Diana Madeira

Influence of temperature in thermal and oxidative stress responses in estuarine fish

The influence of increasing temperatures in thermal and oxidative stress responses were studied in the muscle of several estuarine fish species (Diplodus vulgaris, Diplodus sargus, Dicentrarchus labrax, Gobius niger and Liza ramada). Selected fish were collected in July at the Tagus estuary (24±0.9°C; salinity of 30±4‰; pH=8). Fish were subjected to a temperature increase of 1°C.h(-1) until they reached their Critical Thermal Maximum (CTMax), starting at 24°C (control temperature). Muscle samples were collected during the trial and results showed that oxidative stress biomarkers are highly sensitive to temperature. Results from stress oxidative enzymes show alterations with increasing temperature in all tested species. Catalase (CAT; EC 1.11.1.6) activity significantly increased in L. ramada, D. labrax and decreased in D. vulgaris. Glutathione S-transferase (GST; EC 2.5.1.18) activity increased in L. ramada, D. sargus, D. vulgaris, and D. labrax. In G. niger it showed a cycle of increase-decrease. Lipid peroxidation (LPO) increased in L. ramada, D. sargus and D. labrax. With respect to correlation analysis (Pearson; Spearman r), the results showed that oxidation products and antioxidant defenses were correlated in L. ramada (LPO-CAT and LPO-GST, D. sargus (LPO-CAT), and D. labrax (LPO-CAT). Oxidative biomarkers were correlated with thermal stress biomarker (Hsp70) in L. ramada (CAT-Hsp70), D. vulgaris (LPO-Hsp70), D. labrax (GST-Hsp70) and G. niger (LPO-Hsp70). In conclusion, oxidative stress does occur with increasing temperatures and there seems to be a relation between thermal stress response and oxidative stress response. The results suggest that oxidative stress biomarkers should be applied with caution, particularly in field multi-species/multi-environment studies.

Thermal tolerance of the crab Pachygrapsus marmoratus : intraspecific differences at a physiological (CTMax) and molecular level (Hsp70)

Temperature is one of the most important variables influencing organisms, especially in the intertidal zone. This work aimed to test physiological and molecular intraspecific differences in thermal tolerance of the crab Pachygrapsus marmoratus (Fabricius, 1787). The comparisons made focused on sex, size, and habitat (estuary and coast) differences. The physiological parameter was upper thermal limit, tested via the critical thermal maximum (CTMax) and the molecular parameter was total heat shock protein 70 (Hsp70 and Hsp70 plus Hsc70) production, quantified via an enzyme-linked imunosorbent assay. Results showed that CTMax values and Hsp70 production are higher in females probably due to different microhabitat use and potentially due to different hormonal regulation in males and females. Among females, non-reproducing ones showed a higher CTMax value, but no differences were found in Hsp70, even though reproducing females showed higher variability in Hsp70 amounts. As reproduction takes up a lot of energy, its allocation for other activities, including stress responses, is lower. Juveniles also showed higher CTMax and Hsp70 expression because they occur in greater shore heights and ageing leads to alterations in protein synthesis. Comparing estuarine and coastal crabs, no differences were found in CTMax but coastal crabs produce more Hsp70 than estuarine crabs because they occur in drier and hotter areas than estuarine ones, which occur in moister environments. This work shows the importance of addressing intraspecific differences in the stress response at different organizational levels. This study shows that these differences are key factors in stress research, climate research, and environmental monitoring.

Effect of temperature in multiple biomarkers of oxidative stress in coastal shrimp

Various studies in captivity and in the wild have pointed to the effect of season, and temperature in particular, in the levels of the oxidative stress biomarkers currently used for environmental quality assessment. However, knowledge on how temperature affects the oxidative stress response is unavailable for most species. This study investigated the effect of increasing temperature on lipid peroxidation, catalase activity, superoxide dismutase and glutathione-S-transferase in the shrimps, Palaemon elegans and Palaemon serratus. It was concluded that increasing temperatures significantly affect all the biomarkers tested in both species, with the exception of superoxide dismutase in P. serratus which was not affected by temperature. The oxidative stress response was more intense in P. elegans, than in P. serratus, producing higher peaks of all biomarkers at temperatures between 22°C and 26°C, followed by low levels at higher temperatures. It was concluded that monitoring of ecosystems using oxidative stress biomarkers should take into account the species and thermal history of the organisms. Sampling should be avoided during heat waves and immediately after heat waves.

