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The fight to understand fighting: neurogenetic approaches to the study of aggression in insects
Available online at www.sciencedirect.com
ScienceDirect The fight to understand fighting: neurogenetic approaches to the study of aggression in insects Lewis M Sherer and Sarah J Certel Aggression is an evolutionarily conserved behavior that evolved in the framework of defending or obtaining resources. When expressed out of context, unchecked aggression can have destructive consequences. Model systems that allow examination of distinct neuronal networks at the molecular, cellular, and circuit levels are adding immensely to our understanding of the biological basis of this behavior and should be relatable to other species up to and including man. Investigators have made particular use of insect models to both describe this quantifiable and stereotyped behavior and to manipulate genes and neuron function via numerous genetic and pharmacological tools. This review discusses recent advances in techniques that improve our ability to identify, manipulate, visualize, and compare the genes, neurons, and circuits that are required for the output of this complex and clinically relevant social behavior. Address Division of Biological Sciences, University of Montana, Missoula, MT 59812, United States Corresponding author: Certel, Sarah J ([email protected])
Current Opinion in Insect Science 2019, 36:18–24 This review comes from a themed issue on Neuroscience
injury states, aggression can have destructive physical, emotional, and financial consequences [4,5]. Given both the global health implications posed by pathological levels of aggression and decades of research on aggressive behavior in a range of systems, it is perhaps surprising that many fundamental questions remain unanswered. Such questions include whether there are circuits or neurons dedicated solely to producing or inhibiting aggression and, if so, whether the basic molecular and physiological functions of such neurons or circuits will eventually be understood. The fundamental properties of aggressive behavior itself present unique challenges to researchers. Aggression is not a singular unit but rather the result of interactions between at least two individuals. This interaction requires that members of an interacting pair must decide whether to fight or flee and this decision can be impacted by previous interactions. Addressing such experimental challenges through the isolation of naı¨ve adults has been a strength of insect systems as any previously established encounters that may have established hierarchical relationships are eliminated as well as concerns regarding maternal care variations that can occur in vertebrate systems [6,7].
Edited by Wolf Huetteroth and Dennis Pauls
https://doi.org/10.1016/j.cois.2019.06.004 2214-5745/ã 2019 Elsevier Inc. All rights reserved.
Introduction Aggression is an innate behavior seen in essentially all species of animals where it is commonly used for access to food and shelter, for protection from predation and for access to potential mates [1–3]. Although aggressive behavior between individuals of the same species can improve survival odds and reproductive opportunities, it is an energetically expensive behavior with injury or death as possible outcomes. Therefore, the implementation of this behavior requires an assessment of the costs and benefits as well as physiological mechanisms in place to constrain aggression. This constraint of aggression receives considerable attention in humans as when expressed out of context, due to disease or Current Opinion in Insect Science 2019, 36:18–24
Significant progress in understanding fundamental aspects of aggressive behavior has been made using insects. As a whole, insect models have led the way in demonstrating at the physiological and now circuit level, that aggression-related decision-making and experience-dependent changes in aggression motivation are influenced, regulated, and controlled by genes, neurotransmitters, hormones, and neuromodulators. Specifically, many physiological processes and conserved behaviors including aggression have been shown to be under the control of monoamine neuromodulators including dopamine, serotonin, and octopamine (analogous to norepinephrine) [8,9]. In addition, the aggression versus cooperation phenotype of ant queens has provided an advantage to study behavioral plasticity while crickets have provided a wealth of information regarding the specific pharmacology of neurotransmitters and receptors [10–12]. Finally, the success of behavioral experiments in insects has been significantly impacted by the implementation of an increasing repertoire of sophisticated experimental techniques. Below we focus on advances in new and established genetic tools and approaches that will continue to move the field of insect aggression rapidly forward (Figure 1). www.sciencedirect.com
Neurogenetic tools and aggression: studies in insects Sherer and Certel 19
Figure 1
Genomic Approaches
Circuit Function
Targeting Specific Neurons
Current Opinion in Insect Science
Insect models provide an increasingly diverse toolset with which to examine the neurological basis of aggression. Approaches examining the genomic correlates to aggression are particularly effective in insect models due to highly stereotyped behaviors and well-established mutagenic screening methods. The genetic toolkit available to Drosophila makes it an excellent model to determine the contribution of small, specific groups of neurons to aggression. The simplified nervous system of insects is a critical advantage when identifying neurons within aggression circuits. Fighting crickets were adopted with permission from Rillich and Stevenso, Neuroforum [28] and the fighting Drosophila males from Hoyer et al. [61].
