Trace metals exposure in three different coastal compartments show specific morphological and reproductive traits across generations in a sentinel copepod.

The effect of exposure from several compartments of the environment at the level of individuals was rarely investigated. This study reports the effect of contaminants from varied compartments like sediment resuspension, elutriation from resuspended sediment (extract) and seawater spiked trace metal mixtures (TM) on morphological and reproductive traits of the pelagic bioindicator copepod Eurytemora affinis. At the population level of E. affinis, lowest survival was observed in dissolved exposures (TM and extract) in the first generation (G1), showing some adaptation in the second generation (G2). An opposite trend for resuspended sediment showed higher sensitivity in survival at G2. At the individual level, prosome length and volume proved to be sensitive parameters for resuspended sediments, whereas clutch size and egg diameter were more sensitive to TM and extract. Although the generation of decontamination (G3, no exposure), showed a significant recovery at the population level (survival % along with clutch size) of E. affinis exposed to resuspended sediment, morphological characteristics like prosome length and volume showed no such recovery (lower than control, p < 0.05). To the contrary, dissolved exposure showed no significant recovery from G1 to G3 on neither survival %, clutch size, egg diameter, prosome volume, but an increase of prosome length (p < 0.05). Such tradeoffs in combatting the stress from varied sources of toxicity were observed in all exposures, from G1 to G3. The number of lipid droplets inside the body cavity of E. affinis showed a significant positive correlation with trace metal bioaccumulation (p < 0.01) along with a negative correlation (p < 0.05) with survival and clutch size in each treatment. This confirms the inability of copepods to utilize lipids under stressful conditions. Our study tenders certain morphological and reproductive markers that show specificity to different compartments of exposure, promising an advantage in risk assessment and fish feed studies.

Reproductive trade-offs of the estuarine copepod Eurytemora affinis under different thermal and haline regimes.

Copepod females invest a quantity of resources in their reproduction. Depending on several biotic and abiotic factors and their evolutionary history a trade-off can be commonly observed between producing a large number of smaller offspring or a small number of larger offspring. In this study, a multi-generational approach was applied to determine whether a trade-off between clutch size and egg size existed in the copepod Eurytemora affinis under different controlled conditions of temperature and salinity. This protocol was based on the follow-up of reproductive (Clutch Size 'CS', Egg Diameter 'ED') and morphological (Prosome Length 'PL') traits during several generations. Copepods were acclimated to cold (7 °C) and warm (20 °C) temperatures, and then their reproductive output was tested at the higher temperature of 24 °C. CS and ED were positively correlated to PL, so as a first step linear regressions between each reproductive trait and female PL were performed. The residuals from the regression lines of CS and ED with PL were calculated to remove the effect of female size. When the normalized data (residuals) of CS and ED plotted together a negative relationship between egg size and egg number revealed the existence of a trade-off. Copepod populations initially acclimated to cold temperature are commonly characterized by relatively smaller CS and larger ED. Conversely, warm temperature adapted females produced relatively larger CS and smaller ED. After transfer to a temperature of 24 °C, the ED did not change but the CS showed high variability indicating stressful conditions and no trade-off was observed. These observations suggest that E. affinis is able to modulate its reproduction depending on the encountered temperature. It seems that this copepod species can shift between a K- and an r-strategy in response to colder or warmer conditions. In a late winter-early spring like cold temperature, copepod females seem to invest more on offspring quality by producing relatively larger eggs. This ecological strategy ensures a high recruitment of the spring generation that is responsible for the strength of the maximum population size usually observed in late spring-early summer (May-June). To the contrary, at summer-like temperature, where the population density decreases significantly in the Seine estuary, copepod females seem to switch from K to r strategy by favoring offspring number compared to offspring size. Finally, the use of a higher temperature of 24 °C seems to disrupt the observed reproductive trade-off even after several generations. These results suggest that a switching between K- or r-strategy of E. affinis depends highly on temperature effects. The effect of salinity increase during a summer-like temperature of 20 °C as well as after transfer to 24 °C decreased PL and CS but the ED did not change significantly.

Removal and Biodegradation of Phenanthrene, Fluoranthene and Pyrene by the Marine Algae Rhodomonas baltica Enriched from North Atlantic Coasts.

