Timeless and diapause in Drosophila

Lead Research Organisation: University of Leicester
Department Name: Genetics

Abstract

Hibernation is a protective state of suspended animation that comes around every season in plants and animals that live in the temperate zones of the world. In insects, this seasonal cycle is called diapause, and is stimulated by the reduction in daylength as winter approaches. The other great rhythm on this planet is the circadian cycle which is the daily 24 hour cycle that we all recognise as our 'body clock'. Recently we have found a connection between a gene called timeless that controls the 24 hour rhythm of the geneticists favourite pet, the fruitfly, and the seasonal diapause cycle. A new mutation in the timeless gene, ls-tim, occurred in south-eastern Italy about 8000 years ago, a few thousand years after the last glaciation when flies invaded Europe from Africa This mutation allows the fly to make two types of TIM protein, L-TIM and S-TIM, whereas the ancient gene, s-tim can only make S-TIM. The new mutation, ls-tim, is spreading under natural selection, because it adapts the fly to the seasonal environment of Europe. It does this by reducing the light sensitivity of the ls-tim fly's diapause mechanism. Thus, even in relatively long days, ls-tim flies 'see' shorter days, and this stimulates them diapause earlier than in s-tim flies. The earlier the fly goes into diapause in Europe, the better its chances of surviving the oncoming winter. In addition, the 24 hour circadian clock of ls-tim is also less light-sensitive and this too is adaptive in Europe, which has very exotic photoperiods in summer which causes the fly's biorhythms to creak. Consequently, L-TIM attenuates the fly's photosensitivity for both circadian and seasonal behaviours, and that is perhaps why this new mutation has spread in Europe. Biochemically, the L-TIM protein is less light sensitive because of its weak physical interaction with a circadian photoreceptor called Cryptochrome. The ls-tim mutation creates a site in the L-TIM protein that is not shared in S-TIM. This new site may be phosphorylated, a protein modification that may alter the shape and stability of L-TIM. This may provide the biochemical clue for why ls-tim flies behave differently. We shall study this site to see whether it is indeed phosphorylated. We shall also investigate whether ls-tim flies change the ratios of the L-TIM and S-TIM they produce under the short days and cold temperature that produce diapause. In addition we will study under these conditions how strongly L-TIM and S-TIM 'touch' the photoreceptor Cryptochrome in the fly's brain and eyes, as this physical interaction provides the light input into the circadian mechanism. There has been a long running debate about whether the circadian clock contributes to diapause. Using the timeless gene, we shall genetically dissect the fly's nervous system in order to investigate which groups of neurons are important for diapause. Because the 24 hour clock neurons are well known and easily identified, we should be able to see whether these cells are relevant for the fly's hibernation. We can then visualise L-TIM and S-TIM protein in the relevant cells of ls-tim flies under diapause conditions, to see whether the two proteins behave differently. Insect diapause is a potential target for the control of medical and agricultural pests in temperate regions, and so our work may have practical spin-offs in future.

Technical Summary

A new mutation in the timeless clock gene of Drosophila melanogaster, ls-tim, has spread throughout Europe in the last few thousand years. This variant shows an attenuated photoresponsiveness, both in its circadian clock, and in diapause, the protective response to oncoming winters compared to the ancestral variant s-tim. Both phenotypes are adaptive in seasonal environments. The new variant generates both an L-TIM isoform from an upstream methionine, that is 23 residues longer than S-TIM, which is translated from the downstream methionine. s-tim flies produce only the truncated S-TIM protein. L-TIM shows a reduced physical interaction with the circadian photoreceptor Cryptochrome, which may explain its reduced photosensitivity. The ls-tim mutation generates a de novo phosphorylation site between residues 17-25. We will mutagenise the relevant Serine to an Alanine to see whether this reverts the ls-tim phenotypes to s-tim. If not, we shall randomise the order of the 23 N-terminal residues of L-TIM to examine whether this maintains the ls-tim phenotypes. We will also investigate in ls-tim fly heads the relative levels of L- and S-TIM under temperatures and photoperiods that induce diapause, as well as the dimerization efficiencies of L- and S-TIM with CRY. The tim-null mutant shows a temperature-sensitive diapause which, unlike wild-type, is not suppressed at longer summer photoperiods. We shall restore TIM function to various subsets of neuronal tissues as well as some peripheral tissues using the gal4/gal80/UAS system. We shall determine whether M and E neuronal clocks contribute to the diapause phenotype. Those tissue that our studies deem to be contributing to diapause will then be investigated by ICC in ls-tim flies to examine the dynamics of L- and S-TIM expression, using some of the tools we have generated.

