Current Biology

When undisturbed, Drosophila larvae move forward through their environment with sweeping waves of caudal to rostral muscle contraction [1, 2]. In stark contrast, nociceptive sensory stimuli (such as attacks by parasitoid wasps) trigger the larvae to roll across the substrate by corkscrewing around the long body axis [3, 4]. While studies have described the motor pattern of larval crawling [1, 2], the motor pattern of larval rolling escape locomotion remains unknown. Here, we have determined this pattern. To do so, we developed a high speed confocal time-lapse imaging preparation that allowed us to trigger rolling with optogenetics while simultaneously imaging a genetically encoded calcium sensor that was expressed in the muscles. Of the 30 muscles present in each larval abdominal hemisegment we find that only 11 muscles are consistently and specifically activated across segments during rolling. 8 additional muscles are more sparsely activated. Importantly, the sequential pattern of muscle recruitment during rolling is completely distinct from that of forward or reverse crawling. We discover that a roll involves a wave of muscle activation that propagates around the larval circumference (in the transverse plane of each segment) and involves four coactive muscle groups. A pattern of activation progresses from coactive ventral muscle groups to dorsal groups and then spreads across the midline to the contralateral dorsal muscle groups which then progresses back to the ventral groups. Finally, the direction of a roll (either clockwise or counterclockwise around the body) is determined by the clockwise or counterclockwise order of muscle group activation around the transverse plane.

Perhaps nothing is stronger evidence of the importance of sleep than its conservation across animals [1], but the extent of its regulatory conservation is unknown. The upside-down jelly-fish Cassiopea xamachana sleeps [2], and this behavior is controlled by radially-spaced marginal ganglia. After defining a sleep-wake threshold, we compared gene expression profiles of ganglia from animals sleep-deprived for two nights and found differential expression in many sleep-related genes including GABAergic, melatonergic, and cholinergic receptors. We focused on a nicotinic acetylcholine receptor alpha subunit-like (Chrnal-E), based on its differential expression, and selected animals for a second round of RNAseq that included both light-based and mechanically-based sleep-deprivation. Combining datasets revealed a short list of differentially expressed genes, of which chrnal-E is the most recognizable and well-supported, so we investigated its potential role in sleep regulation. First, we found that chemical cholinergic neuromodulators positively regulate pacemaker activity. Then, we showed by in situ hybridization that chrnal-E is expressed primarily within the ganglia, and that the area of expression expands after sleep deprivation. Next, we developed RNAi for use in Cassiopea and determined that Chrnal-E promotes wakefulness. Finally, we sampled circadian timepoints in the field and found in control conditions, chrnal-E has lowest expression late at night, but in sleep deprived animals, chrnal-E peaks at this time, supporting a link to wakefulness. Our finding that Cassiopea sleep is regulated by the cholinergic system underscores that mechanisms of sleep conservation are deeply conserved in animal evolution.

Dragonflies represent an ancient lineage of visual predators, which last shared a common ancestor with insect groups such as dipteran flies in the early Devonian, 406 million years ago [1,2]. Despite their important evolutionary status, and recent interest in them as a model for complex visual physiology and behavior, the most recent detailed description of the dragonfly optic lobe is itself more than a century old [3]. Many insects process visual information in optic lobes comprising 4 sequential, retinotopically organized neuropils: the lamina, medulla, lobula and a posterior lobula plate devoted to processing information about wide-field motion stimuli [4, 5]. Recent reports suggest that the dragonflies also follow this basic plan, with a divided lobula similar to those of flies, moths and butterflies [6, 7]. Here we refute this claim, showing that dragonflies have an unprecedentedly complex lobula comprising at least 4 sequential synaptic neuropils, in addition to two lobula plate like structures located on opposite sides of the brain. The second and third optic ganglia contain approximately twice as many synaptic layers as any other insect group yet studied. Using intracellular recording and labeling of neurons we further show that the most anterior lobe contains wide-field motion processing tangential neurons similar to those of the posterior lobula plate of dipteran flies. In addition to describing what is probably the most complex and unique optic lobe of any insect to date, our findings provide interesting insights to understanding the evolution of the insect optic lobe and serve as a reminder that the highly studied visual circuits of dipteran flies represent just a single derived form of these brain structures.

