IFT88 maintains sensory function by localising signalling proteins along Drosophila cilia.

Ciliary defects cause several ciliopathies, some of which have late onset, suggesting cilia are actively maintained. Still, we have a poor understanding of the mechanisms underlying their maintenance. Here, we show Drosophila melanogaster IFT88 (DmIFT88/nompB) continues to move along fully formed sensory cilia. We further identify Inactive, a TRPV channel subunit involved in Drosophila hearing and negative-gravitaxis behaviour, and a yet uncharacterised Drosophila Guanylyl Cyclase 2d (DmGucy2d/CG34357) as DmIFT88 cargoes. We also show DmIFT88 binding to the cyclase´s intracellular part, which is evolutionarily conserved and mutated in several degenerative retinal diseases, is important for the ciliary localisation of DmGucy2d. Finally, acute knockdown of both DmIFT88 and DmGucy2d in ciliated neurons of adult flies caused defects in the maintenance of cilium function, impairing hearing and negative-gravitaxis behaviour, but did not significantly affect ciliary ultrastructure. We conclude that the sensory ciliary function underlying hearing in the adult fly requires an active maintenance program which involves DmIFT88 and at least two of its signalling transmembrane cargoes, DmGucy2d and Inactive.

The novel pyridazine pyrazolecarboxamide insecticide dimpropyridaz inhibits chordotonal organ function upstream of TRPV channels.

BACKGROUND: Pyridazine pyrazolecarboxamides (PPCs) are a novel insecticide class discovered and optimized at BASF. Dimpropyridaz is the first PPC to be submitted for registration and controls many aphid species as well as whiteflies and other piercing-sucking insects. RESULTS: Dimpropyridaz and other tertiary amide PPCs are proinsecticides that are converted in vivo into secondary amide active forms by N-dealkylation. Active secondary amide metabolites of PPCs potently inhibit the function of insect chordotonal neurons. Unlike Group 9 and 29 insecticides, which hyperactivate chordotonal neurons and increase Ca2+ levels, active metabolites of PPCs silence chordotonal neurons and decrease intracellular Ca2+ levels. Whereas the effects of Group 9 and 29 insecticides require TRPV (Transient Receptor Potential Vanilloid) channels, PPCs act in a TRPV-independent fashion, without compromising cellular responses to Group 9 and 29 insecticides, placing the molecular PPC target upstream of TRPVs. CONCLUSIONS: PPCs are a new class of chordotonal organ modulator insecticide for control of piercing-sucking pests. Dimpropyridaz is a PPC proinsecticide that is activated in target insects to secondary amide forms that inhibit the firing of chordotonal organs. The inhibition occurs at a site upstream of TRPVs and is TRPV-independent, providing a novel mode of action for resistance management. © 2023 BASF Corporation. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Flonicamid metabolite 4-trifluoromethylnicotinamide is a chordotonal organ modulator insecticide†.

BACKGROUND: The selective aphicide flonicamid is known to cause symptoms in aphids that are like those of chordotonal organ TRPV channel modulator insecticides such as pymetrozine, pyrifluquinazon and afidopyropen. Flonicamid is classified by the Insecticide Resistance Action Committee as a chordotonal organ modulator with an undefined target site. However, although it has been shown not to act on TRPV channels, flonicamid's action on chordotonal organs has not been documented in the literature. RESULTS: Flonicamid causes locusts to extend their hindlegs, indicating an action on the femoral chordotonal organ. In fruit flies, it abolishes negative gravitaxis behavior by disrupting transduction and mechanical amplification in antennal chordotonal neurons. Although flonicamid itself only weakly affects locust chordotonal organs, its major animal metabolite 4-trifluoromethylnicotinamide (TFNA-AM) potently stimulates both locust and fly chordotonal organs. Like pymetrozine, TFNA-AM rapidly increases Ca2+ in antennal chordotonal neurons in wild-type flies, but not iav1 mutants, yet the effect is nonadditive with the TRPV channel agonist. CONCLUSIONS: Flonicamid is a pro-insecticide form of TFNA-AM, a potent chordotonal organ modulator. The functional effects of TFNA-AM on chordotonal organs of locusts and flies are indistinguishable from those of the TRPV agonists pymetrozine, pyrifluquinazon and afidopyropen. Because our previous results indicate that TFNA-AM does not act directly on TRPV channels, we conclude that it acts upstream in a pathway that leads to TRPV channel activation. © 2022 Society of Chemical Industry.

