A pleiotropic chemoreceptor facilitates the production and perception of mating pheromones.

Optimal mating decisions depend on the robust coupling of signal production and perception because independent changes in either could carry a fitness cost. However, since the perception and production of mating signals are often mediated by different tissues and cell types, the mechanisms that drive and maintain their coupling remain unknown for most animal species. Here, we show that in Drosophila, behavioral responses to, and the production of, a putative inhibitory mating pheromone are co-regulated by Gr8a, a member of the Gustatory receptor gene family. Specifically, through behavioral and pheromonal data, we found that Gr8a independently regulates the behavioral responses of males and females to a putative inhibitory pheromone, as well as its production in the fat body and oenocytes of males. Overall, these findings provide a relatively simple molecular explanation for how pleiotropic receptors maintain robust mating signaling systems at the population and species levels.

Genetic and viral approaches to record or manipulate neurons in insects.

The development of genetically encoded tools to record and manipulate neurons in vivo has greatly increased our understanding of how neuronal activity affects behavior. Recent advances enable the use of these tools in species not typically considered genetically tractable. This progress is revolutionizing neuroscience in general, and insect neuroethology in particular. Here we cover the latest innovations and some of their applications in phylogenetically diverse insect species. We discuss the importance and implications of these approaches for both basic and translational research. We focus on genetically encoded and virally encoded tools used for calcium imaging, optogenetics, and synaptic silencing. Finally, we discuss potential future developments of universally applicable, modular, and user-friendly genetic toolkits for neuroethological studies of insect behavior.

The foraging gene as a modulator of division of labour in social insects.

The social ants, bees, wasps, and termites include some of the most ecologically-successful groups of animal species. Their dominance in most terrestrial environments is attributed to their social lifestyle, which enable their colonies to exploit environmental resources with remarkable efficiency. One key attribute of social insect colonies is the division of labour that emerges among the sterile workers, which represent the majority of colony members. Studies of the mechanisms that drive division of labour systems across diverse social species have provided fundamental insights into the developmental, physiological, molecular, and genomic processes that regulate sociality, and the possible genetic routes that may have led to its evolution from a solitary ancestor. Here we specifically discuss the conserved role of the foraging gene, which encodes a cGMP-dependent protein kinase (PKG). Originally identified as a behaviourally polymorphic gene that drives alternative foraging strategies in the fruit fly Drosophila melanogaster, changes in foraging expression and kinase activity were later shown to play a key role in the division of labour in diverse social insect species as well. In particular, foraging appears to regulate worker transitions between behavioural tasks and specific behavioural traits associated with morphological castes. Although the specific neuroethological role of foraging in the insect brain remains mostly unknown, studies in genetically tractable insect species indicate that PKG signalling plays a conserved role in the neuronal plasticity of sensory, cognitive and motor functions, which underlie behaviours relevant to division of labour, including appetitive learning, aggression, stress response, phototaxis, and the response to pheromones.

The genetic architecture of larval aggregation behavior in Drosophila.

Many insect species exhibit basal social behaviors such as aggregation, which play important roles in their feeding and mating ecologies. However, the evolutionary, genetic, and physiological mechanisms that regulate insect aggregation remain unknown for most species. Here, we used natural populations of Drosophila melanogaster to identify the genetic architecture that drives larval aggregation feeding behavior. By using quantitative and reverse genetic approaches, we have identified a complex neurogenetic network that plays a role in regulating the decision of larvae to feed in either solitude or as a group. Results from single gene, RNAi-knockdown experiments show that several of the identified genes represent key nodes in the genetic network that determines the level of aggregation while feeding. Furthermore, we show that a single non-coding variant in the gene CG14205, a putative acyltransferase, is associated with both decreased mRNA expression and increased aggregate formation, which suggests that it has a specific role in inhibiting aggregation behavior. Our results identify, for the first time, the genetic components which interact to regulate naturally occurring levels of aggregation in D. melanogaster larvae.

The gut microbiome defines social group membership in honey bee colonies.

In the honey bee, genetically related colony members innately develop colony-specific cuticular hydrocarbon profiles, which serve as pheromonal nestmate recognition cues. Yet, despite high intracolony relatedness, the innate development of colony-specific chemical signatures by individual colony members is largely determined by the colony environment, rather than solely relying on genetic variants shared by nestmates. Therefore, it is puzzling how a nongenic factor could drive the innate development of a quantitative trait that is shared by members of the same colony. Here, we provide one solution to this conundrum by showing that nestmate recognition cues in honey bees are defined, at least in part, by shared characteristics of the gut microbiome across individual colony members. These results illustrate the importance of host-microbiome interactions as a source of variation in animal behavioral traits.

