes were first incubated for 30 min in extracts and then fertil- ized, as opposed to S. costatum extracts, which did not affect normal elevation of the fertilization membrane. This is the first report that dinoflagellates produce antiproliferative compounds, other than PUSCAs, that can potentially reduce copepod egg production and hatching success.

Four diatoms, Thalassiosira rotula (THA), Thalassiosira weissflogii (TWEI), Skeletonema costatum (SKE) and Phaeodactylum tricornutum (PHA), and one dinoflagellate Prorocentrum minimum (PRO, control diet), were tested during the course of 14-day laboratory experiments examining the effects of these algae on the fecundity and egg viability in the copepod Temora stylifera . All algae were provided at the same mean carbon concentrations of 0.98 μg C ml −1 to normalize the effects due to differences in diatom size and nutrient concentrations, at least with regards to carbon, so that variations in egg production and hatching viability were presumably due to other chemical constituents of the cells. Our results show that only PRO supported high reproductive success, with stable egg production rates and egg viability close to 100%. By contrast, all four diatoms negatively impacted egg production and hatching success, with varying degrees of suppression. The two diets SKE and PHA induced dramatic effects on reproductive success; SKE was the worst diet with naupliar production ceasing altogether within only 3 days. With PHA, fecundity never reached zero, but at the end of the experiments, initial egg production rates had dropped to 6.6% of initial rates, and viability had declined to zero within 7 days. The two Thalassiosira species mainly affected egg viability, so that after 14 days of feeding, a decrease of 90% had occurred. However, egg production rates were high and were reduced only by 15–24% towards the end of the experiments. In terms of potential recruitment rates, even though PRO induced high mean naupliar production, a slight decrease was observed during the first 5 days, likely due to the time required for T. stylifera to acclimate to PRO. After day 5, potential recruitment rate was higher, comparable to in situ values. THA and TWEI inhibited potential recruitment up to 90–100%, but these effects were not evident over a short-term period, denoting the importance of introducing the time factor when evaluating diatom effects on copepod production. With the other two diatom diets, SKE and PHA, recruitment was zero after 2 and 6 days, respectively. Experiments testing the effects of unsaturated aldehydes in THA (2-trans-4-trans-decadienal) and SKE (2-trans-4-trans-octadienal and 2-trans-4-trans-heptadienal) on the hatching viability of T. stylifera indicate that diatom-derived aldehydes may differ in terms of biological activity. Of the three molecules tested, decadienal had somewhat stronger effects on hatching success, with total blockage occurring at concentrations of 2.0 μg ml −1 ; the concentrations to induce total blockage by octadienal and heptadienal were somewhat higher and ≥2.5 μg ml −1 . We suggest that the greater biological activity of SKE in terms of cell blockage may thus be due to the presence of antimitotic compounds other than these unsaturated aldehydes.

Diatoms are small eukaryotic plants with over 10,000 known species, constituting one of the major components of the phytoplankton in freshwater and marine environments. Traditionally, they have been regarded as beneficial to the growth and survival of marine organisms, and to the transfer of organic material through the marine food chain to top consumers and important fisheries. From 1993 onwards, however, evidence has accumulated that has progressively challenged the classic view that diatoms are good and harmless food items for copepods, the dominant constituent of the zooplankton, which sustain the production and growth of larval fish. Laboratory results in recent years have shown that some diatoms potentially reduce copepod egg viability up to 100%; at times, egg production rates are adversely affected as well. Thus, while diatoms may provide a source of energy for copepod larval growth, they often reduce fecundity or hatching success or both. These results constitute the paradox of diatom–co...

Confocal Laser Scanning Microscope techniques have been applied to study the developmental biology of marine copepods and decapod larvae. The lipophylic probes Dil and DiOC(6) were used to study both the external and internal morphology of these crustaceans, whereas the same DiOC(6) and the specific nuclear probe Hoechst 33342 were used to study embryonic development of copepods in vivo. To distinguish viable from non-viable copepod embryos, the vital dye dichlorodihydrofluorescein diacetate (H(2)DCFDA) was used. Major advantages and difficulties in the use of these non-invasive techniques in studies of the reproductive biology of marine crustaceans are discussed. (C) 2003 Wiley-Liss, Inc.

