Ceratophysella sigillata (Collembola, Hypogastruridae) has a life cycle which may extend for >2 years in a temperate climate. It exists in two main morphs, a winter-active morph and a summer-dormant morph in central European forests. The winter-active morph often occurs in large aggregations, wandering on leaf litter and snow surfaces and climbing on tree trunks. The summer-dormant morph is found in the upper soil layers of the forest floor. The cryobiology of the two morphs, sampled from a population near Bern in Switzerland, was examined using Differential Scanning Calorimetry to elucidate the roles of body water and the cold tolerance of individual springtails. Mean (SD) live weights were 62 ± 16 and 17 ± 6 µg for winter and summer individuals, respectively. Winter-active springtails, which were two feeding instars older than summer-dormant individuals, were significantly heavier (by up to 4 times), but contained less water (48% of fresh weight [or 0.9 g g-1 dry weight]) compared with summer-dormant animals (70% of fresh weight [or 2.5 g g-1 dry weight]). Summer-dormant animals had a slightly greater supercooling capacity (mean (SD) -16 ± 6°C) compared with winter-active individuals (-12 ± 3°C), and they also contained significantly larger amounts of both total body water and osmotically inactive (unfrozen) water. In the summer morph, the unfrozen fraction was 26%, compared to 11% in the winter morph. The ratio of osmotically inactive to osmotically active (freezable) water was 1 : 1.7 (summer) and 1 : 3.3 (winter); thus unfrozen water constituted 59% of the total body water during summer compared with only 30% in winter. Small, but significant, levels of thermal hysteresis were detected in the winter-active morph (0.15°C) and in summer-dormant forms (0.05°C), which would not confer protection from freezing. However, the presence of antifreeze proteins may prevent ice crystal growth when feeding on algae with associated ice crystals during winter. It is hypothesised that in summer animals a small decrease in freezable water results in a large increase in haemolymph osmolality, thereby reducing the vapour pressure gradient between the springtail and the surrounding air. A similar decrease in freezable water in winter animals will not have such a large effect. The transfer of free water into the osmotically inactive state is a possible mechanism for increasing drought survival in the summer-dormant morph. The ecophysiological differences between the summer and winter forms of C. sigillata are discussed in relation to its population ecology and survival.
Many insects in temperate regions overwinter in diapause, during which they are cold hardy. In these insects, one of the metabolic adaptations to the unfavorable environmental conditions is the synthesis of cryoprotectants/anhydroprotectants. The aim of this study was to investigate the connection between the antioxidative system and synthesis of cryoprotectants (mainly glycerol) in diapausing larvae of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae). At two periods during diapause (November and February), in three groups of insects (kept under field conditions; -12°C for two weeks; 8°C for two weeks), the activity of key enzymes of the antioxidative system and oxidative part of the hexose monophosphate shunt were measured: superoxide dismutase, catalase, non selenium glutathione peroxidase, glutathione S-transferase, glutathione reductase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, as well that of the antioxidative components: total glutathione and ascorbate, and dehydroascorbate reductase. There was a higher activity of antioxidative enzymes at the beginning of the diapause period (November) compared to late diapause (February), while glutathione and ascorbate were higher in February. Similarly, there was a lower activity of the hexose monophosphate shunt enzymes in February. Exposure of larvae to -12°C resulted in an elevation of hexose monophosphate shunt enzyme activity, especially in November. This was accompanied by a significant increase in glycerol content in February. Changes in ascorbate levels and dehydroascorbate reductase activity in both experimental groups (-12°C and 8°C) suggest a connection between the antioxidative system, metabolism during diapause and cold hardiness. Our results support the notion that antioxidative defense in larvae of Ostrinia nubilalis is closely connected with metabolic changes characteristic of diapause, mechanisms of cold hardiness involved in diapause and the maintenance of a stable redox state.
