Overwintering Strategy and Mechanisms of Cold Tolerance in the Codling Moth (Cydia pomonella)

Background

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. Fully grown last instar larvae overwinter in diapause state. Their overwintering strategies and physiological principles of cold tolerance have been insufficiently studied. No elaborate analysis of overwintering physiology is available for European populations.

Principal Findings

We observed that codling moth larvae of a Central European population prefer to overwinter in the microhabitat of litter layer near the base of trees. Reliance on extensive supercooling, or freeze-avoidance, appears as their major strategy for survival of the winter cold. The supercooling point decreases from approximately −15.3°C during summer to −26.3°C during winter. Seasonal extension of supercooling capacity is assisted by partial dehydration, increasing osmolality of body fluids, and the accumulation of a complex mixture of winter specific metabolites. Glycogen and glutamine reserves are depleted, while fructose, alanine and some other sugars, polyols and free amino acids are accumulated during winter. The concentrations of trehalose and proline remain high and relatively constant throughout the season, and may contribute to the stabilization of proteins and membranes at subzero temperatures. In addition to supercooling, overwintering larvae acquire considerable capacity to survive at subzero temperatures, down to −15°C, even in partially frozen state.

Conclusion

Our detailed laboratory analysis of cold tolerance, and whole-winter survival assays in semi-natural conditions, suggest that the average winter cold does not represent a major threat for codling moth populations. More than 83% of larvae survived over winter in the field and pupated in spring irrespective of the overwintering microhabitat (cold-exposed tree trunk or temperature-buffered litter layer).     

Overwintering conditions affect cold hardiness,survival, and post-overwintering fitness of the pea leaf weevil

Overwintering conditions affect the physiological state of ectotherms, and therefore, their cold hardiness and survival. A measure of the lethal and sublethal impacts of overwintering conditions on pest populations is crucial to predict population dynamics and to manage pests the following spring. The impact of winter conditions can be most intense for invasive insects undergoing range expansion. Insect herbivores can display plastic host use behaviours that depend on their body condition following winter. The pea leaf weevil, Sitona lineatus L. (Coleoptera: Curculionidae), is an invasive pest of field peas, Pisum sativum L., and faba bean, Vicia faba L. (Fabaceae). Pea leaf weevil has expanded its range in North America to include the Prairie Provinces of Canada. This study investigated the effects of temperature and microhabitat on overwintering survival and cold hardiness of pea leaf weevil in its expanded range. Further, we investigated the sublethal effect of overwintering temperature and duration on post-overwintering survival, feeding, and oviposition of pea leaf weevil. We also investigated the role of juvenile hormone in modulating body condition of overwintering weevils. The overwintering survival of pea leaf weevil adults increased with soil temperature and varied with region and microhabitat. More weevils survived winters when positioned near tree shelterbelts compared to open alfalfa fields. The supercooling point of pea leaf weevil varied throughout its expanding range but did not differ for weevils held in the two microhabitats. The average threshold lethal temperature of pea leaf weevil at all three sites was −9.4 °C. Weevils that overwintered for a longer duration and at a higher temperature subsequently fed more on faba bean foliage and laid more eggs compared to those which overwintered for a shorter duration at a lower temperature. Our findings highlight that warm winters would increase overwintering survival and post-overwintering fitness, facilitating further pea leaf weevil invasion northward in the Prairie Provinces of Canada.     

Overwintering of annual bluegrass weevils, Listronotus maculicollis, in the golf course landscape

Annual bluegrass weevil, Listronotus maculicollis (Dietz) (Coleoptera: Curculionidae), adults overwinter in protected areas but their life cycle is completed on highly maintained turfgrass. To better target control tactics on affected golf courses, we need to understand the relationship between overwintering and developmental habitats. Our objectives were to identify factors that influence overwintering site selection and to gather evidence of directional movement. Surveys of natural field populations were conducted in early spring over 2 years to test how abundance of overwintered adults depends on microhabitat (surface composition) and distance from the developmental habitat. The influence of microhabitat on overwintering preference and success was further tested in a multiple‐choice and no‐choice field experiment by relocating overwintering weevils into experimental arenas where four microhabitats were presented together or singly. The timing and direction of dispersal by walking adults was assessed using paired linear pitfall traps. Results showed that adults could overwinter up to 60 m from the fairway and 10 m into the woods. They were most abundant near the tree line; none were detected within 5 m of the fairway. Microhabitat had a significant effect on abundance in 1 year, being highest in mixed tree litter followed by moss, high‐cut grass, and pine litter. Under multiple‐choice conditions, high‐cut grass was preferred over low‐cut grass and leaf litter, followed by pine litter. Pitfall trap captures showed a peak of activity in the spring, and at one site this was directional toward the fairway. There was no directionality or increase in activity in the fall. Based on these results, a new conceptual model of flux between habitats is proposed based on orientation of flying adults to defined tree lines. Defining this behavior will strengthen our ability to target control tactics in space and time against this major turf pest.     