A Markerless Pose Estimator Applicable to Limbless Animals.

The analysis of kinematics, locomotion, and spatial tasks relies on the accurate detection of animal positions and pose. Pose and position can be assessed with video analysis programs, the "trackers." Most available trackers represent animals as single points in space (no pose information available) or use markers to build a skeletal representation of pose. Markers are either physical objects attached to the body (white balls, stickers, or paint) or they are defined in silico using recognizable body structures (e.g., joints, limbs, color patterns). Physical markers often cannot be used if the animals are small, lack prominent body structures on which the markers can be placed, or live in environments such as aquatic ones that might detach the marker. Here, we introduce a marker-free pose-estimator (LACE Limbless Animal traCkEr) that builds the pose of the animal de novo from its contour. LACE detects the contour of the animal and derives the body mid-line, building a pseudo-skeleton by defining vertices and edges. By applying LACE to analyse the pose of larval Drosophila melanogaster and adult zebrafish, we illustrate that LACE allows to quantify, for example, genetic alterations of peristaltic movements and gender-specific locomotion patterns that are associated with different body shapes. As illustrated by these examples, LACE provides a versatile method for assessing position, pose and movement patterns, even in animals without limbs.

Rethinking Opsins.

Opsins, the protein moieties of animal visual photo-pigments, have emerged as moonlighting proteins with diverse, light-dependent and -independent physiological functions. This raises the need to revise some basic assumptions concerning opsin expression, structure, classification, and evolution.

Drosophila Mechanosensory Transduction.

Mechanosensation in Drosophila relies on sensory neurons transducing mechanical stimuli into ionic currents. The molecular mechanisms of this transduction are in the process of being revealed. Transduction relies on mechanogated ion channels that are activated by membrane stretch or the tension of force-conveying tethers. NOMPC (no-mechanoreceptor potential C) and DmPiezo were put forward as bona fide mechanoelectrical transduction (MET) channels, providing insights into MET channel architecture and the structural basis of mechanogating. Various additional channels were implicated in Drosophila mechanosensory neuron functions, and parallels between fly and vertebrate mechanotransduction were delineated. Collectively, these advances put forward Drosophila mechanosensory neurons as cellular paradigms for mechanotransduction and mechanogated ion channel function in the context of proprio- and nociception as well as the detection of substrate vibrations, touch, gravity, and sound.

Auditory DUM neurons in a bush-cricket: inhibited inhibitors.

Thoracic ganglia of many hearing insects house the first level of auditory processing. In bush-crickets, the largest population of local auditory neurons in the prothoracic processing centre are dorsal unpaired median (DUM) neurons. It has been suggested that DUM neurons are inhibitory using γ-aminobutyric acid (GABA) as transmitter. Immunohistochemistry reveals a population of about 35-50 GABA-positive somata in the posterior medial cluster of the prothoracic ganglion. Only very few small somata in this cluster remain unstained. At least 10 neurites from 10 neurons can be identified. Intracellularly stained auditory DUM neurons have their soma in the cluster of median GABA positive cells and most of them exhibit GABA-immunoreactivity. Responses of certain DUM neurons show obvious signs of inhibition. Application of picrotoxin (PTX), a chloride-channel blocker in insects, changes the responses of many DUM neurons. They become broader in frequency tuning and broader or narrower in temporal pattern tuning. Furthermore, inhibitory postsynaptic potentials (IPSPs) may be replaced by excitatory postsynaptic potentials. Loss of an IPSP in the rising graded potential after PTX-application leads to a significant reduction of first-spike latency. Therefore, auditory DUM neurons receive effective inhibition and are the best candidates for inhibition in DUM neurons and other auditory interneurons.

