Show Summary Details

Page of

PRINTED FROM OXFORD HANDBOOKS ONLINE ( © Oxford University Press, 2018. All Rights Reserved. Under the terms of the licence agreement, an individual user may print out a PDF of a single chapter of a title in Oxford Handbooks Online for personal use (for details see Privacy Policy and Legal Notice).

date: 29 February 2020

Identifying Critical Genes, Neurotransmitters, and Circuits for Social Behavior in Invertebrates

Abstract and Keywords

This article examines the use of invertebrates to investigate the genetic and physiological mechanisms that regulate social behavior. A central goal in behavioral neuroscience is to understand how genes encode behavior and how environmental factors influence the expression of these relevant genes. In pursuit of this goal, many scientists who study behavior use a combined ecological, molecular, genomic, and physiological approach. This article discusses the distinct strengths of an approach, species, or finding in the context of two related but unique social behaviors: aggregation and aggression. It considers the genes that control aggregation and aggression by drawing on insights from C. elegans and Drosophila, respectively. It also describes the neurotransmitters, neuromodulators, and receptors that regulate aggregation and aggression.

Keywords: invertebrates, social behavior, genes, aggregation, aggression, C. elegans, neurotransmitters, Drosophila, neuromodulators, receptors

A central goal in behavioral neuroscience is to understand how genes encode behavior and how environmental factors influence the expression of these relevant genes. To reach this goal, many scientists who study behavior use a combined ecological, molecular, genomic, and physiological approach. In addition to the increasing repertoire of techniques, different species or model organisms bring unique advantages in studying a specific behavior, including refined genetic tools, pharmacology, a well-characterized nervous system, optogenetics and electrophysiological manipulations, and/or a robust phenotype within a laboratory setting (De Bono, 2003; Hassanali et al., 2005; Hulme & Whitesides, 2011; Bargmann & Marder, 2013; Gilles & Averof, 2014; Venken & Bellen, 2014; Ernst et al., 2015; Langenhan et al., 2015). Continuing to embrace multiple systems and approaches will increase the possibility of understanding the unifying and unique principles that govern the perception of environmental stimulus to a subsequent behavioral response. In this review, I highlight, when possible, the distinct strengths of an approach, species, or finding in the context of two related but unique social behaviors: aggregation and aggression (Figure 29.1).


Aggregation is a remarkable phenomenon. In wintering sites of coastal California and parts of central Mexico, 50 to 200,000 monarch butterflies (Danaus plexippus) aggregate in appropriate trees (Hill et al., 1976; Wells et al., 1990). On mushroom farms around the world, wild populations of the nematode C. elegans aggregate into columns that contain hundreds of thousands of individuals (Grewal & Richardson, 1991). In parts of Asia and Africa, the devastating effects of desert locust (p. 686) (Schistocerca gregaria) swarms have been revered and feared throughout history (Ellis, 1956; Gillett et al., 1976; Torto et al., 1994). Regardless of its implication for humans, aggregation itself is a basic social phenomenon, and many activities of invertebrates are linked to it, including mating, feeding, and group defense behavior. Although many of the behaviors associated with aggregation versus solitary behavior improve species survival and reproduction, the usage of group life—whether temporary or as part of a social structure—requires flexibility in order to counter risks such as increased predation or resource limitability (Parrish & Edelstein-Keshet, 1999; Riipi et al., 2001; Symonds & Wertheim, 2005; Wertheim et al., 2005).

Identifying Critical Genes, Neurotransmitters, and Circuits for Social Behavior in Invertebrates

Figure 29.1 Internal and external regulators of social behavior. (A) Two Drosophila melanogaster males engaged in aggressive boxing. (Image by S. Certel.) (B) Aggregation behavior displayed by the nematode C. elegans.

(Image courtesy of Ekaterina Voronina.)

For the most part, transitions between gregarious and solitary behavior involve a complex integration of external sensory stimuli, internal signals, and previous experiences. External stimuli, including olfactory, gustatory, visual, and tactile cues, are causal factors for aggregation. Contributing internal signals may involve developmental stage, sex, nutritional state, or hormone levels (Parrish & Edelstein-Keshet, 1999; De Bono, 2003; Ruxton & Sherratt, 2006). These inputs generate a behavioral response by coordinating and inhibiting the activity of two dynamic networks, one of interacting genes and gene products and the other of interconnected neurons.

Although the specific patterns of aggregation vary and the external stimuli environment is rich with diversity among different organisms, common principles and common genes that regulate the transitions of gregarious behavior are emerging. Commonalities regarding gene contributions, second messenger systems, the spatial and temporal regulation of neurotransmitters, and circuit connectivity among different invertebrates will be discussed. First, the genetic tools and screening assays of C. elegans have been instrumental in the identification of critical genes. Second, the rapid transition between solitary and gregarious behavior in the desert locus is providing insight into the coordination and temporal resolution of neurotransmitter function.

Genes That Control Aggregation—Insight From C. elegans

Aggregation in Nematodes

The soil nematode C. elegans is an attractive model to investigate the innate basis of behavior. Mutations that disrupt distinct actions can be readily isolated following a range of mutagenic options and the altered genes identified by genetic and genomic approaches. In addition to mutagenic strategies, at least 30 natural isolates have been collected from all over the world (Hodgkin & Doniach, 1997), providing a source of environmentally driven genetic variation. Animals from about half of these isolates feed alone on a bacterial lawn of E. coli, whereas animals from the other wild isolates aggregate strongly and feed in groups (De Bono & Bargmann, 1998; De Bono, 2003). Thus, aggregation variation within species can provide a robust portal with which to uncover the genetic basis of behavioral diversity that reflects the forces of natural selection.


Using these foraging differences between social and solitary wild strains of C. elegans, de Bono and (p. 687) Bargmann characterized EMS-induced mutations generated in the solitary strain N2 that now resulted in these animals exhibiting social behavior (De Bono & Bargmann, 1998). The behavioral differences were determined to be the result of a single amine acid change in a neuropeptide receptor, npr-1 (neuropeptide receptor resemblance) gene (Figure 29.1) (De Bono & Bargmann, 1998). The two isoforms of Npr-1, differing at a single residue, occurred in the wild. One isoform, Npr-1 215F, was identified exclusively in social strains, whereas the other isoform, Npr-1 215V, occurred in solitary strains. The functionality of this amino acid change was verified at the whole organism level by the demonstration that an Npr-1 215V transgene can indeed induce solitary feeding behavior in a wild social strain. Subsequent studies in numerous systems determined Npr-1 encodes a G protein–coupled receptor that activates heterotrimeric guanine nucleotide–binding proteins (G proteins) to modulate the activity of enzymes, ion channels, and second messenger pathways within a cell (Strader et al., 1994; Gudermann et al., 1997; Riedl & Sokolowski, 2004; Altstein & Nassel, 2010; Hanlon & Andrew, 2015). Changes in residue 215 in the predicted Npr-1 protein have been proposed to alter the strength or the specificity of G protein coupling (Burstein et al., 1996; Gudermann et al., 1997; Hamm, 2001; Riedl & Sokolowski, 2004; Tobin, 2008).

To understand how the Npr-1 215F and Npr-1 215V alleles differentially alter solitary versus group feeding behavior, the de Bono group sought to identify the Npr-1 ligand. Using a genetic screen, Rogers et al. determined FMRFamide-related neuropeptides (FaRPs) encoded by the flp-18 and flp-21 genes function as Npr-1 ligands and furthermore determined these peptides differentially activate the Npr-1 215F and Npr-1 215V receptors (Rogers et al., 2003). This key finding occurred through the heroic effort of testing FaRP peptides in vivo and demonstrated how the intracellular context of a G protein–coupled receptor can alter the in vivo ligand/receptor specificity and, in this example, alter social behavior as well.

The npr-1 story is significant for several reasons. First, it is a classic example of a single gene contributing to a single behavior; second, the work expanded on results from other species; and third, it offered the possibility of understanding social behavior at the neuronal and physiological level. For example, subsequent experiments determined that mutations in other genes, namely osm-9 and ocr-2, restore solitary feeding behavior in npr-1 mutant animals (De Bono et al., 2002). Osm-9 and Ocr-2 were predicted to form a transduction channel in sensory neurons—specifically a TRPV channel—that could convert external stimuli into neural activity (De Bono et al., 2002; Tobin et al., 2002). Conversely, the Npr-1 isoform associated with solitary feeding was found to function in neurons exposed to the body fluid, thus providing a mechanism that responds to internal information and influences aggregative feeding (Coates & De Bono, 2002). NPR-1 activity also suppressed feeding in specific neurons by antagonizing signaling through a cyclic GMP-gated ion channel encoded by tax-2 and tax-4 (Coates & De Bono, 2002).

Signaling through cGMP has also been extensively associated with normal variation in food- related behavior in the fruit fly Drosophila melanogaster and the honeybee Apis mellifera. Drosophila larval foraging behavior is determined by natural variation in a single gene, foraging (for). The for gene encodes a mediator of cGMP action, the protein kinase enzyme PKG. Flies with two for variants, rover or sitter, differ in their PKG levels as well as their locomotive behavior on food (Osborne et al., 1997). In honeybees, the behavioral effect occurs as a result of changes in gene regulation rather than variation: When bees shift from working in the hive to foraging, the expression of for is up-regulated (Ben-Shahar et al., 2002). Together the foraging and npr-1 studies provide two mechanisms by which behavioral diversity can occur: changes in gene regulation and changes in the function of a gene product.

