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
Sandrelli F
(2008)
Comparative analysis of circadian clock genes in insects.
in Insect molecular biology
Kyriacou CP
(2008)
Clines in clock genes: fine-tuning circadian rhythms to the environment.
in Trends in genetics : TIG
Benna C
(2010)
Drosophila timeless2 is required for chromosome stability and circadian photoreception.
in Current biology : CB
Rosato E
(2011)
The role of natural selection in circadian behaviour: a molecular-genetic approach.
in Essays in biochemistry
Schiesari L
(2011)
The hormonal and circadian basis for insect photoperiodic timing.
in FEBS letters
Kyriacou C
(2013)
Animal Behaviour: Monarchs Catch a Cold
in Current Biology
Hut RA
(2013)
Latitudinal clines: an evolutionary view on biological rhythms.
in Proceedings. Biological sciences
Meireles-Filho AC
(2013)
Circadian rhythms in insect disease vectors.
in Memorias do Instituto Oswaldo Cruz
Bartok O
(2013)
Adaptation of molecular circadian clockwork to environmental changes: a role for alternative splicing and miRNAs.
in Proceedings. Biological sciences
Kyriacou CP
(2014)
Sex and rhythms in sandflies and mosquitoes: an appreciation of the work of Alexandre Afranio Peixoto (1963-2013).
in Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases
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 | 03/2013 |
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 | Plenary lecture at public meeting |
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 | Darwin Day, a one day public meeting held in Haifa, Israel, 12th February 2020. This received considerable press and radio coverage. Talk entitled 'From circadian clocks to Nobel Prizes: making Darwin proud' |
Year(s) Of Engagement Activity | 2020 |
URL | http://deeb.haifa.ac.il/2019/12/18/darwin-day-2020/ |
Description | Public lecture University of Padova, Italy |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Public lecture for retirement of Professor Rudi Costa, Dec 6th 2019 at University of Padova |
Year(s) Of Engagement Activity | 2020 |
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-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014 |