The Orphan Cytokine Receptor CRLF3 Emerged With the Origin of the Nervous System and Is a Neuroprotective Erythropoietin Receptor in Locusts.

The orphan cytokine receptor-like factor 3 (CRLF3) was identified as a neuroprotective erythropoietin receptor in locust neurons and emerged with the evolution of the eumetazoan nervous system. Human CRLF3 belongs to class I helical cytokine receptors that mediate pleiotropic cellular reactions to injury and diverse physiological challenges. It is expressed in various tissues including the central nervous system but its ligand remains unidentified. A CRLF3 ortholog in the holometabolous beetle Tribolium castaneum was recently shown to induce anti-apoptotic mechanisms upon stimulation with human recombinant erythropoietin. To test the hypothesis that CRLF3 represents an ancient cell-protective receptor for erythropoietin-like cytokines, we investigated its presence across metazoan species. Furthermore, we examined CRLF3 expression and function in the hemimetabolous insect Locusta migratoria. Phylogenetic analysis of CRLF3 sequences indicated that CRLF3 is absent in Porifera, Placozoa and Ctenophora, all lacking the traditional nervous system. However, it is present in all major eumetazoan groups ranging from cnidarians over protostomians to mammals. The CRLF3 sequence is highly conserved and abundant amongst vertebrates. In contrast, relatively few invertebrates express CRLF3 and these sequences show greater variability, suggesting frequent loss due to low functional importance. In L. migratoria, we identified the transcript Lm-crlf3 by RACE-PCR and detected its expression in locust brain, skeletal muscle and hemocytes. These findings correspond to the ubiquitous expression of crlf3 in mammalian tissues. We demonstrate that the sole addition of double-stranded RNA to the culture medium (called soaking RNA interference) specifically interferes with protein expression in locust primary brain cell cultures. This technique was used to knock down Lm-crlf3 expression and to abolish its physiological function. We confirmed that recombinant human erythropoietin rescues locust brain neurons from hypoxia-induced apoptosis and showed that this neuroprotective effect is absent after knocking down Lm-crlf3. Our results affirm the erythropoietin-induced neuroprotective function of CRLF3 in a second insect species from a different taxonomic group. They suggest that the phylogenetically conserved CRLF3 receptor may function as a cell protective receptor for erythropoietin or a structurally related cytokine also in other animals including vertebrate and mammalian species.

Chromophore-Independent Roles of Opsin Apoproteins in Drosophila Mechanoreceptors.

Rhodopsins, the major light-detecting molecules of animal visual systems [1], consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue [1, 2]. In addition to capturing photons, this chromophore contributes to rhodopsin maturation [3, 4], trafficking [3, 4], and stabilization [5], and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy [6-9]. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light [10, 11]. Light-independent sensory roles have been documented for Drosophila opsins [12-15], yet also these unconventional opsin functions are thought to require chromophore binding [12, 13, 15]. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks [16], yet chromophore-independent roles of opsin apoproteins outside of eyes have, to the best of our knowledge, hitherto not been described. Drosophila chordotonal mechanoreceptors require opsins [13, 15], and we find that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. Our results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components.

Proprioceptive Opsin Functions in Drosophila Larval Locomotion.