The Chinese pine caterpillar Dendrolimus tabulaeformis is an important destructive leaf borer in boreal coniferous forests in China. This species overwinters in the larval stage. Changes in supercooling capacity and physiological-biochemical parameters of D. tabulaeformis larvae from a natural population were evaluated at different stages during the overwintering period. Cold hardiness of overwintering larvae collected in January was significantly greater than that of larvae collected in other months. January larvae survived for 15 days at -10°C and for approximately 2 days at -15°C. By contrast, larvae collected in September survived for no more than 4 h at -5°C and those in November and March no more than 1 day at -15°C. Supercooling point gradually decreased from -5.9 ± 0.3°C in September to a minimum of -14.1 ± 1.0°C in November, then gradually increased to the original value with the advent of spring. Water content gradually decreased from September to November, remained at approximately 74.5% until March and then gradually increased to levels similar to those in September. The lipid content gradually decreased from September to November, remained stable at approximately 3.2% until March and then gradually increased to levels similar to those in September. Glycogen content increased to a peak in November and then decreased. The concentrations of several metabolites showed significant seasonal changes. The most prominent metabolite was trehalose with a seasonal maximum in November. Glucose levels were highest in January and then gradually decreased until in May they were at levels similar to those in September. Glycerol levels remained relatively stable during winter but increased significantly in May. This study indicates that D. tabulaeformis is a freeze-avoidant insect. Larvae increase their supercooling capacity by regulating physiological-biochemical parameters during overwintering., Yuying Shao, Yuqian Feng, Bin Tian, Tao Wang, Yinghao He, Shixiang Zong., and Obsahuje bibliografii
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 of diapause (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.
Larval diapause development and termination and some characteristics of cold hardiness in Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) were studied under field conditions in northern Greece. P. gossypiella overwintering larvae were sampled at 20 to 30 day intervals and subjected to two photoperiodic regimes at 20°C. In larvae kept under a long-day photoperiod (16L : 8D) diapause development was accelerated compared to those kept under a short-day photoperiod (8L : 16D). There was no difference in response to the two photoperiods after February. Mean number of days to pupation of P. gossypiella overwintering larvae decreased progressively through the sampling period, from November to April. Chilling is not a prerequisite but does accelerate diapause development. Supercooling points for P. gossypiella overwintering larvae ranged from -14 to -17°C with the majority dying after freezing.
Diapausing larvae of Aphidoletes aphidimyza (Diptera: Cecidomyiidae) had relatively low supercooling points (SCP) ranging from -19.0 to -26.4°C. None of the specimens that froze at this temperature survived. A high survival rate (up to 87%) at -10°C for 10 days was observed in supercooled larvae. Such features are characteristic for insects that use a chill-tolerance strategy of cold hardiness. However, the cocoons formed by the diapausing larvae were penetrable by external ice crystals and the larvae showed a relatively high survival rate (23 - 34%) at -10°C for 10 days also in the frozen state caused by inoculation by external ice at high subzero temperatures. Such a duality with respect to cold hardiness strategies seems to be ecologically relevant to overwintering in soil habitats where there may be unpredictable contact with external ice.
When puparia of the onion maggot Delia antiqua were preexposed to 5°C for 5 days starting at different time points after pupariation, a large increase in survival after exposure to -20°C for 5 days was observed only when pre-exposure was initiated at 3-6 days after pupariation. The increase in cold hardiness was not associated with a large increase in the trehalose content of the puparia. The supercooling point of the puparia naturally decreased from -18 to -27°C in the first three days after pupariation, and pre-exposure to 5°C did not have an additional effect. Thus, factors responsible for the enhancement of cold hardiness by acclimation other than trehalose and supercooling point should be sought. The period of responsiveness to cold acclimation coincided with the time soon after head evagination, which corresponds to "pupation" in lepidopteran insects. The puparia appear to be physiologically flexible for a short time after head evagination, and able to adapt their physiology to the contemporary cold environment.