GABA Receptor (exp-1)

More than a decade after the Npr-1 discovery, the Bargmann group returned to the same naturally occurring isolates that differed in their aggregation behavior and asked if these lines contained additional aggregation-regulating loci (Bendesky et al., 2012). Using quantitative genetic techniques, including a survey of chromosome substitution strains and quantitative trait loci (QTL) analysis of recombinant inbred lines, three new QTLs affecting aggregation were identified. Fine-mapping localized one QTL, to the vicinity of the gamma-aminobutyric acid (GABA) neurotransmitter receptor gene exp-1 (Bendesky et al., 2012). Further experiments demonstrated that this QTL altered the function of Exp-1, thus identifying Exp-1 and GABA signaling as new players in the regulation of aggregation. These results add to an increasing number of reports that (p. 688) changes in neurotransmitter receptor genes contribute to behavioral diversity (Bendesky et al., 2012; Williams et al., 2014; Yuan et al., 2014; Tachibana et al., 2015) and constitute a source of natural behavioral variation (Wnuk et al., 2014; Shorter et al., 2015).

To connect neurotransmitter signaling with the outside environment as well as to internal signals, Bendesky et al. asked if GABA function connects to the TGF-β pathway because mutations in daf-7 also effect aggregation (Bendesky et al., 2012). The daf-7 TGF-β pathway integrates sensory information related to population density, food availability, and temperature to maximize survival (Ren et al., 1996; Gumienny & Savage-Dunn, 2013), and the authors determined that exp-1 mutations affect the level of daf-7 expression. This result led to the hypothesis that the Exp-1 GABA neurotransmitter receptor may regulate daf-7 expression and neuroendocrine function by playing a role in detecting environmental stress. A cooperative role for the daf-7 pathway and the GABA neurotransmitter system in the regulation of C. elegans dauer development has also been proposed (Hobert et al., 1999).


Chemical signaling is an ancient form of communication between conspecifics. Ascarosides are small molecule signals defined as glycosides of the dideoxysugar ascarylose and were first detected more than 100 years ago in the parasitic roundworms of the family Ascaridia (Flury, 1912). Golden and Riddle demonstrated that metabolite extracts of C. elegans cultures induce dauer formation and results from an EMS mutagenesis screen identified a mutant strain, daf-22, that did not produce dauer-inducing activity (Golden & Riddle, 1982, 1985). Identification of daf-22 provided a genetic entryway into understanding the production and function of ascarosides in behaviors, including aggregation, dauer entry and exit, olfactory plasticity, and mating in C. elegans.

daf-22 encodes a nonspecific lipid-transfer protein that catalyzes the final step in peroxisomal fatty acid beta-oxidation and is an ortholog of the human sterol carrier protein SCP2 (Butcher et al., 2009, Golden & Riddle, 1985). Through the use of comparative metabolomics, the family of hydrophilic ascarosides with short, three- to- nine-carbon side chains was identified as the major components of the dauer pheromone (Jeong et al., 2005; Butcher et al., 2009; Pungaliya et al., 2009). Recent work reveals the nematode is able to alter the composition of the ascaroside pheromones produced under different environmental conditions through specific acyl-CoA oxidases (Zhang et al., 2015). This modulation is accomplished by changes in the levels of acox-2 and acox-3, which act as gatekeepers for the production of shorter-chain ascarosides. Changes in food availability down-regulate the transcript levels of acox-3, while increases in temperature are responsible for increases in both acox-2 and -3 expression (Zhang et al., 2015). These results suggest new layers of communication flexibility in response to environmental factors than previously appreciated.

Following their identification as constituents of the dauer pheromone, several studies found that ascarosides serve a range of important signaling functions, including functioning as highly potent aggregation signals that attract both males and hermaphrodites (Srinivasan et al., 2012; Von Reuss et al., 2012). For example, the pheromone ascaroside C9 (ascr#3) is repulsive to wild-type hermaphrodites, attractive to wild-type males, and usually neutral to "social" hermaphrodites that carry a hypomorphic mutation in the npr-1 neuropeptide receptor gene (Srinivasan et al., 2012). How are chemical communication, in general, and this sophisticated chemical language, in particular, received and integrated into different neuronal circuits to mediate social behavior? A role for G protein–coupled receptors in receiving ascaroside signaling has been suggested due to the ability of G protein alpha subunits gpa-2 and gpa-3 to induce dauer formation upon up-regulation (Zwaal et al., 1997; Lans & Jansen, 2007) and the identification of srg-36 and srg-37, two G protein–coupled receptors that encode redundant receptors for the ascaroside C3 (Mcgrath et al., 2011). To summarize, the work described in this section demonstrates how environmental conditions influence a sophisticated chemical messenger system used by nematodes to communicate to other roundworms in a population. Further information regarding the properties of ascarosides and their role as metabolism-derived structures can be found in reviews (Schroeder, 2015; Von Reuss & Schroeder, 2015).

Neurotransmitters That Regulate Aggregation

Like other invertebrates, the desert locust (Schistocerca gregaria) and the migratory locust (Locusta migratoria) exhibit a population density-dependent transition between solitary and gregarious (p. 689) phases (Ellis, 1956; Uvarov, 1966; Hassanali et al., 2005; Ott et al., 2009; Pener & Simpson, 2009; Buhl et al., 2011; Topaz et al., 2012). Solitarious locusts occur at low population densities and are repelled by other locusts. They are usually bigger, cryptically colored to blend in with their surroundings, and walk with a slow, creeping gait. Locusts in the gregarious phase have a bright body color, exhibit a rapid walking pace and, most important, are attracted to conspecifics and can form large aggregates. The solitary to gregarious phase change is driven by large-scale changes in population density and subsequently limited resources. Desert locusts inhabit arid and semiarid regions where rains are infrequent and sparse. When habitats receive widespread, heavy seasonal rain, population growth is supported; however, upon a return to dry conditions, the declining resources are fought over by solitarious locusts (Buhl et al., 2011; Despland & Simpson, 2000; Ott et al., 2009). The resultant crowding causes a rapid transition to gregarious behavior due to the exposure of visual and olfactory cues from conspecifics as well as mechanosensory stimuli of a hind femur (Leo Lester et al., 2005; Simpson et al., 2001).

Although full phase reversal can take several generations, behavioral gregarization can occur within hours, which is a striking phenomenon considering the extensive differences in behavior, physiology, and morphology that separate the solitary versus gregarious phases (Uvarov, 1966; Pener & Yerushalmi, 1998; Rogers et al., 2003; Ott et al., 2009; Pener & Simpson, 2009; Verlinden et al., 2009). This initial phenotypic plasticity does not allow time for extensive changes in gene expression to be the primary source of behavioral change. Therefore, investigating aggregation behavior in the desert and migratory locus offers a unique system to examine the neurochemical mechanisms that can reconfigure distinct yet connected behavioral changes. Recent studies suggest these changes occur due to the coordinating and inhibiting aspects of distinct neurotransmitters and, not surprisingly, biogenic amines are key players in this behavioral transformation.

Biogenic Amines

A role for the catecholamines dopamine (DA) and octopamine (OA), the insect equivalent of norepinephrine, and serotonin (5-HT) in the regulation of many behaviors, including feeding, sleep and wakefulness, learning and memory, arousal, and aggression, have been established by numerous studies over many decades (Stern, 1999; Homberg, 2002; Roeder, 2002; Verlinden et al., 2010; Waddell, 2010; Nall & Sehgal, 2014; Yamamoto & Seto, 2014; Kravitz & Fernandez Mde, 2015; Langenhan et al., 2015; Rogers & Ott, 2015). In terms of aggregation, aminergic signaling has been confirmed as central to behavioral phase change in the migratory and desert locust (De Bono, 2003; Anstey et al., 2009; Ott et al., 2012; Rogers & Ott, 2015). In recent studies, Simpson and colleagues found that serotonin is required for the initial switch from strong mutual aversion in solitary locusts to the increased activity and coherent group formation in gregarious locusts (Anstey et al., 2009). Inhibition of serotonin synthesis decreased activity and also resulted in 5-HT-depleted locusts avoiding the stimulus group (Anstey et al., 2009; Rogers et al., 2014). In solitarious locusts, gregarizing stimuli caused an increase in 5-HT levels in the thoracic ganglia. Interestingly, distinct differences in the expression responses of the 5-HT neurons were revealed by quantitative analysis of cell body immunofluorescence (Rogers & Ott, 2015). For example, a subset of neurons showed increased serotonin expression only in response to intense visual and olfactory stimuli from conspecifics (Rogers & Ott, 2015), while in a separate neuronal subset, the expression levels of 5-HT in long-term gregarious locusts were lower than solitarious levels. These results suggest a possible two-tiered role of the serotonergic system.

