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Effects of seasonal acclimation on cold tolerance and biochemical status of the carob moth, Ectomyelois ceratoniae Zeller, last instar larvae
Abstract
The carob moth, Ectomyelois ceratoniae, a pest of Punica granatum, overwinters as a larva. In this study, physiological changes, water content, cold hardiness and supercooling points (SCPs) in relation to ambient temperature in the overwintering period (October to March) and changes of these factors between diapausing (February) and non-diapausing (September) larvae were studied. Pupae that were derived from diapausing larvae (April) and from non-diapausing larvae (August) were also compared. Total body sugar, lipid and protein contents increased with decrease in the temperature and reached the highest levels (12.82, 1.99 and 6.11 mg g-1 body weight, respectively) in February, but glycogen content decreased and reached the lowest level (1.12 mg g-1 body weight) in February. There were significant differences in the levels of these compounds between diapausing and non-diapausing larvae, and pupae that were derived from diapausing and non-diapausing larvae. Trehalose and myo-inositol contents increased during diapause and reached the highest levels (0.50 and 0.07 mg g-1 body weight, respectively) in February. There were significant differences in the levels of these compounds between diapausing and non-diapausing larvae, but the differences between pupae that were derived from diapausing and non-diapausing larvae were not significant. The SCP of diapausing larvae (-17.3 °C) was significantly lower than in the non-diapausing larvae (-12.0 °C). SCP decreased gradually in autumn and reached the lowest level in the middle of winter. Changes of cold hardiness were inversely proportional to SCP changes. The lowest levels of water (65%) and weight (43.13 mg) were recorded in January and March, respectively. Most probably, lipids play a role as energy reserve, and low-molecular weight carbohydrates and polyols provide cryoprotection for overwintering larvae of the carob moth. Since the overwintering larvae die at temperatures above the SCP, the carob moth larvae were found to be a chill-intolerant insect.
... Diapausing insects synthesize and hoard various need-based biochemical constituents like polyhydric alcohols, sugars, amino acids, lipids, and fatty acids (Goto et al., 1998;Koštal et al., 2007;Mohammadzadeh et al., 2017). These biochemical compounds provide tolerance against adverse environmental conditions (Heydari & Izadi, 2014;Teets & Denlinger, 2013), through changes in cuticular composition and enzymatic activities (Chown & Terblanche, 2007;Clarke & Worland, 2008). ...
... The higher amounts of amino acids namely, serine, glycine, histidine, arginine, proline, tyrosine, and methionine in estivation, while lower in hibernation as compared with nondiapause larvae of C. partellus suggest that these amino acids could be constituents of certain heat-shock proteins to enhance stress tolerance and maintain development during estivation. The increase in protein content particularly the heatshock proteins during winter diapause have also been reported in some insect species (Ding et al., 2003;Hahn & Denlinger, 2007;Heydari & Izadi, 2014;Rinehart et al., 2007;Yocum, 2001). ...
... In some insects, the diapause larvae have the ability to reserve energy in the form of lipids (Behroozi et al., 2012;Heydari & Izadi, 2014;Sadeghi et al., 2012), while others do not rely on lipids as source of energy during the winter diapause (Goto et al., 1998;Khanmohamadi et al., 2016;Koštal et al., 1998;Mollaei et al., 2016). ...
The Chilo partellus (Crambidae: Lepidoptera) larvae undergoes both hibernation and estivation in India. Although, much has been done on reproductive physiological aspects, little is known about biochemical changes happening during hibernation and estivation in C. partellus. Thus, we mapped changes in amino acid and lipophilic profiles of C. partellus larvae while undergoing hibernation and estivation using high-performance liquid chromatography and gas chromatography mass spectroscopy. The studies revealed higher amounts of amino acids namely, serine, glycine, histidine, arginine, proline, tyrosine, and methionine in estivation, while lower in hibernation as compared with nondiapause larvae of C. partellus. Furthermore, the amounts of aspartic acid, glutamic acid, and alanine in hibernation, and threonine, valine, isoleucine, phenylalanine, and leucine in estivation were on par with nondiapause larvae. The lipophilic compounds namely, linoleic acid, stearic acid, eicosanoic acid, and n-pentadecanol were lower in hibernation than estivation and nondiapause larvae of C. partellus. Palmitoleic acid and methyl 3-methoxytetradecanoate contents were higher in hibernation than estivation and nondiapause, while myristic acid and lathosterol contents were higher in estivation than hibernation and nondiapause larvae of C. partellus. Cholesterol content was higher, while squalene and gamma-ergostenol were lower in hibernation and estivation as compared with nondiapause larvae of C. partellus. These findings suggest that certain amino acids may be constituents of heat-shock proteins and help C. partellus during estivation. However, the lipophilic compounds could be helpful in maintaining development during hibernation and estivation in C. partellus.
... The relationship between these two strategies has remained unclear. Cold hardiness may be achieved independent of diapause (Khanmohamadi et al. 2016;Mollaei et al. 2016;Mohammadzadeh et al. 2017), or it can be a component of the diapause syndrome (Bemani et al. 2012;Heydari and Izadi 2014;Lee 1991;Milonas and Savopoulou-Soultani 1999). ...
... Insects usually sequester sufficient energy reserves, such as carbohydrates and lipids, before entering diapause (Hahn and Denlinger 2010;De Barro et al. 2011). The accumulation of food resources has well been documented in many overwintering insects (Behroozi et al. 2012;Sadeghi et al. 2012;Bemani et al. 2012;Heydari and Izadi 2014). ...
... The same results were reported regarding the pistachio white leaf borer, Ocneria terebinthina Strg. (Lepidoptera: Lymantriidae) (Behroozi et al. 2012), the pistachio fruit hull borer, Arimania comaroffi (Ragonot) (Lep.: Pyralidae) (Bemani et al. 2012), the common pistachio psylla, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae) (Sadeghi et al. 2012), the carob moth, Ectomyelois ceratoniae Zeller (Lep.: Pyralidae) (Heydari and Izadi 2014), the almond wasp, Eurytoma amygdali (Hymenoptera: Eurytomidae) (Khanmohamadi et al. 2016), and the pistachio twig borer, Kermania pistaciella (Lepidoptera: Tineidae) (Mollaei et al. 2016). Based on the results, it seems that the diapausing eggs of the L. malicola used glycogen as the source of the energy reserve and the main source of total simple sugars and polyols. ...
As the key pest of apple fruits, the oystershell scale, Lepidosaphes malicola Borchsenius (Hem.:
Diaspididae), overwinters as diapausing eggs under the protective, waxy cover of females. In this
research, the effects of diapause development, cold acclimation, and rapid cold hardening were studied
on the cold hardiness of the eggs. The changes in some physiological components were also investigated.
The results indicated cold exposure to be a prerequisite for the survival of the diapausing eggs of L.
malicola. No eggs hatched without exposure to cold. In addition, a direct relationship was observed among
cold hardiness, cold acclimation, and diapause of the eggs based on the results. The highest level of
hatching (the highest cold hardiness) of the eggs (80%) occurred in the cold-acclimated eggs at the end
of diapause (March). Rapid cold hardening also influenced the cold hardiness of the eggs with diapause
development. At the end of diapause, the lowest (61%) and the highest (77%) rates of egg survival were
observed when the eggs were exposed to 5 and -10°C for 24 h, respectively. Cold hardiness of the
diapausing eggs of L. malicola was also accompanied by some physiological changes, i.e., a decrease in
glycogen content and an increase in simple sugar, lipid, and protein contents. The lowest glycogen content
(about 50 µg/g) and the highest amounts of total simple sugars (454 µg/g) of lipids (542 µg/g) and proteins
(84 µg/g) were observed in the cold-acclimated eggs at the end of diapause.
... However, the relationship between these two strategies is not clear. In some insects, these two are independent phenomena (Goto et al., 2001;Khanmohamadi et al., 2016;Mollaei et al., 2016;Mohammadzadeh et al., 2017), while in some other insects, cold hardiness is a component of the diapause syndrome (Milonas, 1999;Bemani et al., 2012;Heydari and Izadi, 2014;Cira et al., 2018). In most insects, development of cold hardiness is highly associated with some physiological adaptations, e.g., synthesis and accumulation of low-molecularweight carbohydrates and polyols (cryoprotection) (Behroozi et al., 2012;Vrba et al., 2017;Mohammadzadeh and Izadi, 2018a,b;Izadi et al., 2019), synthesis of antifreeze proteins or carbohydrates (Costanzo and Lee, 2013;Khanmohamadi et al., 2016;Mollaei et al., 2016), use of cryoprotective dehydration (Elnitsky et al., 2008;Clark et al., 2009;Worland et al., 2010), and regulation of ice nucleation (Costanzo and Lee, 2013). ...
... The adults (n = 6 per month) were separately weighed and dried in an oven at 65 • C for 72 h. The water content (w/w) was obtained from subtracting dry weight from fresh weight and then dividing the result by fresh weight (Lehmann et al., 2012;Heydari and Izadi, 2014). ...
... Glucose (Sigma) was used as a standard. This experiment was carried out per month using six adults, each as a replicate (Heydari and Izadi, 2014;Mohammadzadeh and Izadi, 2016). ...
As a serious pest of wheat, the Sunn pest, Eurygaster integriceps Puton (Hem.: Scutelleridae), is prevalent in Iran. This pest belongs to univoltine species and tends to estivate and overwinter in high altitudes of nearby mountains as diapausing adults. The economic importance of the crop was attacked by this pest, i.e., wheat led the authors to study the physiological adaptations of these diapausing adults, that is, changes in the supercooling point (SCP), in the accumulation of cryoprotectants, and in the activities of the related enzymes in relation to diapause development. The mean SCP of the diapausing adults was found to be −8°C. The lowest SCP, i.e., approximately −11°C, was observed in the middle of diapause, October, when the highest cold hardiness was also interestingly recorded. This finding proposed that SCP depression could be a feasible cold-tolerance strategy for diapausing adults. The sugar content was high in the initiation and at the termination of diapause and was low during diapause maintenance. These sugar reserves were most likely utilized to be converted to glycogen and lipid during diapause maintenance as a survival strategy. The changes in the glycogen and lipid contents were inversely proportional to the changes in the total sugar content. The authors also found that the changes in the glycogen content were directly proportional to those in the low-molecular-weight carbohydrates (e.g., glycerol and trehalose) and in the diapause development. This finding underlined the role of the low-molecular-weight carbohydrates, such as the cryoprotectants, in enhancing the cold tolerance of the given insect. In this study, the diapause-associated changes in the activities of α-amylases and proteases were also investigated. The results showed that the enzyme activities were related to diapause development and cold-tolerance enhancement. The highest enzyme activity was observed in September. Since the overwintering adults of the Sunn pest could not tolerate temperatures below their SCPs, they were grouped in the freeze-intolerant species.
... accumulation of low molecular weight carbohydrate that act as a cryoprotectants) to mitigate detrimental effects of cold on cell organelle and membrane injuries. Cryoprotectants have a series of functions, i.e. prevention of intracellular volume from falling below a critical minimum, stabilization of the membrane bilayer structure and lowering the supercooling point (SCP) of some insect species (Storey, 1997;Holmstrup et al., 2002;Wang and Kang, 2005;Bemani et al., 2012;Heydari and Izadi, 2014;Mohammadzadeh and Izadi, 2016). ...
... The whole-body glycogen and polyol content of five larvae from each acclimation regime were measured. Glycogen content was estimated using a modified anthrone method as described in Heydari and Izadi (2014). Briefly, larvae were weighed and homogenized in 200 µl of 2% Na 2 SO 4 and 1300 µl of chloroform-methanol (1:2) was added to the homogenate. ...
... Sugar alcohol content was 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, Ireland) as described by Heydari and Izadi (2014). Briefly, larvae were weighed and homogenized in 1.5-2 ml of 80% ethanol and centrifuged for 15 min at 12000 ×g. ...
... The glycogen content of the larvae was estimated using the modified anthrone method as described by Heydari and Izadi (2014). The larvae were weighed and homogenized in 200 µl of 2% Na 2 SO 4 . ...
... They showed several metabolic adaptations in freeze-tolerant larvae of the goldenrod gall fly for subzero survival. Several other studies reported this sugar as a cryoprotectant in cold hardy insect species (Behroozi et al., 2012;Bemani et al., 2012;Sadeghi et al., 2012;Heydari and Izadi, 2014;Mohammadzadeh and Izadi, 2016). In agreement with our results, Mohammadzadeh et al. (2017) showed that high cold tolerance of larvae of Eurytoma plotnikovi (Hym.: Eurytomidae) was not associated with accumulation of cryoprotectants during overwintering. ...
... In agreement with our results, Mohammadzadeh et al. (2017) showed that high cold tolerance of larvae of Eurytoma plotnikovi (Hym.: Eurytomidae) was not associated with accumulation of cryoprotectants during overwintering. Enhancement of cold tolerance in insects mostly relies on colligative effects through accumulation of high concentration of cryoprotectants such as trehalose and glycerol to depress SCP (Bemani et al., 2012;Hayward et al., 2014;Heydari and Izadi, 2014). In some insects, if cold exposure induces accumulation of cryoprotectants and subsequently elevates cold hardiness of the insect, SCP remains unchanged. ...
