Abstract
A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into proteome changes that underlie the remodeling of tilapia gill epithelium in response to environmental salinity change.
Highlights
IntroductionTilapia have evolved in Africa but have spread to subtropical and tropical freshwater and marine habitats throughout the world as a result of escaping from aquaculture farms and their high environmental adaptability
From the ‡Physiological Genomics Group, Department of Animal Sciences, University of California Davis, One Shields Avenue, Davis, California 95616
Acclimation from hyposmotic to hyperosmotic environments is accompanied by extensive remodeling of gill epithelium, the most prominent feature of which is an increase in the number and size of salt-secretory, mitochondria-rich ionocytes [2]
Summary
Tilapia have evolved in Africa but have spread to subtropical and tropical freshwater and marine habitats throughout the world as a result of escaping from aquaculture farms and their high environmental adaptability These cichlids tolerate salinities ranging from fresh water to almost 4ϫ seawater (120 ppt) and they inhabit freshwater and hypersaline desert lakes as well as coastal marine and brackish habitats [9]. The other approach for LFQ, quantitation of ion current intensity, is used for relative quantitation of protein abundances in gill tissue from salinity stressed fish compared with FW handling controls. Because peak area provides a more accurate measure of peptide (and correspondingly protein) quantity this approach is used in the present study [21, 22]
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