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.

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.

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.

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.

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.

Sensory Conflict Disrupts Activity of the Drosophila Circadian Network.

Periodic changes in light and temperature synchronize the Drosophila circadian clock, but the question of how the fly brain integrates these two input pathways to set circadian time remains unanswered. We explore multisensory cue combination by testing the resilience of the circadian network to conflicting environmental inputs. We show that misaligned light and temperature cycles can lead to dramatic changes in the daily locomotor activities of wild-type flies during and after exposure to sensory conflict. This altered behavior is associated with a drastic reduction in the amplitude of PERIOD (PER) oscillations in brain clock neurons and desynchronization between light- and temperature-sensitive neuronal subgroups. The behavioral disruption depends heavily on the phase relationship between light and temperature signals. Our results represent a systematic quantification of multisensory integration in the Drosophila circadian system and lend further support to the view of the clock as a network of coupled oscillatory subunits.

Comparative Aspects of Hearing in Vertebrates and Insects with Antennal Ears.

The evolution of hearing in terrestrial animals has resulted in remarkable adaptations enabling exquisitely sensitive sound detection by the ear and sophisticated sound analysis by the brain. In this review, we examine several such characteristics, using examples from insects and vertebrates. We focus on two strong and interdependent forces that have been shaping the auditory systems across taxa: the physical environment of auditory transducers on the small, subcellular scale, and the sensory-ecological environment within which hearing happens, on a larger, evolutionary scale. We briefly discuss acoustical feature selectivity and invariance in the central auditory system, highlighting a major difference between insects and vertebrates as well as a major similarity. Through such comparisons within a sensory ecological framework, we aim to emphasize general principles underlying acute sensitivity to airborne sounds.

Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing.

In mammals, the membrane-based protein Prestin confers unique electromotile properties to cochlear outer hair cells, which contribute to the cochlear amplifier. Like mammals, the ears of insects, such as those of Drosophila melanogaster, mechanically amplify sound stimuli and have also been reported to express Prestin homologs. To determine whether the D. melanogaster Prestin homolog (dpres) is required for auditory amplification, we generated and analyzed dpres mutant flies. We found that dpres is robustly expressed in the fly's antennal ear. However, dpres mutant flies show normal auditory nerve responses, and intact non-linear amplification. Thus we conclude that, in D. melanogaster, auditory amplification is independent of Prestin. This finding resonates with prior phylogenetic analyses, which suggest that the derived motor function of mammalian Prestin replaced, or amended, an ancestral transport function. Indeed, we show that dpres encodes a functional anion transporter. Interestingly, the acquired new motor function in the phylogenetic lineage leading to birds and mammals coincides with loss of the mechanotransducer channel NompC (=TRPN1), which has been shown to be required for auditory amplification in flies. The advent of Prestin (or loss of NompC, respectively) may thus mark an evolutionary transition from a transducer-based to a Prestin-based mechanism of auditory amplification.

A mechanosensory pathway to the Drosophila circadian clock.

Circadian clocks attune the physiology of virtually all living organisms to the diurnal cycles of their environments. In metazoan animals, multiple sensory input pathways have been linked to clock synchronization with the environmental cycle (entrainment). Extrinsic entrainment cues include light and temperature. We show that (12-hour:12-hour) cycles of vibration and silence (VS) are sufficient to synchronize the daily locomotor activity of wild-type Drosophila melanogaster. Behavioral synchronization to VS cycles required a functional clock and functional chordotonal organs and was accompanied by phase-shifts of the daily oscillations of PERIOD protein concentrations in brain clock neurons. The feedback from mechanosensory-and particularly, proprioceptive-organs may help an animal to keep its circadian clock in sync with its own, stimulus-induced activities.

Sensory transduction: confusing the senses.

Two new studies in the fruit fly Drosophila demonstrate unexpected molecular, and mechanistic, overlaps between the different senses. In the centre stand two long-established families of sensory proteins--rhodopsins and TRP channels.

Temperature entrainment of Drosophila's circadian clock involves the gene nocte and signaling from peripheral sensory tissues to the brain.

Circadian clocks are synchronized by the natural day/night and temperature cycles. Our previous work demonstrated that synchronization by temperature is a tissue autonomous process, similar to synchronization by light. We show here that this is indeed the case, with the important exception of the brain. Using luciferase imaging we demonstrate that brain clock neurons depend on signals from peripheral tissues in order to be synchronized by temperature. Reducing the function of the gene nocte in chordotonal organs changes their structure and function and dramatically interferes with temperature synchronization of behavioral activity. Other mutants known to affect the function of these sensory organs also interfere with temperature synchronization, demonstrating the importance of nocte in this process and identifying the chordotonal organs as relevant sensory structures. Our work reveals surprising and important mechanistic differences between light- and temperature-synchronization and advances our understanding of how clock resetting is accomplished in nature.