1
2
Supporting information S2: Analyses exploring predation risk effects on oxidative
damage
3
4
Methods
5
We performed t-tests to test for the effects of predation risk on the different response
6
variables. MDA levels were log-transformed in order to meet the assumptions of normality
7
and equal variances. After transformation all variables were normally distributed (SOD: W =
8
0.97, p = 0.35, CAT: W = 0.97, p = 0.35, superoxide anion: W = 0.95, p = 0.28, log(MDA): W
9
= 0.98, p = 0.39. Furthermore, variances between both treatment groups did not differ (F-ratio
10
tests, SOD: F1, 48 = 1.96, p = 0.11, CAT: F1, 48 = 0.49, p = 0.49, superoxide anion: F1, 48 =
11
0.93, p = 0.34, log(MDA): F1, 48 = 0.67, p = 0.42). As MDA levels did not correlate with fat
12
both in the absence of predation risk (r = 0.31, N = 25, p = 0.13) and in the presence of
13
predation risk (r = 0.26, N = 25, p = 0.21), we did not correct MDA levels for fat content.
14
Similarly, as levels of SOD and CAT did not correlate with protein concentration in the
15
absence of predation risk (SOD: r = -0.29, p = 0.16; CAT: r = 0.21, p = 0.33) and in the
16
presence of predation risk (SOD: r = 0.08, p = 0.70; CAT: r = 0.24, p = 0.24), we did not
17
correct SOD and CAT levels for protein concentration.
18
To explore relationships at the individual level between all response variables we
19
calculated Pearson correlation coefficients separately for control larvae and larvae exposed to
20
predation risk.
21
The t-tests revealed significant effects of predation risk on oxidative damage to lipids
22
(measured as MDA levels) and associated underlying variables, the SOD activity and the
23
superoxide anion concentration (see below). The likely way how SOD affects MDA levels is
24
through shaping the net concentration of the superoxide anion. This is because the net
25
concentration of the superoxide anion at any given moment (hence the level that will cause
1
26
the damage to lipids) is the net balance of its production and its dismutation by SOD [1]. Note
27
that the superoxide anion concentration that we measured in the larvae reflects this net
28
balance at a given moment (hence is not the amount of superoxide anion produced). To
29
explore the relationships at the individual level between SOD activity, the superoxide anion
30
concentration and the MDA levels for the two predation risk groups, we performed three
31
ANCOVAs testing for the covariation patterns (1) between SOD and the superoxide anion,
32
(2) between the superoxide anion and MDA, and (3) between SOD and MDA. Each
33
ANCOVA included predation risk, one covariate (1: SOD activity, 2: superoxide anion
34
concentration, 3: SOD activity) and its interaction with predation risk. When the interaction
35
was not significant it was removed from the final model when testing the significance of the
36
main effect. Note that we did not run this analysis for CAT because the activity of this
37
enzyme did not differ between predation risk treatments (see below).
38
The raw data used in the analysis can be found in the electronic supplementary material S3.
39
Results
40
Larvae exposed to predation risk had higher MDA levels than larvae not exposed to predation
41
risk (t48 = 6.82, p < 0.001). Additionally, they also showed lower SOD activity levels (t 48 = -
42
3.53, p < 0.001) and higher superoxide anion concentrations (t48 = 2.33, p = 0.024), but did
43
not differ in CAT activity (t48 = -0.11, p = 0.91) (see figure 2, main manuscript).
44
45
None of the correlations between the response variables were significant in the control
larvae (all p > 0.13) and in the larvae exposed to predation risk (all p > 0.16) (Table S1).
46
The ANCOVA to explore the relationship at the individual level between the levels of
47
SOD and the superoxide anion concentrations showed significant effect of predation risk (F1,
48
47
49
interaction with predation risk (F1, 46 = 0.071, p = 0.79). This means that for a given SOD
= 4.26, p = 0.045) and no significant effects of the SOD level (F1, 47 < 0.001, p = 0.98) or its
2
50
level (hence a given dismutation rate of the superoxide anion) the larvae reared under
51
predation risk showed a higher net superoxide anion concentration than the control larvae
52
(Figure S1a).
53
The second ANCOVA testing for effects of the superoxide anion, predation risk and
54
their two-way interaction on MDA levels showed a significant effect of predation risk (F1, 47 =
55
48.77, p < 0.001) and no effects of the superoxide anion (F1, 47 = 1.99, p = 0.16) or the
56
predation risk × superoxide anion concentration interaction (F1, 46 = 0.055, p = 0.46) (Figure
57
S1b). For the same concentration of superoxide anion, larvae exposed to predation risk
58
showed more lipid peroxidation than the control larvae.
59
The third ANCOVA testing for effects of SOD, predation risk and their two-way
60
interaction on MDA levels showed a significant effect of predation risk (F1, 47 = 4.67, p <
61
0.001 ) and no effects of SOD (F1, 47 = 3.09, p = 0.081) or the predation risk × SOD
62
interaction (F1, 46 = 0.0036, p = 0.95) (Figure S1c). This suggests that MDA levels remained
63
constant across a range of individual SOD defence levels within each treatment group.
64
Discussion
65
At the individual level there does not seem to be a link between SOD, the superoxide anion
66
and lipid peroxidation suggesting that an organism’s response to oxidative stress is complex.
