Periodic Reporting for period 2 - CINCHRON (Comparative INsect CHRONobiology)
Reporting period: 2020-06-01 to 2022-11-30
ADVANCED KNOWLEDGE IN INSECT CHRONOBIOLOGY
Insects and mammals share molecular machinery that generates circadian rhythms in behaviour and physiology. Biological timing has a major impact in human physical and psychological well-being, as well as in the economy of the ’24-hour’ society. Studying insect clocks is a faster, cost-effective and ethical way of learning about mammalian clocks.
During the action substantial advances were made that contributed to the publication of 16 Open Access papers.
PEST AND DISEASE CONTROL
Insects cause serious damage to crops and compromise food production. Due to climate change, tropical insects are expanding their ranges to temperate regions, including Europe, bringing diseases (eg malaria and Dengue fever). The seasonal timekeeping determines when the insects enter diapause, the equivalent of mammalian hibernation and shares molecular components with the 24 hour circadian clock. Manipulating this seasonal response would provide a tool to control these insects, so several of the species we study are agricultural pests or have implications for pest control.
The implementation of our projects in the non-traditional model organisms, Pyrrhocoris apterus, Drosophila suzukii, Acyrthosiphon pisum, and Nasonia vitripennis has led to the identification of new molecules and neurotransmitters involved in seasonal timekeeping. Furthermore, in pea aphids the first physiological link between the central clock and the photoperiodic system was identified.
DEVELOPMENT OF FUTURE RESEARCHERS
Our ESRs became familiar with an extensive set of multidisciplinary skills, and, together with their exposure to the private sector, will enhance the pool of highly skilled, young European researchers.
The outbreak of COVID-19 affected our activities for most of the second reporting period. Despite this, at the end of the action, all our students (except ESR10 who was re-appointed mid 2020) have defended their PhD thesis and been awarded their doctorate, or are in the process of writing up their thesis to be examined by the end of 2023. Our ESRs have been exposed to a wide range of courses and presented and brainstormed their data regularly to all partners and scientific advisers, with each contributing a different skillset. Finally, they have been exposed to the commercial sector by attending webinars organised by our Associate Partners, and some have undergone an internship at Oxitec, UK.
Insects and mammals share molecular machinery that generates circadian rhythms in behaviour and physiology. Biological timing has a major impact in human physical and psychological well-being, as well as in the economy of the ’24-hour’ society. Studying insect clocks is a faster, cost-effective and ethical way of learning about mammalian clocks.
During the action substantial advances were made that contributed to the publication of 16 Open Access papers.
PEST AND DISEASE CONTROL
Insects cause serious damage to crops and compromise food production. Due to climate change, tropical insects are expanding their ranges to temperate regions, including Europe, bringing diseases (eg malaria and Dengue fever). The seasonal timekeeping determines when the insects enter diapause, the equivalent of mammalian hibernation and shares molecular components with the 24 hour circadian clock. Manipulating this seasonal response would provide a tool to control these insects, so several of the species we study are agricultural pests or have implications for pest control.
The implementation of our projects in the non-traditional model organisms, Pyrrhocoris apterus, Drosophila suzukii, Acyrthosiphon pisum, and Nasonia vitripennis has led to the identification of new molecules and neurotransmitters involved in seasonal timekeeping. Furthermore, in pea aphids the first physiological link between the central clock and the photoperiodic system was identified.
DEVELOPMENT OF FUTURE RESEARCHERS
Our ESRs became familiar with an extensive set of multidisciplinary skills, and, together with their exposure to the private sector, will enhance the pool of highly skilled, young European researchers.
The outbreak of COVID-19 affected our activities for most of the second reporting period. Despite this, at the end of the action, all our students (except ESR10 who was re-appointed mid 2020) have defended their PhD thesis and been awarded their doctorate, or are in the process of writing up their thesis to be examined by the end of 2023. Our ESRs have been exposed to a wide range of courses and presented and brainstormed their data regularly to all partners and scientific advisers, with each contributing a different skillset. Finally, they have been exposed to the commercial sector by attending webinars organised by our Associate Partners, and some have undergone an internship at Oxitec, UK.
