Proteomics of osmoregulatory responses in threespine stickleback gills

Abstract

The gill proteome of threespine sticklebacks (Gasterosteus aculeatus) differs greatly in populations that inhabit diverse environments characterized by different temperature, salinity, food availability, parasites, and other parameters. To assess the contribution of a specific environmental parameter to such differences it is necessary to isolate its effects from those of other parameters. In this study the effect of environmental salinity on the gill proteome of G. aculeatus was isolated in controlled mesocosm experiments. Salinity-dependent changes in the gill proteome were analyzed by LC/MSMS data-independent acquisition (DIA) and Skyline. Relative abundances of 1691 proteins representing the molecular phenotype of stickleback gills were quantified using previously developed MSMS spectral and assay libraries in combination with DIA quantitative proteomics. General stress responses were distinguished from osmoregulatory protein abundance changes by their consistent occurrence during both hypo- and hyper-osmotic salinity stress in six separate mesocosm experiments. If the abundance of a protein was consistently regulated in opposite directions by hyper- versus hypo-osmotic salinity stress, then it was considered an osmoregulatory protein. In contrast, if protein abundance was consistently increased irrespective of whether salinity was increased or decreased, then it was considered a general stress response protein. KEGG pathway analysis revealed that the salivary secretion, inositol phosphate metabolism, valine, leucine and isoleucine degradation, citrate cycle, oxidative phosphorylation, and corresponding endocrine and extracellular signaling pathways contain most of the osmoregulatory gill proteins whose abundance is directly proportional to environmental salinity. Most proteins that were inversely correlated with salinity map to KEGG pathways that represent proteostasis, immunity, and related intracellular signaling processes. General stress response proteins represent fatty and amino acid degradation, purine metabolism, focal adhesion, mRNA surveillance, phagosome, endocytosis, and associated intracellular signaling KEGG pathways. These results demonstrate that G. aculeatus responds to salinity changes by adjusting osmoregulatory mechanisms that are distinct from transient general stress responses to control compatible osmolyte synthesis, transepithelial ion transport, and oxidative energy metabolism. Furthermore, this study establishes salinity as a key factor for causing the regulation of numerous proteins and KEGG pathways with established functions in proteostasis, immunity, and tissue remodeling. We conclude that the corresponding osmoregulatory gill proteins and KEGG pathways represent molecular phenotypes that promote transepithelial ion transport, cellular osmoregulation, and gill epithelial remodeling to adjust gill function to environmental salinity.

Osmoregulatory strategies of estuarine fish Scatophagus argus in response to environmental salinity changes

“…To investigate the differences in gill response mechanisms induced by different environmental salinity stresses, osmoregulatory, non-directional, hypo-and hypersaline stress response DEPs were identified as described by Li and Kültz [15]. In the present study, a total of 22 DEPs were consistently positively or inversely correlated with a salinity change and were considered as osmoregulatory proteins (Fig.…”

“…However, the lack of specific antibodies has been the major drawback for physiological studies in non-model fish. Comparative proteomics study provides an alternative approach to detect proteome changes in physiological responses to environmental stresses in some non-model animals, such as Oreochromis mossambicus, Ctenopharyngodon idellus and Gasterosteus aculeatus [14][15][16]. Isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/ MS) have become popular proteomics technology for the direct quantitation and comparison of protein expression levels with great efficiency and accuracy [17].…”

“…High salinity adaptation is primarily achieved via branchial osmoregulation, which is a common trait found in estuarine residents [20]. The fish gill is a multipurpose organ that directly contacts the external milieu and is considered the major tissue for osmoregulation during rapid shifts to hypo-or hypersaline conditions [15,21,22]. In the osmoregulatory process, fish gills are critical to maintain the ionic compositions of body fluids encountering with salinity fluctuations in aquatic environments [15].…”

“…The fish gill is a multipurpose organ that directly contacts the external milieu and is considered the major tissue for osmoregulation during rapid shifts to hypo-or hypersaline conditions [15,21,22]. In the osmoregulatory process, fish gills are critical to maintain the ionic compositions of body fluids encountering with salinity fluctuations in aquatic environments [15]. Fish in hypersaline water passively lose water and gain salt and need to replenish water by drinking and actively excreting the majority of monovalent ions back to the environment, whereas in hyposaline water they actively ingest ions across branchial epithelia to compensate for monovalent ion loss, primarily sodium ion (Na + ) loss [21][22][23][24].…”

Searching for intra-locus sexual conflicts in the three-spined stickleback (Gasterosteus aculeatus) genome

“…ANO4 is associated with various biological processes, including aldosterone secretion, which appears to be associated with osmoregulation in three-spined stickleback (Li and Kültz 2020). ANO4 has also been found in association with adaptation to altitude in Tibetan chicken (Zhang et al 2016).…”