Protein Kinases

Aminergic signaling follows the basic process of cell signaling. An outside stimulus, in this example serotonin, binds and activates its G protein–coupled receptor, thereby inducing a conformational change with the activated GPCR acting as a guanine exchange factor (GEF) for Gα (Rosenbaum et al., 2009; Katritch et al., 2013; Syrovatkina et al., 2016). Activated Gα can then interact with an effector (E), such as adenylyl cyclase, resulting in the activation of cAMP-dependent protein kinase (protein kinase A [PKA]) (Castellucci et al., 1982; Muller & Carew, 1998; Huang & Kandel, 2007). Adenylyl cyclase/PKA signaling plays a central role in diverse forms of plasticity, making it a prime candidate to play a role in the population density–dependent transition to gregarious behavior (Skoulakis et al., 1993; Abel et al., 1997; Muller, 2000; Michel et al., 2008).

Ott and colleagues manipulated the function of two protein kinases—PKA and the foraging gene product, a cGMP-dependent PK (PKG) described earlier—and analyzed four behavioral (p. 690) characteristics of the gregarious phase (Ott et al., 2012). The behavior of long-term gregarious locusts did not change after injecting the PKA inhibitor KT5720 or the PKG inhibitor KT5823. However, solitarious locusts with an RNAi-induced reduction in PKA catalytic subunit C1 expression behaved less gregariously after crowding, and reducing expression of the inhibitory R1 subunit promoted more extensive gregarization following a brief crowding period (Ott et al., 2012). A central role for PKA is consistent with the role of serotonin in mediating locust gregarization as PKA activation may provide the link between a transient 5-HT signal and a later phase of consolidation that entails changes in gene expression (Kang et al., 2004; Badisco et al., 2011). With the identification of serotonin as necessary and sufficient for inducing gregarious behavior, additional attention to the role of other amines in mediating the phenotypic plasticity of solitary versus gregarious transitions was analyzed.

Gene Regulation of Dopamine Synthesis

Using genome-wide gene expression profiling, Ma et al. (2011) determined genes involved in dopamine biosynthesis and synaptic release, including pale, henna, and vat1, were up-regulated in the gregarious phase. DA depletion by RNAi reduction and pharmacological interventions resulted in behavioral shifts toward the solitary state, confirming the importance of the dopamine metabolic pathway. How did the up-regulation of pale, henna, and vat1 expression occur? A role for microRNAs in posttranscriptionally regulating henna and pale was recently described (Yang et al., 2014). Using bioinformatics prediction algorithms, qRT-PCR, and reporter assays, miR-133 was found to affect DA production by binding to the coding region and 3' untranslated region of henna and pale, respectively (Yang et al., 2014). Increasing miR-133 expression suppressed Henna and Pale expression, which consequently decreased DA production, and, vice versa, miR-133 inhibition increased Henna and Pale expression. Both manipulations resulted in a behavioral shift of locusts within the gregarious phase to the solitary phase. Determining how environmental changes interact with molecular mechanisms to regulate biogenic amine production is a critical piece in the puzzle of phenotypic plasticity and phase transition.

At this point we have examined the solitary-to-gregarious behavioral reconfiguration in response to environmental cues at the level of individual genes or gene products related to neurotransmitter function. Although multiple sensory modalities are involved in sensing population density, a simplified version of the process can be thought of as conspecific attraction versus conspecific repulsion. As the role of neurotransmitters in mediating attractive and repulsive behavior in other organisms has yielded insight into neurophysiology and circuit construction (Joseph & Heberlein, 2012; Ko et al., 2015; Leinwand et al., 2015), recent experiments on phase transitions in the migratory locust are providing insight into the neuromodulatory mechanisms linking attraction and repulsion between individuals to large-scale population changes. Results from a previously reported transcriptome analysis indicated genes involved in tyrosine metabolism were up-regulated in stage-specific gregarious locusts (Chen et al., 2010), and phenylalanine and tyrosine are common precursors of tyramine (TA) and OA. In addition, OA and TA have been shown to regulate olfactory-driven behaviors and neuronal responses in cockroaches, moths, and Drosophila. To examine the role of OA and TA signaling in modulating the attraction and repulsion response to gregarious volatiles, Ma et al. combined expression-level quantification, RNA interference against OA and TA receptors, and neuropharmacological injections with a Y-tube behavioral assay (Ma et al., 2011).

Locusts in the gregarious phase avoid gregarious volatiles after 32 hours of isolation, whereas the attraction of solitary locusts to gregarious volatiles occurs after 1 hour of crowding (Guo et al., 2011). The authors determined that activation of octopamine-OARα signaling in solitary locusts resulted in the behavioral change from repulsion to attraction. In contrast, in gregarious locust the attraction to repulsion behavioral changed required the activation of tyramine-TAR signaling. The proposed model suggests the two pathways antagonistically modulate olfactory preference during the locust phase change as both receptors are expressed in several olfactory centers, including the antennal lobes, mushroom bodies, and superior protocerebrum (Ma et al., 2011). How then do solitary and gregarious locusts perceive the same olfactory cue (gregarious volatiles) yet subsequently reach opposite behavioral decisions? One possibility is information on food status could be integrated with olfactory cues to drive a behavioral output. TA signaling responds to the putative juvenile hormone binding protein, Takeout, which is expressed in the insect antenna and is postulated to interact with circulating hormones to convey food status (p. 691) information (Sarov-Blat et al., 2000; Meunier et al., 2007; Chamseddin et al., 2012). In conclusion, examining the solitary versus gregarious phase transition in locusts is providing insight into the coordinating and inhibiting aspects of neurotransmitters and how amine-mediated behavioral plasticity can result in an environmentally driven reorganization of a complex phenotype.


Aggregation between members of a species can provide direct benefits as described earlier, as well as facilitate or inhibit separate activities such as reproduction, defensive behaviors, or aggression (Seidelmann & Ferenz, 2002; Robinson et al., 2005; Ruxton & Sherratt, 2006). Because aggregation can increase competition for local resources, the likelihood of conspecific aggression may also increase. Phase change in locusts and subordinate behavior following defeat are all manifestations of the nervous system’s capacity for behavioral remodeling in response to social experience (Stevenson et al., 2000, 2005; Geva et al., 2010). How is the shift from tolerance of conspecifics to aggression achieved? Studies in a variety of organisms indicate that changes in an individual’s interaction with the environment are effected by the transduction of sensory cues, neuronal function, circuit formation, and neuromodulation (Figure 29.2). Key molecular players that drive or reinforce a new behavioral state include transcription factors, pheromonal receptors, biogenic amines, altered second messenger signaling, and neurotransmitters.

Identifying Critical Genes, Neurotransmitters, and Circuits for Social Behavior in Invertebrates

Figure 29.2 Pathway versus dynamic approaches to behavioral choice. (A) Examining the regulation of external information that drives aggregation versus aggressive behavior can be considered in a linear manner. (B) Understanding behavioral choice at the individual and population levels may occur as a result of a dynamic behavioral transition rather than a defined set point.

Genes That Control Aggression—Insight From Drosophila

Methyl-Binding-Domain Proteins

One key aspect of implementing a new behavioral state is flexibility in gene expression. Epigenetic mechanisms, including chromatin remodeling and DNA methylation, alter gene activity and are responsive to external pressures. Proteins containing a methyl-CpG-binding domain (MBD) bind methylated DNA and play a major role in determining the transcriptional state of the genome by coordinating crosstalk between DNA methylation, histone modifications, and chromatin organization (Marhold et al., 2004; Zilberman, 2008; Boffelli et al., 2014; Tognini et al., 2015). We recently asked if the Drosophila MBD proteins, dMBD-R2 and dMBD2/3, are required for male aggression. Males with an RNAi-based reduction in dMBD-R2 (p. 692) or dMBD2/3 specifically in OA neurons exhibited a decrease in aggression toward a conspecific male, whereas conspecific male-male courtship increased (Gupta et al., 2017). Hypermethylation of the male OA neurons genome also resulted in a decrease in male aggression; however, male–male courtship was not altered (Gupta et al., 2017). Neurons expressing this particular amine were analyzed because previous results demonstrated a subset of OA neurons directly receive male pheromone information and function to promote aggression (Hoyer et al., 2008; Zhou et al., 2008; Andrews et al., 2014). These results suggest epigenetic mechanisms interpreted by MBD proteins are required for male social behavior and offer a specific neuronal genome to examine how potential shifts in gene expression, due at least in part in response to sensory stimuli, are coordinated at the epigenome level.


A second layer of gene expression control is through gene-specific transcription factors that often interact with cofactors to activate or repress expression. Transcription factors can function to control neuronal developmental processes that lead to alterations in behavior, regulate behavior itself due to the control of neuron function, or use a combination of developmental and behavioral processes. Work by the Dierick group has demonstrated that reducing the expression of the conserved Drosophila transcriptional repressor, tailless (tll), results in a strong increase in male aggression (Davis et al., 2014). To confirm the increase in fighting frequency was independent of developmental defects, the authors reduced tll expression only in the adult and found the adult knockdown is sufficient to increase aggression. What target genes might Tll and its corepressor Atrophin regulate? One possible underlying mechanism that may be repressed is neuropeptide function because blocking neuropeptide processing or release suppresses the tll knockdown-induced aggression response (Davis et al., 2014).


