Periodic Reporting for period 4 - EVOCLOCK (From Evolution to Clockworks:Unravelling the molecular basis of circalunar clocks)
Reporting period: 2023-07-01 to 2024-12-31
Nature is structured in time by the recurring cycles of night and day, seasons, tides and lunar phases. Most organisms possess so-called "biological clocks" for anticipating these regular changes in their environment and for adjusting their behaviour and body functions accordingly. The daily clock ("circadian clock", 24 hours) is well understood at the molecular level. But the mechanisms of tidal clocks (12.4 hours), lunar clocks (29.5 days) and annual clocks (1 year) are still largely unknown.
In this project we aim to uncover molecular mechanisms underlying the lunar clock in the marine insect Clunio marinus. Clunio has timed its reproduction to the lowest low tides, which always occur around new moon and full moon. As the tides differ along the coastline, Clunio has adjusted its lunar clock accordingly. By comparing Clunio strains with differing lunar clocks, we can identify candidate lunar clock molecules. This endeavour cannot be undertaken in classical biological model organisms like fruit fly or mouse, because these do not possess a lunar clock. For Clunio, in turn, many of the molecular tools and resources that are available for model organisms, are still lacking. Hence, the objectives of this project are to (1) establish tools to experimentally manipulate Clunio at the molecular level, (2) get a global overview of how Clunio uses its gene repertoire over the lunar cycle, and (3) characterize Clunio's nervous system and the receptors by which it perceives the tides and lunar phases. Finally, we will try and bring together all these lines of research for identifying and describing some lunar clock molecules and mechanisms.
Studying the lunar clock means exploring uncharted territory. Therefore, this project is clearly basic research. In the long term, understanding the lunar clock of Clunio will give insights into insect development and maturation, which may also highlight new opportunities for insect pest control. Knowledge on the lunar clock may further aid marine aquaculture and thus contribute to securing sustainable food supplies for a growing world population. Controlled breeding of marine organisms is often difficult, and much of this may have to do with the lack of understanding of the complex and often lunar-regulated reproduction of marine organisms.
In this project we aim to uncover molecular mechanisms underlying the lunar clock in the marine insect Clunio marinus. Clunio has timed its reproduction to the lowest low tides, which always occur around new moon and full moon. As the tides differ along the coastline, Clunio has adjusted its lunar clock accordingly. By comparing Clunio strains with differing lunar clocks, we can identify candidate lunar clock molecules. This endeavour cannot be undertaken in classical biological model organisms like fruit fly or mouse, because these do not possess a lunar clock. For Clunio, in turn, many of the molecular tools and resources that are available for model organisms, are still lacking. Hence, the objectives of this project are to (1) establish tools to experimentally manipulate Clunio at the molecular level, (2) get a global overview of how Clunio uses its gene repertoire over the lunar cycle, and (3) characterize Clunio's nervous system and the receptors by which it perceives the tides and lunar phases. Finally, we will try and bring together all these lines of research for identifying and describing some lunar clock molecules and mechanisms.
Studying the lunar clock means exploring uncharted territory. Therefore, this project is clearly basic research. In the long term, understanding the lunar clock of Clunio will give insights into insect development and maturation, which may also highlight new opportunities for insect pest control. Knowledge on the lunar clock may further aid marine aquaculture and thus contribute to securing sustainable food supplies for a growing world population. Controlled breeding of marine organisms is often difficult, and much of this may have to do with the lack of understanding of the complex and often lunar-regulated reproduction of marine organisms.
Achieving our first goal, to establish molecular manipulation of Clunio has proven difficult, both due to specific properties of the organism and due to the Covid-19 pandemic, which impeded laboratory work and insect culture maintenance. However, we were able to establish injection into eggs and larvae, allowing us to administer drugs, tracers and other molecules. We also achieved first steps towards genome editing in Clunio. Finally, we developed experimental setups to test the function of Clunio genes in bacteria, insect cell cultures and fruit flies.
