Weak differences in sensitivity to major ions by different larval stages of the mayfly Neocloeon triangulifer

Freshwater salinization is a global ecological concern because of the alarming biodiversity declines associated with increases in major ion concentrations. Loss of mayfly diversity appears to be a common ecological response to anthropogenic salinization worldwide. Remarkably few regulatory standards exist to protect aquatic life from major ions, and antiquated approaches for setting such standards rely on traditional laboratory toxicity tests, which do not address sensitivities of mayflies at different larval stages. The lab-reared mayfly Neocloeon triangulifer (McDunnough, 1931) has emerged as one of the very few useful aquatic insect models for studying the effects of environmental stressors, including salinity, in the laboratory. Here, we asked if different larval life stages are differentially sensitivity to ion concentrations by conducting traditional 96-h toxicity tests with NaCl, CaCl2, and Ca/MgSO4. We used a general linear model to determine if survivorship differed among larval stages as well as ion type and concentration. We also calculated median lethal concentrations (LC50) for each larval stage. Larval sensitivity to NaCl decreased slightly with age (2–6, 9–13, and 17–21 d, with LC50 values of 401, 441, and 570 mg/L, respectively, when expressed as Na concentrations). Similarly, larval sensitivity to Ca/MgSO4 differed slightly among age groups (LC50 = 748, 1503, and 1439 mg/L, respectively, when expressed as SO4 concentrations). Reliable confidence intervals on LC50 values for CaCl2 could not be calculated because of high survivorship. However, our general linear model revealed that age played a moderate role in survival (p = 0.0065) across all salts of interest. To assess the potential changes in ion flux between larval stages, we used radiotracers (22Na, 35SO4, or 45Ca) in 18- and 25-d-old larvae and found no strong differences in ion uptake rates. We also qualitatively examined morphological differences between larval life stages, including the appearance of gills and number of ionocytes. Our results indicate that younger N. triangulifer larvae may be more sensitive to major ions than mature larvae. These results should be considered when experimentally using larger, late-stage N. triangulifer larvae to study the physiological effects and acute toxicity of salinity.