Advances in genomic approaches The relatively simple genomes and stereotyped behaviors of insects provide formidable strengths when identifying genes that regulate aggression. The model insect, Drosophila melanogaster, is a mainstay in behavioral studies that focus on gene identification due in part to its powerful genetic toolbox and its comprehensively sequenced genome. For example, different screening methods have identified that loss-of-function mutations in Cyp6a20 [13], tailless [14], and a putative transmembrane transporter, CG13646 [15] increase aggression levels. However, the genetic analysis of aggression is not limited to Drosophila and insects across orders including Coleoptera (beetles), Hymenoptera (bees, ants, etc.), and Orthoptera (locusts, crickets) bring unique and overlapping strengths as described below. Forward genetic chemical mutagenic screens in Drosophila have historically been a powerful way to identify genes critical for a diverse array of biological processes. However, screening for behavioral changes such as www.sciencedirect.com
differences in aggression levels in individual lines of mutagenized lines is incredibly time consuming. In a recent study, Davis et al., used a physical phenotype, the presence of wing damage in group-housed Drosophila males, as a rapid visual proxy of high levels of aggression [16]. After screening 1400 independent mutant strains, five lines that exhibited a significant amount of wing damage were selected for whole-genome sequencing to identify the causal mutations. A novel mutation in the Shaker locus (which encodes a voltage-gated potassium channel [17]) was identified. This approach is noteworthy for two reasons. First, a rapid forward genetic screen combined with whole genome sequencing can be efficiently used to identify genes that control a complex behavior. Second, novel mutations in known genes may be uncovered that provide new information on gene function which is a strength of chemical mutagenesis. Just as changes in gene function can alter aggression, so can changes in gene expression. While the innate behavior of aggression is genetically ‘hard-wired’, this complex behavior is also socially regulated [18–20]. Queens of the harvester ant Pogonomyrmex californicus display the alternate social behavior of cooperation and aggression, making this insect an ideal system to examine how context-dependent variation in behavior is controlled. By sequencing whole-head RNA from aggressive and non-aggressive ant queens, variations in gene expression patterns were identified [21]. This gene expression data were then compared to the sequenced genome datasets of D. melanogaster to identify a potential group of crossspecies regulatory genes that may be altered in the states of cooperation versus aggression. By examining the variables of social environment, aggressive behavior, and population of origin, the authors identified modules of co-regulated genes involved in metabolism, gene regulation, immune response, and neuron function [22,23]. While the impact of the gene expression changes in the harvester ant are currently only correlative, it may soon be possible to directly test the requirement of these candidates for context-dependent behavior as genomeediting has been successfully used to generate mutations in the clonal raider ant, Ooceraea biroi [24]. Although gene networks controlling social behavior are likely to be different between unrelated species, a correlative system-genetics approach can be used to uncover conserved nodes or modules of transcriptionally interconnected genes that correlate with complex behaviors including aggression. The underlying molecular mechanisms of aggression are of central interest to a wide range of disciplines including behavioral genetics, evolution, neuroscience, medicine, psychology, and criminology, among others. An oft-asked question is whether the molecular mechanisms described in insects are conserved more broadly. The emerging field of sociogenomics explores the relations between Current Opinion in Insect Science 2019, 36:18–24
20 Neuroscience
social behavior and genome structure-function. A key external stimulus that induces a decision to flee or fight, is the response to alarm pheromones (a conspecific signal that induces aggressive behavior across the eusocial Hymenoptera [25]), Using a computational approach, Liu et al., used an orthology analysis of previously published microarray data on honey bee brain gene expression that changed due to exposure to alarm pheromones [26]. A disproportionately large number of genes within the bee dataset were also found in mammals. Genes that exhibited differential expression in response to alarm pheromones had a high degree of orthology with placental mammals, but not with nonsocial insects. A portion of these highly conserved genes are orthologous to genes linked to human neural and behavioral disorders in which altered aggression levels can occur [26]. Regardless of any potential impact on human disease, adaptations of classical methods as well as computational approaches to compare genomes will likely provide opportunities to uncover previously unknown genes and genetic networks that further our understanding of the molecular underpinnings of aggression.
Advances in targeting specific neurons Aggression in all systems is influenced by numerous neurotransmitters, modulators and hormones [27,28]. Assigning a specific role in aggression to a neurotransmitter or neuromodulator has been challenging due to at least two complications: one, separating the function of a neurotransmitter in the brain versus at the neuromuscular junction and two, a single neuron, for example, a monoamine-expressing neuron, may have widespread arborization patterns that could impact many downstream targets. Pharmacological manipulation of various neurotransmitters and their multiple, functionally distinct receptors have provided significant progress in deciphering these questions. However, the precision of genetic tools will be key to determining whether neurons solely dedicated to promoting or inhibiting aggression exist in the nervous system. In Drosophila, the Gal4–UAS system has been used for more than 25 years to manipulate neuron function with temporal control and varying levels of cell-type specificity. This section reviews recent modifications to the Gal4–UAS system that allow further refinement of gene expression or neuron activity that can or are being used in aggression studies (Table 1). Briefly, Gal4–UAS is a modular expression system, in which the yeast transcription factor, Gal4, is placed under control of an endogenous gene promotor or regulatory region [29]. When expressed, Gal4 activates transcription of any gene product of interest placed downstream of the Gal4-binding upstream activating sequence (UAS). To increase spatial control, Gal4-mediated transcription can be suppressed in specific cells by incorporating the inhibitor Gal80 [30]. Gal4–UAS-compatible site-directed recombination strategies have also been developed, Current Opinion in Insect Science 2019, 36:18–24