Sleep in animals plays roles that appear specific to the brain, including synaptic homeostasis [1], neurotransmitter regulation [2], cellular repair [3], memory consolidation [4], and neural plasticity [5,6]. Would any of these functions of sleep be relevant to an animal without a brain? The upside-down jellyfish Cassiopea xamachana, like other cnidarians, lacks a centralized nervous system, yet the animal sleeps [7]. By tracking the propensity of the radially spaced ganglia to initiate muscle contractions over several days we determined how neural activity changes between sleep and wake in a decentralized nervous system. Ganglia-network sleep/ wake activity patterns range from being highly specialized to a few ganglia, to being completely unspecialized. Ganglia specialization also changes over time, indicating a high degree of plasticity in the neural network. The ganglia that lead activity can persist or switch between sleep/wake transitions, signifying a level of local control of the behavioral state in a decentralized nervous system. Following sleep deprivation, ganglia usage becomes far more sleep specialized, demonstrating reduced network plasticity. Together, these findings identify a novel behavioral control system that is decentralized and yet displays temporal specialization and centralization, and show a role for sleep in maintaining neural network plasticity, revealing a conserved function of sleep in this brain-less animal.

The discovery of place cells within the hippocampus has pointed to the importance of the hippocampus for navigation. The more recent discovery of hippocampal time cells has broadened the perspective of encoding in the hippocampus. An alternative hypothesis to the existence of time cells is based on the notion that hippocampal cells deduce location by integrating travelled distance ("path integration"). According to this alternate hypothesis, time cells, which fire at particular times when animals are running on a treadmill without changing location, actually encode accumulated distance on the treadmill. To examine this hypothesis, Kraus et al.1 performed treadmill experiments in which animals either ran for a fixed time or a fixed distance with varying velocities. Two distinct coding modes of hippocampal principal cells were found. Some cells encoded travelled distance and others elapsed time, thus refuting the notion that all hippocampal cells were performing path integration. Using the data from these experiments, we asked whether the two populations depended on the type of task the rats were engaged in. We show that the type of experiment determined the cells encoding, such that in fixed-distance experiments distance-encoding cells dominated, while on fixed-time experiments time-encoding cells dominated. These results suggest that the cells encoding contains a predictive element, dependent on the important variables of the experiment.

Visual navigation in ants has long been a focus of experimental study [1-3], but only recently have explicit hypotheses about the underlying neural circuitry been proposed [4]. Indirect evidence suggests the mushroom bodies (MB), a known site of olfactory learning [5-10], may also be the substrate for visual memory in navigation tasks [11-14]. Computational modelling shows that MB neural architecture could support this function [15, 16], though there is no direct evidence that ants require MBs for visual navigation. Here we show that lesions of MB calyces impair ants visual navigation to a remembered food location whilst leaving their innate responses to visual cues unaffected. Ants are innately attracted to a large visual cue but we trained them to locate a food source at a specific angle to this visual cue. Subsequent bilateral or unilateral lesioning (through procaine hydrochloride injection) of the MB calyces, caused ants to revert to their innate cue attraction whilst control (saline) injected ants still approached the feeder. The ants path straightness and walking speed were unaffected by lesions. Reversion towards the cue direction occurred irrespective of whether it was ipsi-or contralateral to the lesion site, showing this is not due simply to an induced motor bias. Monocular occlusion did not diminish ants ability to locate the feeder, suggesting the lesion is not merely interrupting visual input to the calyx. The demonstrated dissociation between innate and learnt visual responses provides direct evidence for a specific role of the MB in navigational memory.

Spiders can maintain a wide range of free-running periods while still being entrained to a 24-hour day. To investigate the underlying mechanism of this entrainment, we constructed the phase-response curve (PRC) for the orb weaver, Metazygia wittfeldae, by subjecting the spiders to one-hour light pulses at various times throughout the circadian day. The resulting type 0 PRC showed high amplitude (> 6 hour) phase advance and delays when the light pulse was applied during circadian time (CT) 16-18, with a break point of advances to delays at CT 17. We then investigated the genetic mechanism of the phase response to light by splitting M. wittfeldae adult females entrained to 12 hours light:12 hours dark (LD 12:12) into two groups. One group received a 1-hour light pulse 5 hours after lights off (CT17), and one group did not. We then sacrificed spiders for RNA isolations 1 and 10 hours after the light pulse. We identified numerous genes that were downregulated by the light pulse 1 hour after the pulse relative to no pulse group. Intriguingly, many of these genes had a flipped pattern of expression 9 hours later - the pulse group had higher expression than the no pulse group. This pattern is consistent with the shifted phase of locomotor activity expected after the light pulse application. We also identified clock gene homologs in M. wittfeldae that had distinct expression patterns from other arthropods.

