Larval feeding activity and use of embryonic resources determine juvenile performance of the common prawn Palaemonserratus.

Phenotypic links are the potential for "carryover" of effects of experience during one life history stage into performance and selection at subsequent stages. They reflect plastic responses to the environment experienced during an early phase on the phenotype of subsequent phases. We are studying these effects by following individuals of the shrimp Palaemon serratus from the embryonic (eggs carried by females) through the larval phase (pelagic) to the juvenile phase (benthic). In experiment 1, we investigated the effects of larval prey concentration (10, 4 and 2 Artemia/mL) and larval incubation temperature (16 and 22 °C) on larval performance (metamorphosis rate, developmental duration and growth) and then on juvenile performance (survival and Specific Growth Rate, SGR, at 18 and 24 °C in 14 days). In experiment 2, we investigated the effects of embryonic incubation temperature (larval biomass and lipid content of newly hatched larvae from embryos incubated at 12 and 18 °C) and larval prey concentration on larval performance and then on juvenile performance. In both experiments, the larvae plastically increased their development time in response to the reduction in temperature and prey concentration, whereas their survival decreased with temperature and prey concentration. The quantity of lipids available at hatching decreased with decreasing embryonic incubation temperature, which reduced the larval performance, particularly with a low concentration of prey. Survival at 14 days post-metamorphosis was significantly reduced when the embryos were incubated at 12 °C compared with those incubated at 18 °C, regardless of the subsequent larval incubation conditions, revealing phenotypic links between overconsumption of embryonic yolk reserves and post-metamorphic fitness. Overall, juveniles had a better SGR at 24 than at 18 °C, and even better when incubated under stressful embryo-larval conditions (temperature and prey concentration). This study highlighted phenotypic links between developmental stages and over developmental periods of several months.

Cadmium Highlights Common and Specific Responses of Two Freshwater Sentinel Species, Dreissena polymorpha and Dreissena rostriformis bugensis.

Zebra mussel (ZM), Dreissena polymorpha, commonly used as a sentinel species in freshwater biomonitoring, is now in competition for habitat with quagga mussel (QM), Dreissena rostriformis bugensis. This raises the question of the quagga mussel's use in environmental survey. To better characterise QM response to stress compared with ZM, both species were exposed to cadmium (100 µg·L-1), a classic pollutant, for 7 days under controlled conditions. The gill proteomes were analysed using two-dimensional electrophoresis coupled with mass spectrometry. For ZM, 81 out of 88 proteoforms of variable abundance were identified using mass spectrometry, and for QM, 105 out of 134. Interestingly, the proteomic response amplitude varied drastically, with 5.6% of proteoforms of variable abundance (DAPs) in ZM versus 9.4% in QM. QM also exhibited greater cadmium accumulation. Only 12 common DAPs were observed. Several short proteoforms were detected, suggesting proteolysis. Functional analysis is consistent with the pleiotropic effects of the toxic metal ion cadmium, with alterations in sulphur and glutathione metabolisms, cellular calcium signalling, cytoskeletal dynamics, energy production, chaperone activation, and membrane events with numerous proteins involved in trafficking and endocytosis/exocytosis processes. Beyond common responses, the sister species display distinct reactions, with cellular response to stress being the main category involved in ZM as opposed to calcium and cytoskeleton alterations in QM. Moreover, QM exhibited greater evidence of proteolysis and cell death. Overall, these results suggest that QM has a weaker stress response capacity than ZM.

Advancing environmental monitoring across the water continuum combining biomarker analysis in multiple sentinel species: A case study in the Seine-Normandie Basin (France).

