Date Published: June 28, 2017
Publisher: Public Library of Science
Author(s): Franziska Ruf, Martin Fraunholz, Konrad Öchsner, Johann Kaderschabek, Christian Wegener, Henrik Oster.
Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems.
Many holometabolous insect species time their eclosion (i.e. the emergence of the adult insect from the pupa or adult ecdysis) not only to a specific time of season, but also to a specific time of day. This leads to overt daily eclosion rhythms on the population level, well known to entomologists and fly fishermen since centuries. Eclosion assays offer an ideal behavioural read-out to study molecular and cellular mechanisms of circadian timing, as eclosion timing continues under constant conditions and is basically unaffected by the masking influence of motivational or physiological states of the animal such as hunger, memory, age or reproductive state. Classic chronobiological experiments used eclosion assays with fruit flies (genus Drosophila) to demonstrate that behavioural rhythms can be influenced by abiotic factors such as light and temperature [1–3] and can be driven by an endogenous light-entrainable timing system [1,3]. A Drosophila eclosion assay was also used in the ground-breaking identification of period, the first clock gene which was later on also found in vertebrates including humans . For proper eclosion timing in Drosophila, both a central clock in the brain and a peripheral clock in the prothoracic gland are required [6,7]. Eclosion (ecdysis) behaviour itself is orchestrated by a peptidergic signalling cascade comprising hormonal cells as well as peptidergic neurons [8,9]. Results from Drosophila and moths indicate that the release of ecdysis-triggering hormone (ETH) from the epitracheal glands [10–12] initiates this cascade and activates subsets of down-stream peptidergic neurons in a stereotyped sequence [13–16]. How the clock in the brain and prothoracic gland times ETH release and the start of the ecdysis-orchestrating peptidergic cascade is, however, largely unknown.
Our results show that the new WEclMon system is suitable to analyse Drosophila eclosion under different entrainment regimes as well as under natural abiotic conditions. Under standard light entrainment in the laboratory, WEclMons showed a similar performance as the widely used Trikinetics eclosion monitors which suggests that the mechanical agitation in the funnel-type Trikinetics monitors is not influencing eclosion rhythmicity. Moreover, as the WEclMon is a physically open system, puparia are immediately and directly exposed to all abiotic changes in the environment, and are easily accessible for optogenetic manipulations. The monitors come for a reasonable price since they are largely made of non-expensive commercially or freely available components and software. As the system is camera-based, there was no data loss, for example by blocking of funnels or by “clever flies” like D. littoralis  that are able to stay some time inside the funnel. By comparing the empty puparia on the plate to the outcome from the image analysis, it was possible to check for and largely exclude false positive results.