Acclimations of Oreochromis mossambicus to elevated salinity were conducted with multiple rates of salinity increase and duration of exposure to determine the rate-independent maximum salinity limit and the incipient lethal salinity. A data-independent acquisition (DIA) assay library was created for quantitative analysis of over 3000 gill proteins simultaneously in treatments representative of important key zones in the salinity level/duration landscape. From these DIA data, protein networks that represent complex molecular phenotypes associated with salinity acclimation were generated. Acclimations of Oreochromis mossambicus to elevated salinity were conducted with multiple rates of salinity increase and duration of exposure to determine the rate-independent maximum salinity limit and the incipient lethal salinity. A data-independent acquisition (DIA) assay library was created for quantitative analysis of over 3000 gill proteins simultaneously in treatments representative of important key zones in the salinity level/duration landscape. From these DIA data, protein networks that represent complex molecular phenotypes associated with salinity acclimation were generated. Protein fold change (FC) and organismal level performance indicators of salinity tolerance were then correlated. Gill protein networks impacted at extreme salinity levels both above and below the incipient lethal limit include increased energy metabolism, especially upregulation of electron transport chain complex proteins, and significant downregulation of a previously uncharacterized protein which bears strong amino acid sequence similarity to fucolectin. Proteins networks strongly impacted by crossing into the zone of resistance include cell adhesion and extracellular matrix regulation.

Four distinct acclimation protocols were used in this study including; (1) continuous increase in salinity from FW with increases occurring once every 24 hours up to the Morbidity Point (MP) at six different rates of change per day (32 ppt/day, 24 ppt/day, 12 ppt/day, 8ppt/day, 6 ppt/day, and 3 ppt/day), (2) increase from FW to target hypersaline salinity level (85 ppt, 95 ppt, and 105 ppt) at a rate of 6 ppt/day followed by continuous exposure at constant salinity until MP, (3) acclimation from FW to the target salinity at different rates to achieve the target salinity at the same time after 14 days with four target salinities (21 ppt at 1.5ppt/day, 55 ppt at 4 ppt/day, 85 ppt at 6 ppt/day, and 105 at 7.5 ppt/day), followed by 24 hours at the final salinity, and (4) acclimation from FW to 75 ppt at 6 ppt/day followed by maintenance at the final salinity for 10 weeks. Fish used for the twelve-week acclimation to 75 ppt salinity were 8 months old at the commencement of the experiment taken from the same two brood populations as the previous experiments. 15 fish each were placed in two replicate tanks from each brood population for a total of 60 fish in 4 tanks for treatment and the same number for controls. Handling controls were included in parallel for all experiments.

Gill epithelium was sampled to compare proteome changes based on time and absolute salinity levels, including eight samples each from 14-day acclimations to 85 ppt and 105 ppt as well as the FW control and the 12-week 75 ppt treatment and FW control, and six samples from the extended constant salinity to MP at 85ppt and 105ppt. Extraction and in-solution trypsin digestion of proteins were performed as previously reported (Root et al., 2021b).