Discussion
We found strong support for Circadian Rhythm and Thermal Acclimation
Hypotheses, indicating that colonies of Ectatomma ruidumdynamically manage constitutive and induced mechanisms of thermal
tolerance. When we altered the thermal regime experienced by these ant
colonies, the thermal persistence of workers responded accordingly,
consistent with the Thermal Acclimation Hypothesis. Simultaneously,
colonies demonstrated overt diel shifts in thermal persistence in the
absence of external temporal cues, supporting the Circadian Rhythm
Hypothesis. While we found some differences in thermal performance
associated with behavioral roles, the Division of Labor Hypothesis could
not account for differences in thermal persistence based on time of day,
even though that was the prior finding that led us to conduct the
present study (Esch et al., 2017; Nelson et al., 2018). The authors of
the earlier studies wondered how colonies of E. ruidum managed
the thermal tolerance of foragers associated with diel changes in
temperature. Our answer to that question is that a constitutive division
of labor is not as important of a mechanism as circadian rhythms and
proximate acclimation to recent thermal exposure.
We were surprised by the small magnitude of the findings. The
statistical significance of the results is robust, so we have little
doubt that these are real biological phenomena. Nevertheless, the
differences among sample populations were not so great. This is
consistent with the hypothesis that hotter and less seasonal
environments result in narrow thermal limit ranges (Kaspari et al.,
2015). It does not appear that the changes are driven by a number of
outlying individuals with extreme differences, because in Figures 1 and
2, it appears that the entire distributions are shifted. We think that
the biological significance of our findings is not that these tropical
ants are responding to their thermal environment with massive
physiological changes, but rather that these ants have the capacity to
demonstrate flexibility in a relatively complex manner. We did not press
hard on thermal levers to exert stresses on these ants. The marking
experiment simply measured existing differences among ants occurring in
the wild, and the colony fragments in the circadian rhythm experiment
were only held in the laboratory for up to four days. The only treatment
with a more-than-slight thermal stress (cooling a tropical rain forest
species to a constantly air-conditioned environment) produced an effect
of substantial magnitude. We expect that subjecting these ants to
greater thermal extremes will result in more marked effects.
The discovery of a circadian rhythm appears to be a relatively novel
result. Thermal tolerance is known to follow circadian rhythms in some
prokaryotes and plants (Rensing & Monnerjahn, 1996), but to our
knowledge has not yet been documented in any insects. Because some
animals regulate heat shock proteins following the seasons (Arad,
Mizrahi, Goldenberg, & Heller, 2010; Bujan, Roeder, Yanoviak, &
Kaspari, 2020), it makes sense that tropical rain forest insects are
also capable of recapitulating this activity over a 24 hour period, =he
time scale during which they experience the greatest range of thermal
challenges (Janzen, 1967).
In all treatments, thermal persistence was greater at dawn (the daily
thermal minima) than the early afternoon (the daily thermal maxima). We
were primed to expect the opposite result, because Esch et al. (2017)
and Nelson et al. (2018) reported that ants foraging in the heat of the
afternoon demonstrated higher thermal tolerances. In the circadian
rhythm experiment, our controls were foragers freshly sampled from the
field, which constitutes a near-replication of those earlier studies
with a greater sample size, and we found a non-significant result in the
opposite direction. After having had an opportunity to digest the
results from the treatments in the Circadian Rhythm experiment, we have
landed on a working hypothesis. The well documented mechanism of thermal
tolerance for these ants is the production of heat shock proteins, which
are both energetically expensive and are consumed as they are used to
prevent tissue damage from heat (Feder & Hofmann, 1999; Moseley, 1997).
The production of heat shock proteins must happen prior to the moment of
heat exposure, so their production must be made in anticipation of
future need. Ants living in a tropical rain forest, including E.
ruidum , live with absolute certainty that evenings will bring cool
temperatures and the following day will bring heat. Based on our
findings, we expect that these ants upregulated heat shock proteins at
the start of the day, which effectively anticipates the thermal
challenges of a new day. While they know that it will get hot, the
extent of the heat and the timing of it throughout the day are not as
predictable (Sanford Jr, Paaby, Luvall, & Phillips, 1994), so
acclimation matters too. This working hypothesis is consistent with our
finding that ant colonies subjected to a constant cool temperature have
extremely low thermal persistence in the afternoon. We interpret this to
mean that level of heat shock protein production, while governed in part
by a circadian rhythm, can be downregulated once colonies experience
consistently low temperatures. To gain a greater understanding of how
this circadian rhythm works, further work using more extreme thermal
manipulations and more frequent time steps will flesh out the nature of
this daily rhythm, as well as work to measure heat shock protein and
heat shock protein gene expression. Other ant species have already been
shown to upregulate heat shock protein gene expression in response to
heat exposure (Nguyen, Gotelli, & Cahan, 2016), so this is a tenable
future line of investigation.
While our finding of higher thermal tolerance in the early morning
contradicts earlier published results (Esch et al., 2017; Nelson et al.,
2018), we believe this makes sense in light of our other findings. While
we conducted our project in the same field site as Nelson et al. (and
even used the same heating/cooling device), both earlier papers were
conducted over a single 24 hour period during the dry season (which has
cooler temperatures), while the present study was conducted over a
longer duration in the wet season (which is characteristically hotter).
Moreover, the low-temperature sampling by Esch et al. (2017) happened
several hours before the thermal maxima, and the sampling by Nelson et
al. (2018) was between 04:00 and 04:30, whereas our sampling occurred
04:30-06:30. Our slightly later sampling was marginally closer to the
thermal minima, but not to an extent that we think the temperature
difference would matter. Nonetheless, if our working hypothesis that
heat shock protein production is upregulated at the start of the day is
correct, then this time difference could possibly explain why our early
morning ants demonstrated greater thermal tolerance.
We found that foragers have greater thermal persistence than
non-foragers. However, is not the essential prediction of our Division
of Labor hypothesis, which was that there would be differences thermal
persistence between foragers that ventured into the heat of the
afternoon and those that foraged in the cool of the early morning. The
difference between foragers and non-foragers is more parsimoniously
explained by Thermal Acclimation. In the marking experiment, we noticed
that the mean difference in thermal persistence between foragers and
non-foragers was three minutes, which is same magnitude of difference in
the thermal minima assays for the circadian rhythm experiment, between
freshly caught control foragers and laboratory colony ants. This
suggests that both the thermal treatment on colonies as well as the
artifacts of the laboratory manipulation did not alter thermal
persistence in the thermal minima time step, and that the effect of the
treatment was contingent on the circadian rhythm.
While our experiment was not designed with the purpose of assessing
intercolonial differences in thermal persistence, these comparisons were
possible with our sampling design. We separately compared the thermal
tolerances of colonies from the two experiments, and found that workers
from 3 of 21 colonies demonstrated significantly greater thermal
persistence. By random chance, 1 out of 20 colonies is expected to
generate a p-value below 0.05, and we don’t think that adding just 2
more colonies constitutes compelling evidence, as it looks more like a
few lucky rolls on a 20-sided die. We selected our colonies
intentionally so that they were in as similar environments to one
another as possible (with respect to light exposure in particular),
because this was a lurking variable that we sought to control. That we
detected any differences between colonies even though we sought to
prevent them from occurring suggests there is greater ambient variation,
especially considering the abundance and extremely broad geographic
range of this species. Perhaps if we intentionally sampled for thermal
heterogeneity in the field and subjected colonies to thermal treatments,
this would not only affect colony movement behavior as found in an
earlier study (McGlynn et al., 2010), but also shifts in the thermal
persistence of colony members.
With respect to climate change, one central concern is the extent of
behavioral flexibility, ecological plasticity, and evolutionary lability
available to animals facing new thermal challenges. Heat genuinely
prevents ants from foraging, as ants will avoid resource-rich areas if
they cannot withstand the thermal stresses (Spicer et al., 2017).
Thermal limits are a major factor in structuring species distributions
across broad environmental gradients (Diamond et al., 2012). Ants also
adapt their thermal tolerance to local conditions (Bujan & Kaspari,
2017; Diamond, Chick, Perez, Strickler, & Martin, 2017; Villalta et
al., 2020); we have known this ever since Mary Talbot invented equipment
and protocols to measure critical thermal maxima (Talbot, 1943). In
temperate environments, it appears that the ability to withstand cold
may be more predictive of distributions than the ability to withstand
heat (Bishop, Robertson, Van Rensburg, & Parr, 2017; Bujan, Roeder, de
Beurs, Weiser, & Kaspari, 2020). In the tropical climates where the
most species occur, however, we have many open questions about how
biodiversity will respond to rising temperatures (Jenkins et al., 2011).
We argue that an integrative understanding of organisms, including the
functional ecology at the colony level, will be critical for developing
more informative models as the world continues to heat up.
Animals living in social groups are capable of leveraging the modularity
of their colonies to efficiently organize behavioral responses to
thermal challenges. While this is well known from other social insects,
particularly honey bees (for example, Cook & Breed, 2013; Cook et al.,
2016; Kaspar et al., 2018; Stabentheiner, Kovac, & Brodschneider,
2010), we think that a greater research investment into intracolonial
mechanisms of thermal tolerance in ants is necessary to understand the
distribution of thermal tolerance at higher levels of organization. With
the projected levels of warming that we will experience over at least
the next few generations (IPCC, 2014), and the criticality of ants for
ecosystem processes (Del Toro, Ribbons, & Pelini, 2012), we need to
understand colony-level processes of thermal management appears
foundation to conservation planning for these animals.