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


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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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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@
Subject Editor: Thomas Phillips
Received 21 December 2018; Editorial decision 21 March 2019
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
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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
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.
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
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).
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
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,
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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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.
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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Table 2. LT30, LT 50, and LT90 values of Tribolium castaneum adults following different thermal regimes
Treatments naSlope ± SE
Lethal time (hours)
LT30 (95% FL) LT50 (95% FL) LT90 (95% FL)
C 1,305 2.83± 0.21 43.2 (37.5–48.2) 66.1 (60.6–71.2) 187.0 (166.4–217.3)
CA 1,935 4.55± 0.29 103.5 (96.4–109.7) 134.9 (129.0–140.5) 257.8 (241.0–280.2)
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)
Lethal times and 95% ducial limits (FLs) were estimated using logistic regression (SAS Institute 2011).
aThe total number of larval used for bioassay test.
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... Insects are typical poikilothermous animals, in that their body temperature is profoundly affected by the ambient climate [10,11]. Temperature is a key parameter that strongly determines the dynamics of insect survival, abundance, and geographical distribution [3,[12][13][14]. ...
... Too-high temperatures may positively or negatively impact their mating, oviposition, and thermotactic behavior [16], while low temperature stress may inhibit the activity of bio-macromolecules in vivo, reduce rates of metabolism, affect both development and reproduction, and even disturb the sex ratio balance of insects [17]. In general, cold and heat tolerance are important eco-physiological traits related directly to the fitness, survival, and distribution of invasive insects [11,18,19]. Notably, cold tolerance is a vital strategy to prevent cold injuries in insects during harsh winter conditions [13,14,19]. ...
... Notably, cold tolerance is a vital strategy to prevent cold injuries in insects during harsh winter conditions [13,14,19]. The supercooling point (SCP), defined as the temperature at which point an insect's body fluids spontaneously freeze, is a key indicator for evaluating the cold tolerance of many insect species [11,13,14,[20][21][22]. The SCP is predetermined by the insect's inherent biological characteristics; these are closely linked to their body composition and physiological status, e.g., feeding stage, diapause, life stage, and overwintering by freezing avoidance [23]. ...
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Bactrocera tau (Walker) (Diptera: Tephritidae) is a serious, economically important invasive pest that has spread and been established in many regions worldwide. Temperature is a crucial abiotic factor governing insect activity, fitness, and geographical distribution. Yet, surprisingly, the tolerance of B. tau to extreme cold and heat stress remains unclear. Here, we measured the supercooling point (SCP) of different life stages of B. tau. Further, several life stages of B. tau (egg, 1st, 2nd, and 3rd instar larvae, 1-day-old pupae, and 3-day-old adult) were subjected to six low temperatures (−9, −7, −5, −3, −1, and 0 °C) and six high temperatures (39, 40, 41, 42, 43, and 44 °C) for various durations (0.5, 1.0, 2.0, and 4.0 h), and three-way survival–time–temperature relationships were investigated. We found that the SCPs differed significantly among different life stages of B. tau, being the lowest for SCP of eggs, at −25.82 ± 0.51 °C. There was no significant effect of sex on the mean SCPs of B. tau adults, except for 45- to 50-day-old flies. In addition, an interaction effect was uncovered between tested temperatures and exposure duration upon B. tau mortality at different life stages. Eggs exhibited the strongest cold tolerance, yet the weakest heat tolerance. The 3rd instar larvae were the most heat- and cold tolerant among larval stages, followed by the 2nd and 1st instar larvae. The upper limit of the chill injury zone (ULCIZ) for 3-day-old adult and 1-day-old pupae was −2.51 °C and −2.50 °C, respectively, while their corresponding lower limit of thermal injury zone (LLTIZ) was 39.39 °C and 38.29 °C. This paper presents valuable data to provide an integrated knowledge for understanding the cold and heat tolerance potential of B. tau and ensure the proper implementation of post-harvest phytosanitary protocols for this pest’s disinfestation.