consisting of gene sequences flanked by flippase recognition target (FRT) sites along with the recombinase flippase (FLP) placed under Gal4 control. To increase the refinement of gene or neuron manipulation or simultaneously manipulate two neuronal populations, additional independent modular expression systems including the bacterial LexA-lexAop and the Neurospora Q-system have been developed [31,32]. Although hundreds of Gal4 driver lines are publicly available, many lines have widespread expression patterns that prohibit the manipulation of small numbers of neurons. In addition, many drivers express Gal4 not only in the brain but in other tissues as well, making it difficult to conclusively determine the neuronal contribution to a specific behavior. To solve these problems, a number of intersectional modifications have been developed to refine the expression patterns of the Gal4, LexA and Q systems. One modification that increases the spatial and temporal specificity of Gal4 is the Split-Gal4 system. In the SplitGal4 system, the Gal4 transcription factor is separated into its two basic components: the DNA-binding-domain (DBD) and the transcriptional activation domain (AD) [33]. Both of these domains are fused to a leucine zipper motif and separately expressed using two different enhancers or promoters. When co-expressed in the same cell, the leucine zipper motifs bring together the Gal4–DBD and Gal4–AD components to form a functional Gal4 protein that can activate a UAS-reporter. While the Split-Gal4 system is not new, this modification has been recently used to address a significant gap in the study of aggression, namely the identification of neurons that are required for a distinct behavioral module. The vast majority of gene or neuron manipulations in any insect system have resulted in either increased or decreased overall levels of aggression. Duistermars et al., screened many Split-Gal4 lines and identified one combination, R20E08-DBD plus R22D03-AD, that allowed the manipulation of a very small number of neurons in the adult brain whose activity is required for the wing threat module but not for other aggressive patterns [34]. This result indicates that specific aggressive modules can be activated from a multifunctional neuronal network. Refining the Drosophila intersectional strategy to restrict gene or activity levels has also been achieved by combining multiple FLP-recombinase expressing lines. When meeting an individual of the same species, males must decide whether to engage in aggression (if the second fly is a male) or courtship. In an elegant study, Koganezawa et al., used multiple FLP lines to identify neurons required for these mutually exclusive behaviors [36]. ocelliless (Otd)-FLP, described in an earlier study [35], was used to restrict neurons spatially, and fru (the male-specific isoform of fruitless)-FLP was used to manipulate neurons with a male-specific signature [36]. Fruitless was chosen as it has previously been shown that sex-specific splicing of www.sciencedirect.com
Neurogenetic tools and aggression: studies in insects Sherer and Certel 21
Table 1 Neurogenetic tools in insects Tool
Description (currently for use with D. melanogaster unless otherwise noted)
Neurogenetic applications
Gal4/UAS
Binary expression system
lexA/lexAop
Binary expression system. Can be used in conjunction with Gal4/UAS and/or QF/QUAS for intersectional genetic manipulations Binary expression system. Can be used in conjunction with Gal4/UAS and/or lexA/lexAop for intersectional genetic manipulations Binary expression system repressors. Gal80 and QS inhibit the actions of Gal4 and QF, respectively FLP is a recombinase that excises DNA sequences flanked by two identical FRT sites
Reduce or overexpress genes of interest or manipulate neuronal activity Used to manipulate gene expression or neuronal activity. Target smaller neuronal subsets when used with other binary systems
QF/QUAS
Gal80/QS FLP–FRT
Split-Gal4
Tet-off Gal80
trans-Tango Immediate early-gene (IEG) promotor-driven reporter
Ternary expression system that makes Gal4mediated transcriptional activity contingent upon two enhancers, rather than one Tetracycline-inducible expression system that inhibits expression of Gal80, permitting Gal4mediated gene expression Trans-synaptic circuit mapping tool Activity-dependent reporter system in Gryllus bimaculatus
the fruitless gene (FruM) plays a critical role in Drosophila male aggression and courtship behavior [37,38]. In invertebrates and vertebrates, it is well accepted that sexually dimorphic circuits contribute to the regulation of sexually dimorphic behaviors such as aggression and courtship [39–41]. In addition to D. melanogaster, sex-specific aggression patterns have been reported in the stalk-eyed fly [42] and female field cricket (Teleogryllus oceanicus) [43]. The male-specific function of FruM may be conserved as similar sex-specific gene products of the fruitless homolog have been identified in other holometabolous insects such as the mosquito and hymenopteran insects [44–46], and fruitless is required for male courtship behavior in the German cockroach, Blattella germanica [47]. How might the output of two mutually exclusive sexually dimorphic behaviors be coordinated? In the Koganezawa et al., study, flipping the switch between male aggression and courtship required the activity of distinct FruM+ neuronal clusters acting on FruM (for aggression) and FruM+ (for courtship) subsets within a doublesex+ neuronal cluster, pC1. Specifically, activation of the aggression versus courtship centers occurred through a doublelayered inhibitory switch composed of two fru singlepositive clusters, LC1 and mAL. In this circuit, two fru-positive GABAergic neuron clusters, LC1 and mAL, act as a layered inhibitory switch to either activate aggression and suppress courtship via suppression of mAL (in the case of the LC1 cluster) or suppress both www.sciencedirect.com
Manipulation of gene expression or neuronal activity
Restrict Gal4/QF-mediated gene expression or neuron activity Site-directed recombination system that restricts gene expression to increasingly refined neuronal subsets when used with a binary system Manipulation of gene expression or neuronal activity. Two enhancer/promotor-driven Gal4 components restrict genetic manipulations to smaller subsets of neurons This tool permits the restriction of Gal4-mediated gene expression during specific life stages without altering temperature Visualization and manipulation of post-synaptic targets to identify downstream targets within a behavioral circuit Identification and visualization of downstream neurons in response to neuron activation
aggression and courtship via suppression of pC1 neurons (in the case of the mAL cluster). It was proposed that layering GABAergic inhibition between the two classes of inhibitory interneurons could potentially increase the probability that the appropriate behavior will be selected as well as increase the capacity for modulation by external and internal cues [36]. It should be noted that, while the developmental effects of activation were prevented through the use of a temperature-sensitive cation channel, behaviors such as aggression and courtship are sensitive to heat. Optogenetic effectors [48] or a recently developed tetracycline-inducible Gal80 [49] could be used in future aggression and courtship studies to mitigate the effects of temperature on adult social behavior. Regardless, restricting neurons through this layered approach functionally separated small subsets of neurons in a behavioral pathway as well as parsed out the complex nature of neurotransmitter function within a circuit that would not have been possible by activating fru-positive neurons alone.