Brain microRNAs among social and solitary bees.

Evolutionary transitions to a social lifestyle in insects are associated with lineage-specific changes in gene expression, but the key nodes that drive these regulatory changes are unknown. We examined the relationship between social organization and lineage-specific microRNAs (miRNAs). Genome scans across 12 bee species showed that miRNA copy-number is mostly conserved and not associated with sociality. However, deep sequencing of small RNAs in six bee species revealed a substantial proportion (20-35%) of detected miRNAs had lineage-specific expression in the brain, 24-72% of which did not have homologues in other species. Lineage-specific miRNAs disproportionately target lineage-specific genes, and have lower expression levels than shared miRNAs. The predicted targets of lineage-specific miRNAs are not enriched for genes with caste-biased expression or genes under positive selection in social species. Together, these results suggest that novel miRNAs may coevolve with novel genes, and thus contribute to lineage-specific patterns of evolution in bees, but do not appear to have significant influence on social evolution. Our analyses also support the hypothesis that many new miRNAs are purged by selection due to deleterious effects on mRNA targets, and suggest genome structure is not as influential in regulating bee miRNA evolution as has been shown for mammalian miRNAs.

The neurogenetics of sexually dimorphic behaviors from a postdevelopmental perspective.

Most sexually reproducing animal species are characterized by two morphologically and behaviorally distinct sexes. The genetic, molecular and cellular processes that produce sexual dimorphisms are phylogenetically diverse, though in most cases they are thought to occur early in development. In some species, however, sexual dimorphisms are manifested after development is complete, suggesting the intriguing hypothesis that sex, more generally, might be considered a continuous trait that is influenced by both developmental and postdevelopmental processes. Here, we explore how biological sex is defined at the genetic, neuronal and behavioral levels, its effects on neuronal development and function, and how it might lead to sexually dimorphic behavioral traits in health and disease. We also propose a unifying framework for understanding neuronal and behavioral sexual dimorphisms in the context of both developmental and postdevelopmental, physiological timescales. Together, these two temporally separate processes might drive sex-specific neuronal functions in sexually mature adults, particularly as it pertains to behavior in health and disease.

The Drosophila ERG channel seizure plays a role in the neuronal homeostatic stress response.

Neuronal physiology is particularly sensitive to acute stressors that affect excitability, many of which can trigger seizures and epilepsies. Although intrinsic neuronal homeostasis plays an important role in maintaining overall nervous system robustness and its resistance to stressors, the specific genetic and molecular mechanisms that underlie these processes are not well understood. Here we used a reverse genetic approach in Drosophila to test the hypothesis that specific voltage-gated ion channels contribute to neuronal homeostasis, robustness, and stress resistance. We found that the activity of the voltage-gated potassium channel seizure (sei), an ortholog of the mammalian ERG channel family, is essential for protecting flies from acute heat-induced seizures. Although sei is broadly expressed in the nervous system, our data indicate that its impact on the organismal robustness to acute environmental stress is primarily mediated via its action in excitatory neurons, the octopaminergic system, as well as neuropile ensheathing and perineurial glia. Furthermore, our studies suggest that human mutations in the human ERG channel (hERG), which have been primarily implicated in the cardiac Long QT Syndrome (LQTS), may also contribute to the high incidence of seizures in LQTS patients via a cardiovascular-independent neurogenic pathway.

RNA editing is abundant and correlates with task performance in a social bumblebee.

Colonies of the bumblebee Bombus terrestris are characterized by wide phenotypic variability among genetically similar full-sister workers, suggesting a major role for epigenetic processes. Here, we report a high level of ADAR-mediated RNA editing in the bumblebee, despite the lack of an ADAR1-homolog. We identify 1.15 million unique genomic sites, and 164 recoding sites residing in 100 protein coding genes, including ion channels, transporters, and receptors predicted to affect brain function and behavior. Some edited sites are similarly edited in other insects, cephalopods and even mammals. The global editing level of protein coding and non-coding transcripts weakly correlates with task performance (brood care vs. foraging), but not affected by dominance rank or juvenile hormone known to influence physiology and behavior. Taken together, our findings show that brain editing levels are high in naturally behaving bees, and may be regulated by relatively short-term effects associated with brood care or foraging activities.

The cuticular hydrocarbon profiles of honey bee workers develop via a socially-modulated innate process.

