Are mummy characteristics reliable indicators of diapause and cold tolerance in the parasitoid wasp Aphidius rhopalosiphi (Braconidae, aphidiinae) ?

Many insect species have evolved different overwintering survival strategies such as cold tolerance or diapause. This study investigated the relationship between Aphidius rhopalosiphi mummy colour and cold tolerance and diapause. Mummy colour was insufficient to discriminate diapausing from non-diapausing individuals. This phenotypic character seems to reflect environmental conditions rather than direct developmental time and cocoon thickness (identification criteria of diapause). There is, however, a relationship between cold tolerance and mummy colour. Dark mummies exhibited significantly higher water content, survival at low temperature and lower supercooling point values than pale mummies. Mummy colour in Aphidius rhopalosiphi seems to be a phenotypic indicator of the cold tolerance.     

Some attributes of cold hardiness of the gregarious ectoparasitoid Colpoclypeus florus (Hymenoptera: Eulophidae)

Cold hardiness of diapause and non-diapause larvae of the parasitoid Colpoclypeus florus Walker (Hymenoptera: Eulophidae) was examined in the laboratory. Mean supercooling point (SCP) for diapausing larvae was -26.7 degree C and for non-diapausing larvae immediately after their larval development, was -16.2 degree C. Mean SCP for non-diapause larvae at the prepupa stage was -19.1 degree C. A short period of acclimation (1 week at 5 degree C) had no influence on the mean SCPs of both diapause and non-diapause larvae. Pre-freeze mortality for diapause and non-diapause larvae was also studied. A constant exposure of diapause larvae to -6 degree C resulted in high mortality (70.7 percent) after a period of 40 days. In contrast, 6 days at -6 degree C were sufficient to cause the same level of mortality in non-diapause larvae. After exposure of 15 days at -9 degree C, mortality for non-diapause larvae was 70 percent, whereas after 20 days at the same temperature mortality of diapause larvae was 25 percent. The importance of these findings for the cryobiology of C. florus is discussed.     

Chill tolerance in the tropical beetle Stenotarsus rotundus

The tropical beetle Stenotarsus rotundus (Endomychidae) survived chilling at mildly low temperatures (above +5 degree C). With upper limit of cold injury zone (ULCIZ, the highest temperature that causes cold injury) well above freezing point, the supercooling ability (mean supercooling point - SCP; -11 degree C) has no cryoprotective importance. Mortality increases rapidly between -9 and +5 degree C, dependent on accumulated dose of chilling (sum of injurious temperatures - SIT; 2 degree-days below ULCIZ). The cold hardiness traits found in this species are by-products of deep diapause, and may serve as pre-adaptation for expansion into cooler regions.     

Survival at low temperatures in insects: what is the ecological significance of the supercooling point?

Many freezing-intolerant insects may die during long or even brief exposures to temperatures above their supercooling point (SCP). Consequently, the real ecological value of the SCP remains ambiguous, particularly for tropical species that never experienced cold exposures. The bimodal distribution of SCP is discussed in the light of sexual dimorphism. The importance of sex in insect cold hardiness has been regularly neglected and although we admit that in some species sex may be uneasy to determine, it should be taken into account in further studies. We suggest that supercooling ability may be, at least partially, a result of adaptations to other functions unrelated to cold, including the desiccation resistance. The potential causes of insect death at low temperatures during survival experiments have also been examined. Prolonged exposures at lethal low temperatures can produce deleterious effects (including death) even if the insect does not freeze; during long-term exposure to low temperatures the organisms may finally die from the exhaustion of energy reserves.     

Cold hardiness and supercooling capacity in the pea leafminer Liriomyza huidobrensis

Pupal SCP (supercooling point) of Liriomyza huidobrensis showed no variation with age, with an average of -20.9 degree C. Low temperature survival of different ages of pupae showed no correlation with their SCP. Nonlinear regression analysis found that the response of L. huidobrensis pupae to exposure time under different low temperature regimes above -5 degree C was best fitted by a logistic equation. Both low temperature and exposure time had significant effects on pupal mortality. Temperatures above 5 degree C do not prevent pupae from emergence. L. huidobrensis was shown to be a freeze susceptible, and at the same time, a chill tolerant insect. It can tolerate subzero temperatures by supercooling. Compared with L. sativae, another dominant leafminer in China, L. huidobrensis is more cold tolerant. Our results explain differences between the species in geographic distribution and phenology.     

