Thermal acclimation has little effect on tadpole resistance to Batrachochytrium dendrobatidis

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Vol. 133: 207–216, 2019                  DISEASES OF AQUATIC ORGANISMS
                                                                                                           Published online March 28
 https://doi.org/10.3354/dao03347                      Dis Aquat Org

     Thermal acclimation has little effect on tadpole
      resistance to Batrachochytrium dendrobatidis
                                    Karie A. Altman1, 2,*, Thomas R. Raffel1
                     1
                      Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA
         2
          Present address: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

       ABSTRACT: Given that climate change is predicted to alter patterns of temperature variability, it
       is important to understand how shifting temperatures might influence species interactions, includ-
       ing parasitism. Predicting thermal effects on species interactions is complicated, however, be-
       cause the temperature-dependence of the interaction depends on the thermal responses of both
       interacting organisms, which can also be influenced by thermal acclimation, a process by which
       organisms adjust their physiologies in response to a temperature change. We tested for thermal
       acclimation effects on Lithobates clamitans tadpole susceptibility to the fungus Batrachochytrium
       dendrobatidis (Bd) by acclimating tadpoles to 1 of 3 temperatures, moving them to 1 of 5 perform-
       ance temperatures at which we exposed them to Bd, and measuring Bd loads on tadpoles post-
       exposure. We predicted that (1) tadpole Bd load would peak at a lower temperature than the tem-
       perature for peak Bd growth in culture, and (2) tadpoles acclimated to intermediate temperatures
       would have overall lower Bd loads across performance temperatures than cold- or warm-acclimated
       tadpoles, similar to a previously published pattern describing tadpole resistance to trematode
       metacercariae. Consistent with our first prediction, Bd load on tadpoles decreased with increasing
       performance temperature. However, we found only weak support for our second prediction, as
       acclimation temperature had little effect on tadpole Bd load. Our results contribute to a growing
       body of work investigating thermal responses of hosts and parasites, which will aid in developing
       methods to predict the temperature-dependence of disease.

       KEY WORDS: Chytridiomycosis · Temperature · Green frog · Beneficial acclimation · Dormancy
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                  1. INTRODUCTION                                       due to nonlinear and thermal acclimation responses
                                                                        to temperature (Rohr et al. 2013). Thermal acclima-
   Temperature is one of the most important abiotic                     tion, which here refers to adaptive and plastic pheno-
factors influencing organism performance (any phys-                     typic adjustments made in response to temperature
iological trait of interest: e.g. metabolism, sprint                    changes, implies that organism performance at a
speed; Angilletta 2009, Huey & Kingsolver 2011).                        given temperature depends on which temperatures
Given the changing climate, a primary goal of wild-                     the organism experienced in the recent past
life managers is to predict organism performance                        (Angilletta 2009). Species interactions such as para-
under new temperature scenarios. However, several                       sitism compound this complexity, because both para-
factors complicate this, including organism responses                   sites and hosts might have nonlinear and thermal
to temperature variability. Temperature shifts on var-                  acclimation responses to temperature that could
ious timescales (e.g. day to day or month to month)                     drive disease outcomes in variable-temperature
make predicting organism performance difficult                          environments.

*Corresponding author: karie.altman@pitt.edu                            © Inter-Research 2019 · www.int-res.com
208                                        Dis Aquat Org 133: 207–216, 2019

