The sex of organ geometry.

Organs have a distinctive yet often overlooked spatial arrangement in the body1-5. We propose that there is a logic to the shape of an organ and its proximity to its neighbours. Here, by using volumetric scans of many Drosophila melanogaster flies, we develop methods to quantify three-dimensional features of organ shape, position and interindividual variability. We find that both the shapes of organs and their relative arrangement are consistent yet differ between the sexes, and identify unexpected interorgan adjacencies and left-right organ asymmetries. Focusing on the intestine, which traverses the entire body, we investigate how sex differences in three-dimensional organ geometry arise. The configuration of the adult intestine is only partially determined by physical constraints imposed by adjacent organs; its sex-specific shape is actively maintained by mechanochemical crosstalk between gut muscles and vascular-like trachea. Indeed, sex-biased expression of a muscle-derived fibroblast growth factor-like ligand renders trachea sexually dimorphic. In turn, tracheal branches hold gut loops together into a male or female shape, with physiological consequences. Interorgan geometry represents a previously unrecognized level of biological complexity which might enable or confine communication across organs and could help explain sex or species differences in organ function.

Evolution of central neural circuits: state of the art and perspectives.

The wide variety of animal behaviours that can be observed today arose through the evolution of their underlying neural circuits. Advances in understanding the mechanisms through which neural circuits change over evolutionary timescales have lagged behind our knowledge of circuit function and development. This is particularly true for central neural circuits, which are experimentally less accessible than peripheral circuit elements. However, recent technological developments - including cross-species genetic modifications, connectomics and transcriptomics - have facilitated comparative neuroscience studies with a mechanistic outlook. These advances enable knowledge from two classically separate disciplines - neuroscience and evolutionary biology - to merge, accelerating our understanding of the principles of neural circuit evolution. Here we synthesize progress on this topic, focusing on three aspects of neural circuits that change over evolutionary time: synaptic connectivity, neuromodulation and neurons. By drawing examples from a wide variety of animal phyla, we reveal emerging principles of neural circuit evolution.

Tsetse flies (Glossina morsitans morsitans) choose birthing sites guided by substrate cues with no evidence for a role of pheromones.

Tsetse flies significantly impact public health and economic development in sub-Saharan African countries by transmitting the fatal disease African trypanosomiasis. Unusually, instead of laying eggs, tsetse birth a single larva that immediately burrows into the soil to pupate. Where the female chooses to larviposit is, therefore, crucial for offspring survival. Previous laboratory studies suggested that a putative larval pheromone, n-pentadecane, attracts gravid female Glossina morsitans morsitans to appropriate larviposition sites. However, this attraction could not be reproduced in field experiments. Here, we resolve this disparity by designing naturalistic laboratory experiments that closely mimic the physical characteristics found in the wild. We show that gravid G. m. morsitans were neither attracted to the putative pheromone nor, interestingly, to pupae placed in the soil. By contrast, females appear to choose larviposition sites based on environmental substrate cues. We conclude that, among the many cues that likely contribute to larviposition choice in nature, substrate features are a main determinant, while we failed to find evidence for a role of pheromones.

Leveraging open hardware to alleviate the burden of COVID-19 on global health systems.

With the current rapid spread of COVID-19, global health systems are increasingly overburdened by the sheer number of people that need diagnosis, isolation and treatment. Shortcomings are evident across the board, from staffing, facilities for rapid and reliable testing to availability of hospital beds and key medical-grade equipment. The scale and breadth of the problem calls for an equally substantive response not only from frontline workers such as medical staff and scientists, but from skilled members of the public who have the time, facilities and knowledge to meaningfully contribute to a consolidated global response. Here, we summarise community-driven approaches based on Free and Open Source scientific and medical Hardware (FOSH) as well as personal protective equipment (PPE) currently being developed and deployed to support the global response for COVID-19 prevention, patient treatment and diagnostics.

Olfactory receptor pseudo-pseudogenes.