Supercooling capacity and cold hardiness of the adults of the sycamore lace bug, corythucha ciliata (Hemiptera:Tingidae)

Supercooling point (SCP) of female adults of Corythucha ciliata was significantly lower than that of male adults, with an average being -11.49 degrees C and -9.54 degrees C, respectively. Low temperature survival of adults of different ages indicated that there were differences in cold survival ability among age groups of adults. Nonlinear regression analysis found that the response of C. ciliata adults to exposure time under different low temperature regimes (above -5 degrees C) was best fitted by a logistic equation. Both low temperature and exposure time had significant effects on mortality of adults. Temperatures above 5 degrees C did not prevent C. ciliata adults from surviving. C. ciliata was shown to be a freeze-intolerant but chill-tolerant insect. C. ciliata could tolerate subzero temperatures by supercooling. Temperature around -8 degres C is a critical point for successful overwintering of C. ciliata adults, which can establish in the whole areas where Platanus trees are planted in China.     

The tolerance of the field slug Deroceras reticulatum to freezing temperatures

Cold hardiness of ectotherms has been widely studied in arthropods, but there is a more limited literature on the survival of molluscs at low temperatures. A number of intertidal species have been examined in detail, but terrestrial molluscs have largely been overlooked until recently. This paper reports results of laboratory experiments to evaluate the cold hardiness of the terrestrial slug, Deroceras reticulatum. The mean supercooling point (SCP) rose from -4.2 degree C in summer to -3.6 degree C in winter. The SCP that caused 50 percent mortality (LSCP50) remained constant at -4.7 to -4.8 degree C in both seasons, but slugs were able to survive the frozen state for longer in winter (LD50 of 31.8 minutes compared with 17.0 minutes in summer). Slug survival at freezing temperatures was prolonged to at least five hours when placed on a moist, absorbent substrate. D. reticulatum exhibits partial freeze tolerance, with an increased survival in winter. The results are discussed in relation to the natural environment of slugs.     

Rapid cold hardening in young hoppers of the migratory locust Locusta migratoria L. (Orthoptera: Acridiidae)

This paper describes a rapid cold hardening process for first instar hoppers of the migratory locust Locusta migratoria L. First instar hoppers of this species are often subjected to subzero temperatures or frosts in early April or May after their emergence from the soil. The mean supercooling point of hoppers is -13.0 +/- 1.4 degrees C; the fact that none could survive this temperature suggests they are freezing intolerant. When young hoppers were transferred directly from 30 degrees C to -7 degrees C for 2 h, there was only 35.8% survival. However, exposure to 0 degrees C for 2 h prior to transfer to -7 degrees C increased the apparent survival to 75%. A similar rapid cold hardening response can also be induced by gradual cooling at rates of between 0.05 and 0.1 degreess C min(-1). Rapid cold hardening also elevates the Ltime50 of first instar hoppers at -7 degrees C by approximately 3 fold, and reduces the lethal temperature from -10 degrees C to -12 degrees C. However, the protection from cold shock gained through rapid cold hardening was transient and easily lost within 2 h of hoppers being returned to 30 degrees C. The rapid cold hardening response is possibly advantageous to first instar hoppers that are often exposed to large temperature fluctuations in spring or early summer.     

Sub-zero cooling synchronizes post-diapause development of codling moth,Cydia pomonella

Sub-zero cooling treatments of -10 degree C and -15 degree C for 2-6 days were evaluated as a means of meeting the chilling requirement of diapause and to synchronize post-diapause development in larvae which were held in diapause for less than 6 months. Sub-zero cooling did not affect male longevity. After six months in diapause, diapause/cooled females were longer lived than diapause control females but not longer than non-diapause females. In general, diapause-cooled males passed more spermatophores than males from control diapause and non-diapause groups. In general, sub-zero cooling did not consistently affect fecundity and % egg hatch. Non-diapause females laid the most eggs after six months in diapause, and diapause-cooled females laid the most eggs after seven months in diapause. The duration of sub-zero cooling had a significant effect on post-diapause emergence in relation to the duration that the larvae were in diapause. Sub-zero cooling for 4 days at -10 degree C significantly reduced the number of days to adult emergence of larvae which had been in diapause for 0-4 months. Sub-zero cooling at -15 degree C for durations of 2, 4, and 6 days had more variable effects on emergence, but in most cases, sub-zero cooling reduced the amount of time to and span of adult emergence.     