Supercooling point (SCP), survival at low temperatures, rate of water loss in dry air at 20°C and survival under desiccating conditions of eggs of Polydesmus angustus (Diplopoda) were determined. The results were compared with those obtained previously for the eight post-embryonic stadia, to obtain an overview of the changes in resistance to cold and desiccation throughout the species' development. The SCP temperatures of egg batches ranged from -14.8 to -30.6°C and were significantly lower than those of the active stadia. Eggs were not affected by prolonged exposure to low temperature above 0°C and survived much better than active stadia when cooled to -6 and -10°C. This indicates that the cold hardiness of P. angustus is highest in the egg stage and decreases during development. On the other hand, the rate of water loss was significantly higher from eggs than from active stadia. When eggs were taken out of their protective nest, they lost water at the high rate of 7% min-1 in dry air. They also survived for a shorter time than active stadia at 76% RH and 20°C. The resistance to desiccation of P. angustus is lowest at the egg stage and increases during development. The results suggest that the life cycle of P. angustus may have responded to selection pressures other than cold and drought, and do not support the hypothesis that cold hardiness and resistance to desiccation are overlapping adaptations in terrestrial arthropods.
Pine caterpillar, Dendrolimus tabulaeformis Tsai et Liu, is a major pine pest in North China. The larvae enter diapause in the third or fourth instar before winter. Supercooling points (SCP) and cold hardiness of the diapausing larvae were investigated and compared in non-acclimated, acclimated and de-acclimated larvae. A bimodal frequency distribution was observed with a break point of -14°C in the SCP. Larvae in the low group (LG, SCP <= -14°C) were more cold tolerant with lower lethal temperatures than those in the high group (HG, SCP > -14°C). This bimodality occurred in three patterns, LG (> 60% of individuals in LG), LG-HG (< 60% of individuals in LG and HG) and HG (> 60% of individuals in HG), in response to cold acclimation and de-acclimation. The cold hardiness was ranked as: LG > LG-HG > HG pattern. Cold hardiness was enhanced by an increase in concentrations of trehalose, galactose, glucose and mannose in the haemolymph as well as by decrease in metabolism after cold acclimation, but was lost after de-acclimation. Loss of cold hardiness was correlated with decrease in sugars and increase in metabolic rate. In conclusion, the species is a chill tolerant insect, adopting the strategy of depressing SCP through accumulation of low molecular weight sugars in the haemolymph, concomitant with metabolic depression.
Insects and other terrestrial arthropods are widely distributed in temperate and polar regions and overwinter in a variety of habitats. Some species are exposed to very low ambient temperatures, while others are protected by plant litter and snow. As may be expected from the enormous diversity of terrestrial arthropods, many different overwintering strategies have evolved. Time is an important factor. Temperate and polar species are able to survive extended periods at freezing temperatures, while summer adapted species and tropical species may be killed by short periods even above the freezing point.
Some insects survive extracellular ice formation, while most species, as well as all spiders, mites and springtails are freeze intolerant and depend on supercooling to survive. Both the degree of freeze tolerance and supercooling increase by the accumulation of low molecular weight cryoprotectant substances, e.g. glycerol. Thermal hysteresis proteins (antifreeze proteins) stabilise the supercooled state of insects and may prevent the inoculation of ice from outside through the cuticle. Recently, the amino acid sequences of these proteins have been revealed.
Due to potent ice nucleating agents in the haemolymph most Freeze tolerant insects freeze at relatively high temperatures, thus preventing harmful effects of intracellular freezing. Doe to the low water vapour pressure in frozen environments, supercooled terrestrial arthropods are at a risk of desiccation. Glycerol and other low molecular weight substances may protect against dehydration as well as against cold. In the arctic springtail Onychiurus arcticus, freezing is avoided due to dehydration in equilibrium with the ambient freezing temperature. Tn some frozen habitats terrestrial arthropods are enclosed by ice and survive an oxygen deficiency by anaerobic metabolism.
Suggestions for further research include investigating the nature of freeze tolerance, the physiology of prolonged exposures to cold, and the relation between desiccation, anaerobiosis and cold hardiness.