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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.

Numerous biological activities such as grazer defense and intraspecific signaling have been described for diatom oxylipins, fatty acid derived secondary metabolites produced by some diatom species. As the function and importance of these compounds are still controversial, the production of a subclass of these molecules, nonvolatile oxylipins, was studied during an induced bloom of Skeletonema marinoi (Samo et Zingone) in a mesocosm setup. Reproductive parameters of one of the main grazers, Calanus finmarchicus, were also examined during the bloom. Oxylipins detected during the bloom were the same as those previously described for S. marinoi and were detected predominantly in the mesocosm inoculated with this diatom. Reproductive success of C. finmarchicus remained unaffected during the course of the bloom. This may have been due to a dilution effect by the availability of alternative suitable prey or to the limited exposure of the copepods to the oxylipins generated during the short bloom. Follow up laboratory studies showed that oxylipin composition changed both when the S. marinoi clone used for inoculation was grown in the laboratory and in comparison to the well-studied Adriatic clone of S. marina These results highlight the necessity of quantitatively measuring oxylipin concentrations during diatom blooms at sea to be able to correctly evaluate their ecological significance. (C) 2013 Elsevier B.V. All rights reserved.

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.

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.

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 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.

Failure of female reproductive capacity in the copepod Calanus helgolandicus was related to number and combination of the phytoplankton species in the diets. The maternal food effects were detectable at different levels: fecundity, oogenesis and hatching. Fecundity and hatching were normal with two single (ca. Isochrysis galbana and Prorocentrum minimum) and one mixed (Phaeodactylum tricornutum+Dunaliella tertiolecta+Pavlova lutherii+I. galbana+P. minimum) diets. With the single P. lutherii diet, fecundity decreased, but hatching remained optimal. The daily egg production and hatching rates decreased significantly in females fed the other single P. tricornutum, D. tertiolecta and mixed (P. tricornutum+D. tertiolecta+P. lutherii+I. galbana) diets, or starved. The fecundity decrease coincided with gonad atresia, which was reversible when P. tricornutum and P. lutherii diets were replaced by P. minimum diet. It was irreversible when D. tertiolecta was replaced by P. minimum, leading to female sterilization expressed by the deterioration of OS3 and OS2 oocytes, as a function of the feeding duration. We assume that atresia of female gonads was caused by the limitation of essential nutrients in food, such as fatty acids, which induced catabolism and recycling of yolk reserves and thus, maintenance of gonad integrity and low spawning rates. With the D. tertiolecta diet, abnormally high increase of ornithine concentrations in eggs showed that the ornithine metabolism and polyamine pathway were affected during oogenesis, leading atresia of oocytes to be deeply disturbed and followed up by necrosis of the gonads.