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The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. It overwinters as a diapausing fifth instar larva. The overwintering is often a critical part of the insect life-cycle in temperate zone. This study brings detailed analysis of seasonal changes in lipid composition and fluidity in overwintering larvae sampled in the field. Fatty acid composition of triacylglycerol (TG) depots in the fat body and relative proportions of phospholipid (PL) molecular species in biological membranes were analyzed. In addition, temperature of melting (Tm) in TG depots was assessed by using differential scanning calorimetry and the conformational order (fluidity) of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles. We observed a significant increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) in TG depots during the larval transition to diapause accompanied with decreasing melting temperature of total lipids, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. The fluidity of membranes was maintained very high irrespective of developmental mode or seasonally changing acclimation status of larvae. The seasonal changes in PL composition were relatively small. We discuss these results in light of alternative survival strategies of codling moth larvae (supercooling vs. freezing), variability and low predictability of environmental conditions, and other cold tolerance mechanisms such as extending the supercooling capacity and massive accumulation of cryoprotective metabolites., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The seasonal development of physiological features underlying gradual acquisition of relatively high cold tolerance in overwintering adults of the bark beetles, Pityogenes chalcographus was described. Prior to overwintering, the beetles accumulated carbohydrate reserves in the form of glycogen and trehalose. These reserves were partially converted to glycerol during peaking winter so that glycerol concentration reached 1.4M in average, which corresponds to approximately one quarter of the beetle dry mass. Whole body supercooling points decreased from -12.8°C in average at the beginning of dormancy (August) to -26.3°C in average during peaking winter (January). More than 75% of January-collected beetles survived at -5°C for 30days, at -15°C for 60days and more than 40% of them survived at -26°C for 12h. High resistance against inoculation of body fluids with external ice crystals, and low mortality, was observed when January-collected beetles were encased in an ice block for 14days. Thus, the physiological limits of cold tolerance measured at individual level in laboratory were safely sufficient for survival of P. chalcographus at any conceivable cold spell that may occur in Central Europe. In contrast, the field experiment showed that winter survival fluctuated between 23.8% and 69.2% at a population level depending on microclimatic conditions in different altitudes and overwintering locations (standing tree trunk or ground level). The meaning of laboratory-assessed physiological limits of cold tolerance for predictions of population winter survival in the field is discussed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ectotherm animals including insects are known to undergo seasonal restructuring of the cell membranes in order to keep their functionality and/or protect their structural integrity at low body temperatures. Studies on insects so far focused either on fatty acids or on composition of molecular species in major phospholipid classes. Here we extend the scope of analysis and bring results on seasonal changes in minor phospholipid classes, lysophospholipids (LPLs), free fatty acids, phytosterols and tocopherols in heteropteran insect, Pyrrhocoris apterus. We found that muscle tissue contains unusually high amounts of LPLs. Muscle and fat body tissues also contain high amounts of β-sitosterol and campesterol, two phytosterols derived from plant food, while only small amounts of cholesterol are present. In addition, two isomers (γ and δ) of tocopherol (vitamin E) are present in quantities comparable to, or even higher than phytosterols in both tissues. Distinct seasonal patterns of sterol and tocopherol concentrations were observed showing a minimum in reproductively active bugs in summer and a maximum in diapausing, cold-acclimated bugs in winter. Possible adaptive meanings of such changes are discussed including: preventing the unregulated transition of membrane lipids from functional liquid crystalline phase to non-functional gel phase; decreasing the rates of ion/solute leakage; silencing the activities of membrane bound enzymes and receptors; and counteracting the higher risk of oxidative damage to PUFA in winter membranes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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).

Coping with seasonal changes in temperature is an important factor underlying the ability of insects to survive over the harsh winter conditions in the northern temperate zone, and only a few drosophilids have been able to colonize sub-polar habitats. Information on their winter physiology is needed as it may shed light on the adaptive mechanisms of overwintering when compared with abundant data on the thermal physiology of more southern species, such as Drosophila melanogaster. Here we report the first seasonal metabolite analysis in a Drosophila species. We traced changes in the cold tolerance and metabolomic profiles in adult Drosophila montana flies that were exposed to thermoperiods and photoperiods similar to changes in environmental conditions of their natural habitat in northern Finland. The cold tolerance of diapausing flies increased noticeably towards the onset of winter; their chill coma recovery times showed a seasonal minimum between late autumn and early spring, whereas their survival after cold exposure remained high until late spring. The flies had already moderately accumulated glucose, trehalose and proline in autumn, but the single largest change occurred in myo-inositol concentrations. This increased up to 400-fold during the winter and peaked at 147 nmol mg(-1) fresh mass, which is among the largest reported accumulations of this compound in insects.

Overwintering adults of Pyrrhocoris apterus do not tolerate freezing of their body fluids and rely on a supercooling strategy and seasonal accumulation of polyols to survive at subzero body temperatures. We sampled the adults monthly in the field during the cold season 2008-2009 and found active thermal hysteresis factors (THFs) in hemolymph of winter-sampled adults. The hysteresis between the equilibrium melting and freezing points ranged from 0.18°C to 0.30°C. No signs of THFs activity were found in the autumn- and spring-sampled insects. The total free amino acid pool almost doubled during winter time. The sum concentrations of 27 free amino acids ranged between 35 and 40mM in whole body water and 40-45mM in hemolymph during December-February. Two amino acids, Pro and α-Ala most significantly contributed to the seasonal increase, while Gln showed the most dramatic seasonal decrease. Moderate levels of amino acid accumulation in overwintering P. apterus suggest that they are by-products of protein degradation and pentose pathway activity during the state of metabolic suppression imposed by diapause and low body temperature. Potential colligative effects of accumulated amino acids, extending the supercooling capacity of overwintering P. apterus, are negligible. Non-colligative effects require further study., (Copyright © 2011 Elsevier Inc. All rights reserved.)