Animals rely on mechanosensory feedback from proprioceptors to control locomotory body movements. Unexpectedly, we found that this movement control requires visual opsins. Disrupting the Drosophila opsins NINAE or Rh6 impaired larval locomotion and body contractions, independently of light and vision. Opsins were detected in chordotonal proprioceptors along the larval body, localizing to their ciliated dendrites. Loss of opsins impaired mechanically evoked proprioceptor spiking and cilium ultrastructure. Without NINAE or Rh6, NOMPC mechanotransduction channels leaked from proprioceptor cilia and ciliary Inactive (Iav) channels partly disappeared. Locomotion is shown to require opsins in proprioceptors, and the receptors are found to express the opsin gene Rh7, in addition to ninaE and Rh6. Besides implicating opsins in movement control, this documents roles of non-ciliary, rhabdomeric opsins in cilium organization, providing a model for a key transition in opsin evolution and suggesting that structural roles of rhabdomeric opsins preceded their use for light detection.

Mechanical Properties of a Drosophila Larval Chordotonal Organ.

Proprioception is an integral part of the feedback circuit that is essential for locomotion control in all animals. Chordotonal organs perform proprioceptive and other mechanosensory functions in insects and crustaceans. The mechanical properties of these organs are believed to be adapted to the sensory functions, but had not been probed directly. We measured mechanical properties of a particular chordotonal organ-the lateral pentascolopidial (lch5) organ of Drosophila larvae-which plays a key role in proprioceptive locomotion control. We applied tension to the whole organ in situ by transverse deflection. Upon release of force, the organ displayed overdamped relaxation with two widely separated time constants, tens of milliseconds and seconds, respectively. When the muscles covering the lch5 organ were excised, the slow relaxation was absent, and the fast relaxation became faster. Interestingly, most of the strain in the stretched organ is localized in the cap cells, which account for two-thirds of the length of the entire organ, and could be stretched by ∼10% without apparent damage. In laser ablation experiments we found that cap cells retracted by ∼100 µm after being severed from the neurons, indicating considerable steady-state stress and strain in these cells. Given the fact that actin as well as myosin motors are abundant in cap cells, the results point to a mechanical regulatory role of the cap cells in the lch5 organ.

Neuroligins Nlg2 and Nlg4 Affect Social Behavior in Drosophila melanogaster.

The genome of Drosophila melanogaster includes homologs to approximately one-third of the currently known human disease genes. Flies and humans share many biological processes, including the principles of information processing by excitable neurons, synaptic transmission, and the chemical signals involved in intercellular communication. Studies on the molecular and behavioral impact of genetic risk factors of human neuro-developmental disorders [autism spectrum disorders (ASDs), schizophrenia, attention deficit hyperactivity disorders, and Tourette syndrome] increasingly use the well-studied social behavior of D. melanogaster, an organism that is amenable to a large variety of genetic manipulations. Neuroligins (Nlgs) are a family of phylogenetically conserved postsynaptic adhesion molecules present (among others) in nematodes, insects, and mammals. Impaired function of Nlgs (particularly of Nlg 3 and 4) has been associated with ASDs in humans and impaired social and communication behavior in mice. Making use of a set of behavioral and social assays, we, here, analyzed the impact of two Drosophila Nlgs, Dnlg2 and Dnlg4, which are differentially expressed at excitatory and inhibitory central nervous synapses, respectively. Both Nlgs seem to be associated with diurnal activity and social behavior. Even though deficiencies in Dnlg2 and Dnlg4 appeared to have no effects on sensory or motor systems, they differentially impacted on social interactions, suggesting that social behavior is distinctly regulated by these Nlgs.

Hearing regulates Drosophila aggression.

Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly's auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level.

A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy.

A consanguineous family from Pakistan was ascertained to have a novel deafness-dystonia syndrome with motor regression, ichthyosis-like features and signs of sensory neuropathy. By applying a combined strategy of linkage analysis and whole-exome sequencing in the presented family, a homozygous nonsense mutation, c.4G>T (p.Glu2*), in FITM2 was identified. FITM2 and its paralog FITM1 constitute an evolutionary conserved protein family involved in partitioning of triglycerides into cellular lipid droplets. Despite the role of FITM2 in neutral lipid storage and metabolism, no indications for lipodystrophy were observed in the affected individuals. In order to obtain independent evidence for the involvement of FITM2 in the human pathology, downregulation of the single Fitm ortholog, CG10671, in Drosophila melanogaster was pursued using RNA interference. Characteristics of the syndrome, including progressive locomotor impairment, hearing loss and disturbed sensory functions, were recapitulated in Drosophila, which supports the causative nature of the FITM2 mutation. Mutation-based genetic counseling can now be provided to the family and insight is obtained into the potential impact of genetic variation in FITM2.