Nowadays, biomarkers are recognized as valuable tools to complement chemical and ecological assessments in biomonitoring programs. They provide insights into the effects of contaminant exposures on individuals and establish connections between environmental pressure and biological response at higher levels. In the last decade, strong improvements in the design of experimental protocols and the result interpretation facilitated the use of biomarker across wide geographical areas, including aquatic continua. Notably, the statistical establishment of reference values and thresholds enabled the discrimination of contamination effects in environmental conditions, allowed interspecies comparisons, and eliminated the need of a reference site. The aim of this work was to study freshwater-estuarine-coastal water continua by applying biomarker measurements in multi-species caged organisms. During two campaigns, eight sentinel species, encompassing fish, mollusks, and crustaceans, were deployed to cover 25 sites from rivers to the sea. As much as possible, a common methodology was employed for biomarker measurements (DNA damage and phagocytosis efficiency) and data interpretation based on guidelines established using reference values and induction/inhibition thresholds (establishment of three effect levels). The methodology was successfully implemented and allowed us to assess the environmental quality. Employing multiple species per site enhances confidence in observed trends. The results highlight the feasibility of integrating biomarker-based environmental monitoring programs across a continuum scale. Biomarker results align with Water Framework Directive indicators in cases of poor site quality. Additionally, when discrepancies arise between chemical and ecological statuses, biomarker findings offer a comprehensive perspective to elucidate the disparities. Presented as a pilot project, this work contributes to gain insights into current biomonitoring needs, providing new questions and perspectives.

Combined effects of temperature and diet on the performance of larvae produced by young and old Palaemon serratus females.

Seasonal variations in environmental conditions determine the success of decapod larval development, and females transmit more energy in sub-optimal conditions to maximise the fitness of their offspring. The objective of this study was to focus on the combined effects of temperature (14, 18 and 22 °C) and food quality on the performance of larvae produced by 5 young (0+) and 5 old (I+) Palaemon serratus females. We prepared 3 diets based on Artemia, in decreasing order of total fatty acid content: freshly hatched nauplii (N), unenriched metanauplii (M) and metanauplii enriched with a mixture of microalgae (ME). At hatching, the larvae produced by I+ females had a higher biomass but a similar fatty acid concentration to those produced by 0+ females. Larvae survived better and developed relatively faster as temperature increased, and the longer they waited to metamorphose, the greater their weight at metamorphosis. These performances were diet-dependent, with more survival and more growth in less time with diet N than with the other two. Larvae from I+ females performed better than those from 0+ females, especially under the most stressful conditions. The greater biomass of the larvae of I+ females seems to have enabled them to follow a shorter, and therefore faster, development path than those of 0+ females. The larvae's diet also had an impact on post-metamorphic composition: larvae eating a diet richer in fatty acids produced richer juveniles and those eating a poorer diet produced juveniles with slightly more essential fatty acids. This study supports the high plasticity of caridean shrimp larval development and the importance of maternal effects on the fitness of offspring.

Modulation of haemocyte motility by chemical and biological stresses in Mytilus edulis and Dreissena polymorpha.

Mussels are constantly exposed to various pollutants in the environment, which can impair their immune defences against microbes and thus threaten their survival. In this study, we expand the insight into a key parameter of immune response in two mussel species by exploring the impact of exposure to pollutants or bacteria or simultaneous chemical and biological exposure on haemocyte motility. Basal haemocyte velocity in primary culture was high and increasing over time in Mytilus edulis (mean cell speed of 2.32 µm/min ± 1.57) whereas Dreissena polymorpha showed a constant and rather low cell motility with time (mean cell speed of 0.59 µm/min ± 0.1). In the presence of bacteria, the motility of haemocytes was instantly enhanced and slowed down after 90 min for M. edulis. In contrast, in vitro exposure of haemocytes to chemicals, either Bisphenol A, oestradiol, copper, or caffeine, induced an inhibition of cell motility in both mussel species. Finally, the cellular activation observed during bacterial challenges was inhibited by simultaneous exposure to bacteria and pollutants. Overall, our results indicate that chemical contaminants can alter haemocyte migration in mussels which can weaken their response to pathogens and therefore increase their susceptibility to infectious diseases.

Temperature-mediated developmental plasticity in winter and summer larvae of Palaemon serratus.

In a seasonal environment, variation in larval phenotype and developmental plasticity allow crustacean larvae to maximise survival by lengthening or shortening their development. The aim of this study is to investigate the effects of temperature, laying season and their interaction on larval developmental pathways (larval instars and larval stages). We monitored the different larval stages and calculated the number of larval instars reached during the development of winter and summer larvae of Palaemon serratus incubated at 12, 16 or 20 °C. We observed a great variability in the larval development (6-13 larval instars and 6 to 11 larval stages). A higher temperature decreases the development time and the number of larval instars. At a given temperature, the development time of winter and summer larvae was not different. Two larval stages were considered supernumerary (zoea 4 and 6), as they were more frequent at low temperatures. At higher temperatures, some larvae started to develop pleopods as early as the third instar, larval stage which had never been described (named here zoea 3'). This phenomenon was more common in winter larvae than in summer larvae. These results provide new insights into the expression of developmental plasticity in decapod larvae.