No trade-off between high and low temperature tolerance in a winter acclimatized Danish Drosophila subobscura population

Coping with cold winter conditions is a major challenge for many insects.In early spring we observed newly emerged Drosophila subobscura, which had overwintered as larvae and pupae. As temperatures increase during spring these flies are faced with higher minimum and maximum temperatures in their natural microhabitat. Thus, there is a potential costly mismatch between winter and early spring acclimatization and the increased ambient temperatures later in adult life.We obtained individuals from a natural Danish population of D. subobscura and acclimated them in the laboratory to 20 °C for one generation, and compared critical thermal maximum (CTmax) and minimum (CTmin) to that of individuals collected directly from their natural microhabitat. The two populations (laboratory and field) were subsequently both held in the laboratory at 20 °C and tested for their CTmax and CTmin every third day for 28 days.At the first day of testing, field acclimatized D. subobscura had both higher heat and cold resistance compared to laboratory flies, and thereby a considerable larger thermal scope. Following transfer to the laboratory, cold and heat resistance of the field flies decreased over time relative to the laboratory flies. Despite the substantial decrease in thermal tolerances the thermal scope remained larger for field acclimatized individuals for the duration of the experiment.We conclude that flies acclimatized to their natural microhabitat had increased cold resistance, without a loss in heat tolerance. Thus while a negative correlation between cold and heat tolerance is typically observed in laboratory studies in Drosophila sp., this was not observed for field acclimatized D. subobscura in this study. We suggest that this is an adaptation to juvenile overwintering in temperate cold environments, where developmental (winter) temperatures can be much lower than temperatures experienced by reproducing adults after emergence (spring). The ability to gain cold tolerance through acclimatization without a parallel loss of heat tolerance affects thermal scope and suggests that high and low thermal tolerance act through mechanisms with different dynamics and reversibility.     

Linking energetics and overwintering in temperate insects

Overwintering insects cannot feed, and energy they take into winter must therefore fuel energy demands during autumn, overwintering, warm periods prior to resumption of development in spring, and subsequent activity. Insects primarily consume lipids during winter, but may also use carbohydrate and proteins as fuel. Because they are ectotherms, the metabolic rate of insects is temperature-dependent, and the curvilinear nature of the metabolic rate-temperature relationship means that warm temperatures are disproportionately important to overwinter energy use. This energy use may be reduced physiologically, by reducing the slope or elevation of the metabolic rate-temperature relationship, or because of threshold changes, such as metabolic suppression upon freezing. Insects may also choose microhabitats or life history stages that reduce the impact of overwinter energy drain. There is considerable capacity for overwinter energy drain to affect insect survival and performance both directly (via starvation) or indirectly (for example, through a trade-off with cryoprotection), but this has not been well-explored. Likewise, the impact of overwinter energy drain on growing-season performance is not well understood. I conclude that overwinter energetics provides a useful lens through which to link physiology and ecology and winter and summer in studies of insect responses to their environment.     