This study is focused on the removal, accumulation and degradation of three environmental ubiquitous polycyclic aromatic hydrocarbons (PAHs), phenanthrene (PHE), fluoranthene (FLA) and pyrene (PYR), by the marine alga Rhodomonas baltica enriched from the English Channel. After separation, purification and culture in several phases, R. baltica was exposed to PAH concentrations that are frequently encountered in the field in several anthropized environments. The results showed that R. baltica can grow under PAH stress, efficiently remove up to 70% of these compounds from the medium by 216 h of culture and selectively bioaccumulate PAHs by their hydrophobicity. Between PHE, FLA and PYR, phenanthrene was the compound with higher degradation rates throughout incubation. The equilibrium partitioning theoretical approach showed that physico-chemical partitioning, rather than active bioconcentration, was the major factor governing the bioaccumulation, outlying a potential application in decontamination processes for this species.

Myofibril Changes in the Copepod Pseudodiaptomus marinus Exposed to Haline and Thermal Stresses.

Copepods are small crustaceans capable to survive in various aquatic environments. Their responses to changes in different external factors such as salinity and temperature can be observed at different integration levels from copepod genes to copepod communities. Until now, no thorough observation of the temperature or salinity effect stresses on copepods has been done by optical microscopy. In this study, we used autofluorescence to visualize these effects on the morphology of the calanoid copepod Pseudodiaptomus marinus maintained during several generations in the laboratory at favorable and stable conditions of salinity (30 psu) and temperature (18°C). Four different stress experiments were conducted: at a sharp decrease in temperature (18 to 4°C), a moderate decrease in salinity (from 30 to 15 psu), a major decrease in salinity (from 30 to 0 psu), and finally a combined stress with a decrease in both temperature and salinity (from 18°C and 30 psu to 4°C and 0 psu). After these stresses, images acquired by confocal laser scanning microscopy (CLSM) revealed changes in copepod cuticle and muscle structure. Low salinity and/or temperature stresses affected both the detection of fluorescence emitted by muscle sarcomeres and the distance between them. In the remaining paper we will use the term sarcomeres to describe the elements located within sarcomeres and emitted autofluorescence signals. Quantitative study showed an increase in the average distance between two consecutive sarcomeres from 2.06 +/- 0.11 µm to 2.44 +/- 0.42 µm and 2.88 +/- 0.45µm after the exposure to major haline stress (18°C, 0 psu) and the combined stress (4°C, 0 psu), respectively. These stresses also caused cuticle cracks which often occurred at the same location, suggesting the cuticle as a sensitive area for osmoregulation. Our results suggest the use of cuticular and muscle autofluorescence as new biomarkers of stress detectable in formalin-preserved P. marinus individuals. Our label-free method can be easily applied to a large number of other copepod species or invertebrates with striated musculature.

Genetic and morphological heterogeneity among populations of Eurytemora affinis (Crustacea: Copepoda: Temoridae) in European waters.

Our understanding of the systematics of the Eurytemora affinis complex developed at a fast pace over the last decades. Formerly considered as a complex of cryptic species, it is now believed to include three valid species: E. affinis, Eurytemora carolleeae, and Eurytemora caspica. American and European representatives have been studied in detail with respect to fine-scale geographic distribution, levels of genetic subdivision, evolutionary and demographic histories. Morphological components have been less explored. In this study, an analysis of the phylogeny and morphology of E. affinis was done, with a special focus on European populations. A total of 447 individuals of E. affinis from Europe were analyzed with genetic tools and 170 individuals according to morphological criteria. Common and new morphological and genetic features were analyzed. For this, we used ML and Bayesian methods to analyze the bar coding mt-DNA gene cytochrome c oxidase I subunit. Both genetic and morphological analyses showed high heterogeneities among the E. affinis populations from Europe. As a result, three local populations of E. affinis in Western Europe, including the European part of Russia, were established. Their genetic and morphological heterogeneity corresponded to the subspecies level.

Evaluation of the Copepod Eurytemora affinis Life History Response to Temperature and Salinity Increases.