Advances in connectomics How the animal brain processes environmental and internal signals to generate any innate behavior is a fundamental question in neuroscience. Although a ‘wiring diagram’ or ‘connectome’ alone cannot fully explain how a circuit or network generates complex behavior, it does provide a roadmap that can be systematically interrogated. The numerical simplicity of the insect nervous system, the variety of sophisticated genetic tools that Current Opinion in Insect Science 2019, 36:18–24
22 Neuroscience
have and are being developed, and importantly the ease with which connecting neurons can be manipulated, make insects ideal models to study the neural circuitry controlling behavior. This section reviews circuit mapping advances within insect models. Identifying synaptic connections between neurons is crucial for identifying and mapping circuits that promote or regulate aggression. In Drosophila, methods such as tracer dyes or GFP Reconstitution Across Synaptic Partners (GRASP) have been successfully used to identify putative synaptic connections between neurons required for aggressive behavior [50–52]. However, these methods are hindered by low-throughput, low-resolution, and importantly in the case of GRASP, some level of a priori knowledge regarding postsynaptic partners is required. Recently, the trans-Tango system has been developed to permit the visualization of synaptic interactions [53]. Key improvements of the trans-Tango system over GRASP include the ability to visualize the connecting neuron rather than just the reconstituted GFP at the putative synapse as well as the flexibility to identify synaptic partners without any assumptions or previous knowledge about the nature of these connections. Additionally, by combining trans-Tango with other binary reporter systems such as LexA-LexAop, it is possible to identify both the regions of the Drosophila brain that are important for a behavior and the identity of the neurons within those regions. To date, trans-Tango has been successfully used to examine the modulation of postfeeding physiology and behavior as well as identify putative second-order taste neurons [53,54]. We anticipate this powerful tool for anterograde trans-synaptic labeling of neurons is being applied to aggression circuit mapping. Only electron microscopy (EM) provides the complete, unbiased mapping of synaptic connectivity in any organism. In the past, whole-brain connectomes have been limited to a few small organisms, such as the nematode C. elegans, the Drosophila larva, and the tadpole larva of the tunicate Ciona intestinalis [55–57]. Even the small Drosophila adult brain at 100 000 neurons was large for conventional EM analysis. However, Zheng et al., recently developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution [58]. The initial results from this wealth of information has been the identification of a new type of neuronal cell in the Drosophila mushroom body as well as three classes of never-before-seen synapses. Access to the full adult fly brain dataset and analysis code is publicly available [58] and we anticipate exciting new insights using this EM-level brain-spanning resolution into the circuit-based control of complex behaviors including aggression will occur in the next few years. Even with a comprehensive connectome of an organism, the question remains whether a synaptic connection is functionally relevant for a behavior. With this issue in Current Opinion in Insect Science 2019, 36:18–24
mind, a novel transgenic reporter system for whole-brain activity mapping in the cricket Gryllus bimaculatus has recently been developed (Table 1) [59]. Neuronal immediate-early activity regulated genes in the cricket were first identified to allow for the characterization of an activity-regulated promoter. Then using the promoter region of Gryllus egr-B and a nuclear-targeted destabilized EYFP as a reporter, a retroactive, whole-mount, singlecell-resolution activity mapping system was generated. Using this system, expression of the transgenic reporter was induced as a result of feeding and agonistic interactions. The activated neurons are part of the octopaminergic circuitry which plays a major role in promoting aggression in the cricket as well as other insects providing an important proof-of-principle verification of this technique [11,60]. In summation, the use of this activity mapping system will allow for the identification of behaviorally relevant neuronal circuits in the cricket brain and potentially other non-Drosophila insects.
Conclusions The French author Antoine de Saint Exupe´ry observed that the development of tools “ . . . does not isolate [us] from the great problems of nature but plunges [us] more deeply into them.” Indeed, current as well as new approaches in the coming years will need to be applied across models and disciplines to identify circuits or neurons dedicated solely to producing aggression and, importantly, how disturbances in such circuits lead to unchecked or pathological aggression. As work progresses, it will be exciting to see the path addressing the fundamental mechanisms of aggression and the connected path examining the modification of aggression by pharmacologic interventions converge and ultimately impact the study and treatment of pathological aggression in humans.
Conflict of interest statement Nothing declared.
Acknowledgements We thank members of the Certel lab and two anonymous referees for helpful suggestions. The Certel laboratory is funded by N.I.H.R01 GM115510 grant.