Pseudogenes are generally considered to be non-functional DNA sequences that arise through nonsense or frame-shift mutations of protein-coding genes. Although certain pseudogene-derived RNAs have regulatory roles, and some pseudogene fragments are translated, no clear functions for pseudogene-derived proteins are known. Olfactory receptor families contain many pseudogenes, which reflect low selection pressures on loci no longer relevant to the fitness of a species. Here we report the characterization of a pseudogene in the chemosensory variant ionotropic glutamate receptor repertoire of Drosophila sechellia, an insect endemic to the Seychelles that feeds almost exclusively on the ripe fruit of Morinda citrifolia. This locus, D. sechellia Ir75a, bears a premature termination codon (PTC) that appears to be fixed in the population. However, D. sechellia Ir75a encodes a functional receptor, owing to efficient translational read-through of the PTC. Read-through is detected only in neurons and is independent of the type of termination codon, but depends on the sequence downstream of the PTC. Furthermore, although the intact Drosophila melanogaster Ir75a orthologue detects acetic acid-a chemical cue important for locating fermenting food found only at trace levels in Morinda fruit-D. sechellia Ir75a has evolved distinct odour-tuning properties through amino-acid changes in its ligand-binding domain. We identify functional PTC-containing loci within different olfactory receptor repertoires and species, suggesting that such 'pseudo-pseudogenes' could represent a widespread phenomenon.

The €100 lab: A 3D-printable open-source platform for fluorescence microscopy, optogenetics, and accurate temperature control during behaviour of zebrafish, Drosophila, and Caenorhabditis elegans.

Small, genetically tractable species such as larval zebrafish, Drosophila, or Caenorhabditis elegans have become key model organisms in modern neuroscience. In addition to their low maintenance costs and easy sharing of strains across labs, one key appeal is the possibility to monitor single or groups of animals in a behavioural arena while controlling the activity of select neurons using optogenetic or thermogenetic tools. However, the purchase of a commercial solution for these types of experiments, including an appropriate camera system as well as a controlled behavioural arena, can be costly. Here, we present a low-cost and modular open-source alternative called 'FlyPi'. Our design is based on a 3D-printed mainframe, a Raspberry Pi computer, and high-definition camera system as well as Arduino-based optical and thermal control circuits. Depending on the configuration, FlyPi can be assembled for well under €100 and features optional modules for light-emitting diode (LED)-based fluorescence microscopy and optogenetic stimulation as well as a Peltier-based temperature stimulator for thermogenetics. The complete version with all modules costs approximately €200 or substantially less if the user is prepared to 'shop around'. All functions of FlyPi can be controlled through a custom-written graphical user interface. To demonstrate FlyPi's capabilities, we present its use in a series of state-of-the-art neurogenetics experiments. In addition, we demonstrate FlyPi's utility as a medical diagnostic tool as well as a teaching aid at Neurogenetics courses held at several African universities. Taken together, the low cost and modular nature as well as fully open design of FlyPi make it a highly versatile tool in a range of applications, including the classroom, diagnostic centres, and research labs.

In vivo assembly and trafficking of olfactory Ionotropic Receptors.

BACKGROUND: Ionotropic receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors (iGluRs) that are expressed in diverse peripheral sensory neurons and function in olfaction, taste, hygrosensation and thermosensation. Analogous to the cell biological properties of their synaptic iGluR ancestors, IRs are thought to form heteromeric complexes that localise to the ciliated dendrites of sensory neurons. IR complexes are composed of selectively expressed 'tuning' receptors and one of two broadly expressed co-receptors (IR8a or IR25a). While the extracellular ligand-binding domain (LBD) of tuning IRs is likely to define the stimulus specificity of the complex, the role of this domain in co-receptors is unclear. RESULTS: We identify a sequence in the co-receptor LBD, the 'co-receptor extra loop' (CREL), which is conserved across IR8a and IR25a orthologues but not present in either tuning IRs or iGluRs. The CREL contains a single predicted N-glycosylation site, which we show bears a sugar modification in recombinantly expressed IR8a. Using the Drosophila olfactory system as an in vivo model, we find that a transgenically encoded IR8a mutant in which the CREL cannot be N-glycosylated is impaired in localisation to cilia in some, though not all, populations of sensory neurons expressing different tuning IRs. This defect can be complemented by the presence of endogenous wild-type IR8a, indicating that IR complexes contain at least two IR8a subunits and that this post-translational modification is dispensable for protein folding or complex assembly. Analysis of the subcellular distribution of the mutant protein suggests that its absence from sensory cilia is due to a failure in exit from the endoplasmic reticulum. Protein modelling and in vivo analysis of tuning IR and co-receptor subunit interactions by a fluorescent protein fragment complementation assay reveal that the CREL N-glycosylation site is likely to be located on the external face of a heterotetrameric IR complex. CONCLUSIONS: Our data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family.

