Wolbachia infection negatively impacts Drosophila simulans heat tolerance in a strain- and trait-specific manner.

The susceptibility of insects to rising temperatures has largely been measured by their ability to survive thermal extremes. However, the capacity for maternally inherited endosymbionts to influence insect heat tolerance has been overlooked. Further, while some studies have addressed the impact of heat on traits like fertility, which can decline at temperatures below lethal thermal limits, none have considered the impact of endosymbionts. Here, we assess the impact of three Wolbachia strains (wRi, wAu and wNo) on the survival and fertility of Drosophila simulans exposed to heat stress during development or as adults. The effect of Wolbachia infection on heat tolerance was generally small and trait/strain specific. Only the wNo infection significantly reduced the survival of adult males after a heat shock. When exposed to fluctuating heat stress during development, the wRi and wAu strains reduced egg-to-adult survival but only the wNo infection reduced male fertility. Wolbachia densities of all three strains decreased under developmental heat stress, but reductions occurred at temperatures above those that reduced host fertility. These findings emphasize the necessity to account for endosymbionts and their effect on both survival and fertility when investigating insect responses to heat stress.

Developmental neuroscience: Building sex-specific adult circuitry from common larval origins.

The development of sex-specific neural circuitry is critical for reproductive behaviors. A new study traces the developmental origin of female-specific neurons that underlie an adult mating behavior to larval neurons common to both sexes in Drosophila.

Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.

The ability of neurons to rapidly remodel their synaptic structure and strength in response to neuronal activity is highly conserved across species and crucial for complex brain functions. However, mechanisms required to elicit and coordinate the acute, activity-dependent structural changes across synapses are not well understood, as neurodevelopment and structural plasticity are tightly linked. Here, using an RNAi screen in Drosophila against genes affecting nervous system functions in humans, we uncouple cellular processes important for synaptic plasticity and synapse development. We find mutations associated with neurodegenerative and mental health disorders are 2-times more likely to affect activity-induced synaptic remodeling than synapse development. We report that while both synapse development and activity-induced synaptic remodeling at the fly NMJ require macroautophagy (hereafter referred to as autophagy), bifurcation in the autophagy pathway differentially impacts development and synaptic plasticity. We demonstrate that neuronal activity enhances autophagy activation but diminishes degradative autophagy, thereby driving the pathway towards autophagy-based secretion. Presynaptic knockdown of Snap29, Sec22, or Rab8, proteins implicated in the secretory autophagy pathway, is sufficient to abolish activity-induced synaptic remodeling. This study uncovers secretory autophagy as a transsynaptic signaling mechanism modulating synaptic plasticity.

Thermal limits of survival and reproduction depend on stress duration: A case study of Drosophila suzukii.

Studies of ectotherm responses to heat extremes often rely on assessing absolute critical limits for heat coma or death (CTmax), however, such single parameter metrics ignore the importance of stress exposure duration. Furthermore, population persistence may be affected at temperatures considerably below CTmax through decreased reproductive output. Here we investigate the relationship between tolerance duration and severity of heat stress across three ecologically relevant life-history traits (productivity, coma and mortality) using the global agricultural pest Drosophila suzukii. For the first time, we show that for sublethal reproductive traits, tolerance duration decreases exponentially with increasing temperature (R2 > 0.97), thereby extending the Thermal Death Time framework recently developed for mortality and coma. Using field micro-environmental temperatures, we show how thermal stress can lead to considerable reproductive loss at temperatures with limited heat mortality highlighting the importance of including limits to reproductive performance in ecological studies of heat stress vulnerability.

Inter-organ steroid hormone signaling promotes myoblast fusion via direct transcriptional regulation of a single key effector gene.