   Thermal acclimation is an important adaptation for        of acclimation effects for another component of tad-
many organisms that live in variable-temperature             pole resistance to trematode infection — their ability
environments. However, whether and how organ-                to prevent initial parasite encystment — that was con-
isms acclimate can vary widely among populations             sistent with the beneficial acclimation hypothesis but
and species (Stillman 2003, Seebacher et al. 2012),          not the dormancy hypothesis. These results indicate
and several hypotheses have been proposed to ex-             that host thermal responses can have distinct effects
plain this variation. For the host species of interest in    on different aspects of host resistance to infection.
this study (green frogs Lithobates [Rana] clamitans),           Building on the work of Altman et al. (2016), the goal
2 hypotheses were found to be potentially important          of the present study was to determine whether and
for driving the temperature-dependence of tadpole            how thermal acclimation influences L. clamitans tad-
resistance to encysted trematode parasites (Altman           pole resistance to the amphibian chytrid fungus Batra-
et al. 2016). (1) According to the ‘beneficial acclimation   chochytrium dendrobatidis (Bd). Bd has been used in
hypothesis’, an organism acclimated to a particular          multiple studies of host thermal acclimation, the re-
temperature will outperform unacclimated organisms           sults of which have generally supported the beneficial
at that temperature (Leroi et al. 1994). (2) According       acclimation hypothesis (Raffel et al. 2013, 2015). How-
to the ‘dormancy hypothesis’, an organism acclimated         ever, these past studies have only used 2 acclimation
to a particular temperature might adaptively decrease        temperatures, making it impossible to detect potential
its performance across temperatures, for example, to         nonlinear effects of thermal acclimation. We therefore
conserve energy during hibernation or aestivation            used 3 acclimation temperatures in the current study
(Geiser 2004, Storey & Storey 1990).                         to address whether host thermal acclimation can have
   These hypotheses are not mutually exclusive, and          nonlinear effects on Bd susceptibility. We predicted
the 2 mechanisms combined could lead to nonlinear            that L. clamitans thermal acclimation responses might
acclimation effects (Altman et al. 2016). For example,       have nonlinear effects on tadpole resistance to Bd in-
a species that undergoes a dormancy response when            fection, similar to tadpole clearance of R. ondatrae
acclimated to low temperatures might also experience         metacercariae (i.e. intermediate-acclimated tadpoles
a beneficial acclimation response when acclimated to         would have the lowest average Bd loads overall rela-
high temperatures. In this scenario, we would expect         tive to cold- and warm-acclimated tadpoles). Alterna-
both warm- and cold-acclimated organisms to have             tively, thermal acclimation effects on Bd infection
relatively poor performance at cold performance tem-         might more closely resemble acclimation effects on
peratures. This could lead to a nonlinear acclimation        tadpole resistance to initial R. ondatrae encystment
response in which organisms acclimated to intermedi-         (i.e. beneficial acclimation). Importantly, peak Bd
ate temperatures have the highest average perform-           growth tends to occur at lower temperatures on am-
ance relative to cold- or warm-acclimated organisms          phibians (in vivo) than in culture (in vitro: optimum
when all performance temperatures are considered             temperature ~17−25°C; Piotrowski et al. 2004), pre-
(note that warm-acclimated organisms undergoing              sumably due to increased amphibian resistance at
beneficial acclimation in this example could still have      higher temperatures (Andre et al. 2008, Raffel et al.
the highest peak performance at warm performance             2013, Sonn et al. 2017, but see Cohen et al. 2017). We
temperatures relative to cold- and intermediate-accli-       therefore also predicted that Bd load on L. clamitans
mated organisms). This is exactly the pattern Altman         tadpoles would peak at a temperature lower than that
et al. (2016) found for the ability of L. clamitans tad-     of optimum Bd growth in vitro. To test these predic-
poles to clear encysted trematode metacercariae.             tions, we acclimated L. clamitans tadpoles to 1 of 3
Tadpoles acclimated to intermediate temperatures             temperatures, exposed each tadpole to Bd at 1 of 5 per-
cleared a larger proportion of Ribeiroia ondatrae            formance temperatures, and then quantified Bd infec-
metacercariae than cold- or warm-acclimated tad-             tion on each tadpole weekly for 4 wk post-exposure.
poles at low and intermediate performance temp-
eratures (tadpoles cleared similar proportions of
metacercariae at high performance temperatures, re-                    2. MATERIALS AND METHODS
gardless of acclimation temperature; Altman et al.
2016). Such nonlinear acclimation effects are seldom                 2.1. Frog collection and maintenance
reported, perhaps in part because most experimental
studies of thermal acclimation use only 2 acclimation          Green frog tadpoles Lithobates clamitans were col-
temperatures (e.g. Raffel et al. 2013, 2015). Interest-      lected from ponds in Evart and Rochester, Michigan,
ingly, Altman et al. (2016) observed a different pattern     USA in August 2015 and October 2016, respectively.
Altman & Raffel: Tadpole thermal acclimation and parasite resistance                  209