Auditory Efferent System Modulates Mosquito Hearing.

The performance of vertebrate ears is controlled by auditory efferents that originate in the brain and innervate the ear, synapsing onto hair cell somata and auditory afferent fibers [1-3]. Efferent activity can provide protection from noise and facilitate the detection and discrimination of sound by modulating mechanical amplification by hair cells and transmitter release as well as auditory afferent action potential firing [1-3]. Insect auditory organs are thought to lack efferent control [4-7], but when we inspected mosquito ears, we obtained evidence for its existence. Antibodies against synaptic proteins recognized rows of bouton-like puncta running along the dendrites and axons of mosquito auditory sensory neurons. Electron microscopy identified synaptic and non-synaptic sites of vesicle release, and some of the innervating fibers co-labeled with somata in the CNS. Octopamine, GABA, and serotonin were identified as efferent neurotransmitters or neuromodulators that affect auditory frequency tuning, mechanical amplification, and sound-evoked potentials. Mosquito brains thus modulate mosquito ears, extending the use of auditory efferent systems from vertebrates to invertebrates and adding new levels of complexity to mosquito sound detection and communication.

Sensing pH with TMCs.

Transmembrane channel-like (TMC) proteins have been implicated in hair cell mechanotransduction, Drosophila proprioception, and sodium sensing in the nematode C. elegans. In this issue of Neuron, Wang et al. (2016) report that C. elegans TMC-1 mediates nociceptor responses to high pH, not sodium, allowing the nematode to avoid strongly alkaline environments in which most animals cannot survive.

Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion.

Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs.

Hearing in Insects.

Insect hearing has independently evolved multiple times in the context of intraspecific communication and predator detection by transforming proprioceptive organs into ears. Research over the past decade, ranging from the biophysics of sound reception to molecular aspects of auditory transduction to the neuronal mechanisms of auditory signal processing, has greatly advanced our understanding of how insects hear. Apart from evolutionary innovations that seem unique to insect hearing, parallels between insect and vertebrate auditory systems have been uncovered, and the auditory sensory cells of insects and vertebrates turned out to be evolutionarily related. This review summarizes our current understanding of insect hearing. It also discusses recent advances in insect auditory research, which have put forward insect auditory systems for studying biological aspects that extend beyond hearing, such as cilium function, neuronal signal computation, and sensory system evolution.

Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

Ankyrin Repeats Convey Force to Gate the NOMPC Mechanotransduction Channel.

How metazoan mechanotransduction channels sense mechanical stimuli is not well understood. The NOMPC channel in the transient receptor potential (TRP) family, a mechanotransduction channel for Drosophila touch sensation and hearing, contains 29 Ankyrin repeats (ARs) that associate with microtubules. These ARs have been postulated to act as a tether that conveys force to the channel. Here, we report that these N-terminal ARs form a cytoplasmic domain essential for NOMPC mechanogating in vitro, mechanosensitivity of touch receptor neurons in vivo, and touch-induced behaviors of Drosophila larvae. Duplicating the ARs elongates the filaments that tether NOMPC to microtubules in mechanosensory neurons. Moreover, microtubule association is required for NOMPC mechanogating. Importantly, transferring the NOMPC ARs to mechanoinsensitive voltage-gated potassium channels confers mechanosensitivity to the chimeric channels. These experiments strongly support a tether mechanism of mechanogating for the NOMPC channel, providing insights into the basis of mechanosensitivity of mechanotransduction channels.