WP2 (circadian and seasonal chronobiology) focuses on mechanisms that mediate circadian, seasonal and developmental timing in Drosophila, housefly, black soldier fly, the linden bug, butterfly, migratory moth, and Madeira cockroach.
WP2 (DC1; BCAS) Non-Drosophila insect neuropeptides: their role in reproduction and seasonality
Objectives: To study neuropeptides lost in ‘higher’ Diptera such as Drosophila and define their roles in circadian, seasonal and life cycle phenotypes. We will take advantage of our recent genomic and transcriptomic data from the linden bug (Pyrrhocoris apterus),from which we have identified neuropeptide candidates present in basal insect lineages but lost in the fruit fly. Wewill further identify and validate these Heteroptera-specific neuropeptides. We will localize these candidates in brains using in situ hybridization and/or immunohistochemistry (in collaboration with Pyza, JU). Any role in major physiological processes, such as circadianrhythms, reproduction, diapause, mating and the life cycle will be tested using well-established RNA interference. We foresee in-depth analysis of two to three such neuropeptides with potential to reveal important novel insight into insect circadian and seasonal physiology.
Supervisor(s):
Dr. David Doležel
co-supervisor: Dr. Vlastimil Smýkal
Planned secondment(s):
UNI KASSEL, Prof. Monika Stengl, to learn techniques for primary cell cultures;
Jagiellonian University, Prof. Ela Pyza, to perform immunohistochemical experiments.
WP2 (DC2; BCAS) / New and optimized reverse genetic tools for diapause research in non-model insects
Objectives: To develop more efficient methods for gene silencing and gene overexpression in insects. While CRISPR/Cas9 is
an extremely versatile method for gene modification that should in principle work in all organisms, the efficient delivery of gRNA/Cas9 complex is species-specific and requires species-tailored protocols for embryo microinjection. Therefore, many groups of organisms, including insect pests and species established as models in physiology, are not practically accessible to reverse genetics.
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To solve this problem, we will test and optimize recently developed maternal gRNA/Cas9 delivery methods in the linden bug, where gene editing by embryonic gRNA/Cas9 delivery resulted in a robust phenotype. In a parallel research direction, we will explore the use of viral systems, i.e., Negev virus species that we have recently identified in the linden bug and test its expression efficiency using EGFP. The next steps will address if the system, primarily tested on the linden bugs, is accessible to selected species of our consortium (pea aphids, bumblebees, BSF, Musca). Finally, we will overexpress either dominant-negative forms of clock proteins or dsRNA in selected species and test the involvement of the circadian clock in diapause regulation and seasonality.
Supervisor(s):
Dr. Vlastimil Smýkal
co-supervisor: Dr. David Doležel
Planned secondment(s):
University Valencia, Prof. Martines-Torres to learn aphid biology
University of Leicester, Prof Rosato, to learn the biology of the black soldier fly
WP2 (DC3; UWUERZ) / Clock, photoperiodic and neuroendocrine control of fly body size.
Objectives: The aim of the project is to characterise the contribution of growth-related neuroendocrine pathways to the effect of photoperiod on developmental time and body weight. Developmental time and final weight are important parameters for livestock production. In insects, developmental time and final body weight are tightly linked and are under control of two major neuroendocrine pathways: insulin- and prothoracicotropic hormone (PTTH) signalling. Signals from these pathways are integrated in the prothoracic gland (PG) and modulate the production of the key developmental hormone, the steroid ecdysone. In insects (including Drosophila), the photoperiod during development has a strong influence on developmental time and body weight. The underlying neuroendocrine and physiological mechanisms are, however, unclear.