Large social insect colonies exhibit a remarkable ability for recognizing group members via colony-specific cuticular pheromonal signatures. Previous work suggested that in some ant species, colony-specific pheromonal profiles are generated through a mechanism involving the transfer and homogenization of cuticular hydrocarbons (CHCs) across members of the colony. However, how colony-specific chemical profiles are generated in other social insect clades remains mostly unknown. Here we show that in the honey bee (Apis mellifera), the colony-specific CHC profile completes its maturation in foragers via a sequence of stereotypic age-dependent quantitative and qualitative chemical transitions, which are driven by environmentally-sensitive intrinsic biosynthetic pathways. Therefore, the CHC profiles of individual honey bees are not likely produced through homogenization and transfer mechanisms, but instead mature in association with age-dependent division of labor. Furthermore, non-nestmate rejection behaviors seem to be contextually restricted to behavioral interactions between entering foragers and guards at the hive entrance.

The synaptic action of Degenerin/Epithelial sodium channels.

Degenerin/Epithelial Sodium Channels (DEG/ENaCs) are a large family of animal-specific non-voltage gated ion channels, with enriched expression in neuronal and epithelial tissues. While neuronal DEG/ENaCs were originally characterized as sensory receptor channels, recent studies indicate that several DEG/ENaC family members are also expressed throughout the central nervous system. Human genome-wide association studies have linked DEG/ENaC-coding genes with several neurologic and psychiatric disorders, including epilepsy and panic disorder. In addition, studies in rodent models further indicate that DEG/ENaC activity in the brain contributes to many behaviors, including those related to anxiety and long-term memory. Although the exact neurophysiological functions of DEG/ENaCs remain mostly unknown, several key studies now suggest that multiple family members might exert their neuronal function via the direct modulation of synaptic processes. Here, we review and discuss recent findings on the synaptic functions of DEG/ENaCs in both vertebrate and invertebrate species, and propose models for their possible roles in synaptic physiology.

The Impact of Environmental Mn Exposure on Insect Biology.

Manganese (Mn) is an essential trace element that acts as a metal co-factor in diverse biochemical and cellular functions. However, chronic environmental exposure to high levels of Mn is a well-established risk factor for the etiology of severe, atypical parkinsonian syndrome (manganism) via its accumulation in the basal ganglia, pallidum, and striatum brain regions, which is often associated with abnormal dopamine, GABA, and glutamate neural signaling. Recent studies have indicated that chronic Mn exposure at levels that are below the risk for manganism can still cause behavioral, cognitive, and motor dysfunctions via poorly understood mechanisms at the molecular and cellular levels. Furthermore, in spite of significant advances in understanding Mn-induced behavioral and neuronal pathologies, available data are primarily for human and rodents. In contrast, the possible impact of environmental Mn exposure on brain functions and behavior of other animal species, especially insects and other invertebrates, remains mostly unknown both in the laboratory and natural habitats. Yet, the effects of environmental exposure to metals such as Mn on insect development, physiology, and behavior could also have major indirect impacts on human health via the long-term disruptions of food webs, as well as direct impact on the economy because of the important role insects play in crop pollination. Indeed, laboratory and field studies indicate that chronic exposures to metals such as Mn, even at levels that are below what is currently considered toxic, affect the dopaminergic signaling pathway in the insect brain, and have a major impact on the behavior of insects, including foraging activity of important pollinators such as the honey bee. Together, these studies highlight the need for a better understanding of the neuronal, molecular, and genetic processes that underlie the toxicity of Mn and other metal pollutants in diverse animal species, including insects.

The Drosophila Postsynaptic DEG/ENaC Channel ppk29 Contributes to Excitatory Neurotransmission.

The protein family of degenerin/epithelial sodium channels (DEG/ENaCs) is composed of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. Some family members are also enriched in neural tissues in both vertebrates and invertebrates. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. The fruit fly Drosophila melanogaster is an excellent model for deciphering the functions of DEG/ENaC genes because its genome encodes an exceptionally large number of DEG/ENaC subunits termed pickpocket (ppk) 1-31 Here we demonstrate that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction. Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates that the observed phenotypes are likely due to defects in glutamate receptor function. Together, our data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission.SIGNIFICANCE STATEMENT Members of the degenerin/epithelial sodium channel (DEG/ENaC) family are broadly expressed in epithelial and neuronal tissues. To date, the neurophysiological functions of most family members remain unknown. Here, by using the power of Drosophila genetics in combination with electrophysiological and behavioral approaches, we demonstrate that the DEG/ENaC-encoding gene pickpocket 29 contributes to baseline neurotransmission, possibly via the modulation of postsynaptic glutamate receptor functionality.

The neural basis for insect pheromonal communication.