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ScienceDirect The fight to understand fighting: neurogenetic approaches to the study of aggression in insects Lewis M Sherer and Sarah J Certel Aggression is an evolutionarily conserved behavior that evolved in the framework of defending or obtaining resources. When expressed out of context, unchecked aggression can have destructive consequences. Model systems that allow examination of distinct neuronal networks at the molecular, cellular, and circuit levels are adding immensely to our understanding of the biological basis of this behavior and should be relatable to other species up to and including man. Investigators have made particular use of insect models to both describe this quantifiable and stereotyped behavior and to manipulate genes and neuron function via numerous genetic and pharmacological tools. This review discusses recent advances in techniques that improve our ability to identify, manipulate, visualize, and compare the genes, neurons, and circuits that are required for the output of this complex and clinically relevant social behavior. Address Division of Biological Sciences, University of Montana, Missoula, MT 59812, United States Corresponding author: Certel, Sarah J ([email protected])
Current Opinion in Insect Science 2019, 36:18–24 This review comes from a themed issue on Neuroscience
injury states, aggression can have destructive physical, emotional, and financial consequences [4,5]. Given both the global health implications posed by pathological levels of aggression and decades of research on aggressive behavior in a range of systems, it is perhaps surprising that many fundamental questions remain unanswered. Such questions include whether there are circuits or neurons dedicated solely to producing or inhibiting aggression and, if so, whether the basic molecular and physiological functions of such neurons or circuits will eventually be understood. The fundamental properties of aggressive behavior itself present unique challenges to researchers. Aggression is not a singular unit but rather the result of interactions between at least two individuals. This interaction requires that members of an interacting pair must decide whether to fight or flee and this decision can be impacted by previous interactions. Addressing such experimental challenges through the isolation of naı¨ve adults has been a strength of insect systems as any previously established encounters that may have established hierarchical relationships are eliminated as well as concerns regarding maternal care variations that can occur in vertebrate systems [6,7].
Edited by Wolf Huetteroth and Dennis Pauls
https://doi.org/10.1016/j.cois.2019.06.004 2214-5745/ã 2019 Elsevier Inc. All rights reserved.
Introduction Aggression is an innate behavior seen in essentially all species of animals where it is commonly used for access to food and shelter, for protection from predation and for access to potential mates [1–3]. Although aggressive behavior between individuals of the same species can improve survival odds and reproductive opportunities, it is an energetically expensive behavior with injury or death as possible outcomes. Therefore, the implementation of this behavior requires an assessment of the costs and benefits as well as physiological mechanisms in place to constrain aggression. This constraint of aggression receives considerable attention in humans as when expressed out of context, due to disease or Current Opinion in Insect Science 2019, 36:18–24
Significant progress in understanding fundamental aspects of aggressive behavior has been made using insects. As a whole, insect models have led the way in demonstrating at the physiological and now circuit level, that aggression-related decision-making and experience-dependent changes in aggression motivation are influenced, regulated, and controlled by genes, neurotransmitters, hormones, and neuromodulators. Specifically, many physiological processes and conserved behaviors including aggression have been shown to be under the control of monoamine neuromodulators including dopamine, serotonin, and octopamine (analogous to norepinephrine) [8,9]. In addition, the aggression versus cooperation phenotype of ant queens has provided an advantage to study behavioral plasticity while crickets have provided a wealth of information regarding the specific pharmacology of neurotransmitters and receptors [10–12]. Finally, the success of behavioral experiments in insects has been significantly impacted by the implementation of an increasing repertoire of sophisticated experimental techniques. Below we focus on advances in new and established genetic tools and approaches that will continue to move the field of insect aggression rapidly forward (Figure 1). www.sciencedirect.com
Neurogenetic tools and aggression: studies in insects Sherer and Certel 19
Figure 1
Genomic Approaches
Circuit Function
Targeting Specific Neurons
Current Opinion in Insect Science
Insect models provide an increasingly diverse toolset with which to examine the neurological basis of aggression. Approaches examining the genomic correlates to aggression are particularly effective in insect models due to highly stereotyped behaviors and well-established mutagenic screening methods. The genetic toolkit available to Drosophila makes it an excellent model to determine the contribution of small, specific groups of neurons to aggression. The simplified nervous system of insects is a critical advantage when identifying neurons within aggression circuits. Fighting crickets were adopted with permission from Rillich and Stevenso, Neuroforum [28] and the fighting Drosophila males from Hoyer et al. [61].