... Rapid cold hardening (RCH) and long-term cold acclimation (LCA) are potential treatments for achieving improved locomotion, survival, and reproduction in mass-reared beneficial insects when released into low-temperature environments (Kristensen et al., 2007;Boersma et al., 2019). Physiological adaptations to cold include RCH and LCA, which are induced by exposure to appropriate non-lethal low temperatures, and can improve both critical thermal limits and performance at low temperatures Shearer et al., 2016;Gerken et al., 2018;Izadi et al., 2019). For example, acclimation at non-lethal low temperature for either 7 days during adulthood or the whole life stage of Drosophila suzukii adults decreased the chill coma recovery time and increased survival to acute cold stress (Enriquez et al., 2018). ...
... In Gryllus pennsylvanicus, ion-regulatory tissues had reduced chilling injury after cold acclimation (Des Marteaux et al., 2017). As for RCH, the increased survival to acute cold stress seems to be mediated by mechanisms shared with those of cold acclimation (Izadi et al., 2019), e.g. ion transport mechanisms (Armstrong et al., 2012;MacMillan et al., 2014;Coello Alvarado et al., 2015;Overgaard and MacMillan, 2017) and accumulation of cryoprotectants (Storey et al., 1981;Park and Kim, 2014). ...
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... A low SCP is necessary to resist damage caused by low temperatures (Izadi et al., 2019). At the end of diapause, the environmental soil temperature dropped to the lowest value (−4.23°C), while the average SCP of locust eggs rose to −22.46°C, and the SCP of more than 20% of locust eggs rose to −11.00°C, which may be related to individual development. ...
... The inositol content under cold conditions varies among different insect species. For example, the accumulation of trehalose and inositol in Tribolium castaneum acclimated to low temperatures increases the supercooling capacity (Izadi et al., 2019). After 90 days of low-temperature acclimation, the inositol content of Oedaleus asiaticus eggs increases significantly (Li, 2014). ...
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Gomphocerus sibiricus L., the dominant insect species in the alpine and subalpine grassland, overwinters with diapause at egg stage. In this study, cold tolerance and related cryoprotectants of G. sibiricus eggs were investigated. In particular, the supercooling point (SCP), water content, carbohydrates (trehalose, glucose, fructose, glycogen), polyols (glycerol, inositol, sorbitol), fat, and amino acids contents were evaluated at different developmental stages of G. sibiricus eggs collected under natural conditions. The SCPs of eggs were very low (−32.83 to −22.61°C) at mid-diapause. Water content gradually increased during development. The fructose, glycerol, and sorbitol contents were significantly higher in diapausing eggs than in early embryogenesis stage and post-diapause development stage. Glycogen content was high throughout the whole developmental period. The trehalose, glucose, and inositol contents were low during diapause compared to that in early embryogenesis stage and post-diapause development stage. There were no significant differences in the fat content of eggs among all development stages. The total amino acid contents in eggs in the early embryogenesis and at the start of diapause were higher than that in post-diapause eggs. The contents of Glu, Asp, Leu, Pro and Arg during diapause were significantly higher than those during post-diapause development. Results indicate that G. sibiricus eggs have a high supercooling capacity. Successful overwintering can be attributed to the accumulation of glycerol, fructose, sorbitol, and amino acids (Glu, Asp, Leu, Pro and Arg). These findings provide insight into the mechanisms underlying the adaptation of G. sibiricus to cold conditions. Research Highlights Gomphocerus sibiricus eggs have a high supercooling capacity. G. sibiricus eggs have strong cold resistance.
... Chill-Coma Recovery Time: Chill-coma induced by each thermal shock was recorded for each species and developmental stage tested following Izadi et al. (2019). Briefly, each set of 15 adults of T. castaneum and S. oryzae was transferred from each thermal shock to controlling chamber in sterile Petri dishes (6 cm in diameter × 1 cm height) for observation, under a stereomicroscope for the time required for coordinated movement, which was recorded with a stop watch. ...
