WP4 (chronobiology and food security)

Induction of the sexual phase in aphids can be regarded as a diapause induction, since the result of sexual reproduction is the production of cold resistant eggs, that are able to overcome the harsh winter conditions. How the induction of the sexual phase is controlled at the molecular and cellular levels in aphids is mostly unknown, as it is the molecular processes governing the induction of diapause in insects, in general. Thus, learning how aphids regulate diapause at the molecular level will provide new elements to consider in the design of pest management strategies, not only of aphids but also on other pest or vector species. Previous work by our group in A. pisum revealed that key circadian clock gene expression was modified by changes in photoperiod, suggesting clock genes are candidates in the gene network controlling aphid diapause and life cycles. However, no direct evidence of their involvement in the control of the aphid life cycle has been attained yet. To bridge this important gap in knowledge, we will optimize Virus Induced Gene Silencing (VIGS) and establish CRISPR/DIPA-CRISPR methodologies for circadian gene editing of A. pisum. Our previous analyses of clock and diapause-relevant neuropeptides suggest genes timeless, period, cryptochrome 1, ilp4 and Pdf as good candidates for manipulation. To assess the impact of these genetic manipulations, we will analyze the reproductive phenotype and the expression of other selected genes by means of RT-qPCR and/or by immunohistochemistry.

Prof. David Martinez-Torres

Biology Centre, Ceske Budejovice, Czech Republic, Dr. David Doležel/Dr. Vlastimil Smýkal to learn mutagenesis for non-model insects

Similarities in locomotor behaviour between housefly and D. melanogaster have been observed but with differences in the underlying molecular regulation of clock genes. These studies focused on individuals, but social context, such as population density and sex ratio can affect activity patterns. Thus, there is a need to study how temperature and light regimes affect activity patterns reproduction and growth under mass rearing conditions. In addition, selection lines of houseflies are currently being generated for commercial use that can grow efficiently on particular kinds ofwaste streams that are, for example, rich in starch or sugar. Such selection for nutrient conversion can affect gene expression and physiology/ metabolism of flies, which in turn will affect behaviour. The interaction between food composition and the circadian clockrequires further investigation in the mass rearing context. We will first establish basic properties of circadian light entrainment in houseflies for larvae and adults. The circadian system of adults entrains to light-dark cyclesbut as larvae live within their feeding substrate, they may be less sensitive to light than adults who rely on visual cues for mating and oviposition. We will measure circadian clock gene transcription in response to light and temperature stimulation in both developmental stages. Photoperiod is expected to influence larval growth and adult reproduction. We will test whether shortened or lengthened light-dark cycles, as well as constant light, affect larval growth, to find the optimal combination of temperature and photoperiod for mass rearing. We will also test whether particular light wavelengths and intensities can improve larval and adult performance under high densities. Finally, as flies of different geographical origin may be adapted to local conditions, we will investigate populations from various latitudes for photoperiodic response and timing of growth and reproduction with the aim to develop optimal mass rearing conditions. We shall be closely interacting with DC7 (ULEIC) who will work on the BSF and share information and ideas.

Prof. Leo Beukeboom

Prof.  Bregje Wertheim

Prof. Jean-Christophe Billeter

AMUSCA

Jansen, to learn the practicalities of insect mass culturing;

University of Leicester, UK, Prof. Charalambos Kyriacou and Prof. Ezio Rosato for clock gene expression studies and results comparison with BSF

Our specific objectives are: 1) Establish a Radio Frequency Identification Tags (RFID) system which allows high resolution tracking of individuals freely foraging bumble bees in the field. This system will complement lab-based systems already established in our lab. 2) We will establish an in vitro system for artificial rearing of bumble bees that will be used to rear embryos subjected to DNA editing. 3) We will develop RNAi and CRISPR/Cas9 DNA editing protocols to knockdown/knockout clock genes in the bumble bee Bombus terrestris. 4) We will use our CRISPR/Cas9 protocol to edit clock genes in the haploid bumble bee male embryos, and characterize their circadian behaviour when reaching the adult stage. 5) We will use immunocytochemical (ICC) and molecular approaches to study the influence of clock gene mutations on the organization and function of the molecular clockwork. 6) We will use genetic crossing to generate clock mutant queens. We will study time memory and other circadian behaviours in mutant offspring workers of these queens. Additional secondments are planned to BCAS to learn mutagenesis in non-model insects and UNI KASSEL for ICC experiments.

