Molecular assessment of wild populations across marine taxa: importance of taxonomic, seasonal and habitat patterns in environmental monitoring

Abstract

Introduction: Scientists are currently faced with the challenge of assessing the effects of anthropogenic stressors on aquatic ecosystems. Cellular stress response (CSR) biomarkers are ubiquitous and phylogenetically conserved among metazoans [1] and have been successfully applied in environmental monitoring [2–4] but the composition, rate of up-regulation and posttranslational regulation of each protein class can vary [5], not only according to species but also extrinsic (stress levels, previous exposures, season, geographic location) and intrinsic factors (tissue, gender, age-class, nutritional status).The reported variability may thus limit the wide application of such biomarkers in environmental monitoring, imposing the need to identify baseline variability levels in the field.

Materials and methods: Based on this framework, our aim was to carry out a comprehensive and integrative in situ assessment of the CSR (Heat shock protein 70kDa, ubiquitin, antioxidant enzymes quantified via ELISA and kinetic assays [6]) and oxidative damage (lipid peroxidation assessed via TBARS method [6]) in wild populations across marine taxa by collecting fish (n = 2 species), crustaceans (n = 3 species), mollusks (n = 6 species including gastropods, bivalves, polyplacophores) and cnidarians (n = 2 species of sea anemones) during 2 different seasons (spring and summer of 2013) and 2 locations (coast and estuary). Number of collected specimens per species ranged between 7 and 26, depending on abundance.

Results: CSR end-point patterns were different between taxons with mollusks having higher biomarker levels, followed by the cnidarians, while fish and crustaceans showed lower biomarker levels. Mollusks have the highest amounts of heat shock protein 70kDa and cnidarians have the greatest catalase activity and lipid peroxides’ concentration. Ubiquitin levels were higher in fish and cnidarians. Intraspecific variability in the CSR measured by the coefficient of variation (% CV) (including data from all seasons and sites) was elevated (35-94%). Moreover, when species were grouped by mobility (mobile vs sessile/sedentary) there were clear clusters in the principal components analysis, with sessile/sedentary organisms having higher levels of CSR proteins. Overall, there was a seasonal differentiation in biomarker patterns across taxonomic groups, especially evident in fish and cnidaria. A differentiation in biomarker patterns between locations was also observed in mussels M. galloprovincialis and sea anemones A. equina, which showed clear habitat clusters. Overall, specimens collected in the estuary had lower biomarker levels when compared to specimens collected in the coast.

Discussion and conclusions: This work highlights the importance of assessing baseline biomarker levels across taxons, seasons and locations prior to applying biomarker analyses in environmental monitoring. Selecting bioindicator species, defining sampling strategies, and identifying confounding factors are crucial processes to the success of biomarkers as powerful tools for environmental monitoring within the EU Water Framework Directive.

Acknowledgements

Thanks are due to the Portuguese Fundação Calouste Gulbenkian [Research Stimulus Program 2012 award, # 126739], Portuguese Fundação para a Ciência e a Tecnologia (FCT) [SFRH/BPD/117491/2016 to DM, researcher position to CV, UID/Multi/04378/2013, UID/MAR/04292/2013, UID/AMB/50017] and ERDF under the PT2020 Partnership Agreement and Compete 2020 [POCI-01-0145-FEDER-007728 and POCI-01-0145-FEDER-007638].