Anissa Souissi, Sami Souissi, and Jiang-Shiou Hwang (2016) Zooplankton and particularly copepods have a key role in the functioning of aquatic ecosystems. However, the mechanisms involved in the physiological responses of copepods to temperature and salinity increases are little understood, and the role of plasticity involved in facing environmental changes has rarely been demonstrated experimentally. In this study, the copepod Eurytemora affinis, widely distributed in the northern hemisphere, was selected as a biological model to test the effect of a 4°C temperature increase at two salinities. In addition to the optimal salinity (15 psu), a stressful condition of salinity 25 psu was also verified. Copepods from the Seine estuary were acclimated in laboratory to their optimal temperature of 15°C at salinity 15 PSU and then they were acclimated during several generations to their upper thermal limit (20°C) at two salinities (15 and 25 PSU), after which the temperature was raised by 4°C. This experiment revealed that after long-term acclimation and under unlimited food conditions, E. affinis maintained good fitness at 20°C and at both optimal and stressful salinities. After temperature increase to 24°C, the population remained viable but copepod size was significantly decreased as well as female's fecundity. The decrease of fitness was accentuated under the additional stressful condition of salinity 25 psu. This study demonstrated that the mechanisms of response to temperature and salinity increases (i.e. global warming) are complex, and should be investigated through experimental studies that consider acclimation and multigenerational factors. Our results will enrich the development of Individual-Based Models (IBMs) capable to test the role of microevolution and plasticity of E. affinis in the framework of future climate scenarios.

Acute toxicity, uptake and accumulation kinetics of nickel in an invasive copepod species: Pseudodiaptomus marinus.

Pseudodiaptomus marinus is a marine calanoid copepod originating of the Indo-Pacific region, who has successfully colonized new areas and it was recently observed in the European side of the Mediterranean Sea as well as in the North Sea. Actually, many questions were posed about the invasive capacity of this copepod in several non-native ecosystems. In this context, the main aim of this study was to investigate the tolerance and the bioaccumulation of metallic stress in the invasive copepod P. marinus successfully maintained in mass culture at laboratory conditions since 2 years. In order to study the metallic tolerance levels of P. marinus, an emergent trace metal, the nickel, was chosen. First, lethal concentrations determination experiments were done for 24, 48, 72 and 96 h in order to calculated LC50% but also to select a relevant ecological value for the suite of experiments. Then, three types of experiments, using a single concentration of nickel (correspond the 1/3 of 96 h-LC50%) was carried in order to study the toxico-kinetics of nickel in P. marinus. Concerning lethal concentrations, we observed that P. marinus was in the same range of sensitivity compared to other calanoid copepods exposed to nickel in the same standardized experimental conditions. Results showed that the uptake of nickel in P. marinus depends from the pathways of entrance (water of food), but also that Isochrysis galbana, used as a food source, has an important bioaccumulation capacity and a rapid uptake of nickel.

Lipid nanocapsules for behavioural testing in aquatic toxicology: Time-response of Eurytemora affinis to environmental concentrations of PAHs and PCB.

The increasing interest for behavioural investigations in aquatic toxicology has heightened the need for developing tools that allow realistic exposure conditions and provide robust quantitative data. Calanoid copepods dominate the zooplankton community in marine and brackish environments. These small organisms have emerged as attractive models because of the sensitivity of their behaviour to important environmental parameters and the significance of self-induced motion in their ecology. Estuarine copepods are particularly relevant in this context because of their incessant exposure to high levels of pollution. We used lipid nanocapsules to deliver sub-lethal concentrations of PAHs (pyrene, phenanthrene and fluoranthene) and PCB 153 into the digestive track of males and females Eurytemora affinis. This novel approach enabled us to achieve both contact and trophic exposure without using phytoplankton, and to expose copepods to small hydrophobic molecules without using organic solvent. We reconstructed the motion of many copepods swimming simultaneously by means of three-dimensional particle tracking velocimetry. We quantified the combined effects of contact and trophic toxicity by comparing the kinematic and diffusive properties of their motion immediately and after 3h and 24h of exposure. Despite the lack of toxicity of their excipients, both empty and loaded capsules increased swimming activity and velocity immediately after exposure. Laser microscopy imaging shows adhesion of nanocapsules on the exoskeleton of the animals, suggesting contact toxicity. The behavioural response resembles an escape reaction allowing copepods to escape stressful conditions. The contact toxicity of empty capsules and pollutants appeared to be additive and nanocapsules loaded with PCB caused the greatest effects. We observed a progressive accumulation of capsules in the digestive track of the animals after 3h and 24h of exposure, which suggests an increasing contribution of systemic toxicity. Nanocapsules filled with PAHs caused a smaller response compared to empty capsules, which we attribute to the narcotic properties of these toxicants. The sharp decrease in velocity after 24h of exposure to capsules loaded with PCB suggests physiological incapacitation following systemic toxicity. Clear differences are visible between genders in their response to empty and loaded capsules, for all exposure durations. Females appear to be less sensitive than males, suggesting different tolerance to stress conditions. Our results confirm the feasibility of using lipid nanocapsules to identify pollutant-induced behavioural alteration in the plankton. They also add new insights into the contact and systemic toxicity of common pollutants. We expect that our results will assist and evoke further research to develop suitable nanocarrier systems for behavioural testing.

