© 2014. Published by The Company of Biologists Ltd

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A GENETIC REDUCTION IN ANTIOXIDANT FUNCTION CAUSES 1

ELEVATED AGGRESSION IN MICE 2

Michael Garratt1,2

& Robert C Brooks1 3

4

1Evolution & Ecology Research Centre and School of Biological, Earth and Environmental 5

Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australia. 6

2 Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, 7

United States. 8

9

Corresponding author: Michael.Garratt@unsw.edu.au 10

11

SUMMARY 12

Male-male aggression can have a large influence on access to mates, particularly in highly 13

territorial animals such as mice. It has been suggested that males with impaired antioxidant 14

defence and a consequential increased susceptibility to oxidative stress may have a reduced 15

ability to invest in aggressive behaviours, which could limit their mating opportunities and 16

reproductive success. Oxidative stress occurs as a result of an uncontrolled over-production 17

of reactive oxygen species (ROS) in relation to defence mechanisms (such as antioxidants), 18

and can cause damage to a variety of different cellular components. Impairments in specific 19

aspects of antioxidant defence, leading to oxidative stress, can limit investment in some 20

reproductive traits in males, such as sperm quality and the production of sexual signals to 21

attract mates. However, a direct effect of impaired antioxidant defence on aggressive 22

behaviour has not, to our knowledge, been reported. In this study we demonstrate that mice 23

with experimentally elevated sensitivity to oxidative stress (through inhibition of copper-zinc 24

superoxide dismutase (Sod1)) actually show the opposite response to previous predictions. 25

Males completely deficient in Sod1 are more aggressive than both wild-type males and males 26

that express 50% of this antioxidant enzyme. They are also faster to attack another male. The 27

cause of this increased aggression is unknown, but this result highlights that aggressive 28

http://jeb.biologists.org/lookup/doi/10.1242/jeb.112011Access the most recent version at J Exp Biol Advance Online Articles. First posted online on 18 December 2014 as doi:10.1242/jeb.112011http://jeb.biologists.org/lookup/doi/10.1242/jeb.112011Access the most recent version at

First posted online on 18 December 2014 as 10.1242/jeb.112011

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behaviour in mice is not highly constrained by inhibited Sod1 expression, in contrast to other 29

reproductive traits known to be impaired in this mouse model. 30

Key words: antioxidant, fitness, dominance, mouse 31

INTRODUCTION 32

An organism’s ability to survive and reproduce is governed by a range of environmental and 33

physiological conditions (McNamara and Houston, 1996). One potentially important 34

physiological factor is oxidative stress (Bize et al., 2008; Metcalfe and Alonso-Alvarez, 35

2010; Monaghan et al., 2009), a process that can cause damage to proteins, lipids and DNA 36

as a result of uncontrolled production of reactive oxygen species (ROS) (Halliwell and 37

Gutteridge, 1999). ROS are produced through normal metabolic processes and organisms 38

have a variety of defence systems, such as various antioxidants, that protect against ROS 39

induced damage (Valko et al., 2007). However, under certain conditions defences can be 40

overwhelmed and oxidative damage can result (Halliwell and Whiteman, 2004). Oxidative 41

stress may play a major role in the ageing process (Beckman and Ames, 1998; Harman, 42

1956), can contribute to the onset of degenerative diseases (Droge, 2002) and may limit 43

investment in life history traits (Monaghan et al., 2009). 44

Oxidative stress impairs some aspects of male reproduction, particularly sperm 45

quality (Agarwal et al., 2003; Saleh and Agarwal, 2002; Sikka et al., 1995), and, in some 46

species, investment in aspects of sexual signalling used to attract mates (Garratt and Brooks, 47

2012; Metcalfe and Alonso-Alvarez, 2010). It has also been suggested that oxidative stress 48

may limit a male’s ability to invest fully in aggressive behaviours, and thus attain dominance 49

and gain access to mates (Garratt and Brooks, 2012; Metcalfe and Alonso-Alvarez, 2010). 50

Because engaging in aggressive behaviours can elevate metabolic rate (Briffa and Sneddon, 51

2007; DeCarvalho et al., 2004; Haller, 1995; Smith and Taylor, 1993), Metcalfe & Alonso-52