Correction: open labware: 3-d printing your own lab equipment.

[This corrects the article DOI: 10.1371/journal.pbio.1002086.].

Open Labware: 3-D printing your own lab equipment.

The introduction of affordable, consumer-oriented 3-D printers is a milestone in the current "maker movement," which has been heralded as the next industrial revolution. Combined with free and open sharing of detailed design blueprints and accessible development tools, rapid prototypes of complex products can now be assembled in one's own garage--a game-changer reminiscent of the early days of personal computing. At the same time, 3-D printing has also allowed the scientific and engineering community to build the "little things" that help a lab get up and running much faster and easier than ever before.

Embryonic origin of olfactory circuitry in Drosophila: contact and activity-mediated interactions pattern connectivity in the antennal lobe.

Olfactory neuropiles across different phyla organize into glomerular structures where afferents from a single olfactory receptor class synapse with uniglomerular projecting interneurons. In adult Drosophila, olfactory projection interneurons, partially instructed by the larval olfactory system laid down during embryogenesis, pattern the developing antennal lobe prior to the ingrowth of afferents. In vertebrates it is the afferents that initiate and regulate the development of the first olfactory neuropile. Here we investigate for the first time the embryonic assembly of the Drosophila olfactory network. We use dye injection and genetic labelling to show that during embryogenesis, afferent ingrowth pioneers the development of the olfactory lobe. With a combination of laser ablation experiments and electrophysiological recording from living embryos, we show that olfactory lobe development depends sequentially on contact-mediated and activity-dependent interactions and reveal an unpredicted degree of similarity between the olfactory system development of vertebrates and that of the Drosophila embryo. Our electrophysiological investigation is also the first systematic study of the onset and developmental maturation of normal patterns of spontaneous activity in olfactory sensory neurons, and we uncover some of the mechanisms regulating its dynamics. We find that as development proceeds, activity patterns change, in a way that favours information transfer, and that this change is in part driven by the expression of olfactory receptors. Our findings show an unexpected similarity between the early development of olfactory networks in Drosophila and vertebrates and demonstrate developmental mechanisms that can lead to an improved coding capacity in olfactory neurons.

Neuroblast lineage-specific origin of the neurons of the Drosophila larval olfactory system.

The complete neuronal repertoire of the central brain of Drosophila originates from only approximately 100 pairs of neural stem cells, or neuroblasts. Each neuroblast produces a highly stereotyped lineage of neurons which innervate specific compartments of the brain. Neuroblasts undergo two rounds of mitotic activity: embryonic divisions produce lineages of primary neurons that build the larval nervous system; after a brief quiescence, the neuroblasts go through a second round of divisions in larval stage to produce secondary neurons which are integrated into the adult nervous system. Here we investigate the lineages that are associated with the larval antennal lobe, one of the most widely studied neuronal systems in fly. We find that the same five neuroblasts responsible for the adult antennal lobe also produce the antennal lobe of the larval brain. However, there are notable differences in the composition of larval (primary) lineages and their adult (secondary) counterparts. Significantly, in the adult, two lineages (lNB/BAlc and adNB/BAmv3) produce uniglomerular projection neurons connecting the antennal lobe with the mushroom body and lateral horn; another lineage, vNB/BAla1, generates multiglomerular neurons reaching the lateral horn directly. lNB/BAlc, as well as a fourth lineage, vlNB/BAla2, generate a diversity of local interneurons. We describe a fifth, previously unknown lineage, BAlp4, which connects the posterior part of the antennal lobe and the neighboring tritocerebrum (gustatory center) with a higher brain center located adjacent to the mushroom body. In the larva, only one of these lineages, adNB/BAmv3, generates all uniglomerular projection neurons. Also as in the adult, lNB/BAlc and vlNB/BAla2 produce local interneurons which, in terms of diversity in architecture and transmitter expression, resemble their adult counterparts. In addition, lineages lNB/BAlc and vNB/BAla1, as well as the newly described BAlp4, form numerous types of projection neurons which along the same major axon pathways (antennal tracts) used by the antennal projection neurons, but which form connections that include regions outside the "classical" olfactory circuit triad antennal lobe-mushroom body-lateral horn. Our work will benefit functional studies of the larval olfactory circuit, and shed light on the relationship between larval and adult neurons.

