Genome editing retraces the evolution of toxin resistance in the monarch butterfly.

Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness1. Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits2-4, and new genome editing technology is facilitating functional validation of these mutations in whole organisms1,5. We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins6-11. Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na+/K+-ATPase)7-11, the physiological target of cardiac glycosides12. Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization13,14. We then performed CRISPR-Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage11,15. We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides16, culminating in triple mutant 'monarch flies' that were as insensitive to cardiac glycosides as monarch butterflies. 'Monarch flies' retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators17-19. The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis13,14,20-22. Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities2,6-11,15,17-19.

Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae.

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.

Gap Junction-Mediated Signaling from Motor Neurons Regulates Motor Generation in the Central Circuits of Larval Drosophila.

In this study, we used the peristaltic crawling of Drosophila larvae as a model to study how motor patterns are regulated by central circuits. We built an experimental system that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). We then investigated the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 in a middle segment (A4, A5, or A6), but not other segments, dramatically decreased the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 in a posterior segment (A6 or A7) increased the frequency of the motor waves. Since peripheral nerves mediating sensory feedback were severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. Our results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation were lost in shaking-B2 (shakB2 ) or ogre2 , gap-junction mutations in Drosophila, or upon acute application of the gap junction blocker carbenoxolone, implicating electrical synapses in the signaling from MNs. Cell-type-specific RNAi suggested shakB and ogre function in MNs and interneurons, respectively, during the signaling. Our results not only reveal an unexpected role for MNs in motor pattern regulation, but also introduce a powerful experimental system that enables examination of the input-output relationship among the component neurons in this system.SIGNIFICANCE STATEMENT Motor neurons are generally considered passive players in motor pattern generation, simply relaying information from upstream interneuronal circuits to the target muscles. This study shows instead that MNs play active roles in the control of motor generation by conveying information via gap junctions to the central pattern-generating circuits in larval Drosophila, providing novel insights into motor circuit control. The experimental system introduced in this study also presents a new approach for studying intersegmentally coordinated locomotion. Unlike traditional electrophysiology methods, this system enables the simultaneous recording and manipulation of populations of neurons that are genetically specified and span multiple segments.

Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genomic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families-genes directly mediating interactions with plant chemical defenses-underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many insect lineages are ancient (>150 million years ago (mya)), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several nonherbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza has among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant-binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on living plants (bitter or electrophilic phytotoxins) or their ancestral diet (fermenting plant volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight gene candidates that have also been linked to other dietary transitions in Drosophila.

Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genetic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families - genes directly mediating interactions with plant chemical defenses - underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many lineages are ancient (>150 million years ago [mya]), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several non-herbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza have among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on plants (bitter or electrophilic phytotoxins) or their ancestral diet (yeast and fruit volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight strong gene candidates that have also been linked to other dietary transitions in Drosophila .

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Highly contiguous assemblies of 101 drosophilid genomes.

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

Drosophila: Where the Wild Flies Are.

Drosophila melanogaster is a human commensal and dietary generalist. A new study in its ancestral range in Africa finds that wild Drosophila melanogaster are specialists on marula fruit - fruits cached in caves by Pleistocene humans.

Rate control of cell sheet recovery by incorporating hydrophilic pattern in thermoresponsive cell culture dish.

Thready stripe-polyacrylamide (PAAm) pattern was fabricated on a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) surface, and their surface properties were characterized. A PIPAAm surface spin-coated with positive photoresist was irradiated through a 5 µm/5 µm or a 10 µm/10-µm black and white striped photomask, resulting in the radical polymerization of AAm on the photoirradiated area. After staining with Alexa488 bovine serum albumin, the stripe-patterned surface was clearly observed and the patterned surface was also observed by a phase contrast image of an atomic force microscope. NIH-3T3 (3T3) single cells were able to be cultured at 37°C on the patterned surfaces as well as on a PIPAAm surface without pattern, and the detachment of adhered cells was more rapidly from the patterned surface after reducing temperature. Furthermore, the rate of detachment of 3T3 confluent cell sheet on the patterned surface was accelerated, compared with on a conventional PIPAAm surface under the static condition. The rate control of cell sheet recovery should contribute the preservations of cell phenotype and biological functions of cell sheet for applying to clinical trials.

Optogenetic perturbation of neural activity with laser illumination in semi-intact drosophila larvae in motion.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits(1-6). Application of optogenetics(7,8) in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways(9-13). Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system(14,15). In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin(16,17) or halorhodopsin(18-20), respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optical dissection of neural circuits responsible for Drosophila larval locomotion with halorhodopsin.

Halorhodopsin (NpHR), a light-driven microbial chloride pump, enables silencing of neuronal function with superb temporal and spatial resolution. Here, we generated a transgenic line of Drosophila that drives expression of NpHR under control of the Gal4/UAS system. Then, we used it to dissect the functional properties of neural circuits that regulate larval peristalsis, a continuous wave of muscular contraction from posterior to anterior segments. We first demonstrate the effectiveness of NpHR by showing that global and continuous NpHR-mediated optical inhibition of motor neurons or sensory feedback neurons induce the same behavioral responses in crawling larvae to those elicited when the function of these neurons are inhibited by Shibire(ts), namely complete paralyses or slowed locomotion, respectively. We then applied transient and/or focused light stimuli to inhibit the activity of motor neurons in a more temporally and spatially restricted manner and studied the effects of the optical inhibition on peristalsis. When a brief light stimulus (1-10 sec) was applied to a crawling larva, the wave of muscular contraction stopped transiently but resumed from the halted position when the light was turned off. Similarly, when a focused light stimulus was applied to inhibit motor neurons in one or a few segments which were about to be activated in a dissected larva undergoing fictive locomotion, the propagation of muscular constriction paused during the light stimulus but resumed from the halted position when the inhibition (>5 sec) was removed. These results suggest that (1) Firing of motor neurons at the forefront of the wave is required for the wave to proceed to more anterior segments, and (2) The information about the phase of the wave, namely which segment is active at a given time, can be memorized in the neural circuits for several seconds.