Regarding our second goal, we achieved a thorough characterization of how Clunio uses its gene repertoire not only over the lunar cycle, but also during a developmental arrest which synchronizes development with the lunar cycle, and during the subsequent stereotypic development into pupae. This resulted in a large gene expression database, the identification of co-regulated genes and hints towards transcription factors which may mediate lunar clock outputs.
With respect to our third goal, we obtained a 3D reconstruction of Clunio’s larval morphology including the nervous system, based on µCT scans, antibody staining and in-situ hybridization. This also includes basic circuits of the circadian clock. Furthermore, we were able to identify some molecules likely involved in the perception of moonlight and tides. We also performed a thorough assessment of which aspects of moonlight and the tides are informative for setting Clunio’s lunar clock, and how this information is integrated.
Finally, we obtained the fundamental insight that in Clunio the lunar clock is based on counting circadian clock cycles. This explains our repeated finding that differences in the lunar clock of specific Clunio strains seem to rely on changes in circadian clock genes. We also obtained novel insights into the function of one of these genes, the period gene. These results led to the conclusions that in Clunio the circalunar clock is likely derived from a photoperiodic diapause mechanism, that lunar clocks have different mechanisms in different organisms, and that they likely evolved several times independently.
Regarding our second goal, we achieved a thorough characterization of how Clunio uses its gene repertoire not only over the lunar cycle, but also during a developmental arrest which synchronizes development with the lunar cycle, and during the subsequent stereotypic development into pupae. This resulted in a large gene expression database, the identification of co-regulated genes and hints towards transcription factors which may mediate lunar clock outputs.
With respect to our third goal, we obtained a 3D reconstruction of Clunio’s larval morphology including the nervous system, based on µCT scans, antibody staining and in-situ hybridization. This also includes basic circuits of the circadian clock. Furthermore, we were able to identify some molecules likely involved in the perception of moonlight and tides. We also performed a thorough assessment of which aspects of moonlight and the tides are informative for setting Clunio’s lunar clock, and how this information is integrated.
Finally, we obtained the fundamental insight that in Clunio the lunar clock is based on counting circadian clock cycles. This explains our repeated finding that differences in the lunar clock of specific Clunio strains seem to rely on changes in circadian clock genes. We also obtained novel insights into the function of one of these genes, the period gene. These results led to the conclusions that in Clunio the circalunar clock is likely derived from a photoperiodic diapause mechanism, that lunar clocks have different mechanisms in different organisms, and that they likely evolved several times independently.
In this ERC project we identified part of the morphological and neuronal context of the lunar clock, as well as major transcriptional and metabolic outputs. We also found that the lunar clock itself relies on counting days in Clunio, which integrates well with our other genomic and molecular findings. This mechanism was not expected and is different from the lunar clock mechanism in other organisms. This allowed us to conclude that lunar clocks have different mechanisms in different organisms and therefore likely evolved several times independently. These findings are major advancements in the study of lunar clocks and open ways for many new lines of research.
We also established several molecular, genomic and morphological resources for Clunio, an organism that has been barely studied at these levels. This opens novel experimental opportunities for the undertaking to try and understand the molecular basis of the lunar clock.
As outlined above, our insights into the lunar clock and development of Clunio may in the long term result in novel strategies for insect pest control and aquaculture of marine organisms, both contributing to food security for a growing world population. Finally, lunar clocks are a highly fascinating phenomenon and our studies in Clunio attract the attention of both scholars and the lay public, fostering the awe and love for nature that is required to tackle the current biodiversity crisis.
We also established several molecular, genomic and morphological resources for Clunio, an organism that has been barely studied at these levels. This opens novel experimental opportunities for the undertaking to try and understand the molecular basis of the lunar clock.
As outlined above, our insights into the lunar clock and development of Clunio may in the long term result in novel strategies for insect pest control and aquaculture of marine organisms, both contributing to food security for a growing world population. Finally, lunar clocks are a highly fascinating phenomenon and our studies in Clunio attract the attention of both scholars and the lay public, fostering the awe and love for nature that is required to tackle the current biodiversity crisis.