THE ROLE OF SEROTONIN IN FLY AGGRESSION: A SIMPLIFIED SYSTEM TO INVESTIGATE A
COMPLEX BEHAVIOR
by
ANDREW NOEL BUBAK
B.S., University of South Dakota, 2010
M.S., University of South Dakota, 2012
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Neuroscience Program
2017 This thesis for the Doctor of Philosophy degree by
Andrew Noel Bubak
has been approved for the
Neuroscience Program
by
Tania Reis, Chair
John Swallow, Advisor
Thomas Finger
Abigail Person
Michael Greene
Date: ___5-19-2017___
ii Bubak, Andrew Noel (Ph.D., Neuroscience)
The Role of Serotonin in Fly Aggression: A Simplified System to Investigate a Complex Behavior
Thesis directed by Professor John G. Swallow.
ABSTRACT
The use of aggressive behavior for the obtainment of food resources, territory, and reproductive mates is ubiquitous across animal taxa. The appropriate perception and performance of this highly conserved behavior towards conspecifics is critical for individual fitness and thus a product of evolutionary selection in species as diverse as mammals to insects. The serotonergic (5-HT) system, in particular, is a well-known neurochemical modulator of aggression in both vertebrates and invertebrates. However, the underlying proximate mechanisms of 5-HT receptor subtypes and their role in mediating other neurochemical systems also involved in aggression is not well understood in invertebrate species.
Collectively, this work describes the role of 5-HT in the context of game-theory models, sex differences, and interactions with other aggression-mediating neurochemical systems in a novel invertebrate model, the stalk-eyed fly. Specifically, we investigated the role of monoamines in assessing opponent resource- holding potential in aggressive interactions. Using large, published data sets, we aimed to distinguish between two commonly used models of assessment strategies, self-determined persistence and mutual rival assessment, and found that non-morphological factors such as central 5-HT activity can influence individual aggression, potentially altering contest intensity or outcome despite initial size discrepancies. This hypothesis was tested by altering 5-HT levels in size-mismatched opponents and found that while elevated 5-HT increased aggression in the smaller opponent, contest outcome was unaltered with the smaller opponent losing the majority of contests.
Finally, we describe a stage- and sex-specific, inhibitory role for the 5-HT2 and neuropeptide F receptors that is reminiscent of vertebrate species. Additionally, we discuss and present our findings that global 5-HT activity influences the neuropeptide tachykinin, resulting in significant changes in high-intensity aggressive behaviors in males only. Together, our results describe a conserved, context- and sex-dependent role for 5-HT in aggressive behavior, providing valuable insight into the evolutionary origins of this complex behavior.
The form and content of this abstract are approved. I recommend its publication.
Approved: John G. Swallow
iii TABLE OF CONTENTS CHAPTER
I. NEUROCHEMISTRY AS A BRIDGE BETWEEN MORPHOLOGY AND BEHAVIOR: PERSPECTIVES ON AGGRESSION IN INSECTS ...... 1
Abstract ...... 1
Introduction ...... 1
Morphological Correlates of Aggression ...... 3
Neurochemical and Pheromonal Modulation of Aggression ...... 6
Octopamine (OA) ...... 6
Serotonin (5-HT) ...... 7
Pheromones and Neuropeptides ...... 9
The Stalk-Eyed Fly as a Model System for Studying Aggression ...... 12
Perspectives ...... 17
Contributions ...... 18
II. ASSESSMENT STRATEGIES AND FIGHTING PATTERNS IN ANIMAL CONTESTS: A ROLE FOR SEROTONIN? ...... 19
Abstract ...... 19
Introduction ...... 19
Materials and Methods ...... 23
Subjects ...... 23
Forced-Fight Paradigm ...... 23
Drug Administration Studies ...... 24
Serotonin Quantification ...... 24
Statistical Analysis ...... 25
Results ...... 25
Relationships with Preexisting Game Theory Models ...... 26
Relationships between Fight Strategy and Body Size ...... 26
Relationships of Fighting Strategy with 5-HT ...... 28
Discussion ...... 28
iv
Contributions ...... 31
III. DAVID VS. GOLIATH: SEROTONIN MODULATES OPPONENT PERCEPTION BETWEEN SMALLER AND LARGER RIVALS ...... 32
Abstract ...... 32
Introduction ...... 33
Materials and Methods ...... 35
Subjects ...... 35
Pharmacological Manipulation ...... 36
Forced-Fight Paradigm ...... 36
Dissection and Sample Preparation ...... 36
Data Acquisition ...... 37
Behavioral Analysis ...... 37
Statistical Analysis ...... 38
Results ...... 39
Pharmacological Manipulation of 5-HT ...... 39
Aggressive Behavior and Fight Outcome in Treated Flies ...... 39
Aggressive Responses of Larger Opponents Toward Treated Rivals ...... 41
Discussion ...... 43
Contributions ...... 46
IV. SEX DIFFERENCES IN AGGRESSION: DIFFERENTIAL ROLES OF 5-HT2, NEUROPEPTIDE F, AND TACHYKININ ...... 47
Abstract ...... 47
Introduction ...... 47
Materials and Methods ...... 51
Subjects ...... 51
Drug Administration ...... 51
Forced-Fight Paradigm and Behavioral Analysis ...... 51
5-HT Quantification ...... 52
v
RNAi Design and Administration ...... 52
RNA Isolation and Quantification ...... 53
Statistical Analysis ...... 54
Results ...... 54
Expression of 5-HT1A, 5-HT2, 5-HT7 Receptors and the Serotonin Transporter in Male and Female Brains ...... 54
Expression of the Neuropeptide Tk and the NPF Receptor in Male and Female Brains ...... 55
Increased Aggression Corresponds with Reduced 5-HT2 and Heightened Tk Expression in Males but not in Females ...... 55
5-HT2 Knock-Down Increases Aggression in Males but not in Females ...... 58
5-HT2 Activity Modulates NPFr Expression in Males but not Females ...... 61
Elevating 5-HT Concentrations Increases Tk Expression in Males but not Females...... 61
Discussion ...... 63
Contributions ...... 67
V. IS THERE A CONSERVED ROLE FOR 5-HT IN AGGRESSION BETWEEN VERTBRATES AND INVERTEBRATES? ...... 68
Introduction ...... 68
The Stalk-Eyed Fly as a Case Study ...... 70
Interactions Between 5-HT and Neuropeptides ...... 72
Perspective on Future Research ...... 74
REFERENCES ...... 76
vi LIST OF TABLES
TABLE
2.1 Range and mean of eyestalk length between winners and losers ...... 25
3.1 Whole brain 5-HT concentrations in 5-HTP pretreated Davids, untreated Davids, and Goliaths .... 39
4.1 List of primer and probe sequences ...... 53
vii LIST OF FIGURES
FIGURE
1.1 Representative images of both male and female stalk-eyed flies from a sexually dimorphic species, T. dalmanni, and a sexually monomorphic species, T. quinqueguttata ...... 13
1.2 Flow chart of aggressive behaviors exhibited by T. dalmanni ...... 14
2.1 Performance of low intensity behaviors separated by status and size ...... 26
2.2 Escalation patterns differ between status and size...... 27
2.3 Relationship between escalation patterns and neural 5-HT differs between winners and losers ...... 28
3.1 Effects of 5-HTP treatment on smaller opponents during aggressive interactions ...... 40
3.2 Effects of 5-HTP treatment on the initiation of a fight in smaller opponents ...... 41
3.3 5-HTP treated smaller opponents engaged in more high-intensity aggressive behaviors ...... 41
3.4 Fighting a smaller, treated opponent alters escalation patterns ...... 42
3.5 Larger opponents fighting smaller, treated opponents escalated more interactions ...... 42
4.1 Males and females have different expression levels of 5-HT receptor subtypes and the 5-HT transporter ...... 54
4.2 Social isolation increases initiations and high-intensity aggressive behaviors in males only ...... 57
4.3 Social isolation alters 5-HT2 and Tk expression levels in males only ...... 58
4.4 Designed 5-HT2 siRNA is specific ...... 59
4.5 5-HT2 siRNA increases high-intensity behaviors but not initiations in males only ...... 60
4.6 5-HT2 siRNA results in a reduction of NPFr expression in males only ...... 61
4.7 5-HTP treatment increases Tk and NPFr expression in males only ...... 62
5.1 Typical fight progression for male stalk-eyed flies ...... 74
viii CHAPTER I
NEUROCHEMISTRY AS A BRIDGE BETWEEN MORPHOLOGY AND BEHAVIOR:
PERSPECTIVES ON AGGRESSION IN INSECTS1
Abstract
Aggression is a common behavioral trait shared in many animals, including both vertebrates and invertebrates. However, the type and intensity of agonistic encounters and displays can vary widely both across and within species, resulting in complicated or subjective interpretations that create difficulties in developing theoretical models that can be widely applied. The need to easily and objectively identify quantifiable behaviors and their associated morphologies becomes especially important when attempting to decipher the neurological mechanisms underlying this complex behavior. Monoamines, neuropeptides, and pheromones have been implicated as important neuromodulators for agonistic displays in both invertebrates and vertebrates.
Additionally, recent breakthroughs in insect research have revealed exciting proximate mechanisms important in aggression that may be broadly relevant, due to the relatively high conservation of these neurochemical systems across animal taxa. In this review, we present the latest research demonstrating the importance of monoamines, neuropeptides, and pheromones as neuromodulators for aggression across a variety of insect species. Additionally, we describe the stalk-eyed fly as a model system for studying aggression, which integrates physiological, morphological, and neurochemical approaches in exploring detailed mechanisms responsible for this common yet complex behavior. We conclude with our perspective on the most promising lines of future research aimed at understanding the proximate and ultimate mechanisms underlying aggressive behaviors.
Introduction
Due to the potentially profound fitness benefits of gaining access to limited resources through competitions, both vertebrates and invertebrates commonly exhibit aggressive behavior. Precisely because of this central fitness role, aggression presents a valuable model to explore evolutionary connections between behavior, morphology, and physiology. Since fighting can be costly, animals have evolved a variety of
1 The material in Chapter I was originally published in Current Zoology and is included with permission: Bubak AN, Grace JL, Watt MJ, Renner KJ, Swallow JG (2014) Neurochemistry as a bridge between morphology and behavior: perspectives on aggression in insects. Curr Zool. 60(6): 778-790.
1 morphological ornaments and armaments that may be employed in intricate signaling displays to convey aggressive intent and fighting ability without engaging physically (Emlen, 2008; Geist, 1966). Insects provide an important, underutilized model to study the connections between behavior, morphology, and physiology, because sexual selection has resulted in the evolution of extraordinary secondary sexual characters that are used in aggressive confrontations as both signals and weapons. Furthermore, research focusing on the neurobiological aspects involved in aggression has uncovered intrinsic factors such as monoamines, neuropeptides, and pheromones as important modulators of this complex behavior. Importantly, many of these underlying neurochemical modulatory mechanisms appear to have similar functions in other taxa, including vertebrates.
To obtain a better understanding of the mechanisms driving complex behaviors, such as aggression, it is imperative to incorporate morphological and physiological information while also acknowledging underlying neurobiological factors that have a significant modulatory role on behavioral expression. However, gathering these three elements of information within a given taxonomic group can prove difficult, leading to omission of critical data that cannot be supplied by extrapolation from different species. This seems especially pertinent when considering factors such as neuromodulation that may shape proximate expression of individual behavior, which could in turn explain why an individual’s responses can differ depending on the particular circumstance, e.g., facing a familiar versus unknown opponent. Given that the outcome of aggressive encounters often determines reproductive success, the variables mediating agonistic behavior in the proximate sense will provide the substrate for evolutionary selection. Therefore, the ability to quantify the interaction between morphology and neurophysiology in directing behavioral expression has the potential to allow rigorous testing of currently held hypotheses, such as why certain morphological traits continue to be selected for despite apparent cost in some species but have been lost in relatives. The aim of this review is to present the information from insect studies to demonstrate how neurochemistry can be leveraged to investigate the link between morphology and behavior. We show how these elements can be studied simultaneously by utilizing the stalk-eyed fly as a model system, which incorporates morphological and aggressive variability with highly specific neurochemical detection and manipulation methods. We then provide a perspective on possible future approaches using this model that could benefit our current understanding of complex animal behaviors.
2 Morphological Correlates of Aggression
The diversity and conspicuous nature of morphologies associated with aggressive displays has long attracted the attention of evolutionary biologists. The overwhelming majority of these examples come from sexually dimorphic species, which often experience strong sexual selection (Andersson, 1994). We will use the terms armament to refer to morphologies shaped by intrasexual selection (typically male-male competition) and ornament to refer to morphologies shaped by intersexual selection (usually female choice). The most plausible explanation for the evolution of armaments is their use in combat, and typically the individual with the largest armament is more likely to win aggressive encounters (Eberhard, 1987; Moczek and Emlen, 2000; Wcislo and
Eberhard, 1989). However, when armaments are an honest signal of status, they may actually reduce combat, since males can assess one another and an inferior male may choose not to fight in order to avoid unnecessary costs when winning is unlikely. Furthermore, while an ornament does not have to be used in combat, it may either directly or indirectly signal condition, and could potentially be used for assessment of fighting ability by rival males. Therefore, armaments and ornaments are not necessarily mutually exclusive traits. This is supported by the observation that within many species that experience strong sexual selection, females benefit from choosing males bearing morphological features that also predict high success in intrasexual competition
(Berglund et al., 1996; Conner, 1988; Suzaki et al., 2013; Watson and Simmons, 2010). Morphological traits conveying individual status to both sexes may also encompass different sensory modalities in insects, such as auditory information. This is exemplified by the songs of male crickets, which not only communicate competitive ability to rival males but also attractiveness to potential mates (Brown et al., 1996; Brown et al.,
2006). Thus, perception of a range of morphological characters, such as armaments and ornaments, by individuals can be a straightforward way to assess fighting ability of potential rivals.
Developmental history is critical for the optimal growth of armaments that will withstand the rigors of combat and, thus, have value as an aggressive signal. Appropriately, this topic has been extensively studied (for review see Emlen and Nijhout, 2000). In the case of holometabolous insects (those that pupate, e.g., flies, beetles, wasps), all the resources necessary to express these traits must be obtained during a discrete larval developmental window, followed by the simultaneous formation of all adult structures during the quiescent pupal phase. Once emergent, adult individuals have little opportunity to alter the expression of morphological structures, which are static upon final eclosion to the adult form due to the rigid nature of the exoskeleton. Thus,
3 resource limitations during development can have important fitness consequences and may ultimately prove the deciding factor for future agonistic encounters.
Two critical periods of development have been associated with the condition-dependent growth of secondary sexual characters in insects (Emlen et al., 2006). The first critical period applies to all insects, and occurs during the end of larval feeding, just before either the final molt to adulthood for hemimetabolous species (e.g., crickets) or the transition to the pupal stage for holometabolous species (e.g., flies). During this time, levels of juvenile hormone (JH) and ecdysone interact to signal the developmental transition from either preadult nymph or larva to the next stage (Emlen and Allen, 2003). Recent experiments indicate that JH regulates condition-dependent expression of mandibles in male stag beetles (Gotoh et al., 2011) and horned flour beetles (Okada et al., 2012). Since mandibles in these beetle species also function as armaments, the fluctuation in circulating JH, as influenced by resources available during larval feeding, may also be a proximate factor in mediating intrasexual competition. For holometabolous insects, a second critical developmental period occurs once the larva has ceased feeding and has entered pupation. This developmental period spans the time during which growth and development of the adult structures actually occurs. During the second critical period, activity of the insulin/insulin-like growth factor signaling (IIS) pathway is associated with development of secondary sexual characters, including ornaments and armaments (Emlen et al., 2006). In beetles, horn growth during the pupal phase (following cessation of larval feeding) is particularly sensitive to circulating insulin levels compared to other developing organs, suggesting another opportunity for larval condition to influence armament expression in the adult (Emlen et al., 2012; Warren et al., 2013). In other words, nutrition accrued during larval feeding can continue to influence developmental signaling pathways in a condition-dependent manner when feeding is no longer occurring. The IIS pathway has also been implicated in determining adult polyphenism in hemimetabolous insects, as demonstrated by its role in the differentiation of termite soldier castes (Hattori et al., 2013). Most likely, the activities of JH and IIS influence each other, with both being affected by either nutrition or condition that will, in turn, depend on larval feeding experience
(Abrisqueta et al., 2014; Perez-Hedo et al., 2014).
While most variation in insect armaments appears to be heavily condition-dependent, expression is also heritable to some degree (Gotoh et al., 2012; Unrug et al., 2004), as is social dominance (Moore et al.,
2002). Candidate gene studies have revealed that the developmental gene doublesex (dsx), which is involved in
4 many aspects of insect sexual dimorphism, has a dramatic impact on the development of armaments both within and between sexes of horned beetles (Kijimoto et al., 2012). Recent investigations have also shown that the dsx gene in male stag beetles is more sensitive to circulating JH levels, which promotes growth of enlarged male- type mandibles that are used during intrasexual competition (Gotoh et al., 2014). Sequencing of RNA suggests that several other undescribed genes involved in the expression of scarab beetle horns are under recent positive selection, most likely caused by sexual selection favoring larger armaments (Warren et al., 2014). Combined, this raises the possibility that genes such as dsx will infer some intrasexual heritability in armaments to influence future success during agonistic conflicts. However, the degree to which genetic makeup will affect armament growth appears to be intimately linked with larval experience and expression of condition-dependent factors such as JH.
If competitors have similar developmental and/or genetic histories and so have comparable morphology, how can one predict the victor? In this case, it may be beneficial to consider individual social and environmental factors that affect adulthood. Within insects, sexually mature males typically show the most aggressive behaviors, and aggression is usually increased in larger males (Dixon and Cade, 1986; Moore et al.,
2014). In cases where males defend territories, resident males often have an advantage over non-resident males in aggressive disputes (Simmons, 1986). Additionally, older individuals are more likely to show increased aggression in contests, both with age-matched and younger opponents, and have an increased probability of winning fights against younger opponents (Stockermans and Hardy, 2013; Tsai et al., 2014). Levels of aggression have also been shown to increase with social density (Wang and Anderson, 2010). Another contributing factor could be motivational asymmetry. For example, male crickets that have experienced restricted access to mating opportunities are both more aggressive and more likely to win contests against males that have continuous access to females (Brown et al., 2007). Furthermore, the act of winning or losing a contest in itself can affect future outcomes, although experimental evidence supporting either winner or loser effects has been mixed (see Chase et al., 1994) and may depend upon opponent familiarity as demonstrated for some vertebrate species (Forster et al., 2005; Ling et al., 2010). However, to fully understand the utilization of armaments by different species during actual competition, it is imperative to consider other intrinsic factors that have a role in the success of competitions. For example, what neurobiological factors influence the motivational state of opponents, and can this be enough to overcome morphological biases?
5 Neurochemical and Pheromonal Modulation of Aggression
Much of the pioneering work investigating the roles of various neurochemicals, namely monoamines, on invertebrate aggression was completed in arthropods, specifically crustaceans (Livingstone et al., 1980;
Harris-Warrick and Kravitz, 1984; Huber et al., 1997). More recently, the powerful genetic tools available to alter brain function in Drosophila (Baier et al., 2002; Miczek et al., 2007), as well as pharmacological interventions that alter the highly ritualized and characterized fighting behavior of crickets (Adamo and Hoy,
1995; Stevenson et al., 2000; Adamo et al., 1995), have provided insight into the neuromodulation of this complex behavior in insects. Taken as a whole, these studies have sometimes yielded contradictory or ambiguous results, which may arise from such factors as species differences, rearing conditions, subjective behavioral scoring, receptor subtype expression, and genetic background. Nevertheless, there appears to be a close relationship between the effects of the monoamines octopamine (OA) and serotonin (5-HT), along with other less studied neuropeptides and pheromones in the modulation of aggressive displays, indicating a high degree of evolutionary conservation in the behavioral function of these systems across arthropods.