Publications

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Bartok O (2013) Adaptation of molecular circadian clockwork to environmental changes: a role for alternative splicing and miRNAs in Proceedings of the Royal Society B: Biological Sciences

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Dissel S (2014) The Logic of Circadian Organization in Drosophila in Current Biology

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Hut R (2013) Latitudinal clines: an evolutionary view on biological rhythms , in Proceedings of the Royal Society B: Biological Sciences

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Kyriacou CP (2013) Animal behaviour: monarchs catch a cold. in Current biology : CB

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Kyriacou CP (2008) Clines in clock genes: fine-tuning circadian rhythms to the environment. in Trends in genetics : TIG

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Meireles-Filho A (2013) Circadian rhythms in insect disease vectors in Memórias do Instituto Oswaldo Cruz

 
Description We have mutagenised the N-terminal of the clock protein TIMELESS (TIM) to investigate which properties confer the reduced circadian and photoperiodic light responses of the ls-tim variant. We observe that it is the length of the N-terminal region rather than the specific sequence which is important. As this N-terminal fragment mediates the interaction with the photoreceptor CRY, this suggests that the TIM N-terminal is involved in a steric hindrance effect which prevents the normal light responses. We observed that very little PER or TIM could be detected at diapausing temperatures, suggesting that the circadian clock is arrested during diapause. Thus the diapausing effects of the N-terminal of TIM are likely to be developmental in origin.



An extensive neurogenetic analysis of diapause using clock mutants revealed that all clock mutants affected diapause hence there is clearly a modular association between the circadian clock and diapause. We also discovered that a clock in the eye of the fly is responsible for these effects, because stopping the eye clock prevents diapause



Because of the very low TIM levels at low temperatures we were unable to perform some of the biochemical experiments we had planned, so we embarked on a series of studies that focused on some of the many contradictions that exist about diapause in the literature. We were able to solve many of these problems and also clarify the role of some other non-clock genes that contribute to diapause. Finally, and in contradiction to the literature, we observed that diapause in Drosophila melanogaster is an ancient adaptation, not the recently evolved one others have claimed. Thus we have made a significant contribution to understanding the biology of diapause.
Exploitation Route Diapause is a very topical subject because it is potentially a target that can be used against temperate insects of agricultural and medical importance. It is also a biomarker for climate change. We will exploit our findings with high impact papers (several in prep) and further grant applications.
Sectors Agriculture, Food and Drink,Education,Environment,Healthcare

 
Description SUSTAIN -Academy of Medical Sciences
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact SUSTAIN is a programme run by the Academy of Medical Sciences (AMS) which aims to mentor young female bio-medical researchers into negotiating the pitfalls of their early careers. It has been running about 3 years and I am a mentor and on the advisory committee. From the feedback we have received it has been very successful.
URL https://acmedsci.ac.uk/grants-and-schemes/mentoring-and-other-schemes/sustain
 
Description INsecTIME
Amount € 4,000,000 (EUR)
Funding ID 316790 
Organisation Marie Sklodowska-Curie Actions 
Department Initial Training Networks (ITN)
Sector Charity/Non Profit
Country Global
Start 04/2013 
End 03/2017
 
Description Major collaboration with group of Prof Rodolfo Costa, Padova, Italy 
Organisation University of Padova
Country Italy 
Sector Academic/University 
PI Contribution Longterm, Diapause collaboration involving several shared researchers
Start Year 2008
 
Description Genes and behaviour 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research or patient groups
Results and Impact An invited talk given to the Leicester Secular Society, Leicester, February 3 2013

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Insect behavioural genetics 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach regional
Primary Audience Participants in your research or patient groups
Results and Impact Invited guest lecture to Oxford MSc Neurobiology students, Oxford, May 14 2012

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Insect clocks 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach local
Primary Audience Participants in your research or patient groups
Results and Impact Public Talk at 'Neuroday', University of Leicester, May 3 2012

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Open Day neurogenetics in Wurzburg, Germany 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact An open day with collaborators at the University of Wurzburg, Feb 18th 2017 involving lectures and practical demonstrations of arthropod behaviour and genetics
Year(s) Of Engagement Activity 2017
 
Description Public lecture as part of award ceremony - SCNi Institute Prize 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was awarded the Sleep and Circadian neuroscience Institute Prize in 2017 and gave a public lecture to ~200 people. This was recorded and is on youtube
Year(s) Of Engagement Activity 2017
URL https://www.youtube.com/watch?v=Pf994HDsylU
 
Description school visits, London, Leicestershire, 2-3 per year 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact Children and young adults get excited about Biorhythms, ask lots of questions, visit the laboratory, and invite me to give further school talks

schoolchildren visit my lab regularly and schools re-invite me year after year
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,