Both populations were maintained in the lab under                         2.2. Temperature treatments
similar conditions until the experiment began in
November 2016. Similar proportions of tadpoles from             We refer to the temperature at which an organism
each collection location were assigned to each exper-        was maintained before the temperature switch as its
imental treatment, and tadpole population was in-            ‘acclimation temperature’ and the temperature fol-
cluded as a covariate in all analyses. Note that the         lowing a temperature switch as its ‘performance tem-
tadpole population term described both where and             perature’. In a natural setting, both Bd and tadpoles
when the tadpoles were collected (e.g. all individuals       would undergo temperature fluctuations simultane-
from the first population were collected at the same         ously. However, the goal of this experiment was to
time, and all individuals from the second population         detect acclimation effects on tadpole resistance to
were collected at another time).                             Bd; therefore, tadpoles were the only organisms to
   Before the experiment, tadpoles were housed in            undergo a temperature shift during the experiment.
groups within 14 l plastic boxes filled with aerated         In this experimental design, any effects of acclima-
artificial spring water (ASW; Cohen et al. 1980) that        tion temperature can be attributed to the tadpole
was changed weekly. During this time, tadpoles were          acclimation treatments and not to Bd acclimation
fed ground fish flakes and were maintained at room           responses (Raffel et al. 2013, 2015). Onset HOBO
temperature (~22°C) on a 12 h light:12 h dark cycle.         temperature loggers (Bourne) recorded the tempera-
To ensure that tadpoles were Bd negative at the              tures within the incubators every 30 min throughout
beginning of the experiment, tadpoles were main-             the experiment and were used to verify that the incu-
tained at 30°C for 7 d before the experiment to clear        bators maintained our target treatment temperatures
any existing Bd infection (Chatfield & Richards-             throughout the experiment (see Fig. S1 in Supple-
Zawacki 2011). A subset of the tadpoles (72 of 124           ment 1 at www.int-res.com/articles/suppl/d133p207_
total individuals) was also swabbed before the exper-        supp.pdf).
iment, and all were Bd negative.
   During the experiment, tadpoles were maintained
individually in 0.5 l glass canning jars filled with                           2.3. Bd maintenance
300 ml of ASW. Tadpoles were raised on a diet of
ground fish flakes before the experiment; however,             Twenty-five days before exposing frogs to Bd, cry-
fish flakes quickly fouled the water at high treatment       opreserved stock of Bd strain JEL 423 was removed
temperatures. Therefore, tadpoles were fed thawed            from −80°C storage and thawed according to Boyle et
frozen spinach ad libitum during the experiment, and         al. (2003). The thawed aliquot was divided between 2
soiled water was replaced weekly with ASW of the             glass bottles, each containing 200 ml of 1% tryptone
appropriate treatment temperature. When perform-             broth, which were then incubated at 23°C for 18 d.
ing water changes, nets were disinfected by soaking          After this time, replicate 30 ml Bd cultures were pre-
in 2% potassium permanganate for at least 5 min              pared by transferring 3 ml of active Bd culture to
after each use to prevent Bd transmission between            27 ml of sterile 1% tryptone broth in 50 ml centrifuge
tadpoles (nets were rinsed in tap water after disin-         tubes. The new Bd cultures were then divided among
fection and before using them again). Incubators             performance temperature treatments and placed into
constructed from Styrofoam containers, heat tape,            experimental incubators of the appropriate tempera-
and adjustable thermostats were used to house tad-           tures. Bd cultures were allowed to acclimate to their
poles to ensure proper replication of temperature            respective performance temperatures for 7 d before
treatments (Raffel et al. 2013, Greenspan et al. 2016).      being used to inoculate tadpoles.
Tadpoles were checked daily for mortality through-
out the experiment, and jars were rotated daily
within the incubators to account for any within-incu-                       2.4. Experimental design
bator variation. The mass of each tadpole was meas-
ured twice during the experiment: the day tadpoles             L. clamitans tadpoles were randomly assigned to
were exposed to Bd (or sham inoculum) and 28 d               1 of 3 acclimation temperatures (10, 16, or 22°C),
after exposure. The tadpole mass predictor included          where they were maintained for 14 d, which prior
in statistical models was the average of these 2 val-        results suggest is an adequate amount of time for
ues. Results were unchanged when either initial or           the tadpole immune system to acclimate to a new
final mass was included in the models rather than            temperature (e.g. all tadpoles performed similarly
average mass.                                                regardless of acclimation temperature by 14 d post-
210                                     Dis Aquat Org 133: 207–216, 2019