Octopamine (OA)
An important aspect in animal aggression is the decision to engage in a potentially costly, although sometimes valuable, antagonistic interaction. The physiological activity accompanying initial opponent assessment is generally referred to as the fight or flight response, mediated in mammals and other vertebrates by the sympathetic adrenergic/noradrenergic systems (Nelson and Trainor, 2007; Watt et al., 2007). In insects, however, there does not appear to be a physiological role for these catecholamines. Instead, insects rely on two other monoamines, tyramine (TA) and its hydroxylated metabolite, the norepinephrine analog octopamine
(OA), to work in a physiologically similar manner (Roeder et al., 2003; Roeder, 2005; Verlinden et al., 2010;
Farooqui, 2012).
The octopaminergic system has been convincingly implicated in the motivation and escalation of aggressive behavior in some insect species (Adamo et al., 1995; Stevenson et al., 2000; Baier et al., 2002;
Stevenson et al., 2005; Hoyer et al., 2008; Zhou et al., 2008; Rillich and Stevenson, 2011; Stevenson and
Schildberger, 2013). Much of this research was conducted in crickets and fruit flies, utilizing both pharmacological and genetic approaches. In male crickets, one of the first suggestions for the role of OA in
6 modulating insect aggression came from the finding that OA increased in the hemolymph following agonistic encounters (Adamo et al., 1995). Subsequently, depletion of dopamine (DA) and OA in male crickets by hemocoel injections of the tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine (AMT), was found to reduce the initiation, level and duration of aggressive behaviors (Stevenson et al., 2000). The reduced aggressive responses were linked to the depletion of OA, rather than DA, since the behavior was rescued by treatment with the OA receptor agonist chlordimeform in OA-depleted crickets and suppressed in non OA-depleted crickets treated with the OA antagonist epinastine (Stevenson et al., 2005). However, it should be noted that while OA depletion in crickets reduces the intensity of fights, expression of aggressive behavior is not totally abolished
(Stevenson et al., 2000). This suggests that for male crickets, the primary role of OA is to increase the individual’s willingness to escalate the level of aggression once the fight has actually been initiated. In contrast, recent evidence suggests that DA, rather than OA, is necessary for the recovery of aggression in crickets that have been socially defeated (Rillich and Stevenson, 2014), possibly by modulating the motivation to initiate future agonistic encounters.
A similar role for OA in modulating Drosophila aggression has been suggested from work conducted in mutant flies. Drosophila mutants lacking the enzyme tyramine-β-hydroxylase (TβH), which catalyzes the synthesis of OA from TA, exhibit reduced aggression (Baier et al., 2002; Hoyer et al., 2008, Zhou et al., 2008).
Conversely, both treatment with the OA receptor agonist chlordimeform and overexpression of TβH, independently increased aggression in socially reared flies (Zhou et al., 2008). These responses appear to be governed by a specific population of octopaminergic neurons found in the suboesophageal ganglion (Zhou et al., 2008). The role of OA in Drosophila aggression may involve neuromodulatory regulation of contextually appropriate behavioral responses to sensory cues conveyed by the opponent, since the absence of OA induces courtship behavior between males rather than aggression (Certel et al., 2007).
Serotonin (5-HT)
The evolutionarily ancient monoamine, serotonin (5-HT), has fundamental roles in a variety of physiological processes in both vertebrates and invertebrates. Although widely studied in other taxa, relatively few investigations have examined the role of 5-HT in insect aggression. As with OA, much of the pioneering work suggesting that 5-HT enhances aggression in arthropods was completed in crustaceans (Livingstone et al.,
7 1980; Edwards and Kravitz, 1997; Huber et al., 1997). For example, both acute and constant infusion of 5-HT into the hemolymph of crayfish Astacus astacus increases the likelihood and duration for a smaller individual to fight, in a potentially costly agonistic interaction, with a larger, dominant opponent (Huber et al., 1997).
However, elevated 5-HT levels did not affect either the outcome of fights or the escalation pattern of fighting behaviors. In contrast, in a different species of crayfish Procambarus clarkia, reduced levels of aggression were observed following 5-HT injections (Tierney and Mangiamele, 2001) suggesting species specificity in neuromodulatory effects of 5-HT. Interpretations of these findings, along with others, led to the initial suggestion that 5-HT does not have a direct effect on arthropod aggression, but instead may have a modulatory role in the decision to retreat from a fight (Peeke et al., 2000; Kravitz and Huber, 2003).
In insects, specifically crickets and Drosophila, 5-HT was initially reported to have little influence on aggressive behavior (Stevenson et al., 2000; Baier et al., 2002). Pharmacologically depleting 5-HT in crickets by administration of the 5-HT synthesis inhibitor α–methyltryptophan (AMTP) failed to alter the expression of either aggressive or submissive behaviors (Stevenson et al., 2000). Similarly, aggressive behavior in Drosophila was not significantly altered either by selective depletion of 5-HT using the irreversible tryptophan hydroxylase inhibitor, p-chlorophenylalanine, or by enhancing 5-HT levels via administration of the 5-HT precursor, 5- hydroxytryptophan (5-HTP; Baier et al., 2002).
In contrast, other more recent studies using similar methods to alter 5-HT suggest that it may actually play a significant role in modulating aggressive behaviors in both crickets and Drosophila. Pretreatment of crickets with 5-HTP increased some components of cricket fighting behavior, such as fight duration, but decreased the number of attacks and did not appear to alter fight outcome (Dyakonova and Krushinsky, 2013).
Drosophila either pretreated with 5-HTP or genetically modified to overexpress tryptophan hydroxylase to increase 5-HT, exhibit increased aggression (Dierick and Greenspan, 2007). However, depletion of 5-HT, either through genetic manipulation or by the administration of AMTP, did not significantly reduce aggressive behavior when compared to controls, suggesting that 5-HT modulates but is not necessary for the expression of aggression in Drosophila (Dierick and Greenspan, 2007). This role for serotonergic modulation of aggression is supported by the demonstration that Drosophila in which 5-HT neurons were selectively inhibited can exhibit aggression but show a limited ability to escalate the fight (Alekseyenko et al., 2010). Conversely, both fight intensity and rate of fight escalation were increased by selective serotonergic activation (Alekseyenko et al.,
8 2010).
Studies in our laboratory suggest an important role for 5-HT in aggression in a different dipteran species, the stalk-eyed fly Teleopsis dalmanni. Pharmacologically administering 5-HTP markedly increases the probability of winning an aggressive contest in sized-matched pairs, as well as increasing the incidence of high- intensity aggressive behaviors (Bubak et al., 2014b). In accordance with the previously mentioned crayfish work, we also saw a reduction in the motivation to retreat with increases in brain 5-HT concentrations (Bubak et al., 2014b). The function of 5-HT in influencing individual aggression and opponent assessment in stalk-eyed flies is discussed in more detail in section 1.5.
Overall, these results suggest that increased activity in arthropod serotonergic systems contributes to species-specific expression of particular components of aggressive behaviors, but may not be required for the absolute display of aggression. These studies, particularly those using pharmacological manipulations to globally increase or decrease 5-HT, represent net serotonergic effects and do not provide the ability to differentiate more subtle serotonergic actions mediated by different 5-HT receptor subtypes. An approach that will be informative in future research will be to test behavioral outcomes following targeted manipulations of specific serotonergic receptors using pharmacological or genetic methods. Indeed, pharmacologically targeting different 5-HT receptors in socially isolated Drosophila indicates that 5-HT2-like receptor activation decreases a subset of aggressive behaviors such as lunging, while activation of 5-HT1A-like receptors increases specific behaviors such as wing threats (Johnson et al., 2009). This finding is important, revealing insights into potential molecular mechanisms responsible for specific aspects of aggression not provided by broader experiments focusing on whole system deprivation or elevation of 5-HT. Similarly, selective manipulation of each monoamine and of aggressive behavior, (e.g., OA activity escalates aggressive intensity, while 5-HT activity either reduces motivation to retreat or modulates use of specific agonistic signals depending on receptor subtype) may be fruitful in unraveling how complex interactions among neurotransmitter systems can modulate proximate behavioral expression, which may in turn dictate fight outcome and reproductive fitness.
9 Pheromones and Neuropeptides
Another critical component in aggressive behavior is recognition of potential opponents. The ability of insects to discriminate between potential rivals or mates is mediated, in part, by chemosensory communication.
For example, detection of sex-specific pheromones by Drosophila males directs subsequent expression of social behavior, with either aggressive or courtship behavior evoked by male and female pheromones, respectively
(Fernandez et al., 2010; Ferveur, 2005). The sexually dimorphic nature of these pheromones is evidenced by the elicitation of male attacks on females after masculinizing the females’ pheromones (Fernandez et al., 2010). In
Drosophila males, the volatile pheromone, 11-cis vaccenyl acetate (cVA), is associated with the stimulation of male-male aggression, acting through olfactory receptor neurons (Wang and Anderson, 2010; Liu et al., 2011).
Interestingly, acute exposure of socially-naïve male flies to cVA increases aggression, while chronic exposure such as prolonged social housing suppresses the behavior (Liu et al., 2011). These bidirectional actions of cVA are mediated through activation of two distinct olfactory receptor neurons, with aggression increased and suppressed by Or67d and Or65a receptors, respectively (Liu et al., 2011). This finding suggests a role for cVA in regulation of behavior according to social experience (Liu et al., 2011), and highlights the need to identify specific ligand substrates when determining mechanisms by which aggression is modulated, as discussed above for 5-HT.
Although the mechanisms by which pheromones modulate aggression are not completely understood, it is likely that pheromone reception is intimately linked to biogenic amine systems in some insect species. As mentioned above, recognition of male-specific sensory cues in Drosophila and subsequent expression of contextually appropriate behavior is disrupted by decreasing OA activity (Certel et al., 2007). Moreover, neurons expressing taste receptors that differentiate male and female pheromones connect functionally and synaptically with distinct OA neurons in male Drosophila, and both ablation of the taste receptor neurons and decreasing OA synthesis reduce male-male aggression (Andrews et al., 2014). Recent work in our laboratory has found different monoamine profiles in the brains of pavement ants Tetramorium caespitum, a species that exhibits social warfare, following exposure to hydrocarbons from nest-mate or non nest-mate colonies (Bubak et al., 2016b). Specifically, when ants came in contact with a nest-mate or were exposed to beads saturated in nest-mate hydrocarbons, 5-HT was significantly elevated (Bubak et al., 2016b). Conversely, exposure of ants to beads saturated in non nest-mate hydrocarbons induced both mandible biting directed towards the beads and
10 increased levels of brain OA (Bubak et al., 2016b). Combined, the studies with Drosophila (Certel et al., 2007;
Andrews et al. 2014) and our work with pavement ants suggest that identification of potential rivals and subsequent behavioral responses in these species is mediated by activation of 5-HT and OA systems following pheromone detection.
Neuropeptides have also been implicated in the expression of invertebrate aggression. Using the genetic tools available for Drosophila, studies have focused on the role of neuropeptides in modulating insect aggression, in an attempt to uncover phylogenetic similarities. For example, Drosophila possess male-specific neurons that express a gene encoding for the neuropeptide, tachykinin (Tk) (Asahina et al., 2014). This peptide is homologous to Substance P, a mammalian peptide associated with increased aggression in a number of vertebrate species (Halasz et al., 2009; Katsouni et al., 2009; Siegel et al., 1997). Activation of Tk-containing neurons in Drosophila increases male-male aggression, while silencing Tk neurons decreases aggression
(Asahina et al., 2014). Notably, neither manipulation altered male-female courtship behaviors (Asahina et al.,
2014). Additionally, the invertebrate homolog to neuropeptide Y, neuropeptide F (NPF), has been posited to have an inhibitory role in aggression in Drosophila, with elevated aggression observed following genetic silencing of NPF circuits (Dierick and Greenspan, 2007). Aggression in male mice is similarly increased by decreasing neuropeptide Y activity through genetic deletion of neuropeptide Y receptors (Karl et al., 2004). In line with the conservation of neuropeptide function between vertebrates and invertebrates, transcription factors regulating neuropeptide signaling have been identified in the pars intercerebralis (PI) of the Drosophila brain
(Davis et al., 2014). This brain area is thought to be functionally and structurally similar to the mammalian hypothalamus, which has been shown to play a role in aggression through modulation of neuropeptide signaling in several mammalian species (Hartenstein, 2006; Kruk et al., 1984; Gregg and Siegel, 2001).
The general interactive functions of monoamines, chemosensory signals and neuropeptide systems in modulating aggressive behavior appear to share a high degree of conservation across both arthropods and vertebrates. This highlights the value of utilizing animals with relatively simplified neural circuitries, such as insects, for future investigations directed towards gaining more in-depth perspectives on neurobiological factors underlying this complex social behavior. Information gained from such studies can then be applied to understanding mechanisms governing aggression in higher order, more complex nervous systems. Similarly, using insect models to elucidate how a given neurobiological factor may have different behavioral effects
11 depending on social ecology could provide insight into what factors will come under selection pressure to ultimately produce species differences in expression of behavior in other taxa, as has been proposed for applying parallels between neuropeptide signaling and sociality in birds to other vertebrates (Goodson et al.,
2005). The ease of gathering such information in insect models can thus direct whether generalizations about neurochemical modulation of aggression may be applied broadly because of conservation in systems across taxa, or if specific nuances in natural history need to be taken into account to explain why departures from observed patterns have evolved.
The Stalk-Eyed Fly as a Model System for Studying Aggression
Stalk-eyed flies (Diptera; Diopsidae) have emerged as an excellent model system for understanding how sexual selection drives the evolution of showy male traits and associated behaviors (Burkhardt and de la
Motte, 1988; Wilkinson et al., 1998), particularly for studies of morphology (Hingle et al., 2001; Wilkinson and
Reillo, 1994; Worthington et al., 2012; Husak et al., 2013), physiology and performance (Swallow et al., 2000;
Swallow et al., 2009; Ribak and Swallow, 2007; Ribak et al., 2009a), and neurobiology (Buschbeck and Hoy,
1998; Worthington and Swallow, 2010; Egge et al., 2011; Egge and Swallow 2011; Bubak et al., 2013; Bubak et al., 2014b). Males and females of all species in the family Diopsidae are hypercephalic, with the eye bulbs displaced on the ends of long stalks. Recognized as an important feature in diopsid mating systems, eyestalks are used extensively as ornamental signals in both intra and intersexual interactions (Wilkinson and Dodson,
1997; Wilkinson and Johns, 2005). In sexually dimorphic species of stalk-eyed flies, such as Teleopsis dalmanni (Fig. 1.1), male mating success is positively correlated with eye span (Burkhardt et al., 1994;
Wilkinson and Reillo, 1994; Cotton et al., 2010). Females also show a preference for males with longer eye spans (Wilkinson et al., 1998; Burkhardt and de la Motte, 1988). In addition, males compete for access to the limiting resources of food and mates by following a stereotyped fighting repertoire that typically begins with the lining up of eyestalks (Lorch et al., 1993; Panhuis and Wilkinson, 1999, Egge et al., 2011). Such observations indicate that eye span is under current sexual selection.
12
Figure 1.1. Representative images of both male and female stalk-eyed flies from a sexually dimorphic species, T. dalmanni, and a sexually monomorphic species, T. quinqueguttata.
Use of eye span as a communicative signal during male-male competition in stalk-eyed flies is best characterized in sexually dimorphic species such as T. dalmanni and T. whitei. Larger males with broader eye spans typically win agonistic contests, thereby excluding smaller rivals and gaining access to the contested limiting resources (Burkhardt and de la Motte 1987; Cotton et al 2010; Egge and Swallow 2011; Panhuis and
Wilkinson, 1999). Males use multiple displays that escalate from lower to higher intensity behaviors in a stereotyped manner (Egge et al., 2011, but see Brandt and Swallow, 2009) in what appears to be mutual assessment, in order to compare asymmetries in ornament size and fighting ability. Conflicts typically begin with males facing each other and lining up their eyestalks in a parallel manner (de la Motte and Burkhardt,
1983; Panhuis and Wilkinson, 1999). In encounters where opponent eyestalks are evenly matched in size, the
13 individuals will gradually progress to expression of low-intensity behaviors consisting of non-combative actions such as forearm flexing or rearing (see Fig. 1.2). If an opponent does not retreat at this point, high-intensity aggressive behaviors typically follow. These involve physical contact, and comprise elements such as lunges, jump attacks, or tussles (Fig. 1.2). High-intensity physical fights are non-lethal and usually result in one opponent departing uninjured. Aggressive interactions can deescalate at any stage of the conflict, and are terminated when one of the rivals capitulates and retreats (Egge et al., 2011).
Figure 1.2. Flow chart of aggressive behaviors exhibited by T. dalmanni.
Aggression in sexually monomorphic stalk-eyed flies has not been as well studied, but appears to differ from dimorphic species in terms of frequency and components. For example, males of T. quinqueguttata are morphologically indistinguishable from females in terms of eye span (Fig. 1.1), and initiate far less aggressive interactions than any of the dimorphic species that have been measured to date (Panhuis and Wilkinson, 1999).
In addition, the repertoire of behaviors displayed by T. quinqueguttata includes a larger array of low intensity, non-contact displays including wing threats and bobbing (P Johns unpublished). This difference in morphology and behavioral repertoires between sexually dimorphic and monomorphic species is useful for comparative analyses and may provide valuable insight into the evolution of signals used in this complex behavior.
14 Aggressive interactions in stalk-eyed flies are easily characterized and quantifiable. This offers an ideal platform for understanding how the neurobiological factors governing individual differences in behavioral expression and ornament use may provide a proximate mechanism to be acted upon by sexual selection. To investigate the neural mechanisms underlying individual variation in aggression in stalk-eyed flies, we developed a method sufficiently sensitive to detect and quantify a variety of different monoamines (including 5-
HT, DA, and OA) from the brain of a single fly using high performance liquid chromatography (HPLC) with electrochemical detection (Bubak et al., 2013). This quantification from an individual subject contrasts with other methods using smaller insects such as Drosophila, where it is common to pool brain samples to get accurate measurements. This ability to measure monoamines from a single fly has proven to be an important tool for understanding how individual differences in concentrations of monoamines, such as 5-HT, contribute to contest intensity and outcome. For example, we conducted a study in which some males had 5-HT levels elevated via oral administration of the 5-HT precursor, 5-HTP. When pitted in dyadic interactions against non- treated size-matched controls, 5-HTP-treated flies won 70% of the fights (Bubak et al., 2014b), in line with the relationship between elevated 5-HT and increased aggression reported in Drosophila (Dierick and Greenspan,
2007; Alekseyenko et al., 2010). Consistent with this, a portion of the 30% of treated males that lost fights actually had 5-HT levels that were lower than the endogenous concentrations of their control opponents, despite having being treated earlier with 5-HTP. Reanalysis of fight outcome and brain 5-HT concentrations further showed that winners had higher mean brain 5-HT concentrations when compared to losers, regardless of pretreatment. In addition, high intensity aggression was negatively correlated with the difference in 5-HT concentrations between opponents, such that individuals with more closely matched 5-HT engaged in more high-intensity behaviors and longer interactions before the conflict was resolved (Bubak et al., 2014b). These results demonstrate that contest intensity and outcome are not simply a function of absolute levels in 5-HT, but are instead dependent upon the relative difference in 5-HT concentrations between size-matched opponents.