Bridging the Gap: establishing the necessary infrastructure and knowledge for teaching and research in neuroscience in Africa.

Advances in neuroscience research over the last few decades have increased our understanding of how individual neurons acquire their specific properties and assemble into complex circuits, and how these circuits are affected in disease. One of the important motives driving neuroscience research is the development of new scientific techniques and interdisciplinary cooperation. Compared to developed countries, many countries on the African continent are confronted with poor facilities, lack of funding or career development programs for neuroscientists, all of which deter young scientists from taking up neuroscience as a career choice. This article highlights some steps that are being taken to promote neuroscience education and research in Africa.

Divergent evolution of sleep in Drosophila species.

Living organisms synchronize their biological activities with the earth's rotation through the circadian clock, a molecular mechanism that regulates biology and behavior daily. This synchronization factually maximizes positive activities (e.g., social interactions, feeding) during safe periods, and minimizes exposure to dangers (e.g., predation, darkness) typically at night. Beyond basic circadian regulation, some behaviors like sleep have an additional layer of homeostatic control, ensuring those essential activities are fulfilled. While sleep is predominantly governed by the circadian clock, a secondary homeostatic regulator, though not well-understood, ensures adherence to necessary sleep amounts and hints at a fundamental biological function of sleep beyond simple energy conservation and safety. Here we explore sleep regulation across seven Drosophila species with diverse ecological niches, revealing that while circadian-driven sleep aspects are consistent, homeostatic regulation varies significantly. The findings suggest that in Drosophilids, sleep evolved primarily for circadian purposes. The more complex, homeostatically regulated functions of sleep appear to have evolved independently in a species-specific manner, and are not universally conserved. This laboratory model may reproduce and recapitulate primordial sleep evolution.

Sensory neuron population expansion enhances odor tracking without sensitizing projection neurons.

The evolutionary expansion of sensory neuron populations detecting important environmental cues is widespread, but functionally enigmatic. We investigated this phenomenon through comparison of homologous neural pathways of Drosophila melanogaster and its close relative Drosophila sechellia , an extreme specialist for Morinda citrifolia noni fruit. D. sechellia has evolved species-specific expansions in select, noni-detecting olfactory sensory neuron (OSN) populations, through multigenic changes. Activation and inhibition of defined proportions of neurons demonstrate that OSN population increases contribute to stronger, more persistent, noni-odor tracking behavior. These sensory neuron expansions result in increased synaptic connections with their projection neuron (PN) partners, which are conserved in number between species. Surprisingly, having more OSNs does not lead to greater odor-evoked PN sensitivity or reliability. Rather, pathways with increased sensory pooling exhibit reduced PN adaptation, likely through weakened lateral inhibition. Our work reveals an unexpected functional impact of sensory neuron expansions to explain ecologically-relevant, species-specific behavior.

Neuroscience: Hacking development to understand sensory discrimination.

Fine sensory discrimination abilities are enabled by specific neural circuit architectures. A new study reveals how manipulating particular network parameters in the fly's memory centre, the mushroom body, alters sensory coding and discrimination.

