Isolation of new genes involved in temperature synchronization of the circadian clock of Drosophila melanogaster

PhDCircadian clocks regulate behaviour and physiology of many organisms and keep them in synchrony with the environment. Drosophila's circadian clock is mainly synchronized by natural light-dark cycles and temperature fluctuations, both at molecular and behavioural levels. The mechanisms underlying temperature entrainment are poorly understood, but previous studies have shown that this process can be genetically dissected. In this work, I isolated several mutants which interfere with the temperature synchronization of Drosophila's circadian clock. Three variants were isolated in a chemical EMS-mutagenesis screen monitoring putative second- and thirdchromosomal mutations. The mutants behave normal in light-dark cycles suggesting that they specifically interfere with temperature entrainment. In a different, RNAi-based screen, a Forkhead-domain transcription factor encoding gene was isolated, which shows defective circadian activity of per expression and PER accumulation in temperature-entrainment condition, when down-regulated. Finally, a candidate approach led me to identify three genes encoding proteins belonging to the TRP family of ion channels. Mutations in the pyrexia, trpM and trpA1 genes show abnormal temperature synchronization of locomotor behaviour, similar to our EMS-candidates. The isolation and analysis of those mutations are described, as well as a behavioural analysis of the already-known "temperature-mutant" nocte. In particular, I discuss the involvement of chordotonal organs as structures required for temperature entrainment of the clock and the role of nocte for signalling the temperature information from the periphery to the brain. The rest-activity pattern is a well-studied circadian output behaviour; the pupal emergence, named eclosion, is another behaviour strictly regulated by the circadian clock. Here we show that genes important for entrainment of adult locomotor behaviour to temperature do not play the same role in regulating the synchronization of eclosion. To gain insight into the synchronization mechanisms of eclosion, I studied how different entrainment conditions affect the phase and free-running period of eclosion

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster.

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as 'homing' similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species

A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.

Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission

A Code of Ethics for Gene Drive Research

Gene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity. We have developed a code of ethics for gene drive research with the hope that this code will encourage the development of an international framework that includes ethical guidance of gene drive research and is incorporated into scientific practice by gaining broad agreement and adherence

Genetic control strategies for population suppression in the Anopheles gambiae complex: a review of current technologies

Malaria continues to pose a critical public health threat, with mosquitoes from the Anopheles gambiae complex acting as the main vectors of the disease in sub-Saharan Africa, where approximately 95% of malaria-related deaths occur. Despite significant advancements in vector control, such as insecticide-treated bed nets and indoor spraying, the effectiveness of these interventions is increasingly compromised by various challenges, including rising levels of insecticide and pathogen resistance, mosquito behavioural adaptations, and persistent funding gaps. In this context, genetic vector control strategies have shown considerable promise, primarily based on findings from controlled laboratory studies. This review explores the development of these genetic approaches within the Anopheles gambiae complex and outlines future directions for their advancement and potential integration into malaria control efforts

Cryptochrome Antagonizes Synchronization of Drosophila’s Circadian Clock to Temperature Cycles

SummaryBackgroundIn nature, both daily light:dark cycles and temperature fluctuations are used by organisms to synchronize their endogenous time with the daily cycles of light and temperature. Proper synchronization is important for the overall fitness and wellbeing of animals and humans, and although we know a lot about light synchronization, this is not the case for temperature inputs to the circadian clock. In Drosophila, light and temperature cues can act as synchronization signals (Zeitgeber), but it is not known how they are integrated.ResultsWe investigated whether different groups of the Drosophila clock neurons that regulate behavioral rhythmicity contribute to temperature synchronization at different absolute temperatures. Using spatially restricted expression of the clock gene period, we show that dorsally located clock neurons mainly mediate synchronization to higher (20°C:29°C) and ventral clock neurons to lower (16°C:25°C) temperature cycles. Molecularly, the blue-light photoreceptor CRYPTOCHROME (CRY) dampens temperature-induced PERIOD (PER)-LUCIFERASE oscillations in dorsal clock neurons. Consistent with this finding, we show that in the absence of CRY very limited expression of PER in a few dorsal clock neurons is able to mediate behavioral temperature synchronization to high and low temperature cycles independent of light.ConclusionsWe show that different subsets of clock neurons operate at high and low temperatures to mediate clock synchronization to temperature cycles, suggesting that temperature entrainment is not restricted to measuring the amplitude of such cycles. CRY dampens temperature input to the clock and thereby contributes to the integration of different Zeitgebers

Temperature Entrainment of Drosophila's Circadian Clock Involves the Gene nocte and Signaling from Peripheral Sensory Tissues to the Brain

SummaryCircadian clocks are synchronized by the natural day/night and temperature cycles. Our previous work demonstrated that synchronization by temperature is a tissue autonomous process, similar to synchronization by light. We show here that this is indeed the case, with the important exception of the brain. Using luciferase imaging we demonstrate that brain clock neurons depend on signals from peripheral tissues in order to be synchronized by temperature. Reducing the function of the gene nocte in chordotonal organs changes their structure and function and dramatically interferes with temperature synchronization of behavioral activity. Other mutants known to affect the function of these sensory organs also interfere with temperature synchronization, demonstrating the importance of nocte in this process and identifying the chordotonal organs as relevant sensory structures. Our work reveals surprising and important mechanistic differences between light- and temperature-synchronization and advances our understanding of how clock resetting is accomplished in nature

A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.

Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission

Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field

AbstractCRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology.Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours.The gene-drive element spreads rapidly through the populations, fully supresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings.Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive.</jats:p