Such a finding is reliant upon the ability to measure central monoamine levels from individual subjects, which can then be combined with pharmacology and correlated with behavioral expression. Issues such as this could have large implications in studies where the species is too small to be analyzed on the individual level, and further highlight the use of stalk-eyed flies as a model system for detecting potential relationships between neurophysiological mechanisms, morphology of armaments, and aggressive behavior.
15 While relative differences in 5-HT concentrations appear to dictate contests between size-matched stalk-eyed flies, a major component in winning an aggressive encounter between two conspecifics is size discrepancy, where in most cases, the larger individual is victorious. This remains true in T. dalmanni, where the larger opponent predominantly excludes smaller rivals from resources (Burkhardt and de la Motte, 1987;
Wilkinson et al., 1998). One way to understand how a proximate neurobiological mechanism may be acted upon evolutionarily to determine behavioral phenotypes, would be to show that intrinsic factors such as elevated neural 5-HT can influence fight outcomes that would otherwise be decided by morphological discrepancies, and that differences in 5-HT activity are primarily involved in modulation of aggression rather than with factors that may otherwise indirectly influence reproductive success. To investigate how changing 5-HT concentrations could alter a morphologically biased fight, we designed a “David vs. Goliath” experiment where the smaller
“Davids” had pharmacologically elevated 5-HT. Although the fight outcome did not significantly change, with larger untreated opponents still winning the majority of the fights, the behaviors of both participants were altered (Bubak et al., 2015). The treated “Davids” performed more high-intensity aggressive behaviors, and retreated less, compared to untreated controls. Interestingly, the “Goliaths” facing treated “Davids” altered their behavior by more quickly escalating to higher intensities, as well as initiating more high-intensity aggressive behaviors. This increased frequency in initiations and decreased latency to high-intensity behavior by the larger, untreated opponents suggests an assessment of their rivals, with the larger male changing behavioral expression to match the increased aggressiveness of the smaller, treated opponent. In what normally presents as a lopsided fight in favor of the larger participant, altering intrinsic neurobiological factors, such as 5-HT in the smaller opponent, creates a contest closely resembling morphologically size-matched opponents.
Applying the stalk-eyed fly as a model system to study aggression is becoming an increasingly attractive opportunity. The species diversity with respect to mono- and dimorphic phenotypes allows researchers to investigate behavioral differences associated with morphological characteristics. Furthermore, the reliable non-invasive ability to manipulate endogenous 5-HT and other neurochemicals from a single animal can be correlated with the well-described and easily quantifiable aggressive behavioral expression by the same individual. Overall, the stalk-eyed fly creates an exciting model system to investigate how central nervous system chemicals modulate behavior and may play an intricate role in shaping overall morphology.
16 Perspectives
To obtain a more complete understanding of aggression, it is imperative to incorporate neurobiological, morphological, and behavioral information. However, obtaining this information, particularly the physiological and neurological data, can prove difficult in organisms with complex central nervous systems and behavioral responses, such as vertebrates. Many of the molecular and physiological processes involved in generating complex behaviors in both vertebrates and invertebrates are highly conserved. Therefore, focusing on invertebrates, with their relatively simple nervous system and often well-characterized aggressive behavioral patterns, could prove beneficial (Bubak et al., 2014a). Insects are extremely well suited to aggression models, with significant behavioral (e.g., aggressive and nonaggressive) and morphological (e.g., weapon possessing and non-possessing) diversity providing a rich resource to test larger evolutionary questions. Understanding how the neural circuitries and underlying genetic factors mediate the behavioral processes and development of specific distinct morphological features in individual insect species will help elucidate the evolutionary and ecological connections between morphology, behavior, and physiology, and may provide insights into these same processes in vertebrates. Currently, much of this work being conducted in insects focuses heavily on just a few species such as crickets and Drosophila. Obviously, in depth investigations of the physiological and genetic underpinnings of aggression in these species is invaluable. However, it is also vital to apply these findings across a wider range of taxa, with distinctive ornaments, mating systems, and behavioral repertoires, to achieve a more comprehensive understanding of this widely expressed behavior.
A specific advantage to using the stalk-eyed fly as a model system for studying aggression, as mentioned earlier, is the utilization of the vast morphological and behavioral differences we see among species.
For instance, sexual dimorphism in eyestalk length is a trait that has arisen and been lost multiple times in the
Diopsid family (Baker and Wilkinson, 2001), and appears to correlate with differences in the frequency and components of aggressive behavior. A recent molecular phylogenetic analysis of over 30 (Baker et al., 2009) of the estimated 150+ species in the family (Steyskal, 1972; Feijen, 1983; Feijen, 1989) provides a robust framework for mapping differences in neurochemical modulation of aggression according to species relatedness. In turn, this may illustrate how selection can shift from emphasis on static morphological signals to favoring more plastic physiological mechanisms, and hence explain continued emergence of dimorphism versus monomorphism in this family. The differences in behavior and morphology among stalk-eyed flies also lends
17 well to large comparative studies. This strategy has been successfully used to study a variety of evolutionary questions at the organismal level, including speciation in closely related populations of T. dalmanni (Swallow et al., 2005; Christianson et al., 2005) and the co-evolution of ornaments with morphology and locomotor performance (Husak et al., 2011a; Husak et al., 2011b; Ribak et al., 2009b). Similarly, a comparative genomic approach has been used to investigate genes underlying the development of the sexually selected and sexually dimorphic ornaments, eye span, that define stalk-eyed flies (Baker et al., 2009; Wilkinson et al., 2013). It will be interesting to identify genes responsible for aggressive behavior that have been gained and lost, along with eye span length, throughout evolution of the stalk-eyed fly family. Furthermore, the application of genomic techniques in the field, becoming increasingly more accessible by nucleic acid preservation methods, provides an exciting opportunity to tease out specific proximate mechanisms of aggressive displays by studying gene expression differences in geographically separated wild populations, which also show differences in fighting repertoires.
As research into proximate mechanisms responsible for mediating aggression progresses, it will be interesting to determine the level of conservation between invertebrates, such as insects, and vertebrates. Tools such as next-generation sequencing are invaluable for understanding the degree of conservation at the gene level (see Toth and Robinson 2007 for a review on using “genetic toolkits” for elucidating mechanistic conservation underlying behavioral expression), while the ability to manipulate and measure neurochemical activity within individual insects offers a powerful means of examining why variation exists in aggressive behavior, signal use, and contest outcome. Identifying and comparing such proximate mechanisms among behaviorally and morphologically distinct insect species could provide valuable insight into the evolution of the extensive, often bizarre, variety of morphological features and behavioral repertoires used during aggressive competition.
Contributions
I served as the primary author of this published review, conducting an extensive literature search, synthesis of material, and writing of the initial draft of the manuscript.
18 CHAPTER II
ASSESSMENT STRATEGIES AND FIGHTING PATTERNS IN ANIMAL CONTESTS: A ROLE FOR
SEROTONIN?2
Abstract
Accurate assessment of the probability of success in an aggressive confrontation with a conspecific is critical to the survival and fitness of the individuals. Various game theory models have examined these assessment strategies under the assumption that contests should favor the animal with the greater resource- holding potential (RHP), body size typically being the proxy. Mutual assessment asserts that an individual can assess their own RHP relative to their opponent, allowing the inferior animal the chance to flee before incurring unnecessary costs. The model of self-determined persistence, however, assumes that an individual will fight to a set personal threshold, independent of their opponent’s RHP. Both models have been repeatedly tested using size as a proxy for RHP, with neither receiving unambiguous support. Here we present both morphological and neurophysiological data from size-matched and mismatched stalk-eyed fly fights. We discovered differing fighting strategies between winners and losers. Winners readily escalated encounters to higher intensity, physical contact and engaged in less low-intensity, posturing behaviors compared with losers. Although these fighting strategies were largely independent of size, they were associated with elevated levels of 5-HT.
Understanding the neurophysiological factors responsible for mediating the motivational state of opponents could help resolve the inconsistencies seen in current game theory models. Therefore, we contend that current studies using only size as a proxy for RHP may be inadequate in determining the intricacies of fighting ability and that future studies investigating assessment strategies and contest outcome should include neurophysiological data.
Introduction
Engaging in aggressive conflicts can be risky, with the potential of serious injury or death. Although not all conflicts result in injury, with some fighting styles being incapable of inflicting physical damage, prolonged aggressive posturing can waste time and energy, especially if unsuccessful, as well as increase
2 The material within Chapter II was originally published in Current Zoology and is included with permission: Bubak AN, Gerken AR, Watt MJ, Costabile JD, KJ Renner, JG Swallow (2016) Assessment strategies and fighting patterns in animal contests: a role for serotonin? Curr. Zool. zow040.
19 exposure to predators. For these reasons and others, animals have developed impressive, sometimes complex, fighting strategies and signaling mechanisms to resolve conflicts. Signals exchanged during aggressive interactions are of great functional importance because they mediate access to resources while minimizing the costs associated with fighting (Geist 1966; Emlen 2008). Body size relationships, in particular, exert a strong influence on contest outcome, and many signals appear to function to advertise individual size.
Many theoretical models that have been developed to determine contest outcome use body size as the primary indicator of an animal’s fighting ability, typically termed resource-holding potential (RHP). For example, the model of mutual rival assessment predicts that opponents assess each other’s relative body size as a proxy for RHP to aid in the decision to either fight or flee (Parker 1974; Smith and Parker 1976). This assessment provides the smaller opponent, typically inferior in fighting ability, the opportunity to avoid a potentially costly contest in which they have a low probability of success. In contrast, the model of self- determined persistence proposes that the individual engages in a contest to a set personal threshold, hypothesized to be dictated by their own size, independent of their opponent’s size (Taylor and Elwood 2003).
Both models predict that as the size disparity between opponents increases the contest duration will decrease, because the bigger opponent will always fight for longer regardless of whether its size is being appraised by the smaller individual. However, Taylor and Elwood (2003) suggest that separate regressions of contest duration against either the size of the loser or against the size of the winner can distinguish between the
2 models. With mutual rival assessment, the regression coefficients of winner and loser size should be similar in magnitude but opposite directions, negative for winners and positive with losers (Arnott and Elwood 2009). In contrast, the self- determined persistence model predicts that the coefficients will be characterized by a significant positive relationship with loser size and a weakly positive relationship with winner size (Arnott and
Elwood 2009). However, neither model, which represents the 2 extremes of animal assessment strategies, receives unambiguous support. Experimental tests of the predictions of each of the models have yielded inconsistent or contradictory results (Morrell et al., 2005; Stuart-Fox 2006; Brandt and Swallow 2009). A more realistic “partial mutual assessment” strategy in which individuals have reliable knowledge of themselves but limited information about their opponent may better capture the strategy actually employed during assessment
(Prenter et al., 2006).
20 We contend that empirical attempts to gauge the validity of different assessment strategies using only body size as a proxy measure of RHP and contest duration as a measure for cost may be overly simplistic and inadequate to the task. Contest duration alone may be a poor representation of the cost of a contest given the variability in the types of displays or interactions that take place, e.g., 10 min of low-intensity (LI; non- physical) behaviors will have a much lower associated cost compared with 10 min of high-intensity (HI) behaviors. Similarly, size alone does not appear to sufficiently capture the intricacies of individual fighting ability (i.e., motivation, experience, etc.) and should be just 1 factor used to assess and understand contest outcomes. This is especially the case in contests that are resolved predominantly through signal exchange rather than physical infliction of injury. Given the complex behaviors and signals animals engage in during contests, it is reasonable to suggest that they are capable of transmitting and receiving more information about RHP than simply body size that will determine how willing each opponent is to actually commit to fighting.
The neural, sensory, and cognitive mechanisms that may permit assessment not only of rival size but also other factors associated with RHP remain largely unexplored and have the potential to resolve inconsistencies predicted from simple models of contest outcome that rely only on body size. Many of the non- morphological factors responsible for mediating aggressive contests involve an altered motivational state, most likely facilitated by neurobiological factors such as biogenic amines (Bubak et al., 2014a). For example, octopamine (OA) has been demonstrated to be a key element in the rewarding experience of territory possession in crickets, with previously defeated individuals pharmacologically diminished of OA losing the aggressive enhancing effect of occupying a shelter (Rillich et al., 2011). Brain serotonin (5-HT) in invertebrates has been implicated in both overall elevated aggression, including higher intensity aggressive behaviors and escalation patterns, as well as reduced willingness to retreat (Huber et al., 1997; Bubak et al., 2014a, 2014b). Altering brain concentrations of certain biogenic amines in 1 opponent of size-matched pairs has been shown to significantly influence the progression and outcome of fights in several invertebrate species (for review, see
Bubak et al. 2014a). Individual differences in levels of biogenic amines between opponents could account for some of the unexplained variation in contest outcome seen in experiments that only use size as a proxy for RHP.
Obtaining morphological, behavioral, and neurophysiological data in a single species can be difficult.
However, to understand the intimate interactions between morphology and neurophysiology and how this relationship directs behavioral output, experiments that can simultaneously account for all of these variables
21 need to be conducted. Stalk-eyed flies (Diopsidae: Teleopsis dalmanni) provide such a model species, where researchers can incorporate selective neurochemical detection and manipulation techniques while simultaneously assessing individual variance in both morphology and behavior. This species is characterized as having eye bulbs displaced laterally on long stalks, which males use as aggressive signals during confrontations over mates and food resources (Wilkinson and Dodson 1997; Wilkinson and Johns 2005). Males follow a stereotyped escalation pattern starting with lining up of eyestalks, progressively moving toward more intense behaviors (de la Motte and Burkhardt 1983; Panhuis and Wilkinson 1999). Because eye span is an accurate indicator for size in males, individuals with longer eye span typically defeat smaller males (Burkhardt and de la
Motte 1983, 1987; Small et al., 2009; Egge and Swallow 2011). However, although size is a significant factor, smaller males win aggressive encounters against larger conspecifics as much as 10–30% of the time (Small et al., 2009; Egge et al., 2011). Thus, taken alone, size does not entirely explain the outcome of a fight. Instead, a more detailed experimental approach combining analyses of differing fighting strategies with endogenous neurochemicals mediating such behaviors may be more consistent with actual outcomes.
The purpose of this study was to utilize the stalk-eyed fly as a model system to test the theoretical hypotheses of proposed contest models using a large previously collected dataset of intraspecific aggressive contests. Specifically, we aimed to determine whether simultaneously measuring behavioral, morphological, and neurophysiological variables could better explain contest outcome and structure than the simplified models using only morphological data. We show that although size plays an important role in winning a fight, it imperfectly predicts which males are victorious in size-mismatched contests. Interestingly, the data show a significant difference in fighting strategy between winners and losers, which appears largely independent of size. Rather, winners readily escalated encounters to higher intensity, physical contact and engaged in less LI, posturing behaviors compared with losers, no matter what the size disparity between opponents. To determine whether neurophysiological variables could account for these differing fighting strategies shown by winners and losers, we reanalyzed an additional dataset that contained size-matched opponents where half the males had pharmacologically elevated brain 5-HT. In doing so, we discovered that underlying discrepancies in brain 5-HT concentrations between winners and losers are important in directing expression of these differing fighting strategies. Moreover, the strategies associated with individuals possessing higher 5-HT than their opponents are reminiscent of those shown by winners in untreated contests. Therefore, factors such as biogenic amines should
22 be considered in future game-theory models for predicting individual expression of aggression and contest outcome.
Materials and Methods
Subjects
Teleopsis dalmanni is a sexually dimorphic species of stalk-eyed fly native to South East Asia. All laboratory-housed individuals are descendants of pupae obtained from the University of Maryland, College
Park. Flies are reared communally in cages (45cm x 22cm x 19cm) on a 12-h light:dark cycle with free access to food, water, and mating opportunities. Each cage is kept between 25–27 °C at ~80% humidity. All adult males used in the study were between 4–8 weeks post-occlusion. Eye span was measured to the nearest 0.01mm using Scion Image (National Institutes of Health, Bethesda, USA) after brief anesthetization with CO2 (Ribak and Swallow 2007). Eye span is highly correlated with body length, making it an accurate representation of body size (Burkhardt and de la Motte 1983; Wilkinson 1993). Individuals were given an identifying mark between their thoracic spines using an opaque paint pen and transferred to smaller cages (14cm x 14cm x 14cm) containing ~10 individuals. Predetermined opponents were housed separately.
Forced-Fight Paradigm
All behavioral data presented were obtained from previously published studies (Egge et al., 2011; Egge and Swallow 2011) that used the same forced-fight paradigm, details of which can be found in Egge and
Swallow (2011). Briefly, T. dalmanni were measured for eyestalk length to the nearest 0.01mm and given an identifying mark between their thoracic spines. Twenty-four hours prior to the fight, flies were placed in a wood and glass arena, lined with moist filter paper. Opponents were kept separated by an opaque barrier, and no food was given. After 24 h of acclimation to the arena, the barrier was removed and a drop of corn media was presented in the center of the arena, which provided a contestable resource. Each fight was recorded by a digital video camera for 10 min. Behaviors were scored manually using JWatcher (Blumstein et al., 2007) with each fly’s behavior scored independently of the other fly in the arena (i.e., each video was scored twice). Flies were assigned to be a winner or loser based on the number of retreat behaviors (turned away or quickly ran away) exhibited over the entire 10-min fight.
23 Drug Administration Studies
Data reanalyzed for investigation of the relationship of 5-HT and behavior were obtained from Bubak et al., (2014b). In all these studies, treated adult males (n = 20) were administered 3 g of the 5-HT precursor 5- hydroxy-L-tryptophan (5-HTP; H9772; Sigma, St. Louis, MO, USA) in 100 mL of food media containing pureed sweet corn, 25 mg of ascorbic acid, and 1 mL of methylparaben (Wilkinson 1993) as a mold inhibitor.
Flies were fed ad libitum for 4 days. This treatment regime reliably elevates individual 5-HT concentrations in stalk-eyed fly brain tissue (Bubak et al., 2013). Opponents of treated flies (n = 20) were fed the same food media, sans 5-HTP, for 4 days.
Serotonin Quantification
Serotonin (5-HT) in a single whole brain sample was detected by high-performance liquid chromatography with electrochemical detection as previously described (Bubak et al., 2013). Brain samples were frozen immediately after the fight, thawed, and centrifuged at 17,000 rpms. The supernatant was removed, and 45 μL of the sample was injected into the chromatographic system. The amines were separated with a C18
4-μm NOVA-PAK radial compression column (Waters Associates, Inc., Milford, MA, USA) and detected using an LC 4 potentiostat and a glassy carbon electrode (Bioanalytical Systems, West Lafayette, IN, USA). The sensitivity was set at either 0.5 or 1 n/V with an applied potential of +0.9 V vs. a Ag/AgCl reference electrode.
The mobile phase initially was made by dissolving 8.6 g sodium acetate, 250 mg EDTA, 11 g citric acid, 330 mg octanylsulfonic acid, and 160 mL of methanol (all chemicals were obtained from Sigma-Aldrich, St. Louis,
MO, USA) in 1 L of distilled water. In order to obtain the desired separation, additional increments of octanylsulfonic acid and methanol were added to the mobile phase. After removal of the supernatant for monoamine analysis, 60 μl of 0.4 M NaOH was added to the pellet to solubilize the remaining tissue for protein analysis (Bradford 1976). The CSW32 data program (DataApex Ltd., Prague, Czech Republic), set in internal standard mode, calculated 5-HT concentrations based on peak height values obtained from standards (all standards were obtained from Sigma-Aldrich). The resulting amine concentration was divided by microgram protein in the sample to yield picogram amine/microgram protein after appropriate corrections for injection volume vs. preparation volume were carried out.