OptoPi: An open source flexible platform for the analysis of small animal behaviour.

Behaviour is the ultimate output of neural circuit computations, and therefore its analysis is a cornerstone of neuroscience research. However, every animal and experimental paradigm requires different illumination conditions to capture and, in some cases, manipulate specific behavioural features. This means that researchers often develop, from scratch, their own solutions and experimental set-ups. Here, we present OptoPi, an open source, affordable (∼ £600), behavioural arena with accompanying multi-animal tracking software. The system features highly customisable and reproducible visible and infrared illumination and allows for optogenetic stimulation. OptoPi acquires images using a Raspberry Pi camera, features motorised LED-based illumination, Arduino control, as well as irradiance monitoring to fine-tune illumination conditions with real time feedback. Our open-source software (BioImageProcessing) can be used to simultaneously track multiple unmarked animals both in on-line and off-line modes. We demonstrate the functionality of OptoPi by recording and tracking under different illumination conditions the spontaneous behaviour of larval zebrafish as well as adult Drosophila flies and their first instar larvae, an experimental animal that due to its small size and transparency has classically been hard to track. Further, we showcase OptoPi's optogenetic capabilities through a series of experiments using transgenic Drosophila larvae.

Cracking the encoding of human scent in the mosquito brain.

In a recent study, Zhao et al. decipher how the olfactory system encodes human versus animal odors in the mosquito Aedes aegypti. By combining genome engineering, invivo calcium imaging, advanced chemistry, and behavioral analysis, the authors provide compelling evidence that the discriminatory coding of host odors is surprisingly simple - and bridges labeled line with combinatorial coding.

Internal state affects local neuron function in an early sensory processing center to shape olfactory behavior in Drosophila larvae.

Crawling insects, when starved, tend to have fewer head wavings and travel in straighter tracks in search of food. We used the Drosophila melanogaster larva to investigate whether this flexibility in the insect's navigation strategy arises during early olfactory processing and, if so, how. We demonstrate a critical role for Keystone-LN, an inhibitory local neuron in the antennal lobe, in implementing head-sweep behavior. Keystone-LN responds to odor stimuli, and its inhibitory output is required for a larva to successfully navigate attractive and aversive odor gradients. We show that insulin signaling in Keystone-LN likely mediates the starvation-dependent changes in head-sweep magnitude, shaping the larva's odor-guided movement. Our findings demonstrate how flexibility in an insect's navigation strategy can arise from context-dependent modulation of inhibitory neurons in an early sensory processing center. They raise new questions about modulating a circuit's inhibitory output to implement changes in a goal-directed movement.

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals.

Alternative splicing increases neuronal transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. In nonvertebrate bilaterians, this splicing program is restricted to neurons by the posttranscriptional processing of the enhancer of microexons (eMIC) domain in Srrm234. In Drosophila, this processing is dependent on regulation by Elav/Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. We found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient posttranscriptional programs can evolve independently in different phyla to affect distinct cellular modules while maintaining cell-type specificity.

Why flies? Inexpensive public engagement exercises to explain the value of basic biomedical research on Drosophila melanogaster.

Invertebrate model organisms are powerful systems for uncovering conserved principles of animal biology. Despite widespread use in scientific communities, invertebrate research is often severely undervalued by laypeople. Here, we present a set of simple, inexpensive public outreach exercises aimed at explaining to the public why basic research on one particular invertebrate, the insect Drosophila melanogaster, is valuable. First, we designed seven teaching modules that highlight cutting-edge research in Drosophila genetics, metabolism, physiology, and behavior. We then implemented these exercises in a public outreach event that included both children and adults. Quantitative evaluation of participant feedback suggests that these exercises 1) teach principles of animal biology, 2) help laypeople better understand why researchers study fruit flies, and 3) are effective over a wide range of age groups. Overall, this work provides a blueprint for how to use Drosophila as a vehicle for increasing public awareness and appreciation of basic research on genetically tractable insects in particular and invertebrates in general.