Adaptations to cold in Canadian arctic insects

The survival of insects that inhabit Canadian arctic regions depends on a number of factors which have important ecological, behavioral, physiological, and biochemical components. The ability to withstand low winter temperatures is one of the most conspicuous adaptations of northern insects and the one most closely studied in the laboratory. Most species studied so far conform to one or other of the two major overwintering strategies, namely, frost susceptibility, the ability to avoid freezing by supercooling to a considerable degree, or frost tolerance, the survival of actual ice formation within the body. The Arctic beetle, Pytho americanus Kirby, is frost tolerant in both larval and adult stages, a situation which would be congruous with its northern distribution and allow it to spread its life cycle over a number of growing seasons. The main biochemical correlates during the cold-hardening process in this species are increasing glycerol and decreasing glycogen concentrations. In addition to its normally assumed roles in cryoprotection there is evidence to suggest that glycerol may further serve to minimize dehydration in the overwintering insect by increasing the level of bound water. P. americanus larvae and adults have narrow supercooling ranges and maintain their supercooling points in the region of ?4 to ?8 °C. It is hypothesized that these elevated supercooling points are a result of the presence in the hemolymph of nucleating agents which ensure ice formation at high subzero temperatures.Low temperature tolerance strategies of some other arctic and alpine species have been examined and compared with those of relatives from more southerly latitudes. P. americanus has been collected in the Canadian Rockies at elevations of over 6000′, and its frost-tolerant attributes are identical to those of the population collected in the Arctic. A closely related species, P. deplanatus, from the Rockies, however, although it too exhibits frost tolerance in the larval stage, differs markedly from P. americanus in its ability to depress its supercooling range to ?54 °C. It appears that P. deplanatus does not have the ability to synthesize ice-nucleating agents and, therefore, can overwinter in a supercooled condition. Two congeneric species of willow leaf gall sawflies (Pontania spp.), one from Tuktoyaktuk, N.W.T., and the other from southern Vancouver Island have also been compared and contrasted. Pontania sp. on Salix glauca (Tuk., ca. 70 °N) is frost tolerant in its larval stage, has relatively high supercooling points (ca. ?9.0 °C), but does not accumulate glycerol. Pontania sp. from Salix lasiandra (Victoria, ca. 48 °N) has almost identical overwintering properties, indicating the close phylogenetic affinities of cold tolerance in this genus rather than independent adaptation to widely different climatic conditions. Some of the lowest supercooling points ever recorded are from willow stem gall forming insects. Rhabdophaga sp. (Cecidomyiidae) forms potato galls on the stems of Salix lanata in the Inuvik area, N.W.T. After low temperature acclimation, supercooling points down to ?66 °C have been recorded from individual larvae. This is a record, and it indicates that we may be dealing with a system in which most water is in a metabolically bound state. Glycerol levels reach 20% of the fresh body weight during this period. Diastrophus kincaidii Cynipidae) forms stem galls on Thimble Berry (Rubus parviflorus) on southern Vancouver Island. Both of the forementioned species overwinter as larvae in their galls and are, therefore, exposed to ambient air temperatures. A more benign winter climate on Vancouver Island is reflected in the fact that D. kincaidii has supercooling points only in the ?30 to ?33 °C range at the peak of low temperature acclimation, and glycerol levels just below 4% of fresh body weight. Both species are frost susceptible and depend on their supercooling abilities to survive low winter temperatures.     

Cold adaptation in insects of Central Yakutia

The mode of cold hardening was for the first time assessed for 20 insect species living in the extremely cold climate of Yakutia. All insects tested were found to adapt through freeze tolerance, producing ice-nucleating agents that cause the hemolymph to freeze at high subzero temperatures. For the first time ice-nucleating agents were demonstrated in Lepidoptera. Pieris rapae exemplified the possibility of switchover in the survival strategy depending on the climatic conditions.     

Cold tolerance of the maize orange leafhopper,Cicadulina bipunctata

Cicadulina bipunctata was originally distributed in tropical and subtropical regions of the Old World. This leafhopper recently expanded its distribution area to southern parts of temperate Japan. In this study, factors affecting the overwintering ability of C. bipunctata were examined. A series of laboratory experiments revealed that cold acclimation at 15 °C for 7 days enhanced the cold tolerance of C. bipunctata to the same level as an overwintering population, adult females were more tolerant of cold temperature than adult males, and survival of acclimated adult females was highly dependent on temperature from −5 to 5 °C and exposure duration to the temperature. The temperature of crystallization of adult females was approximately −19 °C but temperatures in southern temperate Japan rarely dropped below −10 °C in the winter, indicating that overwintering C. bipunctata adults in temperate Japan are not killed by freezing injury but by indirect chilling injury caused by long-term exposure to moderately low temperatures. An overwintering generation of C. bipunctata had extremely low overwinter survival (<1%) in temperate Japan; however, based on winter temperature ranges, there are additional areas amenable to expansion of C. bipunctata in temperate Japan.     

Cold-hardiness of the arctic beetle, Pytho americanus Kirby Coleoptera,Pythidae (Salpingidae)

The arctic beetle, Pytho americanus Kirby, is frost tolerant in both larval and adult stages. This is the first demonstration that an insect can tolerate freezing in more than one life stage, a situation which would be congruous with its northern distribution and allow it to spread its life cycle over a number of growing seasons. The main biochemical correlates during the cold hardening process of low temperature acclimation are increasing glycerol and decreasing glycogen concentrations. Glycerol is the only polyol to be synthesized during acclimation, and it accumulates to a maximum of 8.2 and 12.2% of the fresh body weight in larvae and adults respectively. This coincides with the peak of frost tolerance. In addition to its normally assumed roles in cryoprotection it is suggested that glycerol may further serve to minimize dehydration in the overwintering insect by increasing the level of ‘bound’ water. Evidence is presented that indicates that glycerol is synthesized mainly from carbohydrate reserves, especially glycogen, but it does not rule out the possibility that a proportion of free glycerol comes from glyceride sources.P. americanus larvae and adults have low supercooling potential and maintain their supercooling points in the region of ?4° to ?8°C. It is hypothesized that these elevated supercooling points are a result of the presence in the haemolymph of nucleating agents which ensure ice formation at high sub-zero temperatures. It is believed that this beetle overwinters in a frozen state within its microhabitat, which is under bark of fallen spruce which is, in turn, covered by an insulating blanket of snow. The advantages of this overwintering strategy are discussed.     