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.
... For the unfavorable thermal conditions, an insect synthesizes and accumulates various types of low-molecular-weight compounds, such as polyhydric alcohols (polyols), sugars, and some of the amino acids (Teets and Denlinger 2013). The synthesis of these compounds whose main task is to inhibit the growth of ice lattice has been reported in some insects for resistance to future hazardous changes of environmental conditions (Hodkova and Hodek 2004, Andreadis et al. 2011, Bemani et al. 2012, Wasielewski et al. 2013, Heydari and Izadi 2014. ...
... According to our findings, lipid content was higher in diapausing larvae than that in nondiapausing larvae, which means that diapausing larvae have the ability to reserve energy in the form of lipids and utilize it during repressed metabolism of overwintering period. Same results have been reported by Behroozi et al. (2012), Sadeghi et al. (2012), Bemani et al. (2012), and Heydari and Izadi (2014). Conversely, some other diapausing insects do not rely on lipids as the source of energy during the overwintering period (Goto et al. 1998, Koštal et al. 1998, Khanmohamadi et al. 2016, Mollaei et al. 2016). ...
... Low-molecular-weight carbohydrates and total simple sugar contents increased with decrease in the glycogen level. This suggests that glycogen in diapausing larvae is mobilized for the production of cryoprotectant molecules to decrease the SCP and increase cold hardiness (Heydari andIzadi 2014, Khanmohamadi et al. 2016). It might be attributed to correlation of glycogen content with supercooling (r = 0.678) and cold hardiness of the larvae (r = −0.729 ...
The pistachio seed wasp, Eurytoma plotnikovi Nikol'skaya (Hymenoptera: Eurytomidae), is one of the main pests in various pistachio growing regions of Iran. This pest passes the winter as diapausing last instar larvae. In this study, the relationship between diapause and cold hardiness and also the physiological and biochemical characteristics the diapausing and nondiapausing larvae of E. plotnikovi were investigated. Digestive α-amylase enzyme showed a high activity (70.41 ± 2.36 µg maltose/min per mg protein) in nondiapausing larvae, but its activity vigorously decreased during the diapause period. Glycogen declined at the beginning of diapause until March. Decrease in glycogen content was proportional to increase in total simple body sugars, trehalose, myo-inositol, and sorbitol contents. Lipid accumulated from the onset of diapause in September until January reaching a high concentration of 28.74 mg/g fresh body weight, but then declined from March to end of diapause in April. The supercooling points were decreased from August (-17.68 ± 0.14°C) to January and reached to its lowest point in January (-23.14 ± 0.27°C), the coldest month of the year, then gradually increased through April (-21.38 ± 0.32°C). The survival rates at low temperature indicate that last instar larvae of E. plotnikovi are most cold tolerant in December-February when total body sugars, trehalose, myo-inositol, and sorbitol concentration is high, suggesting an alternative cryoprotective role for these compounds. The experimental data show that E. plotnikovi is freeze avoidance insect.
... During cold period the biosynthesis of low molecular weight organic solutes, including polyols and sugars, depress the supercooling point (i.e. cryoprotectants), while also stabilizing cellular membranes and proteins (Heydari and Izadi, 2014). Such dynamism is also manifested in the changing responses to cooling rate, relative humidity, starvation and other factors as observed in numerous field and laboratory experiments (Ansart et al., 2002;Hahn et al., 2008;Hemmati et al., 2014). ...
... Optical density was read at 530 nm against a triolein (Sigma) standard in a spectrophotometer (T60U, Harlow Scientific, USA). The glycogen content of 4th instar T. granarium larvae was estimated using modified anthrone method as described in Heydari and Izadi (2014). The larvae were weighed and homogenized in 200 µl of 2% Na 2 SO 4 then, 1300 µl chloroform-methanol (1:2) was added to the homogenate. ...
... Optical density was read at 630 nm in a spectrophotometer (T60U, Harlow Scientific, USA). Trehalose content was measured in vitro using high-performance liquid chromatography (HPLC) as described by Heydari and Izadi (2014). Briefly, the larvae were weighed and homogenized in 1.5-2 ml of 80% ethanol. ...
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.
... In addition, the lack of relationship between SCP and cold hardiness confirms that SCP is not indicative of cold hardiness in this species. Heydari and Izadi (2014) similarly concluded that another (Akbarkooh, Iran) population of A. ceratoniae are chill-intolerant (a synonym of 0chill-susceptibility, Denlinger & Lee 2010); however, they did not expose the larvae to temperatures lower than ¡10 C. Chill-susceptibility has also been reported for overwintering adults of Alphitobius diaperinus (Panzer) (Colinet et al. 2011), larvae of Drosophila melanogaster (Ko st al et al. 2012), pupae of Helicoverpa zea (Boddie) (Morey et al., 2012), and the larvae of Thaumatotibia leucotreta (Meyrick) (Boardman et al. 2012). Even though the mean SCPs did not changed seasonally, the median of the SCPs of overwintering larvae slightly decreased (Figure 2), which suggests that a larger proportion of the population would be capable of surviving. ...
... C) and December 2012 (¡10.7 C). This winter decrease in lower thermal limits was previously reported in last instar A. ceratoniae (Heydari & Izadi 2014), and is typical of insects overwintering in temperate regions Denlinger & Lee 2010;Crosthwaite et al. 2011). However LT 50 of A. ceratoniae was not determined in Akbarkooh population, according to Heydari and Izadi (2014). ...
... This winter decrease in lower thermal limits was previously reported in last instar A. ceratoniae (Heydari & Izadi 2014), and is typical of insects overwintering in temperate regions Denlinger & Lee 2010;Crosthwaite et al. 2011). However LT 50 of A. ceratoniae was not determined in Akbarkooh population, according to Heydari and Izadi (2014). ...
The pomegranate fruit moth, Apomyelois ceratoniae (Zeller), is the most important pest of pomegranate orchards in Iran, where infestations lead to 20%–80% fruit loss. A. ceratoniae overwinters as larvae in several instars. The success in overwintering determines the fruit loss in the following season, thus overwintering physiology of A. ceratoniae could provide insights into population prediction. To this end, overwintering strategy and some seasonal physiological and biochemical changes were investigated in the field-collected larvae of A. ceratoniae. The lowest supercooling point was recorded in November (−14.6 ± 0.9 °C) and the highest in both October and March (−10.2 ± 0.9 °C). The median lethal temperature (LT50) of larvae was higher than supercooling point, suggesting that A. ceratoniae is chill-susceptible. Overwintering larvae had slightly higher concentrations of glycerol and sorbitol compared to summer larvae. There were no significant seasonal changes in body water content or hemolymph osmolality. Current winter temperatures in Iranian orchards are higher than the cold tolerance thresholds of A. ceratoniae, suggesting that overwintering mortality is not a key factor in determining A. ceratoniae populations.
... Frontiers in Physiology frontiersin.org might be converted into simple sugars or sugar alcohol, as reported by several previous studies in Lepidoptera [e.g., Ocneria terebinthina (Behroozi et al., 2012), Ectomyelois ceratoniae (Heydari and Izadi, 2014), and Kermania pistaciella (Mollaei et al., 2016)]. The lipid content remained stable during the early stage of diapause maintenance and decreased significantly after 30 days of pupation. ...
... Cryoprotective dehydration is one strategy to improve cold tolerance in some insects (Kawarasaki et al., 2013;Ciancio et al., 2021). Several studies have shown that the water content of non-diapause insects is significantly higher than that of diapause individuals, such as Pieris melete (Xiao et al., 2015), Ectomyelois ceratoniae (Heydari and Izadi, 2014), and Streltzoviella insularis (Pei et al., 2020). In S. montelus, Wang et al. (2007) reached similar conclusions. ...
The swallowtail butterfly, Sericinus montelus Gray, is endemic to East Asia, has high ornamental value but faces an increased risk of extinction. To understand the overwintering strategies of this species, the dynamic changes in supercooling point (SCP) and water and biochemical contents of diapause-destined and non-diapause S. montelus pupae were investigated. The SCP of laboratory-reared diapause pupae was as low as −26°C compared to −24°C in diapause pupae in the field. Although there was no significant difference in total water content between diapause-destined and non-diapause pupae, the free water of diapause-destined pupae was significantly lower, and the bound water was significantly higher, than that of non-diapause pupae. Lipid, glycogen, and protein contents of diapause-destined pupae showed a downward trend, whereas the total sugar content showed the opposite trend after pupation. The glycogen content decreased rapidly during the initial stage of pupation, whereas the lipid content decreased significantly after 30 days of pupation, suggesting that diapause-destined pupae deplete glycogen stores during the pre-diapause period and then switch to using lipids during the diapause maintenance phase. Trehalose levels in diapause-destined pupae increased significantly and remained high after pupation. Meanwhile, the trehalose content of overwintering pupae during the diapause maintenance period was significantly higher than that of diapause termination pupae in the field. These results suggest that trehalose is the main cryoprotectant for overwintering pupae. Thus, diapausing S. montelus pupae appear to be freeze avoidant, accumulate trehalose as a cryoprotectant, and reduce the free water content to decrease the SCP, enhancing their cold tolerance.
... CA may allow some insects to enhance their cold tolerance. This enhancement involves several physiological and biochemical adjustments, e.g., synthesis 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, Mohammadzadeh and Izadi 2018a). ...
... Ten beetles were weighed and homogenized in 1.5-2 ml of 80% ethanol and centrifuged 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. ...
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.
... Given its importance in insects as an energy source, many studies focused on glycogen function under stress pressures like starvation and cold hardness (Ko st al et al. 2014;Heydari and Izadi 2014;Keshan et al. 2016;Wang et al. 2016a,b). The rate-limiting enzyme of glycogen degradation, GP, was first identified from rabbit skeletal muscle in 1936 (Cori and Cori 1936). ...
... And there were no significant difference in GS activity among dsTPS2, two dsTRE1 and Validamycin injection groups. (Fig. 7B) Glycogen content has been detected under different stress conditions, including starvation, cold hardness, over-winter or seasonal acclimation, and diapauses in previous studies (Ko st al et al. 2014;Heydari and Izadi 2014;Ren et al. 2015;Keshan et al. 2016;Wang et al. 2016a,b), but few studies reported GP and GS expression levels and enzyme activity. Thus, more studies are needed to understand their function in insect physiology, including GP and GS RNAi to study the specific function of these two genes in glycogen and trehalose metabolism regulation, as well as in energy and chitin metabolism regulation in insects. ...
RNA interference has been used to study insects' gene function and regulation. Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in carbohydrates' conversion in insects. Glycogen content and GP and GS gene expression in several tissues and developmental stages of the Brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) were analyzed in the present study, using quantitative reverse-transcription polymerase chain reaction to determine their response to double-stranded trehalases (dsTREs), trehalose-6-phosphate synthases (dsTPSs), and validamycin injection. The highest expression of both genes was detected in the wing bud, followed by leg and head tissues, and different expression patterns were shown across the developmental stages analyzed. Glycogen content significantly decreased 48 and 72 h after dsTPSs injection and 48 h after dsTREs injection. GP expression increased 48 h after dsTREs and dsTPSs injection and significantly decreased 72 h after dsTPSs, dsTRE1-1, and dsTRE1-2 injection. GS expression significantly decreased 48 h after dsTPS2 and dsTRE2 injection and 72 h after dsTRE1-1 and dsTRE1-2 injection. GP and GS expression and glycogen content significantly decreased 48 h after validamycin injection. The GP activity significantly decreased 48 h after validamycin injection, while GS activities of dsTPS1 and dsTRE2 injection groups were significantly higher than that of double-stranded GFP (dsGFP) 48 h after injection, respectively. Thus, glycogen is synthesized, released, and degraded across several insect tissues according to the need to maintain stable trehalose levels. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.
... This is the case of L. migratoria eggs (−26 • C), Liriomyza spp. pupae (−19 • C) (Jing and Kang, 2002), Chrysoperla sinica (−13 • C) , Ectomyelois ceratoniae diapausing larvae (−17.3 • C) (Heydari and Izadi, 2014), and Pityogenes chalcographus (−26.3 • C) (Koštál et al., 2014). In the present study, H. axyridis were divided into black (melanic) and yellow (non-melanic) forms according to the background of their elytra, and into experimental and overwintering populations according to their source. ...
... Thus, overwintering populations seem to decrease their SCP to avoid the damages caused by fluid freezing, which agrees with the previously described seasonal changes in H. axyridis SCP and its significant decrease in winter (Zhao et al., 2008). Some studies reported that insects with low SCP had high levels of trehalose in the winter (Heydari and Izadi, 2014;Koštál et al., 2014;Vallières et al., 2015). This is an important winter adaptation strategy for survival at low temperature. ...
The main function of small heat shock proteins (sHSPs) as molecular chaperones is to protect proteins from denaturation under adverse conditions. Molecular and physiological data were used to examine the sHSPs underlying cold-hardiness in Harmonia axyridis. Complementary DNA sequences were obtained for six H. axyridis sHSPs based on its transcriptome, and the expression of the genes coding for these sHSPs was evaluated by quantitative real-time PCR (qRT-PCR) in several developmental stages, under short-term cooling or heating conditions, and in black and yellow females of experimental and overwintering populations under low-temperature storage. In addition, we measured water content and the super cooling and freezing points (SCP and FP, respectively) of H. axyridis individuals from experimental and overwintering populations. The average water content was not significantly different between adults of both populations, but the SCP and FP of the overwintering population were significantly lower than that of the experimental population. Overall, the six sHSPs genes showed different expression patterns among developmental stages. In the short-term cooling treatment, Hsp16.25 and Hsp21.00 expressions first increased and then decreased, while Hsp10.87 and Hsp21.56 expressions increased during the entire process. Under short-term heating, the expressions of Hsp21.00, Hsp21.62, Hsp10.87, and Hsp16.25 showed an increasing trend, whereas Hsp36.77 first decreased and then increased. Under low-temperature storage conditions, the expression of Hsp36.77 decreased, while the expressions of Hsp21.00 and Hsp21.62 were higher than that of the control group in the experimental population. The expression of Hsp36.77 first increased and then decreased, whereas Hsp21.56 expression was always higher than that of the control group in the overwintering population. Thus, differences in sHSPs gene expression were correlated with the H. axyridis forms, suggesting that the mechanism of cold resistance might differ among them. Although, Hsp36.77, Hsp16.25, Hsp21.00, and Hsp21.62 regulated cold- hardiness, the only significant differences between overwintering and experimental populations were found for Hsp16.25 and Hsp21.00.
... In general, insects are able to survive cold temperature easier where the temperature amplitude is reduced. For this purpose, many insects can gain shelter Heydari and Izadi 2014). During overwintering of the pistachio twig borer, trehalose, glucose, and myo-inositol contents remained unchanged. ...
... This finding is similar to the results obtained by Goto et al (1998) analyzing the overwintering larvae of Enosima leucotaeniella (Ragonot) (Lepidoptera: Pyralidae). On the other hand, lipids have been reported as an energy resource during diapause in some other insects (Han et al 2008, Behroozi et al 2012, Sadeghi et al 2012, Bemani et al 2012, Heydari & Izadi 2014. ...
The pistachio twig borer, Kermania pistaciella Amsel (Lepidoptera: Tineidae), a key pest of pistachio trees, is a monovoltine pest living inside the feeding tunnel of pistachio twigs for almost 10 months in a year and overwinters there as last instar larvae. In this study, we measured some physiological parameters of overwintering field collected larvae of the pest. There were no changes in trehalose, glucose, and myo-inositol contents, but there were differences in the levels of total simple sugar and glycogen during overwintering. Total sugar content at the beginning of overwintering (October) was at the lowest level (24.13 mg/g body weight) and reached to the highest level (55.22 mg/g fresh body weight) in November whereas glycogen content was at the highest level (44.05 mg/g fresh body weight) in October and decreased to 18.42 mg/g fresh body weight in November. Decrease in lipid content during the overwintering period was not significant. The highest and lowest levels of protein content were recorded in January and February, respectively. Supercooling points (SCP) of the overwintering larvae were stable and low (ranged between -17.80 and -25.10°C) throughout the cold season and no larva survived after SCP determination. The lowest cold hardiness (60 and 0.0% survival following exposure to -10 and -20°C/24 h, respectively) was observed for in November-collected larvae. Overwintering larvae of the pistachio twig borer rely mostly on maintaining the high supercooling capacity throughout the overwintering to avoid freezing of their body fluid.
... Goto et al. (1998) and Kostal et al. (1998) reported same results for E. leucotaniella and Cymbalophora pudica, respectively. Conversely for some other diapausing insects lipid has been reported as the source of energy (Han et al. 2008, Behroozi et al. 2012, Sadeghi et al. 2012, Bemani et al. 2012, Heydari and Izadi 2014. ...
... Winter accumulation of low molecular weight carbohydrates (e.g., trehalose and glucose) and/or polyols (e.g., glycerol and myoinositol) has been well documented in many diapausing insects (Goto et al. 2001, Kostal et al. 2007, Han et al. 2008, Behroozi et al. 2012, Sadeghi et al. 2012, Bemani et al. 2012, Heydari and Izadi 2014. In diapausing larvae of the almond wasp, trehalose and glucose levels increased as ambient temperature decreased. ...
The almond wasp, Eurytoma amygdali Enderlein (Hymenoptera: Eurytomidae), a key pest of almond, is a univoltine pest diapausing as last instar larvae inside the damaged fruits for almost nine months in a year. In this study, changes in the amount of total simple sugars, lipid, protein, glycogen, trehalose, glucose, supercooling points (SCPs), and cold hardiness of the diapausing larvae were measured from October to March for first year diapause-destined and in August and September for second year diapause-destined larvae. Changes in glycogen content were reversely proportional to changes in total simple sugars and low molecular weight carbohydrates. These changes reflect the interconversion of glycogen to sugar alcohol in order to increase the insect cold tolerance. We found that cold hardiness and diapause of the last instar larvae of the almond wasp have evolved separately. Cold hardiness was highly associated with physiological changes (accumulation of cryoprotectants), but no physiological changes occurred in early diapause of first year diapause-destined and second year diapause-destined larvae. The almond wasp larvae were found to be a freeze-avoidant insect, as no larva survived after SCP determination and crystallization of its body fluids.
... Hemolymph osmolality could be another determining parameter in the modulation of cryoprotectants (e.g., polyols and low molecular weight carbohydrates) produced by insects to mitigate harmful effects of low temperatures on cell organelle and membrane injuries to survive winter temperatures [126][127][128][129]. Some of the main roles of cryoprotectants are the hindrance of intracellular volume decrease below a critical minimum, the maintenance of the membrane bilayer structure and the decrease in SCP [48,127,[130][131][132][133]. The accumulation of cryoprotectants may also be utilized by medfly during overwintering to increase the potential of its survival during winter [134]. ...
Simple Summary
Ceratitis capitata, also known as the Mediterranean fruit fly, is a notorious pest of fruits and vegetables worldwide. The current expansion of its geographic distribution is limited by the cooler temperate areas of the Northern Hemisphere, which points to a more comprehensive investigation of its thermal biology considering its high invasion capacity. Cold tolerance in adults has been studied thoroughly but the impact of extremely low temperatures on the immature stages remains unexplored. We estimated the acute cold stress response of three geographically divergent populations (two from Greece and one from Croatia). Overall, the populations exhibited different responses to acute cold stress depending on the developmental stage. The northernmost population was the most cold-susceptible at the egg stage, whereas in the southernmost population it was at the larval and pupal stage. The geographically intermediate population was the most cold-tolerant regardless of the developmental stage. The egg stage was the most cold-tolerant, followed by pupae and larvae. Our findings broaden the existing knowledge on cold tolerance of C. capitata and can be used for the development of population and invasion dynamics models.
Abstract
Cold tolerance of adult medflies has been extensively studied but the effect of subfreezing temperatures on the immature stages remains poorly investigated, especially as far as different populations are regarded. In this study, we estimated the acute cold stress response of three geographically divergent Mediterranean fruit fly populations originating from Greece (Crete, Volos) and Croatia (Dubrovnik) by exposing immature stages (eggs, larvae, pupae) to subfreezing temperatures. We first determined the LT50 for each immature stage following one hour of exposure to different temperatures. Then eggs, larvae and pupae of the different populations were exposed to their respective LT50 for one hour (LT50 = −11 °C, LT50 = −4.4 °C, LT50 = −5 °C for eggs, larvae and pupae, respectively). Our results demonstrate that populations responded differently depending on their developmental stage. The population of Dubrovnik was the most cold-susceptible at the egg stage, whereas in that of Crete it was at the larval and pupal stage. The population of Volos was the most cold-tolerant at all developmental stages. The egg stage was the most cold-tolerant, followed by pupae and finally the 3rd instar wandering larvae. This study contributes towards understanding the cold stress response of this serious pest and provides data for important parameters that determine its successful establishment to unfavorable environments with an emphasis on range expansion to the northern, more temperate regions of Europe.
... An example of this is seen in the diapausing larvae of S. crassipalpis, which have been found to contain almost double the concentration of hemolymph protein as the non-diapausing larvae (Adedokun and Denlinger 1985). An increase in protein content of diapausing larvae was also reported in E. ceratoniae (Heydari and Izadi 2014), and the almond wasp, Eurytoma amygdali End. (Hymenoptera: Eurytomidae) (Khanmohamadi et al. 2016). ...
The spotted amber ladybird, Hippodamia variegata (Goeze) (Col.: Coccinellidae), is known to be a potent predator of aphids, psyllids, whiteflies, mealybugs, and some butterfly species. This ladybeetle overwinters in the diapausing adult stage. The current study aimed to evaluate the impact of diapause on the energy resources and cuticular hydrocarbons (CHCs) of the female ladybeetle, specifically comparing the changes in glycogen, lipid and protein contents, and CHCs profile of diapausing and non-diapausing adults. The research utilized gas chromatography-mass spectrometry to analyze whole-body extracts of the beetles. Results showed no significant differences between the amount of glycogen, lipid, and protein contents of diapausing and non-diapausing ladybeetle. The CHCs profile of H. variegata consisted of 24 hydrocarbons categorized into two groups: linear aliphatic hydrocarbons ( n -alkanes) and methyl-branched hydrocarbons (17 molecules), as well as unsaturated cyclic compounds (7 molecules). The n -alkanes, with 14 compounds, were identified as the primary constituents of the CHCs of the ladybeetle. Six molecules were common to non-diapausing and diapausing beetles, five were exclusive to non-diapausing beetles, and 13 were exclusive to diapausing beetles. Moreover, the researchers noted a significant difference in the quantity and quality of CHCs between diapausing and non-diapausing beetles, with diapausing beetles synthesizing more CHCs with longer chains. This disparity in CHC profiles was concluded to be an adaptation of H. variegata to survive harsh environmental conditions during diapause.
... Consistent with these previous findings, we found that the contents of trehalose and glycogen in the overwinter H. axyridis population were higher than those of the experimental population, with a 10-fold increase in trehalose and an approximately 100-fold increase for glycogen (Figure 2). Similar results were reported for the insect Ectomyelois ceratoniae, with an accumulation of trehalose and total sugars from October to February of the next year [55], whereas the contents of trehalose and glycogen in November and March were higher than those of other months in the beetle Pityogenes chalcographus [56]. We found higher trehalose and glycogen contents in the overwinter population than in the experimental population for both yellow and black adults ( Figure 2). ...
Trehalose is a non-reducing disaccharide that plays a key role in the response to cold and other environmental stressors in insects. Harmonia axyridis (Pallas) is an important natural predator of insect pests and has become a cosmopolitan invasive species, causing negative ecological impacts worldwide. In this study, the relative survival ability, trehalose and glycogen contents, trehalose activity and trehalose metabolism-related gene-expression profiles over a cold storage period were compared in a natural overwintering population and an indoor laboratory (experimental) population. Yellow adults were dominant in the overwintering population. The survival rate of the overwintering population during the cold storage period was higher than that of the experimental population after the fifth day. The contents of trehalose and glycogen in the overwinter population were higher than those of the experimental population during cold storage. Trehalose and glycogen contents initially increased and then decreased in the overwinter population, but decreased consistently over the cold storage period in the experimental population. Nevertheless, trehalose levels were relatively higher during the cold storage period in the overwinter population, with higher expression of TPS and TRE and trehalase activity. More importantly, the experiment showed that yellow adults have a better ability to regulate trehalose metabolism under cold storage compared to black adults. A strong resilience against cold stress and long-term cold storage ability could contribute to a better understanding of the invasiveness of H. axyridris.
... Thus, diapausing insects often become more cold-tolerant [19,20]. Under unfavorable environmental conditions, insects synthesize and accumulate distinct types of low-molecular-weight compounds such as polyols, sugars, and some amino acids [21], which function as cryoprotectants and improve their cold hardiness [22,23]. For example, during diapause development, the pupae of Phyllonorycter ringoniella Matsumura gradually accumulate more trehalose, which is used as a cryoprotectant to improve their cold hardiness [24]. ...
Diapause and cold hardiness are essential components of winter survival for most insects in temperate zones. The fall webworm, Hyphantria cunea, overwinters in a pupal diapause. In this study, we investigated the energy consumption and cold hardiness of diapausing pupae. We found that lipid content decreased from October to November and stabilized from November to March. Glycogen content decreased by 61.3% and 52.2% for females and males, respectively, from October to November, and decreased slowly from November to March. We also observed a significant increase in trehalose concentrations as ambient temperatures decreased from October to November and a decrease in trehalose as temperatures increased again in March. We did not observe substantial changes in pupal supercooling points among the dates sampled. In addition, prolonged pupal development time reduced their survival rate and had no significant effect on post-diapause adult body mass and fecundity but reduced egg diameter in females. These results suggest that the energy consumption of H. cunea pupae during early diapause depends on lipid and glycogen, while it shifts to depend on glycogen or other energy stores in the mid- and late diapause stages. Our results also suggest that the prolonged development time of diapausing pupae had a negative effect on post-diapause fitness.
... The content of low-molecular-weight carbohydrates were measured in 300 adults for each diet after 30-day cold storage treatment, freeze-dried for 48 h (2.5L FreeZone, LabconcoInc., USA), ground, and then measured by the method as described by Heydari and Izadi (2014). Briefly, 1 g of dried powder of A. chinensis was extracted with 20 ml of 80% ethanol, and heated at 75 °C for 20 min in a water bath, and then centrifuged for 3 min at 10000 rpm. ...
The preservation and rearing of insect natural enemies is the key for biological control. The biological (survival, longevity and the fecundity) and biochemical (low-molecular-weight carbohydrates, glycerol and fatty acids contents) indexes of Arma chinensis fed by Antherea pernyi (diet AP) or Tenebrio molitor (diet TM) were measured after cold storage treatments. The results showed that the diet affected several biological and biochemical parameters, but varied with the length of cold storage. The survival rate and longevity after 30-day cold storage, and the fecundity after 40-day cold storage were significantly higher for adults reared on TM compared to AP. The super-cooling points and the freezing points were significantly lower for adults reared on TM than AP. Low-molecular-weight carbohydrates, glycerol and unsaturated fatty acids were significantly higher for TM than for AP. The structural equation model showed that diet influenced survival, fertility and SCPs of A. chinensis indirectly through the enzyme activity, and the content of carbohydrates, glycerol and unsaturated fatty acids. These results suggested that the nutrient content of diets affects the accumulation of cold-resistant substances, metabolism level and activities of related enzymes in A. chinensis in a manner that enhances tolerance to cold storage.
... In an investigation on Agonoscena pistaciae collected from the field, total sugar, trehalose, and sorbitol increased from October to January followed by a decrease from January to March, while glycogen had the opposite trend [64], showing that trehalose and total sugars accumulated as the temperature decreased. Similarly, in Ectomyelois ceratoniae, trehalose, and total sugar accumulated from October to February, while glycogen content decreased [65]. Trehalose levels typically increase as the level of glycogen decreases. ...
Harmonia axyridis is a major bio-control agent of pests in agriculture and forest ecosystems. It is also a globally important invasive insect species. To test whether dark elytra colour is associated with greater cold hardiness, we compared the survival rate of prolonged cold exposure in both yellow and black colour morphs of female and male H. axyridis. We determined the trehalose and glycogen content, trehalase activity, and the dynamics of genes associated with the trehalose metabolic pathway. Yellow forms predominated before winter began, however black forms increased from 11.15 to 30.46% after overwintering. There was no significant difference in trehalose content between the females and males during overwintering. Glycogen content in over-wintering yellow females and black males increased significantly, while it decreased in black females. Soluble trehalase activity increased significantly in all the insects except black females. Membrane-bound trehalase activity increased in black males, and decreased in black females. Trehalose and glycogen content and trehalase activity were regulated by differential expression of TRE and TPS genes. Female beetles weighed more than males and survived in low temperatures for longer periods of time, regardless of elytra colour, suggesting that mass is a stronger predictor of overwintering survival rather than colour morph. Our results provide a guide for comparing cold resistance in insects and a theoretical basis for cold storage of H. axyridis for use as natural enemies of pests in biological control programs.
... It is known as a pest and an effective factor in decreasing quantitative and qualitative features of pomegranate in many tropical and subtropical regions ( Warner et al. 1990, Kishani-Farahani et al. 2012, Ramzi et al. 2013). This pest is distributed throughout the pomegranate growing areas of Iran, especially in Yazd, as one of the main pomegranate production regions in Iran ( Heydari and Izadi 2014). During the past 25 yr, E. ceratoniae caused from 10 to 40% damage in the harvestable crop annually ( Nay et al. 2006). ...
Plant-herbivore interactions can be influenced by plant developmental stages. Effects of four different developmental stages of pomegranate fruit (Hazelnut size, Walnut size, Juicy, and Ripe) were investigated on life table parameters, nutritional yield, and energy contents (protein, glycogen, and lipid) of Ectomyelois ceratoniae (Zeller) under controlled conditions in two pomegranate cultivars, namely Shahvare-Danesefid and Esfahani-Daneghermez as susceptible and resistant cultivars, respectively. Biochemical characteristics of the four developmental stages were determined, and a correlation was made with life-history and nutritional responses of E. ceratoniae. Significant differences were observed in developmental time and adult fecundity of E. ceratoniae across various tested stages in both pomegranate cultivars. The highest value of intrinsic rate of increase (rm) was found in Juicy and lowest in Hazelnut-size stage of Shahvare-Danesefid. The highest rm value was on Walnut-size and Juicy stages, and the lower rate was obtained from the feeding of E. ceratoniae on Ripe and Hazelnut-size stages in Esfahani-Daneghermez. A higher relative growth rate (RGR) was observed in Juicy (in Shahvare-Danesefid) and Walnut-size stages (in Esfahani-Daneghermez) when compared with other developmental stages tested. Life-history parameters of E. ceratoniae were correlated with biochemical qualities of pomegranate stages. Results indicated Juicy (Shahvare-Danesefid) and Walnut-size stages (Esfahani-Daneghermez) were relatively susceptible pomegranate stages, and Hazelnut-size and Ripe stages were unsuitable for feeding of E. ceratoniae in Shahvare-Danesefid and Esfahani-Daneghermez, respectively. Findings of this research could aid in the development of integrated management programs of E. ceratoniae in pomegranate orchards.
... Cold hardiness or cold tolerance is an insect's ability to prevent cold injuries during unfavorable winter conditions. Cold hardiness in insects can be achieved by maintaining a supercooled condition colligatively, i.e., synthesis and accumulation of cryoprotectants [1,7,13,29,32], or non-colligatively, i.e., enhancement of the supercooling point (SCP) without any further changes in cryoprotectant concentrations. In the latter group, the changes in SCP values do not follow a particular seasonal trend, and the limit of cold tolerance is regulated by the SCP [20,23]. ...
Ladybird beetles have successfully been used to control different pests. Cheilomenes sexmaculata (Fabricius) and Oenopia conglobata contaminata)Menetries((Coleoptera: Coccinellidae) are two dominant and efficient predators of the common pistachio psylla, Agonoscena pistaciae (Hem.: Psyllidae) in Iran. In the current study, the impact of two diets, i.e., nymphs of A. pistaciae and eggs of Ephestia kuehniella Zeller (Lep.: Pyralidae), were investigated on the cold hardiness, supercooling point (SCP), and lethal temperature of different life stages of the coccinellids. The results suggested that the eggs of E. kuehniella are a suitable diet for both predators. In general, beetles of O. conglobata contaminata were more cold tolerant than those of C. sexmaculata. The SCP of the adults of C. sexmaculata, feeding on psyllid was about -15 °C, whereas the SCP of the beetles, feeding on the eggs of flour moth, was about -19 °C. However, the diets had no significant effects on the SCP of O. conglobata contaminata. For both ladybird beetles, eggs were the most sensitive stage, and adulthood was the most tolerant developmental stage. No eggs survived at zero and subzero temperatures. The survival of C. sexmaculata increased from 6.25% for adults fed on psyllids to 13.75% for those fed on flour moth eggs after 24 h exposure to cold at -4 °C. The survival of O. conglobata contaminata adults after exposure to cold at -4 °C for 24 h raised from 28.75% for adults fed on psyllids to 42.50% for those fed on flour moth eggs. Regardless of the diet, both beetles were considered to be chill-intolerant insects as the most mortality occurred above the SCP.
... Insects usually store energy in the form of lipids for later utilization during overwintering or diapause (Sadeghi et al., 2012). Accumulation of lipids to avoid low-temperature injury has been reported in Agonoscena pistaciae (Sadeghi et al., 2012), Osmia rufa (Dmochowska et al., 2013), Ectomyelois ceratoniae (Heydari and Izadi, 2014), and Spodoptera litura (Zhu et al., 2017). But lipid metabolism in insects under high temperature stress has seldom been reported. ...
Considering the predicted rising temperatures under current climate change and heat wave scenarios, organisms are expected to suffer more intense and frequent thermal stress. Induced heat is accumulated by organisms and can cause a variety of physiological stress responses. Ophraella communa is an effective biological control agent of common ragweed, Ambrosia artemisiifolia, but the responses of this biocontrol agent to heat stress have not been fully elucidated and, therefore, its potential responses to climate change are uncertain. We investigated the physiological metabolism of subsequent O. communa adults after: (1) different developmental stages (egg, larval, pupal, and adult) were exposed to thermal stress for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage); and (2) individuals were exposed to thermal stress throughout the egg-to-adult period for 3 h each day. The high temperatures of 40, 42, and 44°C were used to induce thermal stress. A control group was reared at 28 ± 2°C. The results showed that short- or long-term exposure to daily phasic high temperatures significantly decreased water and lipid contents and significantly increased glycogen and glycerol contents in all adults (i.e., after exposure of different stages or throughout the egg-to-adult period). However, the total sugar content significantly increased in adults after the eggs and larvae were exposed to brief short-term thermal stress. Compared to the control, the total sugar content was also significantly higher in the adults and pupae exposed to 44°C. Total sugar content in females increased significantly in response to long-term phasic thermal stress at 40°C. However, sugar content of males exposed to 44°C decreased significantly. After long-term phasic thermal stress, water and glycogen contents in males were significantly higher than in females; however, females had higher total sugar and lipid contents. Therefore, our study provides a basic understanding of the metabolic responses of O. communa to thermal stress and offers insights into its potential as a natural biocontrol agent against A. artemisiifolia during the summer season and under predicted climate change scenarios.
... The beetle, Harmonia axyridis (Pallas), is an important natural enemy of pests, such as aphids, whiteflies, psyllids, mites, and the eggs and larvae of some lepidopterans and coleopterans (Luciana et al. 2015;Zappalà et al. 2013); it is the focus of pest control strategies in agriculture and forestry in Asia, including China (Tang et al. 2014), but is notorious for its invasiveness of the ecological niche of other ladybird beetles in Europe (Adriaens et al. 2008;Brown et al. 2008a, b). Cold hardiness and diapause are essential components of winter survival for most insects in temperate zones, but in many cases the relationship between these two are not clear (Heydari and Izadi 2014). The coldhardiness of H. axyridis represents an important characteristic and has been widely studied with respect to biological control applications and aspects of invasiveness (Bazzocchi et al. 2004;Berkvens et al. 2010;Pervez and Omkar 2006;van Lenteren et al. 2008). ...
Trehalose is the main blood sugar in insects. To study the function of trehalase during exposure to low temperatures, three other novel cDNAs of trehalase were cloned from Harmonia axyridis by transcriptome sequencing and rapid amplification of cDNA ends. One of the cloned cDNAs encoded a soluble trehalase, the second trehalase cDNA encoded a transmembrane-like domain, and the third cDNA encoded a membrane-bound protein. Therefore, these cDNAs were, respectively, named HaTreh1-5, HaTreh2-like, and HaTreh2. HaTreh1-5, HaTreh2-like, and HaTreh2 cDNAs encoded proteins containing 586, 553, and 633 amino acids with predicted masses of approximately 69.47, 63.46, and 73.66 kDa, and pIs of 9.20, 5.52, and 6.31, respectively. All three novel trehalases contained signal motifs “PGGINKESYYLDSY”, “QWDYPNAWPP”, and a highly conserved glycine-rich (GGGGEY) region. The expression levels of HaTreh1-5 and HaTreh2 mRNAs were high during adult stages, whereas HaTreh2-like was expressed in low amounts in the fourth larval stage. The results showed that the activity of membrane-bound trehalases decreased from 25 to 10 °C and from 5 to − 5 °C during cooling. The results also revealed a decreasing trend in expression of the three HaTreh mRNAs during the cooling treatment, and an initial decrease followed by an increase during the process of re-warming.
... Several insects like Colaphellus bowringi (Tan et al., 2017), Pieris napi (Lehmann et al., 2016), Arimania comaroffi (Bemani et al., 2012), and Ectomyelois ceratoniae (Heydari and Izadi, 2014) accumulate lipids in diapause whereas others like Cymbalophora pudica (Kostál et al., 1998) and Eurytoma amygdali (Khanmohamadi et al., 2016) do not. In group D, fatty acid synthase (FAS, K7IM26_NASVI) and acetyl-CoA carboxylase (ACC, E2B9B3_HARSA) were non-significantly downregulated at the protein level (0.88-and 0.89-fold change, respectively). ...
Aphidius gifuensis Ashmead (Hymenoptera: Aphidiidae) is a solitary endoparasitoid used in the biological control of various aphids. Diapause plays an important role in the successful production and deployment of A. gifuensis. Diapause can effectively extend the shelf life of biological control agents and solve several practical production problems like long production cycles, short retention periods, and discontinuities between supply and demand. In recent years, studies have been conducted on the environmental regulation and physiological and biochemical mechanisms of diapause in A. gifuensis. Nevertheless, the molecular mechanism of diapause in this species remains unclear. In this study, we compared the transcriptomes and proteomes of diapause and non-diapause A. gifuensis to identify the genes and proteins associated with this process. A total of 557 transcripts and 568 proteins were differentially expressed between the two groups. Among them, (1) genes involved in trehalose synthesis such as glycogen synthase, glycogen phosphorylase, and trehalose 6-phosphate synthase were upregulated in diapause at mRNA or protein level while glycolysis and gluconeogenesis-related genes were downregulated, suggesting that A. gifuensis stores trehalose as an energy resource and cryoprotectant; (2) the expression of immune-related genes like C-type lectins, hemocyanin, and phenoloxidase was increased, which helps to maintain immunity during diapause; (3) a chitin synthase and several cuticular protein genes were upregulated to harden the cuticle of diapausing A. gifuensis larval. These findings improve our understanding of A. gifuensis. diapause and provide the foundation for further pertinent studies.
... Trehalose, glucose, glycerol and myo-inositol of last instar larvae of T. granarium were measured by high-performance liquid chromatography (HPLC) as described by Heydari and Izadi (2014). Briefly, one larva was homogenized in 1.5e2 ml of 80% ethanol and the mixture was centrifuged for 10 min at 7150 g. ...
Biology, some physiological processes and cold hardiness of Trogoderma granarium Everts (Coleoptera: Dermestidae) on ten diets (barley, corn, millet, rice, rye, sorghum, triticale, wheat groundnut, and walnut) were studied under laboratory conditions (33 ± 1 °C with 65 ± 5% RH, 14L: 10D). According to the results, the insects reared on triticale had shortest development time, and the highest fecundity and fertility. By contrast, the longest development time, and the lowest fecundity and fertility were on groundnut. The survival rates ranged from 40 to 87% with the lowest values observed on groundnut. T. granarium larvae fed on triticale and millet had the highest amylolytic activity. In contrast, groundnut-fed larvae possessed the lowest amylolytic activity. The highest and lowest proteolytic activity was on rice and millet, respectively. Different diets had a significant effect on larval energy reserves (total body sugars, glycogen, lipid, and protein), which were at the highest levels on triticale and rye, and lowest levels on sorghum. The highest level of trehalose was on triticale, groundnut and rye, and the lowest level was on barley and sorghum. The supercooling point (SCP) of larvae reared on triticale was −20.6, which was significantly lower than on the other diets. Exposures to −5 and −10 °C/24 h were somewhat endured by larvae fed on triticale, rye, walnut, and groundnut whilst exposure to −20 °C/24 h caused 100% mortality in these food groups. These results suggest that larval food quality can affect biological and physiological characteristics and influence the supercooling point and cold hardiness of T. granarium.
... A long period of storage under these temperatures retards the growth of date worms, but does not result in their death. It is also known that dates freezing temperature can be lower than −18 °C [26] and the temperature of −15 °C is lethal for Apomyelois ceratoniae [27]. Thus, low temperature refrigeration can be used as an anti-pest treatment of dates. ...
Large amounts of dates are produced and consumed around the world each year. Apomyelois ceratoniae causes main losses during storage and export of date fruit. Fumigation is the most widely used treatment to prevent postharvest losses. However, this treatment negatively affects the biological value of dates, environmental and economic parameters of production. Widespread fumigation agent methyl bromide is toxic to a human body and contributes to the destruction of the ozone layer. The aim of the current work was to find a new method based on the use of low temperature refrigeration to reduce the pest and insect infestation preserving the nutritive value of dates during cold storage at the same time. A. ceratoniae mortality under different temperatures and dates respiration rate, sugar, organic acids, pectic substances content and sensorial characteristics were studied. The results indicate that at −18 °С the A. ceratonie larva dies within 2 h, the eggs and larvae – within 24 h respectively. Thus, it is clearly shown that there is no need in using chemicals or irradiation to prevent dates pests. Physicochemical and organoleptic proprieties of dates were satisfactory during 360 d at −18 °С and during 180 d at +4 °С. The low temperature refrigeration and storage at −18 °С is recommended for dates to prevent pests and quality losses. It is apparently advantageous for environment and climate if compared with other anti-pest treatment variants.
... The observed trehalose accumulation in particular is reminiscent of diapause-associated increase in levels of this sugar in many species ( Guo et al., 2015;Rozsypal et al., 2013;Hodkova and Hodek. 2004;Xu et al., 2009;Su et al., 1994;Sasibhushan et al., 2013;Heydari and Izadi, 2014;Lu et al., 2014). Our results largely overlap with those previously reported by Kubrak et al. (2014) where both glycogen and trehalose levels rose for the first 3 weeks of diapause induction. ...
D. melanogaster enters a state of reproductive arrest when exposed to low temperatures (12°C) and shorter photoperiods. A number of studies have suggested that diapause has recently evolved in European D. melanogaster populations, that it is not present in the sibling species D. simulans, that it is non-photoperiodic in American D. melanogaster populations, and that it spontaneously terminates after 6-8 weeks. We have studied the overwintering phenotype under different conditions and observe that American, European and, surprisingly, African D. melanogaster populations can show photoperiodic diapause, as can European, but not African D. simulans. Surprisingly other Drosophila species from pan-tropical regions can also show significant levels of photoperiodic diapause. We observe that spontaneous termination of diapause after a few weeks can be largely avoided with a more realistic winter simulation for D. melanogaster, but not D. simulans. Examining metabolite accumulation during diapause reveals that the shallow diapause of D. melanogaster has similar features to that of other more robustly-diapausing species. Our results suggest that diapause may be an ancient character that emerged in the tropics to resist unfavourable seasonal conditions and which has been enhanced during D. melanogaster’s colonisation of temperate regions. Our results also highlight how different methodologies to quantify diapause can lead to apparently conflicting results that we believe can now largely be resolved.
... photoperiod. In addition Heydari & Izadi (2014) reported that the last instar larvae of pomegranate fruit moth are able to tolerate harsh conditions during winter. Our observation during collecting infested pomegranate indicated that only 20% of larvae overwinter as fully grown non feeding larvae.. Objectives of the current study were; 1) to determine the difference in supercooling point (SCP) and cold tolerance of small (2 nd and 3 rd ) and large (4 th and 5 th ) overwintering larvae, 2) how do small larvae compensate energy drain during winter? ...
Low temperatures are a major survival challenge for insects living in temperate zones. The most common features that guaranty survival in the most overwintering insects are diapause and cold hardiness. In spite of obvious advantages, damages due to diapause associated stresses such as cold shock and depletion of energy reserves make diapause a costly life history strategy. Apomyelois ceratoniae (Zeller), a major pest of pomegranate in Iran, overwinters as different larval instars. In comparison to larger counterparts, early instars of pomegranate fruit moth must have sequestered fewer energy reserves, thus they might be less tolerant to cold. By designing experiments similar to natural conditions in December, we demonstrated that overwintering inside pomegranate fruits provides opportunity for larvae to feed and compensates energy depletion. Our results also indicated that there is no significant difference between supercooling point and cold tolerance of the small and the large field collected larvae during autumn and winter, thus overwintering mortality might not be a key driver in population dynamics.
... Although the exact definition of diapause termination is controversial, Kostal (2006) argued that this term should refer to the period where ''specific changes in environmental conditions stimulate the decrease of diapause intensity to its minimum level" which is the definition adopted here. This process which can take several months has been studied in several insects (Arrese and Soulages, 2010;Denlinger and Armbruster, 2014;Irwin and Lee, 2000); however such studies remain challenging because investigations in the field, where the appropriate cues to terminate diapause and initiate development occur (Damos and Savopoulou-Soultani, 2010;Snodgrass, 2003), tend to have long sampling intervals that make it difficult to follow the detailed phases of diapause processes (Heydari and Izadi, 2014;Polavarapu, 2001, 2005). On the other hand, laboratory studies may not capture the fluctuating temperatures and extremes encountered in microhabitats in nature (Bosch and Kemp, 2003;Burgi and Mills, 2010). ...
Insect response to cold stress is often associated with adaptive strategies and chemical variation. However, low-temperature domestication to promote the cold tolerance potential of Bactrocera dorsalis and transformation of main internal substances are not clear. Here, we use a series of low-temperature exposure experiments, supercooling point (SCP) measurement, physiological substances and cryoprotectants detection to reveal that pre-cooling with milder low temperatures (5 and 10°C) for several hours (rapid cold hardening) and days (cold acclimation) can dramatically improve the survival rate of adults and pupae under an extremely low temperature (-6.5°C). Besides, the effect of rapid cold hardening for adults could be maintained even 4 h later with 25°C exposures, and SCP was significantly declined after cold acclimation. Furthermore, content of water, fat, protein, glycogen, sorbitol, glycerol and trehalose in bodies were measured. Results showed that water content was reduced and increased content of proteins, glycogen, glycerol and trehalose after two cold domestications. Our findings suggest that rapid cold hardening and cold acclimation could enhance cold tolerance of B. dorsalis by increasing proteins, glycerol, trehalose and decreasing water content. Conclusively, identifying a physiological variation will be useful for predicting the occurrence and migration trend of B. dorsalis populations.
Temperature is one of the critical abiotic factors influencing the range, richness, activity, life history and viability of insects. The persistence trend of thermal stress on biological and reproductive traits were studied for invasive fall armyworm, Spodoptera frugiperda (J. E. Smith) by exposing early fourth instar larvae to thermal stress treatments (42° and 45 °C) for single and multiple times along with control (27 °C) and reared them till F2 generation. Survival of larvae was minimum in 45 °C single (88%) and multiple thermal stress (92.5%). The carry over effect of thermal stress was evident up to F2 generation for developmental attributes with prolonged developmental duration, reduced maturation success and female biased sex ratio. Reproductive parameters such as gonadosomatic index, eupyrene sperm count, fecundity and mating frequency reduced drastically as a result of thermal stress in F0 generation and the effect persisted up to F2 generation on fecundity and gonadosomatic index. The biochemical response due to thermal stress was investigated by analyzing metabolic reserves in the adult stage. The reduction in water content and accumulation of total sugar, glycogen, lipid and protein content was observed in adults of all three generations. A trade-off between reproduction and survival strategies was evident under thermal stress with the allocation of resources between them. Thus, our study provides a basic understanding on persistence of thermal stress across generations both at biological and biochemical level and offers insights into further studies on demography of fall armyworm under current and future climate change scenario.
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 European grapevine moth, Lobesia botrana (Denis et Schiffermuller), is a serious invasive pest that causes significant losses to the flowers and fruits of grapes in most of the world. This multivoltine pest passes the winter as the third-generation diapausing pupa. The current study was designed to compare nondiapausing (first and second generations) and diapausing pupae (third generation) and to investigate the relationship among cold tolerance, the supercooling point (SCP), and diapause development of the third-generation diapausing pupae. The lethal temperatures (LTs) for the three generations were determined using 24-h exposure at subzero temperatures. The mean SCP of the pupae was estimated at approx. −22.6°C, the lowest level of which (−23.7°C) was recorded in the well-developed diapausing pupae in February. The highest level of cold tolerance was also recorded in February. There were no significant differences among the temperatures required to kill 30, 50, and 90% of the pupae. The temperatures significantly decreased from October onward and reached the lowest levels in February during which the lowest SCP and the highest cold tolerance were observed in the diapausing pupae. No significant differences were found in the cryoprotectant levels, among the diapausing and nondiapausing pupae, and the diapause development. The highest activity of cAMP-dependent protein kinase (AMPK) was recorded in the late diapause in February. The findings suggested a relationship among SCP depression, cold tolerance enhancement, and diapause development. A bimodal cold-tolerance strategy (freeze-intolerant and freeze-tolerant) was found to be a feature of the pupae.
The carob moth, Ectomyelois ceratoniae (Zeller), is a polyphagous pest in hot and dry climates, such as the Mediterranean region, and occurs on citrus in South Africa, however, its pest status is not known. The aim of this research was to investigate its seasonal activity patterns and damage potential on citrus crops, in comparison to a pest of phytosanitary concern and one it can be confused with in the larval stage, the false codling moth, Thaumatotibia leucotreta (Meyrick). The study was carried out over 20 months in two regions, one with predominantly citrus and one with mixed agricultural production. Carob moth activity patterns indicated two periods of activity (summer and early spring), with damage taking place in citrus during autumn, close to harvest. The results showed that there were no significant differences in carob moth trap catches between the two study sites (F1,4 = 0.00661, P = 0.94) and no significant differences in wild false codling moth catches between the two study sites (F1,4 = 1.2960, P = 0.32). While traps in both regions caught carob moths in equal abundances , damage only occurred in the region dominated by citrus crops. Assessing pest status, relative to the false codling moth, indicated that carob moth is not currently a major economic pest.
Chill tolerance (time of survival at -5°C) increased in non-diapausing (reproducing) adults of Pyrrhocoris apterus after a gradual, 4-week-long decrease in ambient temperature from 25° to 0°C. The level of chill tolerance attained after cold-acclimation was considerably lower than that in similarly cold-acclimated diapausing adults. Some physiological changes accompanied the cold-acclimation, irrespective of developmental state (diapause vs. reproduction). They were: A decreased oxygen consumption, loss of body water, an increased haemolymph osmolality, an increased proportion of phosphatidylethanolamines vs. a decreased proportion of phosphatidylcholines in membrane phospholipids, and an increased proportion of linoleic vs. a decreased proportion of oleic acid in phosphatidylethanolamines. Such changes could contribute to the limited potential for cold-acclimation found in non-diapausing insects. Other physiological changes appeared to require the induction of diapause prior to cold-acclimation. They were: Down regulation of ice nucleators resulting in a lowering of the individual supercooling point, synthesis and accumulation of specific "winter" polyols, an increased proportion of palmitic acid in membrane phospholipids; and regulation of the concentrations of Na+ and K+ in the haemolymph. The potential contributions of these changes to the cold hardiness of P. apterus are discussed.
The Carob moth (Ectomyelois ceratoniae) is the most important pest of pomegranates in Iran. In order to evaluate the morphological variation of the pest among different geographic populations, the larvae were collected from infested pomegranates at 14 localities in Iran and reared to adult. Using the landmark-based geometric morphometrics method, variation in wing shape and size and allometric relationships were documented. Mantel tests showed a general correlation between geographic and morphological distances. Some morphological distances with the greatest deviation from this correlation were detected using regression fitted line, and the probable causal factors (e.g. reduced gene flow, isolation) on morphological distances are discussed.
Recent studies dealing with adult (reproductive) diapause in the Coleoptera are reviewed, as a kind of supplement to the classic compendia. In the first two sections, the general characteristics of adult diapause are described and principal terms explained. Original articles dealing with 19 species from nine coleopteran families (Coccinellidae, Chrysomelidae, Bruchidae, Curculionidae, Carabidae, Silphidae, Scolytidae, Scarabaeidae, and Endomychidae) are reviewed. Finally attempts are made at generalisations from the papers reviewed, and hypotheses on diapause evolution are inferred. A polyphenic character of diapause is a prominent feature in C. septempunctata and L. decemlineata, but has been found also in other Coleoptera and in insects generally and often generates voltinism heterogeneity within populations.
Ostrinia nubilalis larvae reared under both nondiapause and diapause-inducing conditions were chilled at 5°C for various periods and their haemolymph glycerol concentrations were measured enzymatically. The ability of fifth (final) instars to accumulate glycerol was dependent upon cold stress but not the diapause state. Furthermore this response was independent of any cold-induced release of cephalic or thoracic hormones. The capacity of O. nubilalis larvae to express cold-induced glycerol accumulation was found to require ecdysis from the fourth to fifth instar. Eggs as well as second, third and fourth instars were completely incompetent. These results indicate that, at the biochemical level, a specific developmental programme or sequence is required for O. nubilalis to demonstrate this response to cold stress.
Seasonal variations in the supercooling point, survival at low temperatures and sugar content were studied in field-collected codling moth larvae. The supercooling point of field-collected larvae decreased significantly from a mean value of –13.4°C in August 2004 (feeding larvae) to –22.0°C in December 2004 (overwintering larvae). Survival at –20°C/24 h was 0% during early autumn, whereas it increased to approximately 60% during winter. The survival at low temperature was well correlated with the supercooling point. The supercooling point of the diapause destined larvae decreased from –16.9 to –19.7°C between September and October as the larvae left the food source and spun a cocoon. For early-diapause larvae, exposure to 5°C/30 days has an additional effect and decreased the supercooling point from –19.7 to –21.3°C. One-month exposure of overwintering larvae to 5°C led to a mortality of 23% in early diapause larvae, while only 4% of diapause larvae died after acclimation. Overwintering larvae accumulated trehalose during winter. There was approximately a threefold increase in trehalose content between larvae at the onset ofdia-pause (5.1 mg/g fresh weight) and larvae in a fully developed diapause (18.4 mg/g fresh weight) collected in January. Trehalose content was correlated with supercooling capacity, survival at low temperatures and chilling tolerance, suggesting that trehalose may play some role in the development of cold tolerance in this species
An insect's cold hardiness affects its potential to overwinter and outbreak in different geographic regions. In this study, we characterized the response of Helicoverpa zea (Boddie) pupae to low temperatures by using controlled laboratory measurements of supercooling point (SCP), lower lethal temperature (LT(50)), and lower lethal time (LLTime). The impact of diapause, acclimation, and sex on the cold hardiness of the pupae also were evaluated. Sex did not significantly affect the SCP, LT(50), or LLTime. However, the mean SCP of diapausing pupae (-19.3°C) was significantly lower than nondiapausing pupae (-16.4°C). Acclimation of nondiapausing pupae to constant temperatures from 10 to 20°C before supercooling also produced a significantly lower SCP than nondiapausing pupae held at 25°C. The LT(50)s of nondiapausing and diapausing were not significantly different, but confirmed that H. zea pupae are chill-intolerant because these lethal temperatures are warmer than the corresponding mean SCPs. Diapausing pupae survived longer than nondiapausing pupae at the same, constant, cold temperatures, a finding consistent with the SCP results. Both of these results suggest enhanced cold hardiness in diapausing pupae. When laboratory results were compared with field temperatures and observed distributions of H. zea in the contiguous United States, the laboratory results corroborated what is currently perceived to be the northern overwintering limit of H. zea; approximately the 40(th) parallel. Moreover, our research showed that areas north of this limit are lethal to overwintering pupae not because of low temperature extremes, but rather the length of time spent at near-zero temperatures.
Managing metabolic resources is critical for insects during diapause when food sources are limited or unavailable. Insects accumulate reserves prior to diapause, and metabolic depression during diapause promotes reserve conservation. Sufficient reserves must be sequestered to both survive the diapause period and enable postdiapause development that may involve metabolically expensive functions such as metamorphosis or long-distance flight. Nutrient utilization during diapause is a dynamic process, and insects appear capable of sensing their energy reserves and using this information to regulate whether to enter diapause and how long to remain in diapause. Overwintering insects on a tight energy budget are likely to be especially vulnerable to increased temperatures associated with climate change. Molecular mechanisms involved in diapause nutrient regulation remain poorly known, but insulin signaling is likely a major player. We also discuss other possible candidates for diapause-associated nutrient regulation including adipokinetic hormone, neuropeptide F, the cGMP-kinase For, and AMPK.
The codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae), a worldwide apple pest, is classified as a freeze-intolerant organism and one of the most cold-tolerant pests. The objectives of this study were to examine the supercooling point of overwintering and non-diapausing larvae of C. pomonella as an index of its cold hardiness, and to assess larval mortality following 24 h exposure to extreme low temperatures ranging from -5 to -25 degrees C. The mean (+/-SE) supercooling point for feeding larvae (third through fifth instars) was -12.4 +/- 1.1 degrees C. The mean supercooling point for cocooned, non-diapausing larvae (i.e., non-feeding stages) decreased as the days that the arvae were cocooned increased and changed between -15.1 +/- 1.2 degrees C for one to two day cocooned arvae and -19.2 +/- 1.8 degrees C for less than five day cocooned larvae. The mean (+/-SE) supercooling point for other non-feeding stages containing pupae and overwintering larvae were -19.9 +/- 1.0 degrees C and -20.2 +/- 0.2 degrees C, respectively. Mean supercooling points of C. pomonella larvae were significantly lower during the winter months than the summer months, and sex had no effect on the supercooling point of C. pomonella larvae. The mortality of larvae increased significantly after individuals were exposed to temperatures below the mean supercooling point of the population. The supercooling point was a good predictor of cold hardiness.
The Asiatic rice borer Chilo suppressalis (Walker) occurs mainly on rice Oryza sativa L. and water-oat Zizania latifolia (Turcz). Certain ecological and physiological differentiations between rice and water-oat populations have been shown. To determine whether there is host-associated differentiation in supercooling capacity, seasonal changes in supercooling points, glycerol content, and other physiological parameters of naturally occurring C. suppressalis larvae overwintering in rice and water-oat plants were compared over the winter. Supercooling points were low in the winter (November and December), significantly higher in March, and significantly lower in the water-oat population than in the rice population in the winter. Larvae from the water-oat population experienced a significant weight loss from December to March and were heavier than those from the rice population in the winter. Body water content (percentage of fresh weight) was low in the winter and increased significantly in March; no population differences were detected. Hemolymph glycerol content was high in the winter and dropped significantly in March; interpopulation differences were significant in December. Hemolymph trehalose content increased in the winter, and no population differences were detected. Whole body glucose and lipid content varied differently between the rice and the water-oat populations over the winter. It was found that variations in hemolymph glycerol content were responsible for the host-associated seasonal changes in supercooling capacity of overwintering larvae of the Asiatic rice borer.
The cold hardiness adaptations of natural and laboratory reared populations of the codling moth, Cydia pomonella, were examined. Hemolymph, gut, and whole body supercooling points (SCPs), 24-h LT50s, polyhydroxy alcohol concentrations, hemolymph freezing points, and hemolymph melting points were determined. Nondiapausing codling moth larvae do not have appreciable levels of ice nucleators in the hemolymph or gut. Whole body supercooling points were higher than hemolymph supercooling points. For nondiapausing larvae, LT50s were significantly higher than both the whole body and the hemolymph supercooling points, indicating the presence of chill sensitivity. As the larvae left the food source and spun a cocoon, both hemolymph and whole body SCPs decreased. Diapause destined larvae had significantly lower hemolymph SCPs than nondiapausing larvae, but whole body SCPs were not significantly different from nondiapausing larvae of the same age. The LT50s of diapause destined and diapausing larvae were significantly lower than that of nondiapausing larvae. Codling moths are freezing intolerant, with LT50s close to the average whole body supercooling point in diapause destined and diapausing larvae. The overwintering, diapausing larvae effectively supercool to avoid lethal freezing by removal of ice nucleators from the gut and body without appreciable increase of antifreeze agents such as polyols or antifreeze proteins.
Prior to this study, antifreeze proteins (AFPs) had not been identified in terrestrial arthropods from the Arctic or anywhere in Alaska. The hemolymph of 75 species of insects and six spiders from interior and arctic Alaska were screened for thermal hysteresis (a difference between the freezing and melting points), characteristic of the presence of AFPs. Eighteen species of insects and three spiders were shown to have AFPs. Ten of the insects with AFPs were beetles including the first species from the families Chrysomelidae, Pythidae, Silphidae and Carabidae. In addition, the first Neuropteran to have AFPs was identified, the lacewing Hemerobius simulans together with the second and third Diptera (the first Tipulids) and the second and third Hemiptera, the stinkbug Elasmostethus interstinctus (the first Pentatomid), and the water strider Limnoporus dissortis (the first Gerrid). Prior to this study, 33 species of insects and three spiders had been reported to have AFPs. Most AFP-producing terrestrial arthropods are freeze avoiding, and the AFPs function to prevent freezing. However, some of the AFP- producing insects identified in this study are known to be freeze tolerant (able to survive freezing) to very low temperatures (-40 to -70 degrees C).
Low temperature is a major environmental constraint impacting the geographic distribution and seasonal activity patterns of insects. Written for academic researchers in environmental physiology and entomology, this book explores the physiological and molecular mechanisms that enable insects to cope with a cold environment and places these findings into an evolutionary and ecological context. An introductory chapter provides a primer on insect cold tolerance and subsequent chapters in the first section discuss the organismal, cellular and molecular responses that allow insects to survive in the cold despite their, at best, limited ability to regulate their own body temperature. The second section, highlighting the evolutionary and macrophysiological responses to low temperature, is especially relevant for understanding the impact of global climate change on insect systems. A final section translates the knowledge gained from the rest of the book into practical applications including cryopreservation and the augmentation of pest management strategies.
We investigated the physiological adaptations for winter survival in a freeze-intolerant chrysomelid, Aulacophora nigripennis, in warm-temperate regions. The adults showed a decreased supercooling point (SCP), increased chill tolerance and high myo-inositol content during winter. Chill tolerance at 0°C appears to be a more suitable indicator of their cold hardiness than SCP because they die at 0°C without freezing and normally are not exposed to subzero temperatures below their SCP (<-4°C). The temporal pattern for changes in chill tolerance correlated closely with that for fluctuations in myo-inositol content, indicating that this compound has a role in chill tolerance. Topical application of a juvenile hormone analogue, pyriproxyfen, at the dose which can terminate their diapause decreased both chill tolerance and myo-inositol content, suggesting that cold tolerance of this beetle is linked to the diapause program. The thermal response in relation to myo-inositol metabolism changed seasonally. This may in part contribute to forming a seasonal profile of myo-inositol pool in a natural population. This study provides one model for the cold tolerance strategy of freeze-intolerant insects in relatively mild temperate regions.
The recent isolation of several diapause up regulated and down regulated genes suggests two key features of diapause that are likely to prevail across species. First, far fewer genes are being expressed during diapause, thus suggesting that diapause does represent a shut-down in gene expression. Second, the fact that certain genes are expressed uniquely in diapause indicates that diapause is more than a shut-down in gene expression and represents a distinct developmental pathway. Only few diapause down regulated and up regulated genes have so far been identified, and among them are potentially important genes coding for cell cycle regulators and stress proteins. It should then be possible to determine whether common patterns of gene expression dictate diapause in different species and different developmental stages.
Low temperature affects insects differently based on the severity of the cold and the duration of exposure. Life stage and acclimation state also have a major impact on an insect's response to low temperature. Many temperate and polar insects enhance their cold-tolerance seasonally in preparation for winter, as short, cool days in autumn trigger cold acclimatization, as well as entry into the metabolic depression of diapause. However, insects also have the capacity to make significant and rapid adjustments to even slight changes in environmental temperature, as would occur on a summer's day. This introductory chapter seeks to provide a short primer on the physiology of insect cold-tolerance that will be useful to students and others new to the area of study. This overview of basic concepts in insect cold-tolerance intends to provide a context for later chapters providing in-depth reviews of specific areas. Specifically, this primer focuses on regulation of supercooling and ice nucleation, and basic adaptations promoting cold-tolerance. Suggestions for conducting and clearly reporting experimental results on insect cold-tolerance are also included. Since this volume is intended to update and complement our previous book, Insects at Low Temperature (Lee and Denlinger, 1991), this synoptic chapter will emphasize articles published during the past 20 years and topics not covered elsewhere in this volume.
