Cyril Turies

Multi-biomarker approach in wild European bullhead, Cottus sp., exposed to agricultural and urban environmental pressures : practical recommendations for experimental design

In freshwater ecosystems, a large number of chemical substances are able to disturb homeostasis of fish by modulating one or more physiological functions including the immune system. The aim of this study was to assess multi-biomarker responses including immunotoxicity induced by urban and agricultural pressure in European bullheads living in a small French river basin. For this purpose, a set of biochemical, immunological, physiological and histological parameters was measured in wild bullheads from five locations characterized by various environmental pressures. Moreover, to address effects of physiological status and contamination level variation on biomarker responses, fish were sampled during three periods (April, July and October). Results revealed a clear impact of environmental pressure on fish health and particularly on immunological status. An increase of EROD activity was recorded between upstream and downstream sites. Upstream sites were also characterized by neurotoxicological effects. Fish exhibited upstream/downstream variations of immunological status but strong differences were observed according to sampling season. Conversely, regarding biochemical and immunological effects, no significant response of physiological indexes was recorded related to environmental pressures. According to these results, the European bullhead appears as a valuable model fish species to assess adverse effects in wildlife due to urban and agricultural pressures.

Localization of steroidogenic enzymes and Foxl2a in the gonads of mature zebrafish (Danio rerio)

In zebrafish, the identification of the cells expressing steroidogenic enzymes and their regulators is far from completely fulfilled though it could provide crucial information on the elucidation of the role of these enzymes. The aim of this study was to better characterize the expression pattern of steroidogenic enzymes involved in estrogen and androgen production (Cyp17-I, Cyp11c1, Cyp19a1a and Cyp19a1b) and one of their regulators (Foxl2a) in zebrafish gonads. By using immunohistochemistry, we localized the steroid-producing cells in mature zebrafish gonads and determined different expression patterns between males and females. All these steroidogenic enzymes and Foxl2a were detected both in the testis and ovary. In the testis, they were all localized both in Leydig and germ cells except Cyp19a1b which was only detected in germ cells. In the ovary, Cyp17-I, Cyp19a1a and Foxl2a were immunolocalized in both somatic and germ cells while Cyp19a1b was only detected in germ cells and Cyp11c1 in somatic cells. Moreover, Cyp19a1a and Foxl2a did not display exactly the same patterns of spatial localization but their expressions were correlated suggesting a possible regulation of cyp19a1a gene by Foxl2a in zebrafish. Comparative analysis revealed a dimorphic expression of Cyp11c1, Cyp19a1a, Cyp19a1b and Foxl2a between males and females. Overall, our study provides a detailed description of the expression of proteins involved in the biosynthesis of steroidal hormones at the cellular scale within gonads, which is critical to further elucidating the intimate roles of the enzymes and the use of the zebrafish as a model in the field of endocrinology.

Involvement of fish immunomarkers in environmental biomonitoring approach: Urban and agri-viticultural context.

The Champagne region (France) is characterized by various chemical environmental pressures which could interfere with the immune status of natural populations of European bullhead, Cottus sp. Some adult fish were caught by electrofishing in spring, summer and autumn to determined immune effect of urban (Muizon), intensive agricultural (Bouy; Prunay) or viticultural (Serzy; Prunay) influences. The major results demonstrated an increase of cellular mortality and a decrease of phagocytosis activity in the stations impacted by agri-viticultural chemicals. These immunomodulations followed the temporal variability due to different treatments (agricultural impacts on spring; viticultural effects on autumn). At the present time, not enough data was provided to confirm the impact of agri-viticultural chemicals on fish immune system without interaction with other environmental factors. For example, in summer, the immunomarkers seems to be not only correlated with water contamination but also with other environmental factors (pathogens, physical field degradation, nutrients, temperature …). Nevertheless, immune parameters give a global view of organism and ecosystem health explaining growing interest for these biomarkers in environmental risk assessment.