Steroid hormones regulate tissue development and physiology by modulating the transcription of a broad spectrum of genes. In insects, the principal steroid hormones, ecdysteroids, trigger the expression of thousands of genes through a cascade of transcription factors (TFs) to coordinate developmental transitions such as larval molting and metamorphosis. However, whether ecdysteroid signaling can bypass transcriptional hierarchies to exert its function in individual developmental processes is unclear. Here, we report that a single non-TF effector gene mediates the transcriptional output of ecdysteroid signaling in Drosophila myoblast fusion, a critical step in muscle development and differentiation. Specifically, we show that the 20-hydroxyecdysone (commonly referred to as "ecdysone") secreted from an extraembryonic tissue, amnioserosa, acts on embryonic muscle cells to directly activate the expression of antisocial (ants), which encodes an essential scaffold protein enriched at the fusogenic synapse. Not only is ants transcription directly regulated by the heterodimeric ecdysone receptor complex composed of ecdysone receptor (EcR) and ultraspiracle (USP) via ecdysone-response elements but also more strikingly, expression of ants alone is sufficient to rescue the myoblast fusion defect in ecdysone signaling-deficient mutants. We further show that EcR/USP and a muscle-specific TF Twist synergistically activate ants expression in vitro and in vivo. Taken together, our study provides the first example of a steroid hormone directly activating the expression of a single key non-TF effector gene to regulate a developmental process via inter-organ signaling and provides a new paradigm for understanding steroid hormone signaling in other developmental and physiological processes.

An automatic system for recognizing fly courtship patterns via an image processing method.

Fruit fly courtship behaviors composed of a series of actions have always been an important model for behavioral research. While most related studies have focused only on total courtship behaviors, specific courtship elements have often been underestimated. Identifying these courtship element details is extremely labor intensive and would largely benefit from an automatic recognition system. To address this issue, in this study, we established a vision-based fly courtship behavior recognition system. The system based on the proposed image processing methods can precisely distinguish body parts such as the head, thorax, and abdomen and automatically recognize specific courtship elements, including orientation, singing, attempted copulation, copulation and tapping, which was not detectable in previous studies. This system, which has high identity tracking accuracy (99.99%) and high behavioral element recognition rates (> 97.35%), can ensure correct identification even when flies completely overlap. Using this newly developed system, we investigated the total courtship time, and proportion, and transition of courtship elements in flies across different ages and found that male flies adjusted their courtship strategy in response to their physical condition. We also identified differences in courtship patterns between males with and without successful copulation. Our study therefore demonstrated how image processing methods can be applied to automatically recognize complex animal behaviors. The newly developed system will largely help us investigate the details of fly courtship in future research.

Evolution of compound eye morphology underlies differences in vision between closely related Drosophila species.

BACKGROUND: Insects have evolved complex visual systems and display an astonishing range of adaptations for diverse ecological niches. Species of Drosophila melanogaster subgroup exhibit extensive intra- and interspecific differences in compound eye size. These differences provide an excellent opportunity to better understand variation in insect eye structure and the impact on vision. Here we further explored the difference in eye size between D. mauritiana and its sibling species D. simulans. RESULTS: We confirmed that D. mauritiana have rapidly evolved larger eyes as a result of more and wider ommatidia than D. simulans since they recently diverged approximately 240,000 years ago. The functional impact of eye size, and specifically ommatidia size, is often only estimated based on the rigid surface morphology of the compound eye. Therefore, we used 3D synchrotron radiation tomography to measure optical parameters in 3D, predict optical capacity, and compare the modelled vision to in vivo optomotor responses. Our optical models predicted higher contrast sensitivity for D. mauritiana, which we verified by presenting sinusoidal gratings to tethered flies in a flight arena. Similarly, we confirmed the higher spatial acuity predicted for Drosophila simulans with smaller ommatidia and found evidence for higher temporal resolution. CONCLUSIONS: Our study demonstrates that even subtle differences in ommatidia size between closely related Drosophila species can impact the vision of these insects. Therefore, further comparative studies of intra- and interspecific variation in eye morphology and the consequences for vision among other Drosophila species, other dipterans and other insects are needed to better understand compound eye structure-function and how the diversification of eye size, shape, and function has helped insects to adapt to the vast range of ecological niches.

Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila.

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

The anti-aging effect of vitamin D and vitamin D receptor in Drosophila midgut.

Adult stem cells are pivotal for maintaining tissue homeostasis, and their functional decline is linked to aging and its associated diseases, influenced by the niche cells' environment. Age- and cancer-related reduction of vitamin D and its receptor levels are well documented in human clinical studies. However, the mechanisms through which the vitamin D/vitamin D receptor pathway contributes to anti-aging and extends life expectancy are not well understood. In this study, we aimed to determine the protective role of the vitamin D/vitamin D receptor pathway in differentiated enterocytes (ECs) during intestinal stem cell (ISC) aging. By utilizing a well- established Drosophila midgut model for stem cell aging biology, we revealed that vitamin D receptor knockdown in ECs induced ISC proliferation, EC death, ISC aging, and enteroendocrine cell differentiation. Additionally, age- and oxidative stress-induced increases in ISC proliferation and centrosome amplification were reduced by vitamin D treatment. Our findings suggest a direct evidence of the anti-aging role of the vitamin D/vitamin D receptor pathway and provides insights into the molecular mechanisms underlying healthy aging in Drosophila.

