1
Greenhouse lid design description:
2
Greenhouse heating lids were constructed from 1 mm thick Sun-Lite Fiberglass
3
(Solar Components Corp., Manchester, NH) and covered with 1 mm mesh on the open
4
bottom to prevent colonization by unwanted invertebrates (Fig. 1a). We modified the
5
design described in Netten et al. (2008) to fit an oblong-shaped tank. To build the lids,
6
we first constructed a wooden frame consisting of two equilateral triangles (80 cm per
7
side) connected by a 135 cm wooden beam at the tip of each triangle. Next, we used tin
8
snips to cut the fiberglass into triangles and rectangles to fit over each side of the frame,
9
attaching the fiberglass with wood screws. We staked the lids to the ground with
10
clothesline that stretched over the top of the lids to prevent them from blowing away in
11
high winds. We recorded the temperature every three hours in all mesocosms using
12
underwater dataloggers (Onset Computer Corporation, Bourne, MA) that were suspended
13
10 cm from the bottom of each mesocosm.
14
15
16
Experimental design pilot study:
To ensure that our experimental manipulation was not systematically altering the
17
abiotic properties and non-host aquatic community in heated compared to unheated
18
mesocosms, we compared two mesocosms with shade-cloth lids to two with nonheating
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“sham” greenhouse lids. These lids were constructed by cutting vent flaps into the sides
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of the warming lids to eliminate the warming effect. Temperature and light were
21
measured as in the full experiment with a suspended Hobo underwater datalogger. We
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used a handheld 556MPS YSI multiprobe (YSI International, Yellow Springs, Ohio) to
23
measure pH, dissolved oxygen, total dissolved solids, conductivity and salinity at two
24
week intervals for this pilot experiment. We also measured periphyton growth at the end
25
of the month as described for the full experiment.
26
To determine whether lid type (nonheating sham vs. shade cloth) influenced
27
baseline abiotic and biotic variables, we used t-tests for variables with one measure per
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mesocosm (temperature, light, fluorescence and periphyton), and lme models for
29
variables with measurements at two time-points (pH, dissolved oxygen, total dissolved
30
solids, conductivity and salinity).
31
There was no difference in any of the abiotic or biotic variables when tanks with
32
nonheating “sham” greenhouse lids were compared to tanks with shade cloth lids.
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Neither temperature (t = 0.69, P = 0.56) nor light (t = 0.28, P = 0.80) differed between
34
these treatments, nor did pH (t = 0.10, P = 0.93), dissolved oxygen (t = -0.61, P = 0.58),
35
total dissolved solids (t = -0.31, P = 0.78), conductivity (t = -0.31, P = 0.78), or salinity
36
(t = -0.78, P = 0.50). Similarly, there was no difference in periphyton biomass (t = -0.23,
37
P = 0.84) or phytoplankton fluorescence (t = -1.0, P = 0.41).
38
39
40
Seeding of experimental mesocosms:
On July 11, 2011 we added substrate in the form of 11 kg of silica sand, nutrients
41
in the form of 30 g of rabbit chow, microbiota in the form of 20 mL of pond sediment,
42
and 1.5 L of water containing zooplankton collected and concentrated from local pond
43
sources. Communities were given two weeks to establish prior to addition of the
44
treatment conditions and experimental focal species. Communities were composed of a
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wide variety of species found in natural pond environments including zooplankton
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(rotifers, hydras, copepods, cladocerans), algae (periphyton, diatoms, and filamentous
47
algae), and benthic invertebrates such as planarians, nematodes and oligochaetes. We
48
added 50 field-collected H. trivolvis snails (mean ± SE size: 7.35 mm ± 0.15, wet weight:
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7.33 g ± 0.37) to each mesocosm two weeks later. Snails were marked with nail polish to
50
facilitate identification of the original cohort. We examined all snails for current
51
infection prior to adding them to mesocosms and dissected a subset (n=102) to ensure
52
they were free from patent or pre-patent trematode infections. We obtained parasite eggs
53
by filtering the feces of surrogate rat hosts exposed to R. ondatrae and chose realistic
54
doses of parasite eggs (~14,000 in the low infection treatment and 28,000 in the high)
55
following Johnson et al. (2012). We added equal amounts of filtered feces from
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uninfected rats to the unexposed mesocosms.
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58
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Temperature effects on non-host community:
To assess temperature-driven changes to the aquatic community that could have
60
influenced the environment or host food resources within the mesocosm, we sampled
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zooplankton on 31 March 2012, by collecting water samples using a 30 cm long x 5 cm
62
diameter pvc pipe and passing them through a 45 μm sieve. We preserved these samples
63
in 70% ethanol and counted the cladocerans and copepods in each sample. We also
64
compared relative phytoplankton fluorescence among mesocosms using a fluorometer
65
(Turner Designs Instruments, Sunnyvale, CA) once before winter and three times the
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following spring. Finally, we collected periphyton in May and August, 2012 from one of
67
two ceramic tiles (10.5 cm2) placed on the north side of each mesocosm. Samples were
68
filtered, dried and weighed to obtain estimates for periphyton growth over the duration of
69
the experiment.
