J Insect Behav (2014) 27:57–66

DOI 10.1007/s10905-013-9408-2

High Repeatability of Anti-Predator Responses

and Resting Metabolic Rate in a Beetle

Indrikis Krams & Inese Kivleniece & Aare Kuusik &

Tatjana Krama & Todd M. Freeberg & Raivo Mänd &
Ljubova Sivacova & Markus J. Rantala & Marika Mänd

Revised: 19 July 2013 / Accepted: 24 July 2013 /

Published online: 7 August 2013
# Springer Science+Business Media New York 2013

Abstract Measures of repeatability are essential for understanding behavioral consis-

tency and individual differences in behavior, i.e. animal personalities. We studied anti-
predator responses of the yellow mealworm beetle (Tenebrio molitor) and performed
behavioral tests in plastic containers representing a typical laboratory environment of
T. molitor. Behavioral tests were repeated in Eppendorf test tubes where we also
measured resting metabolic rate (RMR). Results show that the response latency to a
threatening/startling stimulus, and the total time spent in the state of tonic immobility,
correlated across the tests. The behavioral responses were repeatable and RMR covaried
phenotypically with personality: we found a negative correlation between response
latency time and time spent immobile, a positive correlation between response latency
and RMR, and a negative correlation between RMR and total time spent immobile.
These correlations were also similar across trials performed in the Eppendorf test tubes
and the plastic containers.

Keywords Anti-predator responses . behavioral syndrome . resting metabolic rate .

Tenebrio molitor . pace-of-life hypothesis

I. Krams (*) : R. Mänd

Institute of Ecology and Earth Sciences, University of Tartu, Vanemuise 46, Tartu 51014, Estonia
e-mail: indrikis.krams@ut.ee

I. Krams : I. Kivleniece : T. Krama : L. Sivacova

Institute of Systematic Biology, University of Daugavpils, Vienības 13, Daugavpils 5401, Latvia

A. Kuusik : M. Mänd
Department of Plant Protection, Institute of Agricultural and Environmental Sciences,
Estonian University of Life Science, Kreutzwaldi 1, Tartu 51014, Estonia

T. M. Freeberg
Department of Psychology and Department of Ecology & Evolutionary Biology,
University of Tennessee, Austin Peay Building 301B, Knoxville, TN 37996, USA

I. Krams : M. J. Rantala
Department of Biology, University of Turku, Turku 20014, Finland
58 J Insect Behav (2014) 27:57–66

Introduction

The study of animal personalities is now at the forefront of contemporary behavioral

ecology because its integrative and interdisciplinary approach provides a better understand-
ing of the evolution of behavior (Sih et al. 2004; Bateson 2003; Stamps 2003; Carere and
Eens 2005). According to the definition of animal personality, suites of correlated behaviors
are consistent and repeatable across time and contexts. The associations between two or
more behaviors are called behavioral syndromes. Theory suggests that personality is tightly
linked to individual life histories and to variation of such environmental factors as predation
pressure (Houston and McNamara 1999; Dall et al. 2004; McElreath and Strimling 2006;
Wolf et al. 2007, 2008; Houston 2010). Existing evidence suggests that individuals can be
divided into shy or bold behavioral types along the reactive–proactive axis (Koolhaas et al.
1999; Sih et al. 2004). Proactive individuals tend to sustain high productivity with a
potential cost to survival, whereas reactive individuals prioritize survival with the cost of
productivity (Biro and Stamps 2008; Smith and Blumstein 2008). To meet the demands of
their faster pace of life (Wolf et al. 2007; Dingemanse and Wolf 2010; Biro and Stamps
2010; Réale et al. 2010a, b), proactive individuals usually are relatively starvation-risk
averse, but as a result may be more predation-risk prone than reactive individuals (Quinn
et al. 2012). Recent research has shown that predation risk related survival and stress
responsiveness can be dependent not only on behavioral type but also on metabolic rates of
animals. For example, individuals with high ventilation rates resumed their rates of feeding
faster than individuals with low ventilation rates in Nile tilapia (Oreochromis niloticus)
(Barreto and Volpato 2011). The relationship between RMR and explorative behavior was
more complex in the common lizard (Zootoca vivipara) where individuals with low
exploration score and high resting metabolic rate (RMR) and individuals with high
exploration rate and low RMR survived better than individuals with other combinations
of RMR and exploration activity (Le Gilliard et al. 2013).
Under the risk of predation, many animals display tonic immobility, which is an
unlearned, reversible, coma-like behavioral response (Gallup 1974; Marx et al. 2008). A
recent study shows that response latency to become immobile, and the duration of
immobile state in yellow mealworm beetles (Tenebrio molitor), can be considered as
personality traits and may be related to metabolic rate because proactive individuals with
shorter latency times had higher RMR rates (Krams et al. 2013a, b). This suggests that T.
molitor may be an excellent model to test various ecological relationships between
individual metabolic rate and energy output (e.g., Rezende et al. 2005, 2009; Speakman
et al. 2003, 2004; Biro and Stamps 2008, 2010).
Studies dealing with behavioral responses and measurements of metabolism to reveal
factors underlining personality often face a difficulty: the repeatability of all manipula-
tions including measurements of metabolism across different ecological conditions and
contexts. Given that repeatable measurements of metabolism in a single individual usually
take several hours, these studies may be extremely time consuming. The aim of this
experimental study was to investigate the consistency of anti-predator responses and to
test whether these responses of individual beetles are linked to their resting metabolic
rates. We also attempted to reduce the number of behavioral trials while studying
behavioral syndromes and respiration of T. molitor.
As it was recently shown, some anti-predator responses of T. molitor can be studied
by placing the beetles individually in plastic containers with bran food substrate, while
J Insect Behav (2014) 27:57–66 59

the presence of predators can be simulated by flicking the container, after which the
beetles usually become immobile on the surface or in the layer of bran (Krams et al.
2013a, b). In this study, we obtained repeatabilities of behavioral responses by
placing insects into the plastic containers and also into Eppendorf test tubes con-
taining no food to see whether the behavioral responses differ under the two different
conditions.

Methods

Beetles

The beetles used in the experiment originated from a natural population living in barns in
southeastern Latvia (Daukste et al. 2012). The stock culture was maintained at 25±2 °C
on bran mixed with wheat flour, fresh carrots and apples. We removed pupae from the
culture on the day of pupation, weighed them, and determined their sex according to
Bhattacharya et al. (1970). We then kept the pupae and newly emerged adults individ-
ually in numbered 200 ml plastic containers filled with a mixture of bran and wheat flour
and with fresh carrot/apple pieces offered ad libitum. The beetles were weighed at the
age of 10 days using a Kern analytical balance (Kern & Sohn, Balingen, Germany). We
performed behavioral trials across two contexts/environments, RMR and obtained
repeatabilities of the measured traits. We used beetles of average body mass (±4 %
average mass). Individuals with different body mass and body size have different life-
history values (e.g. Krams et al. 2011a; Nespolo et al. 2011) and may thus differ in the
expression of behaviors. Therefore, we excluded nearly 20 % consisting of the smallest
and largest beetles from this time-consuming study and to be able collect a sufficient
sample size study, which was restricted to male individuals only (n=60).

Mimicking of Predator Approach in Open Arena

When predators attack, mealworm beetles often feign death by entering a tonic immobil-
ity state. T. molitor is a nocturnal species and we carried out the trials in the beginning of
the night (8:00–11:00 PM). We hit the 200 ml plastic container of each beetle from above
with a stick, applying a mass of about 40 g from the distance of 10 cm (Krams et al.
submitted). The floor of each plastic container (10×6 cm) was covered with a 0.5 cm layer
of bran. We used this depth of bran to provide beetles their usual food supply, to allow the
beetles to locomote without slipping, and to burrow partly as a form of escape mechanism.
We observed the response of the beetles under red light (25 W red incandescent bulb),
which did not affect activity of T. molitor. Since members of Tenebrionidae family likely
cannot see long (red) wavelengths (Briscoe and Chittka 2001), the dim red light should
mimic the nocturnal conditions of the beetles (Kivleniece et al. 2010; Krams et al. 2011a,
b). We recorded individual latency times to become immobile, as well as the total time the
beetles spent motionless. These trials were repeated 10 days later. Nespolo and Franco
(2007) found a linear reduction of repeatabilities with time between two measurements,
suggesting habituation in many animals to experimental procedures. However, 10 days
period between subsequent measurements seems to be sufficient to avoid habituation in
the case of such short-lived organisms as mealworm beetles.
60 J Insect Behav (2014) 27:57–66

Mimicking Predator Approach in Insect Chamber and Flow-Through CO2

Respirometry

Five days after the first test in the 200 ml plastic containers, we handled mealworm
beetles for 2 min and then gave them the opportunity to escape into a conical plastic
Eppendorf test tube (volume 3 mL), which was used as an insect chamber. The insect
chamber was connected to the respirometer by means of rubber tubing (Lighton 2008).
In the insect chamber, the beetles either became less active or completely immobile, as if
they were hiding in a dark and safe crevice, whereas they never adopted an immobile
pose during handling. The beetles were left in the insect chambers. We waited for at least
5 min or maximum 12 min for beetles to resume active struggling movements. As soon
as the beetles became active again, we exposed the chambers to a sudden and a brief
mechanical stimulus by flicking the tube from above with a stick, which simulated
applying of mass of about 40 g dropped from the height of 10 cm. As in the previous
trials this mimicked a jump of a bird or rodent onto the insect chamber (Krams et al.
2013a, b). We recorded individual response latency time to the mechanical stimulus as
time to become immobile, and also recorded the total time beetles spent immobile.
As soon as behavioral trials were done, we started measuring resting metabolic rate
of the beetles. The insects remained in their chambers and we only monitored their
rates of metabolism. All of the beetles reached the lowest rates of CO2 production
within 30 min, when they were motionless. We attempted to continue measurements
of CO2 emissions for more than 2 h. However, this did not affect baselines levels of
CO2 emissions (paired t-test: n=16, P=0.87), and the baselines of recordings roughly
corresponded to the resting metabolic rate of each insect. As soon as the measure-
ments were over, we returned the beetles back to their plastic containers. We repeated
these trials 10 days later. Since behavioral trials may affect values of RMR, we
performed a third measurement of RMR 5 days later. During this trial we just placed
an insect in the insect chamber and measured RMR only.
The gas analyzer and flow-through respirometer were calibrated by means of cali-
bration gases (Trägergase, VEB, Saxon Junkalor GmbH, Dessau) with gas injection
(see also Kuusik et al. 2002; Mänd et al. 2006). When the respirometry was carried out,
the insect chamber was perfused with dry (5–7 % RH) CO2-free air, produced by passing
air over Drierite (W. A. Hammond Drierite Co. Ltd, Xenia, Ohio) and soda-lime
granules at a flow rate of 60 ml min−1. Baseline drift of the analyzer was corrected
during analysis from the measurements at the beginning and end of each trial with the
respirometer chamber empty (Duncan 2003; Duncan and Byrne 2005; Gray and Bradley
2006). The respirometric device was combined with an infrared optical system using IR-
emitting diodes (TSA6203) and IR-sensor diodes (BP104) that were placed on the sides
of the insect chamber. IR-diodes made it possible to record CO2 production and
movements of each beetle simultaneously.

Data Analysis

Values of behavioral data were not distributed normally and were thus square root
transformed in Statistica 8.0 software (StatSoft Inc). To ascertain whether individual test
beetles were consistent in their responses, the repeatabilities of response latency time to
become immobile, the total time beetles spent immobile and RMR were calculated
J Insect Behav (2014) 27:57–66 61

across two trials. Repeatability is the fraction of data variation that is due to differences
between individuals (Bell et al. 2009). We followed a method by Lessells and Boag
(1987) for calculating approximate repeatability values. This is a method for calculating
approximate repeatability (R) values from the F ratio.
Since we measured RMR, response latency time to become immobile and the total
time spent immobile repeatedly, all correlations were calculated from the average param-
eter values across the two measurement occasions. All of the tests used in this study were
two tailed.

Results

Consistency of Behavior and RMR Over Time

The latency to become immobile in response to flicking the plastic container varied
between 3 and 196 s (29.81±7.98, mean ± SD), and the total time spent motionless
after flicking varied between 4 and 256 s (99.47±24.58, mean ± SD). Response
latency after flicking of the plastic container was highly repeatable between two trials
separated by 10 days (R=0.58, F59,60=3.82, p<0.0001), as was the total time spent
immobile (R=0.71, F59,60=5.94, p<0.0001).
The latency to become immobile in response to flicking the insect chamber varied
between 2 and 121 s (31.24±9.16, mean ± SD), and the total time spent motionless
after flicking the insect chamber varied between 5 and 273 s (103.43±34.51, mean ±
SD). Individuals were significantly repeatable both in the response latency after
flicking of the insect chamber (R=0.75, F59,60=6.90, p<0.0001) and in the total time
spent immobile (R=0.75, F59,60=7.06, p<0.0001).
We found that individual beetles did not differ in their average latency to become
immobile when in the plastic containers and insect chambers (paired t-test, df=59, t=−1.30,
p=0.20). The total time spent immobile also did not differ significantly between trials done
in the plastic containers and insect chambers (paired t-test, df=59, t=−0.60, p=0.55).
RMR of mealworm beetles varied between 18.02 and 72.43 VCO2 μl h−1 (42.79
±31.02, mean ± SD). RMR was found to be highly repeatable among individual beetles
(R=0.92, F59,60=77.59, p<0.0001). We compared RMR between second and third trials to
see whether measuring of behavioral responses may influence values of RMR. However,
RMR did not differ significantly between trials when behavioral measurements were done
before measuring of RMR and when we measured RMR only (paired t-test, df=59,
t=0.94, p=0.35).

Correlations Between Behavior and RMR

RMR positively correlated both with response latency to flicking the plastic container
(Pearson’s r=0.46, p<0.001, Fig. 1) and response latency to flicking the insect
chamber (Pearson’s r=0.48, p<0.001, Fig. 1). The RMR negatively correlated both
with the total time of immobility in the plastic container (Pearson’s r=−0.39,
p=0.002) and in the insect chamber (Pearson’s r=−0.29, p=0.021).
The response latency to flicking and the total time spent immobile were negatively
correlated in the plastic containers (Pearson’s r=−0.51, p<0.0001, Fig. 2). Likewise
62 J Insect Behav (2014) 27:57–66

Fig. 1 Correlations between resting metabolic rate and response latency to simulated predator approach
and duration of time spent motionless in the plastic container and insect chamber (all data square-root
transformed)

Fig. 2 Negative correlation between response latency to simulated predator approach and duration of time
spent motionless in the plastic container and insect chamber (all data square-root transformed)
J Insect Behav (2014) 27:57–66 63

we found a negative correlation between the response latency and duration of

immobility when flicking the insect chamber (Pearson’s r=−0.47, p<0.0001, Fig. 2).
We found that behavioral responses were consistent between the two environ-
ments. This is shown by a significant positive correlation between individual mea-
surements of response latency to become immobile in the insect chamber and plastic
container (Pearson’s r=0.84, p<0.0001). There was also a significant positive corre-
lation in the total time spent immobile between the two environments (Pearson’s
r=0.89, p<0.0001).

Discussion

Our study revealed a significant consistency of anti-predator responses of semi-natural

T. molitor over time. Our data suggest that the latency to become immobile and the total
duration of immobility to flicking the plastic container or the insect chamber represent
valuable parameters for further studies of personality-related behavior in this species
(Gyuris et al. 2011; Kortet and Hedrick 2007; Pinter-Wollman 2012).
We supported some previous studies by showing that rapid interruption in activity by
flicking the substrate was associated with longer periods of immobility in T. molitor
(Nakayama and Miyatake 2010; Nakayama et al. 2012; Nishi et al. 2010; Nakayama and
Miyatake 2009; Krams et al. submitted). The results obtained within and between
contexts suggests a behavioral syndrome in anti-predator behavior of T. molitor, where
behavioral correlations arise as a consequence of individual variation in two or more
behaviors (e.g., Bell et al. 2009; Kortet and Hedrick 2007; Réale et al. 2010a, b; Sih et al.
2004). Moreover, we also found associations of RMR with anti-predator responses:
reduced rates of RMR were related to longer periods of immobility. This important result
shows that anti-predator behavior is part of a more complex syndrome involving
metabolic capabilities of an organism.
The most important finding of this study is that anti-predator responses of T. molitor
were not only highly repeatable across time but also across contexts (environments). Since
the response latency and the total time spent immobile did not significantly differ between
the trials done in plastic containers and insect chamber, it means that one of those
measurements may be omitted, which may save substantial amounts of time in future
work on RMR and behavioral anti-predator responses. However, we do not recommend
avoiding measurement of repeatability, because consistency of the two behavioral re-
sponses might be influenced also by genetic differences between animals (Foster and
Endler 1999a, b; Buczkowski and Silverman 2006; Yamada et al. 2009; Pölkki et al.
2012) and properties of their environment such as ambient temperature (Prokkola et al.
2013) or parasites (Ilvonen et al. 2011). The high consistency of individual responses and
the correlation between the responses might be explained by predation, which probably
shapes the behavior of our semi-natural beetles. It was shown that T. molitor with higher
levels of RMR, longer latency to become immobile and shorter total time spent immobile
were more exposed to the risk of bird and rodent predation (Krams et al. 2013a, b).
However, this could not be the case in long-term laboratory populations of T. molitor
where predation as a selective force has not played any role for many generations.
The results of this study show that while studying individual consistency of anti-
predator behavior and RMR of T. molitor, all of the necessary measurements can be
64 J Insect Behav (2014) 27:57–66

done by placing an individual beetle in an insect chamber/Eppendorf tube to perform

behavioral assays and measure RMR. We suggest this approach because behavioral
assays did not affect subsequent measurements of RMR in any detrimental way, and also
because the latency to reach the state of tonic immobility and the total duration of
immobility did not differ between trials done in the plastic containers and the insect
chamber. Overall, the results suggest T. molitor as an excellent species for the studies of
associations between metabolism and anti-predator behavior, and also show that com-
plexity of experimental procedures can be reduced to save much time in the laboratory.
Our results are in line with the pace-of-life (POLS) hypothesis (e.g. Réale et al. 2010a,
b; Vainikka et al. 2011; Niemelä et al. 2012). The concept of POLS is supported by the
finding that such important anti-predator responses as the latency to become immobile
and the total duration spent immobile under the risk of predation are under the control of
individual metabolic rate. It is known that bold and active behavior increases energy
intake rates (Biro and Stamps 2008; Kortet et al. 2010). We show that individuals with
shorter latencies to become immobile and longer immobile periods are shy and their RMR
is lower than that of bold individuals. This is an important evidence on the association
between anti-predator behavior and metabolism. We suggest the further research should
be focused on searching for genetic correlations between anti-predator behavior and RMR
for a better understanding of coevolution between life-history and personality.

Acknowledgments This work has been supported by the European Social Fund within the Project
“Support for the implementation of doctoral studies at Daugavpils University” (Agreement Nr. 2009/
0140/1DP/1.1.2.1.2./09/IPIA/VIAA/015) to Inese Kivleniece. We are thankful to Tuul Sepp for her help
with the figure.

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