A burden shared: The evolutionary case for studying human deafness in Drosophila.

Hearing impairment is the most prevalent sensory disease in humans and can have dramatic effects on the development, and preservation, of our cognitive abilities and social interactions. Currently 20 % of the world's population suffer from a form of hearing impairment; this is predicted to rise to 25 % by 2050. Despite this staggering disease load, and the vast damage it inflicts on the social, medical and economic fabric of humankind, our ability to predict, or prevent, the loss of hearing is very poor indeed. We here make the case for a paradigm shift in our approach to studying deafness. By exploiting more forcefully the molecular-genetic conservation between human hearing and hearing in morphologically distinct models, such as the fruit fly Drosophila melanogaster, we believe, a deeper understanding of hearing and deafness can be achieved. An understanding that moves beyond the surface of the 'deafness genes' to probe the underlying bedrock of hearing, which is shared across taxa, and partly shared across modalities. When it comes to understanding the workings (and failings) of human sensory function, a simple fruit fly has a lot to offer and a fly eye might sometimes be a powerful model for a human ear. Particularly the use of fly avatars, in which specific molecular (genetic or proteomic) states of humans (e.g. specific patients) are experimentally reproduced, in order to study the corresponding molecular mechanisms (e.g. specific diseases) in a controlled yet naturalistic environment, is a tool that promises multiple unprecedented insights. The use of the fly - and fly avatars - would benefit humans and will help enhance the power of other scientific models, such as the mouse.

Hearing of malaria mosquitoes is modulated by a beta-adrenergic-like octopamine receptor which serves as insecticide target.

Malaria mosquitoes acoustically detect their mating partners within large swarms that form transiently at dusk. Indeed, male malaria mosquitoes preferably respond to female flight tones during swarm time. This phenomenon implies a sophisticated context- and time-dependent modulation of mosquito audition, the mechanisms of which are largely unknown. Using transcriptomics, we identify a complex network of candidate neuromodulators regulating mosquito hearing in the species Anopheles gambiae. Among them, octopamine stands out as an auditory modulator during swarm time. In-depth analysis of octopamine auditory function shows that it affects the mosquito ear on multiple levels: it modulates the tuning and stiffness of the flagellar sound receiver and controls the erection of antennal fibrillae. We show that two α- and ß-adrenergic-like octopamine receptors drive octopamine's auditory roles and demonstrate that the octopaminergic auditory control system can be targeted by insecticides. Our findings highlight octopamine as key for mosquito hearing and mating partner detection and as a potential novel target for mosquito control.

Electrophysiological Measurements of Compound Action Potential Responses from the Antennal Nerve in Response to Stimulation.

Electrophysiological recordings taken from the antennal nerve can provide essential information on the general auditory condition of the mosquito tested. Furthermore, electrophysiological recordings provide detailed information on what types of stimulation induce the largest nerve responses. When these are used in conjunction with a vibrometer to measure the corresponding movement of the antennal ear during stimulation, a comprehensive overview of hearing function can be obtained. This protocol can be applied to male and female adults from any mosquito strain and can be scaled relative to available resources.

Acoustic Physiology in Mosquitoes.

The acoustic physiology of mosquitoes is perhaps the most complex within the entire insect class. Past research has uncovered several of its-sometimes stunningly unconventional-principles, but many mysteries remain. Their solution necessitates a concerted transdisciplinary effort to successfully link the neuroanatomical and biophysical properties of mosquito flagellar ears to the behavioral ecology of entire mosquito populations. Neuroanatomically, mosquito ears can rival those of humans in both complexity and sheer size. The approximately 16,000 auditory hair cells within the human organ of Corti, for example, are matched by the approximately 16,000 auditory neurons in the Johnston's organ of a male Anopheles mosquito. Both human and mosquito ears receive very extensive efferent innervation, which modulates their function in ways that are as yet poorly understood. Different populations of neuronal and nonneuronal cell types divide the labor of the mosquito ear amongst themselves. Yet, what exactly this labor is, and how it is achieved, is at best vaguely known. For the majority of mosquitoes, biologically relevant sounds are inextricably linked to their flight tones. Either these flight tones are (directly) the sounds of interest or they contribute (indirectly) to the production of audible sound through a process called nonlinear distortion. Finally, male ears can generate tones themselves: The generation of an internal "phantom copy" of a female flight tone (or self-sustained oscillation) is believed to aid the male hearing process. Here, we introduce protocols that target the mosquitoes' auditory neuroanatomy, electrophysiology, and behavior to help shed light on some of these issues.

Recording and Extraction of Mosquito Flight Tones.

Despite the artificial conditions, flight tone recordings taken from tethered mosquitoes can provide valuable information on the acoustic signals produced by male and female mosquitoes. Although auditory responsiveness appears to be largely (and possibly exclusively) restricted to males, the flight tones of both sexes have sensory-ecological relevance, as it is the mixing of the two tones that produces audibility in males and thereby facilitates reproduction. This protocol describes how to record wing flapping from mounted mosquitoes and how to estimate wingbeat frequencies from those recordings.

Mosquito Phonotaxis Assay.

Phonotaxis experiments can provide information on the spectrum of sounds relevant to mosquito acoustic behaviors. It is widely known that males of disease-transmitting species are attracted to tones with frequencies resembling the wingbeat frequencies of their conspecific females. Thus, phonotaxis experiments can be coupled with wingbeat frequency measurements to inform the development of vector control tools such as acoustic traps and lures. This protocol describes how to set up and execute a phonotaxis experiment.

Immunohistochemical Staining of the Mosquito Ear.

Immunohistochemistry has played a major role in improving our understanding of the anatomy and function of the nervous system. The use of fluorescent dyes that label different antigens reveals how biological tissues are built and how interactions between cells take place. Obtaining this information is particularly important in the case of the mosquito ear given its highly complex anatomy. This protocol describes an immunohistochemical technique to stain the mosquito ear. The first steps of the procedure include the embedding of the tissue in albumin-gelatin and its sectioning into thin slices to allow antibody penetration. The immunohistochemical procedure can be exploited to detect protein expression and localization by using antibodies specifically raised against the protein of interest or that recognize epitope tags fused to proteins using genome editing methods.

MR Enterography in Crohn's Disease: Comparison of Contrast Imaging with Diffusion-weighted Imaging and a special Form of Color Coding. / MR-Enterografie bei Morbus Crohn: Vergleich der Kontrastbildgebung mit diffusionsgewichteter Bildgebung und einer besonderen Form der Farbcodierung.

PURPOSE: We compared contrast-enhanced MR enterography with diffusion-weighted sequences to evaluate the validity of diffusion-weighted sequences for activity assessment in Crohn's disease compared to endoscopy. In addition, we investigated a new color-coded image post-processing technique in comparison with standard sequences and endoscopy. MATERIALS AND METHODS: Included were 197 MR enterographies (2015-2017) performed by using standardized examination protocols. The intestine was divided into 7 segments, which were compared separately. The accuracy of the MR examinations with regard to disease activity was validated using the Seo and MaRIA score and endoscopy findings. In addition, the image data were post-processed using a color-coded evaluation method (DCE tool on OsiriX). RESULTS: The comparison between contrast-enhanced and diffusion-weighted sequences showed a highly significant correlation for all bowel sections with a mean Spearman correlation coefficient of 0.876 (0.809-0.928). The color-coded image post-processing showed a sensitivity of 83.2 % and a specificity of 70.5 % in comparison with the MaRIA score. In comparison to endoscopy, a sensitivity of 81.3 % and a specificity of 70.5 %. In comparison with endoscopy, the MaRIA score showed a sensitivity of 80.2 % and a specificity of 84.0 % at a cut-off of 7. The visual score according to Seo showed a sensitivity of 85.7 % with a specificity of 77.0 % in the contrast-weighted examination and a sensitivity of 87.9 % and a specificity of 71.8 % for diffusion weighted images. CONCLUSION: Diffusion-weighted sequences are as good as contrast-weighted sequences for assessing inflammatory activity in Crohn's disease. Contrast is often helpful for assessing complications, but this was not the purpose of this study. Visual imaging using color-coded data sets was similarly good at detecting inflammation. KEY POINTS: · MR enterography is possible without contrast using diffusion-weighted imaging with comparable high informative value.. · Inflammatory activity in MR correlates sufficiently well with endoscopy. · The MaRIA and Seo scores are comparable in their validity.. · The colored representation of inflamed bowel sections is a promising technique.. CITATION FORMAT: · Jakob M, Backes M, Schaefer C et al. MR Enterography in Crohn's Disease: Comparison of Contrast Imaging with Diffusion-weighted Imaging and a special Form of Color Coding. Fortschr Röntgenstr 2022; 194: 1119 - 1131.

Hitting the right note at the right time: Circadian control of audibility in Anopheles mosquito mating swarms is mediated by flight tones.

Mating swarms of malaria mosquitoes form every day at sunset throughout the tropical world. They typically last less than 30 minutes. Activity must thus be highly synchronized between the sexes. Moreover, males must identify the few sporadically entering females by detecting the females' faint flight tones. We show that the Anopheles circadian clock not only ensures a tight synchrony of male and female activity but also helps sharpen the males' acoustic detection system: By raising their flight tones to 1.5 times the female flight tone, males enhance the audibility of females, specifically at swarm time. Previously reported "harmonic convergence" events are only a random by-product of the mosquitoes' flight tone variance and not a signature of acoustic interaction between males and females. The flight tones of individual mosquitoes occupy narrow, partly non-overlapping frequency ranges, suggesting that the audibility of individual females varies across males.

Telemetric capsule-based upper gastrointestinal tract - blood detection - first multicentric experience.

INTRODUCTION: Risk stratification in upper gastrointestinal bleeding (UGIB) currently relies on clinical parameters and risk scores. HemoPill® acute (Ovesco Endoscopy, Tuebingen, Germany) is a pill-shaped, orally administered sensor capsule for real-time blood detection. The aim of this study was to evaluate the system in clinical routine. MATERIAL AND METHODS: Sixty-one consecutive patients in whom the HemoPill® had been used at 12 international hospitals between July 2019 and March 2020 were retrospectively analysed. Indications for application were the clinical suspicion of UGIB, small bowel bleeding, of rebleeding after hemostasis. Primary endpoints were technical success and bleeding detection/exclusion. Secondary endpoints included adverse events and change of clinical course. RESULTS: The capsule was used in 45 (73%) patients with UGIB, in 12 (20%) patients with small bowel bleeding and in four (7%) patients for exclusion of rebleeding. Technical success was 98%. 35/60 (58%) cases were capsule-positive and among these, endoscopy showed bleeding in 20/35 (57%) cases. None of the 25 capsule-negative patients rebled. Emergency endoscopy could be avoided in 18/25 (72%) cases. Serious adverse events did not occur. CONCLUSION: HemoPill®-based blood detection is feasible and safe. Negative capsule results might 'downgrade' the need for urgent endoscopy.

Turnover and activity-dependent transcriptional control of NompC in the Drosophila ear.

Across their lives, biological sensors maintain near-constant functional outputs despite countless exogenous and endogenous perturbations. This sensory homeostasis is the product of multiple dynamic equilibria, the breakdown of which contributes to age-related decline. The mechanisms of homeostatic maintenance, however, are still poorly understood. The ears of vertebrates and insects are characterized by exquisite sensitivities but also by marked functional vulnerabilities. Being under the permanent load of thermal and acoustic noise, auditory transducer channels exemplify the homeostatic challenge. We show that (1) NompC-dependent mechanotransducers in the ear of the fruit fly Drosophila melanogaster undergo continual replacement with estimated turnover times of 9.1 hr; (2) a de novo synthesis of NompC can restore transducer function in the adult ears of congenitally hearing-impaired flies; (3) key components of the auditory transduction chain, including NompC, are under activity-dependent transcriptional control, likely forming a transducer-operated mechanosensory gain control system that extends beyond hearing organs.

Assessing the acoustic behaviour of Anopheles gambiae (s.l.) dsxF mutants: implications for vector control.

BACKGROUND: Release of gene-drive mutants to suppress Anopheles mosquito reproduction is a promising method of malaria control. However, many scientific, regulatory and ethical questions remain before transgenic mosquitoes can be utilised in the field. At a behavioural level, gene-drive carrying mutants should be at least as sexually attractive as the wildtype populations they compete against, with a key element of Anopheles copulation being acoustic courtship. We analysed sound emissions and acoustic preference in a doublesex mutant previously used to collapse Anopheles gambiae (s.l.) cages. METHODS: Anopheles rely on flight tones produced by the beating of their wings for acoustic mating communication. We assessed the impact of disrupting a female-specific isoform of the doublesex gene (dsxF) on the wing beat frequency (WBF; measured as flight tone) of males (XY) and females (XX) in homozygous dsxF- mutants (dsxF-/-), heterozygous dsxF- carriers (dsxF+/-) and G3 dsxF+ controls (dsxF+/+). To exclude non-genetic influences, we controlled for temperature and wing length. We used a phonotaxis assay to test the acoustic preferences of mutant and control mosquitoes. RESULTS: A previous study showed an altered phenotype only for dsxF-/- females, who appear intersex, suggesting that the female-specific dsxF allele is haplosufficient. We identified significant, dose-dependent increases in the WBF of both dsxF-/- and dsxF+/- females compared to dsxF+/+ females. All female WBFs remained significantly lower than male equivalents, though. Males showed stronger phonotactic responses to the WBFs of control dsxF+/+ females than to those of dsxF+/- and dsxF-/- females. We found no evidence of phonotaxis in any female genotype. No male genotypes displayed any deviations from controls. CONCLUSIONS: A prerequisite for anopheline copulation is the phonotactic attraction of males towards female flight tones within mating swarms. Reductions in mutant acoustic attractiveness diminish their mating efficiency and thus the efficacy of population control efforts. Caged population assessments may not successfully reproduce natural mating scenarios. We propose to amend existing testing protocols to better reflect competition between mutants and target populations. Our findings confirm that dsxF disruption has no effect on males; for some phenotypic traits, such as female WBFs, the effects of dsxF appear dose-dependent rather than haplosufficient.

Buzzkill: targeting the mosquito auditory system.

Sound plays an important role in mosquito sensory ecology. Acoustic perception and acoustically driven behaviours therefore represent potentially effective control targets. Previous scientific efforts around acoustic-based control and surveillance have not been systematic and ambiguity around the exact role of acoustic communication in conspecific interactions remains. Here, we briefly review recent advances in mosquito auditory physiology and behavioural ecology as well as ongoing activities to incorporate sound into control and surveillance tools. We highlight areas where increased collaboration between physiologists, molecular biologists, behavioural ecologists and control experts is needed to capitalize on this progress and realize the potential of sound-based technologies and strategies.

Homeostatic maintenance and age-related functional decline in the Drosophila ear.

Age-related hearing loss (ARHL) is a threat to future human wellbeing. Multiple factors contributing to the terminal auditory decline have been identified; but a unified understanding of ARHL - or the homeostatic maintenance of hearing before its breakdown - is missing. We here present an in-depth analysis of homeostasis and ageing in the antennal ears of the fruit fly Drosophila melanogaster. We show that Drosophila, just like humans, display ARHL. By focusing on the phase of dynamic stability prior to the eventual hearing loss we discovered a set of evolutionarily conserved homeostasis genes. The transcription factors Onecut (closest human orthologues: ONECUT2, ONECUT3), Optix (SIX3, SIX6), Worniu (SNAI2) and Amos (ATOH1, ATOH7, ATOH8, NEUROD1) emerged as key regulators, acting upstream of core components of the fly's molecular machinery for auditory transduction and amplification. Adult-specific manipulation of homeostatic regulators in the fly's auditory neurons accelerated - or protected against - ARHL.

How Many Clocks, How Many Times? On the Sensory Basis and Computational Challenges of Circadian Systems.

A vital task for every organism is not only to decide what to do but also when to do it. For this reason, "circadian clocks" have evolved in virtually all forms of life. Conceptually, circadian clocks can be divided into two functional domains; an autonomous oscillator creates a ~24 h self-sustained rhythm and sensory machinery interprets external information to alter the phase of the autonomous oscillation. It is through this simple design that variations in external stimuli (for example, daylight) can alter our sense of time. However, the clock's simplicity ends with its basic concept. In metazoan animals, multiple external and internal stimuli, from light to temperature and even metabolism have been shown to affect clock time. This raises the fundamental question of cue integration: how are the many, and potentially conflicting, sources of information combined to sense a single time of day? Moreover, individual stimuli, are often detected through various sensory pathways. Some sensory cells, such as insect chordotonal neurons, provide the clock with both temperature and mechanical information. Adding confusion to complexity, there seems to be not only one central clock in the animal's brain but numerous additional clocks in the body's periphery. It is currently not clear how (or if) these "peripheral clocks" are synchronized to their central counterparts or if both clocks "tick" independently from one another. In this review article, we would like to leave the comfort zones of conceptual simplicity and assume a more holistic perspective of circadian clock function. Focusing on recent results from Drosophila melanogaster we will discuss some of the sensory, and computational, challenges organisms face when keeping track of time.

Sex and species specific hearing mechanisms in mosquito flagellar ears.