exposure to a parasite; Altman et al. 2016). After       Table 1. Regression statistics from linear mixed-effects mod-
the acclimation period, tadpoles were moved to 1 of      els (function lmer in R package lme4) describing the effects of
                                                         acclimation temperature (acc. temp.) and performance tem-
5 performance temperatures (13, 16, 19, 22, or           perature (perf. temp.) on the average number of Batra-
25°C), where they were exposed to Bd zoospores           chochytrium dendrobatidis (Bd) zoospores on frogs at each of
that had been acclimated to the performance tem-         the 4 swab time points: 7, 14, 21, and 28 d post-exposure. Re-
perature (Fig. S2 in Supplement 1). This tempera-        sults are from the simplest model at each time point (p < 0.1
                                                         for inclusion in the model). Block coefficients are reported as
ture range is consistent with pond temperatures
                                                         NA (not applicable) because block was a categorical variable
in which L. clamitans tadpoles live (Altman et           with more than 2 treatment levels and therefore generated
al. 2016) and supports Bd growth both in vitro           multiple-coefficients. Bold indicates significance (p < 0.05)
(Piotrowski et al. 2004, Voyles et al. 2017) and in
vivo (e.g. Raffel et al. 2015). Only 3 acclimation         Swab time point             Coefficient      df        F       p
temperatures were used as opposed to 5 so that the          and predictor
experiment could be conducted with a smaller sam-
                                                           7 d post-exposure
ple size, and acclimation temperatures were chosen
                                                           Mass                        −1.10 × 10−1   1, 64.9    2.12    0.150
to represent the lower, middle, and upper range of         Tadpole population          −2.41 × 10−2   1, 65.0    0.03    0.869
performance temperatures tested here. Note that a          Block                           NA         2, 25.6    0.16    0.851
10°C performance treatment was intended for this           Acc. temp.                  −1.50 × 10−4   1, 24.6   < 0.01   0.999
                                                           Acc. temp.2                  4.56 × 10−3   1, 25.0    2.28    0.143
experiment as well; however, it was eliminated be-
                                                           Perf. temp.                 −9.51 × 10−3   1, 25.4   13.39    0.001
cause the Bd zoospore concentration in 10°C cul-           Perf. temp.2                 9.28 × 10−3   1, 24.9    8.01    0.009
tures was not high enough on the day of exposure           Acc. temp. × perf. temp.    −1.34 × 10−3   1, 50.2    0.31    0.581
to prepare inocula. Each acclimation by perform-           Acc. temp.2 × perf. temp.   −1.36 × 10−3   1, 52.0    3.81    0.056
ance temperature cross included 3 control tadpoles         14 d post-exposure
and at least 5 Bd-exposed tadpoles (Table S1). Tad-        Mass                         2.30 × 10−1   1, 63.9    8.30    0.005
                                                           Tadpole population          −6.50 × 10−2   1, 67.2    0.19    0.667
poles were maintained for 6 h in their new per-            Block                           NA         2, 25.6    1.36    0.276
formance incubators before being exposed to Bd             Acc. temp.                   4.04 × 10−4   1, 33.2   < 0.01   0.972
zoospores to ensure their water was at the new             Perf. temp.                 −4.12 × 10−2   1, 25.1   11.32    0.002
performance temperature prior to adding the zoo-           Perf. temp.2                 1.21 × 10−2   1, 25.2   12.73    0.001
spore inoculum.                                            21 d post-exposure
                                                           Mass                         9.74 × 10−2   1, 65.5   1.50     0.225
  To accommodate time constraints, the experiment
                                                           Tadpole population           2.10 × 10−2   1, 62.1   0.02     0.886
was conducted in 3 temporal blocks. Temporal               Block                           NA         2, 30.2   0.67     0.520
blocks had the same experimental timeline but              Acc. temp.                  −1.34 × 10−2   1, 46.1   2.38     0.129
began on 3 consecutive days (e.g. Day 0 for Block          Perf. temp.                 −1.66 × 10−2   1, 29.7   0.69     0.414
A occurred the day before Day 0 for Block B).              Acc. temp. × perf. temp.     6.30 × 10−3   1, 45.3   8.80     0.005

Total sample sizes for each treatment (Table S1 in         28 d post-exposure
                                                           Mass                         2.87 × 10−1   1, 64.3   7.20     0.009
Supplement 1) were spread out over the 3 blocks.           Tadpole population           1.97 × 10−1   1, 67.9   0.95     0.334
Blocks contained similar numbers of tadpoles from          Block                           NA         2, 26.4   0.23     0.799
each temperature treatment and tadpole collection          Acc. temp.                  −1.97 × 10−2   1, 29.4   1.87     0.182
population. Tadpoles in each block were of similar         Perf. temp.                 −2.77 × 10−2   1, 25.9   2.79     0.107
size (average mass ± SE: Block A, 0.82 ± 0.11 g;
Block B, 0.78 ± 0.11 g; and Block C, 1.20 ± 0.17 g;
p = 0.096 for the effect of block on average tad-                               2.5. Bd exposure
pole mass using simple linear regression). Note
that there was a marginally nonsignificant effect of         On the day of exposure, one 30 ml Bd culture from
block on tadpole developmental stage (p = 0.055),         each performance temperature was passed through a
which reflects that tadpoles in Block C tended to         20 µm nylon filter (Spectrum Laboratories) to isolate
be more developed (mean Gosner stage ± SE:                zoospores. The zoospore concentration of each ino-
31.17 ± 0.99) than those in Blocks A (28.28 ± 0.81)       culum was determined by counting zoospores using
and B (28.63 ± 0.91). However, block was included         a hemocytometer, and an appropriate volume of ster-
as a covariate in all analyses and was not a signifi-     ile broth was added to dilute each culture to a con-
cant predictor of Bd load in any analysis (Table 1,       centration of 106 zoospores ml−1. Three ml of inocu-
Tables S2−S5 in Supplement 1); therefore, this            lum of the appropriate temperature was added to each
marginal difference was unlikely to dramatically          tadpole’s water so that each tadpole was exposed to
influence our results.                                    3 × 106 zoospores. Three control tadpoles at each
Altman & Raffel: Tadpole thermal acclimation and parasite resistance                     211