24 Statistical Analysis
Separate unpaired Student t-tests were used to test for differences in the means of eyestalk length, flexing behaviors, HI (physical contact) to LI (non-physical) behavioral ratios, and 5-HT levels between winners and losers as well as larger and smaller opponents. To test for differences within and between groups in
HI to LI behavioral ratios at different time points during the fight according to either status (winner/loser) or on body size relative to opponent, separate 2-way ANOVA (Analysis of Variance; time x either status or size) was applied, with Sidak’s multiple comparison tests used to compare behavior between groups at each time point.
Significant effects of time were followed by Sidak’s tests to compare time point 1 against all subsequent time points to reveal differences within groups in HI/LI expression as the fight progressed. For these particular analyses, the same individuals did not contribute to data for every time point, precluding the use of a repeated measures ANOVA. All statistical tests were set at a 0.05 alpha level and conducted using Prism 6 (GraphPad software, La Jolla, USA).
Results
A total of 63 fights from previous size-matched and mismatched studies were quantified, with a distribution in size disparity between opponents ranging from 0% to 13% (measured by eyestalk length; Table
2.1). Winners were determined by fewest amounts of retreats relative to their opponent in a 10-min forced-fight paradigm.
Table 2.1. Range and mean of eyestalk length between winners and losers
Size range (mm) Mean size (mm ± SEM)
Winners (63) 7.25-8.75 8.03 ± 0.04
Losers (63) 7.23-8.66 7.86 ± 0.03
p < 0.001 (Student’s t-test)
Numbers of subjects contributing to these datasets are indicated in brackets. SEM: standard error of mean
25 Relationships with Preexisting Game Theory Models
Both models of self-determined persistence and mutual assessment predict a strong positive relationship with contest duration and loser RHP. Indeed, our data also support this prediction when eyestalk length is used as a measurement of RHP (linear regression; R2 = 0.064; p < 0.05; Y = 64.2x - 383.5). However, our data did not indicate a significant relationship between contest duration and RHP difference between opponents (linear regression; R2 = 0.002; p = 0.72; Y = -10.9x + 126.3), a prediction of both self-determined persistence and mutual assessment. Furthermore, our data did not suggest a significant relationship with winner
RHP and contest duration (linear regression; R2 = 0.007; p = 0.51; Y = 18.7x - 26.6), typically used as a key analysis to differentiate between both models. Therefore, our data do not seem to be consistent with either model when size is used as a proxy for RHP.
Relationships between Fight Strategy and Body Size
The fighting strategy of winners and losers differed in both LI (non- physical) and escalation patterns of aggressive behaviors. Losers engaged in significantly more flexing behaviors compared with their winning counterpart (unpaired Student’s t-test, p < 0.05, df = 62; Figure 2.1A). To test whether this posturing effect was a result of size relative to opponent, we analyzed flexing as a function of smaller and larger competitors, regardless of winning or losing, and found no significant difference between opponents (unpaired Student’s t- test, p = 0.94, df = 61; Figure 2.1B). There was also no significant difference between relative size differences and frequency of flexing behaviors (linear regression; R2 = 0.006; p = 0.57; Y = -1.51x + 10.77). Together, this suggests that flexing is independent of relative size differences.
Figure 2.1. (A) When competitors were separated by status (winners and losers), losers performed significantly more flexing behaviors compared with winners (unpaired Student’s t-test, p < 0.05, df=62). (B) Separation by size demonstrates no significant difference between flexing behaviors (unpaired Student’s t-test, p = 0.94, df = 61). Numbers presented as means ± SEM.
26 Next, we analyzed escalation patterns and total fight intensity between winners and losers by measuring the ratio of HI behaviors over LI behaviors expressed during the entire fight. The mean total time spent performing aggressive behaviors was approximately 120 s ± 6.1 s, no flies engaged in aggressive behaviors beyond 300 s. To examine how aggression was distributed across the 10-min forced-fight paradigm, we divided each contest into discrete time bins, 2-way ANOVA revealed effects of status (F1,116 = 19.98, P <
0.001), time (F4,116 = 34.04, p < 0.001) and an interaction between both factors (F4,116 = 12.56, p < 0.001).
Subsequent pairwise comparisons showed that winners or losers engaging in aggressive behaviors for a total of
4 min or shorter (time points 1–4) in the 10-min fights did not differ in escalation patterns (Sidak P > 0.05;
Figure 2.2A). However, when opponents engaged in aggressive behaviors for a total duration between 4–5min
(time point 5), winners performed significantly more HI than LI behaviors compared with losers in the latter stages of the fight (2-way ANOVA, p < 0.001, df = 116; Figure 2.2B). To test whether this was an effect of size, we ran the same analysis for larger and smaller opponents regardless of fight outcome and discovered that this difference in escalation pattern was abolished (2-way ANOVA, p > 0.05, df = 116; Figure 2.2B), suggesting that escalation patterns are largely independent of size. Additionally, there was no relationship between relative size difference and HI to LI behavior ratios (linear regression; R2 = 0.009; p = 0.46; Y = 0.25x
+ 0.44). Although opponent size was not a reliable predictor of fighting strategy with respect to either escalation patterns or posturing, winners were significantly larger on average compared with losers (unpaired t-test, P <
0.001, df = 124; Table 2.1), suggesting that size still plays a crucial role in fight outcome.
Figure 2.2 (A) Winners performed significantly more HI to LI behaviors compared with losers in fights that lasted between 4 and 5 min (time point 5). Both winners and losers had significantly higher HI/LI behavioral ratios at time point 5 compared with all other time points within their groups. (B) When separated by size, regardless of fight outcome, significantly higher HI/LI behavioral ratios are again seen at time point 5 compared with all other time points, but there is no difference between the groups. Asterisk (*) indicates differences between winners and losers at that time point; has (#) indicates a significant within group difference compared with time point 1. Time points correlate with minutes spent fighting. Dashed line signifies a 1:1 ratio of HI behaviors to LI behaviors.
27 Relationships of Fighting Strategy with 5-HT
To investigate other potential factors besides size that influence fight intensity, we reanalyzed data from our previous studies where we had pharmacologically elevated neural 5-HT concentrations in half the male opponents in size-matched fights. Winners in these size-matched fights had significantly higher levels of
5-HT (unpaired t-test, p < 0.01, df = 38; Figure 2.3) as well as a greater HI to LI behavior ratio over the entire
10-min fight (unpaired t-test, p < 0.05, df = 38; Figure 2.3), indicating elevated intensity. In the absence of size differences, this mismatch in neural 5-HT concentrations appears sufficient to account for discrepancies in fight intensity between otherwise equal opponents. Thus, when taken together, neural 5-HT and size may represent a better predictor of a winner’s fighting strategy than absolute or relative size alone.
Losers Winners 0.8
0.6
0.4
HI/LI Behaviors HI/LI 0.2
0.0 0 20 40 60 80 100 5-HT (pg/mg protein)
Figure 2.3. In size-matched fights, winners had significantly higher brain 5-HT levels (x axis; unpaired t-test, p < 0.01, df = 38; mean ± SEM) as well as a higher HI/LI behavioral ratio (y axis; unpaired t-test, p < 0.05, df = 38; mean ± SEM) compared with losers.
Discussion
Stalk-eyed flies engage in both LI (non-physical) and HI (physical) aggressive behaviors during fights.
Males follow a predictable, stereotyped escalation pattern starting with lining up of eyestalks, presumably for rival assessment, then progressively moving toward more intense behaviors (de la Motte and Burkhardt 1983;
Egge et al., 2011; Bubak et al., 2014b). Similar to other animals, size seems to play an important role in the outcome of these aggressive encounters, with the larger male typically succeeding in routing his opponent.
28 However, when taken alone, size cannot completely predict specific winning and losing strategies in this species, and should be but 1 factor in determining fighting ability of individual animals. This is demonstrated by our reanalysis of data obtained from previous studies investigating the relationship between central monoamine levels and aggressive motivation and fighting strategy in size-matched contests. Therefore, we propose that future models attempting to measure assessment strategy (e.g., self-determined persistence vs. mutual rival assessment) and fight outcome based on individual RHP should incorporate neurophysiological information.
We hypothesized that losers engaged in significantly more flexing behaviors compared with winners as a technique for smaller males to “bluff” their opponent without actually engaging in physical contact behaviors.
Use of such a strategy would be in accordance with mutual rival assessment theory, with smaller males exhibiting less intense aggression after perceiving a larger opponent. However, we saw no difference in flexing between smaller vs. larger opponents, with losing males always displaying more flexing. This suggests that individual males employing this ultimately losing strategy do so regardless of size discrepancies. In contrast, winners engaged in more HI, physical contact behaviors, but this only became evident if the fight lasted more than 4 min. Again, this was independent of opponent size. The retention of LI behaviors by losers despite body size differences argues against predictions made by either the mutual rival assessment or self-determined persistence models when using size as the sole measure of RHP (e.g., Arnott and Elwood 2009). Similarly, the fact that size-independent differences in aggressive behavior by winning males only appeared after the contest had exceeded a certain duration suggests factors other than physical indicators of RHP are modulating individual responses to social challenge in this species.
To understand what other factors may be influencing these differing escalation patterns, we reanalyzed a dataset of size-matched males with half the opponents containing pharmacologically exaggerated endogenous levels of brain 5-HT. In doing so, we discovered that elevated brain concentrations of 5-HT were sufficient to mimic the winning strategy of the randomly paired opponents. Specifically, treated males with higher 5-HT but identical in size to their opponent engaged in significantly more HI behaviors than LI behaviors. This is remarkably similar to the pattern shown by winning opponents in contests of varying size disparity, and suggests that endogenous brain levels of 5-HT, and possibly other biogenic amines such as OA, may be responsible for the escalation patterns shown by winners during longer duration fights. We have previously demonstrated that raising 5-HT in a smaller competitor will lead to increased willingness to engage in HI
29 encounters with larger opponents in stalk-eyed fly males (Bubak et al., 2015). However, smaller opponents treated with 5-HT were not more likely to win the fight compared with their control counterpart, because the larger opponent preemptively escalated the contest to physical contact behaviors. This suggests that the larger opponent, when faced with a treated, hyper-aggressive smaller opponent, switches his escalation pattern after gathering specific information about its rival’s aggressive state. This may be an explanation for the sudden switch in escalation patterns seen in this study. Taken in this respect, applying 5-HT as the predicting variable to existing theoretical models may produce different outcomes for winners and losers depending on the model employed. For instance, our data suggest that losers trend toward fitting the self-determined persistence model, with aggression increasing as 5-HT levels approach those of the opponent. In contrast, 5-HT only predicts HI aggression as the fight continues and the winners have had the opportunity to perceive the opponent’s aggression, which is more in agreement with predictions of the mutual rival assessment model.
Perception of size for opponent assessment is still clearly playing a role in contest outcome, as the majority of winning males were larger than their opponents. Size can predict escalation and intensity patterns within the first 4 min of a fight, so it may be possible that initial perception of size is the primary determining factor for short-lived encounters in stalk-eyed flies, especially for opponents that go on to lose the contest.
However, it does not explain why winners would only escalate intensity as fights progressively become more costly and time-consuming. Although possessing higher 5-HT may partly account for this change in behavioral expression, as discussed above, perception of other cues may also contribute to the motivation to stay in the contest or escalate physically, usurping size as the determining factor in fight outcome. This possibility is suggested by studies showing that the fighting strategy of smaller crickets changes when visual perception of the opponent is not possible, with blinded crickets fighting for longer and at higher intensity against a larger opponent with disabled mandibles that could not inflict physical damage (Rillich et al., 2007). Given that smaller crickets will normally flee from a larger rival in the opening stages of an interaction (Rillich et al.,
2007), this implies that opponent assessment and potential risk of engagement is based on a combination of both size perception and damage accrual as the contest progresses. We found that winning flies exhibited less flexing and more HI behaviors than losers, even when opponents were matched in size, suggesting similar cumulative perception of cues and risk assessment as the fight continued. Specifically, eventual winners may be gathering information throughout the fight about their opponent in addition to perception of size alone, possibly from the
30 excess of posturing (flexing) behaviors performed by the losers. This collated information then conveys a lower threat level to winners, promoting the confidence to escalate the fight. Consistent with this idea, fights shorter than the 4-min mark may not provide sufficient time to gather necessary information about the opponent, resulting in both winners and losers engaging in a similar ratio of HI to LI behaviors.
In summary, we have described 2 different fighting strategies between winning and losing male stalk- eyed flies. Additionally, we have found that these strategies are largely independent of relative body size, one of the most commonly used proxies of RHP for game theory modeling and assessment strategies. Brain levels of
5-HT appear to provide ability to predict which individuals will display less intense aggression and go on to lose the fight, whereas a combination of size, opponent assessment and higher 5-HT could explain the escalation pattern of the winning fighting strategies. The contribution of endogenous 5-HT to both fighting strategies suggests that it is imperative that future game theory and assessment models include neurochemical factors when attempting to decipher the mechanisms of animal contests.
Contributions
Collection of the first large behavioral data set presented in this manuscript was conducted by Dr.
Gerken, a former MS student of Dr. Swallow. I subsequently reanalyzed the data to test longstanding game theory models. I also collected the second data set containing neurochemical information, designed the questions and arguments presented, and wrote the manuscript.
31 CHAPTER III
DAVID VS. GOLIATH: SEROTONIN MODULATES OPPONENT PERCEPTION BETWEEN
SMALLER AND LARGER RIVALS3
Abstract
During agonistic encounters, the perception of a larger opponent through morphological signaling typically suppresses aggression in the smaller individual, preventing contest intensity escalation. However, non- morphological factors such as central serotonin (5-HT) activity can influence individual aggression, potentially altering contest intensity despite initial size discrepancies. When male stalk-eyed flies (Teleopsis dalmanni) fight, contest escalation is directly proportional to similarity in body size, with escalation being lower in size- mismatched contests. We have shown that both high-intensity aggression and the probability of winning are increased in males with pharmacologically elevated 5-HT relative to size-matched non-treated opponents. Here, we hypothesized that, in size-mismatched contests, increasing brain 5-HT in the smaller opponent could similarly increase aggression and counteract the low contest intensity normally driven by size discrepancy.
Size-mismatched male pairs (greater than 5% difference in eyestalk length) engaged in a forced fight paradigm, with the smaller fly either untreated or with pharmacologically elevated 5-HT levels. The expression of high- intensity aggressive behaviors was significantly increased in smaller treated opponents, but the probability of winning was not altered. This suggests that while elevated serotonergic activity can increase aggression and intensity despite perception of a larger opponent, this is not sufficient to overcome size biases with respect to contest outcome. However, the fact that larger opponents continued to win against smaller treated flies was not simply a function of size. Instead, untreated larger males adjusted their fighting strategy to match the increased aggression of their smaller treated opponent, suggesting contextual flexibility in behavior based on individual opponent assessment.
3 The material within Chapter III was originally published in Behavioral Brain Research and is included with permission: Bubak AN, Rieger NS, Watt MJ, Renner KJ, Swallow JG (2015) David vs. Goliath: serotonin modulates opponent perception between smaller and larger rivals. Behav. Brain Res. 292: 521-527.
32 Introduction
Aggressive contests are seen across many taxa and have important fitness implications for the individuals involved, resolving conflicts among conspecifics over access to limited resources such as territory, food and mates (Archer, 1988). Size discrepancy between competitors is an important factor in deciding conflict outcome (Huntingford and Turner, 1987). A large discrepancy in body size, or ornament size in sexually selected species, often leads to quick contest resolution in favor of the larger competitor, because body size and ornament size can serve as reliable indices of resource holding potential (Parker, 1974). Relative body size provides a ready means of opponent assessment and, in a variety of species, larger competitors often experience a greater likelihood of winning a direct encounter. Absolute body size is a dependable predictor of winning contests in crickets (Dixon and Cade, 1986), crayfish (Huber et al., 1997), swimming crabs (Glass and
Huntingford, 2010) and cichlid fish (Enquist et al., 1990; Turner, 1994). Similarly, ornament size reliably predicts contest outcome in many species. Examples of ornament size that have been positively associated with increased odds of winning contests include the head plume in male quail (Hagelin, 2001), casque and pink coloration size of Cape dwarf chameleons (Stuart-Fox et al., 2006), the red spot of the American rubyspot
(Contreras-Garduno et al., 2008) and comb size (along with body size) in the red jungle fowl (Ligon et al.,
1990).
Opponent assessment, aggression, and conflict resolution are modulated by central monoaminergic activity (Turner, 1994; Schwarzer et al., 2013) altering of which may be sufficient to overcome initial size- biased disparities. In some arthropods, increased activity of the monoamine serotonin (5-HT) is associated with either greater expression of high-intensity aggression or decreased likelihood of retreating from an agonistic encounter, depending on the species (Huber et al., 1997; Momohara et al., 2013; Dierick and Greenspan, 2007;
Johnson et al., 2009; Kravitz, 2000; Summers et al., 2005). This finding is supported by studies in which individual aggressive behavior is heightened by increasing 5-HT levels through either pharmacological or genetic manipulations (Huber et al., 1997; Dierick and Greenspan, 2007; Livingstone et al., 1980; Pedetta et al.,
2010; Alekeseyenko et al., 2010). While the role of 5-HT in arthropods in aggressive expression and contest outcome between size-matched opponents is relatively well studied and clear, with some conflicting results, the effects of 5-HT manipulation on opponent assessment and contest outcome in size-mismatched contests is equivocal and understudied. For example, Huber (Huber et al., 1997) found that increased 5-HT in one crayfish
33 species decreased the willingness of smaller subordinate animals to retreat but did not increase the chance of winning a contest. In contrast, Momohara (Momohara et al., 2013) reported that elevating 5-HT in another species of crayfish increased not only levels of aggression but also likelihood of winning. However, both studies suggest that 5-HT is important for modulating aggression, including decisions regarding initiation, escalation or retreat, and can promote smaller individuals to overcome initial opponent assessments based on body size.
We have been developing the sexually dimorphic stalk-eyed fly, Teleopsis dalmanni, as a model for investigating mechanisms underlying aggression, escalation, and conflict resolution, because males use their elongated eyestalks as communicative signals in two different aggressive contexts (Wilkinson and Johns, 2005); male flies defend both diurnal feeding sites (De la Motte and Burkhardt, 1983; Panhuis and Wilkinson, 1999) and nocturnal roosting sites that allow mating access to females (Burkhardt and de la Motte, 1985; Burkhardt and de la Motte, 1987; Small et al., 2009). Larger males with broader eye spans (i.e., longer eyestalks) typically win contests in both situations (Burkhardt and de la Motte, 1988; Cotton et al., 2010). However, smaller males do win a small percentage of these interactions (Panhuis and Wilkinson, 1999; Egge and Swallow, 2011).
Agonistic encounters between males follow a stereotyped escalated progression from lower to higher intensity actions consistent with sequential assessment (but see Brandt and Swallow, 2009) that are terminated when one of the rivals capitulates and retreats (Egge et al., 2011), typically without injury.