Hearing is essential for the courtship of one of the major carriers of human disease, the mosquito. Males locate females through flight-tone recognition and both sexes engage in mid-air acoustic communications, which can take place within swarms containing thousands of individuals. Despite the importance of hearing for mosquitoes, its mechanisms are still largely unclear. We here report a multilevel analysis of auditory function across three disease-transmitting mosquitoes (Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus). All ears tested display transduction-dependent power gain. Quantitative analyses of mechanotransducer function reveal sex-specific and species-specific variations, including male-specific, highly sensitive transducer populations. Systemic blocks of neurotransmission result in large-amplitude oscillations only in male flagellar receivers, indicating sexually dimorphic auditory gain control mechanisms. Our findings identify modifications of auditory function as a key feature in mosquito evolution. We propose that intra-swarm communication has been a driving force behind the observed sex-specific and species-specific diversity.

Sequencing of intraductal biopsies is feasible and potentially impacts clinical management of patients with indeterminate biliary stricture and cholangiocarcinoma.

BACKGROUND: Definite diagnosis and therapeutic management of cholangiocarcinoma (CCA) remains a challenge. The aim of the current study was to investigate feasibility and potential impact on clinical management of targeted sequencing of intraductal biopsies. METHODS: Intraductal biopsies with suspicious findings from 16 patients with CCA in later clinical course were analyzed with targeted sequencing including tumor and control benign tissue (n = 55 samples). A CCA-specific sequencing panel containing 41 genes was designed and a dual strand targeted enrichment was applied. RESULTS: Sequencing was successfully performed for all samples. In total, 79 mutations were identified and a mean of 1.7 mutations per tumor sample (range 0-4) as well as 2.3 per biopsy (0-6) were detected and potentially therapeutically relevant genes were identified in 6/16 cases. In 14/18 (78%) biopsies with dysplasia or inconclusive findings at least one mutation was detected. The majority of mutations were found in both surgical specimen and biopsy (68%), while 28% were only present in biopsies in contrast to 4% being only present in the surgical tumor specimen. CONCLUSION: Targeted sequencing from intraductal biopsies is feasible and potentially improves the diagnostic yield. A profound genetic heterogeneity in biliary dysplasia needs to be considered in clinical management and warrants further investigation. TRANSLATIONAL IMPACT: The current study is the first to demonstrate the feasibility of sequencing of intraductal biopsies which holds the potential to impact diagnostic and therapeutical management of patients with biliary dysplasia and neoplasia.

Light Dominates Peripheral Circadian Oscillations in Drosophila melanogaster During Sensory Conflict.

In Drosophila, as in other animals, the circadian clock is a singular entity in name and concept only. In reality, clock functions emerge from multiple processes and anatomical substrates. One distinction has conventionally been made between a central clock (in the brain) and peripheral clocks (e.g., in the gut and the eyes). Both types of clock generate robust circadian oscillations, which do not require external input. Furthermore, the phases of these oscillations remain exquisitely sensitive to specific environmental cues, such as the daily changes of light and temperature. When these cues conflict with one another, the central clock displays complex forms of sensory integration; how peripheral clocks respond to conflicting input is unclear. We therefore explored the effects of light and temperature misalignments on peripheral clocks. We show that under conflict, peripheral clocks preferentially synchronize to the light stimulus. This photic dominance requires the presence of the circadian photoreceptor, Cryptochrome.

Evolutionary changes in transcription factor coding sequence quantitatively alter sensory organ development and function.

Animals are characterized by a set of highly conserved developmental regulators. Changes in the cis-regulatory elements of these regulators are thought to constitute the major driver of morphological evolution. However, the role of coding sequence evolution remains unresolved. To address this question, we used the Atonal family of proneural transcription factors as a model. Drosophila atonal coding sequence was endogenously replaced with that of atonal homologues (ATHs) at key phylogenetic positions, non-ATH proneural genes, and the closest homologue to ancestral proneural genes. ATHs and the ancestral-like coding sequences rescued sensory organ fate in atonal mutants, in contrast to non-ATHs. Surprisingly, different ATH factors displayed different levels of proneural activity as reflected by the number and functionality of sense organs. This proneural potency gradient correlated directly with ATH protein stability, including in response to Notch signaling, independently of mRNA levels or codon usage. This establishes a distinct and ancient function for ATHs and demonstrates that coding sequence evolution can underlie quantitative variation in sensory development and function.