Most diatom species produce at least two classes of oxylipins, polyunsaturated aldehydes (PUAs) and non-volatile oxylipins (NVOs). These compounds can act as grazer deterrents by reducing the reproductive success of copepod grazers. The aim of the present study was to better understand the impact of late-winter diatom blooms and of diatom-derived PUAs and NVOs on the reproduction of two dominant copepod species in the northern Adriatic Sea, in two consecutive years. Higher diatom abundance in 2005 compared to 2004 coincided with higher egg-production rates in both Acartia clausi and Calanus helgolandicus. The increased contribution of diatoms to the phytoplankton assemblage in 2005 also led to a higher phytoplankton production of NVOs, whereas PUA levels were low in both years. These three-times-higher oxylipin levels correlated with a decrease in average hatching success in both A. clausi and C helgolandicus from similar to 80% in 2004 to similar to 60% in 2005, indicating that the observed negative effects on copepod recruitment in the latter year were related to the greater production of diatom NVOs. Our findings reveal the importance of these metabolites and offer an explanation as to why diatom blooms may be more toxic for copepods in some years compared to others. (C) 2015 Elsevier B.V. All rights reserved.

Ecologically-relevant marine diatoms produce a plethora of bioactive oxylipins deriving from fatty acid oxidation, including aldehydes, hydroxy-fatty acids, epoxy-hydroxy-fatty acids, and oxo-acids. These secondary metabolites have been related to the negative effect of diatoms on copepod reproduction, causing low hatching success and teratogenesis in the offspring during periods of intense diatom blooms. The common intermediates in the formation of oxylipins are fatty acid hydroperoxides. The quantitative measurement of these intermediates can fundamentally contribute to understanding the function and role of lipoxygenase metabolites in diatom-copepod interactions. Here, we describe the successful adaptation of the ferrous oxidation-xylenol orange 2 (FOX2) assay to diatom samples, which showed several advantages over other spectrophotometric and polarographic methods tested in the present work. Using this method we assessed fatty acid hydroperoxide levels in three diatom species: Skeletonema marinoi, Thalassiosira rotula, and Chaetoceros affinis, and discuss results in light of the literature data on their detrimental effects on copepod reproduction.

The bloom-forming alga Phaeocystis is ingested by a variety of zooplankton grazers, but is thought to be a poor source of food. We examined copepod grazing on solitary Phaeocystis cells by adult females of Temora stylifera, and survival, fecal pellet production, egg production and egg hatching success in Calanus helgolandicus and T. stylifera over periods of 15 consecutive days. Phaeocystis cell concentrations were high (1.2-3.6 X 10 4 cells ml -1 for C. helgolandicus and 2.5-7.9 × 10 4 cells ml -1 for T. stylifera), but within the range of maxima recorded for natural blooms. Both copepods survived well and continuously produced fecal pellets (indicating continuous grazing) on a diet of Phaeocystis. However, egg production rates for both copepods were low, even though hatching success of the few eggs produced was high. Clearance rates for T stylifera were higher than for most previous measurements of other copepods feeding on Phaeocystis solitary cells at lower cell concentrations. We conclude that even though copepods feed well upon Phaeocystis, resulting poor fecundity on this diet may inhibit copepod population increases during blooms, thereby contributing to the perpetuation of blooms. However, the high egg hatching success on this diet argues against Phaeocystis containing chemical compounds that act as mitotic inhibitors reducing copepod egg viability, such as those found in some other phytoplankters.

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.

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.

A review of the life history traits of subtemperate copepod genera indicates a number of common features. Most dominant species are continuous spawners with maximum levels of egg production reached at 15–20°C, depending on the species. Egg development times are quite rapid, about 24–72 h, within a temperature range of 15–30°C, and are largely temperature dependent. The potential fecundity seems to range widely among species; the most fecund species belong to the genera Centropages whereas the least fecund are those bearing egg sacs. The factors affecting production levels are both food quantity and food quality dependent but may also be regulated by metabolic processes that are in turn temperature dependent. Also potential egg viability seems to range widely during the year, from 0 to 60% in most cases, indicating that recruitment to the adult population may largely be controlled by physiological mortality rather than to predation or advective processes. There are probably several causes leading to reduced hatching success including an absence of remating, poor food conditions and/or the presence feeding deterrents such as the recently described inhibitory compounds blocking normal copepod embryogenesis. Another common feature of subtemperate species is that they show relatively constant development times from egg to adult, ranging generally from 18 to 25 d, depending on temperature and food conditions. Adult longevity then ranges from 2 to 3 months for adult females maintained in the laboratory which is much shorter than the 1 yr lifespan reported for north-temperate to boreal species.