The exact mechanisms underlying opponent assessment in stalk-eyed flies that eventually lead to conflict resolution are not well understood. However, we have recently developed methods to quantify and to pharmacologically manipulate 5-HT in the brains of individual stalk-eyed flies (Bubak et al., 2013), allowing us to investigate its proximate role in regulating and constraining aggression. In previous studies, we found that stalk-eyed flies which had higher brain 5-HT relative to an opponent showed increases in the expression of aggressive behaviors, decreases in the number of retreats and that higher 5-HT resulted in a markedly increased likelihood of winning against a size-matched competitor (Bubak et al., 2014b). These results indicate an important role of 5-HT in both conflict escalation and conflict resolution in this species. In this study, we determined whether increasing brain 5-HT could overcome the escalation pattern and outcome of an aggressive contest normally predicted by a size discrepancy. We hypothesized that pharmacologically increasing 5-HT in smaller males, “David”, would increase both aggressive behaviors and contest escalation while decreasing willingness to retreat despite the initial assessment of a larger untreated opponent, “Goliath”. Concurrently, we
34 observed whether the larger opponent’s behavior changed as a result of the pharmacologically-enhanced aggression of smaller flies. Increased aggressive responses by larger males toward treated smaller males would indicate that agonistic reactions can be modulated according to the perceived level of opponent aggression, surpassing assessment of a supposedly weaker competitor based initially on body size alone. Thus, we predicted that if larger flies were assessing their opponents, in part by the opponent’s level of aggression, then they would react with escalated aggression in a manner more reminiscent of size-matched contests.
Materials and Methods
Subjects
Male stalk-eyed flies, Teleopsis dalmanni, used in this experiment were all descendants of pupae collected by Dr. Gerald Wilkinson (University of Maryland-College Park) from Gomback Field Station near
Kuala Lumpur, Malaysia in 2012. Flies in this study were housed communally ( 100) in clear plastic cages (45
× 22 × 19 cm) with access to food and water ad libitum on a 12 h light/dark cycle∼ at constant temperature
(25◦C) and humidity ( 70%) (Wilkinson, 1993). Cages included hanging string to mimic rootlets found in the natural habitat, which ∼promotes natural mating and competitive behavior among males. Study males were between three weeks and 2 months post-eclosion. Males were anesthetized with CO2, positioned on their thoracic spines, and eye span was measured to the nearest 0.01 mm using scion image (Ribak et al., 2009). Eye span differences in this species serve as a reliable index for body size difference (Wilkinson, 1993; Burkhardt and de la Motte, 1983). Therefore, opposing pairs of size-mismatched males were established based on a minimum of 5% difference in eye span. The males were assigned to 3 treatment groups, comprised of untreated smaller opponents (n = 20), pharmacologically treated smaller opponents (n = 20), and untreated larger opponents (facing either a treated or untreated smaller opponent, n = 20 per group). Respective groups were then transferred to smaller cages (14 × 14 × 14 cm) housing between 2 and 6 flies. Food and water were provided ad libitum.
35 Pharmacological Manipulation
Sterilized and pureed sweet corn kernels were prepared according to (Bubak et al., 2013). Both untreated and treated food media included 1 mL/100 mL methylparaben as a mold inhibitor, 25 mg of ascorbic acid to act as a stabilizer, and food coloring to ensure uniform mixing. Treated food media also included 3 g/100 mL of 5-hydroxy-L-tryptophan (5-HTP; Sigma–Aldrich, St Louis, MO) (Bubak et al., 2013). Flies were allowed access to the treated corn ad libitum for 4 days prior to trials, which reliably produces a minimum 8- fold increase of brain 5-HT content relative to non-treated flies (Bubak et al., 2013).
Forced-Fight Paradigm
After the 4-day feeding period, size mismatched fighting pairs were placed in an arena (11×6.5×5 cm rectangle containing a removable glass ceiling and glass wall) containing moist filter paper as a floor and a removable central cardboard barrier that separated the individuals, but no food was provided (Egge and
Swallow, 2011; Egge et al., 2011). Twelve hours after placement in the arena, the cardboard divider was removed and a small piece of pureed corn (approximately 5 mm in diameter) was introduced to initiate the contest (Bubak et al., 2013). This 12-h starvation period promotes willingness to engage in aggressive behaviors over a food resource (Egge and Swallow, 2011; Egge et al., 2011). The interactions were then recorded for 10 min using a digital video recorder for later scoring of behavior.
Dissection and Sample Preparation
Immediately following the behavioral contest, flies were aspirated out of the arena and anesthetized with CO2. The heads were immediately removed under a microscope at 40× magnification using micro-scissors.
Eye stalks and mouthparts were both removed, as these contain residual food, tissue, and pigments that can interfere with analysis of brain tissue. Micro-tweezers were inserted into the oral cavity to split open the exoskeleton and expose the brain. The exposed neural tissue and surrounding exoskeleton were then placed into
60 μL of acetate buffer containing the internal standard α-methyl-dopamine and stored at −80 ◦ C. The dissection process took less than 1 min per fly.
36 Data Acquisition
5-hydroxytryptophan (5-HTP) and 5-HT were measured in single whole brain samples using high performance liquid chromatography with electrochemical detection using a method adapted from (Bubak et al.
2013). Frozen samples were thawed and then centrifuged at 17,000 rpm at 4◦C. The supernatant was removed and 45μL of the sample was injected into the chromatographic system. Amines were separated using a C18 4
μm NOVA-PAK radical compression column (Waters Associates Inc., Milford, MA) and detected using an LC
4 potentiostat and a glassy carbon electrode (Bioanalytical Systems, West Lafayette, IN). The sensitivity was set at 1 nA/V with an applied potential of +0.9 V vs. an Ag/AgCl reference electrode. The mobile phase was made by dis- solving 8.6 g sodium acetate, 250 mg EDTA, 11 g citric acid, 130 mg octylsulfonic acid in water, and adding 160 mL of methanol to a final volume of 1L distilled water. The pH was adjusted to 4.1. All chemicals were obtained from Sigma–Aldridge (St. Louis, MO). The remaining tissue pellet was solubilized in 60 μL of
0.4 M NaOH and analyzed for protein using the Bradford method (Bradford, 1976). Amine concentrations were determined using a CSW32 data program set in internal standard mode using peak height values relative to standards. The resulting concentrations were divided by μg protein in the sample yielding pg amine/μg protein and corrected for injection vs. preparation volume.
Behavioral Analysis
For each individual, the number and duration of separate interactions (beginning with lining up eyestalks to conflict resolution) occurring within the 10 min forced fight were measured. Specific behaviors of each opponent were also measured, based on an ethogram adapted from (Egge et al., 2011). The behaviors included within the ethogram were mutually exclusive so that only one behavior during an aggressive interaction could occur at any given time. Any behaviors occurring outside an aggressive interaction were not scored. An aggressive interaction began with the approach of a fly toward its opponent or by the mutual lining up of eyestalks between opponents. The behaviors scored for each opponent fall into three categories; low- intensity behaviors, high-intensity behaviors, and conflict resolution behaviors. Low-intensity behaviors consist of aggressive behaviors that do not include physical contact with an opponent, such as contest initiation
(approaching an opponent that ultimately results in an aggressive confrontation), flexing, and rearing. Only one of the opponents was awarded a score for contest initiation at the beginning of each interaction, if both flies
37 appeared to approach their opponent and line up simultaneously no initiation score was awarded. High-intensity behaviors are denoted by physical contact between opponents, including swiping motions with the forelegs toward the opponent, tussling in which the competitors interlock forelegs, and jump attacking where a competitor jumps or climbs onto the back of its opponent. Conflict resolution was marked by behaviors which led to the aggressive interaction being halted for a minimum of 3 s. Behaviors that could cause this resolution included slowly walking away from an opponent, retreating (swiftly moving in opposite direction from opponent) from or pursuing an opponent, or becoming unaligned from the opponent. Behaviors were scored using the behavioral software Jwatcher (Blumstein et al., 2007). Along with these behaviors, duration of individual interactions (beginning with lining up eyestalks to conflict resolution) was also measured. Fights were scored by experimenters blind to treatment, with each fly’s behavior measured separately. As fights consist of multiple aggressive interactions, the contest outcome was determined by the number of retreats such that the fly engaging in more retreat behaviors was designated to be the loser and its opponent the winner (Egge et al., 2011).
Statistical Analysis
A one-way ANOVA with a Tukey’s multiple comparisons test was used to test for differences in 5-HT concentrations between all groups. Separate unpaired Student’s t-tests were used to test for differences in means for number of interactions, initiations, high- intensity behaviors, or escalations between treated and untreated smaller opponents, or between larger competitors facing treated vs. untreated opponents. The likelihood of being the first to either escalate or initiate an interaction was determined within each treatment group using separate Fisher’s exact tests employing a two-tailed contingency table. The probability of either treated or untreated smaller opponents winning the entire 10 min forced fight was also determined using a Fisher’s exact test. All statistical tests were conducted using Prism 6 (GraphPad Software) with a set 0.05 alpha level.
38 Results
Pharmacological Manipulation of 5-HT
Pretreatment with 5-HTP resulted in a significant elevation of brain 5-HT in treated smaller flies when compared to all other groups (one-way ANOVA, F (3, 76) = 21.61, p < 0.001; Table 3.1). Treated competitors had an approximately 12-fold increase compared to untreated competitors (180.8 ± 35.1 vs. 15 ± 0.5 pg/μg protein; Table 3.1). The 5-HT concentrations in larger opponents of both treated and untreated smaller rivals were not significantly different (19.8 ± 1.2 vs. 17.2 ± 2 pg/μg protein; Table 3.1). In previous studies, using the same dose of 5-HTP, the increase in 5-HT was approximately two-fold (Bubak et al., 2013; Bubak et al.,
2014b). We have no explanation for this discrepancy.
Table 3.1. Whole brain 5-HT concentrations in 5-HTP pretreated Davids, untreated Davids, and Goliaths. 5-HT (pg/μg protein)
Smaller untreated 15 ± 0.5
Smaller treated 180.8 ± 35.1
Larger (untreated opponent) 17.2 ± 2
Larger (treated opponent) 19.8 ± 1.2
(Values presented as mean ± SEM).
Aggressive Behavior and Fight Outcome in Treated Flies
Smaller treated males engaged in significantly more aggressive interactions during the 10 min forced fight with their larger opponent compared to dyads containing untreated smaller opponents (mean ± SEM; 4.95
± 0.6 vs. 3.4 ± 0.39, respectively; Student’s t-test, df (38), p = 0.0361; Fig. 3.1A). Furthermore, smaller treated flies won or tied significantly more of these aggressive interactions compared to smaller untreated flies (mean ±
SEM; 3.5 ± 0.43 vs. 1.9 ± 0.3, respectively; Student’s t-test, df (38), p = 0.0041; Fig. 3.1B). The mean duration of interactions within a fight involving treated and untreated opponents remained essentially unchanged (13.19 s
± 1.14 vs. 13.03 s ± 2.13; Student’s t-test, p = 0.92).
39
Figure 3.1. (A) Treated smaller opponents engaged in significantly more interactions within the 10-min fight compared to untreated, smaller opponents. (B) Treated smaller opponents had significantly more non-losing interactions (winning or tie) within the 10-min fight compared to untreated, smaller opponents. (Values presented as mean ± SEM; Student’s t-test).
Treated smaller opponents were also more likely than untreated smaller opponents to initiate aggression during the 10 min forced fight, with 18 out of 20 treated flies initiating at least one aggressive interaction. In contrast, only 7 of the 20 untreated smaller flies initiated aggressive behaviors (Fisher’s exact test p = 0.0079, Fig. 3.2A). Furthermore, treated males performed approximately four times as many initiations in a fight compared to untreated individuals (mean ± SEM; 1.75 ± 0.32 vs. 0.4 ± 0.11; Student’s t-test, df (38), p =
0.0003; Fig. 3.2B). Treated, smaller opponents also produced twice as many high-intensity behaviors (physical contact) during their interactions compared to untreated, smaller opponents (mean ± SEM; 2.45 ± 0.44 vs. 1.2 ±
0.33; Student’s t-test, df (38), p = 0.0282; Fig. 3.3). However, smaller, treated flies did not show an increased likelihood of escalating an encounter to high-intensity levels, judged by being the first to engage in swiping, tussling, or jumping (mean ± SEM; 0.75 ± 0.22 vs. 0.45 ± 0.18; Student’s t-test, df (38), p = 0.31). Out of 20 treated, smaller flies, 8 escalated a contest compared to 5 untreated smaller flies (Fisher’s exact test p = 0.5).
Smaller treated competitors, despite having more successful interactions within the 10min contest, also did not show a greater probability of winning the entire 10 min forced fight when compared to untreated competitors, winning 4 of 20 fights with 7 draws while their untreated counterparts won 3 of 20 fights with 6 draws (Fisher’s
Exact Test, p = 0.67).
40 Figure 3.2. (A) There was a significantly higher percentage of smaller treated opponents that initiated an aggressive encounter over the entire duration of the 10-min fight compared to untreated smaller opponents (Two-tailed contingency table, Fisher’s exact test). (B) Treated smaller opponents also performed significantly more initiations on average compared to untreated smaller opponents (values presented as mean ± SEM; Student’s t-test).
Figure 3.3. Treated smaller opponents engaged in significantly more high-intensity interactions compared to untreated smaller opponents (values presented as mean ± SEM; Student’s t-test).
Aggressive Responses of Larger Opponents toward Treated Rivals
Larger males facing treated smaller opponents performed a greater number of high-intensity behaviors during their interactions than those facing untreated, smaller opponents (mean ± SEM; 5.5 ± 1.1 vs. 1.94 ± 0.84;
Student’s t-test, df (36), p = 0.0137; Fig. 3.4A). The mean number of escalations for larger flies facing treated opponents was also significantly higher than larger flies facing untreated opponents (mean ± SEM; 1.7 ± 0.33 vs. 0.75 ± 0.32; Student’s t-test, df (38), p = 0.045; Fig. 3.4B). Likewise, individual larger competitors facing treated opponents were more likely to escalate fights, defined as being the first to perform a high intensity behavior (make physical contact) such as swiping, tussling or jump attack in an already ongoing aggressive contest, than larger competitors facing an untreated counterpart (16 vs. 7 individuals, respectively; Fisher’s exact test, p = 0.0095, Fig. 3.5). However, the number of aggressive interactions initiated by larger males
41 toward either treated or untreated smaller males during the 10-min fight did not differ (mean ± SEM; 2.65 ±
0.35 vs. 2.05 ± 0.27; Student’s t-test, df (38), p = 0.18). The likelihood of larger individuals initiating at least one interaction was also not significantly different when facing either a treated or untreated opponent (20 vs. 18 individuals, respectively; Fisher’s exact test, p = 0.49). Thus, larger flies needed to be in an already progressing aggressive interaction before they altered their behavior by escalating to higher intensities; an effect that is amplified when facing treated opponents.
Figure 3.4. (A) Larger males facing smaller treated opponents performed significantly more high-intensity behaviors compared to larger males facing untreated smaller opponents. (B) Larger males facing smaller treated opponents also performed significantly more escalations compared to larger males facing untreated smaller opponents. (Values presented as mean ± SEM; Student’s t-test).
Figure 3.5. A significantly higher percentage of larger males that fought treated smaller opponents escalated an interaction during the 10-min fight compared to larger males that fought untreated smaller opponents (Two- tailed contingency table, Fisher’s exact test).
42 Discussion
In this study, we examined the effects of 5-HT manipulation on smaller competitors to determine whether elevated 5-HT levels would be able to overcome the normal size biases of opponent assessment and contest escalation. Previous studies have shown that larger male stalk-eyed flies win more than 75% of contests in which the combatants differed by more than 5% in eye span (Panhuis and Wilkinson, 1999; Egge and
Swallow, 2011). Additionally, in a size-matched contest, males with 5-HT pharmacologically elevated by roughly 2-fold won 85% of the time against control males (Bubak et al., 2013) and displayed greater levels of high-intensity behaviors and fewer retreat behaviors (Bubak et al., 2014b). Following the procedures developed by (Bubak et al., 2013), we manipulated brain 5-HT levels of smaller competitors by oral administration of its metabolic precursor, 5-HTP, resulting in a nearly 12-fold 5-HT increase in the treated smaller flies relative to all other groups. Despite the large increase in 5-HT, pre-treatment with 5-HTP did not affect over-all contest outcomes; that is, the treatment did not result in the smaller competitor, “David”, slaying the larger “Goliath”.
However, it did significantly alter the progression of the contest and the types of aggressive behaviors displayed by both the larger and smaller opponents, consistent with our predictions.
Treated smaller flies showed an increased willingness to engage and persist in aggressive contests, corroborating the findings of Bubak (Bubak et al., 2014b), which showed that flies with elevated 5-HT were less likely to retreat in contests with size-matched opponents. Consistent with our results, Huber (Huber et al.,
1997) also demonstrated that subordinate crayfish treated with 5-HT were less willing to retreat, but that overall contest outcome remained unchanged, with subordinate competitors still more likely to lose. In our study, increasing brain 5-HT also increased the likelihood that smaller flies would engage in aggressive contests, promoting both the initiation and number of aggressive interactions. This may indicate that heightened 5-HT either increased motivation to engage in an aggressive contest or artificially enhanced self-perception of resource holding potential (i.e., fighting ability). Finally, treated males engaged in more high-intensity behaviors (swiping, tussling, and jump attacks) than untreated males. The increased high-intensity interactions and behaviors in treated flies suggests that 5-HT plays an important role in the decision to not only remain in a contest but also to persist in expressing high levels of aggression despite discrepancies in body size.
43 Interestingly, despite the increased number of high-intensity behaviors exhibited in contests including treated smaller flies, contest duration did not change, indicating that the escalation and continuation of high- intensity encounters was not simply a byproduct of the contest lasting longer. Together, these behavioral differences between treated and untreated flies indicate that 5-HT may either be involved in rival assessment or in controlling the willingness to engage in risky behaviors, rather than being absolutely necessary for the expression of aggression. Although it was initially reported that neural 5-HT had a limited or absent role in insect aggression (e.g., crickets and Drosophila; (Stevenson et al., 2000; Baier et al., 2002)), more recent studies suggest, at least for some species, the contrary (reviewed in (Bubak et al., 2014a)). For example, decreasing 5-
HT activity in male Drosophila reduces fight escalation but does not abolish aggression (Dierick and
Greenspan, 2007; Alekeseyenko et al., 2010) while increasing 5-HT increases aggression (Dierick and
Greenspan, 2007). However, the precise physiological mechanism by which 5-HT modulates aggression is still unknown. It is likely that particular 5-HT receptor subtypes contribute to the expression and inhibition of distinct aggressive behaviors as seen in Drosophila (Johnson et al., 2009). This mechanism could be extended further by the possibility of these receptors controlling the release of other neurochemicals known to increase aggression in insects such as octopamine (Stevenson et al., 2000; Adamo et al., 1995; Hoyer et al., 2008; Zhou et al., 2008; Rillich and Stevenson, 2011; Rillich and Stevenson, 2015). The underlying proximate mechanisms by which 5-HT modulates aggression as well as the correlation with other biochemical systems should be the focus of future studies.
Intriguingly, 5-HTP treated, smaller flies were not the only competitors to show a change in aggressive behavior. The larger flies competing against smaller, treated males performed significantly more high-intensity behaviors than their counterparts facing untreated, smaller opponents (Fig. 3.4A), and also engaged in a higher number of interactions that escalated in intensity. However, it was only the larger competitor, and not their treated opponents, that showed greater likelihood of being the first to escalate a contest by expressing a high-intensity behavior. The number of contests initiated by larger males remained the same regardless of whether the opponent was treated. This suggests that the larger flies are driving the escalation of these aggressive contests in response to the greater frequency of aggressive behaviors displayed by the smaller opponents with elevated 5-HT levels. Since treated flies but not their opponents initiated more aggressive interactions, the heightened aggressive reaction by the larger flies suggests that the
44 smaller opponents may have evoked an overt aggressive response from the larger opponent, which normally would have won the fight simply by posturing. In essence, smaller, treated flies appear to be “poking a sleeping giant” and forcing an escalation response from their larger opponent despite the potential negative consequences.