Advances in genomic approaches The relatively simple genomes and stereotyped behaviors of insects provide formidable strengths when identifying genes that regulate aggression. The model insect, Drosophila melanogaster, is a mainstay in behavioral studies that focus on gene identification due in part to its powerful genetic toolbox and its comprehensively sequenced genome. For example, different screening methods have identified that loss-of-function mutations in Cyp6a20 [13], tailless [14], and a putative transmembrane transporter, CG13646 [15] increase aggression levels. However, the genetic analysis of aggression is not limited to Drosophila and insects across orders including Coleoptera (beetles), Hymenoptera (bees, ants, etc.), and Orthoptera (locusts, crickets) bring unique and overlapping strengths as described below. Forward genetic chemical mutagenic screens in Drosophila have historically been a powerful way to identify genes critical for a diverse array of biological processes. However, screening for behavioral changes such as www.sciencedirect.com
differences in aggression levels in individual lines of mutagenized lines is incredibly time consuming. In a recent study, Davis et al., used a physical phenotype, the presence of wing damage in group-housed Drosophila males, as a rapid visual proxy of high levels of aggression [16]. After screening 1400 independent mutant strains, five lines that exhibited a significant amount of wing damage were selected for whole-genome sequencing to identify the causal mutations. A novel mutation in the Shaker locus (which encodes a voltage-gated potassium channel [17]) was identified. This approach is noteworthy for two reasons. First, a rapid forward genetic screen combined with whole genome sequencing can be efficiently used to identify genes that control a complex behavior. Second, novel mutations in known genes may be uncovered that provide new information on gene function which is a strength of chemical mutagenesis. Just as changes in gene function can alter aggression, so can changes in gene expression. While the innate behavior of aggression is genetically ‘hard-wired’, this complex behavior is also socially regulated [18–20]. Queens of the harvester ant Pogonomyrmex californicus display the alternate social behavior of cooperation and aggression, making this insect an ideal system to examine how context-dependent variation in behavior is controlled. By sequencing whole-head RNA from aggressive and non-aggressive ant queens, variations in gene expression patterns were identified [21]. This gene expression data were then compared to the sequenced genome datasets of D. melanogaster to identify a potential group of crossspecies regulatory genes that may be altered in the states of cooperation versus aggression. By examining the variables of social environment, aggressive behavior, and population of origin, the authors identified modules of co-regulated genes involved in metabolism, gene regulation, immune response, and neuron function [22,23]. While the impact of the gene expression changes in the harvester ant are currently only correlative, it may soon be possible to directly test the requirement of these candidates for context-dependent behavior as genomeediting has been successfully used to generate mutations in the clonal raider ant, Ooceraea biroi [24]. Although gene networks controlling social behavior are likely to be different between unrelated species, a correlative system-genetics approach can be used to uncover conserved nodes or modules of transcriptionally interconnected genes that correlate with complex behaviors including aggression. The underlying molecular mechanisms of aggression are of central interest to a wide range of disciplines including behavioral genetics, evolution, neuroscience, medicine, psychology, and criminology, among others. An oft-asked question is whether the molecular mechanisms described in insects are conserved more broadly. The emerging field of sociogenomics explores the relations between Current Opinion in Insect Science 2019, 36:18–24
20 Neuroscience
social behavior and genome structure-function. A key external stimulus that induces a decision to flee or fight, is the response to alarm pheromones (a conspecific signal that induces aggressive behavior across the eusocial Hymenoptera [25]), Using a computational approach, Liu et al., used an orthology analysis of previously published microarray data on honey bee brain gene expression that changed due to exposure to alarm pheromones [26]. A disproportionately large number of genes within the bee dataset were also found in mammals. Genes that exhibited differential expression in response to alarm pheromones had a high degree of orthology with placental mammals, but not with nonsocial insects. A portion of these highly conserved genes are orthologous to genes linked to human neural and behavioral disorders in which altered aggression levels can occur [26]. Regardless of any potential impact on human disease, adaptations of classical methods as well as computational approaches to compare genomes will likely provide opportunities to uncover previously unknown genes and genetic networks that further our understanding of the molecular underpinnings of aggression.
Advances in targeting specific neurons Aggression in all systems is influenced by numerous neurotransmitters, modulators and hormones [27,28]. Assigning a specific role in aggression to a neurotransmitter or neuromodulator has been challenging due to at least two complications: one, separating the function of a neurotransmitter in the brain versus at the neuromuscular junction and two, a single neuron, for example, a monoamine-expressing neuron, may have widespread arborization patterns that could impact many downstream targets. Pharmacological manipulation of various neurotransmitters and their multiple, functionally distinct receptors have provided significant progress in deciphering these questions. However, the precision of genetic tools will be key to determining whether neurons solely dedicated to promoting or inhibiting aggression exist in the nervous system. In Drosophila, the Gal4–UAS system has been used for more than 25 years to manipulate neuron function with temporal control and varying levels of cell-type specificity. This section reviews recent modifications to the Gal4–UAS system that allow further refinement of gene expression or neuron activity that can or are being used in aggression studies (Table 1). Briefly, Gal4–UAS is a modular expression system, in which the yeast transcription factor, Gal4, is placed under control of an endogenous gene promotor or regulatory region [29]. When expressed, Gal4 activates transcription of any gene product of interest placed downstream of the Gal4-binding upstream activating sequence (UAS). To increase spatial control, Gal4-mediated transcription can be suppressed in specific cells by incorporating the inhibitor Gal80 [30]. Gal4–UAS-compatible site-directed recombination strategies have also been developed, Current Opinion in Insect Science 2019, 36:18–24