... Survival after low-temperature shocks: The insect survival after the exposure was recorded after 24 h maintained under controlled conditions of 30 • C and 65% RH, which allowed eventual recovery, or confirmed death (Izadi et al., 2019). The adults or larvae were considered dead if no movement was observed when prodded with a small brush. ...
Insects face several (environmental) abiotic stressors, including low temperature, which cause the failure of neuromuscular function. Such exposure leads insects toa reversible comatose state termed chill-coma, but the consequences of this state for the organism biology were little explored. Here, the consequences of the chill-coma phase were investigated in two of the main stored product pest species-the red flour beetle Tribolium castaneum (larvae and adults) and the rice weevil Sitophilus oryzae (adults). For this purpose, a series of low-temperature shocks were used to estimate the chill-coma recovery time (CCRT), survival, nutrition and weight gain/ growth of T. castaneum (larvae and adults) and S. oryzae, as well as the development of T. castaneum life stages. The relatively long CCRT was characteristic of beetle larvae, at different low-temperature shocks, and CCRT increased with decreasing temperatures and increasing exposure intervals for both pest species. The survival was little affected by the low-temperature shocks applied, but such shocks affected insect feeding and growth. Tri-bolium castaneum larvae was more sensitive than adults of both insect species. Moreover, the relative consumption and weight gain of S. oryzae adults were lower than those of T. castaneum adults and mainly larvae, while feeding deterrence was not affected by low temperature shocks, unlike food conversion efficiency. Low-temperature shocks, even under short duration at some temperatures, significantly delayed development. The lower the temperature and the higher the exposure period, the more delayed the development. Thus, the physiological costs of chill-coma are translated into life-history consequences, with potential implications for the management of this insect pest species in stored products and even more so on red flour beetles and rice weevils.
... Cold acclimation is a physiological strategy that is widely adopted by ectotherms to adapt to sudden changes in temperature and has been documented in numerous insects within a temperature range of between 0 and 10 °C [31][32][33][34][35]. Among coleopteran species, it has been established that in several species, a period of cold acclimation can contribute to enhancing their cold tolerance [36][37][38], and studies that examine plastic responses such as cold acclimation will contribute to predicting the effects of climate change on species distribution and survival [39]. In the present study, we found that both the males and females of A. hygrophila undergo cold acclimation and that individuals thus acclimated are characterized by significant reductions in mortality on subsequent exposure to low temperatures. ...
Full-text available
Agasicles hygrophila Selman and Vogt is used in the biological control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, with the northward establishment of A. philoxeroides in China, the weak adaptivity of A. hygrophila to cold weather has resulted in the ineffective control of A. philoxeroides in northern China. Cold acclimation can significantly enhance insect cold tolerance, enabling them to cope with more frequent climate fluctuations. To improve the biological control efficacy of A. hygrophila in cold climates, we compared the effects of rapid cold hardening and acclimation on A. hygrophila under laboratory conditions. On initially transferring adults from 26 to −10 °C for 2 h, mortality reached 80%. However, when pre-exposed to 0 °C for 2 h and then transferred to −10 °C for 2 h, adult mortality was reduced to 36.67%. These findings indicate that cold acclimation can enhance the cold tolerance of A. hygrophila under laboratory conditions. However, the beneficial cold acclimation effects waned after more than 15 min of recovery at 26 °C. Exposure to 15 °C for 24 h or gradual cooling from 0 to −10 °C at 1 °C·min−1 also induced cold acclimation, indicating that long-term cold and fluctuating cold acclimation are also potentially effective strategies for enhancing low-temperature tolerance.
... Stress, in the form of starvation, heat or cold shock, or combinations thereof (Shostak et al. 2015), has been experimentally applied to show negative effects on reproductive output and behaviour, movement patterns, and immune response (Sbilordo and Gräzer 2011;Eggert et al. 2015;. Alternatively, effects of tolerance to stress can be the response variable, and this has been used to show that the ability to tolerate stress is affected by a range of factors including parental age , thermal acclimation regime (Izadi et al. 2019) and rearing conditions . Later-life effects of natal/juvenile stress have also been shown in Tribolium, with the natal environment affecting adult dispersal (Van Allen and Bhavsar 2014), competitive dynamics (Van Allen and Rudolf 2015), productivity and rates of cannibalism (Boyer 1976). ...
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Flour beetles of the genus Tribolium have been utilised as informative study systems for over a century and contributed to major advances across many fields. This review serves to highlight the significant historical contribution that Tribolium study systems have made to the fields of ecology and evolution, and to promote their use as contemporary research models. We review the broad range of studies employing Tribolium to make significant advances in ecology and evolution. We show that research using Tribolium beetles has contributed a substantial amount to evolutionary and ecological understanding, especially in the fields of population dynamics, reproduction and sexual selection, population and quantitative genetics, and behaviour, physiology and life history. We propose a number of future research opportunities using Tribolium , with particular focus on how their amenability to forward and reverse genetic manipulation may provide a valuable complement to other insect models.
The cowpea weevil, Callosobruchus maculatus (F.) (Col.: Bruchidae) is a cosmopolitan field-to-store pest ranked as the major post-harvest pest of cowpea in tropical regions. The cold tolerance of an insect species can vary as a result of abiotic features including food resources. In this study, C. maculatus larvae were fed with proline and trehalose (10, 20, and 40 mmol) treated cowpea seeds to determine the effects of these potential cryoprotectants on the supercooling (SCP) and cold hardiness of the upcoming adult beetles. The SCPs of the control, proline-fed and trehalose-fed adults non-significantly changed from −18.2 °C for the control to −17.2 °C for trehalose-fed adults. The cold hardiness (24 h at 0, -5.0, −7.5, −10.0, and −12.5 °C) of the adults was almost the same for control and treatments. Median lethal times (LT50; lethal time for 50% mortality) were 6.3, 6.0, and 5.4 h, respectively. Moreover, feeding the larvae with proline and trehalose-treated seeds did not affect the proline and trehalose contents of the adult beetles. Our results showed that C. maculatus could not tolerate subzero temperatures well above their SCP, indicating that this species might be a chill-susceptible insect.
The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae) is a very destructive polyphagous insect pest of cereal crops accounting for up to 100% yield losses. To survive winter low temperatures in the Americas, adults FAW are known to migrate south for warmer climates and then re‐invade northern USA and Canada the following summer. Since its African invasion, no studies have looked at its overwintering biology. Specifically, there is no information on in situ ontogenetic low temperature tolerance, despite its significance in explaining overwintering survival. Here, we thus investigated low temperature tolerance of FAW larvae (3rd, 4th, 5th and 6th) and adults from field populations through assessing basal stress tolerance (critical thermal minima [CTmin], supercooling point [SCP] and chill coma recovery time [CCRT]) and plasticity using standardized protocols. Our results showed significant life stage effects on low temperature tolerance, although all were chill‐susceptible. Adults had lower CTmin and CCRT than larvae (higher cold tolerance). However, early instar larvae had significantly depressed SCPs than later instars and adults. All larval instars tested showed no plastic responses to CTmin, while for adults, cold hardening appeared to come at a cost of CTmin. These results suggest ontogenetic differences in S. frugiperda cold hardiness albeit all are susceptible to chilling. Second, the absence or cost of hardening confirms FAW's maladaptation to low temperatures. However, Botswana microclimate records show that severe low temperature stress is limited, and thus, in situ overwintering is possible. These results are important in developing informed pest management options for effective management of FAW in Africa.