Prof. Guy Bloch

Biology Centre, Ceske Budejovice, Czech Republic, Dr. David Doležel to learn CRISPR

University Kassel, Germany, Prof. Monika Stengl to perform ICC experiments

We will uncover the female and male-released pheromonal cues in Spodoptera and Drosophila, and characterize their effect on the clock. We will investigate the impact of chemical communication on a multitude of rhythmic outputs including calling (pheromone release), oviposition, and larval hatching, and determine how internal state (e.g., mated vs. non-mated females) gates the access of specific chemosensory cues to the clock. The location of the central clock in the moth brain will be defined through immunocytochemistry and FISH, and the neuroanatomical pathways that link the higher-order chemosensory neurons with the central clock will be mapped. Taking advantage of neural activity-induced immediate-early gene expression (e.g., hr38/Nr4a1) we will define when during the day and how chemical cues change the neurophysiological activity of the central clock neurons. We will employ comparative neural circuit analysis to reveal what makes the moth clock exquisitely sensitive to pheromones while the fruitfly clock supposedly displays relative recalcitrance to chemosensory entrainment.

Dr. Abhishek Chaterjee

University of Münster, Germany, Prof. Ralf Stanewsky, The PhD student will focus on clock cell-specific bioluminescence imaging in Drosophila to reveal if specific nodes in the clock neuronal network are the preferential targets of pheromone-mediated social entrainment.

We will study 1) Can the circadian environment enhance the fitness and health of the BSF? 2) Can we use the circadian environment to synchronise the pupation stage and emergence? 3) Is there is a seasonal factor in the life cycle of the BSF even under controlled conditions which affects industrial productivity. Can we smooth this out by modulating the circadian/seasonal environment? 4) What is the diapausing stage(s) for this insect? 5) Can we shorten the BSF life cycle by manipulating its circadian clock? 6) When during the day/night do adult flies mate and when do females oviposit? By applying our circadian/seasonal expertise we will be able to solve these issues and build a basic understanding of the circadian and seasonal biology of this extraordinarily useful insect. We know from our own studies in Drosophilaand from others in the melon flythat mutations in clock genes such as period can shorten the generation time so a CRISPR/Cas9 gene editing approach will also be applied to period (the sequence is known from the BSF genome) to examine whether we can do the same for the BSF. This will require a careful cost/benefit analysis because shortening the life cycle may lead to smaller flies and this might lead to smaller larvae (larvae produce the protein), so the costs may outweigh the benefits of a faster throughput. Nevertheless, it is a very interesting developmental question and will add a molecular dimension to DC17s research project. Any results we obtain will be fed back to our APs immediately once they have beenvalidated and the APs will be free to implement these in their production lines.

Prof. Charalambos Kyriacou

Nasekomo and Freeze-M to learn the basic biology of BSF

RUG & AMUSCA to compare housefly and BSF biology and production lines

1. We will build genetic tools in BSFs, adding to the toolkit already available. Specifically, we will use existing techniques in BSFs (such as the PiggyBac transposase system) to develop a more efficient and site-specific transgenic method based on the Phi31C integrase. We will use such a method to establish bipartite (GAL4/UAS, LexA/LexAop, and QF/QUAS) expression systems and to optimise protocols for gene editing (for instance, by producing lines expressing CAS9 either constitutively or after induction).  2. We will investigate the interplay between daily timing, circadian rhythmicity, and seasonality with the physiology of different developmental stages and tissues. The goal is to identify the conditions for achieving the highest yield of expressed proteins with the lowest impact on development and metabolism. We will explore the production of both foreign proteins, such as nanobodies (used in research, diagnostic or treatment), and endogenous enzymes (such as lipases, amylases and proteases that have potential industrial applications). For the former we will overexpress tagged variants using the bipartite systems we will build. We will identify by Western Blot the combinations of tissues, developmental stages, and environmental conditions that work best. We will use chromatography approaches for protein purification. To predict which developmental/environmental conditions and tissues are optimal for the expression of endogenous enzymes, we will use RNAseq. Using genome editing we will add tags to the most promising enzymes to validate their expression by Western Blot and for purification. This approach is distinct from and complementary to that carried out by DC16, which is focused on overall physiology, growth and behavior.

Prof. Ezio Rosato

Biology Centre, Ceske Budejovice, Czech Republic, Dr. David Doležell to learn transformation in non-model insects

Nasekomo to learn intensive industrial rearing of BSFs