Alvarez (2010) predicted that males with good antioxidant defences may be able to sustain 53

the highest levels of aggression. Garratt & Brooks (2012) offered similar suggestions, and 54

further highlighted that oxidative stress can impair bioenergetic function, which may reduce 55

energy allocation to metabolically demanding reproductive behaviours. 56

In this study we manipulate antioxidant defence through molecular genetic 57

techniques, to provide the first direct test of whether susceptibility to oxidative stress 58

influences an individual male’s ability/willingness to invest in aggressive behaviours, and 59

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attain dominance over another male. We used a genetically modified strain of mouse that 60

does not express copper-zinc superoxide dismutase (Sod1), an antioxidant that has an 61

important in vivo role in protecting against oxidative stress. We examined aggressive 62

behaviours in homozygous fully deficient Sod1 males (Sod1 -/-), heterozygous deficient Sod1 63

males (Sod1 +/-; with 50% Sod1 enzymatic activity) and wild-type (Sod1 +/+) males. Sod1 -64

/- males have been most frequently studied. When housed in standard laboratory conditions 65

they show an elevation of oxidative damage in various tissues throughout adult life (Elchuri 66

et al., 2005; Muller et al., 2006) and have a shorter lifespan (Elchuri et al., 2005). They have 67

reduced sperm motility (Garratt et al., 2013; Tsunoda et al., 2012) and are subfertile when 68

mated with a least one different strain of female (Garratt et al., 2013). Males of this strain are 69

also less able to invest in some molecular and morphological components of olfactory sexual 70

signalling known to be important in attraction of mates (Garratt et al., 2014). Less is known 71

about the phenotype of Sod1 +/- males. No differences in oxidative damage or lifespan 72

(Elchuri et al., 2005) have been detected in these animals in comparison to wild-type males. 73

However, cells from Sod1 +/- mice have a slightly increased sensitivity to paraquat, a 74

molecule that increases production of free radicals, and these mice also suffer lower motor 75

neuron survival after induced injury (Reaume et al., 1996). 76

In mice acts of overt aggression, such as trying to bite, chase and even kill competitor 77

individuals, help males to attain dominance over other individuals and monopolise matings 78

within a territory area (Bronson, 1979). To explore the degree to which aggression is 79

sensitive to impaired antioxidant defence, we used previously published, standard protocol 80

for assessing aggression and the ability to attain dominance in mice (Jones and Nowell, 1974; 81

Jones and Nowell, 1989). This involved pairing two mice in a divided cage over three weeks, 82

and allowing them to interact aggressively regularly over this period (see methods). To 83

minimise animal numbers and maximise the comparisons we could make between genotypes, 84

Sod1 +/+ and Sod1 -/- males were each paired with Sod1 +/- males. We could then determine 85

whether Sod1 +/- males were more likely to win or lose fights with either of the other 86

genotypes. Because Sod1 -/- and Sod1 +/+ males were both paired with the same genotype of 87

male (Sod1 +/-), we could also compare the aggression of these two genotypes of males 88

against a standardised, equivalent competitor male. 89

90

RESULTS 91

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Pairs of Sod1 +/+ and Sod1 +/- males were allowed to interact eight times over three weeks. 92

Over this period, Sod1 +/+ and Sod1 +/- males initiated a similar number of aggressive 93

behaviours (attacks, chases or attempts to bite the other male) (2

= 0.15, 1 d.f., p = 0.70; 94

Figure 1a). Both genotypes of male were also just as likely to initiate the first aggressive 95

behaviour in a given experimental interaction (2

= <0.1, 1 d.f., p = 0.96; Figure 2a). The 96

number of aggressive behaviours exhibited decreased over the successive trials (2

= 13.43, 1 97

d.f., p = 0.0002; Figure 1a), to a similar extent in both genotypes (interaction between 98

genotype and trial number: 2

= 0.07, 1 d.f., p = 0.79). 99

When 10 Sod1 -/- males were allowed to interact with paired Sod1 +/- males over three 100

weeks, Sod1 -/- males initiated a greater number of aggressive behaviours in comparison to 101