Lipid nanocapsules as a new delivery system in copepods: Toxicity studies and optical imaging.

In this paper, we investigated the potential of lipid nanocapsules (LNCs) as a delivery system of small hydrophobic molecules, polycyclic aromatic hydrocarbons (PAHs) - pyrene, fluoranthene, phenanthrene, in the copepod Acartia tonsa. The LNCs were produced by a phase inversion process with a nominal size of 50 nm. These nanocapsules were obtained without organic solvent and with pharmaceutically acceptable excipients. The PAHs-LNCs displayed a stable monodisperse size distribution and a good stability in sea water for 7 days. By using fluorescent LNCs, it was possible to evidence LNCs ingestion by the copepods using confocal laser scanning microscopy. While blank LNCs are not toxic to copepods at tested concentrations, PAH-loaded LNCs were found to be very toxic on A. tonsa with a high mortality rate reaching 95% after 72 h exposure to 200 nM pyrene-loaded LNCs. On the other hand, when acetone is used to dissolve an equivalent concentration of PAHs in sea water, the copepod mortality is 10 times lower than using LNCs as nano-delivery system. This confirms the efficiency of using LNCs to deliver molecules directly in the gut or copepod carapace. The small size and non toxicity of these delivery nano-systems make them suitable for drug delivery to copepods.

Label-free microscopy and stress responses reveal the functional organization of Pseudodiaptomus marinus copepod myofibrils.

Pseudodiaptomus marinus copepods are small crustaceans living in estuarine areas endowed with exceptional swimming and adaptative performances. Since the external cuticle acts as an impermeable barrier for most dyes and molecular tools for labeling copepod proteins with fluorescent tags are not available, imaging cellular organelles in these organisms requires label free microscopy. Complementary nonlinear microscopy techniques have been used to investigate the structure and the response of their myofibrils to abrupt changes of temperature or/and salinity. In contrast with previous observations in vertebrates and invertebrates, the flavin autofluorescence which is a signature of mitochondria activity and the Coherent Anti-Stokes Raman Scattering (CARS) pattern assigned to T-tubules overlapped along myofibrils with the second harmonic generation (SHG) striated pattern generated by myosin tails in sarcomeric A bands. Temperature jumps from 18 to 4 °C or salinity jumps from 30 to 15 psu mostly affected flavin autofluorescence. Severe salinity jumps from 30 to 0 psu dismantled myofibril organization with major changes both in the SHG and CARS patterns. After a double stress (from 18 °C/30 psu to 4° C/0 psu) condensed and distended regions appeared within single myofibrils, with flavin autofluorescence bands located between sarcomeric A bands. These results shed light on the interactions between the different functional compartments which provide fast acting excitation-contraction coupling and adequate power supply in copepods muscles.

Chemical structure and biological activity of a highly branched (1 → 3,1 → 6)-ß-D-glucan from Isochrysis galbana.

A highly branched (1 → 3,1 → 6)-ß-D-glucan was isolated from the microalga Isochrysis galbana Parke (Isochrysidales, Haptophyta). The polysaccharide structure was analyzed by methylation and Smith degradation, as well as by ESI and MALDI TOF mass spectrometry and NMR spectroscopy. The glucan was shown to contain a (1 → 6)-linked backbone, where every residue is substituted at position 3 by Glc, which in turn may be substituted at C-6 by a single Glc or by rather short (up to tetrasaccharide) oligosaccharide chains. All the 3-linked Glc residues are present in these side chains. In the biological activity experiments it was demonstrated that the polysaccharide directly inhibits the proliferation of U937 human leukemic monocyte lymphoma cells and therefore has potential anti-tumor activity.