Effects of 17α-Ethinylestradiol (EE2) exposure during early life development on the gonadotropic axis ontogenesis of the European sea bass, Dicentrarchus labrax.

Exposure of young organisms to oestrogenic endocrine disrupting chemicals (EDCs) can elicit adverse effects, particularly on the reproductive function. In fish, as in other vertebrates, reproduction is controlled by the neuroendocrine gonadotropic axis, whose components are mainly regulated by sex steroids and may then be targets for EDCs. In the present study, we investigated the effects of a xenoestrogen exposure on the ontogenesis of the gonadotropic axis in European sea bass. After exposure of hatching larvae for 8 days to 17α-ethinylestradiol (EE2) (0.5 nM and 50 nM), gene expression for kisspeptins (kiss1, kiss2), gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), gonadotropin beta subunits (lhß and fshß) and brain type aromatase (cyp19a1b) were measured using quantitative real-time PCR. Our results demonstrate that EE2 strongly stimulated the expression of brain type aromatase (cyp19a1b) in sea bass larvae. In addition, EE2 exposure also affected the mRNA levels of kiss1, gnrh1 and gnrh3 by inducing a downregulation of these genes during the early developmental stages, while no effect was seen in gnrh2, lhß and fshß. These results reinforce the idea that the larval development is a sensitive critical period in regard to endocrine disruption and that the gonadotropic axis in the developing sea bass is sensitive to xenoestrogen exposure.

N-acetyl-ß-d-glucosaminidase activity in Palaemon serratus - Methodological optimisation and intrinsic variability.

Chitinolytic enzymes fulfil a key role in the moulting process of crustaceans, in degrading the endocuticle during apolysis. Measuring the enzyme activity is an interesting manner to monitor the moult process at sub-individual level, complementary to the classical observation of the integument morphogenesis, ecdysis success, or moult cycle duration. The present study aimed to optimise the methodology of using N-acetyl-ß-D-glucosaminidase (NAGase) activity to monitor moulting in the marine prawn Palaemon serratus, and to compare NAGase activity levels along the moult cycle of both male and female specimens. First, to optimise protocols for five different organs, different reaction medium compositions were tested, considering the type buffer, concentration of the substrate, and the load in enzymatic extract. Second, levels of NAGase activity were closely monitored during eight moulting stages in male prawns. Variations in NAGase activity were observed during the moult cycle, with an increase in activity in the late premoult phase of approximately 2.4-fold the level of the intermoult phase. This response profile was observed for each tested organ. The levels of NAGase activity of male and female specimens were compared during three stages of the premoult phase. The patterns observed for both sexes were similar for all the tested organs.

Signification of DNA integrity in sperm of Palaemon serratus (Pennant 1777): Kinetic responses and reproduction impairment.

The study of the effects of contamination on sperm quality not only provides an early, specific and integrative response to the fraction of bioavailable pollutants, but also has been shown to predict the potential of this fraction to modify an organism's capacity to reproduce. In addition, fertility damage in invertebrates has been addressed as a major problem that may pose a threat to the maintenance of populations. In this context, the present study proposes a methodology based on the measurement of sperm DNA integrity to evaluate the impact of paternal damaged DNA on the reproductive success of Palaemon serratus. A preliminary methodological optimization step was carried out to assess the kinetics of response of spermatozoa as well as the sensitivity of the spermatozoa according to their location in the genital tract. Spermatozoa appeared to be sensitive to a short in vivo exposure to the direct acting agent methyl methanesulfonate (i.e. MMS; 2 days), with a persistence of damage even after a 30 days' recovery in a clean environment, suggesting a probable lack of DNA repair machinery. Moreover, our results revealed no difference in the level of DNA damage in mature spermatozoa whatever the exposure in spermatophore located in the terminal ampulla or in the proximal and distal part of the vas deferens. Finally, a significant decrease in the percentage of naturally bred prawns has been observed at the highest concentration of MMS (i.e. 100 µM). Nevertheless, no reproduction impairment (i.e. fertilization rate and early embryo development) following a paternal exposure has been shown in spite of very high levels of sperm DNA damage. In regard to the literature, this result raises questions concerning the kinetics of expression of genotoxic damage on progeny in the Palaemon model and future work will be led in this way.

