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.

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.

Maintenance of Cell Fate by the Polycomb Group Gene Sex Combs Extra Enables a Partial Epithelial Mesenchymal Transition in Drosophila.

Epigenetic silencing by Polycomb group (PcG) complexes can promote epithelial-mesenchymal transition (EMT) and stemness and is associated with malignancy of solid cancers. Here we report a role for Drosophila PcG repression in a partial EMT event that occurs during wing disc eversion, an early event during metamorphosis. In a screen for genes required for eversion we identified the PcG genes Sexcombs extra (Sce) and Sexcombs midleg (Scm) Depletion of Sce or Scm resulted in internalized wings and thoracic clefts, and loss of Sce inhibited the EMT of the peripodial epithelium and basement membrane breakdown, ex vivo. Targeted DamID (TaDa) using Dam-Pol II showed that Sce knockdown caused a genomic transcriptional response consistent with a shift toward a more stable epithelial fate. Surprisingly only 17 genes were significantly upregulated in Sce-depleted cells, including Abd-B, abd-A, caudal, and nubbin Each of these loci were enriched for Dam-Pc binding. Of the four genes, only Abd-B was robustly upregulated in cells lacking Sce expression. RNAi knockdown of all four genes could partly suppress the Sce RNAi eversion phenotype, though Abd-B had the strongest effect. Our results suggest that in the absence of continued PcG repression peripodial cells express genes such as Abd-B, which promote epithelial state and thereby disrupt eversion. Our results emphasize the important role that PcG suppression can play in maintaining cell states required for morphogenetic events throughout development and suggest that PcG repression of Hox genes may affect epithelial traits that could contribute to metastasis.

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.

Deletion of the nicotinic acetylcholine receptor subunit gene Dα1 confers insecticide resistance, but at what cost?

Nicotinic acetylcholine receptors (nAChRs) have vital functions in processes of neurotransmission that underpin key behaviors. These pentameric ligand-gated ion channels have been used as targets for insecticides that constitutively activate them, causing the death of insect pests. In examining a knockout of the Dα1 nAChR subunit gene, our study linked this one subunit with multiple traits. We were able to confirm previous work that had identified Dα1 as a target of the neonicotinoid class of insecticides. Further, we uncovered roles for the gene in influencing mating behavior and patterns of sleep. The knockout mutant was also observed to have a significant reduction in longevity. This study highlighted the severe fitness costs that appear to be associated with the loss of function of this gene in natural populations in the absence of insecticides targeting the Dα1 subunit. Such a fitness cost could explain why target site resistances to neonicotinoids in pest insect populations have been associated specific amino acid replacement mutations in nAChR subunits, rather than loss of function. That mutant phenotypes were observed for the two behaviors examined indicates that the functions of Dα1, and other nAChR subunits, need to be explored more broadly. It also remains to be established whether these phenotypes were due to loss of the Dα1 receptor and/or to compensatory changes in the expression levels of other nAChR subunits.

Pleiotropic Effects of Loss of the Dα1 Subunit in Drosophila melanogaster: Implications for Insecticide Resistance.

Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, our previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. We used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. To our knowledge, this is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance.

Transcriptome Analysis of Drosophila melanogaster Third Instar Larval Ring Glands Points to Novel Functions and Uncovers a Cytochrome p450 Required for Development.

In Drosophila melanogaster larvae, the ring gland (RG) is a control center that orchestrates major developmental transitions. It is a composite organ, consisting of the prothoracic gland, the corpus allatum, and the corpora cardiaca, each of which synthesizes and secretes a different hormone. Until now, the RG's broader developmental roles beyond endocrine secretion have not been explored. RNA sequencing and analysis of a new transcriptome resource from D. melanogaster wandering third instar larval RGs has provided a fascinating insight into the diversity of developmental signaling in this organ. We have found strong enrichment of expression of two gene pathways not previously associated with the RG: immune response and fatty acid metabolism. We have also uncovered strong expression for many uncharacterized genes. Additionally, RNA interference against RG-enriched cytochrome p450s Cyp6u1 and Cyp6g2 produced a lethal ecdysone deficiency and a juvenile hormone deficiency, respectively, flagging a critical role for these genes in hormone synthesis. This transcriptome provides a valuable new resource for investigation of roles played by the RG in governing insect development.

In vivo functional analysis of the Drosophila melanogaster nicotinic acetylcholine receptor Dα6 using the insecticide spinosad.

The vinegar fly, Drosophila melanogaster, has been used to identify and manipulate insecticide resistance genes. The advancement of genome engineering technology and the increasing availability of pest genome sequences has increased the predictive and diagnostic capacity of the Drosophila model. The Drosophila model can be extended to investigate the basic biology of the interaction between insecticides and the proteins they target. Recently we have developed an in vivo system that permits the expression and study of key insecticide targets, the nicotinic acetylcholine receptors (nAChRs), in controlled genetic backgrounds. Here this system is used to study the interaction between the insecticide spinosad and a nAChR subunit, Dα6. Reciprocal chimeric subunits were created from Dα6 and Dα7, a subunit that does not respond to spinosad. Using the in vivo system, the Dα6/Dα7 chimeric subunits were tested for their capacity to respond to spinosad. Only the subunits containing the C-terminal region of Dα6 were able to respond to spinosad, thus confirming the importance this region for spinosad binding. A new incompletely dominant, spinosad resistance mechanism that may evolve in pest species is also examined. First generated using chemical mutagenesis, the Dα6(P146S) mutation was recreated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, the first use of this technology to introduce a resistant mutation into a controlled genetic background. Both alleles present with the same incompletely dominant, spinosad resistance phenotype, proving the P146S replacement to be the causal mutation. The proximity of the P146S mutation to the conserved Cys-loop indicates that it may impair the gating of the receptor. The results of this study enhance the understanding of nAChR structure:function relationships.

Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction.

Insecticide research has often relied on model species for elucidating the resistance mechanisms present in the targeted pests. The accuracy and applicability of extrapolations of these laboratory findings to field conditions varies but, for target site resistance, conserved mechanisms are generally the rule rather than the exception (Perry et al., 2011). The spinosyn class of insecticides appear to fit this paradigm and are a pest control option with many uses in both crop and animal protection. Resistance to spinosyns has been identified in both laboratory-selected and field-collected pest insects. Studies using the model insect, Drosophila melanogaster, have identified the nicotinic acetylcholine receptor subunit, Dα6 as an important target of the insecticide spinosad (Perry et al., 2007; Watson et al., 2010). Field-isolated resistant strains of several agricultural pest insects provide evidence that resistance cases are often associated with mutations in orthologues to Dα6 (Baxter et al., 2010; Puinean et al., 2013). The expression of these receptors is difficult in heterologous systems. In order to examine the biology of the Dα6 receptor subunit further, we used Drosophila as a model and developed an in vivo rescue system. This allowed us to express four different isoforms of Dα6 and show that each is able to rescue the response to spinosad. Regulatory sequences upstream of the Dα6 gene able to rescue the resistance phenotype were identified. Expression of other D. melanogaster subunits revealed that the rescue phenotype appears to be Dα6 specific. We also demonstrate that expression of pest insect orthologues of Dα6 from a variety of species are capable of rescuing the spinosad response phenotype, verifying the relevance of this receptor to resistance monitoring in the field. In the absence of a robust heterologous expression system, this study presents an in vivo model that will be useful in analysing many other aspects of these receptors and their biology.