temperature were also exposed to 3 ml of sterile             plement 2 at www.int-res.com/articles/suppl/d133
broth at the appropriate temperature. An extra water         p207_supp.pdf).
change was performed 3 d after tadpoles were ex-
posed to Bd to prevent water fouling due to the pres-
ence of extra nutrients from the added broth.                                2.7. Statistical analyses

                                                                Most of the tadpoles that died during the experi-
      2.6. Bd swabbing and quantitative PCR                  ment died within 3 d of Bd or sham exposure (before
                                                             water had been changed following exposure). These
  Each tadpole was swabbed 10 times across the               animals were excluded from analyses, because swabs
mouthparts at 7, 14, 21, and 28 d post-exposure using        were likely to pick up transient Bd DNA from zoo-
sterile fine-tip cotton swabs (Advantage Bundling            spores that failed to establish in the tadpoles (Table S1
SP). Vinyl gloves were worn while swabbing, and be-          details the number of tadpoles per treatment in-
tween each animal, gloves were disinfected and               cluded in analyses). The only exception to this was 1
rinsed by successive immersion in 10% bleach, 1%             tadpole in the 22°C acclimation × 19°C performance
AmQuel Plus (a dechlorinator; Kordon), and deion-            temperature treatment, which died between 7 and
ized water (Raffel et al. 2013). Swabs were then             14 d post-exposure. This tadpole was therefore in-
frozen at −20°C until DNA extractions were per-              cluded in the 7 d post-exposure analysis but was
formed. Bd DNA was extracted from swabs using                excluded from subsequent analyses.
40 µl PrepMan Ultra extraction buffer (Applied Bio-             R statistical software v.3.0.5 (R Core Team 2018)
systems), according to Kriger et al. (2006).                 was used to conduct all analyses. Infection data are
  Quantitative PCR (qPCR) was run according to the           usually non-normally distributed and often fit a neg-
protocol of Kriger et al. (2006), using a Bio-Rad CFX        ative binomial distribution, and qPCR data are typi-
Connect system (Bio-Rad Laboratories). Samples were          cally log-normally distributed. Past studies have used
run in singlicate to control costs (Kriger et al. 2006).     zero-inflated negative binomial generalized linear
TaqMan® Exogenous Internal Positive Control Re-              models (e.g. function glmmadmb in package glm-
agents (Applied Biosystems) were added to every              mADMB or the recently updated function glmmTMB
reaction well to assess reaction inhibition (Kriger et       in package glmmTMB) to analyze data similar to
al. 2006). This internal positive control (IPC) system       those from this experiment (e.g. Raffel et al. 2013,
includes a standard concentration of artificial DNA          2015, McMahon et al. 2014); however, these models
with its own primers and a distinct fluorescent probe,       would not converge when fit to our data. Regular
and the strength of the IPC reaction was used to             negative binomial generalized linear models (func-
assess inhibition. All samples were initially run at         tion glm.nb in package MASS) resulted in unreason-
1:10 dilution, and samples that were inhibited were          ably low p-values for many terms in the model, indi-
re-run at 1:100 dilution. A sample was considered            cating possible overfitting. We ultimately decided to
inhibited if its IPC cycle threshold (CT) value was          analyze log-transformed zoospore numbers using
more than 4 cycles greater than the average CT value         linear mixed-effects models assuming log-normally
of the negative control wells on the plate on which          distributed data (function lmer in package lme4,
the sample was run. Furthermore, all sample curves           Bates et al. 2015), which allowed us to include incu-
were visually inspected, and any samples with ab-            bator as a random effect in all analyses. Performance
normally shaped curves were re-run at the original           incubator was the random effect for all models except
dilution.                                                    those testing for main or quadratic effects of acclima-
  Bd standards based on the internal transcribed             tion temperature, where acclimation incubator was
spacer region of the Bd genome (ranging from 2.1 ×           instead specified as the random effect (Altman et al.
101 to 2.1 × 104 gene copies µl−1; Pisces Molecular)         2016).
were included on every qPCR plate. Standards were               For the linear mixed-effects models, the response
not found to vary substantially among plates; there-         variable in each analysis was Bd load, quantified as
fore, all plates from the experiment were combined           the log number of zoospores detected on each swab.
to form a composite standard curve for use in esti-          A 1 was added to zoospore numbers before log-trans-
mating the starting gene copy number of each                 formation to allow for the transformation of zeroes.
sample. Finally, the starting quantity units were con-       Both acclimation and performance temperature data
verted from gene copies to zoospores using a conver-         were centered prior to analyses by subtracting the
sion factor of 63.5 gene copies zoospore−1 (see Sup-         mean temperature value from each data point to en-
212                                       Dis Aquat Org 133: 207–216, 2019