consisting of gene sequences flanked by flippase recognition target (FRT) sites along with the recombinase flippase (FLP) placed under Gal4 control. To increase the refinement of gene or neuron manipulation or simultaneously manipulate two neuronal populations, additional independent modular expression systems including the bacterial LexA-lexAop and the Neurospora Q-system have been developed [31,32]. Although hundreds of Gal4 driver lines are publicly available, many lines have widespread expression patterns that prohibit the manipulation of small numbers of neurons. In addition, many drivers express Gal4 not only in the brain but in other tissues as well, making it difficult to conclusively determine the neuronal contribution to a specific behavior. To solve these problems, a number of intersectional modifications have been developed to refine the expression patterns of the Gal4, LexA and Q systems. One modification that increases the spatial and temporal specificity of Gal4 is the Split-Gal4 system. In the SplitGal4 system, the Gal4 transcription factor is separated into its two basic components: the DNA-binding-domain (DBD) and the transcriptional activation domain (AD) [33]. Both of these domains are fused to a leucine zipper motif and separately expressed using two different enhancers or promoters. When co-expressed in the same cell, the leucine zipper motifs bring together the Gal4–DBD and Gal4–AD components to form a functional Gal4 protein that can activate a UAS-reporter. While the Split-Gal4 system is not new, this modification has been recently used to address a significant gap in the study of aggression, namely the identification of neurons that are required for a distinct behavioral module. The vast majority of gene or neuron manipulations in any insect system have resulted in either increased or decreased overall levels of aggression. Duistermars et al., screened many Split-Gal4 lines and identified one combination, R20E08-DBD plus R22D03-AD, that allowed the manipulation of a very small number of neurons in the adult brain whose activity is required for the wing threat module but not for other aggressive patterns [34]. This result indicates that specific aggressive modules can be activated from a multifunctional neuronal network. Refining the Drosophila intersectional strategy to restrict gene or activity levels has also been achieved by combining multiple FLP-recombinase expressing lines. When meeting an individual of the same species, males must decide whether to engage in aggression (if the second fly is a male) or courtship. In an elegant study, Koganezawa et al., used multiple FLP lines to identify neurons required for these mutually exclusive behaviors [36]. ocelliless (Otd)-FLP, described in an earlier study [35], was used to restrict neurons spatially, and fru (the male-specific isoform of fruitless)-FLP was used to manipulate neurons with a male-specific signature [36]. Fruitless was chosen as it has previously been shown that sex-specific splicing of www.sciencedirect.com
Neurogenetic tools and aggression: studies in insects Sherer and Certel 21
Table 1 Neurogenetic tools in insects Tool
Description (currently for use with D. melanogaster unless otherwise noted)
Neurogenetic applications
Gal4/UAS
Binary expression system
lexA/lexAop
Binary expression system. Can be used in conjunction with Gal4/UAS and/or QF/QUAS for intersectional genetic manipulations Binary expression system. Can be used in conjunction with Gal4/UAS and/or lexA/lexAop for intersectional genetic manipulations Binary expression system repressors. Gal80 and QS inhibit the actions of Gal4 and QF, respectively FLP is a recombinase that excises DNA sequences flanked by two identical FRT sites
Reduce or overexpress genes of interest or manipulate neuronal activity Used to manipulate gene expression or neuronal activity. Target smaller neuronal subsets when used with other binary systems
QF/QUAS
Gal80/QS FLP–FRT
Split-Gal4
Tet-off Gal80
trans-Tango Immediate early-gene (IEG) promotor-driven reporter
Ternary expression system that makes Gal4mediated transcriptional activity contingent upon two enhancers, rather than one Tetracycline-inducible expression system that inhibits expression of Gal80, permitting Gal4mediated gene expression Trans-synaptic circuit mapping tool Activity-dependent reporter system in Gryllus bimaculatus
the fruitless gene (FruM) plays a critical role in Drosophila male aggression and courtship behavior [37,38]. In invertebrates and vertebrates, it is well accepted that sexually dimorphic circuits contribute to the regulation of sexually dimorphic behaviors such as aggression and courtship [39–41]. In addition to D. melanogaster, sex-specific aggression patterns have been reported in the stalk-eyed fly [42] and female field cricket (Teleogryllus oceanicus) [43]. The male-specific function of FruM may be conserved as similar sex-specific gene products of the fruitless homolog have been identified in other holometabolous insects such as the mosquito and hymenopteran insects [44–46], and fruitless is required for male courtship behavior in the German cockroach, Blattella germanica [47]. How might the output of two mutually exclusive sexually dimorphic behaviors be coordinated? In the Koganezawa et al., study, flipping the switch between male aggression and courtship required the activity of distinct FruM+ neuronal clusters acting on FruM (for aggression) and FruM+ (for courtship) subsets within a doublesex+ neuronal cluster, pC1. Specifically, activation of the aggression versus courtship centers occurred through a doublelayered inhibitory switch composed of two fru singlepositive clusters, LC1 and mAL. In this circuit, two fru-positive GABAergic neuron clusters, LC1 and mAL, act as a layered inhibitory switch to either activate aggression and suppress courtship via suppression of mAL (in the case of the LC1 cluster) or suppress both www.sciencedirect.com
Manipulation of gene expression or neuronal activity
Restrict Gal4/QF-mediated gene expression or neuron activity Site-directed recombination system that restricts gene expression to increasingly refined neuronal subsets when used with a binary system Manipulation of gene expression or neuronal activity. Two enhancer/promotor-driven Gal4 components restrict genetic manipulations to smaller subsets of neurons This tool permits the restriction of Gal4-mediated gene expression during specific life stages without altering temperature Visualization and manipulation of post-synaptic targets to identify downstream targets within a behavioral circuit Identification and visualization of downstream neurons in response to neuron activation
aggression and courtship via suppression of pC1 neurons (in the case of the mAL cluster). It was proposed that layering GABAergic inhibition between the two classes of inhibitory interneurons could potentially increase the probability that the appropriate behavior will be selected as well as increase the capacity for modulation by external and internal cues [36]. It should be noted that, while the developmental effects of activation were prevented through the use of a temperature-sensitive cation channel, behaviors such as aggression and courtship are sensitive to heat. Optogenetic effectors [48] or a recently developed tetracycline-inducible Gal80 [49] could be used in future aggression and courtship studies to mitigate the effects of temperature on adult social behavior. Regardless, restricting neurons through this layered approach functionally separated small subsets of neurons in a behavioral pathway as well as parsed out the complex nature of neurotransmitter function within a circuit that would not have been possible by activating fru-positive neurons alone.
