Neural dysfunction correlates with heat coma and CT_max in Drosophila but does not set the boundaries for heat stress survival

Abstract

Running title: Neural dysfunction in heat stress 12 13 Summary statement 14Hyperthermic failure of the Drosophila central nervous system causes heat coma, a phenotype varying in 15 temperature between drosophilids, but neural failure is likely not the primary cause of heat mortality. 16 Abstract 17When heated, insects loose coordinated movement followed by the onset of heat coma (CTmax). These 18 phenotypes are popular measures to quantify inter-and intraspecific differences in insect heat tolerance, and 19CTmax correlate well with current species distributions. Here we examined the function of the central nervous 20 system (CNS) in five species of Drosophila with different heat tolerances, while they were exposed to either 21 constant high temperature or a gradual increasing temperature (ramp). Tolerant species were able to preserve 22 CNS function at higher temperatures and for longer durations than sensitive species and similar differences 23 were found for the behavioral indices (loss of coordination and onset of heat coma). Furthermore, the timing 24 and temperature (constant and ramp exposure, respectively) for loss of coordination or complete coma 25 coincided with the occurrence of spreading depolarisation (SD) events in the CNS. These SD events disrupt 26 neurological function and silence the CNS suggesting that CNS failure is the primary cause of impaired 27 coordination and heat coma. Heat mortality occurs soon after heat coma in insects and to examine if CNS 28 failure could also be the proximal cause of heat death, we used selective heating of the head (CNS) and 29 abdomen (visceral tissues). When comparing the temperature causing 50% mortality (LT50) of each body 30 part to that of the whole animal, we found that the head was not particularly heat sensitive compared to the 31 abdomen. Accordingly, it is unlikely that nervous failure is the principal/proximate cause of heat mortality in 32 Drosophila. 33In the ramping assay, temperature was increased by 0.25 °C min -1 from 19 °C. Two behavioural phenotypes 126 were recorded during this experiment: 1) the temperature at which the fly would lose coordination and fall on 127 its back (Tback) and 2) the temperature at which the fly was completely still (Tcoma). Tcoma was verified by 128 poking the vial lids with a stick to agitate the flies and check for reflexes. The static assay used a similar 129 setup and method to record knockdown, but instead of increasing the temperature gradually, the flies were 130 placed in the bath pre-set to 38 °C, after which the exposure durations causing loss of coordinated movement 131 (tback) and heat coma (tcoma) were noted (here the lowercase "t" represents time). The "static" assay was only 132 static for 1 hour at 38 °C after which the temperature was increased by 0.25 °C min -1 to ensure that more heat

Integrating GWAS and Transcriptomics to Identify the Molecular Underpinnings of Thermal Stress Responses in Drosophila melanogaster

“…These included a cluster of functionally related genes (dar1, fru, NetA, RhoGEF64C, trio, twf) involved in nervous system development, which was reflected in overrepresentation of the nervous system and cell morphogenesis and differentiation GO categories in the CT min GWAS gene set. Neuronal failure operationally defines both CT min and CT max (Andersen et al, 2018;Andersen and Overgaard, 2019;Jørgensen et al, 2019), and dynamic stabilization of the neuromuscular circuit under temperature stress is a likely mechanism for altering thermal limits. Indeed, previous investigation of the genetic architecture of cold hardiness and electrophysiological analyses of the rapid hardening response both suggest an important role for stabilization of ion channels and cytoskeletal structures supporting the synapse and neuromuscular junction (Klose and Robertson, 2004;Robertson and Money, 2012;Freda et al, 2017).…”

“…The few genes that consistently appeared in overrepresented categories, fra, fz2 and ptc, are also functionally associated with the nervous system, including axon and dendrite guidance and synapse organization. Spreading depolarization of the central nervous system (triggered by failure to maintain ion gradients between the intra-and extracellular compartments) is linked with heat tolerance across Drosophila species (Jørgensen et al, 2019), indicating that neuronal failure is an important component of heat tolerance in addition to cold tolerance. An additional set of three genes, Pax, sls and Zasp66, co-occurred in several associated categories of muscle structure and development ( Figure 5).…”

Leaf-cutting ants’ critical and voluntary thermal limits show complex responses to size, heating rates, hydration level, and humidity

“…Within voluntarily tolerated thermal levels, different aspects of physiological performance are optimized at different temperatures (e.g., stamina may be more optimized at lower temperatures than sprint speed, Huey et al 1984). Nonetheless, if body temperatures rise excessively, locomotor and neural processes eventually stop, and the animals reach their Critical Thermal Maximum (CT max , Cowles and Bogert 1944;Jørgensen et al 2020), which can kill them almost immediately (Angilletta et al 2007;Christian and Morton 1992;Ribeiro et al 2012).…”

Brain dysfunction during warming is caused by oxygen limitation in larval zebrafish

“…Temperature-induced neural dysfunction could consequently underlie upper thermal tolerance limits, either via direct thermal effects on neurons (11, 12) or via indirect thermal effects from oxygen limitations (18). Examples of severe heat-induced neural dysfunctions include spreading depolarizations in the brain of fruit flies (26), loss of rhythmic neural activity in the digestive system of the Jonah crab (27), and thermogenic seizures in vertebrates (28–30). However, few studies have directly investigated how neural dysfunction relates to measurements of thermal tolerance.…”

“…However, few studies have directly investigated how neural dysfunction relates to measurements of thermal tolerance. In fruit flies, spreading depolarizations measured in restrained flies occur at similar temperatures as a heat-induced coma in freely moving conspecifics (26). Similarly, goldfish lose equilibrium at temperatures that alters the activity of cerebellar neurons in anesthetized conspecifics (11), and a study on Atlantic cod found that cooling the brain marginally increased CT max , suggesting a causal link between brain function and thermal tolerance (12).…”