Role of intracellular contents to facilitate supercooling capability in beech (Fagus crenata) xylem parenchyma cells

In order to find the possible role of intracellular contents in facilitating the supercooling capability of xylem parenchyma cells, changes in the temperature of supercooling levels were compared before and after the release of intracellular substances from beech xylem parenchyma cells by DTA. Various methods were employed to release intracellular substances from xylem parenchyma cells and all resulted in a reduction of supercooling ability. It was concluded that the reduction of supercooling ability primarily resulted from changes of intracellular conditions, including the release of intracellular contents or their mixing with extracellular solutions, rather than due to changes of cell wall structures. It is therefore suggested that any unidentified intracellular contents may function to facilitate supercooling capability in xylem parenchyma cells.     

Insect cold hardiness: a Canadian perspective

The cold climates and diverse environments of Canada have allowed key studies of insect cold hardiness that developed and widened the understanding of this subject. For example, freezing tolerance, chilling tolerance, freezing resistance, supercooling, cryoprotectants and other features can be combined in many different ways, reflecting a wide range of adaptations. Many other factors interact with and influence cold hardiness, such as habitats and their selection, and water and energy balances. These findings suggest several topics that would be especially fruitful for further study in northern Canada.     

Biochemistry and physiology of overwintering in the mature larva of the pine needle gall midge,Thecodiplosis japonensis (Diptera: cecidomyiidae) in Korea

The pine needle gall midge, Thecodiplosis japonensis, overwinters in the soil as a third instar mature larva. The metabolic and physiological compensations and adjustments during its overwintering and acclimation were studied. Field-sampled larvae in 1997/98 winter showed a significant increase in whole-body trehalose by January (5.71 +/- 0.09 vs. 9.41 +/- 0.42 mg/g wet weight) along with a more significant decrease in whole-body glycogen (16.25 +/- 0.18 vs. 5.65 +/- 0.45 mg/g wet weight). Afterwards, there was a partial reconversion of trehalose to glycogen. Moreover, trace amounts of glycerol and steady content of glucose as potential cryoprotectants were found during the overwintering period. Temperature acclimation of field-sampled larvae affects interconversion between trehalose and glycogen. Trehalose accumulation does not affect the larval supercooling capacity. The mean supercooling point of the larvae remained nearly constant at about -20 degree he winter and was unchanged after temperature acclimation. Low temperature survival experiment suggested that the larvae adopt a freeze-avoiding strategy for overwintering.     

Cryo-scanning electron microscopic study on freezing behavior of xylem ray parenchyma cells in hardwood species

Differential thermal analysis (DTA) has indicated that xylem ray parenchyma cells (XRPCs) of hardwood species adapt to freezing of apoplastic water either by deep supercooling or by extracellular freezing, depending upon the species. DTA studies indicated that moderately cold hardy hardwood species exhibiting deep supercooling in the XRPCs were limited in latitudinal distribution within the −40°C isotherm, while very hardy hardwood species exhibiting extracellular freezing could distribute in colder areas beyond the −40°C isotherm. Predictions based on the results of DTA, however, indicate that XRPCs exhibiting extracellular freezing may appear not only in very hardy woody species native to cold areas beyond the −40°C isotherm but also in less hardy hardwood species native to tropical and subtropical zones as well as in a small number of moderately hardy hardwood species native to warm temperate zones. Cryo-scanning electron microscopic (cryo-SEM) studies on the freezing behavior of XRPCs have revealed some errors in DTA. These errors are originated mainly due to the overlap between exotherms produced by freezing of water in apoplastic spaces (high temperature exotherms, HTEs) and exotherms produced by freezing of intracellular water of XRPCs by breakdown of deep supercooling (low temperature exotherms, LTEs), as well as to the shortage of LTEs produced by intracellular freezing of XRPCs. In addition, DTA results are significantly affected by cooling rates employed. Further, cryo-SEM observations, which revealed the true freezing behavior of XRPCs, changed the previous knowledge of freezing behavior of XRPCs that had been obtained by freeze-substitution and transmission electron microscopic studies. Cryo-SEM results, in association with results obtained from DTA that were reconfirmed or changed by observation using a cryo-SEM, revealed a clear tendency of the freezing behavior of XRPCs in hardwood species to change with changes in the temperature in the growing conditions, including both latitudinal and seasonal temperature changes. In latitudinal temperature changes, XRPCs in less hardy hardwood species native to tropical and subtropical zones exhibited deep supercooling to −10°C, XRPCs in moderately hardy hardwood species native to temperate zones exhibited a gradual increase in the supercooling ability to −40°C from warm toward cool temperate zones, and XRPCs in very hardy hardwood species native to boreal forests exhibited extracellular freezing via an intermediate form of freezing behavior between deep supercooling and extracellular freezing. In seasonal temperature changes, XRPCs in hardwood species native to temperate zones changed their supercooling ability from a relatively low degree in summer to a high degree in winter. XRPCs in hardwood species native to boreal forests changed their freezing behavior from deep supercooling to −10°C in summer to extracellular freezing in winter. These results indicate that the freezing behavior of XRPCs in hardwood species tends to shift gradually from supercooling of −10°C, to a gradual increase in the deep supercooling ability to −40°C or less, and finally to extracellular freezing as a result of cold acclimation in response to both latitudinal and seasonal temperature changes. It is thought that these temperature-dependent changes in the freezing behavior of XRPCs in hardwood species are mainly controlled by changes in cell wall properties, although no distinct changes were detected by electron microscopic observations in cell wall organization between hardwood species or between seasons. Evidence of temperature-dependent changes in the freezing behavior of XRPCs in hardwood species provided by the results of studies using a cryo-SEM has indicated the need for further investigation to clarify cold acclimation-induced cell wall changes at the sub-electron microscopic level in order to understand the mechanisms of freezing adaptation.     