Acoel worms belong to an enigmatic and understudied animal lineage in the phylum Xenacoelomorpha. Sparse taxonomic and histological work suggests that these worms exhibit a diversity of reproductive anatomies and likely a corresponding diversity in reproductive behavior. Here, we study the reproductive life history of the three-banded panther worm Hofstenia miamia, an acoel that is emerging as a lab-tractable model system. Using confocal microscopy and histology, we describe H. miamias reproductive organs, identifying structures previously unknown in acoels. Following a cohort of worms from zygote to adulthood, we quantify the developmental dynamics of their reproductive organs, and find that these organs emerge in a stereotyped sequence as a function of increasing body size. Studying the dynamics of organ growth and de-growth during regeneration and in starvation, we show that reproductive organs follow similar growth rules in these contexts, suggesting that they are regulated by a size-associated program in all growth contexts. Finally, we study egg-laying behavior, finding that H. miamia lay their eggs through their mouths after loading them into their pharynges. Worms lay eggs for multiple months after a single mating, suggesting long-term sperm storage despite lacking a storage organ; we also find that worms can lay viable eggs without mating, indicating a capacity for self-fertilization. Further, we show that worms assess their environment to make decisions about when and where to lay their eggs, and sometimes lay eggs in communal clutches. Together, our work establishes foundational knowledge to enable the experimental study of reproductive anatomy, physiology, and behavior in acoels.

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis, and thus provides a unique system to study the evolution and functional specificity of mitotic tubulins and the resulting spindle. Previous studies showed that Naegleria amoebae express a divergent -tubulin during mitosis and we now show that Naegleria amoebae express a second mitotic - and two mitotic {beta}-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical - and {beta}- tubulins, contain residues that suggest distinct microtubule properties, and may represent drug targets for the "brain-eating amoeba" Naegleria fowleri. Using quantitative light microscopy, we find that Naeglerias mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naeglerias spindle is twisted and its length increases during mitosis suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers, and secondary bundles function as bridging fibers.

Motion vision underpins a wide range of adaptive behaviours essential for individual and species survival. In hoverflies, some visual behaviours are sexually dimorphic, including for example male high-speed pursuit of conspecifics, matched by improved optics, and faster photoreceptors. Other visual behaviours are sexually monomorphic, with for example similar foraging flight speeds in male and female hoverflies. However, whether the descending neurons responsible for sensorimotor transformation of optic flow are sexually dimorphic is unknown. To address this, we combined morphological analysis with electrophysiology of optic flow sensitive descending neurons and compared neural responses to the behavioural output in tethered hoverflies. We found that while optomotor flight behaviour is largely sexually monomorphic, the underlying neural responses are sexually dimorphic, especially at higher optic flow velocities. Additionally, behavioural responses were noticeably slower than neural responses. Together, our findings uncover a nuanced, sex- and stimulus- dependant sensorimotor transformation, shaped by both neural architecture and behavioural demands.

While real-time vocal adjustments are crucial for interactive communication, not much is known about the spectral and temporal vocal flexibility of animals. Here, using a combination of field song recordings and controlled playback experiments, we show that wild nightingales imitate whistle song syllable durations in real time. However, when exposed to playbacks with variations in whistle pitch and duration that are beyond their natural range of vocalizations, nightingales emulated temporal or spectral features, approximating the whistle playbacks within the constraints of their natural whistle song repertoire. Our findings reveal a previously unknown dimension of real-time vocal flexibility in songbirds and suggest complex auditory-motor integration during song interactions.

Cellularization is the coordinated division of a multinucleate cytoplasm into many cells.1-3 Multinucleation is a common life cycle strategy observed across eukaryotic lineages, including in microbial eukaryotes, fungi, plants and animals, and is associated with the ability to transition to a unicellular state through cellularization.4 In the best-studied model for this process, Drosophila melanogaster, cellularization requires the coordinated action of actin and microtubule (MT) networks to bring about the synchronous invagination of plasma membrane furrows, but the extent of conservation of these mechanisms across eukaryotes remains unknown.1,5,6 Here we investigate cellularization in the ichthyosporean Sphaeroforma arctica, a close relative of animals with a multinucleate life cycle stage.7-9 Using live cell imaging, ultrastructure expansion microscopy (U-ExM) and volume electron microscopy, we define the membrane, MT and actin dynamics that accompany cellularization in S. arctica. Using pharmacological inhibitors and centrifugation, we show that MTs, in addition to positioning nuclei, play a role in guiding nascent furrows to sustain equi-partitioning of nuclei and cytoplasm between daughter cells. Our findings indicate that cellularization is regulated through crosstalk between actin and MT networks, exhibiting mechanistic parallels with canonical cytokinesis, and establish S. arctica as a valuable model for investigating general principles of cellularization.