(*peer review publication)
CIRCADIAN CHRONOBIOLOGY
1. D. melanogaster 24 h rhythms, temperature compensation, the ability of the clock to maintain a 24h period at different temperatures was observed to have an intercellular communication component involving two subgroups of clock neurons.
2. A number of transcription factors that distinguish two groups of clock neurons (expressing or not the photoreceptor CRY) were identified, and experimental manipulation of these genes by RNAi revealed that some generated arrhythmicity, and are implicated in the physiology or development of these neurons
3. Rhodopsin, Rh2, plays a role in the clock’s response to light. Rh5 mutants showed defective behaviour during temperature cycles in the cooler range. Removal of the Chordotonal Organ (ChO) neurons affected behavioural entrainment to temperature cycles but both the ChO neurons and the antennae contribute to normal temperature entrainment.***
4. CRY expressing neurons get updates about each other’s CRY status. PDF receptor signalling was also found to be involved in this circadian photoreception pathway. Furthermore, the Rh2 photoreceptor is involved in circadian light input under high intensity light conditions, thereby uncovering a novel circadian light input pathway.
5. 13% of the Nasonia vitripennis transcriptome is regulated by light. Unlike Drosophila, Nasonia can be entrained under red light.
SEASONAL CHRONOBIOLOGY
6. The D. littoralis clock is highly plastic and can adapt to extreme latitudes suggesting that such flexibility is adaptive.
7. Clock neuron projections in the pea aphid were identified via immunohistochemistry using antibodies against different clock proteins and neuropeptides.*
8. Clock neurons expressing CRY in pea aphids contact regions of the brain implicated in controlling diapause providing a link between the measurement of day-length and seasonal responses.*
9. Neuropeptides mediate the robust photoperiodic diapause of linden bugs (P. apterus). Gene silencing technology was developed to disrupt the underlying genes and study their seasonal effects.*
10. Bumblebees injected with dsRNA had a slower adjustment to new light regimes, implicating blue opsin as a circadian photoreceptor of the bumblebee.
11. Genetic variation in the clock genes period, cry-2 and cycle is associated with latitude-dependent variation in the diapause response of N. vitripennis.
METABOLIC CHRONOBIOLOGY
12. Ucp4C expression levels are correlated with the temperature at which flies live, both in D. melanogaster and the pest D. suzukii.
13. Clock cells controlling cyclic expression of heme oxygenase (ho) mRNA in the Drosophila brain have been identified.**
COMMERCIAL CHRONOBIOLOGY
14. B. mori sensitivity to infection varies throughout the day, with animals infected at ZT3 being significantly more sensitive to infection compared to animals infected at night.***
15. The first clock mutant in bumblebees has been generated by gene editing.
CIRCADIAN CHRONOBIOLOGY
1. D. melanogaster 24 h rhythms, temperature compensation, the ability of the clock to maintain a 24h period at different temperatures was observed to have an intercellular communication component involving two subgroups of clock neurons.
2. A number of transcription factors that distinguish two groups of clock neurons (expressing or not the photoreceptor CRY) were identified, and experimental manipulation of these genes by RNAi revealed that some generated arrhythmicity, and are implicated in the physiology or development of these neurons
3. Rhodopsin, Rh2, plays a role in the clock’s response to light. Rh5 mutants showed defective behaviour during temperature cycles in the cooler range. Removal of the Chordotonal Organ (ChO) neurons affected behavioural entrainment to temperature cycles but both the ChO neurons and the antennae contribute to normal temperature entrainment.***
4. CRY expressing neurons get updates about each other’s CRY status. PDF receptor signalling was also found to be involved in this circadian photoreception pathway. Furthermore, the Rh2 photoreceptor is involved in circadian light input under high intensity light conditions, thereby uncovering a novel circadian light input pathway.
5. 13% of the Nasonia vitripennis transcriptome is regulated by light. Unlike Drosophila, Nasonia can be entrained under red light.