sure the interpretability of both linear and quadratic      effect. Furthermore, a binomial generalized linear
effects in each model. Data from each of the 4 swab         mixed-effects model (function glmer in package
time points were analyzed separately. At each time          lme4) was run to assess the temperature effects on
point, both linear and quadratic effects of acclimation     whether a tadpole tested positive for Bd at any point
and performance temperature on Bd load were tested          during the experiment (Table S4). This analysis used
for. Interactions between acclimation and perform-          χ2 rather than F-tests, and tadpoles with Bd loads
Altman & Raffel: Tadpole thermal acclimation and parasite resistance                    213

                   1.8     a                                                       b
                   1.6
                   1.4
                   1.2
                   1.0
                   0.8
                   0.6
                   0.4
Log(Zoospores+1)

                   0.2
                   0.0

                   1.8     c                                                       d
                   1.6
                   1.4
                   1.2
                   1.0
                   0.8
                   0.6
                   0.4
                   0.2
                   0.0
                      10       13   16      19       22       25      28      10       13     16       19     22      25       28
                                                           Performance temperature (°C)
Fig. 1. Mean ± SE number of Batrachochytrium dendrobatidis (Bd) zoospores (log transformed) detected on tadpoles accli-
mated to 10°C (open circles), 16°C (gray circles), and 22°C (black circles) at each performance temperature (tadpoles were ex-
posed to Bd at the same performance temperatures, but points are offset for clarity). Each panel shows data from 1 of the 4
                       swabbing time points: (a) 7 d, (b) 14 d, (c) 21 d, and (d) 28 d post-exposure to Bd

significant interaction between acclimation and per-                       the average number of zoospores per animal detected
formance temperatures (F1, 45.3 = 8.80, p = 0.005). At                     on tadpoles ranged from 0.5 to 2835 zoospores, with
14 and 28 d post-exposure, larger tadpoles had                             an average of 307 zoospores (average log(zoospores)
higher Bd loads (14 d, F1, 63.9 = 8.30, p = 0.005; 28 d,                   = 2.49), indicating that our qPCR methods were able
F1, 64.3 = 7.20, p = 0.009). There was no interaction                      to detect Bd infection and that many of the tadpoles
between mass and acclimation or performance                                simply did not become infected.
temperature.
  Results from the other analyses conducted were
generally consistent with those presented in the main                                        4. DISCUSSION
text (Tables S2−S6). In these analyses, performance
temperature was consistently a stronger predictor of                         Bd load on Lithobates clamitans tadpoles was
Bd load than acclimation temperature (Tables S2−S4),                       consistently highest at the lowest performance tem-
and tadpole mass was positively correlated with Bd                         perature (13°C) relative to the other performance
load when mass significantly contributed to the mod-                       temperatures tested and declined as performance
els (Tables S2 & S3). Results for Bd load averaged                         temperatures increased (Fig. 1). This supports our
through each time point (7−14, 7−21, and 7−28 d post-                      prediction that Bd growth on tadpoles would peak at a
exposure) are shown in Fig. S3 in Supplement 1.                            lower temperature than that at which it peaks in cul-
  The results presented in Fig. 1 and Fig. S3 describe                     ture (17−25°C; Piotrowski et al. 2004). Note, however,
the average log-transformed number of zoospores on                         that we were unable to detect a true peak in Bd load
all Bd-exposed tadpoles, regardless of whether they                        in this study, because our highest observed Bd loads
tested positive for Bd. Because many tadpoles never                        occurred at the lowest temperature we tested. Our re-
became infected, the average Bd load is quite low.                         sults are consistent with those of other studies that
Among tadpoles that tested positive for Bd, however,                       have demonstrated a negative relationship between
214                                        Dis Aquat Org 133: 207–216, 2019