Insects rely on chemosensory signals to drive a multitude of behavioral decisions. From conspecific and mate recognition to aggression, the proper detection and processing of these chemical signals - termed pheromones - is crucial for insects' fitness. While the identities and physiological impacts of diverse insect pheromones have been known for many years, how these important molecules are perceived and processed by the nervous system to produce evolutionarily beneficial behaviors is still mostly unknown. Here we present an overview of the current state of research into the peripheral and central nervous system mechanisms that process and drive behavioral responses to diverse pheromonal cues.

Function and evolution of microRNAs in eusocial Hymenoptera.

The emergence of eusociality ("true sociality") in several insect lineages represents one of the most successful evolutionary adaptations in the animal kingdom in terms of species richness and global biomass. In contrast to solitary insects, eusocial insects evolved a set of unique behavioral and physiological traits such as reproductive division of labor and cooperative brood care, which likely played a major role in their ecological success. The molecular mechanisms that support the social regulation of behavior in eusocial insects, and their evolution, are mostly unknown. The recent whole-genome sequencing of several eusocial insect species set the stage for deciphering the molecular and genetic bases of eusociality, and the possible evolutionary modifications that led to it. Studies of mRNA expression patterns in the brains of diverse eusocial insect species have indicated that specific social behavioral states of individual workers and queens are often associated with particular tissue-specific transcriptional profiles. Here, we discuss recent findings that highlight the role of non-coding microRNAs (miRNAs) in modulating traits associated with reproductive and behavioral divisions of labor in eusocial insects. We provide bioinformatic and phylogenetic data, which suggest that some Hymenoptera-specific miRNA may have contributed to the evolution of traits important for the evolution of eusociality in this group.

Negative impact of manganese on honeybee foraging.

Anthropogenic accumulation of metals such as manganese is a well-established health risk factor for vertebrates. By contrast, the long-term impact of these contaminants on invertebrates is mostly unknown. Here, we demonstrate that manganese ingestion alters brain biogenic amine levels in honeybees and fruit flies. Furthermore, we show that manganese exposure negatively affects foraging behaviour in the honeybee, an economically important pollinator. Our findings indicate that in addition to its direct impact on human health, the common industrial contaminant manganese might also have indirect environmental and economical impacts via the modulation of neuronal and behavioural functions in economically important insects.

Natural antisense transcripts regulate the neuronal stress response and excitability.

Neurons regulate ionic fluxes across their plasma membrane to maintain their excitable properties under varying environmental conditions. However, the mechanisms that regulate ion channels abundance remain poorly understood. Here we show that pickpocket 29 (ppk29), a gene that encodes a Drosophila degenerin/epithelial sodium channel (DEG/ENaC), regulates neuronal excitability via a protein-independent mechanism. We demonstrate that the mRNA 3'UTR of ppk29 affects neuronal firing rates and associated heat-induced seizures by acting as a natural antisense transcript (NAT) that regulates the neuronal mRNA levels of seizure (sei), the Drosophila homolog of the human Ether-à-go-go Related Gene (hERG) potassium channel. We find that the regulatory impact of ppk29 mRNA on sei is independent of the sodium channel it encodes. Thus, our studies reveal a novel mRNA dependent mechanism for the regulation of neuronal excitability that is independent of protein-coding capacity. DOI: http://dx.doi.org/10.7554/eLife.01849.001.

Feminization of pheromone-sensing neurons affects mating decisions in Drosophila males.

The response of individual animals to mating signals depends on the sexual identity of the individual and the genetics of the mating targets, which represent the mating social context (social environment). However, how social signals are sensed and integrated during mating decisions remains a mystery. One of the models for understanding mating behaviors in molecular and cellular terms is the male courtship ritual in the fruit fly (Drosophila melanogaster). We have recently shown that a subset of gustatory receptor neurons (GRNs) that are enriched in the male appendages and express the ion channel ppk23 play a major role in the initiation and maintenance of male courtship via the perception of cuticular contact pheromones, and are likely to represent the main chemosensory pathway that influences mating decisions by males. Here we show that genetic feminization of ppk23-expressing GRNs in male flies resulted in a significant increase in male-male sexual attraction without an apparent impact on sexual attraction to females. Furthermore, we show that this increase in male-male sexual attraction is sensory specific, which can be modulated by variable social contexts. Finally, we show that feminization of ppk23-expressing sensory neurons lead to major transcriptional shifts, which may explain the altered interpretation of the social environment by feminized males. Together, these data indicate that the sexual cellular identity of pheromone sensing GRNs plays a major role in how individual flies interpret their social environment in the context of mating decisions.