Advances in connectomics How the animal brain processes environmental and internal signals to generate any innate behavior is a fundamental question in neuroscience. Although a ‘wiring diagram’ or ‘connectome’ alone cannot fully explain how a circuit or network generates complex behavior, it does provide a roadmap that can be systematically interrogated. The numerical simplicity of the insect nervous system, the variety of sophisticated genetic tools that Current Opinion in Insect Science 2019, 36:18–24
22 Neuroscience
have and are being developed, and importantly the ease with which connecting neurons can be manipulated, make insects ideal models to study the neural circuitry controlling behavior. This section reviews circuit mapping advances within insect models. Identifying synaptic connections between neurons is crucial for identifying and mapping circuits that promote or regulate aggression. In Drosophila, methods such as tracer dyes or GFP Reconstitution Across Synaptic Partners (GRASP) have been successfully used to identify putative synaptic connections between neurons required for aggressive behavior [50–52]. However, these methods are hindered by low-throughput, low-resolution, and importantly in the case of GRASP, some level of a priori knowledge regarding postsynaptic partners is required. Recently, the trans-Tango system has been developed to permit the visualization of synaptic interactions [53]. Key improvements of the trans-Tango system over GRASP include the ability to visualize the connecting neuron rather than just the reconstituted GFP at the putative synapse as well as the flexibility to identify synaptic partners without any assumptions or previous knowledge about the nature of these connections. Additionally, by combining trans-Tango with other binary reporter systems such as LexA-LexAop, it is possible to identify both the regions of the Drosophila brain that are important for a behavior and the identity of the neurons within those regions. To date, trans-Tango has been successfully used to examine the modulation of postfeeding physiology and behavior as well as identify putative second-order taste neurons [53,54]. We anticipate this powerful tool for anterograde trans-synaptic labeling of neurons is being applied to aggression circuit mapping. Only electron microscopy (EM) provides the complete, unbiased mapping of synaptic connectivity in any organism. In the past, whole-brain connectomes have been limited to a few small organisms, such as the nematode C. elegans, the Drosophila larva, and the tadpole larva of the tunicate Ciona intestinalis [55–57]. Even the small Drosophila adult brain at 100 000 neurons was large for conventional EM analysis. However, Zheng et al., recently developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution [58]. The initial results from this wealth of information has been the identification of a new type of neuronal cell in the Drosophila mushroom body as well as three classes of never-before-seen synapses. Access to the full adult fly brain dataset and analysis code is publicly available [58] and we anticipate exciting new insights using this EM-level brain-spanning resolution into the circuit-based control of complex behaviors including aggression will occur in the next few years. Even with a comprehensive connectome of an organism, the question remains whether a synaptic connection is functionally relevant for a behavior. With this issue in Current Opinion in Insect Science 2019, 36:18–24
mind, a novel transgenic reporter system for whole-brain activity mapping in the cricket Gryllus bimaculatus has recently been developed (Table 1) [59]. Neuronal immediate-early activity regulated genes in the cricket were first identified to allow for the characterization of an activity-regulated promoter. Then using the promoter region of Gryllus egr-B and a nuclear-targeted destabilized EYFP as a reporter, a retroactive, whole-mount, singlecell-resolution activity mapping system was generated. Using this system, expression of the transgenic reporter was induced as a result of feeding and agonistic interactions. The activated neurons are part of the octopaminergic circuitry which plays a major role in promoting aggression in the cricket as well as other insects providing an important proof-of-principle verification of this technique [11,60]. In summation, the use of this activity mapping system will allow for the identification of behaviorally relevant neuronal circuits in the cricket brain and potentially other non-Drosophila insects.
Conclusions The French author Antoine de Saint Exupe´ry observed that the development of tools “ . . . does not isolate [us] from the great problems of nature but plunges [us] more deeply into them.” Indeed, current as well as new approaches in the coming years will need to be applied across models and disciplines to identify circuits or neurons dedicated solely to producing aggression and, importantly, how disturbances in such circuits lead to unchecked or pathological aggression. As work progresses, it will be exciting to see the path addressing the fundamental mechanisms of aggression and the connected path examining the modification of aggression by pharmacologic interventions converge and ultimately impact the study and treatment of pathological aggression in humans.
Conflict of interest statement Nothing declared.
Acknowledgements We thank members of the Certel lab and two anonymous referees for helpful suggestions. The Certel laboratory is funded by N.I.H.R01 GM115510 grant.
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