Highly polluted life history and acute heat stress, a hazardous mix for blue mussels.

Intertidal sessile organisms constitute through their life history unintended stress recorders. This study focuses on the impact of pollution on Mytilus edulis ability to cope with an additional stress. For this purpose, two acclimation stages to different temperatures were conducted before an acute stress exposure in mussels collected from a heavily polluted site. Gill proteomes were analyzed by 2DE and regulated proteins identified. Massive mortality was observed for organisms acclimated to colder temperatures. Despite this major difference, both groups shared a common response with a strong representation of proteoforms corresponding to "folding, sorting and degradation" processes. Nevertheless, surviving mussels exhibit a marked increase in protein degradation consistent with the observed decrease of cell defense proteins. Mussels acclimated to warmer temperature response is essentially characterized by an improved heat shock response. These results show the differential ability of mussels to face both pollution and acute heat stress, particularly for low-acclimated organisms.

Neuroendocrine disruption in the shore crab Carcinus maenas: Effects of serotonin and fluoxetine on chh- and mih-gene expression, glycaemia and ecdysteroid levels.

Serotonin, a highly conserved neurotransmitter, controls many biological functions in vertebrates, but also in invertebrates. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are commonly used in human medication to ease depression by affecting serotonin levels. Their residues and metabolites can be detected in the aquatic environment and its biota. They may also alter serotonin levels in aquatic invertebrates, thereby perturbing physiological functions. To investigate whether such perturbations can indeed be expected, shore crabs (Carcinus maenas) were injected either with serotonin, fluoxetine or a combination of both. Dose-dependent effects of fluoxetine ranging from 250 to 750nM were investigated. Gene expression of crustacean hyperglycemic hormone (chh) as well as moult inhibiting hormone (mih) was assessed by RT-qPCR at 2h and 12h after injection. Glucose and ecdysteroid levels in the haemolymph were monitored in regular intervals until 12h. Serotonin led to a rapid increase of chh and mih expression. On the contrary, fluoxetine only affected chh and mih expression after several hours, but kept expression levels significantly elevated. Correspondingly, serotonin rapidly increased glycaemia, which returned to normal or below normal levels after 12h. Fluoxetine, however, resulted in a persistent low-level increase of glycaemia, notably during the period when negative feedback regulation reduced glycaemia in the serotonin treated animals. Ecdysteroid levels were significantly decreased by serotonin and fluoxetine, with the latter showing less pronounced and less rapid, but longer lasting effects. Impacts of fluoxetine on glycaemia and ecdysteroids were mostly observed at higher doses (500 and 750nM) and affected principally the response dynamics, but not the amplitude of glycaemia and ecdysteroid-levels. These results suggest that psychoactive drugs are able to disrupt neuroendocrine control in decapod crustaceans, as they interfere with the normal regulation of the serotonergic system.

Consequences of acclimation on the resistance to acute thermal stress: Proteomic focus on mussels from pristine site.

Climate change constitutes an additional threat for intertidal species that already have to cope with a challenging environment. The present study focuses on the blue mussel Mytilus edulis and aims at investigating the importance of thermal acclimation in heat stress response. Microcosm exposures were performed with mussels submitted to an identical acute thermal stress following two thermal summer acclimations standing for present or future temperature conditions. Gill proteomes were analyzed by 2DE and 96 differentially expressed proteoforms were identified. Our results show that cell integrity appears to be maintained by the rise in molecular protective systems (i.e. Heat Shock Proteins), and by the reallocation of energy production via a switch to anaerobic metabolism and the setting up of alternative energy pathways. Finally, our results indicate that the response of mussels to acute thermal stress is conditioned by the acclimation temperature with an improved response in organisms acclimated to higher temperatures.

2-DE Mapping of the Blue Mussel Gill Proteome: The Usual Suspects Revisited.