temperature and Bd growth on amphibian hosts,                acclimation studies should cover a broader range of
which likely arises from a combination of enhanced           temperatures, including those near organisms’ upper
host immunity and reduced Bd growth (independent             and lower thermal ranges, so that these effects can
of host immunity) at high temperatures (Piotrowski et        be detected if they exist. Note, however, that it is
al. 2004, Rollins-Smith et al. 2011). For example, Row-      unlikely that tadpole thermal acclimation would in-
ley & Alford (2013) found that the probability of natu-      fluence tadpole susceptibility to Bd at temperatures
ral Bd infection in 3 amphibian host species declined        above those tested in this study, because Bd load was
as they spent more time at temperatures above 25°C.          already very low on tadpoles at 25°C (Fig. 1), and Bd
Furthermore, optimal Bd growth on several species of         growth in vitro is typically low at temperatures above
amphibians, or highest Bd-induced host mortality, oc-        25°C (Piotrowski et al. 2004, Voyles et al. 2017).
curred at a lower temperature than that at which Bd             At 7 d post-exposure in the present study, there
growth peaks in vitro, likely because host resistance is     was a marginally nonsignificant interaction between
enhanced at higher temperatures (Andre et al. 2008,          performance temperature and the quadratic effect of
Raffel et al. 2013, Cohen et al. 2017, Sonn et al. 2017).    acclimation temperature (Table 1). When exposed to
However, a recent study showed that a cold-adapted           Bd at 13°C, a trend showed that tadpoles acclimated
amphibian species was instead more susceptible to            to 16°C had lower Bd loads than tadpoles acclimated
Bd infection at high temperatures, due to a thermal          to either 10 or 22°C; however, tadpoles had similar
mismatch between host and Bd thermal performance             (low) Bd loads at higher performance temperatures
curves (Cohen et al. 2017). These studies highlight          (Fig. 1). This might indicate a beneficial acclimation
the need to consider the thermal responses of both           response by tadpoles acclimated to 22°C and a dor-
host and parasite when predicting the temperature-           mancy response by 13°C acclimated tadpoles. How-
dependence of infection.                                     ever, at 14 d post-exposure, tadpoles had similar Bd
   Interestingly, we saw very little evidence for an         loads at 13°C regardless of acclimation temperature;
effect of thermal acclimation of L. clamitans tadpoles       therefore, any possible advantage of being accli-
on their susceptibility to Bd. Performance tempera-          mated to intermediate temperatures might only be
ture was a much stronger predictor of Bd load on tad-        temporary. Altman et al. (2016) found stronger evi-
poles than acclimation temperature, no matter how            dence for a nonlinear pattern in the ability of green
the data were analyzed (Table 1, Tables S2−S5), and          frog tadpoles to clear Ribeiroia ondatrae metacercar-
even so, tadpoles only had appreciable Bd loads at           iae, in which 7 d after parasite exposure, tadpoles
the 13°C performance temperature (Fig. 1). In addi-          acclimated to intermediate temperatures had cleared
tion, there was no effect of performance temperature         a larger proportion of metacercariae than either cold-
on Bd load at 21 and 28 d post-exposure (Table 1).           or warm-acclimated tadpoles (Altman et al. 2016).
The effects of Bd are generally less adverse in tad-         This pattern was especially evident at lower perform-
poles than in adult amphibians (Berger et al. 1998),         ance temperatures (13−23°C); tadpoles cleared simi-
and tadpole species vary in their susceptibility to Bd       lar proportions of metacercariae at the 2 highest per-
(Blaustein et al. 2005). Therefore, low risk of severe       formance temperatures (25 and 28°C), regardless of
disease or low overall tadpole susceptibility to Bd          acclimation temperature (Altman et al. 2016). Al-
could have contributed to the lack of thermal accli-         though the present study revealed only a weak pat-
mation effects observed in our study. However, it is         tern suggesting that acclimation to intermediate tem-
important to note that our study was limited by the          peratures might enhance tadpole resistance to initial
fact that the lowest performance temperature that we         Bd exposure at low temperatures, its similarity to the
were able to test was 13°C. Based on our finding that        results of Altman et al. (2016) is intriguing. Future
tadpoles had high Bd loads at 13°C and the fact that         studies that directly address potential parallels in
Bd is capable of sustaining growth at temperatures as        host thermal acclimation responses to multiple para-
low as 2°C (Voyles et al. 2017), it is possible and even     sites would therefore be valuable.
likely that tadpoles would also exhibit high Bd loads           Acclimation temperature had no effect on tadpole
at temperatures lower than those tested in this exper-       Bd load after 7 d post-exposure in the current study
iment (e.g. 7−10°C). Furthermore, thermal acclima-           (Fig. 1c,d). We did detect a significant interaction be-
tion effects are sometimes evident only at very high         tween acclimation and performance temperatures
or low temperatures (e.g. Kaufmann & Bennett 1989).          when the data from 21 d post-exposure were analyzed
Therefore, tadpole acclimation temperature might             in isolation, with tadpoles acclimated to 10°C exhibit-
influence host Bd susceptibility at low temperatures         ing higher Bd loads at 13°C (performance tempera-
that were untested in this study. Future thermal             ture) than tadpoles acclimated to 16 or 22°C (Fig. 1c).
Altman & Raffel: Tadpole thermal acclimation and parasite resistance                          215