Too much or too little of the neuromodulator OA alters both aggression and mating behavior in numerous insects. Recent work has identified a specific transcription factor required to regulate the enzymatic production necessary for the synthesis of OA. TfAP-2 is a Drosophila homolog of the murine AP-2 transcription family member, TFAP2B, which has been shown to be necessary for noradrenergic neuron development (Hong et al., 2008; Schmidt et al., 2011). By asking if alterations in Tfap-2 expression, specifically in OA neurons, effected Drosophila male aggression, Williams et al. found that TFAP-2 overexpression increased lunge and boxing frequency, two key behavioral patterns (Williams et al., 2014). With the relevance to aggression established, the authors examined the transcript levels of genes that control OA production or release, including Tdc2, Tβh, and Vmat. Levels of Tβh, the enzyme necessary to convert tyramine to octopamine, were decreased in TfAP-2 loss-of-function males, while in TfAP-2 overexpression increased Tβh expression. The transcript levels of the vesicular monoamine transporter (Vmat), which transports amines into synaptic vesicles, was also significantly reduced by a reduction in TfAP-2 and increased by TfAP-2 overexpression (Williams et al., 2014). Finally, a link between OA signaling and the satiation hormone Dsk was proposed, suggesting the internal nutritional state and external food-related chemical information may be ultimate targets of this molecular mechanism.

Olfactory Receptor 67d (Or67d)

Up to this point, controlling gene expression required for the function of neurons that may at least in part receive sensory information has been the focus. However, recent work has identified receptors that respond to aggression-promoting pheromonal information. cVA is a male-specific lipid that has multiple functions in social communication (Ejima, 2015). From a distance, cVA works as an aggregation pheromone (Bartelt et al., 1985; Xu et al., 2005), whereas in close proximity, high densities of male flies are thought to release volatile cVA, thus promoting aggression while inhibiting male–male courtship (Jallon, 1984; Wang & Anderson, 2010). How might the aggregation and the aggression-promoting effect of cVA be received by a male? The odorant receptor, Or67d, was found to respond to cis-vaccenyl acetate (cVA) stimulation (Van Der Goes Van Naters & Carlson, 2007).

Work by Wang and Anderson 2010 demonstrated that increased cVA input through the Or67d receptor or activation of Or67d-expressing olfactory sensory neurons is sufficient to promote male aggression (Wang & Anderson, 2010). In turn, cVA-promoted aggression can promote male fly dispersal from a food resource, in a manner dependent on Or67d-expressing sensory neurons. These data suggest that cVA may provide feedback information on male population density, through its effect on aggression. However, long-term exposure to cVA may (p. 693) desensitize aggression-promoting circuits (Liu et al., 2011), in conjunction with olfactory input through odorant receptor Or65a.

Neuromodulators That Control Aggression—Octopamine

Developmental Role

In the adult nervous system, neurotransmitters released from presynaptic terminals bind to receptors on postsynaptic cells, which lead to either an excitation or inhibition of these neurons. However, during nervous system development, neurotransmitters-neuromodulators, their synthesizing enzymes, and their receptors are often expressed before synapses are being formed (Murrin et al., 2007). In particular, mono- or biogenic amines, which function as neurotransmitters or neuromodulators, are expressed in the very early embryo (Herlenius & Lagercrantz, 2001, 2004; Lagercrantz & Ringstedt, 2001). Because neurotransmitters/neuromodulators are secreted molecules, studies have examined potential roles as developmental signals that influence proliferation, neuronal survival, axon guidance, and synapse maturation (Hodges & Richerson, 2008).

In Drosophila, octopamine can function in synapse formation and plasticity (O’dell et al., 2010; Roeder, 2005) At the larval neuromuscular junction, OA activation of OAβ2R autoreceptors stimulates the development of new synaptic boutons, whereas a separate OA autoreceptor, OAβ1R, functions to brake the positive feedback (Koon et al., 2011; Koon & Budnik, 2012). This autoregulatory mechanism might control the amount of OA released by octopamine neurons and regulate the structure of neuronal arborizations. The presence of a positive feedback that controls the growth of modulatory inputs could provide a mechanism by which the experience of an animal can modify circuitry and subsequently potentially adapt to a changing environment (Koon et al., 2011).

One aspect of a dynamic environment repeatedly mentioned in this chapter is changing pheromonal information. The existence of functional and synaptic connections between male pheromone responsive Gr32a neurons and OA neurons located in the subesophageal zone was recently demonstrated and the authors asked if Gr32a axonal morphology was altered when their synaptic partners lack OA (Andrews et al., 2014). Using Gr32a-Gal4 to drive reporter GFP expression, the stereotypical projections of Gr32a-expressing neurons from control and OA-deficient males were examined. Aberrant targeting of Gr32a axons in OA-deficient brains was observed, suggesting OA itself is required for the correct differentiation and positioning of OA neuron synapses targeted by pheromone-responsive sensory neurons.

Negative Role for Octopamine

In modulating or strengthening a pheromonal or sensory signal, OA can be described as aggression promoting. However, in some insect societies, a signal to start aggressive behaviors must come at the expense of an ongoing behavior: cooperation. When Polyrhachis moesta ant queens establish a colony with genetically unrelated queens, cooperative brood rearing and the exchange of food occur. These interactions are suggested to promote social partnership and can result in the transfer of hydrocarbons between nestmates, which is important in nestmate recognition (Boulay et al., 2000; Hashimoto et al., 2013). In contrast, colonies that contain a single queen exclude other queens via aggressive behavior. Koyama et al. determined that OA levels in the brains of cooperative colony queens were significantly higher than those in noncooperating queens (Koyama et al., 2015). Therefore, in this system, an OA-controlled increase in aggression may be considered a factor that reduces cooperation, and given that social cooperation needs to be continually reinforced by social bonding (Boulay et al., 2000), low OA levels might be necessary to maintain the partnership among cofounding queens (Koyama et al., 2015).

Receptors That Regulate Aggression

More than 40 years ago, a vertebrate norepinephrine-containing neuron was described by “the terminal networks of this system are very diffuse, with more than 100,000 terminals from an individual monoamine-containing cell body” (Omenn, 1976, p. 438). In invertebrates, amine neurons also project extensively throughout the brain (Dacks et al., 2005; Sinakevitch & Strausfeld, 2006; Busch et al., 2009; Certel et al., 2010; Chiang et al., 2010). Amines are released and function, at least in part, via diffusion-mediated signaling, known as volume transmission (Agnati et al., 2010; Fuxe et al., 2012), thus generating the ability to modulate neuronal function in a large number of brain areas, with different timescales, and with a variety of cellular effects. Taking into account this complexity, are the multifaceted roles of biogenic amines in regard to social behavior separable? Recent work examining the role of a dopamine and the neuropeptide tachykinin receptor on aggression (p. 694) suggests the possibility of receptor specificity and circuit elucidation.

Dopamine Receptors

Dopamine has been linked to aggression in various species; however, the precise role or roles served by this amine specifically on aggression have been difficult to define because the release of DA can influence many behaviors (Nieoullon & Coquerel, 2003; Andretic et al., 2005; Mao & Davis, 2009; Riemensperger et al., 2011; Burke et al., 2012). Recently, Kravitz and colleagues were able to inactivate or activate a subset of DA neurons at the single-cell level using a refined version of the GAL4/upstream activating sequence (UAS) binary system. Their results indicate manipulating two distinct subsets of dopaminergic neurons resulted in an increase in male aggression (Alekseyenko et al., 2013). Examining the presynaptic terminals of these aggression-promoting neurons within the T1 and PPM3 cluster revealed projections to different parts of the central complex (a region of the Drosophila brain that is proposed to combine various modalities of sensory to direct behavioral responses), including regions that overlap with the receptor fields of DD2R and DopR DA receptor subtypes, respectively (Alekseyenko et al., 2013). These results suggest that specific DA neurons may influence aggression through direct or indirect communication with two distinct DA receptor subtypes.

The Neuropeptide Tachykinin and Its Receptor

Aggression and courtship are two sexually dimorphic social behaviors. Neurons in Drosophila that express the neuropeptide tachykinin (Tk)/Substance P have been shown be part of a male-specific gustatory circuit that generates courtship suppression toward a female by responding to an antiaphrodisiac pheromone (Shankar et al., 2015). Tachykinin-expressing male-specific neurons that function in aggression have also been identified (Asahina et al., 2014). Tk is coexpressed in a small cluster of FruM(+) neurons that are found only in males, and activating or silencing these neurons increased and decreased conspecific male aggression, respectively, without altering male–female courtship behavior. Mutations in both Tk and a putative receptor, Takr86C, suppressed the increase in male aggression that occurred following Tk + neuronal activation (Asahina et al., 2014). One theme in this review has been the role of sensory information in social behavior, and therefore it is striking that Tk neuron activation promotes aggression even in the absence of a variety of sensory cues (Asahina et al., 2014). This result suggests the motivation to fight can be triggered by manipulating “middle” steps in an aggression-promoting circuit.