The Blue Mussel (Mytilus edulis, L. 1758) is an ecologically important and commercially relevant bivalve. Because of its ability to bioconcentrate xenobiotics, it is also a widespread sentinel species for environmental pollution, which has been used in ecotoxicological studies for biomarker assessment. Consequently, numerous proteomics studies have been carried out in various research contexts using mussels of the genus Mytilus, which intended to improve our understanding of complex physiological processes related to reproduction, adaptation to physical stressors or shell formation and for biomarker discovery. Differential-display 2-DE proteomics relies on an extensive knowledge of the proteome with as many proteoforms identified as possible. To this end, extensive characterization of proteins was performed in order to increase our knowledge of the Mytilus gill proteome. On average, 700 spots were detected on 2-DE gels by colloidal blue staining, of which 122 different, non-redundant proteins comprising 203 proteoforms could be identified by tandem mass spectrometry. These proteins could be attributed to four major categories: (i) "metabolism", including antioxidant defence and degradation of xenobiotics; (ii) "genetic information processing", comprising transcription and translation as well as folding, sorting, repair and degradation; (iii) "cellular processes", such as cell motility, transport and catabolism; (iv) "environmental information processing", including signal transduction and signalling molecules and interaction. The role of cytoskeleton proteins, energetic metabolism, chaperones/stress proteins, protein trafficking and the proteasome are discussed in the light of the exigencies of the intertidal environment, leading to an enhanced stress response, as well as the structural and physiological particularities of the bivalve gill tissue.

The multi-xenobiotic resistance (MXR) efflux activity in hemocytes of Mytilus edulis is mediated by an ATP binding cassette transporter of class C (ABCC) principally inducible in eosinophilic granulocytes.

In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK571) blockers, we show that MXR activity is only carried out by MRP-type transporters in M. edulis hemocytes. In addition, cell-type-gated flow cytometry and calculation of the MXR activity factor indicate that ABCC-efflux activity is higher and more inducible in eosinophilic granulocytes than in other hemocyte subtypes. We conclude that, in the hemocytes of M. edulis, MXR phenotype is mediated by an ABCC/MRP-type transporter activity principally supported by eosinophilic granulocytes. A role for ABC transporters in hemocyte migration is discussed.

Regulation of volume-sensitive Cl- channels in multi-drug resistant MCF7 cells.

The P-glycoprotein (P-gp) is thought to be involved in the regulation of volume-sensitive chloride channels. In this study, the possible coupling between P-gp and swelling-activated chloride channels has been examined in MCF7 cells with sensitive (MDR-), resistant (MDR+), and reversed resistant (MDR(REV)) phenotypes. Western blot analysis showed that incubation of cells with doxorubicin induced P-gp expression in a reversible manner. Exposure of MDR+ cells to hypotonicity resulted in an inhibition of P-gp activity while hypotonic challenges induced swelling-activated chloride currents (I(Cl-swell)) in MDR-, MDR+, and MDR(REV) MCF7 cells. While verapamil inhibited I(Cl-swell) in all cell types, doxorubicin and vincristine rapidly and reversibly inhibited I(Cl-swell) uniquely in MDR+. Intracellular dialysis of MDR+ cells with C219 anti-P-gp antibody abolished the sensitivity of I(Cl-swell) to doxorubicin and led to a response pattern very close to that of MDR- cells. Taken together, these results strongly suggest that the P-glycoprotein regulates I(Cl-swell) in resistant MCF7.

Cell responses to xenobiotics: comparison of MCF7 multi-drug- and mussel blood cell multi-xenobiotic-defense mechanisms.

Multi-drug resistance (MDR) in MCF7 breast cancer cells and multi-xenobiotic resistance (MXR) in mussel (Mytilus edulis) blood cells (MBC) are well known mechanisms that contribute to the decrease in intracellular concentrations of many unrelated but cytotoxic compounds. In the present work, we have carried out comparative investigations of the MDR/MXR protective mechanisms using a rapid colorimetric assay for cell viability and calcein accumulation for MDR/MXR activities. These studies were performed using cultured MCF7 and MBC before and after in vitro exposure to xenobiotics. Our results indicate that a 5-day exposure to doxorubicin or vincristine decreased calcein accumulation in MBC which is consistent with an induction of multi-xenobiotic resistance. The increase in calcein accumulation provoked by 1-h treatment with 50 microM verapamil was much lower in MBC when compared to the P-glycoprotein overexpressing MCF7 cell line. We conclude that such microplate assays could be used in primary cultures of MBC to estimate the effects of various chemicals on MXR activity.