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We have previously shown for pharate Drosophila that PTTH neurons provide a link between the central clock in the brain, and the peripheral clock in the PG. Furthermore, at least in Drosophila, the PTTH neurons likely receive light input via circadian clock neurons. We will 1) use established genetic tools to characterise the role and contribution of insulin and PTTH signalling to photoperiod-dependent developmental time and body weight, and 2) measure the effect on carbohydrate and lipid content. Previously, we have shown that light as well as the circadian clock affect the temporal daily pattern of lipid metabolites in Drosophila, and found that PTTH and likely insulin-signalling is under clock control. Therefore, we will 3) characterise the effect of different photoperiods on molecular clock gene oscillations in growth-relevant and metabolically important peripheral clocks (PG, fat body, gut) in intact larvae and pupae using recently developed bioluminescence reporters. Our results will produce a model of how photoperiod -via different body clocks and neuroendocrine signalling- affects developmental time and body weight. In a next step, this model can be tested by pharmacological manipulations (e.g. injection of hormonal agonists/antagonists) in larger flies (Musca, BSF).
Supervisor(s):
Prof. Christian Wegener
Planned secondment(s):
Nasekomo and/or Amusca to learn methods and research needs of BSF/housefly biology & rearing.
University of Münster, Prof. Ralf Stanewsky to establish real-time luciferase imaging in peripheral metabolic clocks.
WP2 (DC5; JU) / Circadian plasticity of synapses in the visual system of insects
Objectives: The aim of this project is to study circadian plasticity of synapses in the visual system of insects and the effect of different types of synapses on behaviour (locomotor activity and sleep) and diapause. The visual system of insects consists of the retina and three optic neuropils (lamina, medulla and lobula) of the brain. In flies, butterflies, and beetles there is also an additional neuropil called the lobula plate. The retina receives and transmits photic and visual information to the brain through tetrad synapses which are formed in the lamina between R1-R6 photoreceptor terminals and four postsynaptic cells, including L1, L2 interneurons and glial cells.
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The number of tetrad synapses oscillates during the day in light/dark conditions (LD12:12) with two peaks, in the morning and in the evening. The morning peak is mainly regulated by light and the evening one by the circadian clock. Both peaks in the number of synapses are correlated with the peaks of high locomotor activity. Beside tetrad synapses, feedback synapses formed between L2 monopolar cells back onto the photoreceptor terminals oscillate during the day with a peak during the night, but their number does not change after the exposure to light. The mechanisms of circadian plasticity of tetrad and feedback synapses are unknown. In the project we will study: 1) the effect of feedback synapses on circadian plasticity of tetrad synapses and on behaviour (locomotor activity and sleep) 2) genes and proteins involved in the circadian plasticity 3) the effects of disrupting tetrad and feedback synapses on each other, on the circadian clock and behaviour. 4) circadian plasticity of synapses in the visual system of two lepidopteran models, day active Pieris brassicae and night active Autographa gamma, and explore the role of synaptic plasticity in the regulation of diapause in long and short photoperiods.
Supervisor(s):
Prof E. Pyza
Planned secondment(s):
University of Groningen, The Netherlands, Dr. Casper van der Kooi, the biology of Autographa gamma and Pieris brassicae.
WP2 (DC7; RUG) / Clock entrainment is well defined in established insect models, but much less clear in non-model insects, including pests.
Objectives: TBA
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TBA
Supervisor(s):
Prof. Roelof Hut
Dr. Casper van der Kooi
Planned secondment(s):
TBA
WP2 (DC10; CNRS) / Analysis of natural variants to identify novel clock/sleep genes in Drosophila
Objectives: To identify and study neurogenetically, two novel naturally occurring variants that affect sleep-wake rhythms in Drosophila. Natural strains of D. melanogaster have been used to generate isogenic lines whose genome has been sequenced and are available for phenotypic analysis. We have tested 170 lines for sleep-wake cycles in different light-dark conditions (light quality, photoperiod). Several lines that show specific defects of synchronization of the sleep-wake cycle have been isolated
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Behavioural analysis will be extended to new environmental conditions. Genetic mapping will be performed using markers/deletions and complemented by the use of mutants, interfering RNA or CRISPR to identify the candidate loci. In parallel to the search for the candidate genes, the molecular correlates of these behavioural defects will be sought by analysing the expression of clock genes under the same environmental conditions that revealed the sleep-wake phenotype. The insect sleep-wake cycle is an important fitness character because disruptions impact negatively on behaviour, metabolism and longevity. Consequently, using Drosophila as the genetic model will serve to identify the homologous sleep- wake (circadian) genes in the agriculturally/industrially-relevant insects also studied in this consortium and which may impact on the life cycle or longevity.
