Effects of brief chilling and desiccation on ion homeostasis in the central nervous system of the migratory locust, Locusta migratoria

Highlights

• Brief dehydration and chilling both enhanced resistance to anoxic coma.

• Neither chilling nor dehydration enhanced resistance to cold-related neural shutdown in the metathroacic ganglion.

• Chilling enhanced resistance to pharmacological inhibition of Na⁺K⁺ATPase, though dehydration did not have a similar effect.

Abstract

In insects, chilling, anoxia, and dehydration are cues to trigger rapid physiological responses enhancing stress tolerance within minutes. Recent evidence suggests that responses elicited by different cues are mechanistically distinct from each other, though these differences have received little attention. Further, the effects are not well studied in neural tissue. In this study, we examined how brief exposure to desiccation and chilling affect ion homeostatic mechanisms in metathoracic ganglion of the migratory locust, Locusta migratoria. Both desiccation and chilling enhanced resistance to anoxia, though only chilling hastened recovery from anoxic coma. Similarly, only chilling enhanced resistance to pharmacological perturbation of neuronal ion homeostasis. Our results indicate that chilling and desiccation trigger mechanistically distinct responses and, while both may be important for neuronal ion homeostasis, chilling has a larger effect on this tissue.

Summary statement

This is one of few studies to demonstrate the importance of the central nervous system in rapid acclimatory responses in insects.

Introduction

Insects and other small ectotherms may experience drastic changes in body temperature, hydration state, and even oxygen availability as a result of ambient conditions, behavior, and microhabitat effects (Heinrich, 1993; Lehmann, 2001; Hoback and Stanley, 2001; Chown and Nicholson, 2004). Perturbations such as these produce significant physiological challenges through wide-ranging effects on enzymatic activity, metabolic pathways, ion homeostasis, and solute concentrations (Wegener, 1993; Lee, 2010; Edney, 2012; MacMillan and Sinclair, 2011). To overcome these challenges, insects make rapid physiological adjustments that enhance stress tolerance and preserve basic performance and behavior (Lee et al., 1987; Wang and Kang, 2003; Shreve et al., 2004; Bazinet et al., 2010; Bubliy et al., 2012). These acclimatory responses are triggered by brief exposure to environmental stressors, such as high and low temperature, hypoxia, and dehydration (Lee et al., 1987; Coulson and Bale, 1991; Levis et al., 2012).

The underpinning mechanisms of these rapid acclimatory responses are not well characterized, particularly regarding the role of neural tissue. Rapid cold-hardening (RCH), where insects and other invertebrate ectotherms increase their stress tolerance during brief exposure to abiotic stress, is the most thoroughly studied of the rapid acclimatory responses. RCH can occur independently of the central nervous system (CNS), demonstrating that nervous tissue is not an essential factor in the response (Yi and Lee, 2003, Yi and Lee, 2004; Gantz and Lee, 2015). Nevertheless, there is mounting evidence of neuronal modulation during brief chilling. For example, RCH causes changes in protein abundance and phosphorylation states in nervous tissue of flesh flies, Sarcophaga spp. (Li and Denlinger, 2008; Teets and Denlinger, 2016). Neuronal involvement is also implicated in RCH by enhancing resistance to stress-induced coma (Kelty and Lee, 1999; Powell and Bale, 2006; Srithiphaphirom et al., 2019). In the CNS, stress-induced coma manifests as spreading depolarization (SD) within the integrating regions of the ganglia (Robertson et al., 2017) and it is characterized by waves of cellular depolarization and impaired signal generation (Rodgers et al., 2007; Somjen, 2001; Spong et al., 2016). Further, brief exposure to chilling reduces the temperature at which this SD occurs in the ganglia (Srithiphaphirom et al., 2019). Though it is important to recognize that stress-induced coma may be due to depolarization of either neuron or muscle membrane potential (Spong et al., 2016; Overgaard and MacMillan, 2017; Andersen and Overgaard, 2019), RCH prolongs normal activity under stressful conditions in both whole-organism (Kelty and Lee, 1999; Shreve et al., 2004), and neural tissue-specific measurements (Armstrong et al., 2012; Srithiphaphirom et al., 2019). These results suggest that RCH enhances the capacity to maintain ion homeostasis and propagate electrical signals in both skeletal muscles and neurons.

Since neuronal ion homeostasis is maintained, in part, by ion channel and Na⁺/K⁺ ATPase activity (Emery et al., 1998; Hochachka and Somero, 2002; Rodgers et al., 2007), it is possible that rapid acclimatory responses enhance resistance to SD by modifying one or both of these to be more effective at low temperatures. While there is no evidence to suggest that brief pretreatment (i.e. inducing rapid acclimation) can increase total Na⁺/K⁺ ATPase within a tissue (Rodgers et al., 2007), sub-lethal heat exposure for only 3 h causes Na⁺/K⁺ ATPase trafficking into neuronal plasma membranes (Hou et al., 2014). Thus high temperature exposure can rapidly enhance the capacity of neural tissue to maintain ion homeostasis without the production of new proteins. Similarly, RCH enhances resistance to and accelerates recovery from chill-induced SD, suggesting that brief chilling can also increase Na⁺/K⁺ ATPase activity in neuronal plasma membranes (Armstrong et al., 2012). Further, RCH can proceed when protein synthesis is pharmacologically blocked (Misener et al., 2001), which is consistent with the role of Na⁺/K⁺ ATPase trafficking rather than synthesis in this response.

When triggered by sub-lethal heating or chilling, rapid acclimation protects neurons' ability to resist stress-induced SD, though recent evidence suggests that acclimation triggered by brief dehydration (drought-induced rapid hardening) is mechanistically distinct from RCH (Yi et al., 2017). Further, because drought-induced rapid hardening was only recently described (Levis et al., 2012), its underpinning mechanisms are poorly understood and there is only marginal evidence that it enhances the capacity to maintain ion homeostasis (Yi et al., 2017). Further, previous work on drought-induced rapid hardening has mostly focused on Dipterans and little is known about how other taxa respond to acute dehydration. Thus, we investigated the effects of drought-induced rapid hardening on neuronal ion regulation in the migratory locust, Locusta migratoria.

L. migratoria is well-suited to studying neuronal ion regulation because the metathoracic ganglion (MTG), which contains vital neural circuitry such as the ventilatory central pattern generator (CPG), is readily accessible and provides an opportunity to measure membrane potential and [K⁺] surge during experimental application of stressors (Robertson, 2004; Rodgers et al., 2007, Rodgers et al., 2009). Since anoxia produces large surges of K⁺ in the CNS (Rodgers et al., 2007), we used the induction of and recovery from anoxic coma as a proxy for resistance to and clearance of ion surge in whole organisms, which provided an organismal-level comparison for our subsequent CNS-specific analyses. Then, to measure the resistance to chill-induced SD in the CNS, we used both potassium-sensitive and Direct Current (DC) electrodes inserted in the MTG to measure the extracellular [K⁺] surge and changes in DC potential during cooling. Finally, we measured the latency to the negative DC shift in potential in the MTG after application of the Na⁺/K⁺ ATPase inhibitor ouabain (i.e. the time between application of ouabain and the first observed effects on ion homeostasis) to characterize the effects of brief chilling and dehydration on Na⁺/K⁺ ATPase activity.