BACKGROUND For many insects, including invasive species, overwintering survival is achieved behaviourally (e.g. through migration) or physiologically by entering diapause, a state of arrested physiological development that may be accompanied with depressed supercooling points (SCPs). Diapause allows in situ adaptation to adverse environmental conditions, providing sufficient parent propagules for insect pest proliferation when optimal conditions resurface. This phenomenon has however not been observed in the invasive South American tomato pinworm Tuta absoluta in its Mediterranean invaded areas. Moreover, no studies have looked at its overwintering survival in sub‐Saharan Africa. Here, we thus investigated the cold hardiness of T. absoluta larvae and adults to better explain its local overwintering adaptation strategy. RESULTS Larval lower lethal temperatures ranged from ‐1°C to ‐17°C for 0.5‐4h durations. Adults showed lower temperature activity limits than larvae albeit freeze strategy experiments showed neither survived internal freezing. Fasting and dehydration pre‐treatment generally depressed SCPs, although asymmetrically, conferring more negative SCPs for larvae. Ramping rates, synonymic to diurnal temperature changes also significantly affected SCPs while, inoculative freezing significantly compromised freezing temperatures in both larvae and adults. CONCLUSION Our results suggest that (1) T. absoluta larvae and adults are chill‐susceptible and may successfully overwinter, (2) larvae appear more cold hardy than adults and (3) ecological factors e.g. inoculative freezing, cooling rates, feeding‐ and hydration ‐status may affect cold hardiness. These results are important in determining species range limits, population phenology, modelling pest risk status and allows temporal life‐stage specific targeting of management strategies. This article is protected by copyright. All rights reserved.
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In this study, cold hardiness and some physiological characteristics of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae) larvae, were investigated under different thermal regimes, i.e., control, cold-acclimated (CA), fluctuating-acclimated (FA), and rapid cold-hardened (RCH). In all the regimes, the larval survival rate decreased with a decrease in temperature. CA larvae showed the highest cold hardiness following 24 h exposure at -15 and -20°C. Control larvae had the highest glycogen content (34.4 ± 2.3 μg/dry weight). In contrast, CA larvae had the lowest glycogen content (23.0 ± 1.6 μg/dry weight). Change in trehalose content was reversely proportional to changes in glycogen content. The highest myo-inositol and glucose contents were detected in CA larvae (10.7 ± 0.4 μg/dry weight) and control (0.49 ± 0.03 μg/dry weight), respectively. In control and treated larvae, [Na+] decreased, though [K+] increased, with increasing exposure time. The shape of the thermal reaction curve of AMP-depended protein kinase and protein phosphatase 2C followed the same norm, which was different from protein phosphatase 1 and protein phosphatase 2A. Protein phosphatase 2A and 2C showed a complete difference in thermal reaction norms. Indeed, thermal fluctuation caused the highest changes in the activity of the enzymes, whereas the RCH showed the lowest changes in the activity of the enzymes. Our results showed a significant enhancement of larval cold tolerance under CA regime, which is related to the high levels of low molecular weight carbohydrates under this regime. Our results showed that among the different thermal regimes tested, the CA larvae had the lowest supercooling point (about -22°C) and the highest cold hardiness following 24 h exposure at -15 and -20°C.
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Fitness is determined by the ability of an organism to both survive and reproduce; however, the mechanisms that lead to increased survival may not have the same effect on reproductive success. We used nineteen natural Drosophila melanogaster genotypes from the Drosophila Genetic Reference Panel to determine if adaptive plasticity following short-term acclimation through rapid cold-hardening (RCH) affects mating behavior and mating success. We confirmed that exposure to the acclimation temperature is beneficial to survival following cold stress; however, we found that this same acclimation temperature exposure led to less efficient male courtship and a significant decrease in the likelihood of mating. Cold tolerance and the capacity to respond plastically to cold stress were not correlated with mating behavior following acclimation, suggesting that the genetic control of the physiological effects of the cold temperature exposure likely differ between survival and behavioral responses. We also tested whether the exposure of males to the acclimation temperature influenced courtship song. This exposure again significantly increased courtship duration; however, courtship song was unchanged. These results illustrate costs of short-term acclimation on survival and reproductive components of fitness and demonstrate the pronounced effect that short-term thermal environment shifts can have on reproductive success.