Physiological, cellular and biochemical thermal stress response of intertidal shrimps with different vertical distributions: Palaemon elegans and Palaemon serratus

The ability to cope with high temperature variations is a critical factor in intertidal communities. Two species of intertidal rocky shore shrimps (Palaemon sp.) with different vertical distributions were collected from the Portuguese coast in order to test if they were differentially sensitive to thermal stress. Three distinct levels of biological organization (organismal, biochemical, and cellular) were surveyed. The shrimp were exposed to a constant rate of temperature increase of 1°C x h(-1), starting at 20°C until reaching the CTMax (critical thermal maximum). During heat stress, two biomarkers of protein damage were quantified in the muscle via enzyme-linked immunosorbent assays: heat shock proteins HSP70 (hsp70/hsc70) and total ubiquitin. Muscle histopathological alterations caused by temperature were also evaluated. CTMax values were not significantly different between the congeners (P. elegans 33.4 ± 0.5 °C; P. serratus 33.0 ± 0.5 °C). Biomarker levels did not increase along the temperature trial, but P. elegans (higher intertidal) showed higher amounts of HSP70 and total ubiquitin than P. serratus (lower intertidal). HSP70 and total ubiquitin levels showed a positive significant correlation in both species, suggesting that their association is important in thermal tolerance. Histopathological observations of muscle tissue in P. serratus showed no gross alterations due to temperature but did show localized atrophy of muscle fibers at CTMax. In P. elegans, alterations occurred at a larger scale, showing multiple foci of atrophic muscular fascicles caused by necrotic or autolytic processes. In conclusion, Palaemon congeners displayed different responses to stress at a cellular level, with P. elegans having greater biomarker levels and histopathological alterations.

Comparing biomarker responses during thermal acclimation: A lethal vs non-lethal approach in a tropical reef clownfish

Knowledge of thermal stress biology for most tropical fish species in reef ecosystems under climate change is still quite limited. Thus, the objective of this study was to measure the time-course changes of thermal stress biomarkers in the commercially exploited coral reef fish Amphiprion ocellaris, during a laboratory simulated event of increased temperature. Heat shock protein 70kDa (Hsp70) and total ubiquitin (Ub) were determined in the muscle (lethal method) and in the fin (non-lethal alternative method) under two temperature treatments (control - 26°C and elevated temperature - 30°C) throughout one month with weekly samplings. Results suggest that biomarker basal levels are tissue-specific and influence the degree of response under temperature exposure. Responses were highly inducible in the muscle but not in fin tissue, indicating that the latter is not reliable for monitoring purposes. Thermal stress was observed in the muscle after one week of exposure (both biomarkers increased significantly) and Ub levels then decreased, suggesting the animals were able to acclimate by maintaining high levels of Hsp70 and through an effective protein turnover. In addition, the results show that mortality rates did not differ between treatments. This indicates that A. ocellaris is capable of displaying a plastic response to elevated temperature by adjusting the protein quality control system to protect cell functions, without decreasing survival. Thus, this coral reef fish species presents a significant acclimation potential under ocean warming scenarios of +4°C. Monitoring of thermal stress through a non-lethal method, fin-clipping, although desirable proved to be inadequate for this species.

Sea warming affects bream (Sparus aurata) tissues and stress proteins (HSP70)

***** and M.S. Diniz* *REQUIMTE, Departamento de Quimica, Faculdade de Ciencias e Tecnologia, Centro de Quimica Fina e Biotecnologia, Universidade Nova de Lisboa, 2829-516 Caparica. **Laboratorio Maritimo da Guia, Centro de Oceanografia, Faculdade de Ciencias da Universidade de Lisboa, Av. Nossa Senhora do Cabo, 939, 2750-374 Cascais, Portugal ***Centro de Oceanografia, Faculdade de Ciencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal ****IMAR-Instituto do Mar, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa -Departamento de Ciencias e Engenharia do Ambiente. Quinta da Torre, 2829-516 Caparica, Portugal. *****BIOSCOPE Group, Physical-Chemistry Department, Faculty of Science, Campus Ourense, University of Vigo, 32004, Ourense, Spain. ******Unitat d´Enginyeria de Proteines i Proteomica, Institut de Biotecnologia i Biomedicina, Universitat Autonoma de 08193, Barcelona, Spain The increase in CO

Seasonal changes in stress biomarkers of an exotic coastal species – Chaetopleura angulata (Polyplacophora) – Implications for biomonitoring

Knowledge on baseline values of stress biomarkers in natural conditions is urgent due to the need of reference values for monitoring purposes. Here we assessed the cellular stress response of the chiton Chaetopleura angulata in situ. Biomarkers commonly used in environmental monitoring (heat shock protein 70kDa, total ubiquitin, catalase, glutathione-S-transferase, superoxide-dismutase, lipid peroxidation) were analyzed in the digestive system, gills and muscle of C. angulata, under spring and summer conditions in order to assess seasonal tissue-specific responses. Season had an effect on all targeted organs, especially affecting the digestive system which displayed clear seasonal clusters. The respective Integrated Biomarker Response (IBR) showed a 7.2-fold seasonal difference. Muscle and gills showed similar IBRs between seasons making them appropriate organs to monitor chemical pollution as they were less responsive to seasonal variation. The most stable biomarkers in these organs were ubiquitin and superoxide-dismutase thus being reliable for monitoring purposes.