Estimates of the amount of time amphibians require               In conclusion, our study demonstrates the impor-
for immune system acclimation range from days                 tance of accounting for thermal effects on both para-
(Maniero & Carey 1997, Altman et al. 2016, Greenspan          site infectivity and host resistance when predicting
et al. 2017) to weeks (Bly & Clem 1991), and different        the temperature-dependence of disease, as other
aspects of acclimation might require different amounts        authors have previously emphasized (Raffel et al.
of time. It is therefore possible that tadpoles were not      2013, Sonn et al. 2017). Unlike previous studies that
fully acclimated to their performance temperatures by         demonstrated beneficial acclimation effects in am-
21 d post-exposure. However, given that Bd levels of-         phibians exposed to Bd following a temperature shift
ten fluctuate through time (e.g. Briggs et al. 2010, Ger-     (Raffel et al. 2013, 2015), we found little evidence for
vasi et al. 2013, Catenazzi et al. 2017, Daversa et al.       an effect of thermal acclimation on L. clamitans tad-
2018) and that no effect of acclimation temperature on        pole susceptibility to Bd. However, there was an
Bd load was found at any other time point after 7 d           interesting similarity between the weak effect of L.
post-exposure, it seems possible that the apparent ac-        clamitans thermal acclimation on tadpole resistance
climation effect at 21 d post-exposure was caused by          to Bd infection and previously reported effects on R.
random fluctuations in Bd load.                               ondatrae infection (Altman et al. 2016), which might
   At both 14 and 28 d post-exposure in our study,            warrant further study. It is unknown whether other
larger tadpoles had higher Bd loads (Table 1). We             ectotherms might show similar thermal responses
chose to end our study at 28 d post-exposure because          when challenged with various pathogens, but if gen-
we were focused on thermal acclimation effects, which         eralizable, these results could help lead to better pre-
are typically short-term. In a natural setting, larger,       dictions of the temperature-dependence of wildlife
older tadpoles (which have presumably been continu-           and vector-borne diseases.
ously exposed to Bd throughout their larval period)
had increased probabilities of Bd infection (Sapsford
                                                              Acknowledgements. This work was conducted with Oakland
et al. 2018). However, it is unknown whether a similar
                                                              University Institutional Animal Care and Use Committee
pattern might be evident in a single-exposure labora-         (IACUC) approval (IACUC no. 15101). We thank the anony-
tory study. Future studies tracking long-term (> 28 d)        mous reviewers whose input greatly improved the manu-
Bd infection in tadpoles could provide insight into           script. Thank you to the Zielinski family for allowing us to
whether the mass effects on tadpole Bd load that we           collect tadpoles on their property and to Joyce Longcore for
                                                              providing Bd cultures. Finally, thanks to S. Brady, A. Foster,
found were due to random fluctuations in Bd load or if        K. Julius, J. McBride, R. McWhinnie, M. Ostrowski, K. Rose,
they reflect a persistent pattern.                            H. Russell, J. Sckrabulis, R. Stepanian, J. Tituskin, and S.
   Many outstanding questions remain regarding the            Zielinski for assisting with animal maintenance, data collec-
effects of thermal acclimation on parasitism. Although        tion, and qPCR. This work was supported by an NSF
                                                              CAREER award to T.R.R. (IOS-1651888). K.A.A. was sup-
we know that the amphibian immune response is
                                                              ported by a King-Chávez-Parks Future Faculty Fellowship.
temperature dependent (Rollins-Smith et al. 2011),
relatively little is known about how thermal acclima-
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Editorial responsibility: Louise Rollins-Smith,                     Submitted: April 4, 2018; Accepted: January 9, 2019
Nashville, Tennessee, USA                                           Proofs received from author(s): March 22, 2019
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