Evaluation of chlorpyrifos effects, alone and combined with lipopolysaccharide stress, on DNA integrity and immune responses of the three-spined stickleback, Gasterosteus aculeatus

Organism immune defences might be weakened by pollutants, largely detected in aquatic ecosystems, leading to the facilitation for opportunistic pathogens to infect organisms. In this context, destabilization of fish non-specific immune parameters and erythrocyte DNA integrity was tested, on a model fish species, the three-spined stickleback (Gasterosteus aculeatus), after exposure to chlorpyrifos (CPF). Alone, pesticide exposure induced a genotoxic potential (chlorpyrifos at 1.75 and 0.88µg/L) in addition to a decrease in phagocytosis capacity and a stimulation of respiratory burst. Then, to mimic pathogenic infection, fish exposure to chlorpyrifos was combined with lipopolysaccharides (LPS) stress. In this second experiment, an increase of DNA damage was observed in fish exposed to a lower concentration of chlorpyrifos and LPS. Moreover, at the higher concentration of chlorpyrifos, an early destabilization of innate immunity was observed as suggested by the absence of an increase of lysosomal presence in fish injected with LPS. This study highlighted the usefulness of stress on stress responses to better understand the impact of contaminants on the organisms health.

Effects of chronic exposure to cadmium and temperature, alone or combined, on the threespine stickleback ( Gasterosteus aculeatus ): interest of digestive enzymes as biomarkers

The development of predictive, sensitive and reliable biomarkers is of crucial importance for aquatic biomonitoring to assess the effects of chemical substances on aquatic organisms, especially when it comes to combined effects with other stressors (e.g. temperature). The first purpose of the present study was to evaluate the single and combined effects of 90 days of exposure to an environmental cadmium concentration (0.5 μg L-1) and two water temperatures (16 and 21 °C) on different parameters. These parameters are involved in (i) the antioxidant system (superoxide dismutase activity -SOD- and total glutathione levels -GSH-), (ii) the energy metabolism, i.e. energy reserves (glycogen, lipids, proteins) and digestive enzymes (trypsin, amylase, intestinal alkaline phosphatase -IAP-), and (iii) biometric parameters (weight, length, Fultons condition factor, and the gonadosomatic index -GSI-) of threespine stickleback (Gasterosteus aculeatus). The second purpose was to determine the interest of the three digestive enzymes as biomarkers in comparison with the other parameters. The higher temperature (21 °C) impacted the anti-oxidant and energy reserve parameters. In liver, GSH levels increased on day 60, while SOD decreased on days 15 and 90, with a significant decrease of protein and lipid energy reserves on day 90. In muscle, the higher temperature decreased SOD activity only on day 90. G. aculeatus biometric parameters were also impacted by the higher temperature, which limited stickleback growth after 90 days of exposure. In female sticklebacks, the GSI peaked on day 60 and decreased sharply on day 90, while the highest values were reached at day 90 in the control groups, suggesting impaired reproduction in sticklebacks raised at 21 °C. These results suggest that 21 °C is an upper-limit temperature for long-term physiological processes in sticklebacks. In contrast, very low-concentration cadmium exposure had no effect on classical biomarkers (energy reserves, antioxidant parameters, biometric parameters). However, digestive enzymes showed an interesting sensitivity to cadmium, which was emphasized by high temperature. The activity of the three digestive enzymes decreased significantly on day 90 when sticklebacks were exposed to cadmium alone, while the decrease was stronger and was recorded earlier (from day 15) when they were exposed to the cadmium-temperature combination. Compared to conventional measurements, digestive enzymes responded rapidly. This could be an important advantage for them to be used as early warning tools to reflect the health status of organisms, particularly for trypsin and IAP activities.

Modelling the effect of season, sex, and body size on the three-spined stickleback, Gasterosteus aculeatus, cellular innate immunomarkers: A proposition of laboratory reference ranges

Innate immunomarkers reflect both environmental contamination and fish health status, providing useful information in environmental risk assessment studies. Nevertheless, the lack of knowledge about the effect of confounding factors can lead to data misinterpretation and false diagnoses. The aim of this study was to evaluate the impact of three confounding factors (season, sex and body size) on three-spined stickleback innate immunomarkers in laboratory conditions. Results shown strong seasonal variations in stickleback innate immunomarkers, with higher immune capacities in late winter-early spring and a disturbance during the spawning period in late spring-summer. Sex and body size had a season dependant effect on almost all tested immunomarkers. Reference ranges were established in laboratory-controlled conditions (i.e. laboratory reference ranges) and compared with data obtained from in vivo chemical expositions. The predictive power of the statistical model depended on the immunomarker, but the control data of the in vivo experiments, realized in same laboratory conditions, were globally well include in the laboratory reference ranges. Moreover, some statistical effects of the in vivo exposures were correlated with an augmentation of values outside the reference ranges, indicating a possible harmful effect for the organisms. As confounding factors influence is a major limit to integrate immunomarkers in biomonitoring programs, modelling their influence on studied parameter may help to better evaluated environmental contaminations.

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler’s index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass.