Supplementary Information: Methods
Experimental Design: Immune Gene Assay
6:00 AM
replicate 1
18 C
+0C +6C
26 C
+0C +6C
replicate 2
32 C
+0C +6C
18 C
+0C +6C
26 C
+0C +6C
32 C
+0C +6C
unmanipulated
20
20
20
20
20
20
20
20
20
20
20
20
injury
20
20
20
20
20
20
20
20
20
20
20
20
heat-killed E. coli
20
20
20
20
20
20
20
20
20
20
20
20
6:00 PM
replicate 1
18 C
+0C +6C
26 C
+0C +6C
replicate 2
32 C
+0C +6C
18 C
+0C +6C
26 C
+0C +6C
32 C
+0C +6C
unmanipulated
20
20
20
20
20
20
20
20
20
20
20
20
injury
20
20
20
20
20
20
20
20
20
20
20
20
heat-killed E. coli
20
20
20
20
20
20
20
20
20
20
20
20
We randomly assigned a total of 1440 female, 3-day old mosquitoes to remain either unmanipulated (negative control, n =
480) or to be injected with either 0.2 µL of sterile LB broth (positive control, n = 480) or 0.2 µL heat-killed Escherechia
coli (200,000 bacteria per dose, n = 480). After being anesthetized on ice and challenged, mosquitoes were distributed
into cups (n = 20) and placed into one of 12 reach-in incubators consisting of three temperature treatments (18oC, 26oC,
32oC), two diurnal fluctuation treatments (+0oC and +6oC), and two replicates. This experiment was conducted twice,
starting at 6:00 AM and at 6:00 PM, to evaluate the effects of time of day and any interactions between time of day and
our temperature treatments on mosquito immune gene expression and daily mortality.
Experimental Design: Mosquito Resistance Assay
6:00 AM
replicate 1
18 C
+0C +6C
25
25
26 C
+0C +6C
25
25
replicate 2
32 C
+0C +6C
25
25
18 C
+0C +6C
25
25
26 C
+0C +6C
25
25
32 C
+0C +6C
25
25
6:00 PM
replicate 1
18 C
+0C +6C
25
25
26 C
+0C +6C
25
25
replicate 2
32 C
+0C +6C
25
25
18 C
+0C +6C
25
25
26 C
+0C +6C
25
25
32 C
+0C +6C
25
25
We injected a total of 600 female, 3-day old mosquitoes with 0.2 µL live E. coli (2,000 bacteria per dose). After being
anesthetized on ice and infected, mosquitoes were distributed into cups (n = 25) and placed into one of 12 reach-in
incubators consisting of three temperature treatments (18oC, 26oC, 32oC), two diurnal fluctuation treatments (+0oC and
+6oC), and two replicates. This experiment was conducted starting at 6:00 AM and at 6:00 PM to evaluate the effects of
time of day and any interactions between time of day and our temperature treatments on mosquito resistance to bacterial
growth and daily mortality.
Parton-Logan Curves for the Diurnal Temperature Fluctuation Treatments
The diurnal temperature model:
The Parton-Logan model, characterized by a sinusoidal progression during the daytime and a decreasing exponential
curve during the night is a good representation of both the phase and form of natural diurnal temperature rhythms.
𝑇 = 𝑇𝑚𝑖𝑛 + (𝑇𝑚𝑎𝑥 − 𝑇𝑚𝑖𝑛 )𝑠𝑖𝑛 (𝜋
𝐷
𝑡 − 12 + 2
𝐷 + 2𝑝
) ; 𝑡𝑟𝑖𝑠𝑒 ≤ 𝑡 ≤ 𝑡𝑠𝑒𝑡
−𝑁
−(𝑡 − 𝑡𝑠𝑒𝑡 )
𝑇𝑚𝑖𝑛 − 𝑇𝑠𝑒𝑡 exp ( 𝜏 ) + (𝑇𝑠𝑒𝑡 − 𝑇𝑚𝑖𝑛 )𝑒𝑥𝑝 (
)
𝜏
𝑇=
; 𝑡𝑠𝑒𝑡 ≤ 𝑡 ≤ 𝑡𝑟𝑖𝑠𝑒
−𝑁
1 − 𝑒𝑥𝑝 ( 𝜏 )
where Tmin and Tmax (oC) are the minimum and maximum daily air temperatures, t (hrs) the time, D (hrs) the day length, p
(1.5 hr) the time duration between solar noon and Tmax, trise (hrs) the time of sunrise, tset (hrs) the time of sunset, Tset (oC)
the temperature at sunset, N (hrs) the duration of the night, and τ the nocturnal time constant. The following parameters
were set at these values for each fluctuation around a mean of 18oC, 26oC, and 32oC:
Tmin = 12oC, 20oC, and 26oC
trise = 6 hrs; 6:00 AM
Tmax = 24oC, 32oC, and 38oC
tset = 18 hrs; 6:00 PM
D = 12 hrs
p = 1.5 hrs
N = 12 hrs
τ =4
We then programmed the diurnally fluctuating incubators with temperatures generated from the above model across
various time points in a 24 hr period of time (plots for the program of each incubator are included below).
Incubator Programs:
Actual model
40
Incubator program
35
temperature (oC)
30
25
20
mean ambient temperature
18oC
15
10
5
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Actual model
40
Incubator program
35
temperature (oC)
30
mean ambient temperature
26oC
25
20
15
10
5
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Actual model
40
Incubator program
35
mean ambient temperature
32oC
temperature (oC)
30
25
20
15
10
5
0
0
2
4
6
8 10 12 14 16 18
hours during the day (hrs)
20
22
24
Supplementary Information Results:
Table 1 Final results from generalized linear model analysis of mosquito mortality from the gene expression assay.
Significant effects for each factor are in bold (p < 0.05), and dashes indicate higher order interactions that were eliminated
from the full model.
mosquito mortality (n = 72)
Wald X 2
d.f.
p - value
intercept
12.01
1
0.001
time of day
0.22
1
0.638
temperature
9.46
2
0.009
fluctuation
0.62
1
0.432
immune challenge
14.15
2
0.001
replicate
0.07
1
0.793
time of day x temperature
7.49
2
0.024
factors
Overall, mosquito death was minimal in the gene assay experiment. However, immune challenging mosquitoes in general
did increase the number of dead mosquitoes (unmanipulated vs. injury, p < 0.0001; unmanipulated vs. heat-killed E. coli,
p < 0.0001; injury vs. heat-killed E. coli, p = 0.686). Similar to the mortality observed in the mosquito resistance assay,
more mosquitoes died when they were challenged with heat-killed E. coli in the morning and placed into a warm
environment than mosquitoes challenged in the morning and placed into a cool environment (6:00 AM, 18oC vs. 32oC p =
0.026). Further, the effect of mean ambient temperature on mosquito mortality was no longer significant when mosquitoes
were challenged in the evening (Fig SI 1).
Figure SI 1 Mosquito mortality due to immune challenge was significantly affected by changes in mean ambient
temperature and time of day (6:00 AM, black line; 6:00 PM, red line) challenge was administered.
mean no. dead mosquitoes
1.6
1.4
1.2
1.0
0.8
6:00 AM
0.6
6:00 PM
0.4
0.2
0.0
18 C
26 C
temperature
32 C
(oC)