The harlequin ladybird, Harmonia axyridis, is one of the most successful invasive insect species worldwide. We investigated whether (i) chill coma recovery time (CCRt) changes during the ontogenetic development of this species, (ii) CCRt varies in response to repeated cold shocks, and (iii) CCRt could be a good predictor of winter survival ability in adults. CCRt decreased during larval development, the lowest CCRt values were observed in teneral adults (one and four days old), and significantly higher values were observed for older adults (16 and 32 days old). Repeated cold shocks (two hours at −3 °C), interrupted by short (30 min) warm periods (22 °C) resulted in decreased CCRt after the second cold shock, probably depicting an acclimation response, but then CCRt increased with additional cold shocks, likely revealing the accumulation of chill injuries. The CCRt of pre-overwintering individuals was not correlated with their winter survival. This indicates that CCRt is not a reliable measure of cold tolerance in H. axyridis. However, this result could be partially affected by the experimental setup – the use of laboratory-reared individuals who experienced standardized conditions and thus the variability in CCRt of tested animals could be much lower than the variability present in nature. The substantial variation observed over the ontogenetic development of H. axyridis poses important methodological implications for future studies, as animals of the same stage/age should be compared with each other. The observed U-shaped response to repeated cold shocks indicates that the expectation of linearity between cold exposure and insect response is an oversimplification of real situations.
Measuring the supercooling point (SCP) is a standard procedure to describe the cold tolerance of freeze-avoiding arthropods. The SCP of an individual animal is a stochastic event that will occur with increasing probability as the temperature is lowered below the freezing point of that animal. Nevertheless, the repeatability and extent of stochasticity of the SCP has not previously been determined. The repeatability of the SCP in post-diapause, laboratory cold-acclimated and naturally acclimated field-collected linden bugs (Pyrrhocoris apterus; Heteroptera: Pyrrhocoridae) was investigated in this study. Two methods were used: (a) repeated freezing of previously frozen and thawed individuals, and (b) repeated cooling of groups of individuals to the population median SCP. The results showed a significant positive correlation between the SCP and repeated SCP. All individuals died when frozen, whereas none died at temperatures above the SCP. Most of the individuals survived repeated cooling to the population median SCP. Survivorship increased from 85% to 97% (first to fourth repeated cooling to the population median SCP) when individuals were frozen to within 0.5 °C above the population median SCP. The SCP in post-diapause, cold-acclimated insects is a fixed, intrinsic cold tolerance metric with slight individual stochastic variance (SD < 1 °C).
Trogoderma granarium Everts (Coleoptera: Dermestidae) is an important insect pest of stored products. In this study, the survival strategies of T. granarium fourth instar larvae were investigated at different sub-zero temperatures following different cooling rates, acclimation to different relative humidity (RH) and different starvation times. Our results show that larvae of T. granarium are freeze-intolerant. There was a strong link between cooling rates and supercooling point, which means the slower the decrease in temperature, the lower the supercooling point. Trehalose content was greater in insects cooled at a rate of 0.5. °C/min. According to results, the RH did not affect supercooling point. However, acclimation to an RH of 25% increased mortality following exposure to - 10. °C/24. h. The time necessary to reach 95% mortality was 1737. h and 428. h at - 5. °C and - 10. °C. The lowest lipid and trehalose content was detected in insects acclimated to 25% RH, although, the different RH treatments did not significantly affect glycogen content of T. granarium larvae. The supercooling point of larvae was gradually increased following starvation. By contrast, fed larvae had the greatest lipid, glycogen, and trehalose content, and insects starved for eight days had the lowest energy contents. There was a sharp decline in the survival of larvae between - 11 and - 18. °C after 1. h exposure. Our results indicate the effects of cooling rate and starvation on energy reserves and survival of T. granarium. We conclude that T. granarium may not survive under similar stress conditions of the stored products.