Our results stand in contrast to Huber (Huber et al., 1997) who found that the main effect of 5-HT elevation in subordinate crayfish was to increase the duration of contests between smaller and larger opponents, which appeared to be driven by the smaller opponent’s unwillingness to retreat and not by escalation of fighting patterns by either the dominant or subordinate opponent specifically. In this study, we did see increased initiations of aggressive encounters and increased interactions by treated flies, alluding to a willingness to fight, despite the duration of these contests not being increased. Similarly, Momohara (Momohara et al., 2013) found that smaller crayfish of a different species from that examined by Huber (Huber et al., 1997) treated with 5-HT showed an increase in aggressive behaviors toward larger opponents, but not in contest duration. However, in contrast to our study, Momohara (Momohara et al., 2013) demonstrated that 5-HT elevations increased the likelihood of winning contests against larger opponents. A possible explanation of the discrepancy between our study and that of Momohara (Momohara et al., 2013) is that the larger opponent’s perception of their treated opponent was altered, leading to the increase in escalation behaviors and resulting in a more costly fight that would favor a larger competitor. Thus, our study indicates that treatment of the smaller rival results in the alteration of behaviors of both opponents, providing clues to the assessment strategies used by stalk-eyed flies and the degree of behavioral information they perceive during a confrontation.
Overall, we found that increasing brain 5-HT in smaller opponents led not only to an increased willingness to engage in aggressive encounters but also an increased production of high-intensity behaviors, consistent with previous studies (Huber et al., 1997; Momohara et al., 2013; Kravitz, 2000; Bubak et al.,
2014b). Furthermore, while the smaller treated flies increased aggression and were more likely to initiate aggressive contests and engage in risky high-intensity behaviors, it was actually their larger opponents that showed a higher likelihood of being the first to express a high-intensity behavior. This implies that aggressive behavior is not fixed in these individuals, but can be modified in a contextually-appropriate manner beyond initial morphological perception by incorporating assessments of opponent behavior as the interaction proceeds.
45 Contributions
I served as the primary author of this manuscript. I had a critical role in the design of the study, collection of data, and analysis of results. I also wrote the manuscript.
46 CHAPTER IV
SEX DIFFERENCES IN AGGRESSION: DIFFERENTIAL ROLES OF 5-HT2, NEUROPEPTIDE F
AND TACHYKININ
Abstract
Despite the conserved function of aggression in the obtainment of critical resources such as food and mates across taxa, serotonin’s (5-HT) modulatory role on aggressive behavior appears to be largely inhibitory for vertebrates but stimulatory for invertebrates. However, critical gaps exist in our knowledge of invertebrates that need to be addressed before definitively stating opposing roles for 5-HT and aggression. Specifically, the role of 5-HT receptor subtypes are largely unknown as is the interactive role of 5-HT with other neurochemical systems known to play a critical role in aggression. Here, we investigated the independent function of 5-HT2 as well as its interactive role with neuropeptides in an invertebrate model of aggression, the stalk-eyed fly.
Knockdown of 5-HT2 by siRNA revealed a sex-dependent, inhibitory role in males only. Additionally, we provide evidence for 5-HT2’s involvement in regulating neuropeptide F activity, a suspected inhibitor of aggression. However, this function appears to be stage-specific, altering only the initiation stage of aggressive conflicts. Alternatively, pharmacologically increasing systemic concentrations of 5-HT significantly elevated the expression of the aggression-enhancing neuropeptide tachykinin, again, in a stage- and sex-dependent manner. Together, these results demonstrate a more nuanced role for 5-HT in modulating aggression in invertebrates, revealing an important interactive role with neuropeptides that is more reminiscent of vertebrates.
The sex-differences described here also provide valuable insight into the evolutionary contexts of this complex behavior.
Introduction
Serotonin (5-HT) appears to promote aggression in invertebrates (reviewed in Bubak et al., 2014a;
2016b), in contrast to the largely inhibitory effect seen in vertebrates (Nelson and Trainor, 2007, but see Olivier and van Oorschot, 2005). Much of the empirical support for this dichotomy comes from invertebrate studies primarily focusing on actions at the systemic level. For example, crayfish administered 5-HT were more willing to reengage with a dominant opponent (Huber et al., 1997) and dipterans with pharmacologically or genetically elevated 5-HT concentrations demonstrated more aggressive behaviors when paired with
47 unmanipulated opponents (Dierick and Greenspan, 2007; Alekseyenko et al., 2010; Bubak et al., 2014b). While these findings support the presumption that 5-HT has opposing effects on invertebrate aggression from vertebrates, there are critical gaps in knowledge that need to be considered before accurately stating that 5-HT exclusively modulates invertebrate aggression in a positive manner.
A more nuanced role for 5-HT in invertebrate aggression emerges when considering involvement of receptor subtypes. In vertebrates, differential binding of specific 5-HT receptors, predominantly 5-HT1A, 5-
HT1B, and 5-HT2 subtypes, has profound implications for aggressive behavior (Juarez et al., 2013, Takahashi et al., 2011, Popova et al, 2010). Notable sequence and functional homology for these subtypes have been described in invertebrates (Tierney, 2001). Additionally, similarities in their influence on aggression across taxa have been suggested (Johnson et al., 2009), however, these studies are extremely limited. For example, activation of 5-HT2 receptors by the specific agonist 2,5-dimethoxy-4-iodoamphetamine has an anti-aggressive effect in both rodents (Sanchez et al., 1993; Olivier et al., 1995) and Drosophila (Johnson et al., 2009), suggesting 5-HT2 receptor function is conserved evolutionarily. In contrast, a divergent role is indicated for 5-
HT1A receptors, activation of which largely dampens mammalian aggression (Takahashi et al., 2012) while enhancing aggressive behavior in Drosophila (Johnson et al., 2009). Whether these same similarities and differences in subtype function exist in invertebrates other than Drosophila remains to be determined.
It is also possible that 5-HT receptors have distinct functions in mediating the contextual expression of specific aggressive behaviors and their intensity, which in turn will direct how the conflict proceeds (i.e., initiation, escalation, and termination). For instance, while 5-HT1A and 5-HT2 receptors are generally inhibitory, agonists of these receptors can promote high intensity aggression in mammals during certain situations such as maternal, territorial, and self-defense (Karl et al., 2004; Takahashi et al., 2012), demonstrating these subtypes can exert opposing effects according to context. In male Drosophila, overall aggression is heightened or decreased according to 5-HT1A or 5-HT2 receptor activation, respectively, but 5-HT1A predominantly affects expression of low intensity aggression (threat displays) while 5-HT2 mediates high intensity behaviors such as lunging (Johnson et al., 2009). From a systemic view, pharmacologically elevating brain 5-HT also contextually modulates aggression in male stalk-eyed flies (Teleopsis dalmanni). Specifically, smaller males display low intensity aggression and initiate less contests when faced with a larger opponent, but increasing 5-HT causes the smaller male to initiate and escalate fights by promoting high-intensity aggression
48 (Bubak et al., 2015). Interestingly, contest duration remains the same whether the smaller male is treated with
5-HT or not (Bubak et al., 2015). In contrast, in size-matched contests, aggression intensity and contest duration appear to be modulated as a function of the difference in brain 5-HT between opponents (Bubak et al.,
2014b). In other words, opponents with closer brain 5-HT concentrations will engage in prolonged high intensity contests regardless of whether 5-HT has been increased in one male, with this manipulation only making contests shorter and less intense if it causes brain 5-HT to be substantially elevated above that of the opponent (Bubak et al., 2014b). Thus, aggression in T. dalmanni provides a useful model for examining how 5-
HT can discretely modulate behavioral expression according to the specific situational contexts and aggressive stages; initiation, escalation, and termination of conflicts. However, it is not known if these differential effects are dependent on 5-HT receptor specificity. This relationship between 5-HT receptor subtype and aggression in
T. dalmanni was investigated in the current study.
The extent to which 5-HT modulates discrete aggressive behaviors in invertebrates may also be influenced by the actions of neuropeptide systems, as shown for vertebrates. For example, lesioning neurons containing tachykinin (Tk) receptors reduced violent attacks in rats but left milder attacks unaffected (Halasz et al., 2009). Similarly, high-intensity aggressive behavior during intrasexual contests is elicited by activation of
Tk neurons in male Drosophila (Asahina et al., 2016). Overlap in function is also seen with neuropeptide Y
(NPY) and its invertebrate homolog neuropeptide F (NPF), which decrease frequency of high intensity aggression in mice and Drosophila, respectively (Dierick and Greenspan, 2007; Karl et al., 2004). These findings imply evolutionarily conserved roles for these neuropeptides in determining aggression intensity and conflict escalation.
Moreover, NPY has been shown in mammals to exert its effects by modulating 5-HT activity (Karl et al., 2004), and conversely, 5-HT is known to directly influence NPY release in brain regions that mediate aggression (Dryden et al., 1996a,b; Smialowska et al., 2001; Glass et al., 2010). However, unlike mammals, 5-
HT and NPF pathways appear to act independently in regulating Drosophila aggression (Dierick and
Greenspan, 2007). Receptors for Tk are located on both 5-HT and non-5-HT neurons within the mammalian 5-
HT cell body region (dorsal raphe), and can modulate neuronal firing and 5-HT release in terminal regions
(Maejima et al., 2013). In contrast, Tk and 5-HT do not appear to be co-localized to the same neurons in the majority of invertebrates (Osborne et al., 1982; Chamberlain et al., 1986; Langworthy et al., 1997; Ingell, 2001;
49 Boyer et al., 2007; Boyan et al., 2010; Herbert et al., 2010), and while they have been shown to exert similar excitatory effects on crustacean cardiac and gut ganglia (Cruz-Berudez & Marder, 2007; Rehm et al., 2008), it is unclear whether Tk and 5-HT influence each other to regulate invertebrate aggression. Combined, these findings highlight the need to consider multiple discrete measures of aggressive behavior (e.g., frequency and intensity plus contest duration) along with activity of other neuromodulators when determining the role of 5-HT as a whole.
Finally, a seemingly more troubling limitation in our knowledge of how 5-HT mediates aggressive behavior is the lack of studies conducted with female invertebrates. Although several studies have described neural pathways involved in aggression and related behaviors, the almost exclusive use of male subjects precludes determining whether these are sex-specific (for review see Manoli et al., 2013). Given the stark contrast in aggressive and related behaviors between the sexes of many invertebrate species, as well as morphological dissimilarities (Bubak et al., 2014a), it is reasonable to suggest significant differences in mechanisms as well.
Here, we sought to address some of the gaps in knowledge about 5-HT modulation of invertebrate aggression by using a model species, the sexually dimorphic stalk-eyed fly T. dalmanni. Specifically, we determined 1) whether 5-HT receptor subtypes mediate expression of aggressive intensity and contest progression, 2) if there is a direct functional relationship between 5-HT subtypes and the neuropeptides Tk and
NPF in regulating aggression, and 3) how these findings differ between sexes. Using a combination of socio- environmental manipulation, RNA interference (RNAi), and pharmacological treatment, we describe sex- specific roles for 5-HT2 receptors, Tk and NPF in behavioral expression and how this relates to contest progression and outcome. We also demonstrate a direct effect of 5-HT on neuropeptide expression that is posited to modulate male aggression. Our results reveal the impressive level of conservation with respect to neurochemical mechanisms among species as diverse as vertebrates and invertebrates, and challenges the perception of an opposite role for 5-HT in the mediation of aggression.
50 Methods and Materials
Subjects
The sexually dimorphic stalk-eyed fly, Teleopsis dalmanni, is native to South East Asia. All flies used in this study were descendants of pupae collected from wild populations in 2012, housed at the University of
Maryland, College Park. Individuals were housed communally in cages (45 cm x 22 cm x 19 cm) on a 12-h light:dark cycle with free access to food, water, and mates. Sexually mature flies in this study were briefly anesthetized using ice and measured to the nearest 0.01 mm using cellSens standard software (Olympus). Size- matched individuals were determined as being < 1% difference in eye span. The high correlation of eye span to body size in this species makes eye span an accurate representation of body size (Burkhardt and de la Motte
1983; Wilkinson 1993). Predetermined opponents were given identifying paint marks between their thoracic spines and housed separately prior to surgeries and fight contests. Isolated flies were housed separately for 7 days prior to fight contests.
Drug Administration
Flies selected for 5-HT manipulation were fed sterilized, pureed corn containing either 3 g of 5- hydroxy-L-tryptophan (5-HTP; H9772; Sigma, St. Louis, MO) in 100 mL media or vehicle/100 mL media for 4 days as previously described (Bubak et al., 2013, 2014b). Specifically, the vehicle media contained 100 mL of corn, 1 mL of methylparaben (Ward’s Science, Rochester, NY) as a mold inhibitor (Wilkinson 1993), and 25 mg of ascorbic acid (Sigma, St. Louis, MO) to act as a stabilizer (Dierick and Greenspan 2007).
Forced-Fight Paradigm and Behavioral Analysis
Opponent matchups for the experiments described below are as follows: socially isolated vs socially reared, 5-HT2 siRNA vs vehicle siRNA, and 5-HTP treated vs vehicle treated. Size- and sex-matched opponents were placed in an arena (11 cm x 6.5 cm x 5 cm) containing a glass wall and ceiling, for filming, and a removable cardboard barrier that separated the individuals. Flies were starved for 12 hours prior to the contest to increase the incentive to fight over a piece of pureed corn that was placed in the arena center immediately following barrier removal. All contests that occurred during 10 mins of fighting were scored using the behavioral scoring software, JWatcher (UCLA). Scored behaviors for each individual were based on an
51 existing ethogram adapted from Egge et al., 2011. Specific behaviors fall into three categories: contest initiation, escalation, and termination. Contest initiations were determined by one opponent approaching the other, of which ultimately results in an aggressive behavioral exchange. Only one opponent was awarded an initiation per contest, none were awarded if there was ambiguity in determining the initiator. Escalations of fights were determined by high-intensity (HI), physical contact, behaviors. Termination and subsequent winners of contest were determined by retreats; with the individual with the fewest number of retreats after the
10 minute fighting period deemed the winner.
5-HT Quantification
Quantification of brain 5-HT was conducted using high-performance liquid chromatography (HPLC) with electrochemical detection as previously described (Bubak et al., 2013). Briefly, whole brains were dissected and frozen in 60 µL of acetate buffer containing the internal standard α-methyl-dopamine (Merck) and stored at -80oC. Samples were thawed and centrifuged at 17,000 rpms. A portion of the sample (45 µL) was injected into the chromatographic system and the amines were separated with a C18 4 µm NOVA-PAK radial compression column (Waters Associates, Inc. Milford, MA) and detected using an LC 4 potentiostat and a glassy carbon electrode (Bioanalytical systems, West Lafayette, IN). The CSW32 data program (DataApex
Ltd., Czech Republic) calculated monoamine concentrations based on peak height values that were obtained from standards (Sigma-Aldridge, St. Louis, MO). The remaining sample was solubilized for protein analysis with 60 µL of 0.4 M NaOH (Bradford, 1976). The final amine concentrations are expressed as pg amine/ µg protein following appropriate corrections for injection vs preparation volume.
RNAi Design and Administration
Dicer-substrate siRNAs (short interfering RNA) used in this study were synthesized by IDT
(Integrated DNA Technologies, Coralville, IA). Probes targeting 5-HT2 were generated using cDNA sequences of Teleopsis dalmanni. Probes targeting green fluorescent protein (GFP) were generated from cDNA sequences of Aequorea coerulescens. Predetermined size- and sex-matched opponents were injected with 100 nL of a solution containing siRNA generated against either GFP for controls or 5-HT2 for treated groups. The siRNA was coated (1:1 ratio) in the transfection reagent polyethylenimine (PEI) to aid in membrane penetration and
52 stabilization. Injections were administered in the head cavity through the base of the proboscis. The brain was not penetrated with the needle.
Males and females were randomly selected from a social cage and size- and sex-matched to an opponent. Treatment groups were randomly selected; experimental males and females were injected with 100 nL of 5-HT2 siRNA solution and control flies were injected with 100 nL of GFP siRNA solution and allowed to recover within their respective groups for 48 hours with free access to food and water. Flies were then placed in the forced-fight paradigm described above.
RNA Isolation and Quantification
RNA isolation was conducted from dissected whole-brain tissue using the Direct-zolTM RNA MiniPrep
(ZYMO Research, Irvine, CA) according to the manufacturer’s instructions. Isolated RNA was reverse transcribed using the Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA).
Quantitative PCR was performed by an Applied Biosystem’s StepOne machine (Foster City, CA) using
TaqMan master mix (Applied Biosystems), the primer-probe pair for the endogenous control, Gapdh, and one of the following target gene primer-probe pairs: 5-HT1A, 5-HT2, 5-HT7, SERT, Tk, or NPFr (Integrated DNA
Technologies, Coralville, Iowa; Table 1). We intentionally focused on the Tk ligand itself and not the receptor due to ambiguity in the specific receptor responsible for baseline aggression in dipteran species (Asahina et al.,
2014). Relative quantification (RQ) of the PCR product was conducted using the comparative CT method
(Schmittgen and Livak, 2008). Values were presented as target gene expression relative to the endogenous control Gapdh.
Table 4.1. List of RT-qPCR primer and probe sequences.
Gene Primer (Fwd) Primer (Rev) Probe Gapdh -GACCGTGAGTAGAGTCGTATTTG- -TTCTCCGTGCTGCTGTTT- -TTGACTGCTACAACGGAAGCTCCG- 5-HT1A -GGGCTCAATGCTCCTCAATAA- -CGCTGCTCGTATCAGTTTCA- -TGTCAACGGAAGTGACCTTATGGCA- 5’ 3’ 5’ 3’ 5’ 3’ 5-HT2 -TCCGTGTCGCTCTTTCTTTC- -CGTTGCTTTACAGGCGATTTATT- -AGGTTCAGTAGATGCGCGACGTTT- 5’ 3’ 5’ 3’ 5’ 3’ 5-HT7 -AGGTTTGCGGAAGTCTCTATTC- -GCCACAGTCACTTTCCTCATTA- -TCGGATTCAGTAGCGAGTTTGCATAGC- 5’ 3’ 5’ 3’ 5’ 3’ SERT -GCCAAATTCGGTGCTGTTATT- -CTACACTGCACCTTCCTGTATG- -TGCGATCCATAATTGCCCACTTGC- 5’ 3’ 5’ 3’ 5’ 3’ NPFR -CGAATGCAATGGCCGTAATG- -GGTTCGTATGCTGTCTTGTGTA- -AAGCCTGCAACATGGCAATCATCT- 5’ 3’ 5’ 3’ 5’ 3’ TK -TTCTGTGTTGCTGCTCTCTTAT- -GGCGAAGATGACGGTATATCAA- -CGCACACCAACGAAAGCGTTTGAA- 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
53 Statistical Analysis
To test for statistical differences of behaviors performed between groups, separate two-tailed Wilcoxin matched pairs signed-rank test was used. Two-tailed Student’s t-test were applied to test for relative differences between mean mRNA expression between treatment groups as well as differences in mean 5-HT levels as quantified by HPLC. Alpha value was set at 0.05.
Results
Expression of 5-HT1A, 5-HT2, 5-HT7 Receptors and the Serotonin Transporter in Male and Female Brains
Since 5-HT plays a prominent role in modulating invertebrate aggression (Bubak et al., 2014a), including increasing aggressive displays in male T. dalmanni, we first measured relative sex differences in mRNA expression of 5-HT1A, 5-HT2, and 5-HT7 receptors and the 5-HT transporter (SERT; Fig. 4.1). Males had markedly lower expression levels of the 5-HT2 receptor subtype (Student’s t-test, p < 0.001) and SERT
(Student’s t-test, p < 0.001) but significantly higher levels of 5-HT1A (Student’s t-test, p < 0.01; Fig. 4.1) compared to females. Males and females had similar expression levels of the 5-HT7 receptor subtype (Fig. 4.1).