Sod1 +/- males (2

= 6.96, 1 d.f., p = 0.0083; Figure 1b). This effect was consistent over the 102

three week duration of the experiment, with both genotypes of male increasing their levels of 103

aggression over time (effect of trial number: 2

= 5.66, 1 d.f., p = 0.017; interaction between 104

genotype and trial number: 2

= 0.01, 1 d.f., p = 0.89; Figure 1b). Sod1 -/- males were also 105

much more likely to be the first to express an aggressive behaviour in a given experimental 106

trial (effect of genotype: 2

= 9.45, 1 d.f., p = 0.002; Figure 2b). 107

Because Sod1 -/- and Sod1 +/+ males were paired with equivalent Sod1 +/- males over the 108

same time period, in identical conditions, we also compared the aggressive behaviour shown 109

by Sod1 -/- and Sod1 +/+ males in their respective trials. When paired with Sod1 +/- males, 110

Sod1 -/- males expressed a significantly greater number of aggressive behaviours than Sod1 111

+/+ (2

= 9.62, 1 d.f., p < 0.002; Figures 1a and 1b), an effect that became progressively 112

stronger over successive experimental interactions (interaction between genotype and time: 113

2

= 22.54, 1 d.f., p < 0.0001). Sod1 -/- males were also much more likely to express the first 114

aggressive behaviour in their experimental interactions than Sod1 +/+ (2

= 8.73, 1 d.f., p < 115

0.003; Figures 2a and 2b). 116

117

DISCUSSION 118

Our results reveal that males with experimentally impaired antioxidant defence (Sod1 -/-), 119

through complete SOD1 deficiency, are more aggressive than both heterozygous knockout 120

males (Sod1 +/-), which are 50% deficient in SOD1, and wild-type (Sod1 +/+) males. These 121

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results do not support the prediction that an increased susceptibility to oxidative stress 122

reduces a male’s ability to invest in aggressive behaviours, although we cannot exclude the 123

possibility that constraints on fighting ability might be revealed if males are allowed to fight 124

more frequently, or over a longer duration. However, in this experiment males were required 125

to fight with another male eight times over a three week period and there was no evidence 126

that the aggressive Sod1 -/- phenotype abated: on the contrary, Sod1 -/- males continued to 127

become more aggressive, while wild-type males instead reduced their aggressive behavior 128

over progressive experimental trials. 129

Although aggressive behavior is not negatively affected by Sod1 -/- deficiency, at 130

least not in a noticeable manner here, other aspects of reproduction are known to be impaired 131

in this mouse model. Female fertility in Sod1-/- mice is markedly reduced, with females only 132

producing a couple of small sized litters when allowed to breed over three to six months (Ho 133

et al., 1998; Matzuk et al., 1998). Sperm motility and fertilization success are reduced in 134

Sod1 -/- male mice under certain conditions (Garratt et al., 2013; Tsunoda et al., 2012). 135

Particular molecular (MUPs) and morphological (preputial glands) aspects of sexual 136

signalling are also depressed in Sod1 -/- males (Garratt et al., 2014). In contrast to these 137

declines in physiological aspects of reproduction, behavioural investment in scent marking is 138

not noticeably affected by Sod1 deficiency (Garratt et al., 2014), and investment in 139

aggressive behaviour actually seems to increase. There is, therefore, substantial variability in 140

the degree to which different aspects of reproduction are affected by oxidative stress. We 141

speculate that investment in behavioural aspects of reproduction may be less physiologically 142

constrained by oxidative stress, allowing males to increase their investment in such traits 143

more freely than in molecular and morphological aspects of reproductive investment. 144

Although behavioural traits require energy they do not require the synthesis and maintenance 145

of specific molecular components that could be susceptible to oxidative damage (over that 146

required for muscular function). Other reproductive traits, such as sperm production, require 147

efficient generation and maintenance of cellular components that are easily damaged by 148

uncontrolled production of ROS. The degree to which molecular and cellular components 149

involved in a specific aspect of reproduction are susceptible to oxidative damage might 150

influence whether that aspect of reproductive allocation is negatively impacted by oxidative 151

stress. 152

Instead of the predicted decrease in aggression in Sod1 -/- mice, we found that these 153

males exhibit a greater number of attacks and are quicker to attack their opponent in staged 154