The bean bug ( Riptortus pedestris ) is a pest of soybeans and other legumes in Japan and other Asian countries. It enters a facultative adult diapause on exposure to short days. While photoperiodism and diapause are well understood in R. pedestris , knowledge of cold tolerance is very limited, as is information on the effect of diapause on cold tolerance. We examined the effect of photoperiod, cold acclimation, and feeding status on cold tolerance in R. pedestris . We found that cold acclimation significantly increased survival at −10°C in both long- and short-day adult R. pedestris . Since the difference in cold survival between long- and short-day cold-acclimated groups was only marginal, we conclude that entering diapause is not crucial for R. pedestris to successfully pass through cold acclimation and become cold tolerant. We observed similar effects in 5th instar nymphs, with both long- and short-day cold-acclimated groups surviving longer cold exposures compared with non-acclimated groups. Starvation, which was tested only in adult bugs, had only a negligible and negative impact on cold survival. Although cold tolerance significantly increased with cold acclimation in adult bugs, supercooling capacity unexpectedly decreased. Our results suggest that changes in supercooling capacity as well as in water content are unrelated to cold tolerance in R. pedestris . An analysis of metabolites revealed differences between the treatments, and while several metabolites markedly increased with cold acclimation, their concentrations were too low to have a significant effect on cold tolerance.
Spodoptera exigua is a secondary target pest of Bt cotton commercialized in China. With the continuous adoption of Bt cotton, populations of S. exigua have gradually increased. However, the cold tolerance ability of Bt-resistant S. exigua and the effect of continuous Bt diet on anti-cold materials are unknown. In our study, it was found that Bt-resistant S. exigua (Bt10) developed better with shorter larval and pupal duration and higher pupation rate compared to CK at the suboptimal low temperature. The supercooling points and freezing points of the Bt-resistant S. exigua strain were determined, and body water content and anti-cold materials such as total sugar, trehalose and glycogen, glycerol and fat were examined to explore the effect of Bt toxin on overwintering and on population increase. The results showed that the supercooling point and the freezing point of the Bt-resistant S. exigua pupae were both significantly lower than that of the Bt-susceptible strain. No difference was found in the body water content of pupae and adults between the two strains. Total sugar content of the Bt-resistant strain at both the pupal and adult stages was higher than that of the susceptible strain at the corresponding stages, and glycogen content of the Bt-resistant strain at the larval stage was higher than that of the susceptible larval S. exigua. Fat content of the Bt-resistant larvae, pupae and adults was for each higher than that of the susceptible strain, but the difference was not significant except for that of the 3rd instar larvae. Glycerol content of the Bt-resistant strain at larval, pupal and adult stages was for each higher than that of the corresponding life stages of the susceptible strain. It can be seen that more glycerol was accumulated in Bt-resistant S. exigua. The results indicate that Bt-resistant S. exigua has better cold tolerance. The contents of the anti-freeze substances of progeny, especially glycerol, were increased after previous generations were continuously fed on Bt protein, which means that the Bt-resistant secondary target pests could more easily overcome the overwinter season and become a source of crop damage the following year.
The Russian wheat aphid, Diuraphis noxia (Kurdjumov), is one of the most important pests of wheat and barley in most wheat-producing countries. Rapid cold hardiness (RCH) is a capacity of insects to develop, within hours, protection against subfreezing temperatures that plays an important role in aphid survival in response to sudden decreases in air temperature. In this research, we investigated the duration and rate of cooling on the induction of RCH of D. noxia and the costs of RCH on aphid development and fecundity. By transferring aphids directly from 20 °C to a range of subzero temperatures for 2 h, the lower lethal temperature for 80% mortality (LT80) was determined to be - 11.9 °C. Preconditioning the aphids at 0 °C for 1-3 h prior to exposure at (LT80) (-11.9 °C) resulted in a sharp increase in survival, with little change with longer durations of preconditioning. The slowest cooling rate (0.05 °C/min) increased survival fourfold, whereas rates from 0.1 to 1 °C/min increased survival twofold compared with a direct transfer to 0 °C, regardless of aphid stage used. Deleterious effects of RCH were not observed on aphid development, longevity, or fecundity. The present study indicates that RCH is induced in D. noxia in just a few hours in response to sudden lowering of temperatures to freezing, with little or no cost in reproductive capacity.