Threshold shifts and developmental temperature impact trade-offs between tolerance and plasticity.

Mounting evidence suggests that ectotherms are already living close to their upper physiological thermal limits. Phenotypic plasticity has been proposed to reduce the impact of climate change in the short-term providing time for adaptation, but the tolerance-plasticity trade-off hypothesis predicts organisms with higher tolerance have lower plasticity. Empirical evidence is mixed, which may be driven by methodological issues such as statistical artefacts, nonlinear reaction norms, threshold shifts or selection. Here, we examine whether threshold shifts (organisms with higher tolerance require stronger treatments to induce maximum plastic responses) influence tolerance-plasticity trade-offs in hardening capacity for desiccation tolerance and critical thermal maximum (CTMAX) across Drosophila species with varying distributions/sensitivity to desiccation/heat stress. We found evidence for threshold shifts in both traits; species with higher heat/desiccation tolerance required longer hardening treatments to induce maximum hardening responses. Species with higher heat tolerance also showed reductions in hardening capacity at higher developmental acclimation temperatures. Trade-off patterns differed depending on the hardening treatment used and the developmental temperature flies were exposed to. Based on these findings, studies that do not consider threshold shifts, or that estimate plasticity under a narrow set of environments, will have a limited ability to assess trade-off patterns and differences in plasticity across species/populations more broadly.

Drosophila flight: How flies control casts and surges.

In the absence of directional cues, most foraging animals explore space by turning and zigzagging in search of sensory information. Recent progress in the identification of the neural correlates of turns in flies offers exciting new perspectives on the evolution of neural circuits controlling fundamental aspects of orientation responses.

A mismatch in the expression of cell surface molecules induces tissue-intrinsic defense against aberrant cells.

Tissue-intrinsic error correction enables epithelial cells to detect abnormal neighboring cells and facilitate their removal from the tissue. One of these pathways, "interface surveillance," is triggered by cells with aberrant developmental and cell-fate-patterning pathways. It remains unknown which molecular mechanisms provide cells with the ability to compare fate between neighboring cells. We demonstrate that Drosophila imaginal discs express an array of cell surface molecules previously implicated in neuronal axon guidance processes. They include members of the Robo, Teneurin, Ephrin, Toll-like, or atypical cadherin families. Importantly, a mismatch in expression levels of these cell surface molecules between adjacent cells is sufficient to induce interface surveillance, indicating that differences in expression levels between neighboring cells, rather than their absolute expression levels, are crucial. Specifically, a mismatch in Robo2 and Robo3, but not Robo1, induces enrichment of actin, myosin II, and Ena/Vasp, as well as activation of JNK and apoptosis at clonal interfaces. Moreover, Robo2 can induce interface surveillance independently of its cytosolic domain and without the need for the Robo-ligand Slit. The expression of Robo2 and other cell surface molecules, such as Teneurins or the Ephrin receptor is regulated by fate-patterning pathways intrinsic and extrinsic to the wing disc, as well as by expression of oncogenic RasV12. Combined, we demonstrate that neighboring cells respond to a mismatch in surface code patterns mediated by specific transmembrane proteins and reveal a novel function for these cell surface proteins in cell fate recognition and removal of aberrant cells during development and homeostasis of epithelial tissues.

New insights into ginsenoside Rg1 regulating the niche to inhibit age-induced germline stem cells depletion through targeting ECR/BMP signaling pathway in Drosophila.