The history of cold hardiness research in terrestrial arthropods

R.A.F. Reaumur in 1736 was the first entomologist to observe that some insects can tolerate freezing, while others cannot. The first reviews on insect cold hardiness were published by P. Bachmetjew who also discovered supercooling in insects. Authors like W. Robinson realised that piercing the cuticle changed the supercooling capacity and N.M. Payne pointed out that there are great seasonal variations in insect cold hardiness. In the early literature, references were frequently made to plant cold hardiness, in which theories on tolerance to freezing were more elaborated. A modern approach to the studies of insect cold hardiness was initiated by R.W. Salt. The number of reports increased enormously in the 1960's and 1970's. During the last 20 to 30 years new insight has appeared from studies on ice nucleators, antifreeze proteins, ice-nucleating bacteria, desiccation and biochemistry of cryoprotectants.     

Snow white and the seven dwarfs: a multivariate approach to classification of cold tolerance

Two main cold hardiness strategies of insects - freeze tolerance in some species, and overwintering in a supercooled state without tolerance of freezing in many others - were recently reclassified. However, I present several problems with the current systems. My suggested classification is based on clearer definitions of the causes of cold injury. I recognize three main mortality factors: freezing of body liquids, cold shock, and cumulative chill injury. Presence or absence of each of these factors produce eight combinations. I have named the eight classes after Snow White and the Seven Dwarfs to avoid nomenclatural confusion. Some of these classes are probably not used as tactics against cold injury by any insect species. Other classes contain so many species that they might be reclassified in more detail, using values of supercooling point and other quantitative parameters. However, widely comparable parameters, like the upper limit of cold injury zone and the sum of injurious temperatures are still rarely published, thus we still lack comprehensive data for multivariate analyses. Every cold hardiness strategy should be characterized by a meaningful class or subclass together with the physiological, biochemical, and behavioural mechanisms employed by the insects. I also point out the existence of strategies that combine two tactics - either a switching strategy (during preparation for winter, population "chooses" which tactic will be used), or a dual strategy (individuals are ready to use one of the tactics depending on the prevailing environmental conditions).     

Cryopreservation of Arabidopsis thaliana shoot tips

Development of a successful shoot tip cryopreservation method for Arabidopsis thaliana L. will enable researchers to use molecular tools to study processes important for successful cryopreservation in this model organism. We demonstrate that Arabidopsis can be successfully cryopreserved using either plant vitrification solution 2 (PVS2) or plant vitrification solution 3 (PVS3) as cryoprotectants prior to rapidly cooling shoot tips in liquid nitrogen (LN). Shoot tip regrowth after PVS2 cryoprotectant treatment was improved after cold acclimation treatments of 8 or 18 days. All of the shoots tips regrew after LN exposure when cryoprotected with PVS3 for 60 min at 22 degree C. In addition, shoot tips could be cryopreserved using a two-step cooling procedure with PGD (polyethylene glycol-glucose-dimethyl sulfoxide) as a cryoprotectant. The high levels of shoot formation after LN exposure of Arabidopsis shoot tips makes this a desirable system in which molecular tools can be used to examine how alterations in biochemical, metabolic and developmental processes affect regrowth after cryoprotective treatments.     