SEASONAL CHRONOBIOLOGY
6. The D. littoralis clock is highly plastic and can adapt to extreme latitudes suggesting that such flexibility is adaptive.
7. Clock neuron projections in the pea aphid were identified via immunohistochemistry using antibodies against different clock proteins and neuropeptides.*
8. Clock neurons expressing CRY in pea aphids contact regions of the brain implicated in controlling diapause providing a link between the measurement of day-length and seasonal responses.*
9. Neuropeptides mediate the robust photoperiodic diapause of linden bugs (P. apterus). Gene silencing technology was developed to disrupt the underlying genes and study their seasonal effects.*
10. Bumblebees injected with dsRNA had a slower adjustment to new light regimes, implicating blue opsin as a circadian photoreceptor of the bumblebee.
11. Genetic variation in the clock genes period, cry-2 and cycle is associated with latitude-dependent variation in the diapause response of N. vitripennis.
METABOLIC CHRONOBIOLOGY
12. Ucp4C expression levels are correlated with the temperature at which flies live, both in D. melanogaster and the pest D. suzukii.
13. Clock cells controlling cyclic expression of heme oxygenase (ho) mRNA in the Drosophila brain have been identified.**
COMMERCIAL CHRONOBIOLOGY
14. B. mori sensitivity to infection varies throughout the day, with animals infected at ZT3 being significantly more sensitive to infection compared to animals infected at night.***
15. The first clock mutant in bumblebees has been generated by gene editing.
Our work with D. melanogaster made significant progress beyond state-of-the art with findings on temperature compensation, the cellular role of CRY, and the importance of the rhodopsins in light responses. We have a comprehensive view of how different environmental factors influence the output of the clock and how photoreceptors influence the input, information that, within a mammalian context, can suggest optimal design of lighting environments in the workplace. Fundamental work on understanding the circadian and robust seasonal clocks in high latitude Drosophila species and the linden bug, reveal a role of circadian clock genes on diapause. Reagents that disrupt circadian clock proteins have been studied by others and could thus prove useful in a seasonal context for pest control.
Progress has been made in understanding the circadian biology of insect pets and species relevant for biological control. Candidate genes that determine the light sensitivity of the circadian and seasonal clock have been identified in pea aphids and Nasonia respectively.
Some mitochondrial proteins in D. melanogaster show circadian cycles of expression that can be targeted in pest species. Indeed, a pesticide already exists that disrupts the cycling of heme oxygenase (HO), an enzyme that protects the cell. Ucp4C, crucial for cellular heat generation is a further potential target for disruption of cold-adaptation in pest species.
In the silkmoth, we have made significant progress in understanding the circadian nature of bacterial infections with obvious economic implications for silk production. In the pollinator bumblebee, we wish to understand the role of the clock and complex social behaviour. In both species, we have developed gene knock-out techniques for clock genes and observed their effects on infection and social behaviour. These results will inform the silkmoth industry and beekeepers on the relevance of the clock for the health and well-being of these species and how to optimise the insects circadian rearing conditions.
Progress has been made in understanding the circadian biology of insect pets and species relevant for biological control. Candidate genes that determine the light sensitivity of the circadian and seasonal clock have been identified in pea aphids and Nasonia respectively.
Some mitochondrial proteins in D. melanogaster show circadian cycles of expression that can be targeted in pest species. Indeed, a pesticide already exists that disrupts the cycling of heme oxygenase (HO), an enzyme that protects the cell. Ucp4C, crucial for cellular heat generation is a further potential target for disruption of cold-adaptation in pest species.
In the silkmoth, we have made significant progress in understanding the circadian nature of bacterial infections with obvious economic implications for silk production. In the pollinator bumblebee, we wish to understand the role of the clock and complex social behaviour. In both species, we have developed gene knock-out techniques for clock genes and observed their effects on infection and social behaviour. These results will inform the silkmoth industry and beekeepers on the relevance of the clock for the health and well-being of these species and how to optimise the insects circadian rearing conditions.