PURPOSE: The age-induced imbalance in ecological niches leads to the loss of GSCs, which is the main reason for ovarian germline senescence. Ginsenoside Rg1 can delay ovarian senescence. Here, we shed light on new insights of ginsenoside Rg1 in regulating the niche to maintain GSCs self-renewal and discussing related molecular mechanisms. METHODS: The differences among GSC number, reproductive capacity of naturally aging female Drosophila after ginsenoside Rg1 feeding were analyzed by immunofluorescence and behavior monitoring. The expressions of the active factors in the niche and the BMP signaling were analyzed through Western blot and RT-qPCR. The target effect was verified in the ECR mutant and combined with the molecular docking. RESULTS: Ginsenoside Rg1 inhibited the age-induced reduction of the GSCs number and restored offspring production and development. Ginsenoside Rg1 promoted the expression of anchor proteins E-cadherin, stemness maintenance factor Nos and differentiation promoting factor Bam, thereby GSCs niche homeostasis was regulated. In addition, ginsenoside Rg1 was bound to the LBD region of the hormone receptor ECR. Ginsenoside Rg1 promotes the regeneration of GSCs by targeting the ECR to increase pSmad1/5/8 expression and thereby activating the BMP signaling pathway. In addition, ginsenoside Rg1 maintenance of niche homeostasis to promote GSCs regeneration is dependent on ECR as demonstrated in ECR mutants. CONCLUSIONS: Ginsenoside Rg1 regulated the ecological niche homeostasis of GSCs and promoted the regeneration of GSCs by targeting the ECR/BMP signaling pathway in hormone-deficient states in aging ovaries. It is of great significance for prolonging fertility potential and delaying ovarian senescence.

A neuron-glia lipid metabolic cycle couples daily sleep to mitochondrial homeostasis.

Sleep is thought to be restorative to brain energy homeostasis, but it is not clear how this is achieved. We show here that Drosophila glia exhibit a daily cycle of glial mitochondrial oxidation and lipid accumulation that is dependent on prior wake and requires the Drosophila APOE orthologs NLaz and GLaz, which mediate neuron-glia lipid transfer. In turn, a full night of sleep is required for glial lipid clearance, mitochondrial oxidative recovery and maximal neuronal mitophagy. Knockdown of neuronal NLaz causes oxidative stress to accumulate in neurons, and the neuronal mitochondrial integrity protein, Drp1, is required for daily glial lipid accumulation. These data suggest that neurons avoid accumulation of oxidative mitochondrial damage during wake by using mitophagy and passing damage to glia in the form of lipids. We propose that a mitochondrial lipid metabolic cycle between neurons and glia reflects a fundamental function of sleep relevant for brain energy homeostasis.

Organization of an ascending circuit that conveys flight motor state in Drosophila.

Natural behaviors are a coordinated symphony of motor acts that drive reafferent (self-induced) sensory activation. Individual sensors cannot disambiguate exafferent (externally induced) from reafferent sources. Nevertheless, animals readily differentiate between these sources of sensory signals to carry out adaptive behaviors through corollary discharge circuits (CDCs), which provide predictive motor signals from motor pathways to sensory processing and other motor pathways. Yet, how CDCs comprehensively integrate into the nervous system remains unexplored. Here, we use connectomics, neuroanatomical, physiological, and behavioral approaches to resolve the network architecture of two pairs of ascending histaminergic neurons (AHNs) in Drosophila, which function as a predictive CDC in other insects. Both AHN pairs receive input primarily from a partially overlapping population of descending neurons, especially from DNg02, which controls wing motor output. Using Ca2+ imaging and behavioral recordings, we show that AHN activation is correlated to flight behavior and precedes wing motion. Optogenetic activation of DNg02 is sufficient to activate AHNs, indicating that AHNs are activated by descending commands in advance of behavior and not as a consequence of sensory input. Downstream, each AHN pair targets predominantly non-overlapping networks, including those that process visual, auditory, and mechanosensory information, as well as networks controlling wing, haltere, and leg sensorimotor control. These results support the conclusion that the AHNs provide a predictive motor signal about wing motor state to mostly non-overlapping sensory and motor networks. Future work will determine how AHN signaling is driven by other descending neurons and interpreted by AHN downstream targets to maintain adaptive sensorimotor performance.

Sox21b underlies the rapid diversification of a novel male genital structure between Drosophila species.

The emergence and diversification of morphological novelties is a major feature of animal evolution.1,2,3,4,5,6,7,8,9 However, relatively little is known about the genetic basis of the evolution of novel structures and the mechanisms underlying their diversification. The epandrial posterior lobes of male genitalia are a novelty of particular Drosophila species.10,11,12,13 The lobes grasp the female ovipositor and insert between her abdominal tergites and, therefore, are important for copulation and species recognition.10,11,12,14,15,16,17 The posterior lobes likely evolved from co-option of a Hox-regulated gene network from the posterior spiracles10 and have since diversified in morphology in the D. simulans clade, in particular, over the last 240,000 years, driven by sexual selection.18,19,20,21 The genetic basis of this diversification is polygenic but, to the best of our knowledge, none of the causative genes have been identified.22,23,24,25,26,27,28,29,30 Identifying the genes underlying the diversification of these secondary sexual structures is essential to understanding the evolutionary impact on copulation and species recognition. Here, we show that Sox21b negatively regulates posterior lobe size. This is consistent with expanded Sox21b expression in D. mauritiana, which develops smaller posterior lobes than D. simulans. We tested this by generating reciprocal hemizygotes and confirmed that changes in Sox21b underlie posterior lobe evolution between these species. Furthermore, we found that posterior lobe size differences caused by the species-specific allele of Sox21b significantly affect copulation duration. Taken together, our study reveals the genetic basis for the sexual-selection-driven diversification of a novel morphological structure and its functional impact on copulatory behavior.

