A novel thermosensitive escape behavior in Drosophila larvae

Abstract

Animals detect and respond to temperature changes in their environment. Responses can be choice based to achieve an optimum environment for physiological function, or escape reflexes as temperature reaches noxious levels. Both types of response can be observed in Drosophila.1,2 Recent descriptions of temperature responses in Drosophila have uncovered mechanisms and pathways involved in optimal temperature detection in flies 3 that appear to be shared in nociceptive perception in mammals. 4 Mutations in a number of Transient Receptor Potential (TRP) receptors have been identified. Behavioral analysis of TRP receptor mutants and the expression patterns of the TRP proteins have helped to identify components of the underlying neurocircuitry. 1,3,[5][6][7] We describe here a robust behavioral escape response from larvae to rising temperature. The behavior closely resembles previously described nocifensive responses elicited by touch with a heated probe set at 39-41°C producing a corkscrewing avoidance of the stimulus.1 A later modification of this stimulus application employed a temperature regulated thermal probe. 8 For both studies, duration and force of contact between probe and larvae is a function of the testers' judgement and dexterity. In an attempt to remove this potential operator variability, we applied a thermal stimulus to larvae by immersion in a water droplet and subsequent heating of the water to noxious levels. In practical terms, we placed a larva in 30 μl of water at room temperature on a petri dish lid. The lid was then transferred to a hotplate surface set to 70°C. Care was taken to ensure that the petri dish lid consistently came into close contact with the hotplate. As our baseline, we used a fine temperature probe (a thermocouple) to determine the rate of temperature increase in the water droplet so that we have an exact determination of the temperature of the droplet at any time-point ( fig. 1B) Keywords: nociception, nocifensive, TRP receptor, Drosophila larvae dish holding the droplet and larva to the hot plate, we recorded the time at which the larva initiated the characteristic nocifensive corkscrew roll. We use our time/temperature relationship curve to infer the temperature in the droplet of water when the escape response is activated. In our assay, the escape response in wildtype larvae was evoked at 28-29°C ( fig. 1c). We determined the escape response temperature for larvae of our lab stock of w 1118 , and two wild-type stocks, Canton-S and Oregon-R. The three wild-type stocks all responded at a temperature just above 29°C (29.3°C, 29.8°C and 29.4°C respectively). In larvae mutant for the TRP receptor painless (pain 1 ), or functionally blocked by the expression of tetanus toxin light chain 10 in the class IV da sensory neurons, the response occurred at 33-34°C ( fig. 1c). Control animals expressing inactive tetanus toxin light chain respond at 28.1°C. This suggests that the painless TRP receptor may be involved in responses to temperature around 29°C in this behavioral test.In additi...

Teaching report: the use of Drosophila melanogaster larval thermosensitive escape behaviour as a model system to demonstrate sensory function

“…Far more robust responses were observed at 29 and 30°C (60% and above). This was consistent with the findings of Oswald et al (2011). If required, the data can be readily expressed as percentage response.…”

“…This practical is based on a method developed for research purposes (Oswald et al 2011); however, it has proven to be both reliable and robust in use with post-16 biology students.…”

“…Such responses can reflect choice to achieve an optimum environment for physiological function or escape reflexes as temperature reaches noxious levels. We demonstrate here that responses to temperature we have identified in Drosophila larvae (Oswald et al 2011) can be employed to teach an understanding of the purpose and functioning of the peripheral sensory nervous system both at the high school and undergraduate level. Identifying and employing a robust and appropriate behavioural response in a suitable organism can be challenging in the classroom environment.…”

“…When Drosophila larvae are immersed in a droplet of water, and the temperature of the water then increased, the larvae always respond with stereotypical escape behaviour ('head whipping' followed by a violent 'corkscrewing') at a definable temperature (Oswald et al 2011). This response temperature can also be determined by quick immersion of larvae in water baths set over a short range of temperatures and students are then tasked with defining the temperature at which the larvae respond with their escape behaviour.…”

Local and Global Methods of Assessing Thermal Nociception in <em>Drosophila</em> Larvae

“…Although it is not clear when a larva might experience a globally noxious stimulus in the wild, in the lab the behavioral responses to this global exposure are more complex than those observed upon local stimulation. A strength of the heat plate assay, also noted by others 3 , is that it has little user-to-user variability since touching the larva is not a component of the protocol. The only substantial variance seems to be in defining when each behavior commences and this can be minimized with repeated viewing/familiarity.…”