We hypothesized that the stark difference in 5-HT2 expression levels between sexes could be one potential mechanism responsible for the sex differences observed in baseline aggression.
Figure 4.1. Relative mRNA expression between normally reared adult male and female stalk-eyed flies. Males have a significantly lower expression of 5-HT2 (0.41 ± 0.03, n = 9 vs 1.0 ± 0.002, n = 9) and SERT (0.284 ± 0.02, n = 9 vs 1.0 ± 0.001, n = 9) but significantly higher expression levels of 5-HT1A (1.64 ± 0.17, n = 12 vs 1.0 ± 0.001, n = 9) compared to their female counterparts. There was no statistical difference in expression levels of 5-HT7 between males and females. Values are normalized to female expression levels and presented as mean ± SEM. (Two-tailed, Student’s t-test; p < 0.05*, p < 0.01**, p < 0.001***).
54 Expression of the Neuropeptide Tk and the NPF Receptor in Male and Female Brains
We also investigated the expression of the NPF receptor in male and female flies, since NPY and the invertebrate analog NPF inhibits aggressive behavior in other animal species (Dierick and Greenspan, 2007;
Karl et al., 2004). We also measured the baseline expression of the neuropeptide Tk, which has been reported to increase aggression in vertebrates and Drosophila (Asahina et al., 2014; Halasz et al., 2009). Baseline relative mRNA expression levels of the NPF receptor (1 ± 0.07, n = 12 vs 0.86 ± 0.05, n = 12; Student’s t-test, p > 0.05) and Tk (1 ± 0.23, n = 7 vs 0.95 ± 0.17, n = 9; Student’s t-test, p > 0.05) were similar between females and males, respectfully.
Increased Aggression Corresponds with Reduced 5-HT2 and Heightened Tk Expression in Males but not in
Females
Social isolation increases aggression in both invertebrate and vertebrate species (Twenge et al., 2001;
Wongwitdecha and Marsden 1996; Alexander 1961; Johnson et al., 2009). We first tested whether social isolation induced aggression in male stalk-eyed flies. Following 7 days of isolation, males had a higher probability of winning the aggressive confrontation compared to their socially reared opponents (Fig. 4.2D;
85% vs 15%). Isolated males also initiated significantly more confrontations (Fig. 4.2A; Wilcoxon matched- pairs signed rank test, p < 0.01) and performed more high-intensity (physical) behaviors than their socially- reared opponents (Fig. 4.2C; Wilcoxon matched-pairs signed rank test, p < 0.05). When we repeated this experiment with female flies we found no difference in either initiations or escalations of aggression in socially- isolated females when compared to colony-raised females (Fig. 4.2B). The lack of high-intensity behaviors performed and overall alterations in aggressive behaviors in female fights did not allow for easily identified winners, thus high-intensity behaviors and the probability of winning the aggressive encounter was not computed.
Next, we measured brain 5-HT2 and Tk expression to determine if expression levels covaried with the display of increased aggression. In male flies raised in social isolation, there was a decrease in 5-HT2 expression relative to colony-raised flies (Fig. 4.3; Student’s t-test, p < 0.01). In contrast, Tk expression was markedly increased in brains obtained from socially isolated male flies relative to colony-raised males (Fig. 4.3;
Student’s t-test, p < 0.001). When the experiment was repeated in females, we found expression levels of 5-
55 HT2 and Tk in socially isolated flies did not differ significantly from values obtained in colony-raised flies (Fig.
4.3). There were no significant differences in the expression of brain 5-HT1A, 5-HT7, or NPFr expression following isolation in either males or females (Fig. 4.3).
56
Figure 4.2. Social isolation increased initiations, escalations, and winning probability in males but not females. A) Males, socially isolated, performed significantly more initiations compared to their socially reared opponents. B) Social isolation had no effect on initiations in female aggressive contests. C) Males, socially isolated, performed significantly more high-intensity aggressive behaviors compared to their socially reared opponents. Behavior values are normalized to control opponents with connecting black lines representing opponents. Red lines represent the slope with the blue shaded area representing the 95% confidence interval. (Two-tailed, Wilcoxon matched-pairs signed-rank test; n = 20, p < 0.05*, p < 0.01**, p < 0.001***). D) Males that were socially isolated had a significantly higher winning percentage compared to their socially reared opponents (85% vs 15%; error bars represent 95% confidence interval).
57
Figure 4.3. Social isolation reduced 5-HT2 expression and increased Tk expression in males but not females. A) Expression of 5-HT2 reduced ~28% in males following social isolation (1.0 ± 0.074, n = 9 vs .72 ± 0.06, n = 12) whereas Tk expression was increased ~77% compared to socially reared males (1.0 ± 0.13, n = 9 vs 1.77 ± 0.04, n = 6). B) No change in expression value was measured in females following social isolation. Values are normalized to socially reared expression levels and presented as mean ± SEM. (Two-tailed, Student’s t-test; p < 0.05*, p < 0.01**, p < 0.001***).
5-HT2 Knock-Down Increases Aggression in Males but not in Females
To functionally test the role of 5-HT2 in aggression, we designed siRNA to selectively knock-down the receptor subtype. Intracranial injections of 5-HT2 siRNA reduced 5-HT2 receptor expression by approximately
30% 48 hours after the injection compared to control-injected individuals (siRNA generated to target GFP; Fig.
4.4; Student’s t-test, p < 0.05). Importantly, the designed 5-HT2 siRNA was specific, as it did not affect the expression of the closely related 5-HT1A receptor (Fig. 4.4).
58
Figure 4.4. Individual flies injected with 5-HT2 siRNA had significantly reduced 5-HT2 expression levels compared to vehicle injected siRNA (1.0 ± .09, n = 9 vs 0.68 ± 0.06, n = 6). 5-HT2 siRNA did not affect expression levels of the closely related 5-HT1A receptor. Values are normalized to vehicle injected individuals and presented as mean ± SEM. (Two-tailed, Student’s t-test; p < 0.05*, p < 0.01**, p < 0.001***).
Socially-reared males injected with 5-HT2 siRNA initiated more confrontations compared to their vehicle-injected opponents (Fig. 4.5; Wilcoxon matched-pairs signed rank test, p < 0.001). However, high- intensity behaviors did not differ between 5-HT2 siRNA injected and vehicle-injected opponents (Fig. 4.5).
Female opponents did not differ in initiations, despite 5-HT2 siRNA-injected females having significantly lower
5-HT2 expression levels compared to their vehicle-treated opponents (Fig. 4.5). Again, the lack of high- intensity behaviors and overall aggressive alterations in female fights did not allow for confident computation of mean high-intensity behaviors or designation winners.
59
Figure 4.5. 5-HT2 siRNA injections increased initiations and winning probability in males but not females. A) Males, injected with 5-HT2 siRNA, performed significantly more initiations compared to their vehicle injected opponents. B) 5-HT2 siRNA had no effect on initiations in female aggressive contests. C) Males, injected with 5-HT2 siRNA, had no differences in high-intensity aggressive behaviors compared to their vehicle injected opponents. Behavior values are normalized to control opponents with connecting black lines representing opponents. Red lines represent the slope with the blue shaded area representing the 95% confidence interval. (Two-tailed, Wilcoxon matched-pairs signed-rank test; n = 16, p < 0.05*, p < 0.01**, p < 0.001***). D) Males, injected with 5-HT2 siRNA, had a significantly higher winning percentage compared to their vehicle injected opponents (81% vs 19%; error bars represent 95% confidence interval).
60 5-HT2 Activity Modulates NPFr Expression in Males but not Females
To probe for potential interactions between 5-HT and neuropeptides in modulating aggression in males, we next measured brain NPFr and Tk expression levels following the siRNA-induced reduction in 5-
HT2. Reducing expression of 5-HT2 using siRNA significantly reduced NPFr expression in males (Fig. 4.6;
Student’s t-test, p < 0.01). When this experiment was repeated in females, our results showed that reduction of
5HT2 receptors did not significantly affect NPFr expression (Fig. 6). Expression of Tk did not differ in males or females treated with 5-HT2 siRNA (Fig. 4.6).
Figure 4.6. A) Injections of 5-HT2 siRNA did not alter expression levels of Tk in either males or females compared to their vehicle injected counterparts. B) Injections with 5-HT2 siRNA reduced NPFr expression in males (0.69 ± 0.07, n = 9 vs 1.0 ± 0.06, n = 12) compared to vehicle injected individuals but not in females. Values are normalized to female expression levels and presented as mean ± SEM. (Two-tailed, Student’s t-test; p < 0.05*, p < 0.01**, p < 0.001***).
Elevating 5-HT Concentrations Increases Tk Expression in Males but not Females
We have previously reported an increase in high-intensity aggressive behaviors in male stalk-eyed flies following administration of the 5-HT metabolic precursor, 5-HTP (Bubak et al, 2014b). When we repeated this study in female stalk-eyed flies, we found that 5-HTP-treated individuals do not exhibit a significant difference in aggressive behaviors compared to their untreated opponents, despite having significantly elevated brain 5-HT concentrations (18.44 ± 2.99, n = 18 vs 9.8 ± 0.6, n = 19; pg/μg protein; Student’s t-test, p < 0.01). Treated
61 females did not differ in the average number of initiations performed in a fight compared to controls (1.16 ±
0.28 vs 1.47 ± 0.3, n = 19; Student’s t-test, p > 0.05). Additionally, treatment with 5-HTP did not increase high- intensity behaviors, with less than half of the fights containing a single high-intensity behavioral exchange (6 out of 19). We next explored whether this sex-difference in the expression of high-intensity behaviors following 5-HTP administration might be related to changes in Tk expression. Indeed, males treated with 5-
HTP had significantly higher Tk expression compared to vehicle-treated males (Fig. 4.7; Student’s t-test, p <
0.001). In contrast, females treated with 5-HTP did not have a significant difference in Tk expression when compared to controls (Fig. 4.7A). Additionally, expression of NPFr was also significantly raised in males following 5-HTP treatment but not in females (Fig. 4.7B; Student’s t-test, p < 0.001). We did not find significant differences in the expression of brain levels of 5-HT1A, 5-HT2, or 5-HT7, respectively, in either males or females treated with 5-HTP when compared to vehicle-treated controls.
Figure 4.7. A) Treatment with 5-HTP increased expression levels of Tk in males compared to vehicle treated individuals (3.46 ± 0.36, n = 12 vs 1.0 ± 0.09, n = 9) but not in females. B) Treatment with 5-HTP increased expression levels of NPFr in males compared to vehicle treated individuals (1.48 ± 0.07, n = 12 vs 1.05 ± 0.11, n = 9) but not in females. Values are normalized to female expression levels and presented as mean ± SEM. (Two-tailed, Student’s t-test; p < 0.05*, p < 0.01**, p < 0.001***).
62 Discussion
In these studies, we used the sexually dimorphic stalk-eyed fly as a model to study the neurophysiological mechanisms mediating aggression. This model offers several advantages. First, flies contain fewer neurons and neuronal connections compared to vertebrates while simultaneously maintaining the relevant behaviors. Additionally, the monoaminergic and peptidergic systems involved in these behavioral processes are highly conserved across taxa (Blenau and Baumann, 2001; Martin and Krantz, 2014; Vleugels et al., 2013; Asahina et al., 2014; Baker et al., 1991; Halasz et al., 2009; Tierney 2001). This is not surprising because the fitness benefits and selection pressures of engaging in aggressive conflicts are relevant to most living animal species (Lorenz, 1966). Finally, the extreme sexual dimorphism exhibited by this species in addition to significant behavioral sex-differences, provides a valuable evolutionary perspective of aggression.
Studying the conserved mechanisms of aggression in a variety of animal species will allow us to obtain a better grasp, both functionally and evolutionarily, of the origin and regulation of this complex behavior across taxa and sexes.
Males and females of this species differently express central 5-HT1A- and 5-HT2-like receptor subtypes and SERT under colony-raised conditions. In contrast, the distribution of 5-HT7-like receptors was similar in both sexes. These results indicate the likelihood for sex specific roles in serotonergic function that might be related to the regulation of a number of physiological processes including reproduction and aggression.
Based on our previous work (Bubak et al, 2013, 2014b) and the work of others (Dierick and
Greenspan, 2007; Johnson et al., 2009; Alekseyenko et al., 2010) linking elevated 5-HT to enhanced aggressive responses in insects combined with sex differences in aggression (Nilsen et al., 2004), we next tested the effects of social isolation on aggression and expression of 5-HT receptor subtypes in male and female stalk-eyed flies.
Previous studies indicate that social vertebrates and invertebrates that are either reared in isolation or exposed to periods of isolation exhibit increases in aggression to conspecifics (Twenge et al., 2001; Wongwitdecha and
Marsden 1996; Alexander, 1961; Johnson et al., 2009, Stevenson and Rillich, 2013). Our behavioral results indicate that socially isolated male stalk-eyed flies increase conflict initiations, fight escalation, and have a higher probability of winning a fight. This result suggests that the absence of socialization in stalk-eyed flies may impair detection of social cues that dampen normal aggressive responses and prevent the escalation of a fight to potentially injurious intensities. The finding that isolated male stalk-eyed flies exhibit higher levels of
63 aggression is similar to isolation effects on Drosophila behavior (Johnson et al., 2009). Unlike males, socially reared and socially-isolated females exhibited similar levels of initiations and escalations in aggressive behavior. This result differs from findings in female Drosophila, which exhibit heightened aggression when reared in isolation (Ueda and Wu, 2009). When we evaluated the expression of 5-HT-like receptors in these flies, there were no differences in either 5-HT1A or 5-HT7 receptors in either sex when compared to socially reared flies. However, the expression of 5-HT2 receptors decreased in males but not females when compared to socially reared controls. These results differ from earlier work in Drosophila which showed that socially- isolated males had decreased expression of 5-HT1A- and increased expression of 5-HT2-like receptor mRNA
(Johnson et al., 2009). These differences may be species specific, but more likely may be related to the isolation protocol employed. In the Drosophila study, the flies were isolated 2 to 3 days post-eclosion and kept in constant light whereas we isolated sexually mature flies for 7 days and used a 12:12 light dark cycle. Our finding that social isolation decreases 5-HT2-like receptor expression and increases aggression raises the possibility that the increase in aggression in socially-isolated male stalk-eyed flies may be linked to decreased serotonergic activation of 5-HT2-like receptors.
In addition to measuring 5-HT-like receptor subtypes, we also evaluated the expression of Tk and
NPFr expression in socially-isolated and socially reared flies. Expression levels of NPFr in males and NPFr and
Tk in females were similar for both rearing conditions, respectively. However, the expression of Tk was markedly increased in socially-isolated males. Recent work in Drosophila has shown that neurons expressing
Tk regulate male-male aggression (Asahina et al., 2014). Thus, the increased sex-specific increase in Tk found in socially-isolated flies likely contributes to the increase in aggression displayed by these insects similar to what is observed in vertebrate species (Katsouni et al., 2009). Furthermore, the expression changes in 5-HT2- like receptors and Tk were restored to socially-reared levels when male flies raised in isolation were returned to the colony and tested 48 hours later.
The finding that social isolation decreased the expression of 5-HT2-receptors, increased Tk expression, and increased aggression led us to test if knockdown of 5-HT2-like receptors would increase aggression and the expression of Tk. We also tested the effects of knocking down the 5-HT2-like receptor on NPFr expression. Our results, which show that males exhibit increased aggression with 5-HT2-like receptor knockdown, suggest that serotonergic activation of the 5-HT2-like receptor inhibits aggression. This result is consistent with earlier work
64 in Drosophila, which showed that treatments with 5-HT2 agonists decreased aggression (Johnson et al., 2009).
Furthermore, our finding that knockdown of 5-HT2-like receptors increased the initiation but not the intensity of aggression suggests that the 5-HT2-like receptors specifically affect the motivation to engage stage of an aggressive conflict. This result appears to be specific to males. When the 5-HT2-like receptor was knocked down in females, behavioral responses were similar to those of vehicle-treated controls. The knockdown of 5-
HT2-like receptors did not affect expression of Tk in either sex. The absence of a change in male Tk expression suggests that the increase in Tk observed in isolated males is independent of the decrease in the expression of the 5-HT2-like receptor subtype. Knockdown of the 5-HT2-like receptor in females did not significantly alter the expression of NPFr in female brains but, in males, NPFr expression was deceased. In Drosophila, NPF decreases aggression (Dierick and Greenspan, 2007) and the decreased expression of NPFr may contribute to the increased aggression exhibited by male stalk-eyed flies following knockdown of the 5-HT2-like receptor. In
Drosophila, work using genetically modified flies suggests that the serotonergic and NPF contributions to aggression function independently (Dierick and Greenspan, 2007).
Since 5-HTP pretreatment increases 5-HT and aggression in male flies (Bubak et al., 2013, 2014b), we next tested the effects of 5-HTP pretreatment on female aggression and on the expression of 5-HT1A-, 5-HT2-,
5-HT7- like receptors, Tk, and NPFr in male and female brains. In both males and females, 5-HTP pretreatment did not significantly affect the expression of the 5-HT-like receptor subtypes tested. In addition, 5-HTP pretreatment did not affect Tk or NPFr expression in females. However, increasing 5-HT by 5-HTP pretreatment in males increased the expression of both Tk and NPFr. The result for NPFr complements the finding that knockdown of 5-HT2-like receptors decreases NPFr and suggests a potential link between 5-HT2- like receptors and the NPF system. However, the increase in NPFr expression following the increase in aggression may seem counterintuitive since NPF is posited to be inhibitory towards aggression. It is important to note that the increase in aggression following 5-HTP treatment in males is behavior specific, resulting in an increase in high-intensity aggressive behaviors and not initiations (Bubak et al., 2013, 2014b). Therefore, it is possible that the high-intensity behavioral increases following 5-HTP treatment is an effect of the increased Tk activity and may be largely independent of NPFr activity.
65 Sex differences in aggression intensities are common in animal species, with males predominantly being the more aggressive sex (Lorenz, 1966). Our results indicate that this is also true for stalk-eyed flies. In these experiments, males consistently exhibited high levels of aggression in response to serotonergic manipulations as well as increased aggression when raised in isolation; treatments which had negligible effects on female aggression. However, it is important to emphasize that female stalk-eyed flies do engage in aggressive conflicts (Bath et al., 2015). It is possible that the forced-fight paradigm over a food source was not a sufficient stimulus to induce intra-female competition. Perhaps engaging in a fight over egg laying sites would provide females with a more powerful incentive to fight. Nevertheless, our results do provide several lines of evidence, which suggest sexually dimorphic differences in brain serotonergic system contribute to the increased aggression found in males. Our results do not allow for isolating which specific aspect of 5-HT function impacted Tk or NPFr. However, it would be valuable to investigate the sex differences in SERT and 5-HT1A- like receptors and the extent to which they contribute to sex-dependent differences in aggression and serotonergic modulation of neuropeptide function in future studies. Activation of 5-HT1A-like receptors has been linked to male aggression in Drosophila (Dierick and Greenspan, 2007, Johnson et al., 2009) and the lower expression of SERT in male stalk-eyed flies suggests that males have slower 5-HT clearance.
Furthermore, in Drosophila, Tk and NPF neurons are sexually dimorphic (Lee et al., 2006, Asahina, et al.,
2014).