Cold hardiness and diapause are both essential components of winter survival for most insects of the temperate zone. But, in many cases, it is not clear how these two are related. Are they independent events or is cold hardiness a component of the diapause syndrome? Both independence (Lees, 1955; Salt, 1961; Ring, 1972) and dependence (Asahina, 1969; Mansingh, 1971, 1974) of cold hardiness and diapause have been defended vigorously, and indeed evidence for both possibilities can be found in the literature. In this chapter I argue that cold hardiness can be achieved independently of diapause, but cold hardiness is often a component of the diapause syndrome and the expression of diapause frequently extends the insect’s capacity to cold harden.
The pistachio white leaf borer, Ocneria terebinthina Strg., is a minor pest of pistachio trees in the pistachio-producing zone of Rafsanjan, Iran, where it overwinters as fourth larval instar. In this study, the quantities of several metabolites including trehalose, glycogen and lipids were measured each month from October 2009 to March 2010. Physiological changes in relation to ambient temperature were also investigated in field-collected overwintering larvae. The glycogen content decreased as ambient temperature decreased; this decline was proportional to increases in trehalose content. In January, which has a mean ambient temperature of 8.0°C, the glycogen content of the larvae was at its lowest level (17.98 mg/g fresh body weight) whereas trehalose was at its highest level (23.99 mg/g fresh body weight). Total body lipids decreased during overwintering and reached the lowest level at the onset of spring. In conclusion, trehalose may be involved in winter survival and the adaptation of the pistachio white leaf borer to cold, by providing cryoprotection. Larvae of the pistachio white leaf borer may have the ability to reserve energy in the form of lipids and utilize it during overwintering.
The common pistachio psylla, Agonoscena pistaciae Burckhardt & Lauterer (Hemiptera: Psyllidae), is known as the key pest of pistachio orchards in Iran. This pest passes the winter as adults. In this study, energy allocation changes in relation to ambient temperature were investigated in field-collected adults by measuring total body sugar, trehalose, glucose, sorbitol, myoinositol, glycogen, lipid, and protein contents. Glycogen content decreased with decrease in ambient temperature. The decrease in glycogen content was proportional to the increase in total body sugar, trehalose, myoinositol, and sorbitol contents. In January, with mean ambient temperature of 5.4°C, glycogen content was at the lowest level, whereas total body sugar, trehalose, glucose, and sorbitol were at the highest level. Total body sugar, trehalose, myoinositol, and sorbitol contents increased as temperature decreased from 22.7°C in October to 5.4°C in January. In conclusion, low molecular weight carbohydrates and polyols may play a role in winter survival and adaptation to cold of the common pistachio psylla by providing the required cryoprotection. Also, overwintering adults of the common pistachio psylla may store energy in the form of lipid for later utilization during the overwintering.
between diapausing and non-diapausing larvae of the pine caterpillar Dendrolimus tabulaeformis (Lepidoptera: Lasiocampidae). Zoological Studies 47(1): 96-102. We investigated chemical and morphological differences between diapausing and non-diapausing larvae of the pine caterpillar, Dendrolimus tabulaeformis (Lepidoptera: Lasiocampidae). Chemical analyses showed that both the lipid content in the whole body and the concentra- tion of trehalose significantly differed between diapausing and non-diapausing larvae. The lipid, protein, and amino acid contents of the hemolymph of diapausing larvae were higher than those of non-diapausing larvae. The content of free fatty acids of non-diapausing larvae was 4 times higher than that of diapausing larvae. Moreover, diapausing larvae showed lower oxygen consumption than non-diapausing larvae. Differences in the anatomy of incretions according to the physiological condition (diapausing or non-diapausing) were evident, and included the number of cells in the corpora allata, the volume of the nucleus, and the internal structure of the cytoplasm. The prothoracic glands also differed between diapausing and non-diapausing larvae. These results suggest that the pine caterpillars hormonally regulate their body composition to adapt to their surround- ings. http://zoolstud.sinica.edu.tw/Journals/47.1/96.pdf
It is well known that temperature has a pervasive effect on insects. Nearly every aspect of an insect’s life is influenced by temperature, from direct effects on the kinetics of enzymatic reactions, to defining the limits of physiological function and behavior, and ultimately to shaping of evolutionary pathways. As a group, insects, more than any other eukaryotic taxon, have evolved not only to survive but to flourish in a wide variety of thermal environments.
The role of polyhydric alcohols in cryoprotection is probably the most extensively studied feature of insect cold hardiness. The importance of glycerol as a cryoprotectant was first recognized by R. W. Salt after he and others linked the presence of high levels of glycerol with winter hibernation, diapause, or freezing survival (Salt, 1957, 1959, 1961; Wyatt and Kalf, 1957; Chino, 1957). Over the last 30 years, literally hundreds of publications have described the occurrence of glycerol or other polyols in both freeze-tolerant and freeze-avoiding insects (for reviews, see Salt, 1961; Hansen, 1980; Ring, 1980; Sømme, 1982; Miller, 1982; Duman et al., 1982; Baust et al., 1982; Zachariassen, 1985; Lee et al., 1986; Storey and Storey, 1988). Glycerol is by far the most common cryoprotectant, but sorbitol, mannitol, ribitol, erythritol, threitol, and ethylene glycol also occur along with a selection of sugars, including trehalose, sucrose, glucose, and fructose (see Fig. 4.1) (Miller and Smith, 1975; Hayakawa and Chino, 1981; Sømme, 1982; Gehrken, 1984; Zachariassen, 1985; Hamilton et al., 1985; Storey and Storey, 1988). Glycerol contents that range as high as 25% of the fresh weight of the animal have been reported with polyol concentrations in excess of 2 M in the body fluids of many species (Salt, 1961; Ring, 1981; Zachariassen, 1985; Storey and Storey, 1988). The majority of species produce only a single polyol, but dual or even multiple component systems also occur, glycerol plus sorbitol being the most common pairing (Storey and Storey, 1988).
In the field in Iraq Ectomyelois ceratoniae had four generations on an artificial diet and four to five generations on pomegranate fruits. On an artificial diet the mean time for development from egg to adult ranged from 41 to 59 days for the first three generations. Fourth-generation larvae entered diapause in the fall and averaged 241 days. Insect population increased progressively during the fruiting season. Mean percentage of infestation increased from 20% by early generations to more than 80%. High infestation was observed in southcentral areas of Iraq. Orchards in which sanitation was poor were highly infested.
Abstract The apple leaf miner Phyllonorycter ringoniella (Matsumura) (Lepidoptera: Gracillariidae) overwinters as a diapausing pupa. The diapause rate reaches 100% in early October. Diapause intensity decreases gradually from early October and diapause terminates in early February. The fresh body weight of diapausing pupae is 1.6 times that of non-diapausing pupae. The main cryoprotectant in P. ringoniella pupae is trehalose. Three stages are distinguishable as indicated by the correlations between diapause intensity, levels of cold hardiness and the trehalose content: diapause induction occurred in October, diapause development from November to December, and post-diapause quiescence from January to April. During diapause induction, the pupae accumulate low levels of trehalose and do not survive exposure to −15 °C. During diapause development, the pupae gradually accumulate more trehalose and show some ability to survive exposure to −15 °C, but not to −20 °C. During post-diapause quiescence, the pupae accumulate relatively more trehalose and cold hardiness fully develops, but decreases quickly in April. The trehalose content in pupae sampled in December is unaffected by acclimation temperatures in the range 0–30 °C, but decreases in pupae sampled in March after acclimation at temperatures from 5 to 15 °C. These results suggest that overwintering pupae of P. ringoniella have the ability to accumulate trehalose and develop a high level of cold hardiness during diapause development.
Phosphorus metabolism, respiratory rate and carbohydrate changes were measured during diapause termination in pupae of Pieris brassicae in a test of the relationship between the pattern of cryoprotectant synthesis and degradation and changes in diapause metabolism. Field overwintering pupae show a gradual increase in metabolic rate measured by 31P NMR from February with a more rapid increase in early May during the last 2 weeks before eclosion. Sorbitol decreases throughout the period following a trend which begins earlier in the winter. Trehalose declines only in the last 2 weeks as glucose increases. Artificial termination of diapause by injection of 20-hydroxyecdysone condenses these changes into a period of 10–12 days at 20°C allowing a more detailed comparison to be made. There are signs of metabolic rate increase in the first 2 days after injection shown by a small increase in ATP and related decrease in inorganic phosphate. Sorbitol declines rapidly from 1 to 3 days after injection whereas trehalose declines after 3 days. Respiratory rate does not begin to increase until 2 days after injection and continues to increase slowly for another 8 days. The possible relationships between these components of diapause metabolism are discussed.
The relationship between diapause‐associated metabolic suppression and carbohydrate metabolism linked with cold tolerance was investigated in pupae of Pieris brassicae L. Cold tolerance was assessed by measuring the crystallization temperature (T c ) and by estimates of pre‐freeze mortality. Metabolic suppression was measured using ³¹ P nmr and carbohydrates by GLC.
Sorbitol (a possible cryoprotectant) accumulated from the onset of diapause in October until December reaching a concentration of c. 40 mMolal in both years of the study, but then declined from January until adult eclosion in May. The T c remained between ‐23 and ‐25 o C throughout the winter except for a slight rise before eclosion in May. The absence of a significant T c suppression is as predicted from the low concentration of sorbitol accumulated. The pre‐freeze mortality experiments indicate that pupae are most cold tolerant in the period December‐February when sorbitol concentration is high, suggesting an alternative cryoprotective role for sorbitol. Glycogen declined at the beginning of diapause until February after which there was some recovery suggesting that it may be the source of carbon for sorbitol synthesis.
Diapause‐associated metabolic suppression is evident in the low ³¹ P nmr resonances of ATP during November‐February compared with non‐diapause pupae and diapause pupae soon after pupation. The suppression of metabolism at this time may have a direct role in cryoprotection and by itself (rather than sorbitol) account for the increased pre‐freeze cold tolerance. ATP appears to increase slowly from February until a sharp increase occurs shortly before eclosion. Arginine phosphate remains high during diapause until late February‐March when it begins a decline which continues until eclosion. A period of change in energy and carbohydrate metabolism is apparent at the same time which may indicate diapause termination and related changes in cold tolerance mechanisms.
It is argued that in P.brassicae sorbitol accumulates as a result of metabolic suppression and may have no cryoprotective role. However, for species living in, or colonizing, low temperature environments it is a short evolutionary step to exploit this pathway and accumulate high concentrations of polyols as a specialized cold tolerance strategy.
1. Four groups of spruce budworm larvae, Choristoneura fumiferana, of the same physiological stage (at the beginning of diapause) were exposed to natural temperatures, starting in July, August, September and October. Post-diapause emergence and certain metabolites were monitored throughout the overwintering period.
2. Larval exposure to high temperatures for long periods before winter had a significant effect on temperature requirements for subsequent diapause development and apparently caused a certain amount of mortality, especially for those larvae that entered diapause earlier in the season.
3. Substantial loss of lipid and glycogen reserves was related to intensity of high temperature exposure during the pre-winter period, which in turn was associated with the timing of diapause initiation.
4. Patterns of glycerol accumulation during the winter were also influenced by temperature conditions in the early phase of diapause development.
5. Among several possible mortality factors, an extended cool period in spring was suggested as a potential cause of overwintering mortality, which was closely associated with a deficiency in energy reserves caused by high temperature exposure before winter.
6. These results stress the significant impact of pre-winter conditions on the overwintering process and highlight the importance of an ecophysiological approach to insect overwintering biology.
The objective of this study was to associate levels of nutritional reserves (specifically lipids, sugars, and glycogen) in
individual Mediterranean fruit flies, Ceratitis capitata (Diptera: Tephritidae), with observed patterns of behavior in the field. We collected females (n=255) and males (n=181) from the field, recording the time of collection and the activity they were engaged in when collected. Subsequently,
we employed colorimetric biochemical techniques to determine the precise amounts of lipids, sugars, and glycogen in each individual.
Lipid and sugar levels in males varied significantly according to the time of collection and the type of activity. Lipid and
sugar levels in females did not vary in this manner. Sugar levels in both males and females were highest during the evening,
when most feeding occurs. Males that engaged in sexual signaling in leks during the mid-afternoon had relatively low sugar
and high lipid levels. Males engaged in the alternative mating tactic of fruit guarding had relatively high sugar and low
lipid contents. Glycogen levels in males were high in the mornings, and a decline in glycogen content was associated with
participation in leks; however, female glycogen levels did not vary significantly with time of day or activity. Our results
provide quantitative evidence for the role nutrient reserves play in driving patterns of male reproductive behavior, yet suggest
that factors other than sugar and lipid reserves constrain female behavior.
