Amino acid substitutions in the Na ⁺ /K ⁺ ‐ <scp>ATPase</scp> may contribute to salinity tolerance in insects

Abstract Environmental salinity levels vary naturally across terrestrial ecosystems but can be heightened locally by coastal proximity and desertification as well as human activities such as road salt application and agriculture. Since salt is essential for many physiological processes in insects, rising environmental sodium concentrations may drive behavioural changes, where insects select environments and food sources with suitable sodium levels, or evolutionary changes in constitutive or plastic physiological mechanisms to process salt, potentially altering ecological dynamics and species interactions. Numerous hematophagous (blood feeding) insects such as the yellow‐fever mosquito Aedes aeqypti are known to be able to breed in relatively saline environments. Among phytophagous (plant feeding) insects, grasshoppers can be important herbivores in arid and coastal salt‐affected regions, whereas the monarch butterfly ( Danaus plexippus ) appears to perform relatively well on milkweed host plants growing in roadsides influenced by salt runoff. Several of these insects share a common trait: amino acid substitutions in the first extracellular loop of the Na + /K + ‐ATPase (NKA), a sodium pump crucial for maintaining ion balance. For the monarch butterfly, these substitutions confer resistance to toxic cardenolides from milkweeds, but it is unclear whether NKA substitutions may influence salt tolerance. Here, we investigate whether the NKA substitutions found in these insects may contribute to salt tolerance using gene‐edited Drosophila melanogaster mutant strains as models. We show that flies with substitution Q111L (found in Aedes mosquitoes) or a combination of Q111L and A119S (found in grasshoppers) exhibited greater salt tolerance, whereas flies carrying the combination of substitutions found in the monarch (Q111V, A119S and N122H) did not. Our results suggest that the monarch butterfly may rely on alternate mechanisms for salt tolerance and that its NKA substitutions are important primarily for cardenolide resistance. However, substitution Q111L and the combination of Q111L and A119S may be relevant for salt tolerance in a variety of insects. Uncovering mechanisms of salt tolerance enhances our understanding of species distributions, ecological interactions and evolutionary physiology in response to changing environmental salinity levels.