Producing context-specific motor acts requires sensorimotor neural networks to integrate multiple sensory modalities. Some of this integration occurs via presynaptic interactions between proprioceptive afferent neurons themselves,1,2 other by afferents of different modalities targeting appropriate motor neurons (MNs).3-5 How the interneuronal network typically interposed between sensory afferents and MNs contributes to this integration, particularly at single-neuron resolution, is much less understood. In stick insects, this network contains nonspiking interneurons (NSIs) converging onto the posture-controlling slow extensor tibiae motor neuron (SETi). We analyzed how load altered movement signal processing by tracing the interaction of movement (femoral chordotonal organ, fCO) and load (tibial campaniform sensilla, tiCS) signals from the afferents through the NSI network to the motor output. On the afferent level, load reduced movement signal gain by presynaptic inhibition; tiCS stimulation elicited primary afferent depolarization and reduced fCO afferent action potential amplitude. In the NSI network, graded responses to movement and load inputs summed nonlinearly and increased the gain of NSIs opposing movement-induced reflexes. The gain of SETi and muscle movement reflex responses consequently decreased. Gain modulation was movement parameter-specific and required presynaptic inhibition; pharmacologically blocking presynaptic inhibition abolished load-dependent tuning of SETi responses. These data describe sensorimotor gain control at the sensory, premotor, and motor levels. Presynaptic inhibition-mediated nonlinear integration allowed the NSI network to respond to movement sensory input in a context (load)-dependent manner. These findings show how gain changes can allow premotor networks to integrate multiple sensory modalities and thus generate context-appropriate motor activity.

Many animal species are able to return to their nest after a foraging excursion without using familiar visual cues to guide them. They accomplish this by using a navigation competence known as path integration, which is vital in environments that do not have prominent visual features. To perform path integration, an animal maintains a running estimate of the distance and direction to its origin as it moves. This distance and direction estimate needs to be maintained in memory until the animal uses it to return to its nest. However, the neural substrate of this memory remains uncertain. A common hypothesis is that the information is maintained in a bump attractors state. We test the bump attractor hypothesis and find that its predictions are inconsistent with the path integration behaviour of ants, thus highlighting the need for alternative models of path integration memory.

Females control the paternity of their offspring by selectively mating with males they perceive to be of high quality. In species where females mate with multiple males in succession, females may bias offspring paternity by favoring the sperm of one male over another, a process known as cryptic female choice (CFC). While evidence of CFC exists in multiple taxa, the mechanisms underlying this process have remained difficult to unravel. Understanding CFC requires demonstration of a female-driven post mating bias in sperm use and paternity, and a causal link between this bias and male cues. Here, we show that in the vinegar fly Drosophila melanogaster, mated females eject the ejaculate of their first mate faster when exposed to the pheromones of an attractive male than in the presence of an unattractive one. Using transgenic males expressing fluorescent sperm, we show that exposure to attractive males between mating causes twice-mated females to bias sperm storage towards the second male, affecting paternity. Using pheromonal bioassays in combination with genetic manipulation of sensory systems, we show that females modulate ejaculate ejection latency in response to male pheromones heptanal and 11-cis-Vaccenyl acetate (cVA) sensed via olfactory receptor neurons OR35a, Or22a, Or65a and OR67d, demonstrating that polyandrous females use male pheromonal cues to modulate ejaculate ejection timing. We provide the first demonstration to our knowledge of a CFC mechanism allowing a female to increase or decrease the share of paternity of her first mate depending on the sensing of the quality of potential mates in her environment. These findings showcase that paternity can be influenced by events that go beyond copulation and highlights the importance of post-copulatory sexual selection. One Sentence SummaryWe show that females bias sperm use and paternity towards specific males in response to male 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 studies showed 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 characteristics found in the wild. We show that gravid tsetse were neither attracted to the putative pheromone nor, interestingly, to pupae placed in the soil. In contrast, females appear to choose larviposition sites based on environmental cues. We conclude that it is the substrate, rather than larval pheromones, which drives larviposition site selection under naturalistic conditions.

Organisms must focus their attention on ethologically relevant stimuli to maximise survival and reproduction. This is achieved, in part, through a process termed habituation, in which responses to repeated or constant sensory inputs are sequentially dampened, favouring robust responses to novel stimuli. Prior investigations in the fruit fly, Drosophila, have uncovered genes and neural mechanisms that facilitate habituation. However, these works have primarily focused on habituation to visual, olfactory, and gustatory, stimuli. In contrast, neurogenetic processes promoting habituation to mechanosensory stimuli have been less studied. Here we develop an automated system that yields long-term analogue measurements of mechanosensory habituation in adult fruit flies. Using this platform, we reveal a role for the neuronal calcium sensor Neurocalcin in habituation to mechanical stimuli. Loss of Neurocalcin disrupts nighttime but not daytime sleep, particularly under long-night conditions. Mimicking a method used to treat insomnia, we show that compressing nighttime duration restores consolidated night sleep in Neurocalcin mutants. Surprisingly, this manipulation also renormalises mechanosensory habituation in this background, suggesting a link between sleep and mechanosensory habituation. Indeed, we show that limiting night sleep duration in wild type flies similarly impairs habituation to mechanosensory stimuli. Collectively, our findings reveal a previously unappreciated link between nighttime sleep and mechanosensory habituation in Drosophila.