Cold hardiness and biochemical changes were investigated in winter and summer pupae of the cabbage armyworm Mamestra brassicae at the diapause and post-diapause stages under temperature acclimation. Diapause pupae were successively acclimated to 25, 20 and then 10°C (warm-acclimated group). Pupae at the diapause and post-diapause stages were successively acclimated to 5, 0, −5 and then −10°C (cold-acclimated groups). Supercooling point values in winter and summer pupae remained constant regardless of the diapause stages and acclimated temperatures. Warm-acclimated pupae at the diapause stage did not survive the subzero temperature exposure, whereas, cold-acclimated pupae achieved cold hardiness to various degrees. Winter pupae were more cold hardy than summer pupae, and pupae at the post-diapause stage were more cold hardy than those at the diapause stage. Trehalose contents in winter pupae rose under cold acclimation. Summer pupae accumulated far lower trehalose contents than winter pupae, with the maximal level occurring in winter pupae at the post-diapause stage. Glycogen content remained at a high level in diapause pupae after warm acclimation, whereas it decreased after cold acclimation. Alanine, the main free amino acid in haemolymph after cold acclimation, increased at lower temperatures in both diapause and post-diapause pupae, but the increase was greater in the diapause pupae. These results suggest that cold hardiness is more fully developed in winter pupae than in summer pupae, and cold acclimation provides higher cold hardiness in winter pupae at the post-diapause stage than at the diapause stage.
Insect cold survival has drawn increased attention in recent years, prompting a new classification of survival phenotypes that reflect the ecological and physiological differences in the manner in which insects survive the winter. In addition, more is known about the nature of cold injury and its effects on an insect system from the whole body to the subcellular level, although much remains to be discovered. Other than the detection of misfolded protein by the heat shock response, signal transduction of low temperatures by insects is largely unexplored. Recent advances in Drosophila and a few vertebrate systems reveal the potential importance of transient receptor potential ion channels in low temperature signal transduction, and membrane fluidity also possibly plays a role in the detection of low temperatures by insects. Physiological changes that occur in insect cells as a result of low temperatures or in anticipation of low temperatures include the induction of a suite of heat shock proteins, metabolic polyol synthesis, antifreeze protein production, and the induction of extracellular ice nucleation. In addition, a variety of new genes are now known to be induced upon cold exposure, but the roles of these molecules in cold survival are still largely unknown.
Diapausing cold-acclimated adults of the bug Pyrrhocoris apterus accumulate four ‘winter’ polyols, ribitol, sorbitol, mannitol and arabinitol, in total concentrations of up to 100 mM. The accumulation started only when the temperatures dropped below a threshold of +5°C in laboratory acclimated insects. The supercooling capacity (SCP) was not affected by polyol accumulation and remained constant at approximately −17°C. Cold hardiness, measured as survival time (Lt50) at −15°C, increased from approximately 1 day to approximately 1 week in parallel with polyol accumulation. There was a tight correlation (r=0.98) between the concentration of ‘winter’ polyols in haemolymph and Lt50−15. When a mixture of ribitol and sorbitol was injected into the haemolymph of the bugs acclimated to +5°C, the concentration of polyols increased from 2.5 to 83.1 mM in haemolymph, or from 0.07 to 6.61 μg/mg of fresh weight in the whole body, the SCP remained unchanged and survival after exposure to −14°C for 3 days increased approximately three-fold in comparison to untreated controls. Such results were interpreted as evidence for the cryoprotective role of accumulated polyols independent of the depression of SCP. Acclimation protocol using thermoperiod, mimicking daily temperature oscillations, resulted in moderately lower SCP, higher sum of polyols accumulated and significantly longer Lt50−15 than at acclimation protocol with constant temperatures.
Prepupae of the arctiid moth Cymbalophora pudica spend spring and summer months in a summer diapause (aestivation), the duration of which is photoperiodically controlled. Cold hardiness, drought tolerance and some physiological and biochemical parameters were measured in aestivating prepupae. Large amounts of metabolic reserves, in the form of lipids and glycogen, accumulated prior to aestivation. Glycogen served as the main metabolic fuel for aestivating prepupae. Metabolic rate decreased rapidly after the onset of the inactive prepupal stage and remained low (5–15% of the level in active larva) during aestivation. A spontaneous increase of the respiration rate occurred before pupation. Neither low mol. wt sugars or alcohols (polyols) accumulated nor the haemolymph osmotic pressure changed during aestivation. Drought tolerance of aestivating prepupae was high (no decrease in survival after exposure to r.h.<10% at a temperature of 23°C for a substantial part of diapause) owing to their extensive capacity to stabilize the relative body water content irrespective of the r.h. of surrounding air. Cold hardiness was low (>90% decrease in survival after exposure to −7°C for 24 h). Cold and drought acclimations did not lead to significant changes in the measured physiological and biochemical parameters but cold (not drought) acclimation caused a significant increase in cold hardiness. Neither drought tolerance nor the increase in cold hardiness after cold acclimation appear to be related to presence/accumulation of polyols in aestivating C. pudica prepupae.
Calanoid copepods, such as Calanus finmarchicus, are a key component of marine food webs. C. finmarchicus undergo a facultative diapause during juvenile development, which profoundly affects their seasonal distribution and availability to their predators. The current ignorance of how copepod diapause is regulated limits understanding of copepod population dynamics, distribution, and ecosystem interactions. Heat shock proteins (Hsps) are a superfamily of molecular chaperones characteristically upregulated in response to stress conditions and frequently associated with diapause in other taxa. In this study, 8 heat shock proteins were identified in C. finmarchicus C5 copepodids (Hsp21, Hsp22, p26, Hsp90, and 4 forms of Hsp70), and expression of these transcripts was characterized in response to handling stress and in association with diapause. Hsp21, Hsp22, and Hsp70A (cytosolic subfamily) were induced by handling stress. Expression of Hsp70A was also elevated in shallow active copepodids relative to deep diapausing copepodids, which may reflect induction of this gene by varied stressors in active animals. In contrast, expression of Hsp22 was elevated in deep diapausing animals; Hsp22 may play a role both in short-term stress responses and in protecting proteins from degradation during diapause. Expression of most of the Hsps examined did not vary in response to diapause, perhaps because the diapause of C. finmarchicus is not associated with the extreme environmental conditions (e.g., freezing and desiccation) experienced by many other taxa, such as overwintering insects or Artemia cysts.
The original Lowry method of protein determination has been modified by the addition of sodium dodecyl sulfate in the alkali reagent and an increase in the amount of copper tartrate reagent. These alterations allowed the method to be used with membrane and lipoprotein preparations without prior solubilization or lipid extraction and with samples containing 200 mm sucrose or 2.5 mm EDTA.
From the available experimental data a relatively clear picture can be established with regard to the physiological importance of some of the mechanisms involved in insect cold hardening. In freeze-avoiding insects, all potent ice-nucleating agents are removed or inactivated, leading to a depression of the supercooling points to about 20 degrees C. Accumulation of polyols causes a further depression with a magnitude of about twice the corresponding melting-point depression. Production of thermal hysteresis factors causes a stabilization of the supercooled state. In freeze-tolerant insects, potent ice-nucleating agents are produced in the extracellular body fluid, ensuring a protective extracellular freezing at a few degrees below zero. Accumulation of polyols causes a steep drop in the lethal temperature, due to a reduction of the amount of ice by a colligative mechanism. However, there is still much to be learned about the mechanisms by which ice-nucleating agents, polyols, and thermal hysteresis agents are acting. Furthermore, the regulatory mechanisms involved in the production and elimination of these components from the body fluid of the insects are not understood. Also, when it comes to the influence of environmental factors, like photoperiod and temperature, there is much to be learned. In addition to giving attention to these topics, future research should be focused on the possible role of other factors in cold hardening such as bound water, dehydration, low-molecular-weight solutes other than polyols, and the biochemical mechanisms forming the basis of the seasonal changes in the cold hardiness of insects.
Seasonal dynamics of ecophysiological parameters are described which are relevant to overwintering in field-collected adults of a Czech population of the red firebug, Pyrrhocoris apterus. Five life-cycle phases were distinguished using the duration of pre-oviposition period as a criterion: reproductive activity (spring-early summer), intensification of reproductive diapause (RD) (peak of summer), maintenance of RD (late summer-early autumn), termination of RD (late autumn-early winter), and low temperature quiescence (LTQ) (winter). The supercooling capacity and chill tolerance (c.t.) increased simultaneously with the termination of RD and all three processes were triggered/conditioned by autumnal decrease in ambient temperatures. Maximum supercooling capacity and c.t. 'outlived' the end of diapause and persisted throughout the LTQ state. The limits of c.t. were estimated as -15 degrees C/1-2 weeks for 50% survival. Ribitol, sorbitol, arabinitol, and mannitol were accumulated in the winter-sampled insects. Relatively low concentrations of polyols (dominating ribitol reached ca. 1% FW) indicate that they do not function as colligative cryoprotectants. However, because their seasonal occurrence coincided with the highest c.t., their non-colligative cryoprotectant effects would merit further study. Although the overwintering microhabitat of P. apterus is buffered, the temperatures may fall to -13 degrees C during exceptionally cold winters and thus, the parameters of c.t. seem to be just appropriately tuned to the local overwintering conditions.
Diapausing cold-acclimated adults of the bug Pyrrhocoris apterus accumulate four 'winter' polyols, ribitol, sorbitol, mannitol and arabinitol, in total concentrations of up to 100 mM. The accumulation started only when the temperatures dropped below a threshold of +5 degrees C in laboratory acclimated insects. The supercooling capacity (SCP) was not affected by polyol accumulation and remained constant at approximately -17 degrees C. Cold hardiness, measured as survival time (Lt50) at -15 degrees C, increased from approximately 1 day to approximately 1 week in parallel with polyol accumulation. There was a tight correlation (r=0.98) between the concentration of 'winter' polyols in haemolymph and Lt50(-15). When a mixture of ribitol and sorbitol was injected into the haemolymph of the bugs acclimated to +5 degrees C, the concentration of polyols increased from 2.5 to 83.1 mM in haemolymph, or from 0.07 to 6.61 microg/mg of fresh weight in the whole body, the SCP remained unchanged and survival after exposure to -14 degrees C for 3 days increased approximately three-fold in comparison to untreated controls. Such results were interpreted as evidence for the cryoprotective role of accumulated polyols independent of the depression of SCP. Acclimation protocol using thermoperiod, mimicking daily temperature oscillations, resulted in moderately lower SCP, higher sum of polyols accumulated and significantly longer Lt50(-15) than at acclimation protocol with constant temperatures.
Amounts of several metabolites were measured in overwintering larvae of Enosima leucotaeniella acclimated to temperatures between -5 and 15 degrees C for 30days. In the diapausing stage, cold hardiness, as shown by the survival rate, began rising below 15 degrees C. Glycogen content decreased as the temperature decreased from 10 to 0 degrees C. Trehalose content rose as the temperature decreased from 15 to 5 degrees C, but remained unchanged as the temperature decreased from 5 and 0 degrees C. Twenty-eight free amino acids were detected in the haemolymph; levels of proline, glutamine and glutamic acid increased at high temperatures, but alanine increased at low temperatures, especially as temperature decreased from 5 to 0 degrees C. Lipid content was unchanged by the different acclimation temperatures. The effects of temperature, diapause and aerobic conditions on the levels of carbohydrates and amino acids in overwintering larvae were analyzed. Alanine levels rose at low temperature only when the larvae were in the diapausing stage. The level of trehalose rose at low temperature in both the diapausing and post-diapausing stages, although it was higher at aerobic conditions in the post-diapausing stage. These results suggest that efficient trehalose synthesis occurs under the combination of low temperature and aerobic conditions of the post-diapausing stage, so that cold hardiness in overwintering E. leucotaeniella larvae may rise to a high level in winter.
Partial clones for two members of Leptinotarsa decemlineata inducible 70kDa heat shock protein family (LdHSP70A and B) were developed using RT-PCR. LdHSP70A, but not LdHSP70B, was upregulated during adult diapause. The ability of L. decemlineata to express these two genes in response to subzero temperatures depended on the thermal history of the beetles. Chilling diapausing beetles increased the rate at which both LdHSP70A and B were expressed following a cold shock at -10 degrees C. Following cold shock at -10 degrees C, LdHSP70B expression peaked after 3h at 15 degrees C for chilled diapausing individuals, decreasing to near background levels by the sixth hour. In contrast, nonchilled diapausing beetles expressed their highest level of LdHSP70B only after 6h at 15 degrees C. Diapausing beetles exposed to a thermoperiod with a mean temperature of either 0 or -2.5 degrees C expressed significantly higher levels of both LdHSP70A and B than beetles exposed to constant 0 or -2.5 degrees C. These results demonstrate that the expression of LdHSP70A and B is differentially regulated in response to diapause and environmental conditioning.
Environmental adaptations of the rice stem borer, Chilo suppressalis and the blue alfalfa aphid, Acyrthosiphon konodi to seasonal fluctuations
- H Tsumuki
Tsumuki, H. (1990) Environmental adaptations of the rice stem borer, Chilo suppressalis and the blue alfalfa aphid, Acyrthosiphon konodi to seasonal fluctuations. pp. 273–278 in