70
We used linear mixed effects models, which included mesocosm as a random
71
effect, to test for interactive effects of temperature and exposure dose on zooplankton,
72
and interactive effects of temperature, exposure dose, and time on phytoplankton
73
fluorescence, and periphyton (see analysis section for full experiment for more details on
74
approach). When necessary, variables were transformed to meet assumptions of
75
normality.
76
The aquatic community was influenced by temperature differences in the heated
77
and unheated tanks. There were more zooplankton (cladocerans and copepods) and
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phytoplankton (measured through fluorescence) in heated tanks relative to unheated
79
(Cladocerans: t = 2.44, P = 0.02; Copepods: t = 2.80, P = 0.01; Phytoplankton: t = 2.10, P
80
= 0.03). Temperature did not influence the amount of periphyton measured in tanks,
81
however periphyton levels were higher in August than in May (temperature: t = 0.31, P =
82
0.76; time: t = -7.10, P < 0.01). There was no effect of periphyton biomass on the number
83
of snails egg masses per mesocosm (Table A1). There was also no effect of
84
phytoplankton fluorescence on egg masses, and only August fluorescence values related
85
significantly (negatively) to total adult snails (Table A1). Taken together, these results
86
suggest that temperature-driven changes in primary production were unlikely to have
87
contributed significantly to the observed the changes in snail dynamics.
88
Figures:
0.6
0.4
Unheated High Exposure
Heated Unexposed
Unheated Unexposed
Heated High Exposure
Heated Low Exposure
Unheated Low Exposure
0.0
0.2
Proportion Surviving
0.8
1.0
89
0
2
4
6
8
Sample Point
90
91
92
Figure A1. Snail survival curves by treatment.
10
12
14
200
150
100
Unheated
High Exposure
Unheated
Low Exposure
50
Heated
Unexposed
Heated High
Exposure
0
Cumulative Egg Masses
Unheated
Unexposed
Heated Low
Exposure
truncated
Aug
22
Sep
20
Oct
18
Mar
30
May
15
Jun
26
Jul 3
1
Date
93
94
Figure A2 Mean ± SE number of egg masses counted at each sampling date during the
95
experiment. Reduced reproduction by uninfected snails in the heated relative to unheated
96
mesocosms may have been due to thermal stress.
97
35
30
25
20
15
10
0
5
Total number shedding
Adults
Hatchlings
HH
UH
UL
Treatment
98
99
HL
Figure A3. Mean ± SE number of adults (e.g., from the original cohort of added snails)
100
and hatchlings (snails that were >5 mm and not marked as being from the original cohort)
101
with mature infections in each treatment at the end of the experiment (HH=Heated high
102
infection, HL=Heated low infection, UH=Unheated high infection, UL=Unheated low
103
infection).
Unheated Low Exposure
Unheated High Exposure
Heated Low Exposure
Heated High Exposure
0
Number cercariae per snail
50
100
150
104
May
June
July
Date
August
105
106
Figure A4. Mean ± SE number of cercariae released per snail at each of the sampling
107
dates. Note that cercariae were not preserved at the September sampling and samples
108
were counted from 10 randomly selected snails from each mesocosm.
109
80000
60000
40000
20000
0
Mean sum of cercariae
Heated High Exposure
Heated Low Exposure
Unheated High Exposure
Unheated Low Exposure
April
May
June
July
Month
110
111
Figure A5. Mean ± SE estimated number of cercariae released per month by adult snails
112
in each treatment. Note that the experiment concluded on August 14, so we do not
113
include estimates for this partial month.
114
115
Table A1. Values of linear regressions relating measures of phytoplankton
116
fluorescence or periphyton biomass to numbers of snails and numbers of egg
117
masses.
Model:
March Fluorometry
April Fluorometry
October Fluorometry
August Fluorometry
May Periphyton
August Periphyton
118
119
Snails
t-value
-0.74
1.87
1.18
-2.15
-0.41
0.72
Egg Masses
df p-value t-value
34 0.46
-1.05
34 0.07
1.02
34 0.25
-0.06
34 0.04*
-1.3
34 0.68
-1.02
34 0.48
0.91
df
34
34
34
34
34
34
p-value
0.30
0.32
0.96
0.20
0.31
0.37
120
References:
121
Johnson, P.T.J., Preston, D.L., Hoverman, J.T., Henderson, J.S., Paull, S.H., Richgels,
122
K.L.D., et al. (2012). Species diversity reduces parasite infection through cross-
123
generational effects on host abundance. Ecology, 93, 56-64.
124
Netten, J.J.C., van Nes, E.H., Scheffer, M. & Roijackers, R.M.M. (2008). Use of open-
125
top chambers to study the effect of climate change in aquatic ecosystems. Limnol.
126
Oceanogr-Meth., 6, 223-229.