A GENETIC REDUCTION IN ANTIOXIDANT …jeb.biologists.org/content/jexbio/early/2014/12/09/jeb...1 A GENETIC REDUCTION IN ANTIOXIDANT FUNCTION CAUSES ELEVATED AGGRESSION IN MICE2 3 Michael
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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
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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
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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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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