Butterflies have photoreceptor cells that are sensitive to the ultraviolet part of the spectrum due to ultraviolet-sensitive rhodopsin (UVRh), a gene that has been duplicated in the Heliconius genus. In individuals expressing UVRh1 and UVRh2, electrophysiological and behavioral studies demonstrate that these opsin proteins enable discrimination of UV wavelengths. This behavioral trait varies between species, being absent in H. melpomene and limited to females in H. erato. To identify the evolutionary origins of this trait, we first examined UV color vision in H. charithonia, a species related to H. erato in the sara/sapho group. We found that this species also has sexually dimorphic UV color vision. To identify the genetic basis of this trait, we built a reference-grade genome assembly of H. charithonia. We discovered that one duplicate, UVRh1, is present on the W chromosome, making it obligately female-specific. We employed gDNA PCR assays of UVRh1 across the Heliconius genus. In species with sexually dimorphic UVRh1 mRNA expression, UVRh1 gDNA is absent in males, whereas in species with sexually monomorphic UVRh1 mRNA expression, UVRh1 gDNA is found in both sexes. The presence or absence of male UVRh1 expression across the Heliconius phylogeny supports a model where sexual dimorphism was acquired early via movement of a gene duplication to the W-chromosome. We used CRISPR-Cas9 to engineer a deletion in the UVRh1 locus in female H. charithonia and use immunohistochemistry to show that UVRh1 protein expression is absent in mutant tissue, similar to that of males. Our results show that a rare behavioral phenotype, sex-specific UV color vision, was acquired via sex chromosome gene traffic of a duplicated UV rhodopsin.

Target interception is a complex sensorimotor behavior which requires fine tuning of the sensory system and its strategic coordination with the motor system. Despite various theories about how interception is achieved, its neural implementation remains unknown. We have previously shown that hunting dragonflies employ a balance of reactive and predictive control to intercept prey, using sophisticated model driven predictions to account for expected prey and self-motion. Here we explore the neural substrate of this interception system by investigating a well-known class of target-selective descending neurons (TSDNs). These cells have long been speculated to underlie interception steering but have never been studied in a behaving dragonfly. We combined detailed neuroanatomy, high-precision kinematics data and state-of-the-art neural telemetry to measure TSDN activity during flight. We found that TSDNs are exquisitely tuned to prey angular size and speed at ethological distances, and that they synapse directly onto neck and wing motoneurons in an unusual manner. However, we found that TSDNs were only weakly active during flight and are thus unlikely to provide the primary steering signal. Instead, they appear to drive the foveating head movements that stabilize prey on the eye before and likely throughout the interception flight. We suggest the TSDN population implements the reactive portion of the interception steering control system, coordinating head and wing movements to compensate for unexpected prey motion.

Non-recombining regions of the genome often have profound effects on evolution, resulting in phenomena such as sex chromosomes and supergenes. Amongst the strangest examples are balanced lethal systems, such as that found in newts of the genus Triturus. These systems halve reproductive output, and the evolution of such a deleterious trait is difficult to explain. For Triturus an intriguing model proposes that the balanced lethal system evolved from an ancestral Y-chromosome. To test this hypothesis, we identify the Y-chromosome of Triturus and verify whether it, or the balanced lethal system, is homologous to the Y-chromosome of its sister genus Lissotriton, which does not possess the balanced lethal system. We identify a set of candidate Y-linked markers in T. ivanbureschi and place them on a high-density linkage map that we construct with 7,233 RADseq markers. We validate male specificity of the markers across the genus, and then place both the Triturus and Lissotriton Y-linked regions within previously constructed target capture linkage maps that include genes linked to the balanced lethal system. We observe that neither the Triturus balanced lethal system, nor the Triturus Y-chromosome are homologous to the Lissotriton Y-chromosome. This is the first molecular evidence of a transition between Y-chromosome systems within salamanders. However, unless additional sex chromosome turnover events are involved, our data does not support a sex chromosome origin of the balanced lethal system.