Developmental remodeling repurposes larval neurons for sexual behaviors in adult Drosophila.

Most larval neurons in Drosophila are repurposed during metamorphosis for functions in adult life, but their contribution to the neural circuits for sexually dimorphic behaviors is unknown. Here, we identify two interneurons in the nerve cord of adult Drosophila females that control ovipositor extrusion, a courtship rejection behavior performed by mated females. We show that these two neurons are present in the nerve cord of larvae as mature, sexually monomorphic interneurons. During pupal development, they acquire the expression of the sexual differentiation gene, doublesex; undergo doublesex-dependent programmed cell death in males; and are remodeled in females for functions in female mating behavior. Our results demonstrate that the neural circuits for courtship in Drosophila are built in part using neurons that are sexually reprogrammed from former sex-shared activities in larval life.

Heterogeneity of synaptic connectivity in the fly visual system.

Visual systems are homogeneous structures, where repeating columnar units retinotopically cover the visual field. Each of these columns contain many of the same neuron types that are distinguished by anatomic, genetic and - generally - by functional properties. However, there are exceptions to this rule. In the 800 columns of the Drosophila eye, there is an anatomically and genetically identifiable cell type with variable functional properties, Tm9. Since anatomical connectivity shapes functional neuronal properties, we identified the presynaptic inputs of several hundred Tm9s across both optic lobes using the full adult female fly brain (FAFB) electron microscopic dataset and FlyWire connectome. Our work shows that Tm9 has three major and many sparsely distributed inputs. This differs from the presynaptic connectivity of other Tm neurons, which have only one major, and more stereotypic inputs than Tm9. Genetic synapse labeling showed that the heterogeneous wiring exists across individuals. Together, our data argue that the visual system uses heterogeneous, distributed circuit properties to achieve robust visual processing.

Multivariate classification of multichannel long-term electrophysiology data identifies different sleep stages in fruit flies.

Identifying different sleep stages in humans and other mammals has traditionally relied on electroencephalograms. Such an approach is not feasible in certain animals such as invertebrates, although these animals could also be sleeping in stages. Here, we perform long-term multichannel local field potential recordings in the brains of behaving flies undergoing spontaneous sleep bouts. We acquired consistent spatial recordings of local field potentials across multiple flies, allowing us to compare brain activity across awake and sleep periods. Using machine learning, we uncover distinct temporal stages of sleep and explore the associated spatial and spectral features across the fly brain. Further, we analyze the electrophysiological correlates of microbehaviors associated with certain sleep stages. We confirm the existence of a distinct sleep stage associated with rhythmic proboscis extensions and show that spectral features of this sleep-related behavior differ significantly from those associated with the same behavior during wakefulness, indicating a dissociation between behavior and the brain states wherein these behaviors reside.

Nested neural circuits generate distinct acoustic signals during Drosophila courtship.

Many motor control systems generate multiple movements using a common set of muscles. How are premotor circuits able to flexibly generate diverse movement patterns? Here, we characterize the neuronal circuits that drive the distinct courtship songs of Drosophila melanogaster. Male flies vibrate their wings toward females to produce two different song modes-pulse and sine song-which signal species identity and male quality. Using cell-type-specific genetic reagents and the connectome, we provide a cellular and synaptic map of the circuits in the male ventral nerve cord that generate these songs and examine how activating or inhibiting each cell type within these circuits affects the song. Our data reveal that the song circuit is organized into two nested feedforward pathways with extensive reciprocal and feedback connections. The larger network produces pulse song, the more complex and ancestral song form. A subset of this network produces sine song, the simpler and more recent form. Such nested organization may be a common feature of motor control circuits in which evolution has layered increasing flexibility onto a basic movement pattern.