Odorant mixtures elicit less variable and faster responses than pure odorants.

In natural environments, odors are typically mixtures of several different chemical compounds. However, the implications of mixtures for odor processing have not been fully investigated. We have extended a standard olfactory receptor model to mixtures and found through its mathematical analysis that odorant-evoked activity patterns are more stable across concentrations and first-spike latencies of receptor neurons are shorter for mixtures than for pure odorants. Shorter first-spike latencies arise from the nonlinear dependence of binding rate on odorant concentration, commonly described by the Hill coefficient, while the more stable activity patterns result from the competition between different ligands for receptor sites. These results are consistent with observations from numerical simulations and physiological recordings in the olfactory system of insects. Our results suggest that mixtures allow faster and more reliable olfactory coding, which could be one of the reasons why animals often use mixtures in chemical signaling.

Hemocyte Nuclei Isolation from Adult Drosophila melanogaster for snRNA-seq.

In adult Drosophila, most of the hemocytes are macrophage-like cells (so called plasmatocytes), which serve various functions in organ homeostasis and immune defense. Ontogeny and functions are largely conserved between vertebrate and invertebrate macrophages. Hence, Drosophila offers a powerful genetic toolbox to study macrophage function and genetically modulate these cells. Technological advances in high-throughput sequencing approaches allowed to give an in-depth characterization of vertebrate macrophage populations and their heterogenous composition within different organs as well as changes in disease. Embryonic and larval hemocytes in Drosophila have been recently analyzed in single-cell RNA-sequencing (scRNA-seq) approaches during infection and steady state. These analyses revealed anatomical and functional Drosophila hemocyte subtypes dedicated to specific tasks. Only recently, the Fly Cell Atlas provided a whole transcriptomic single-cell atlas via single-nuclei RNA-sequencing (snRNA-seq) of adult Drosophila including many different tissues and cell types where hemocytes were also included. Yet, a specific protocol to isolate nuclei from adult hemocytes for snRNA-seq and study these cells in different experimental conditions was not available. In this chapter, we give a detailed protocol to purify hemocyte nuclei from adult Drosophila, which can be used in subsequent analyses such as snRNA-seq.

Detection of G-Quadruplex DNA Structures in Macrophages.

In addition to the canonical B-DNA conformation, DNA can fold into different secondary structures. Among them are G-quadruplex structures (G4s). G4 structures are very stable and can fold in specific guanine-rich regions in DNA and RNA. Different in silico, in vitro, and in cellulo experiments have shown that G4 structures form so far in all tested organisms. There are over 700,000 predicted G4s in higher eukaryotes, but it is so far assumed that not all will form at the same time. Their formation is dynamically regulated by proteins and is cell type-specific and even changes during the cell cycle or during different exogenous or endogenous stimuli (e.g., infection or developmental stages) can alter the G4 level. G4s have been shown to accumulate in cancer cells where they contribute to gene expression changes and the mutagenic burden of the tumor. Specific targeting of G4 structures to impact the expression of oncogenes is currently discussed as an anti-cancer treatment. In a tumor microenvironment, not only the tumor cells will be targeted by G4 stabilization but also immune cells such as macrophages. Although G4s were detected in multiple organisms and different cell types, only little is known about their role in immune cells. Here, we provide a detailed protocol to detect G4 formation in the nucleus of macrophages of vertebrates and invertebrates by microscopic imaging.

DNA damage signaling in Drosophila macrophages modulates systemic cytokine levels in response to oxidative stress.

Environmental factors, infection, or injury can cause oxidative stress in diverse tissues and loss of tissue homeostasis. Effective stress response cascades, conserved from invertebrates to mammals, ensure reestablishment of homeostasis and tissue repair. Hemocytes, the Drosophila blood-like cells, rapidly respond to oxidative stress by immune activation. However, the precise signals how they sense oxidative stress and integrate these signals to modulate and balance the response to oxidative stress in the adult fly are ill-defined. Furthermore, hemocyte diversification was not explored yet on oxidative stress. Here, we employed high-throughput single nuclei RNA-sequencing to explore hemocytes and other cell types, such as fat body, during oxidative stress in the adult fly. We identified distinct cellular responder states in plasmatocytes, the Drosophila macrophages, associated with immune response and metabolic activation upon oxidative stress. We further define oxidative stress-induced DNA damage signaling as a key sensor and a rate-limiting step in immune-activated plasmatocytes controlling JNK-mediated release of the pro-inflammatory cytokine unpaired-3. We subsequently tested the role of this specific immune activated cell stage during oxidative stress and found that inhibition of DNA damage signaling in plasmatocytes, as well as JNK or upd3 overactivation, result in a higher susceptibility to oxidative stress. Our findings uncover that a balanced composition and response of hemocyte subclusters is essential for the survival of adult Drosophila on oxidative stress by regulating systemic cytokine levels and cross-talk to other organs, such as the fat body, to control energy mobilization.

Muscle function and homeostasis require cytokine inhibition of AKT activity in Drosophila.

Unpaired ligands are secreted signals that act via a GP130-like receptor, domeless, to activate JAK/STAT signalling in Drosophila. Like many mammalian cytokines, unpaireds can be activated by infection and other stresses and can promote insulin resistance in target tissues. However, the importance of this effect in non-inflammatory physiology is unknown. Here, we identify a requirement for unpaired-JAK signalling as a metabolic regulator in healthy adult Drosophila muscle. Adult muscles show basal JAK-STAT signalling activity in the absence of any immune challenge. Plasmatocytes (Drosophila macrophages) are an important source of this tonic signal. Loss of the dome receptor on adult muscles significantly reduces lifespan and causes local and systemic metabolic pathology. These pathologies result from hyperactivation of AKT and consequent deregulation of metabolism. Thus, we identify a cytokine signal that must be received in muscle to control AKT activity and metabolic homeostasis.

Composition of agarose substrate affects behavioral output of Drosophila larvae.

In the last decade the Drosophila larva has evolved into a simple model organism offering the opportunity to integrate molecular genetics with systems neuroscience. This led to a detailed understanding of the neuronal networks for a number of sensory functions and behaviors including olfaction, vision, gustation and learning and memory. Typically, behavioral assays in use exploit simple Petri dish setups with either agarose or agar as a substrate. However, neither the quality nor the concentration of the substrate is generally standardized across these experiments and there is no data available on how larval behavior is affected by such different substrates. Here, we have investigated the effects of different agarose concentrations on several larval behaviors. We demonstrate that agarose concentration is an important parameter, which affects all behaviors tested: preference, feeding, learning and locomotion. Larvae can discriminate between different agarose concentrations, they feed differently on them, they can learn to associate an agarose concentration with an odor stimulus and change locomotion on a substrate of higher agarose concentration. Additionally, we have investigated the effect of agarose concentration on three quinine based behaviors: preference, feeding and learning. We show that in all cases examined the behavioral output changes in an agarose concentration-dependent manner. Our results suggest that comparisons between experiments performed on substrates differing in agarose concentration should be done with caution. It should be taken into consideration that the agarose concentration can affect the behavioral output and thereby the experimental outcomes per se potentially due to the initiation of an escape response or changes in foraging behavior on more rigid substrates.