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trials. The increased aggression with oxidative stress we report was unexpected, but several 155

previous studies have provided evidence that male aggression can correlate with a marker of 156

oxidative stress or antioxidant defence (measured after the assessment of aggression). In a 157

reptile species, White’s skinks (Egernia whitii), male aggression is positively correlated with 158

a marker of oxidative damage in plasma (Isaksson et al., 2011). In laboratory mice, Rammal 159

et al. (2010) demonstrated a negative correlation between latency to attack an intruder and 160

intra-cellular redox status (with a higher status expected to signify greater oxidative stress) of 161

peripheral blood granuloctyes. A recent study in humans has also revealed a positive 162

correlation between a patient’s history of aggression and two markers of oxidative stress in 163

plasma (Coccaro et al., 2014). These studies have been interpreted in various different ways, 164

with oxidative stress being suggested as either a cause (Coccaro et al., 2014) or consequence 165

(Isaksson et al., 2011; Rammal et al., 2010) of differences in aggressive behaviour. Our study 166

provides the first experimental evidence that oxidative stress can directly alter investment in 167

aggressive behaviours, although the underlying cause for increased aggression with oxidative 168

stress requires further investigation. 169

Investment in aggression is influenced by both intrinsic and extrinsic factors. We 170

think that it is unlikely that these males have some elevated physiological function (e.g. 171

intrinsic factor) that increases the capacity for aggressive interactions, particularly as Sod1-/- 172

males show impairments in muscle mass and function that increase with age (Muller et al., 173

2006; Vasilaki et al., 2010). It is possible, however, that impaired Sod1 -/- expression causes 174

some pathophysiological change, in the brain, for example, that increases the expression of 175

aggressive behaviours, or willingness to fight. In this scenario the increased aggression in 176

Sod1-/- males is not an adaptive response, rather a negative consequence of oxidative 177

damage. This could include any hypothetical damage to known pathways directly associated 178

with aggressive behaviour, such serotonin, dopamine or glutamate pathways (Coccaro et al., 179

2014). Oxidative damage in the brain could affect aggression indirectly, by influencing 180

emotional status in some manner, such as by altering anxiety, which has been linked both 181

positively (Konig et al., 1996) and negatively (Nyberg et al., 2003) to offensive aggression. 182

Experimental manipulations that alter oxidative stress have previously been linked to changes 183

in the expression of anxiety-linked behaviours (Berry and Cirulli, 2013; Hovatta et al., 2005), 184

generally suggesting that oxidative stress increases anxiety (Bouayed et al., 2009). As a 185

consequence, an effect of oxidative stress on anxiety, although not previously noted in this 186

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mouse model, offers one plausible pathway through which this aspect of physiology and 187

aggressive behaviour are linked. 188

Extrinsic factors that influence aggression include winner and loser effects, and 189

territory possession (which are controlled for in this experiment), in addition to other factors 190

that increase motivation to fight (Dugatkin, 1997). It is interesting to note that previous 191

experiments have observed increased aggression levels with manipulations that would be 192

expected to reduce male physiological function. Male field crickets (Gryllus integer) (Pölkki 193

et al., 2013) with an experimentally-activated immune response show heightened aggression 194

and dominance, which the authors suggest might be a plastic response to the survival threat 195

presented by the immune challenge: a form of “terminal investment” in reproduction, which 196

has also been observed in relation to other components of reproductive behaviour (Candolin, 197

2000; Clutton-Brock, 1984). In white-footed mice (Peromyscus leucopus), wild caught males 198

that are parasitized with bot flies (Cuterebra fontinella) are more aggressive in staged trials in 199

the laboratory than non-parasited males, another example of increased aggression with 200

reduced survival prospects (Cramer and Cameron, 2007). 201

As Sod1-/- male mice have repeatedly been shown to have a lifespan that is shorter by 202

about 30 % (Elchuri et al., 2005; Perez et al., 2009; Zhang et al., 2013), increased aggressive 203

behaviour by Sod1 -/- males could possibly also reflect a terminal investment strategy, with 204

males investing more in aggressive behaviour in an effort to increase their immediate 205

reproductive success. Another possibility is that Sod1-/- males show elevated levels of 206

aggression because they are compensating for their reduced ability to invest in olfactory 207

signalling. When male mice win fights and become dominant their investment in olfactory 208

signalling changes, with particular volatile molecules increasing in urinary concentration 209