Aggression Circuitry

A second subset of male-specific aggression-promoting neurons was identified in a neuronal activation-based, large-scale screen in Drosophila. From 8 to 10 interneurons known as PI neurons were identified that coexpressed FruM+ and, when thermogenetically activated, enhanced male aggression and courtship toward a female (Hoopfer et al., 2015). To determine if the output of this small subset of FruM+ P1 neurons might require different thresholds of PI activation, optogenetic experiments were performed (Hoopfer et al., 2015). By expressing a genetically encoded far red-shifted channelrhodopsin CsChrimson reporter, the authors found P1 neuronal activation could promote aggression at a threshold below that required for a courtship behavioral pattern, the wing extension or song. These results demonstrating the FruM+ P1 neurons function in both aggression and courtship circuitry based on the activation threshold provide insight into functional mechanisms as well as potentially circuitry output partners that facilitate male aggression and courtship.


Decades of inquiry using invertebrates to examine the genetic and physiological mechanisms that regulate social behavior have provided exceptional contributions. These impacts have been felt on multiple levels; first, in regard to elucidating the principles required to wire behavior into the nervous system of any organism, and second, examining how insects behave has contributed to their management. The continued advancement of genetic-based tools and manipulations is moving the field of behavior forward not just in terms of neuronal communication but with questions concerning epigenetics and the integration of environmental influences. Each new layer of understanding regarding the great capacity of the “simple” invertebrate nervous system to generate complex behaviors strengthens Charles Darwin’s observation that “the brain of an ant is one of the most marvelous atoms of matter in the world, perhaps more so than the brain of man” (Darwin, 1981, p. 147).


Abel, T., Nguyen, P. V., Barad, M., Deuel, T. A., Kandel, E. R., & Bourtchouladze, R. (1997). Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory. Cell, 88, 615–626.Find this resource:

Agnati, L. F., Guidolin, D., Guescini, M., Genedani, S., & Fuxe, K. (2010). Understanding wiring and volume transmission. Brain Research Review, 64, 137–159.Find this resource:

Alekseyenko, O. V., Chan, Y. B., Li, R., & Kravitz, E. A. (2013). Single dopaminergic neurons that modulate aggression in Drosophila. Proceedings of the National Academy of Sciences USA, 110, 6151–6156.Find this resource:

Altstein, M., & Nassel, D. R. (2010). Neuropeptide signaling in insects. Advances in Experimental Medcine and Biology, 692, 155–165.Find this resource:

Andretic, R., van Swinderen, B., & Greenspan, R. J. (2005). Dopaminergic modulation of arousal in Drosophila. Current Biology, 15, 1165–1175.Find this resource:

Andrews, J. C., Fernandez, M. P., Yu, Q., Leary, G. P., Leung, A. K., Kavanaugh, M. P., . . . Certel, S. J. (2014). Octopamine neuromodulation regulates Gr32a-linked aggression and courtship pathways in Drosophila males. PLoS Genetics, 10, e1004356.Find this resource:

Anstey, M. L., Rogers, S. M., Ott, S. R., Burrows, M., & Simpson, S. J. (2009). Serotonin mediates behavioral gregarization underlying swarm formation in desert locusts. Science, 323, 627–630.Find this resource:

Asahina, K., Watanabe, K., Duistermars, B. J., Hoopfer, E., Gonzalez, C. R., Eyjolfsdottir, E. A., . . . Anderson, D. J. (2014). Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell, 156, 221–235.Find this resource:

Badisco, L., Ott, S. R., Rogers, S. M., Matheson, T., Knapen, D., Vergauwen, L., . . . Vanden Broeck, J. (2011). Microarray-arrayed based transcriptomic analysis of differences between long-term gregarious and solitarious desert locusts. PLoS One, 6(11), e28110.Find this resource:

Bargmann, C. I., & Marder, E. (2013). From the connectome to brain function. Nature Methods, 10, 483–490.Find this resource:

Bartelt, R. J., Schaner, A. M., & Jackson, L. L. (1985). cis-Vaccenyl acetate as an aggregation pheromone inDrosophila melanogaster. Journal of Chemical Ecology, 11, 1747–1756.Find this resource:

Ben-Shahar, Y., Robichon, A., Sokolowski, M. B., & Robinson, G. E. (2002). Influence of gene action across different time scales on behavior. Science, 296, 741–744.Find this resource:

Bendesky, A., Pitts, J., Rockman, M. V., Chen, W. C., Tan, M. W., Kruglyak, L., & Bargmann, C. I. (2012). Long-range regulatory polymorphisms affecting a GABA receptor constitute a quantitative trait locus (QTL) for social behavior in Caenorhabditis elegans. PLoS Genetics, 8, e1003157.Find this resource:

Boffelli, D., Takayama, S., & Martin, D. I. (2014). Now you see it: Genome methylation makes a comeback in Drosophila. Bioessays, 36, 1138–1144.Find this resource:

Boulay, R., Soroker, V., Godzinska, E. J., Hefetz, A., & Lenoir, A. (2000). Octopamine reverses the isolation-induced increase in trophallaxis in the carpenter ant Camponotus fellah. Journal of Experimental Biology, 203, 513–520.Find this resource:

Buhl, J., Sword, G. A., Clissold, F. J., & Simpson, S. J. (2011). Group structure in locust migratory bands. Behavioral Ecology and Sociobiology, 65, 265–273.Find this resource:

Burke, C. J., Huetteroth, W., Owald, D., Perisse, E., Krashes, M. J., Das, G., . . . Waddell, S. (2012). Layered reward signalling through octopamine and dopamine in Drosophila. Nature, 492, 433–437.Find this resource:

Burstein, E. S., Spalding, T. A., & Brann, M. R. (1996). Amino acid side chains that define muscarinic receptor/G-protein coupling. Studies of the third intracellular loop. Journal of Biological Chemistry, 271, 2882–2885.Find this resource:

Busch, S., Selcho, M., Ito, K., & Tanimoto, H. (2009). A map of octopaminergic neurons in the Drosophila brain. Journal of Comparitive Neurology, 513, 643–667.Find this resource:

Butcher, R. A., Ragains, J. R., Li, W. Q., Ruvkun, G., Clardy, J., & Mak, H. Y. (2009). Biosynthesis of the Caenorhabditis elegans dauer pheromone. Proceedings of the National Academy of Sciences USA, 106, 1875–1879.Find this resource:

Castellucci, V. F., Nairn, A., Greengard, P., Schwartz, J. H., & Kandel, E. R. (1982). Inhibitor of adenosine 3':5'-monophosphate-dependent protein kinase blocks presynaptic facilitation in Aplysia. Journal of Neuroscience, 2, 1673–1681.Find this resource:

Certel, S. J., Leung, A., Lin, C. Y., Perez, P., Chiang, A. S., & Kravitz, E. A. (2010). Octopamine neuromodulatory effects on a social behavior decision-making network in Drosophila males. PLoS ONE, 5(10), e13248.Find this resource:

Chamseddin, K. H., Khan, S. Q., Nguyen, M. L., Antosh, M., Morris, S. N., Kolli, S., . . . Bauer, J. H. (2012). Takeout-dependent longevity is associated with altered Juvenile Hormone signaling. Mechanisms of Ageing and Development, 133, 637–646.Find this resource:

Chen, S., Yang, P., Jiang, F., Wei, Y., Ma, Z., & Kang, L. (2010). De novo analysis of transcriptome dynamics in the migratory locust during the development of phase traits. PLoS One, 5, e15633.Find this resource:

Chiang, A. S., Lin, C. Y., Chuang, C. C., Chang, H. M., Hsieh, C. H., Yeh, C. W., . . . Hwang, J. K. (2010). Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution. Current Biology, 21, 1–11.Find this resource:

Coates, J. C., & de Bono, M. (2002). Antagonistic pathways in neurons exposed to body fluid regulate social feeding in Caenorhabditis elegans. Nature, 419, 925–929.Find this resource:

Dacks, A. M., Christensen, T. A., Agricola, H. J., Wollweber, L., & Hildebrand, J. G. (2005). Octopamine-immunoreactive neurons in the brain and subesophageal ganglion of the hawkmoth Manduca sexta. Journal of Comparative Neurology, 488, 255–268.Find this resource:

Darwin, C. (1981). The descent of man and selection in relation to sex. (Original work published in 1871). Princeton, NJ: Princeton University Press.Find this resource:

Davis, S. M., Thomas, A. L., Nomie, K. J., Huang, L., & Dierick, H. A. (2014). Tailless and Atrophin control Drosophila aggression by regulating neuropeptide signalling in the pars intercerebralis. Nature Communications, 5, 3177.Find this resource:

de Bono, M. (2003). Molecular approaches to aggregation behavior and social attachment. Journal of Neurobiology, 54, 78–92.Find this resource:

de Bono, M., & Bargmann, C. I. (1998). Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans. Cell, 94, 679–689.Find this resource:

de Bono, M., Tobin, D. M., Davis, M. W., Avery, L., & Bargmann, C. I. (2002). Social feeding in Caenorhabditis elegans is induced by neurons that detect aversive stimuli. Nature, 419, 899–903.Find this resource:

Despland, E., & Simpson, S. J. (2000). The role of food distribution and nutritional quality in behavioural phase change in the desert locust. Animal Behavior, 59, 643–652.Find this resource:

Ejima, A. (2015) Pleitrophic actions of the male pheromone cis-vaccenyl acetate in Drosophila melanogaster. Journal (p. 696) Comparative Physiology A Neuroethology Sensory Neural Behavioral Physiology, 201(9), 927–932.Find this resource:

Ellis, P. E. (1956). Differences in social aggregation in two species of locust. Nature, 178, 1007–1007.Find this resource:

Ernst, U. R., Van Hiel, M. B., Depuydt, G., Boerjan, B., De Loof, A., & Schoofs, L. (2015). Epigenetics and locust life phase transitions. Journal of Experimental Biology, 218, 88–99.Find this resource:

Flury, F. (1912). On the chemistry and toxicology of ascarides. Archives in Experimental Pathology and Pharmacology, 67, 275–392.Find this resource:

Fuxe, K., Borroto-Escuela, D. O., Romero-Fernandez, W., Ciruela, F., Manger, P., Leo, G., . . . Agnati, L. F. (2012). On the role of volume transmission and receptor-receptor interactions in social behaviour: Focus on central catecholamine and oxytocin neurons. Brain Research, 1476, 119–131.Find this resource:

Geva, N., Guershon, M., Orlova, M., & Ayali, A. (2010). Memoirs of a locust: Density-dependent behavioral change as a model for learning and memory. Neurobiology of Learning and Memory, 93, 175–182.Find this resource:

Gilles, A. F., & Averof, M. (2014). Functional genetics for all: Engineered nucleases, CRISPR and the gene editing revolution. EvoDevo, 5, 43.Find this resource:

Gillett, S. D., Packham, J. M., & Papworth, S. J. (1976). Possible pheromonal effects of aggregation and dispersion in desert locust, Schistocerca-Gregaria (Forsk). Acrida, 5, 287–297.Find this resource:

Golden, J. W., & Riddle, D. L. (1982). Caenorhabditis-elegans dauer larva pheromone. Journal of Nematology, 14, 443–443.Find this resource:

Golden, J. W., & Riddle, D. L. (1985). A gene affecting production of the Caenorhabditis-elegans dauer-inducing pheromone. Molecular Genetics and Genomics, 198, 534–536.Find this resource:

Grewal, P. S., & Richardson, P. N. (1991). Effects of Caenorhabditis-elegans (nematoda, rhabditidae) on yield and quality of the cultivated mushroom Agaricus-bisporus. Annals in Applied Biology, 118, 381–394.Find this resource:

Gudermann, T., Schoneberg, T., & Schultz, G. (1997). Functional and structural complexity of signal transduction via G-protein-coupled receptors. Annual Review of Neuroscience, 20, 399–427.Find this resource:

Gumienny, T. L., & Savage-Dunn, C. (2013). TGF-beta signaling in C. elegans. WormBook, 1–34, doi: 10.1895.Find this resource:

Guo, W., Wang, X., Ma, Z., Xue, L., Han, J., Yu, D., & Kang, L. (2011). CSP and takeout genes modulate the switch between attraction and repulsion during behavioral phase change in the migratory locust. PLoS Genetics, 7, e1001291.Find this resource:

Gupta, T., Morgan, H. R., Andrews, J. C., Brewer, E. R., & Certel, S. J. (2017) Methyl-CpG binding domain proteins inhibit interspecies courtship and promote aggression in Drosophila. Scientific Reports, 7, 5844.Find this resource:

Hamm, H. E. (2001). How activated receptors couple to G proteins. Proceedings of the National Academy of Sciences USA, 98, 4819–4821.Find this resource:

Hanlon, C. D., & Andrew, D. J. (2015). Outside-in signaling—A brief review of GPCR signaling with a focus on the Drosophila GPCR family. Journal of Cell Science, 128, 3533–3542.Find this resource:

Hashimoto, A., Sasaki, K., Koyama, S., & Satoh, T. (2013). Food exchange behavior between multiple founding queens of Polyrhachis moesta (Hymenoptera: Formicidae) changes during hibernation. Applied Entomology and Zoology, 48, 141–145.Find this resource:

Hassanali, A., Njagi, P. G., & Bashir, M. O. (2005). Chemical ecology of locusts and related acridids. Annual Review of Entomology, 50, 223–245.Find this resource:

Herlenius, E., & Lagercrantz, H. (2001). Neurotransmitters and neuromodulators during early human development. Early Human Development, 65, 21–37.Find this resource:

Herlenius, E., & Lagercrantz, H. (2004). Development of neurotransmitter systems during critical periods. Experimental Neurology, 190 Suppl 1, S8–S21.Find this resource:

Hill, H. F., Wenner, A. M., & Wells, P. H. (1976). Reproductive behavior in an overwintering aggregation of monarch butterflies. American Midland Naturalist, 95, 10–19.Find this resource:

Hobert, O., Tessmar, K., & Ruvkun, G. (1999). The Caenorhabditis elegans lim-6 LIM homeobox gene regulates neurite outgrowth and function of particular GABAergic neurons. Development, 126, 1547–1562.Find this resource:

Hodges, M. R., & Richerson, G. B. (2008). Contributions of 5-HT neurons to respiratory control: neuromodulatory and trophic effects. Respiratory Physiology & Neurobiology, 164, 222–232.Find this resource:

Hodgkin, J., & Doniach, T. (1997). Natural variation and copulatory plug formation in Caenorhabditis elegans. Genetics, 146, 149–164.Find this resource:

Homberg, U. (2002). Neurotransmitters and neuropeptides in the brain of the locust. Microscopy Research and Technique, 56, 189–209.Find this resource:

Hong, S. J., Lardaro, T., Oh, M. S., Huh, Y., Ding, Y., Kang, U. J., . . . Kim, K. S. (2008). Regulation of the noradrenaline neurotransmitter phenotype by the transcription factor AP-2beta. Journal of Biological Chemistry, 283, 16860–16867.Find this resource:

Hoopfer, E. D., Jung, Y., Inagaki, H. K., Rubin, G. M., & Anderson, D. J. (2015). P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila. Elife, 4, e11346.Find this resource:

Hoyer, S. C., Eckart, A., Herrel, A., Zars, T., Fischer, S. A., Hardie, S. L., & Heisenberg, M. (2008). Octopamine in male aggression of Drosophila. Current Biology, 18, 159–167.Find this resource:

Huang, Y. Y., & Kandel, E. R. (2007). 5-Hydroxytryptamine induces a protein kinase A/mitogen-activated protein kinase-mediated and macromolecular synthesis-dependent late phase of long-term potentiation in the amygdala. Journal of Neuroscience, 27, 3111–3119.Find this resource:

Hulme, S. E., & Whitesides, G. M. (2011). Chemistry and the worm: Caenorhabditis elegans as a platform for integrating chemical and biological research. Angewandte Chemie International Edition in English, 50, 4774–4807.Find this resource:

Jallon, J. M. (1984). A few chemical words exchanged by Drosophila during courtship and mating. Behavior Genetics, 14, 441–478.Find this resource:

Jeong, P. Y., Jung, M., Yim, Y. H., Kim, H., Park, M., Hong, E., . . . Paik, Y. K. (2005). Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone. Nature, 433, 541–545.Find this resource:

Joseph, R. M., & Heberlein, U. (2012). Tissue-specific activation of a single gustatory receptor produces opposing behavioral responses in Drosophila. Genetics, 192, 521–532.Find this resource:

Kang, L., Chen, X., Zhou, Y., Liu, B., Zheng, W., Li, R., . . . Yu, J., (2004). The analysis of large-scale gene expression correlated to the phase changes of the migratory locust. Proceedings of the National Academy of Sciences USA, 101(51), 17611–17615.Find this resource:

Katritch, V., Cherezov, V., & Stevens, R. C. (2013). Structure-function of the G protein-coupled receptor superfamily. Annual Review of Pharmacology and Toxicology, 53, 531–556.Find this resource:

Ko, K. I., Root, C. M., Lindsay, S. A., Zaninovich, O. A., Shepherd, A. K., Wasserman, S. A., . . . Wang, J. W. (2015). (p. 697) Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits. Elife, 4, e08298.Find this resource:

Koon, A. C., Ashley, J., Barria, R., DasGupta, S., Brain, R., Waddell, S., . . . Budnik, V. (2011). Autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. Nature Neuroscience, 14, 190–199.Find this resource:

Koon, A. C., & Budnik, V. (2012). Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling. Journal of Neuroscience, 32, 6312–6322.Find this resource:

Koyama, S., Matsui, S., Satoh, T., & Sasaki, K. (2015). Octopamine and cooperation: Octopamine regulates the disappearance of cooperative behaviours between genetically unrelated founding queens in the ant. Biology Letters, 11, 20150206.Find this resource:

Kravitz, E. A., & Fernandez Mde, L. (2015). Aggression in Drosophila. Behavioral Neuroscience, 129, 549–563.Find this resource:

Lagercrantz, H., & Ringstedt, T. (2001). Organization of the neuronal circuits in the central nervous system during development. Acta Paediatrica, 90, 707–715.Find this resource:

Langenhan, T., Barr, M. M., Bruchas, M. R., Ewer, J., Griffith, L. C., Maiellaro, I., . . . Monk, K. R. (2015). Model organisms in G protein-coupled receptor research. Molecular Pharmacology, 88, 596–603.Find this resource:

Lans, H., & Jansen, G. (2007). Multiple sensory G proteins in the olfactory, gustatory and nociceptive neurons modulate longevity in Caenorhabditis elegans. Developmental Biology, 303, 474–482.Find this resource:

Leinwand, S. G., Yang, C. J., Bazopoulou, D., Chronis, N., Srinivasan, J., & Chalasani, S. H. (2015). Circuit mechanisms encoding odors and driving aging-associated behavioral declines in Caenorhabditis elegans. Elife, 4, e10181.Find this resource:

Leo Lester, R., Grach, C., Paul Pener, M., & Simpson, S. J. (2005). Stimuli inducing gregarious colouration and behaviour in nymphs of Schistocerca gregaria. Journal of Insect Physiology, 51, 737–747.Find this resource:

Liu, W., Liang, X., Gong, J., Yang, Z., Zhang, Y. H., Zhang, J. X., & Rao, Y. (2011). Social regulation of aggression by pheromonal activation of Or65a olfactory neurons in Drosophila. Nature Neuroscience, 14, 896–902.Find this resource:

Ma, Z., Guo, W., Guo, X., Wang, X., & Kang, L. (2011). Modulation of behavioral phase changes of the migratory locust by the catecholamine metabolic pathway. Proceedings of the National Academy of Sciences USA, 108, 3882–3887.Find this resource:

Mao, Z., & Davis, R. L. (2009). Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity. Frontiers in Neural Circuits, 3, 5.Find this resource:

Marhold, J., Kramer, K., Kremmer, E., & Lyko, F. (2004). The Drosophila MBD2/3 protein mediates interactions between the MI-2 chromatin complex and CpT/A-methylated DNA. Development, 131, 6033–6039.Find this resource:

McGrath, P. T., Xu, Y., Ailion, M., Garrison, J. L., Butcher, R. A., & Bargmann, C. I. (2011). Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes. Nature, 477, 321–325.Find this resource:

Meunier, N., Belgacem, Y. H., & Martin, J. R. (2007). Regulation of feeding behaviour and locomotor activity by takeout in Drosophila. Journal of Experimental Biology, 210, 1424–1434.Find this resource:

Michel, M., Kemenes, I., Muller, U., & Kemenes, G. (2008). Different phases of long-term memory require distinct temporal patterns of PKA activity after single-trial classical conditioning. Learning and Memory, 15, 694–702.Find this resource:

Muller, U. (2000). Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees. Neuron, 27, 159–168.Find this resource:

Muller, U., & Carew, T. J. (1998). Serotonin induces temporally and mechanistically distinct phases of persistent PKA activity in Aplysia sensory neurons. Neuron, 21, 1423–1434.Find this resource:

Murrin, L. C., Sanders, J. D., & Bylund, D. B. (2007). Comparison of the maturation of the adrenergic and serotonergic neurotransmitter systems in the brain: Implications for differential drug effects on juveniles and adults. Biochemistry and Pharmacology, 73, 1225–1236.Find this resource:

Nall, A., & Sehgal, A. (2014). Monoamines and sleep in Drosophila. Behavioral Neuroscience, 128, 264–272.Find this resource:

Nieoullon, A., & Coquerel, A. (2003). Dopamine: A key regulator to adapt action, emotion, motivation and cognition. Current Opinion in Neurology, 16 Suppl 2, S3–S9.Find this resource:

O’Dell, T. J., Connor, S. A., Gelinas, J. N., & Nguyen, P. V. (2010). Viagra for your synapses: Enhancement of hippocampal long-term potentiation by activation of beta-adrenergic receptors. Cell Signalling, 22, 728–736.Find this resource:

Omenn, G. S. (1976). Neurochemistry and behavior in man. Western Journal of Medicine, 125, 434–451.Find this resource:

Osborne, K. A., Robichon, A., Burgess, E., Butland, S., Shaw, R. A., Coulthard, A., . . . Sokolowski, M. B. (1997). Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila. Science, 277, 834–836.Find this resource:

Ott, S. R., Verlinden, H., & Rogers, S. M. (2009). The phenotypic plasticity of swarm formation in the Desert locust: Mechanisms and consequences. Comparative Biochemistry and Physics A, 153a, S156–S156.Find this resource:

Ott, S. R., Verlinden, H., Rogers, S. M., Brighton, C. H., Quah, P. S., Vleugels, R. K., . . . Vanden Broeck, J. (2012). Critical role for protein kinase A in the acquisition of gregarious behavior in the desert locust. Proceedings of the National Academy of Sciences USA, 109, E381–E387.Find this resource:

Parrish, J. K., & Edelstein-Keshet, L. (1999). Complexity, pattern, and evolutionary trade-offs in animal aggregation. Science, 284, 99–101.Find this resource:

Pener, M. P., & Simpson, S. J. (2009). Locust phase polyphenism: An update. Advances in Insect Physiology, 36, 1–272.Find this resource:

Pener, M. P., & Yerushalmi, Y. (1998). The physiology of locust phase polymorphism: an update. Journal of Insect Physiology, 44, 365–377.Find this resource:

Pungaliya, C., Srinivasan, J., Fox, B. W., Malik, R. U., Ludewig, A. H., Sternberg, P. W., & Schroeder, F. C. (2009). A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proceedings of the National Academy of Sciences USA, 106, 7708–7713.Find this resource:

Ren, P., Lim, C. S., Johnsen, R., Albert, P. S., Pilgrim, D., & Riddle, D. L. (1996). Control of C. elegans larval development by neuronal expression of a TGF-beta homolog. Science, 274, 1389–1391.Find this resource:

Riedl, C. A., & Sokolowski, M. B. (2004). Behavioral genetics: Guanylyl cyclase prompts worms to party. Current Biology, 14, R657–R658.Find this resource:

Riemensperger, T., Isabel, G., Coulom, H., Neuser, K., Seugnet, L., Kume, K., . . . Birman, S. (2011). Behavioral consequences of dopamine deficiency in the Drosophila central nervous system. Proceedings of the National Academy of Sciences USA, 108, 834–839.Find this resource:

(p. 698) Riipi, M., Alatalo, R. V., Lindstrom, L., & Mappes, J. (2001). Multiple benefits of gregariousness cover detectability costs in aposematic aggregations. Nature, 413, 512–514.Find this resource:

Robinson, G. E., Grozinger, C. M., & Whitfield, C. W. (2005). Sociogenomics: social life in molecular terms. Nature Reviews Genetics, 6, 257–271.Find this resource:

Roeder, T. (2002). Biochemistry and molecular biology of receptors for biogenic amines in locusts. Microscopy Research and Technique, 56, 237–247.Find this resource:

Roeder, T. (2005). Tyramine and octopamine: Ruling behavior and metabolism. Annual Review of Entomology, 50, 447–477.Find this resource:

Rogers, C., Reale, V., Kim, K., Chatwin, H., Li, C., Evans, P., & de Bono, M. (2003). Inhibition of Caenorhabditis elegans social feeding by FMRFamide-related peptide activation of NPR-1. Nature Neuroscience, 6, 1178–1185.Find this resource:

Rogers, S. M., Cullen, D. A., Anstey, M. L., Burrows, M., Despland, E., Dodgson, T., . . . Simpson, S. J. (2014). Rapid behavioural gregarization in the desert locust, Schistocerca gregaria entails synchronous changes in both activity and attraction to conspecifics. Journal of Insect Physiology, 65, 9–26.Find this resource:

Rogers, S. M., & Ott, S. R. (2015). Differential activation of serotonergic neurons during short- and long-term gregarization of desert locusts. Proceedings of the Royal Society B-Biological Sciences, 282(1800), 20142062.Find this resource:

Rosenbaum, D. M., Rasmussen, S. G., & Kobilka, B. K. (2009). The structure and function of G-protein-coupled receptors. Nature, 459, 356–363.Find this resource:

Ruxton, G. D., & Sherratt, T. N. (2006). Aggregation, defence and warning signals: the evolutionary relationship. Proceedings of the Royal Society B-Biological Sciences, 273, 2417–2424.Find this resource:

Sarov-Blat, L., So, W. V., Liu, L., & Rosbash, M. (2000). The Drosophila takeout gene is a novel molecular link between circadian rhythms and feeding behavior. Cell, 101, 647–656.Find this resource:

Schmidt, M., Huber, L., Majdazari, A., Schutz, G., Williams, T., & Rohrer, H. (2011). The transcription factors AP-2beta and AP-2alpha are required for survival of sympathetic progenitors and differentiated sympathetic neurons. Developmental Biology, 355, 89–100.Find this resource:

Schroeder, F. C. (2015). Modular assembly of primary metabolic building blocks: a chemical language in C. elegans. Chemistry and Biology, 22, 7–16.Find this resource:

Seidelmann, K. & Ferenz, H. J. (2002). Courtship inhibition pheromone in desert locusts, Schistocerca gregaria. Journal of Insect Physiology, 48(11), 991–996.Find this resource:

Shankar, S., Chua, J. Y., Tan, K. J., Calvert, M. E., Weng, R., Ng, W. C., Mori, K., & Yew, J. Y. (2015). The neuropeptide tachykinin is essential for pheromone detection in a gustatory neural circuit. Elife, 4, e06914.Find this resource:

Shorter, J., Couch, C., Huang, W., Carbone, M. A., Peiffer, J., Anholt, R. R., & Mackay, T. F. (2015). Genetic architecture of natural variation in Drosophila melanogaster aggressive behavior. Proceedings of the National Academy of Sciences USA, 112, E3555–E3563.Find this resource:

Simpson, S. J., Despland, E., Hagele, B. F., & Dodgson, T. (2001). Gregarious behavior in desert locusts is evoked by touching their back legs. Proceedings of the National Academy of Sciences USA, 98, 3895–3897.Find this resource:

Sinakevitch, I., & Strausfeld, N. J. (2006). Comparison of octopamine-like immunoreactivity in the brains of the fruit fly and blow fly. Journal of Comparative Neurology, 494, 460–475.Find this resource:

Skoulakis, E. M., Kalderon, D., & Davis, R. L. (1993). Preferential expression in mushroom bodies of the catalytic subunit of protein kinase A and its role in learning and memory. Neuron, 11, 197–208.Find this resource:

Srinivasan, J., von Reuss, S. H., Bose, N., Zaslaver, A., Mahanti, P., Ho, M. C., . . . Schroeder, F. C. (2012). A modular library of small molecule signals regulates social behaviors in Caenorhabditis elegans. PLoS Biology, 10, e1001237.Find this resource:

Stern, M. (1999). Octopamine in the locust brain: cellular distribution and functional significance in an arousal mechanism. Microscopy Research and Technique, 45, 135–141.Find this resource:

Stevenson, P. A., Dyakonova, V., Rillich, J., & Schildberger, K. (2005). Octopamine and experience-dependent modulation of aggression in crickets. Journal of Neuroscience, 25, 1431–1441.Find this resource:

Stevenson, P. A., Hofmann, H. A., Schoch, K., & Schildberger, K. (2000). The fight and flight responses of crickets depleted of biogenic amines. Journal of Neurobiology, 43, 107–120.Find this resource:

Strader, C. D., Fong, T. M., Tota, M. R., Underwood, D., & Dixon, R. A. F. (1994). Structure and function of G-protein-coupled receptors. Annual Review of Biochemistry, 63, 101–132.Find this resource:

Symonds, M. R. E., & Wertheim, B. (2005). The mode of evolution of aggregation pheromones in Drosophila species. Journal of Evolutionary Biology, 18, 1253–1263.Find this resource:

Syrovatkina, V., Alegre, K. O., Dey, R., & Huang, X. Y. (2016). Regulation, signaling, and physiological functions of G-proteins. Journal of Molecular Biology, 428(19), 3850–3868.Find this resource:

Tachibana, S., Touhara, K., & Ejima, A. (2015). Modification of male courtship motivation by olfactory habituation via the GABAA receptor in Drosophila melanogaster. PLoS One, 10, e0135186.Find this resource:

Tobin, A. B. (2008). G-protein-coupled receptor phosphorylation: Where, when and by whom. British Journal of Pharmacology, 153(Suppl 1), S167–S176.Find this resource:

Tobin, D. M., Madsen, D. M., Kahn-Kirby, A., Peckol, E. L., Moulder, G., Barstead, R., Maricq, A. V., & Bargmann, C. I. (2002). Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron, 35, 307–318.Find this resource:

Tognini, P., Napoli, D., Tola, J., Silingardi, D., Della Ragione, F., D’Esposito, M., & Pizzorusso, T. (2015). Experience-dependent DNA methylation regulates plasticity in the developing visual cortex. Nature Neuroscience, 18, 956–958.Find this resource:

Topaz, C. M., D’Orsogna, M. R., Edelstein-Keshet, L., & Bernoff, A. J. (2012). Locust dynamics: behavioral phase change and swarming. PLoS Computational Biology, 8, e1002642.Find this resource:

Torto, B., Obengofori, D., Njagi, P. G. N., Hassanali, A., & Amiani, H. (1994). Aggregation pheromone system of adult gregarious desert locust Schistocerca-gregaria (Forskal). Journal of Chemical Ecology, 20, 1749–1762.Find this resource:

Uvarov, B. (1966). Grasshoppers and Locusts, A handbook of general acridology., Cambridge, UK: Cambridge University Press, 1, 332–386.Find this resource:

van der Goes van Naters, W., & Carlson, J. R. (2007). Receptors and neurons for fly odors in Drosophila. Current Biology, 17, 606–612.Find this resource:

Venken, K. J., & Bellen, H. J. (2014). Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster. Methods, 68, 15–28.Find this resource:

Verlinden, H., Badisco, L., Marchal, E., Van Wielendaele, P., & Vanden Broeck, J. (2009). Endocrinology of reproduction and phase transition in locusts. General and Comparitive Endocrinology, 162, 79–92.Find this resource:

(p. 699) Verlinden, H., Vleugels, R., Marchal, E., Badisco, L., Pfluger, H. J., Blenau, W., & Broeck, J. V. (2010). The role of octopamine in locusts and other arthropods. J Insect Physiology, 56, 854–867.Find this resource:

von Reuss, S. H., Bose, N., Srinivasan, J., Yim, J. J., Judkins, J. C., Sternberg, P. W., & Schroeder, F. C. (2012). Comparative metabolomics reveals biogenesis of ascarosides, a modular library of small-molecule signals in C. elegans. Journal of the American Chemistry Society, 134, 1817–1824.Find this resource:

von Reuss, S. H., & Schroeder, F. C. (2015). Combinatorial chemistry in nematodes: modular assembly of primary metabolism-derived building blocks. Natural Products Reports, 32, 994–1006.Find this resource:

Waddell, S. (2010). Dopamine reveals neural circuit mechanisms of fly memory. Trends in Neuroscience, 33, 457–464.Find this resource:

Wang, L., & Anderson, D. J. (2010). Identification of an aggression-promoting pheromone and its receptor neurons in Drosophila. Nature, 463, 227–231.Find this resource:

Wells, H., Wells, P. H., & Cook, P. (1990). The importance of overwinter aggregation for reproductive success of monarch butterflies (Danaus-plexippus L). Journal of Theoretical Biology, 147, 115–131.Find this resource:

Wertheim, B., van Baalen, E. J. A., Dicke, M., & Vet, L. E. M. (2005). Pheromone-mediated aggregation in nonsocial arthropods: An evolutionary ecological perspective. Annual Review of Entomology, 50, 321–346.Find this resource:

Williams, M. J., Goergen, P., Rajendran, J., Klockars, A., Kasagiannis, A., Fredriksson, R., & Schioth, H. B. (2014). Regulation of aggression by obesity-linked genes TfAP-2 and Twz through octopamine signaling in Drosophila. Genetics, 196, 349–362.Find this resource:

Wnuk, A., Kostowski, W., Korczynska, J., Szczuka, A., Symonowicz, B., Bienkowski, P., . . . Godzinska, E. J. (2014). Brain GABA and glutamate levels in workers of two ant species (Hymenoptera: Formicidae): Interspecific differences and effects of queen presence/absence. Insect Science, 21, 647–658.Find this resource:

Xu, P., Atkinson, R., Jones, D. N., & Smith, D. P. (2005). Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons. Neuron, 45, 193–200.Find this resource:

Yamamoto, S., & Seto, E. S. (2014). Dopamine dynamics and signaling in Drosophila: An overview of genes, drugs and behavioral paradigms. Experimental Animals, 63, 107–119.Find this resource:

Yang, M., Wei, Y., Jiang, F., Wang, Y., Guo, X., He, J., & Kang, L. (2014). MicroRNA-133 inhibits behavioral aggregation by controlling dopamine synthesis in locusts. PLoS Genetics, 10, e1004206.Find this resource:

Yuan, Q., Song, Y., Yang, C. H., Jan, L. Y., & Jan, Y. N. (2014). Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila. Nature Neuroscience, 17, 81–88.Find this resource:

Zhang, X., Feng, L., Chinta, S., Singh, P., Wang, Y., Nunnery, J. K., & Butcher, R. A. (2015). Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans. Proceedings of the National Academy of Sciences USA, 112, 3955–3960.Find this resource:

Zhou, C., Rao, Y., & Rao, Y. (2008). A subset of octopaminergic neurons are important for Drosophila aggression. Nature Neuroscience, 11(9), 1059–1067.Find this resource:

Zilberman, D. (2008). The evolving functions of DNA methylation. Current Opinion in Plant Biology, 11, 554–559.Find this resource:

Zwaal, R. R., Mendel, J. E., Sternberg, P. W., & Plasterk, R. H. (1997). Two neuronal G proteins are involved in chemosensation of the Caenorhabditis elegans Dauer-inducing pheromone. Genetics, 145, 715–727. (p. 700) Find this resource: