Cold tolerance of the red flour beetle, Tribolium castaneum (Herbst) (Col.: Tenebrionidae) under different thermal regimes: impact of cold acclimation

Abstract

The red flour beetle, Tribolium castaneum (Herbst), is a serious pest of stored product worldwide. Cold tolerance or cold hardiness is an important ecophysiological trait related directly to survival, fitness, and distribution of insects. In this study, the effects of four thermal regimes, i.e., control (C), cold acclimation (CA), rapid cold hardening (RCH), and fluctuating-acclimation (FA), were examined for their effects on cold tolerance, supercooling point (SCP), lower lethal temperature (LLT), and chill-coma recovery time (CCRT) of the red flour beetle. In addition, changes in cryoprotectant (trehalose, sorbitol, and myo-inositol) levels were investigated under each thermal treatment. The results documented a substantial enhancement in the SCP, cold hardiness, and cryoprotectant levels of the adults of T. castaneum under CA regimes. The lowest SCP, highest trehalose and myo-inositol contents, and, subsequently, the greatest survival rate were observed in cold-acclimated beetles. In addition, coordination between cryoprotectant level, SCP, and cold tolerance of the pest was observed. The highest and lowest CCRT were observed at control and CA, respectively. In RCH regime with the highest impact, LLT reached the lowest level of -22°C. As most of the mortality of T. castaneum occurred at a temperature above the SCP, so this pest could be considered as a chill-susceptible insect.

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Stored-Product
Cold Tolerance of the Tribolium castaneum (Coleoptera:
Tenebrionidae), Under Different Thermal Regimes: Impact
of Cold Acclimation
H. Izadi,1,2, M. Mohammadzadeh,1,2,3 and M. Mehrabian1
1Pistachio Safety Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran, 2Department of Plant Protection,
Faculty of Agriculture, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran, and3Corresponding author, e-mail: m.mohammadzadeh@
stu.vru.ac.ir
Subject Editor: Thomas Phillips
Received 21 December 2018; Editorial decision 21 March 2019
Abstract
The red flour beetle, Tribolium castaneum (Herbst), is a serious pest of stored product worldwide. Cold toler-
ance or cold hardiness is an important ecophysiological trait related directly to survival, fitness, and distribu-
tion of insects. In this study, the effects of four thermal regimes, i.e., control (C), cold acclimation (CA), rapid
cold hardening (RCH), and fluctuating-acclimation (FA), were examined for their effects on cold tolerance,
supercooling point (SCP), lower lethal temperature (LLT), and chill-coma recovery time (CCRT) of the red flour
beetle. In addition, changes in cryoprotectant (trehalose, sorbitol, and myo-inositol) levels were investigated
under each thermal treatment. The results documented a substantial enhancement in the SCP, cold hardiness,
and cryoprotectant levels of the adults of T.castaneum under CA regimes. The lowest SCP, highest trehalose
and myo-inositol contents, and, subsequently, the greatest survival rate were observed in cold-acclimated bee-
tles. In addition, coordination between cryoprotectant level, SCP, and cold tolerance of the pest was observed.
The highest and lowest CCRT were observed at control and CA, respectively. In RCH regime with the highest
impact, LLT reached the lowest level of −22°C. As most of the mortality of T.castaneum occurred at a tempera-
ture above the SCP, so this pest could be considered as a chill-susceptible insect.
Key words: trehalose, cold acclimation, cold tolerance, stored pest
The red our beetle, Tribolium castaneum (Herbst), is a serious
worldwide pest of stored products and particularly food grains.
This pest attacks different food commodities such as our, cereals,
meal, crackers, beans, spices, pasta, cake mix, dried pet food, dried
owers, chocolate, nuts, seeds, and museum specimens (Via 1999,
Weston and Rattlingourd 2000). Different developmental stages of
the pest along with dead bodies, cast skins, and fecal pellets are usu-
ally found in the contaminated stores. Therefore, damage by the pest
is due to feeding and contaminating the commodities. Control of
this pest, as with other stored pests, relies mostly on the application
of fumigants, e.g., phosphine and methyl bromide. Continuous use
of these insecticides results in the development of resistance, side
effects on nontarget organisms, and environmental contamination
(Stejskal 2015). Thus, the replacement of synthetic pesticides with
safe alternatives such as phytosanitary temperature treatment should
be considered in the control of stored product pests (Subramanyam
and Hagstrum 1995).
Body temperature of insects, as poikilothermous animals, is
variable and depends on the environmental temperature. Growth,
development, and survival of these animals are profoundly af-
fected by ambient temperature (Woods etal. 2003). Therefore, in
this group of animals, the ability of thermoregulation has been
developed. Thermoregulation is the maintenance of the body tem-
perature in a natural environment by a behavioral or physiological
process (synthesis and accumulation of cryoprotectant metabolites)
(May 1979, Chidawanyika et al. 2017). However, different biotic
and abiotic factors such as insect species and life stage, relative hu-
midity, acclimation, and particularly the duration of exposure time
may inuence the extent of thermoregulation (Fields 2001, Beckett
et al. 2007, Mohammadzadeh and Izadi 2018a). Thus, measure-
ment of the low-temperature performance or cold hardiness is a way
to determine the relationship between dominant physiological or
ecological effects of temperature and survival of the insect species
(Sinclair etal. 2015). Based on Lee (1989), cold hardiness or cold tol-
erance is the ability of insects to survive long- or short-term exposure
to low temperatures. This ability is a variable function of biotic and
abiotic factors such as insect species and its life stage, age, sex, nutri-
tional and physiological status, diapause development, season of the
Journal of Economic Entomology, XX(XX), 2019, 1–6
doi: 10.1093/jee/toz089
Research
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year, and the magnitude and duration of the cold (Lee 1989, Sinclair
etal. 2015, Mohammadzadeh and Izadi 2018a). Experimentally, a
temperature at which insect body uid freezes and consequently, the
latent heat of fusion releases has been known as the supercooling
point (SCP). In most insects, cold hardiness strategies are usually
determined by preliminary measurement of the SCP (Sinclair etal.
2015, Li 2016, Su etal. 2017, Ditrich 2018, Mohammadzadeh and
Izadi 2018b). Insect’s responses to low temperature have been cat-
egorized in three levels: chill-susceptible (if most of the mortality
occurs above the SCP), freeze-avoidant (if most of the mortality oc-
curs at the SCP), and freeze-tolerant (if most of the mortality occurs
below the SCP) (Sinclair etal. 2015). In addition, when insects are
exposed to low temperatures, many of them enter a reversible phase
of paralysis which is attributed to the disruption of neuromuscular
and homeostatic functions and termed chill-coma (MacMillan and
Sinclair 2011, Findsen etal. 2014, Sinclair etal. 2015, Overgaard
and MacMillan 2017). The temperature at which insects enter chill-
coma is known as the critical thermal minimum (CTmin) (Sinclair
etal. 2015). In chill-susceptible insects, chill-coma usually results in
accumulation of cold injury which is a function of the intensity and
duration of the cold stress (Andersen et al. 2017). Chill coma re-
covery is the ability of an insect to reestablish ion and water homeo-
stasis and exhibit a predetermined behavior. Chill-coma recovery
time (CCRT) is the time that an insect needs to recover from a co-
matose state (Findsen etal. 2014, Sinclair et al. 2015, Knapp etal.
2018). However, uctuation in temperature may be used as a signal
by some insects to alter their cold tolerance (Findsen etal. 2014).
In the current study, the effects of cold acclimation (CA),
uctuating-acclimation (FA), and rapid cold hardening (RCH) on
cold tolerance of T. castaneum were investigated. CA may allow
some insects to enhance their cold tolerance. This enhancement in-
volves several physiological and biochemical adjustments, e.g., syn-
thesis and accumulation of low-molecular-weight carbohydrates and
polyols, upregulation of enzyme activity, and/or expansion of the SCP
(Heydari and Izadi 2014, Khanmohamadi et al. 2016, Overgaard
and MacMillan 2017, Mohammadzadeh and Izadi 2018a).
Materials and Methods
Insect Rearing
A laboratory colony of T.castaneum was started with the larvae col-
lected from stored rice seeds from Karaj, Iran. The insects were fed
on pistachio nut under a controlled environmental chamber at 28±
1°C with 65± 5% RH and a photoperiod of 14:10 (L:D) h.The fth
generation was used for the experiments.
Determination of theSCP
To measure the SCP of T.castaneum, the individual adult (n=15)
with adhesive tape was xed to a thermocouple (NiCr-Ni probe)
connected to an automatic temperature recorder, Testo 177-T4
(Testo, Friedrichshafen, Germany). The specimens were cooled in
a programmable refrigerated test chamber (GT-7005-A, Gotech,
Taichung,Taiwan) at a rate of 0.5°C/min. The lowest temperature
recorded before the onset of release of the latent heat of fusion was
considered as the SCP (Sinclair etal. 2015).
CA Treatments
Adults of T.castaneum were divided into four groups: control (C),
CA, FA, and RCH. For the control treatment, the beetles were put
in a translucent plastic cup containing pistachio nuts and kept in
standard rearing conditions. For CA treatment, the beetles were put
in a translucent plastic cup containing pistachio nuts, cooled in a
programmable refrigerator from rearing conditions to 12°C at a rate
of 0.5°C/min and kept at this temperature for 10 d.After that, the
temperature was lowered to 5°C at the same rate and the beetles
were kept at this temperature for another 10 d.For FA treatment,
the beetles were put in a translucent plastic cup containing pistachio
nuts, cooled in a programmable refrigerator from rearing conditions
in a cycle of 240min at 5°C followed by 20min at −10°C followed
by 240min at 5°C followed by 940min at 33°C. This cycle repeated
10 consecutive days. For RCH treatment, the beetles were directly
transferred from a rearing chamber to a programmable refrigerator
set at 0°C and kept for 4h. Survived beetles were used for subse-
quent experiments.
Cold ToleranceAssay
To investigate the cold tolerance of T.castaneum, ve replicates
and 15 beetles for each replicate were used at each treatment
and temperature point. To estimate the cold tolerance, the insects
treated with different thermal regimes, i.e., control (optimal tem-
perature), CA, FA, and RCH, were kept in a programmable re-
frigerated test chamber, whose temperature was lowered at a rate
of 0.5°C/min from experimental conditions to subzero temperat-
ures, i.e., 0, −5, −10, and −15± 0.5°C. These temperatures were
chosen based on the temperature in which insects can survive in
some treatments. The beetles were held at each temperature for
24 h. Thereafter, the beetles maintained at an optimum growth
temperature for 24h to check for survival. The beetles showing
no movement in their appendages were considered to be dead
(Mohammadzadeh and Izadi 2016).
Survival After Chronic Low-Temperature Exposure
To estimate the effect of chronic cold exposure on T.castaneum, the
beetles were exposed to −5°C, and survival was recorded every 6h
to calculate LT30 (lethal time at which 30% of the beetles died), LT50
(lethal time at which 50% of the beetles died), and LT95 (lethal time
at which 95% of the beetles died). The experiment was repeated ve
times for each thermal regime.
Survival After Acute Low-Temperature Exposure
The lower lethal temperatures (LLTs) of T.castaneum were deter-
mined using an acute exposure (1h) to subzero temperatures. Five
groups of 15 beetles were held at separate test temperatures, i.e., 0 to
−25°C (resulting in mortality from 0 to 100%), for 1h. Temperatures
were recorded using a thermocouple (NiCr-Ni probe) connected to
an automatic temperature recorder, Testo 177-T4 (Testo). The sur-
vival rate of the beetles was assessed after 24h in each temperature.
Finally, LT80–1 h was calculated as the lowest temperature at which
80% of the beetles died after 1-h exposure (Sinclair and Rajamohan
2008).
Chill-Coma RecoveryTime
Chill-coma was induced by transferring T.castaneum adults from
each thermal regime to sterile Petri dishes (6cm in diameter ×
1cm height) placed on a mixture of water and ice (slurry). David
etal. (1998) reported that the placing insects on ice–water slurry
(about 0°C) induces chill coma within a few minutes. After 2h,
the Petri dishes were transferred to room temperature. We docu-
mented the time required for coordinated movement, with all
six legs, when the beetles were still inverted and always in the
same position. The experiments were repeated ve times for each
thermal regime.
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Low-Molecular-Weight Carbohydrate Analysis
The whole-body polyol proles of T. castaneum under different
thermal regimes were repeated ve times with 10 beetles for each
treatment. Sugar alcohol contents were measured in vitro using
high-performance liquid chromatography (HPLC) (Knauer, Berlin,
Germany) equipped with a carbohydrate column with 4µm particle
size (250mm × 4.6mm, I.D., Waters, Dublin,Ireland) and UV-visible
detector. Acetonitrile–water (70:30) was used as eluent. Elution speed
was 1ml/min and separation achieved at 40± 1°C. Ten beetles were
weighed and homogenized in 1.5–2ml of 80% ethanol and centri-
fuged for 15min at 12,000 × g. The supernatant was evaporated at
40°C in a vacuum drying oven and resuspended in 1ml of HPLC grade
water and 20µl of the supernatant were run along with the standard
of each carbohydrate from 1,500 to 5,500ppm (Heydari and Izadi
2014). The amount of sugar was determined from a standard curve,
using glucose (Sigma Chemical Co., St. Louis, MO) as a standard.
Statistical Analysis
Data were initially tested for normality with Kolmogorov−Smirnov
test and homoscedasticity with Levene’s test before subjecting them to
analysis of variance (ANOVA). All the data were analyzed using SAS
ver. 9.2 program (PROC GLM; SAS Institute 2011). Statistical analyses
were performed, based on completely randomized design, using one-
way ANOVA followed by a post hoc Tukey’s test at α=0.05.
Results
SCPs of the Pest Under Different Thermal Regimes
A signicant difference between the SCPs of the adults of
T.castaneum was observed under different thermal regimes. In the
control, the SCP was at the highest level of −10.7°C, but in cold-
acclimated beetles, the SCP reached the lowest level of −18.4°C.
However, the SCPs of the beetles under thermal regimes decreased as
follow: C > RCH > FA > CA (Table 1).
Cold Tolerance of the Pest Under Different Thermal
Regimes
Twenty-four-hour exposure at 0, −5, −10, and −15°C signicantly af-
fects the survival of the adults of T.castaneum. Anyway, the highest
survival rate was observed in cold-acclimated beetles. When the adults
exposed to −15°C for 24h, the survival rates in CA and FA were 25.3
and 10.6%, respectively, but in control and RCH, survival rate reached
to the lowest level of zero percent and no adult survived (Table 1).
Changes in Carbohydrate Contents of the Pest
Under Different Thermal Regimes
Changes in the amounts of trehalose, sorbitol, and myo-inositol
were measured under control, CA, FA, and RCH regimes. Trehalose
followed by myo-inositol was found to be the most abundant cryo-
protectants of the beetle. The changes in trehalose and myo-inositol
followed the same trend in different thermal regimes. The highest
and the lowest levels of these cryoprotectants were observed in cold-
acclimated and control regimes, respectively. No signicant differ-
ence was observed between trehalose and myo-inositol contents of
FA and RCH regimes. However, the highest level of sorbitol was
recorded in FA regime (Fig. 1).
Effect of Thermal Regimes onCCRT
There was a substantial effect of CA on the CCRT of the beetles
(Fig. 2). The highest and lowest CCRT were recorded for control
and CA, respectively. No signicant difference was observed between
control and FA and RCH but the difference between FA and RCH
was signicant.
Effect of Thermal Regimes on Lethal Temperature
In all treatments, mortality began at 0°C and increased rapidly with
decreasing temperature. The LLT (temperature at which 100% mor-
tality occurred) for control and CA was −15°C, whereas for FA was
about −17°C and reached the lowest level of −22°C in RCH regime
(Fig. 3; Table 2).
Discussion
The results of this study demonstrated a substantial effect of the
thermal regimes on the SCP, cold hardiness, and cryoprotectant
levels of adult of T. castaneum. The same results have been re-
ported by Izadi et al. (2019) on Plodia interpunctella (Hübner)
and Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) and
Mohammadzadeh and Izadi (2018a) on Trogoderma granarium
Everts (Coleoptera: Dermestidae).
CA has been dened as ‘a reversible phenotypic change’ in re-
sponse to decreasing the environmental temperature in order to en-
hance cold hardiness of the insects (Rozsypal et al. 2018). Based
on the results of our study, CA regime was found to be the most
effective thermal regime. The lowest SCP, the highest trehalose and
myo-inositol contents, the highest survival rate, and the lowest
CCRT were recorded for cold-acclimated beetles. Therefore, it could
be concluded from our results that elevation of cold hardiness of the
T.castaneum adults in CA regime is a function of even a substantial
depression in SCP and/or a signicant enhancement in cryoprotectant
(trehalose and myo-inositol) level. CA increased the cryoprotectant
level of P.interpunctella and E.ceratoniae (Izadi etal. 2019) and Tro.
granarium (Mohammadzadeh and Izadi 2018a). In cold-acclimated
Schizaphis graminum (Rondani)(Hemiptera: Aphididae), trehalose
and glucose contents increased considerably (Saeidi et al. 2017a).
In overwintering larvae of the banded woolly bear caterpillar,
Pyrrharctia isabella (Smith)(Lepidoptera: Erebidae), CA increased
Table 1. Relationship between low-temperature survival rate and SCPs (mean ± SE) of Tribolium castaneum adults following different
thermal regimes
Treatments SCP (°C)
Survival rate (%)/24 h
0°C −5°C −10°C °15°C
C −10.7± 0.4d 76.2± 2.4c 62.1± 2.5b 16.4± 1.9d 0.0± 0.0c
CA −18.4± 0.7a 92.7± 2.6a 72.4± 1.4a 59.8± 1.6a 25.3± 1.8a
FA −16.1± 0.9b 84.9± 2.7b 71.6± 1.8a 49.4± 1.6b 10.6± 2.7b
RCH −14.2± 0.5c 77.6± 2.8bc 59.8± 2.1b 33.8± 1.7c 0.0± 0.0c
The means followed by different letters in the same columns are signicantly different (Turkey’s test, P<0.05).
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free proline, total amino acids, and proteins levels of hemolymph
(Yi and Lee 2016). CA increased the amounts of trehalose, proline,
asparagine, glutamic, acid, and lysine in Sitophilus granarius (L.)
(Coleoptera: Curculionidae) and Cryptolestes ferrugineus (Stephens)
(Coleoptera: Laemophloeidae) (Fields etal. 1998). Cryoprotectants
such as trehalose play an important role in depressing the SCP and
elevating cold tolerance of insects. So, the lower SCP and the higher
survival rate of T.castaneum in CA regime could be attributed to the
higher levels of cryoprotectants.
Our results documented that different thermal regimes result in
the decrease of the pest survival with the decrease in temperature,
but, again the cold-acclimated beetles always performed better cold
tolerance. In agreement with this nding, previous studies demon-
strated signicant effects of CA on cold tolerance of the insects. For
example, 4-d acclimation at 11°C improved the survival rate and
chill tolerance of Locusta migratoria L. (Orthoptera: Acrididae)
(Andersen et al. 2017). Rozsypal etal. (2018) found that CA sig-
nicantly enhanced cold tolerance of Riptortus pedestris (Fabricius)
(Hemiptera: Alydidae). Mohammadzadeh and Izadi (2018a) re-
sulted in an enhancement in cold tolerance of cold-acclimated larvae
of Tro. granarium. Izadi etal. (2019) indicated a signicant elevation
in the cold tolerance of P.interpunctella and E.ceratoniae under CA
regime.
The results of our study revealed that the changes in the SCP
of the pest under different thermal regimes were signicant but,
in cold-acclimated beetles, the SCP decreased to the lowest level
nearly two times lower than that of the control. In the tested
thermal regimes, the survival rate of the beetles was at the highest
level at 0°C per 24h, decreased with a decrease in temperature and
reached the lowest level at −15°C per 24h. In CA regime, at −15°C
per 24 h survival rate was 25 times more than that of the con-
trol. The adults of T.castaneum increased their cold tolerance by
depressing the SCP and increasing the amount of cryoprotectants.
Several previous studies demonstrated the signicant effect of CA
on depression of the SCP (Hiiesaar etal. 2001, Mohammadzadeh
and Izadi 2018a, Izadi et al. 2019). However, the results of the
current study do not support by the previous results (Hart etal.
2002, Tullett et al. 2004, Hughes et al. 2009, Maes etal. 2012,
Spranghers etal. 2017).
A short-term acclimation that enhances cold tolerance of insects
over immediate exposure to low temperature (minutes to hours) can
be considered as RCH (Saeidi etal. 2017b, Everman etal. 2018).
RCH is an adaptive and transient response to a rapid shift in the
ambient temperature (Everman etal. 2018). In this study, RCH de-
creased the SCP and increased the survival rate of T.castaneum com-
pared with the control. Enhancement of the pest cold tolerance was
coincident with a signicant increase in trehalose and myo-inositol
contents. Therefore, the elevated survival rate of the pest under RCH
regime suggests that the RCH is a rapidly cold tolerance adapta-
tion strategy in response to acute cold shock. These ndings are con-
sistent with those of Wang and Kang (2003) (L.migratoria), Sinclair
Fig. 2. The effects of different thermal regimes on CCRT of Tribolium
castaneum (mean ± SE). Each point is an average of five replications. Mean
values followed by different letters are significantly different (Tukey’s test,
P<0.05).
Fig. 3. Survival of Tribolium castaneum after acute low-temperature
exposure (mean ± SE). The horizontal line indicates 80% mortality (LT80–1h).
Fig. 1. Carbohydrate contents of Tribolium castaneum (mean ± SE) after
exposure to different thermal regimes. Each point is an average of five
replications. Mean values followed by different letters are significantly
different (Tukey’s test, P<0.05).
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5
and Chown (2006) [Afrinus sp. (Coleoptera: Tenebrionidae)], Kelty
(2007) [Drosophila melanogaster Meigen (Dip.: Drosophilidae)],
Terblanche et al. (2007) [(Glossina pallidipes Austin (Dip.:
Glossinidae)], Ju etal. (2011) [Corythucha ciliata (Say) (Hemiptera:
Tingidae)], Overgaard etal. (2011) (D.melanogaster), Findsen etal.
(2013) (L.migratoria), and Everman etal. (2018) (D.melanogaster).
However, these ndings do not support by the results of Izadi etal.
(2019) (P.interpunctella and E. ceratoniae) and Mohammadzadeh
and Izadi (2018a) (Tro. granarium). Results of Shintani and Ishikawa
(2007) indicated that the effect of CA and RCH in Psacothea hilaris
(Pascoe) (Coleoptera: Cerambycidae) eggs persisted for a few weeks
and a few days, respectively.
RCH and CA increase the insect’s tolerance to acute and chronic
cold shocks, respectively. These processes play important roles in the
enhancement of the insect’s survival in response to rapid (RCH) and
gradual decreases (CA) in ambient temperature (Lee et al. 2006).
Our results showed the lowest CCRT for CA and the highest for
control and RCH. In addition, compared with the control, the lowest
and the highest LLTs were recorded for RCH and CA, respectively.
On the other hand, RCH had no signicant effect on CCRT but sub-
stantially decreased the LLT, whereas CA had no signicant effect on
LLT but signicantly reduced the CCRT of the pest. Therefore, it is
obvious from our results that adult T.castaneum have the ability to
rapidly enhance their cold tolerance under RCH regime. In fact, RCH
allows this pest to quickly protect itself against the deleterious effects
of acute cold shock. In agreement with our ndings, Lee etal. (2006)
reported the signicant effect of RCH on the survival of summer-
acclimatized larvae of an Antarctic midge, Belgica antarctica Jacobs
(Diptera: Chironomidae). Ju etal. (2011) documented that RCH in-
creased the survival of the sycamore lace bug, Co. ciliata. Izadi etal.
(2019) showed that RCH had the highest impact on the LLT of the
larvae of P.interpunctella and E. ceratoniae. However, our results
did not support by results of Findsen etal. (2013) who reported that
RCH improved CCRT in the migratory locust, L. migratoria and
Srithiphaphirom (2016) who found that RCH at 4°C reduced CCRT
in L.migratoria.
Our results suggested that CA can improve the chill-coma toler-
ance and reduce the CCRT of T.castaneum. These ndings are con-
sistent with the results of Srithiphaphirom (2016) who found that
acclimation of L.migratoria at 10°C signicantly reduced the CCRT.
Moreover, our results showed that RCH, FA, and CA can improve
cold tolerance of T.castaneum but the impact of CA is substantially
greater than that of the others. Our results indicated that in all the
treatments most of the mortality occurred at a temperature above
the SCP of the beetles. Therefore, T.castaneum could be categorized
as a chill-susceptible insect. In this group of insects, the measure-
ment of the CCRT is a way for the determination of cold tolerance
(Findsen etal. 2013, 2014) and, on the other hand, CCRT is a metric
of cold tolerance (Scharf etal. 2014). However, Knapp etal. (2018)
demonstrated that CCRT was not a reliable indicator of cold toler-
ance in Harmonia axyridis Pallas (Coleoptera: Coccinellidae).
In conclusion, our results indicated that CA substantially re-
duced the SCP and enhanced survival of T.castaneum. The decrease
in the SCP and increase in cold tolerance were accompanied by
an elevation in cryoprotectant (trehalose and myo-inositol) levels.
Moreover, CA reduced the CCRT, whereas the LLT was crucially
affected by RCH.
Acknowledgments
The authors are grateful to the research vice presidency of Pistachio Safety
Research Center, Rafsanjan University of Medical Sciences for a grant to
Dr. Izadi.
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Lethal time (hours)
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FA 1,575 3.28± 20 60.9 (55.0–66.1) 87.9 (82.4–93.2) 216.1 (197.3–241.8)
RCH 1,350 2.98± 0.21 52.1 (46.2–57.3) 78.0 (72.5–83.4) 209.6 (186.8–243.0)
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Journal of Economic Entomology, 2019, Vol. XX, No. XX
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