(Novotny et al., 1990). Several of these volatiles are produced in the preputial glands, which 210

have been found to be smaller in Sod1 -/- males when housed in a competitive environment 211

(Garratt et al., 2014). It is feasible that increased aggression by Sod1 -/- males partially 212

compensates for a reduced ability to produce volatile molecules that signal their dominance 213

to male con-specifics. 214

Our result of increased aggression in Sod1 -/- males adds to a complex picture of how 215

increased susceptibility to oxidative stress influences male reproductive effort. Further 216

studies in additional mouse models and other taxa may help to adjudicate the generality of the 217

result we reveal. There are range of different mouse models with genetically impaired 218

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antioxidant defence and varying degrees of associated pathophysiology (Perez et al., 2009); 219

examination of sexual signalling and aggression in these mice may reveal the general 220

sensitivity of these reproductive traits to perturbations in redox status. Conditional knockouts 221

of antioxidant defence, where the expression of a particular gene in that defence process can 222

be manipulated over a specific period of an animal’s life cycle, may be particularly helpful in 223

determining the impact of oxidative stress on these traits during a specific period of 224

adulthood (Hamilton et al., 2012). Ultimately, however, direct links between oxidative stress 225

and aggression need to be tested for in organisms other than biomedical models, as these 226

model animals show alterations in their behaviour and life history due to selective breeding in 227

laboratory conditions. Further exploration of the direct effects of oxidative stress on 228

aggression in a more diverse range of species, perhaps through manipulation of ROS 229

production (genetic manipulation of antioxidant defence is only available in model 230

organisms), may help to confirm whether oxidative stress has a sufficient impact on 231

aggression in wild animals that it impacts their ability to attain dominance and mating 232

success. 233

234

235

236

MATERIALS AND METHODS 237

Subjects 238

The Sod1 line of mice were maintained on a C57BL/6 background. The generation of this 239

knockout strain (Kostrominova et al., 2007; Muller et al., 2006) and details of our breeding 240

colony (Garratt et al., 2013) have been reported previously. Briefly, the line of Sod1 mice 241

used in these experiments was derived from three pairs of Sod1+/- mice imported from the 242

Jackson Laboratories (Bar Harbor, ME, USA) and used to create a SPF breeding colony at 243

the Australian BioResource Centre in Mossvale, NSW, Australia. Experimental mice were 244

the progeny of matings between Sod1 +/- pairs; the Sod1 genotypes of offspring were 245

determined by genotyping a small sample of ear tissue collected at weaning. Genotyping was 246

conducted by the mouse genotyping service at the Australian Cancer Research Foundation 247

(ACRF), the Garvan Institute, using a combination of real-time PCR and melting curve 248

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analysis. When 6–8 weeks old, experimental mice were transported and housed in 249

conventional facilities at the University of New South Wales (UNSW). The mice were 250

maintained at 22oC under a 12 h light:12 h darkness cycle. All experimental procedures and 251

aggression trials were conducted in the dark period under dim red light. Two weeks prior to 252

the experiment, males were housed singly in cages (53 x 35 x 18 cm) and were regularly 253

exposed to the odour and presence of males and females of the CBA strain (CBA/CaHAusb) 254

to ensure the development of normal reproductive behaviour. Food (stock feed from 255

Gordon’s Specialty Stockfeeds, Yanderra, NSW, Australia) and water were provided ad 256

libitum. All experimental procedures were approved by the UNSW animal ethics committee 257

(approval number: 12/30A). 258

259 260

Aggressive interactions 261

262

We created 11 pairs of Sod1 +/+ males and Sod1 +/- males. At the same time a second set of 263

paired males consisted of 10 Sod1 -/- and Sod1 +/- males. One male in each pair was marked 264

with a fur clip on the back to allow individual identification of the males during aggressive 265

interactions. The genotype of the male that was marked was randomised, and the 266

experimenter that recorded the aggressive interactions was unaware of each male’s genotype 267

(blind to both the genotypes of the males in the cages and which cages contained Sod1 -/- and 268