小菜蛾在温带地区越冬研究进展

小菜蛾是世界性重要害虫。在热带和亚热带地区冬季的十字花科植物上能正常发育繁殖,可见到各种虫态。但在温带冬季十字花科植物不能生长的地区,小菜蛾的越冬成为一个重要生态学问题。综述了亚洲、北美洲和欧洲小菜蛾越冬的研究进展。小菜蛾在日本的北海道、本州岛的北陆和东北大部分地区不能越冬,越冬北限相当于冬季积雪覆盖时间为60d的区域;在中国,小菜蛾不能在寒冷的东北地区越冬,在长江中下游以南地区冬季可见各虫态,但越冬北限尚不清楚;小菜蛾在冬天气候温和的韩国以及澳大利亚东南部继续发生;北美洲的加拿大西部和安大略地区大量的试验证明小菜蛾不能成功越冬,在美国南部小菜蛾冬季可正常发生,北部小菜蛾的越冬尚未见系统研究报道,但确认春季从南部运输的受小菜蛾感染的甘蓝等种苗是美国北部的重要虫源。小菜蛾在欧洲各地越冬的系统研究未见报道,没有证据表明在英国小菜蛾会发生有显著意义的越冬。目前小菜蛾越冬研究主要采用冬季直接试验观察和基于耐寒性试验的越冬预测两种方法。冬季直接试验观察法包括:(1)利用人工饲养的小菜蛾在田间各种潜在的越冬场所的越冬试验;(2)在秋播、野生或残留的十字花科植物上进行冬季种群的系统抽样调查;(3)越冬前后在前茬为十字花科植物的田块广泛搜寻普查小菜蛾的存活个体。基于耐寒性试验的越冬预测法:在获取小菜蛾越冬场所温度的基础上设计低温处理模式,试验低温处理后小菜蛾的存活率及后续发育和生殖。将试验数据和各地气温或小气候相结合,对小菜蛾在的越冬可能性进行推断。     

Complex life cycles and the responses of insects to climate change

Many organisms have complex life cycles with distinct life stages that experience different environmental conditions. How does the complexity of life cycles affect the ecological and evolutionary responses of organisms to climate change? We address this question by exploring several recent case studies and synthetic analyses of insects. First, different life stages may inhabit different microhabitats, and may differ in their thermal sensitivities and other traits that are important for responses to climate. For example, the life stages of Manduca experience different patterns of thermal and hydric variability, and differ in tolerance to high temperatures. Second, life stages may differ in their mechanisms for adaptation to local climatic conditions. For example, in Colias, larvae in different geographic populations and species adapt to local climate via differences in optimal and maximal temperatures for feeding and growth, whereas adults adapt via differences in melanin of the wings and in other morphological traits. Third, we extend a recent analysis of the temperature-dependence of insect population growth to demonstrate how changes in temperature can differently impact juvenile survival and adult reproduction. In both temperate and tropical regions, high rates of adult reproduction in a given environment may not be realized if occasional, high temperatures prevent survival to maturity. This suggests that considering the differing responses of multiple life stages is essential to understand the ecological and evolutionary consequences of climate change.     

Cold tolerance of New Zealand alpine insects

New Zealand has extensive alpine and subalpine habitats where, together with some lowland sites, insects are exposed to subzero temperatures. Studies of cold tolerance in New Zealand insects have centred on an alpine weta (Hemideina maori), which is the world's largest freezing tolerant insect, and an alpine cockroach (Celatoblatta quinquemaculata). Both of these insects are moderately freezing tolerant and have ice nucleating agents in their haemolymph and guts. There is some evidence for the survival of intracellular ice formation in the isolated gut tissue of C. quinquemaculata. Trehalose is a suggested cryoprotectant in both H. maori and C. quinquemaculata whilst proline also provides this role in H. maori. Cells and tissues of both insects maintain viability and physiological function during freezing to moderately low temperatures but viability declines at lower temperatures, the most vulnerable tissue presumably setting the limit to the survival of the animal. Antifreeze proteins are found in the gut tissue of C. quinquemaculata and may protect this tissue when freezing occurs in the gut. Several other New Zealand insects are also moderately freezing tolerant and the apparent dominance of this cold tolerance strategy in the New Zealand fauna may reflect the relatively mild climate but unpredictable exposure to subzero temperatures that is typical of many Southern Hemisphere environments.     