Supervisor(s):
Dr. Francois Rouyer
Planned secondment(s):
University of Münster, Prof. Ralf Stanewsky for experiments with luciferase reporters
WP2 (DC12; UH) / Unraveling the Influence of the Gut Microbiome on Insect Seasonal and Developmental Timing
Objectives: To identify changes in gut microbiome composition accompanying the transition from reproductive to non-reproductive states in insects, while also assessing their causal role. The gut microbiome has been shown to play a pivotal role in processes such as metabolism, immunity, and brain function. Furthermore, it is essential for circadian rhythmicity and establishing diurnal preference. Recent studies have suggested the importance of microbiome diversity in driving seasonal responses, motivating our investigation into the gut microbiomes of three distinct insect species: the black soldier fly (BSF, Hermetia illucens), the fruit fly (Drosophila melanogaster), and the house fly (Musca domestica).
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Using fecal 16S rRNA sequencing, we will characterize the microbiome composition during diapause and non-diapause states, identifying significantly enriched bacterial species in each state. Our methodology involves the use of germ-free, axenic insects, generated through a published protocol, to explore the impact of specific bacteria on diapause response. The top ten bacterial species that differ between reproductive states will be tested. Insects, subjected to either diapause or non-diapause conditions, will be fed a medium spiked with bacteria enriched in the microbiome of the opposite reproductive state. Control groups, not exposed to these specific bacterial species, will also be included. Through these experiments, we aim to establish the causal role of the microbiome in seasonal responses.
To investigate the microbiome’s impact on the circadian system in the gut, immunocytochemistry (ICC) will be employed to assess circadian system function in insects carrying various exogenous bacteria.
Supervisor(s):
Prof. Eran Tauber
Planned secondment(s):
Amusca
Nasekomo
Jagellonian University, Poland Prof. Elzbieta Pyza to perform ICC experiments
WP2 (DC13; UNI KASSEL) / Plasticity of the neuropeptidergic circadian clock in the cockroach: adjustment to different photoperiods
Objectives: Characterization of cellular andmolecular mechanisms of circadian clock plasticity in adjusting to
environmental rhythms at different temperature ranges using the cockroach model. Highly adaptive cockroaches spread readily into different climate zones all over the world providing threats as agricultural pests and as disease vectors. The Madeira cockroach Rhyparobia (Leucophaea) maderae is an established model system in chronobiology, especially suited to cellular and behavioral analysis due to its large neurons, its longevity, and stable behavioral rhythms. The cockroach circadian clock controlling sleep-wake cycles is identified and its brain circuits are currently functionally characterized by combining
transcriptomics, RNAi, electrophysiology, mass spectrometry, Ca2+ imaging combined with pharmacology in vivo and in vitro, next to immunocytochemistry and behavioral analysis.
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We will search for the limits of behavioral/physiological adaptation to different photoperiods, challenged with light pollution and extreme temperature changes, as occurs with anthropomorphic environmental interventions. In a combination of the respective experimental techniques established in our lab we will focus on the pigment-dispersing factor (PDF)-expressing circadian clock neurons in clock circuits that control sleep-wake cycles. We will examine how different photoperiods change sleep-wake behavior and its underlying neuronal network of PDF neurons, focusing on their responsiveness to the neuroactive substances of circadian light entrainment pathways, such as GABA and neuropeptides. With RNAi-dependent knockdown of circadian clock genes we will probe how the molecular circadian clockwork affects the clock´s cellular and molecular plasticity in response to different photoperiods at different levels of complexity. We expect that light as well as temperature will directly affect PDF precursor transcription allowing for cellular plasticity of the PDF-expressing neuronal network of the cockroach circadian clock.
Supervisor(s):
Prof. Dr. Monika Stengl
Prof. Dr. Suzanne Neupert
Planned secondment(s):