Insects, like the model species Drosophila melanogaster, lose neuromuscular function and enter a state of paralysis (chill coma) at a population-and species-specific low temperature threshold that is decreased by cold acclimation. Entry into this coma is related to a spreading depolarization in the central nervous system, while recovery involves restoration of electrochemical gradients across muscle cell membranes. The Na + /K + -ATPase helps maintain ion balance and membrane potential in both the brain and hemolymph (surrounding muscles), and changes in thermal tolerance traits have therefore been hypothesized to be closely linked to variation in the expression and/or activity of this pump in multiple tissues. Here, we tested this hypothesis by measuring activity and thermal sensitivity of the Na + /K + -ATPase at the tagmaspecific level (head, thorax and abdomen) in warm-(25°C) and cold-acclimated (15°C) flies by Na + /K + -ATPase activity at 15, 20, and 25°C. We relate differences in pump activity to differences in chill coma temperature, spreading depolarization temperature, and thermal dependence of muscle cell polarization. Differences in pump activity and thermal sensitivity induced by cold acclimation varied in a tissue-specific manner: While cold-acclimated flies had decreased thermal sensitivity of Na + /K + -ATPase that maintains activity at low temperatures in the thorax (mainly muscle), activity instead decreased in the heads (mainly brain). We argue that these changes may assist in maintenance of K + homeostasis and membrane potential across muscle membranes and discuss how reduced Na + /K + -ATPase activity in the brain may counterintuitively help insects delay coma onset in the cold..

Species from colder climates tend to be more chill tolerant regardless of the chill tolerance trait measured, but for Drosophila melanogaster, population-level differences in chill tolerance among populations are not always found when a single trait is measured in the laboratory. We measured chill coma onset temperature, chill coma recovery time, and survival after chronic cold exposure in replicate lines derived from multiple paired African and European D. melanogaster populations. The populations in our study were previously found to differ in chronic cold survival ability, which is believed to have evolved independently in each population pair; however, they did not differ in chill coma onset temperature and chill coma recovery time in a manner that reflected their geographic origins, even though these traits are known to vary with origin latitude among Drosophila species and are among the most common metrics of thermal tolerance in insects. While it is common practice to measure only one chill tolerance trait when comparing chill tolerance among insect populations, our results emphasise the importance of measuring more than one thermal tolerance trait to minimize the risk of missing real adaptive variation in insect thermal tolerance.

Insects, like the model species Drosophila melanogaster, lose neuromuscular function and enter a state of paralysis (chill coma) at a population- and species-specific low temperature threshold that is decreased by cold acclimation. Entry into this coma is related to a spreading depolarization in the central nervous system, while recovery involves restoration of electrochemical gradients across muscle cell membranes. The Na+/K+-ATPase helps maintain ion balance and membrane potential in both the brain and hemolymph (surrounding muscles), and changes in thermal tolerance traits have therefore been hypothesized to be closely linked to variation in the expression and/or activity of this pump in multiple tissues. Here, we tested this hypothesis by measuring activity and thermal sensitivity of the Na+/K+-ATPase at the tagma-specific level (head, thorax and abdomen) in warm-(25°C) and cold-acclimated (15°C) flies by Na+/K+-ATPase activity at 15, 20, and 25°C. We relate differences in pump activity to differences in chill coma temperature, spreading depolarization temperature, and thermal dependence of muscle cell polarization. Differences in pump activity and thermal sensitivity induced by cold acclimation varied in a tissue-specific manner: While cold-acclimated flies had decreased thermal sensitivity of Na+/K+-ATPase that maintains activity at low temperatures in the thorax (mainly muscle), activity instead decreased in the heads (mainly brain). We argue that these changes may assist in maintenance of K+ homeostasis and membrane potential across muscle membranes and discuss how reduced Na+/K+-ATPase activity in the brain may counterintuitively help insects delay coma onset in the cold.

Plastic pollution is a growing threat to our natural environment. Plastic waste/pollution results from high emissions of both macro (> 5 mm) and microplastics (MPs; < 5 mm) as well as environmental fractioning of macroplastics into microplastics. Microplastics have been shown to have a range of negative impacts on biota. Harmonized methods to accurately measure and count MPs from animal samples are limited, but what methods exist are not ideal for a controlled laboratory environment where plastic ingestion, transformation, and elimination can be quantified and related to molecular, physiological, and organismal traits. Here we propose a complete method for isolating and characterizing fluorescent MPs by combining several previously reported approaches into one comprehensive workflow. We combine tissue dissection, organic material digestion, sample filtering, and automated imaging techniques to show how fluorescently-labelled MPs provided to animals (e.g. in their diet) in a laboratory setting can be isolated, identified, and quantified. As a proof of concept, we fed crickets (Gryllodes sigillatus) a diet of 2.5% (w/w) fluorescently-labelled plastics and isolated and characterized plastic particles within the gut and frass.

Plastic pollution is a growing threat to our natural environment. Plastic waste/pollution results from high emissions of both macro (&gt;5 mm) and microplastics (MPs; &lt;5 mm) as well as environmental fractioning of macroplastics into MPs. MPs have been shown to have a range of negative impacts on biota. Harmonized methods to accurately measure and count MPs from animal samples are limited, but what methods exist are not ideal for a controlled laboratory environment where plastic ingestion, degradation and elimination can be quantified and related to molecular, physiological and organismal traits. Here, we propose a complete method for isolating and quantifying fluorescent MPs by combining several previously reported approaches into one comprehensive workflow. We combine tissue dissection, organic material digestion, sample filtering and automated imaging techniques to show how fluorescently labelled MPs provided to insects (e.g. in their diet) in a laboratory setting can be isolated, identified and quantified. As a proof of concept, we fed crickets (Gryllodes sigillatus) a diet of 2.5% (w/w) fluorescently labelled plastics and isolated and quantified plastic particles within the gut and frass.