Sod1 +/+ males), ensuring unbiased assessment of aggressive behaviour. 269

After two weeks of single housing, each pair of males were housed in cages (53 x 35 x 18 270

cm) divided by a perforated plastic barrier, with a male on either side; this barrier allowed 271

continuous visual and olfactory contact between the males but did not permit direct physical 272

contact between the pairs. Males were housed in these conditions for three weeks, and over 273

this period males were allowed to interact aggressively eight times. There was at least one 274

rest day (e.g. the males did not fight) between each aggressive interaction. 275

On each day of aggressive interactions, the barrier between males was removed. Males were 276

allowed to interact directly for a 15 minute period or until 10 aggressive interactions between 277

the males had occurred, whichever was first. The trial was then stopped and the barrier and 278

bedding returned to the cage. During the trials an experimenter was always present to split up 279

any fights that were persistent (aggressive interactions that continued for more than 10 280

seconds) or involved obvious biting, following previous protocols (Garratt et al., 2012). This 281

ensured none of the males were injured during the experiment. This experimenter also 282

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documented the time until each male initiated an aggressive behaviour and the number of 283

aggressive behaviours initiated and received by each male. An aggressive behaviour was 284

defined when a male attempted or succeeded in biting, chasing or kicking the other male. 285

Separate aggressive interactions were recorded when there was a period of three seconds 286

between any of these behaviours. If one mouse was aggressive towards the other, and the 287

attack was broken up by the experimenter, then the mouse immediately initiated another 288

aggressive interaction, this was counted as two separate events. As 10 aggressive interactions 289

were permitted before the trial was terminated, the maximum number of aggressive 290

interactions each male could initiate or receive was 10. If males mutually initiated an 291

aggressive behaviour, both males were considered to have initiated and neither male was 292

considered to have received an aggressive behaviour. 293

294

Data analysis 295

To test for differences between genotypes in the expression of aggressive behaviours, we 296

constructed Generalised Linear Mixed Effect Models using the lme4 package in R. Models 297

that explored the number of aggressive behaviours expressed by each male were fitted with a 298

Poisson distribution. Models that examined which male was first to initiate an aggressive 299

behaviour were fitted with a binomial distribution, with males initiating the first behaviour 300

scored as “1” and those that did not scored as “0” (if both males initiated a behaviour at the 301

same time they both received “1” and if neither attacked both received “0”). Male genotype 302

and trial number were added as fixed effects. Male ID and pair were added as random effects, 303

to control for repeated assessment of male behaviours and the non-independence between 304

individuals in each pair. The significance of the genotype and trial number effect, and 305

interaction between the two, were tested by comparing models with and without a particular 306

term using a log-likelihood ratio test. 307

308

ACKNOWLEDGEMENTS 309

This project was approved by the UNSW animal ethics committee. We thank BRC animal 310

house staff for assistance with animal maintenance. 311

312

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AUTHOR CONTRIBUTIONS 313

M.G. and R.C.B. conceived the study. M.G. conducted the study and wrote the manuscript. 314

315

COMPETING INTERESTS 316

No competing interests declared. 317

318

FUNDING 319

This study was funded by an Australian Research Council (ARC) Discovery Grant awarded 320

to R.C.B. 321

322

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306. 454

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Figure Legends 457

Figure 1. The number of aggressive interactions initiated by males in each experimental trial. 458

(a) Aggressive behaviours initiated by Sod1+/+ males and Sod1 +/- males that were paired 459

together. (b) Aggressive behaviours initiated by Sod1-/- males and Sod1 +/- males that were 460

paired together. Trial numbers are successive, with trial one being the first trial conducted 461

and trial eight being the last at the end of three weeks. The maximum number of aggressive 462

interactions males could initiate in each trial was 10. 463

Figure 2. The proportion of males that attacked first for each genotype in each experimental 464

interaction. (a) Sod1+/+ males and Sod1 +/- males that attacked in each trial. (b) Proportion 465

of Sod1-/- males and Sod1 +/- males that attacked in each trial. Note that the values for both 466

genotypes in each trial do not always add up to one because in some trials neither male 467

initiated an aggressive interaction, while in others both males mutually initiated an aggressive 468

interaction at the same time. 469

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Figure 1 473

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475 Figure 2 476