How does Drosophila melanogaster overwinter?

In temperate regions low temperatures seem to be the most restrictive factor for survival of Drosophila natural populations, which depends on the capacity of one or more developmental stages to resist unfavourable winter conditions. In this study we have attempted to answer the question of how D. melanogaster overwinters under natural temperature conditions. Only adults overwintered and no diapause was observed in any developmental stage. Thus, developmental duration becomes a decisive component with respect to overwintering potential and, therefore, the preadult stages are unlikely to overwinter. Possible evolutionary steps in adaptation to cold regions are discussed.     

Effects of temperature and drought on early life stages in three species of butterflies: Mortality of early life stages as a key determinant of vulnerability to climate change?

Anthropogenic climate change poses substantial challenges to biodiversity conservation. Well‐documented responses include phenological and range shifts, and declines in cold but increases in warm‐adapted species. Thus, some species will suffer while others will benefit from ongoing change, although the biological features determining the prospects of a given species under climate change are largely unknown. By comparing three related butterfly species of different vulnerability to climate change, we show that stress tolerance during early development may be of key importance. The arguably most vulnerable species showed the strongest decline in egg hatching success under heat and desiccation stress, and similar pattern also for hatchling mortality. Research, especially on insects, is often focussed on the adult stage only. Thus, collating more data on stress tolerance in different life stages will be of crucial importance for enhancing our abilities to predict the fate of particular species and populations under ongoing climate change.     

Comparative analysis of overwintering physiology in nine species of semi‐aquatic bugs (Heteroptera: Gerromorpha)

In semi‐aquatic bugs (Heteroptera: Gerromorpha), the strategies of overwintering in a cryothermic state (i.e. at body temperatures below the equilibrium freezing point) remain largely unexplored. The present study provides an analysis of the ecophysiological aspects of overwintering in nine gerromorphan species. All nine species avoid ice formation by means of a more or less extensive supercooling of their body fluids. There is a tight correlation between the supercooling point (SCP) and the lower lethal temperature. Different species use different physiological adjustments to increase the likelihood of survival in a supercooled state. These include stabilization of the supercooled state by active antifreeze factors that cause thermal hysteresis between equilibrium melting and freezing points, the accumulation of low‐molecular weight sugars and polyols with putative cryoprotective functions, or by having a relatively high body fluid osmolality, combined with a low level of hydration. The majority of species under study overwinter only as adults, whereas Velia caprai Tamanini can overwinter either as an adult or in the egg stage. The supercooling capacity of V. caprai adults is insufficient to prevent the risk of lethal freezing. The adults therefore survive only opportunistically in suitable microhabitats, and/or during mild winters. The survival of V. caprai in winter is assured by extensive supercooling and having overwintering eggs that are highly cold tolerant.     

Evergreen foliage allows early hatching in a pine processionary moth and escape from predation

1. Seasonal variation in leaf quality and climate conditions often imposes constraints on the temporal occurrence of tree‐feeding insect larvae, but the seasonal effects of predation have received limited attention. In temperate climate zones, both the abundance and activity of predators can be expected to vary over time. 2. The study reported herein examined the impact of temporal variation in predator activity levels on the life history of an herbivorous insect feeding on a constant food source: previous‐year needles of Scots pine (Pinus sylvestris L.). In field experiments, the survival and growth rates of colonies of Thaumetopoea pinivora Treitschke larvae that had been manipulated to hatch at three different dates were compared. Eggs of T. pinivora usually hatch by mid‐April in southern Sweden, which is earlier than most other herbivorous insects that overwinter as eggs in this region. 3. Predator exclusion experiments indicated that larvae which hatched later than April experienced a higher level of predator activity, mainly by ants. The final larval size and the timing of pupation were not affected by hatching date. First instar larvae were more extensively preyed on than second instars. 4. The life history of herbivore species can be affected by seasonal variation in predation pressures. This study suggests that early hatching in a lepidopteran species can allow a temporal escape from predation during the vulnerable early life stages.     

Overwintering strategy in Pyrrhocoris apterus (Heteroptera): the relations between life-cycle, chill tolerance and physiological adjustments

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.