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Freshwater salinity varies in natural systems and plays a role in species distribution. Anthropogenic alterations to freshwater salinity regimes include sea level rise and subsequent intrusion of saline waters to inland habitats. While mayflies are generalized to be sensitive to increasing salinity, we still know remarkably little about the physiological processes (and their plasticity) that determine the performance of species in a changing world. Here, we explored life-history outcomes and physiological plasticity in a population of Callibaetis floridanus (Ephemeroptera: Baetidae) from a coastal pond that routinely experiences saltwater intrusion. We reared naiads from egg hatch to adulthood across a gradient of increasing salinities (113, 5,020, 9,921 μS/cm). Radiotracer flux studies (22Na, 35SO4, and 45Ca) were conducted in naiads reared at each salinity, revealing a positive association between ionic concentration and uptake rates. However, the influence of rearing history on ionic influx rates was apparent when naiads were transferred from their respective rearing water to the other experimental conditions. For example, we observed that naiads reared in the low salinity treatment (113 μS/cm) had 10.8-fold higher Na uptake rates than naiads reared at 9,921 μS/cm and transferred to 113 μS/cm. Additionally, naiads acclimated to the higher salinity water exhibited reduced uptake in ion-rich water relative to those reared in more dilute conditions (e.g., in 9,921 μS/cm water, 113 and 5,020 μS/cm acclimated naiads had 1.5- and 1.1-fold higher Na uptake rates than 9,921 μS/cm acclimated naiads, respectively). We found no significant changes in survival (80 ± 4.4%, mean ± s.e.m.) or naiad development time (24 ± 0.3 days, mean ± s.e.m.) across these treatments but did observe a 27% decrease in subimago female body weight in the most dilute condition. This reduction in female weight was associated with higher oxygen consumption rates in naiads relative to the other rearing conditions. Collectively, these data suggests that saline adapted C. floridanus may be more energetically challenged in dilute conditions, which differs from previous observations in other mayfly species.

Temperature dictates the performance of aquatic ectotherms. However, the physiological and biochemical processes that drive thermally-mediated life history patterns (and limits) remain poorly understood because they are rarely studied simultaneously. In our previous work, we have established life history outcomes (e.g., survivorship, development time, growth rates, and fitness) in mayflies (Neocloeon triangulifer) reared at static temperatures ranging from 14 to 30°C at 2°C intervals. In this study, we conducted biochemical measurements (RT-qPCR of select genes and targeted, quantitative metabolomic profiling) on N. triangulifer mature larvae reared at temperatures associated with excellent survival and fitness (22–24°C), compromised survival and fitness (28°C), and chronic lethality (30°C—larvae survived for a few weeks but failed to emerge to adulthood). Patterns of gene expression were similar to those observed in acute ramping experiments reported previously: larvae reared at 30°C resulted in significant upregulation in the thermally responsive gene HEAT SHOCK PROTEIN 90 (HSP90) but no significant changes in hypoxia responsive genes [EGGLAYING DEFECTIVE9 (EGL-9) and LACTATE DEHYDROGENASE (LDH)]. Additionally, primers for genes associated with energy: INSULIN RECEPTOR (IR), mechanistic TARGET OF RAPAMYCIN (mTOR), and TREHALOSE 6 PHOSPHATE SYNTHASE (T6PS) were developed for this study. IR and mTOR were significantly upregulated while T6PS showed trend of downregulation in larvae reared at 30°C. Metabolomic profiles revealed general depletion of lipids and acylcarnitines in larvae exposed to chronic thermal stress, suggesting that larvae were energetically challenged despite continuous access to food. For example, concentrations of lysoPhosphatidylcholine (lysoPC) a C20:3 decreased as fitness decreased with increasing temperature (2.3- and 2.4-fold at 28 and 30°C relative to controls). Tissue concentrations of the biogenic amine histamine increased 2.1- and 3.1-fold with increasing temperature, and were strongly and negatively correlated with performance. Thus, both histamine and lysoPC a C20:3 are potential biomarkers of thermal stress. Taken together, our results primarily associate energetic challenge with thermally mediated fitness reduction in N. triangulifer.