Cold exposure depolarizes cells in insects due to a reduced electrogenic ion transport and a gradual increase in extracellular [K⁺]. Cold-induced depolarization is linked to cold injury in chill-susceptible insects, and the locust, Locusta migratoria, has shown improved cold tolerance following cold-acclimation through depolarization resistance. Here we investigate how cold-acclimation influences depolarization resistance and how this resistance relates to improved cold tolerance. To address this question, we investigated if cold-acclimation affects the electrogenic transport capacity and/or the relative K⁺ permeability during cold exposure by measuring membrane potentials of warm- and cold-acclimated locusts in the presence/absence of ouabain (Na⁺/K⁺ pump blocker) or 4-aminopyridine (4-AP, voltage-gated K⁺ channel blocker). In addition, we compared the membrane lipid composition of muscle tissue from warm- and cold-acclimated locust, and the abundance of a range transcripts related to ion transport and cell injury accumulation. We found that cold-acclimated locusts are depolarization resistant due to an elevated K⁺ permeability, facilitated by opening of 4-AP sensitive K⁺ channels. In accordance, cold-acclimation was associated with an increased abundance of shaker transcripts (gene encoding 4-AP sensitive voltage-gated K⁺ channels). Furthermore, we found that cold-acclimation improved muscle cell viability following exposure to cold and hyperkalemia even when muscles were depolarized substantially. Thus, cold-acclimation confers resistance to depolarization by altering the relative ion permeability, but cold-acclimated locusts are also more tolerant to depolarization.

Methods are described in the associated article

Table S1 – List of primers and their sequences. All primers were created based on sequences from the L. migratoria genome. Primers marked with ¹ are primers based on unannotated transcripts where annotation was inferred by looking for homologues in the Drosophila melanogaster genome. All other primers are based on annotated transcripts in the L. migratoria genome. Table S1 – List of primers and their sequences. All primers were created based on sequences from the L. migratoria genome. Primers marked with ¹ are primers based on unannotated transcripts where annotation was inferred by looking for homologues in the Drosophila melanogaster genome. All other primers are based on annotated transcripts in the L. migratoria genome.

Table S2 – Transcription abundance (TA) from warm- (WA) or cold-acclimated (CA) locusts. The transcription abundance is presented in the format 1-2-4 days of acclimation and displayed as transcription relative to the common average (both sexes combined) transcription level for locusts acclimated under warm conditions for 1 day. For the transcripts marked with ^u, their primers promoted unspecific/uneven amplification. AS, AD or S represent significant effects of acclimation status (AS), acclimation duration (AD) or sex (S) and x denotes a significant interaction between two effects. Statistical analysis was done as 3-way ANOVAs, N was 4/5/2 (WA 1/2/4 days, males), 8/7/5 (CA 1/2/4 days males), 12/5/5 (WA 1/2/4, females), or 13/11/6 (CA 1/2/4 , females) animals.

Table S3 – Analysis of lipid content of muscles from warm- (WA) or cold-acclimated (CA) locusts. The lipids are named by their head group with chain length and number of saturated bonds in the parenthesis. The head groups are: FA (fatty acyl), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (Pser), sphingomyelin (SM) and cardiolipin (CL). The p-values reported are calculated from a 2-tailed student’s t-test. To be considered significant, p-values must be less than the corresponding threshold calculated using the holm-bonferroni method. Lipids with a p-value below their threshold for statistical significance are marked with bold font.