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.

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.

Experiments were conducted to test the effect of overcrowding on egg production rates in the copepod Centropages typiciis. Fertility was not significantly reduced by the presence of other conspecifics when the volume of water was the same for females incubated alone or in the presence of other females and males. There was no evidence of chemical cues affecting egg production when females were kept in large (1 female in 100 ml sea water [SW] or small (1 female in 25 ml SW) volumes of water, with or without partitions, and in the presence or absence of food or males. Also, spawning rate was not reduced when females were incubated in flow-through experimental chambers which allowed diffusion of pheromones between females incubated alone or in high densities (100 ind. in 50 ml SW). Only when females were placed at unnaturally high densities (1 female ml) was egg production significantly reduced, probably due to physical disturbance and increased number of collisions rather than chemical communication among individuals. The results show that chemical cues do not affect spawning behaviour when population densities are close to field conditions. By contrast, overcrowding induced by swarming or aggregation may potentially limit reproductive success due to reduced feeding, and production and cannibalism of eggs.

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.

Aquatic organisms are constantly exposed to both physical (e.g. temperature and salinity variations) and chemical (e.g. endocrine disruptor chemicals, heavy metals, hydrocarbons, diatom toxins, and other toxicants) stressors which they react to by activating a series of defense mechanisms. This paper reviews the literature on the defense systems, including detoxification enzymes and proteins (e.g. glutathione S-transferases, heat shock proteins, superoxide dismutase and catalase), studied in copepods at the molecular level. The data indicate high inter- and intra-species variability in copepod response, depending on the type of stressor tested, the concentration and exposure time, and the enzyme isoform studied. Ongoing -omics approaches will allow the identification of new genes which will give a more comprehensive overview of how copepods respond to specific stressors in laboratory and/or field conditions and the effects of these responses on higher trophic levels.

Several species and strains of diatoms were tested for their effects on egg production, hatching success, faecal pellet production, ingestion rate and food selection in the copepod Temora stylifera. Two of the diatom species tested were well known polyunsaturated aldehyde (PUA) producers: Skeletonema marinoi (SM) and Thalassiosira rotula (TR1), while the other species tested, Skeletonema pseudocostatum, was a non-PUA producing species. Another T. rotula strain (TR2) was also tested since previous studies had reported contradictory results, due either to absence of PUA production or production in small amounts. Our results showed strong inhibitory effects on reproductive parameters of copepods with all diatom species tested. Although we confirm that S. pseudocostaum did not produce PUAs, this diatom produced large quantities of other oxylipins such as (5Z,8Z,11Z,13E,15S,17Z)-15-hydroxy-5,8,11,13,17-eicosapentaenoic acid (15S-HEPE) and 13,14-13R-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,11Z,17Z-tetraenoic acid (13,14-HEpETE) and 15-oxo-5Z,9E,11E,13E-pentadecatetraenoic acid (15-oxoacid), all of which have already been found in S. marinoi (Fontana et al., 2007a) and Pseudonitzschia delicatissimia (d'Ippolito et al., 2009). Another unidentified oxylipin was also present in high quantities in LC–MS profiles. Some of these oxygenated fatty acid derivatives were also found, together with PUAs, in TR1 and in TR2. Both PUA-producing and non-producing diatoms caused negative effects on hatching success with induction of apoptosis in newly hatched nauplii. This suggests that negative diatom effects can also depend on the production of non volatile oxylipins. Copepod feeding behaviour was found to be non-selective when each diatom species was offered together with the non toxic dinoflagellate Prorocentrum minimum suggesting that grazers are not aware of the toxicity of their food.

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.