67
We see three non-exclusive explanations for the absence of covariation between the activity
68
of SOD, and the superoxide anion and MDA concentrations:
69
1) This can be the result of individual fine-scale adjustment, meaning that each
70
individual larva adjusted its level of defence in order to maintain a certain fixed level of the
71
superoxide anion and MDA. The absence of a link between SOD and superoxide anion
72
concentrations may indicate that within each treatment group (control and predation risk)
73
larvae tried to keep the superoxide anion concentration at a certain fixed level irrespective of
3
74
their SOD defence level, hence larvae individually adjusted their SOD activity level to not
75
exceed a certain net superoxide anion level. The observation that larvae exposed to predation
76
risk were not able to keep superoxide anion concentrations as low as the control animals may
77
indicate that they did not further increase their SOD levels to balance the higher superoxide
78
anion production. This is likely because energetic constraints due to a shunting of energy to
79
the fight-or-flight response, precluded a further increase of energetically costly [4-5] SOD
80
activity. The absence of an effect of the net concentration of the superoxide anion on MDA in
81
both treatment groups may indicate that within each treatment group larvae tried to keep the
82
MDA level at a certain level irrespective of the level of this potent ROS, hence larvae
83
adjusted their defence and repair mechanisms to not exceed a certain MDA level.
84
2) This may indicate that, in addition to SOD, larvae also used other mechanisms to
85
defend themselves against the radicals and prevent damage. Candidate mechanisms are the
86
use of scavenger molecules (such as glutathione) and enzymes which repair oxidative damage
87
to lipids [6]. For the same concentration of superoxide anion, larvae exposed to predation risk
88
showed more lipid peroxidation than the control larvae. This may indicate that larvae relied
89
on other mechanisms (in addition to SOD) to reduce MDA levels and that larvae under
90
predation risk invested less in these other mechanisms, again probably because they were
91
more energy-limited due to their investment in the fight-or-flight response.
92
3) The absence of covariation patterns at the individual level after 7 days of exposure
93
may be due to the transient nature of the antioxidant response. Possibly, the damselfly larvae
94
initially responded to predation risk by increasing their antioxidant defence to prevent
95
accumulation of ROS. Due to energetic constraints, the larvae may not have been able to
96
maintain this high enzymatic activity when they were exposed to the predation risk for a
97
longer period, resulting in a decrease in SOD activity. This finally may have resulted in higher
98
MDA levels under predation risk. Such transient responses in SOD activity have been
4
99
observed in response to pesticides. For example, Zhang et al. [7] reported an increase in SOD
100
activity after 3 and 7 days of exposure to the herbicide fomesafen, yet a decrease in SOD
101
activity after 14 days of exposure.
102
103
References
104
1 Monaghan, P., Metcalfe, N.B. & Torres, R. 2009 Oxidative stress as a mediator of life
105
106
107
108
history trade-offs: mechanisms, measurements and interpretation. Ecol. Lett. 12, 75-92.
2 Slos, S. & Stoks, R. 2008 Predation risk induces stress proteins and reduces antioxidant
defense. Funct. Ecol. 22, 637-642.
3 Burton, T., Killen, S.S., Armstrong, J.D. & Metcalfe, N.B. 2011 What causes intraspecific
109
variation in resting metabolic rate and what are its ecological consequences? Proc. R. Soc.
110
B 278, 3465-3473.
111
112
113
114
115
116
4 De Block, M. & Stoks, R. 2008 Compensatory growth and oxidative stress in a damselfly.
Proc. R. Soc. B 275, 781-785.
5 Slos, S., De Meester, L. & Stoks, R. 2009 Food level and sex shape predator-induced
physiological stress: immune defence and antioxidant defence. Oecologia 161, 461-467.
6 Korsloot, A., van Gestel, C.A.M. & van Straalen, N.M. 2004 Environmental stress and
cellular response in arthropods. Boca Raton: CRC.
117
7 Zhang, Q., Zhu, L., Wang, J., Xie, H., Wang, J., Han, Y. & Yang, J. 2013 Oxidative stress
118
an lipid peroxidation in the earthworm Eisenia fetida induced by low doses of fomesafen.
119
Environ. Sci. Pollut. Res. 20, 201-208.
5
120
Table S1: Pearson correlations between growth rate, MDA level, activities of SOD and of
121
CAT and superoxide anion concentrations. Correlations above the diagonal are those of the
122
control larvae, correlations below the diagonal are those of the larvae exposed to predation
123
risk. MDA levels were log-transformed. The sample size is 25 for every correlation.
growth
growth
MDA
SOD
CAT
O2-
r = -0.21
p = 0.30
r = -0.031
p = 0.88
r = -0.36
p = 0.36
r = 0.19
p =0.37
MDA
r = 0.16
p = 0.43
r = 0.28
p = 0.17
r = 0.088
p = 0.68
r = -0.28
p = 0.17
SOD
r = 0.15
p = 0.48
r = 0.17
p = 0.41
r = -0.21
p = 0.30
r = -0.18
p = 0.93
124
125
6
CAT
r = 0.084
p = 0.69
r = 0.16
p = 0.43
r = -0.30
p = 0.14
r = 0.073
p = 0.73
O2r = 0.23
p = 0.27
r = -0.05
p = 0.81
r = 0.06
p = 0.76
r = 0.026
p = 0.90
(a) 1.0
Predation risk absent
Predation risk present
Superoxide anion (M)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
1.2
(b)
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
SOD (units)
1.5
Log MDA (log nmol/ml)
Predation risk absent
Predation risk present
1.0
0.5
0.0
-0.5
-1.0
-1.5
0.3
(c)
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Superoxide anion (M)
1.5
Log MDA (log nmol/ml)
Predation risk absent
Predation risk present
1.0
0.5
0.0
-0.5
-1.0
-1.5
1.2
126
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
SOD (units)
127
Figure S1: Relationships at the individual level between (a) SOD activity and superoxide
128
anion levels, (b) superoxide anion levels and MDA levels, and (c) SOD activity and MDA
129
levels. Striped lines represent the relationship for control larvae, solid lines represent the
130
relationship for larvae exposed to predation risk.
7