Abstract
To assess the influence of food type on biomarkers, mussels (Mytilus galloprovincialis) were maintained under laboratory conditions and fed using 4 different microalgae diets ad libitum for 1 week: (a) Isochrysis galbana; (b) Tetraselmis chuii; (c) a mixture of I. galbana and T. chuii; and (d) a commercial food (Microalgae Composed Diet, Acuinuga). Different microalgae were shown to present different distribution and fate in the midgut. I. galbana (≈4 μm Ø) readily reached digestive cells to be intracellularly digested. T. chuii (≈10 μm Ø and hardly digestible) was retained in stomach and digestive ducts for long times and extracellularly digested. Based on these findings, it appeared likely that the presence of large amounts of microalgal enzymes and metabolites might interfere with biochemical determinations of mussel’s biomarkers and/or that the diet-induced alterations of mussels’ digestion could modulate lysosomal and tissue-level biomarkers. To test these hypotheses, a battery of common biochemical, cytological and tissue-level biomarkers were determined in the gills (including activities of pyruvate kinase, phosphoenolpyruvate carboxykinase and cytochrome c oxidase) and the digestive gland of the mussels (including protein, lipid, free glucose and glycogen total content, lysosomal structural changes and membrane stability, intracellular accumulation of neutral lipids and lipofuscins, changes in cell type composition and epithelial thinning, as well as altered tissue integrity). The type of food was concluded to be a major factor influencing biomarkers in short-term experiments though not all the microalgae affected biomarkers and their responsiveness in the same way. T. chuii seemed to alter the nutritional status, oxidative stress and digestion processes, thus interfering with a variety of biomarkers. On the other hand, the massive presence of I. galbana within digestive cells hampered the measurement of cytochemical biomarkers and rendered less reliable the results of biochemical biomarkers (as these could be attributed to both the mussel and the microalgae). Research to optimize dietary food type, composition, regime and rations for toxicological experimentation is urgently needed. Meanwhile, a detailed description of the food type and feeding conditions should be always provided when reporting aquatic toxicological experiments with mussels, as a necessary prerequisite to compare and interpret the biological responses elicited by pollutants.
Highlights
Mussels are widely used sentinel organisms in pollution monitoring programs to assess the biological effects of pollutants
Mussels were divided in 4 experimental groups and fed ad libitum for a week using four different microalgae diets commonly used as food for mussels in laboratory experimentation: (a) I. galbana, (b) T. chuii, (c) a mixture of I. galbana and T. chuii, and (d) a commercial food (Microalgae Composed Diet, Acuinuga SL, A Coruña, Spain)
With the exception of a previous study [109], the massive presence of microalgae-like spherical bodies in mussel digestive cells has not been previously reported in laboratory experiments which used I. galbana as food [36,41] even though several of these studies were based on microscopic observations of digestive gland tissue sections
Summary
Mussels are widely used sentinel organisms in pollution monitoring programs to assess the biological effects of pollutants. Laboratory experiments are crucial to gain understanding of the biological effects of pollutants and to develop a reliable toolbox of biomarkers for environmental monitoring and assessment. Some experimental variables such as temperature, photoperiod, salinity, water renewal, and dosing are recognized as key conditions to correctly perform laboratory experiments and routinely reported in publications and research reports. A large variety of food types and feeding strategies are used in aquatic toxicological experiments with mussels; these include absence of additional food supply, supply of diverse commercial food products or a variety of live microalgae either in monocultures or in mixtures. The food type and feeding strategy influence the levels and responsiveness of biomarkers, the generalizations and comparisons between these experiments would be difficult
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