Cold resistance in the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

We have investigated cold resistance, measured by the supercooling point (SCP) temperature, in life stages of the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae), collected in Brittany poultry houses. Mean SCP values drastically increased during the insect ontogeny: egg (-26.1 C), first instar larvae (-21.6 C), last instar larvae (-15.5 C), pupae (-11.6 C), teneral adults (-12.0 C) and mature adults (-13.1 C). Nymphal metamorphosis and adult maturation did not promote substantial decrease of freezing resistance. The SCP values reflect the physiological states of the developmental stages especially the absence of ice nucleating agents: (i) lower SCP values in egg and unfed newly-emerged larvae I (i.e. -25.1 C), (ii) higher SCP values in fed larvae (i.e. -14.7 C), pupae and adults most likely due to the presence of ice nucleation sites in the gut. A tropical species, A. diaperinus, seems not to use its potential cold hardiness even in winter to remain in this warm habitat in temperate regions.     

Purification and partial characterization of thermal hysteresis proteins from overwintering larvae of pine needle gall midge,Thecodiplosis japonensis (Diptera: cecidomiidae)

The pine needle gall midge of Thecodiplosis japonensis is a serious forest pest and overwinters as a 3rd instar larva at soil surface in Korea. The time necessary for killing 50% of larvae at -15 degree C is 160 min. During overwintering period, T. japonensis larvae accumulate relatively high content of trehalose as the main cryoprotectant. In this paper, the proteinaceous cryoprotectants were identified. Two thermal hysteresis proteins (THP-1S and 2S) were purified from overwintering larvae by ethanol fractionation, trichloroacetic acid precipitation, ion-exchange chromatography (DEAE-Sephadex A-25) and gel permeation chromatography (Sephadex G-100). Their molecular weights are 34.9 and 37.8 kD respectively. T. japonensis THPs cannot be stained by periodic acid-Schiffs' reagent, suggesting no carbohydrate in them. The thermal hysteresis activity of THP-2 at the concentration of 50 mg/ml is 11.02 +/- 0.08 degree C (mean +/- SD, n=10), perhaps the highest active insect THP. It is the first report of purified T. japonensis THPs in Diptera.     

Refining the risk of freezing mortality for antarctic terrestrial microarthropods

In studies of three common, freezing susceptible, Antarctic microarthropods, the springtail Cryptopygus antarcticus and the mites Alaskozetes antarcticus and Halozetes belgicae, we report (i) the consequences on cold tolerance of cooling in contact with water, and (ii) the risk of freezing when held at temperatures above the typical freezing point (measured using standard techniques) for up to 12 h. The springtail showed no change in SCP distribution when in contact with freezing water while, in contrast, the mites showed clear shifts towards decreased cold tolerance, in addition to death of c. 33% of individuals during the freezing of the water. The springtail showed a bimodal SCP distribution, with the population divided into "high"(typically -8 to -12 degree C) and "low" (typically below -20 degree C) groups. Some animals held at temperatures above these values froze, over a timescale between minutes and several hours. These results highlight the danger of equating standard cold tolerance measures with mortality risk under more realistic water and thermal regimes.     

Antioxidant systems in supporting environmental and programmed adaptations to low temperatures

Hetero and endothermic adaptive responses arising as a result of natural responses to environmental cues include antioxidant systems that support adaptations to environmental low temperatures in the broadest sense. These temperatures induce phase changes in energy production and consequently changes in the concentration of reactive oxygen species (ROS). The latter may lead to oxidative stress and the impairment of cellular homeostasis and antioxidant defence systems (ADS) scavenge the ROS so generated. In endotherms the ADS responds to oxidative pressure during acute cold stress conditions, this response is tissue specific and does not extend to prevent other oxidative damage. The early acute phase of cold exposure is accompanied by a significant depletion in redox equivalents. Under such conditions it is questionable if ADS has the capacity to neutralize elevated levels of ROS since there is also an increased energy demand and enhanced ATP consumption. Prolonged exposure to cold leads to ADS adaptation. Hibernators and freeze-tolerant species elevate their ADS before hibernation or freezing in order to prepare for and cope with re-awakening. The involvement of ROS and the role of the ADS in organisms subjected to low temperatures are features intercalated into physiological mechanisms of homestasis. The exact mechanisms for ADS regulation have not been fully defined and are the subject of many ongoing intriguing scientific investigations.