It is generally presented that 5-HT has opposing roles in aggression between vertebrates, including humans, and invertebrates with a largely inhibitory role for the former (Brown et al., 1979; Linnoila et al., 1983;
Mann, 1995 and 1999; Ferrari et al., 2005; Summers and Winberg, 2006) and a facilitatory role for the latter
(Livingstone et al., 1980; Edwards and Kravitz, 1997; Huber et al., 1997; Dierick and Greenspan, 2007;
Alekseyenko et al., 2010; Alekseyenko et al., 2014; Bubak et al., 2013, 2014a, 2014b). Here, we show a more nuanced role for 5-HT and the receptor subtypes in aggression in a novel invertebrate species that is sex- dependent as well as conflict stage-dependent. These findings are largely dependent on 5-HT’s interactive role with neuropeptides and under different contexts can be inhibitory, stimulatory, or even absent depending on sex.
Thus, stating opposing roles for 5-HT between vertebrates and invertebrates overlooks the intricate and conserved interactive role this monoaminergic system has with other neurochemical systems known to influence aggressive behavior. Investigating whether these interactions exist and function similarly in a range
66 of animal species undergoing diverse selection pressures will aid in uncovering the origins and, by extension, mechanisms of this complex behavior.
Contributions
I served as the primary author of this manuscript. I designed the experiments, developed the siRNA protocol, collected the data, and wrote the paper. J. Costabile and K. Hoover played a valuable role in the statistical analysis and presentation of the data.
67 CHAPTER V
IS THERE A CONSERVED ROLE FOR 5-HT IN AGGRESSION BETWEEN VERTEBRATES AND
INVERTEBRATES?
Introduction
Aggressive behavior is ubiquitous in both vertebrates and invertebrates and is used to gain access to desirable resources such as territory, food, and access to mates (Edwards and Herberholz, 2005; Summers et al.,
2005). The appropriate perception and execution of aggression from and towards conspecifics is critical for individual fitness and is thus a product of natural selection; as a consequence, efficient behavioral and assessment strategies have evolved in species as diverse as mammals and insects. Across all taxa, aggression is modulated by monoaminergic activity (Alekseyenko et al., 2013; Bubak et al., 2014a; Overli et al., 2007;
Rillich and Stevenson, 2014), with 5-HT playing a key role (Alekseyenko et al., 2014; Alekseyenko et al., 2010;
Bubak et al., 2014a, 2014b; Bubak et al., 2015; Kravitz, 1988; Nelson and Trainor, 2007; Summers et al., 2005;
Takahashi et al., 2012; Takahashi et al., 2011). Serotonin, 5-HT receptor structure and function, and the 5-HT transporter (SERT) are phylogenetically conserved (Blenau and Baumann, 2001; Martin and Krantz, 2014).
However, 5-HT appears to exert opposing roles in the generation of the complex behaviors associated with aggression in invertebrates and vertebrates (see discussion below), which is seemingly counterintuitive. We propose that the gaps in our knowledge of invertebrate aggression makes such a conclusion premature and, while there is much to learn with regard to proximate mechanisms mediating aggression in invertebrates, the serotonergic mechanisms may be more reminiscent of their vertebrate relatives than previously thought.
In most vertebrates, including humans, 5-HT is largely viewed as acting as an inhibitory neuromodulator of aggression (de Almeida et al., 2015; de Boer et al., 2016; de Boer et al., 2015; Summers et al., 2005; Summers et al., 2006). This is revealed by studies showing that genetic or pharmacological reductions in 5-HT are associated with increased aggression (Audero et al., 2013; Caramaschi et al., 2008;
Cervantes and Delville, 2007; Manuck et al., 1999; Mosienko et al., 2012; Perez-Rodrigues et al., 2010; van Erp and Miczek, 2000; Vergnes and Kempf, 1982). Conversely, augmenting 5-HT through either dietary supplementation (Hoglund et al., 2005; Winberg et al., 2001) or reducing SERT function suppresses aggression
(Caldwell and Miczek, 2008; Deckel, 1996; Kohlert et al., 2012; Larson and Summers, 2001; Perreault et al.,
2003). The majority of work on vertebrate aggression has focused on male behavior. However, there is
68 evidence suggesting that, unlike the effects of 5-HT on male aggression in rodents, 5-HT may increase or have minimal effects on aggression in females (Joppa et al., 1997; Terranova et al., 2016; Villalba et al., 1997), but see (Heiming et al., 2013).
In contrast to vertebrates, the majority of empirical studies suggest 5-HT increases aggression in invertebrates. Acute 5-HT injections into the hemolymph of crustaceans induces subordinate males to reengage in confrontations with dominant opponents while decreasing the willingness to retreat (Antonsen and Paul,
1997; Huber et al., 1997; Livingstone et al., 1980; Panksepp et al., 2003), and in some species, increases the probability of winning a fight (Momohara et al., 2013). These effects can also be induced with the blockade of
SERT (Huber et al., 1997; Momohara et al., 2013; Panksepp and Huber, 2002). A similar relationship between
5-HT and aggressive behavior is seen in insects, with pharmacological or genetic manipulations that elevate 5-
HT increasing aggression in Drosophila, stalk-eyed flies, crickets, and ants (Alekseyenko et al., 2010; Bubak et al., 2014b; Bubak et al., 2015; Bubak et al., 2013; Dierick and Greenspan, 2007; Dyakonova and Krushinsky,
2013; Kostowski and Tarchalska, 1972; Szczuka et al., 2013). These studies, in which 5-HT is experimentally increased prior to a conflict, may be consistent with the rapid and transient increase in endogenous 5-HT observed in highly aggressive individuals during vertebrate interactions (de Boer et al., 2015; Matter et al.,
1998; Summers et al., 2005; Takahashi et al., 2012). In contrast to vertebrates, decreasing 5-HT function does not appear to affect aggression in either male crickets or male Drosophila, suggesting that while 5-HT modulates aggressive interactions in a permissive manner, it is not essential to the expression of invertebrate aggression (Dierick and Greenspan, 2007). The role of 5-HT in female invertebrate aggression is largely unknown. Thus, there is a clear need for further empirical studies before conclusions about the activational or sex-specific role of 5-HT in invertebrate aggression can be drawn with confidence.
In vertebrates, progress has been made in understanding how 5-HT modulates aggression through differential binding of specific 5-HT receptors, with 5-HT1A, 5-HT1B, and 5-HT2 subtypes being strongly implicated (Juarez et al., 2013; Popova et al., 2010; Takahashi et al., 2012; Takahashi et al., 2011). However, the contribution of 5-HT receptor subtypes to invertebrate aggression is not as well understood. Of the 7 known
5-HT receptor families in mammals, 3 (5-HT1, 5-HT2, and 5-HT7) have been described with notable sequence and functional homology in insects (Vleugels et al., 2013), with the limited available evidence implicating 5-
HT1-like and 5-HT2-like receptor subtypes in aggression (Johnson et al., 2009). Importantly, specific receptors
69 appear to modulate expression of discrete aggressive behaviors in Drosophila, being enhanced or reduced by activation of 5-HT1A and 5-HT2 receptors, respectively (Johnson et al., 2009). Whether this same pattern of receptor mediation of aggression holds true for other invertebrates is currently unknown. As such, the role of 5-
HT receptor subtypes in regulating specific components of aggressive behavior, beyond a general enhancing or reducing of aggression, cannot be compared between vertebrates and invertebrates. Further, there is a distinct gap in understanding how 5-HT activity contributes to sex-based differences in expression of aggressive behavior in invertebrates. Such knowledge is crucial for understanding not only how individual or sex-specific responses to agonistic interactions can be discretely modulated by 5-HT activity, but also why functional homologies or differences in such a conserved neurotransmitter system would have evolved across vertebrates and invertebrates.
The Stalk-Eyed Fly as a Case Study
Stalk-eyed flies (Diptera; Diopsidae) serve as an exceptional model system for understanding how sexual selection drives the evolution of showy male traits (Baker and Wilkinson 2001; Burkhardt and de la
Motte, 1988; Hingle et al., 2001; Husak et al., 2013; Wilkinson and Reillo, 1994; Worthington et al., 2012;) and communicative behaviors associated with their display. Males and females of all species in the family have eye bulbs displaced on the ends of eyestalks, with significant length variation between and within species. These eyestalks are used extensively as ornamental signals in both intra and intersexual interactions (Wilkinson and
Dodson, 1997; Wilkinson and Johns, 2005). In sexually dimorphic species of stalk-eyed flies, females prefer males with longer eye spans (Wilkinson et al., 1998; Burkhardt and de la Motte, 1988). In addition, males compete amongst each other for access to the limiting resources of food and mates, with larger males typically emerging victorious (Lorch et al., 1993; Panhuis and Wilkinson, 1999, Egge et al., 2011). Consequently, male success is positively correlated with eye span (Burkhardt et al., 1994; Wilkinson and Reillo, 1994; Cotton et al.,
2010).
The sexually dimorphic stalk-eyed fly (Teleopsis dalmanni) provides an ideal model to study aggression, from both a neurophysiological and evolutionary perspective. As described above, males use their elongated eyestalks in aggressive conflicts against rival males. Larger males with broader eye spans typically win these contests and gain access to limiting resources such as food or roosting sites (Burkhardt and de la
70 Motte 1987; Small et al., 2009; Cotton et al 2010; Egge and Swallow 2011; Panhuis and Wilkinson, 1999).
Conflicts follow a stereotypical progression of behaviors that can be broken into 3 distinct stages: initiation, escalation, and termination. A contest typically begins with one individual approaching the other, effectively initiating the fight (de la Motte and Burkhardt, 1983; Panhuis and Wilkinson, 1999, Egge et al., 2011).
Approach is always followed by lining up of eyestalks, which appears to be mutual assessment (Bubak et al.,
2016a; but see Brandt and Swallow, 2009) in order to compare asymmetries in ornament size and fighting ability. This is followed immediately by low-intensity (LI), non-physical posturing behaviors that then escalate to higher intensity (HI), physical contact exchanges. This transition from posturing to potentially injurious behaviors is deemed the escalation stage. Fights are terminated when one rival capitulates and retreats (Egge et al., 2011). Females of this species also engage with conspecifics, however, they do so to a much lower intensity, rarely escalating to HI exchanges (Bath et al., 2015).
We can take advantage of the fact that aggressive interactions in stalk-eyed flies are easily characterized and quantifiable to uncover proximate neurobiological mechanisms governing individual differences in behavioral expression. To investigate these neural mechanisms, we developed a method to detect and quantify biogenic amines (including 5-HT, dopamine (DA), and octopamine (OA)) from the brains of individual flies using high performance liquid chromatography with electrochemical detection (Bubak et al.,
2013). Additionally, we developed a short interfering RNA (siRNA) method to specifically target 5-HT receptor subtypes. Combined, these methods allow us to investigate the effects of global concentrations of 5-
HT itself along with the underlying activational role of the specific 5-HT receptor subtypes, which represents an important advancement because most research investigating 5-HT’s role in aggression in invertebrates has focused primarily on global concentration approaches.
We have previously shown that pharmacologically increasing neural 5-HT through the metabolic precursor, 5-HTP (5-hydroxytryptophan), resulted in a significant increase in HI behaviors in males (Bubak et al., 2014b). This is in line with previous studies demonstrating a positive relationship with global concentrations of 5-HT and aggression in invertebrate species (Antonsen and Paul, 1997; Huber et al., 1997;
Livingstone et al., 1980; Panksepp et al., 2003; Momohara et al., 2013; Dierick and Greenspan, 2007).
However, when we repeated this experiment with females we found no difference in either behavioral output or outcome of a fight. This surprising result turned our attention to investigating the expression profiles of 5-HT
71 receptor subtypes between males and females of this species. As a consequence, we found a markedly lower expression of 5-HT2 in males compared to females at normal, untreated levels. We next hypothesized that 5-
HT2 was inhibitory towards aggression which could then explain, in part, the difference in aggressive behavior seen between sexes of this species.
Social isolation has been shown to increase aggressive behavior in both vertebrates and invertebrates
(Twenge et al., 2001; Wongwitdecha and Marsden 1996; Alexander 1961; Johnson et al., 2009). To test the role of 5-HT2 on aggression in a non-pharmacological or genetic manipulation, we socially isolated males and females, pitted them against conspecifics and measured their receptor expression changes. Males, following social isolation, were significantly more aggressive performing more HI behaviors and initiating more contests than their socially reared opponent. Females, however, maintained normal aggression levels and did not differ in any behaviors compared to their socially reared opponents. By quantifying 5-HT2 expression levels, we found that socially isolated males had a significant reduction in the receptor subtype compared to their socially reared opponents. Females that were socially isolated did not have altered 5-HT2 expression levels. Why males are more responsive, in terms of 5-HT receptor expression changes, compared to females in the absence of social interactions is intriguing. One speculative explanation could be that the presence of females suppresses aggression in males to potentially counteract excessive aggression towards potential mating partners.
To functionally test the role of 5-HT2 on aggression, we selectively knocked down the receptor subtype using siRNA. By reducing 5-HT2 expression by approximately 30% (a similar reduction observed in isolated males), we found that males initiated significantly more fights but performed the same amount of HI behaviors compared to their vehicle treated opponents. This suggests a possible specific role for 5-HT2 in controlling the willingness to engage in a fight whereas escalations to potentially injurious levels are mediated by a separate independent mechanism. Female aggression, again, did not change following successful reduction of 5-HT2.
Interactions between 5-HT and Neuropeptides
5-HT has been demonstrated to modulate a variety of other neurochemical systems, including the neuropeptides tackykinin (Tk; the invertebrate equivalent to substance P) and neuropeptide F (NPF; the invertebrate equivalent to neuropeptide Y), which, by themselves have been linked to aggressive behavior in both vertebrates and invertebrates. For example, activating Tk/substance P, increases aggression across both
72 taxa (Asahina et al., 2014; Baker et al., 1991; Halasz et al., 2009; Katsouni et al., 2009). In contrast, NPY /
NPF act to suppress aggression (Dierick and Greenspan, 2007; Karl et al., 2004). Importantly, both neuropeptides are influenced by 5-HT activity (Guiard et al., 2007; Hennessy et al., 2017; Karl et al., 2004;
Sergeyev et al., 1999). The interactions between the serotonergic system and other neurochemical systems known to influence aggression represents a significant knowledge gap in invertebrate aggression research.
These results make it clear that investigating 5-HT’s role in aggression in isolation will lead to an incomplete understanding of the multilayered regulatory control of this complex behavior. Therefore, to uncover the interacting role of 5-HT with other neurochemical systems, we investigated changes in the two aforementioned neuropeptides following 5-HTP dosing, social isolation, and 5-HT2 siRNA delivery in both males and females.
Following social isolation, we saw an increase in HI behaviors and conflict initiations in males but not females. As mentioned above, social isolation resulted in a significant decrease in 5-HT2 in males only, but we also observed an increase in Tk expression as well. When we compared Tk expression following 5-HTP manipulation, we saw a similar increase; which corresponds with the elevated HI behavioral output. This suggests a possible specific control of HI, escalatory behaviors by Tk and not necessarily other less intense behaviors of an aggressive confrontation, as described in vertebrates. For example, lesioning neurons containing Tk receptors reduced HI attacks but left milder aggressive behaviors unaffected in rats (Halasz et al.,
2009). The increase in HI aggressive behaviors following global manipulations of 5-HT may be a byproduct of its interaction with Tk. Our data suggest the increase in HI aggressive behaviors is more directly a function of increased Tk. It is possible that the earlier studies in other invertebrate species that demonstrated a positive correlation with 5-HT and heightened aggression are observing the same interactive mechanism between 5-HT and Tk. Nonetheless, a direct link between increased 5-HT and Tk activity with aggression can now be made for both invertabrates and vertebrates. Furthermore, there is evidence for 5-HT and Tk corelease from the same neurons in mammals (Chan-Palay et al., 1978). Whether this relationship exists in invertebrates or is sex- dependent is relatively unknown and should be the focus of future studies as this could reveal why we do not observe any changes in females following identical treatments.
The willingness to engage in a fight was significantly altered following administration of 5-HT2 siRNA in males but not females, despite females having similarly reduced expression levels as males. We discovered this may be due to a sex-dependent interactive role of 5-HT2 and the NPF receptor. Following successful
73 reduction of 5-HT2 we also recorded a reduction in NPFr in males but not females. This is in line with NPF’s inhibitory role in aggression observed in other invertebrate and vertebrate species (Dierick and Greenspan,
2007; Karl et al., 2004). This is suggestive again of another stage- and sex-specific regulation of aggression by serotonergic and neuropeptidergic interactions. A schematic illustrating a typical progression of an aggressive encounter for male stalk-eyed flies and the proposed role of 5-HT2, NPFr, Tk, and 5-HT on various stages is shown in Fig. 5.1.
Figure 5.1. A typical fight progression for male stalk-eyed flies and the proposed roles of 5-HT2, NPFr, Tk, and 5-HT.
Perspective on Future Research
The regulation of stage-specific behaviors by independent mechanisms described here emphasize the importance of measuring behaviors during all stages of an aggressive conflict rather than focusing on one or two proxies of aggression (i.e., latency to attack). Using the stalk-eyed fly system, we were able to demonstrate a more nuanced role for 5-HT activity in the regulation of aggression that is more reminiscent of vertebrate species. Altering global levels of the ligand itself does not seem adequate to accurately depict a ‘positive relationship’ between 5-HT and aggression in invertebrate species. Future work should focus on 5-HT receptor subtypes and their interactions with other systems known to influence aggression, such as neuropeptides and other biogenic amines like DA and OA.
Physiological mechanisms mediating aggressive displays and the behavioral outputs are influenced heavily by selective pressures. Sexual dimorphism is likely a consequence of increased levels of sexual selection derived from intrasexual competition over territory, access to mates or intersexual selection favoring exaggerated traits. If elongated eyestalks result in evolutionarily significant changes in behaviors including but
74 not limited to mating systems and aggression, then patterns of correlated evolution should be reflected in the family’s phylogenetic history as it evolved and diversified. Genetic drift, while capable of producing interspecific variation, will not tend to produce patterns of correlated evolution. Interspecific comparisons and correlations, a trade-mark of comparative physiology, allow us to investigate the product of ‘natural experiments (Feder et al., 2000).’ Our understanding of the evolutionary pattern generated by these comparisons would be enhanced by the application of phylogenetically-based methods of statistical analysis (Felsenstein,
1985; Garland, 2001). In the case of stalk-eyed flies, because eye span dimorphism has been gained and lost multiple times in the family (Baker and Wilkinson, 2001), this natural experiment has been, in essence, repeated multiple times. Increased aggression in species with greater sexual dimorphism could have many selective causes and a rigorous comparative analysis should help reveal the mechanistic underpinnings of aggression, including complex interactions between the serotonergic and other neurochemical systems. Comparative transcriptome analyses (RNA-seq) should reveal which genes are commonly up- or downregulated and the complex interactions between genes in monomorphic vs. dimorphic species.
The degree to which behavioral outputs differ between species may depend on the context in which the aggressive confrontation is taking place (i.e., food resource vs mate access), making it difficult to parse out specific mechanisms with whole transcriptome approaches. Therefore, it is critical to follow-up these broad findings with carefully designed behavioral studies directed at aggression under different contexts and at different stages. For example, pitting female opponents in a forced-fight paradigm with food being the incentive may be a less powerful stimulus to provoke aggressive confrontations than would access to egg laying sites. Additionally, behaviors representing the decision to engage an opponent should be distinct from those that represent the willingness to escalate to potentially dangerous intensities or those that represent contest termination (i.e., retreating). In order to get a more fundamental understanding of the origin and control of this complex behavior, it is imperative that research is conducted in a range of different animal species, under several contexts, and in the view of diverse evolutionary pressures.
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