Basking improves but winter warming worsens overwinter survival in the linden bug.

The present study investigates the effects of rare winter basking behavior (observed in wild populations of the Linden bug, Pyrrhocoris apterus) and the effects of winter warming (predicted by climate models) on overwinter survival and physiology of P. apterus. The insects were exposed to scenarios simulating basking and winter warming in the laboratory. Part of the insects were exposed to real winters under semi-natural conditions in the field for comparison. The results show a clear positive effect of winter basking, implying that basking behavior is critical for overwinter survival in P. apterus. In contrast, winter warming was found to have a strong negative effect on overwinter survival, potentially representing a threat to central European populations of P. apterus. Physiological parameters (mass, water content, SCP, energy reserves) measured in this study cannot fully explain all the results. Further study is needed to better understand the mechanisms behind the positive effects of winter basking and the negative effects of winter warming on overwintering P. apterus.

Estimating the Potential of Insects from Warmer Regions to Overwinter in Colder Regions under a Warming Winter Scenario Using Simulation Experiments: A Case Study in Sesamia nonagrioides.

Ongoing climate change and anthropogenic pressure are having a profound influence on insects, causing species diversity to decline and populations to shrink. Insect pests invade new areas and cause economic and human health problems. Low temperatures in winter are thought to be one of the main barriers to the successful colonization of higher latitudes. Climate models predict that winter temperatures will increase more than summer temperatures in temperate and polar regions, potentially allowing species from warmer climates to colonize higher latitudes. Understanding how climate change will affect the distribution of insects is critical to many areas of human activity. One possible but seldom used way to predict likely range shifts of insects due to climate change is through simulation experiments. Here, I present and test a method to assess the potential of insect species from warmer regions to survive winters in colder regions under a warming winter scenario. The method is based on laboratory simulations of warming winters. The applicability of the method is demonstrated using the example of a Mediterranean pest, Sesamia nonagrioides, whose ability to survive Central European winters under a warming winter scenario is assessed. The method presented here is relatively simple, with potentially high accuracy of estimates.

Stabilization of insect cell membranes and soluble enzymes by accumulated cryoprotectants during freezing stress.

Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.

A mixture of innate cryoprotectants is key for freeze tolerance and cryopreservation of a drosophilid fly larva.

Insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on the mechanisms of individual CP interactions, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Here, we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata, which survives long-term cryopreservation in liquid nitrogen. The CPs proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine and sarcosine accumulate in hemolymph in a ratio of 313:108:55:26:6:4:2.9:0.5 mmol l-1. Using calorimetry, we show that artificial mixtures, mimicking the concentrations of major CPs in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we show that during slow extracellular freezing trehalose becomes concentrated in partially dehydrated hemolymph where it stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it probably forms a layer of dense viscoelastic liquid. We propose that amorphous glass and viscoelastic liquids may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen.

Do energy reserves and cold hardiness limit winter survival of Culex pipiens?

The risks of depletion of energy reserves and encountering lethally low temperatures are considered as two important mortality factors that may limit winter survival of mosquito, Culex pipiens f. pipiens populations. Here we show that the autumn females carry lipid reserves, which are safely sufficient for at least two overwintering periods, provided the females diapausing at temperatures typical for underground spaces (0 °C - 8 °C) would continuously rest at a standard metabolic rate (SMR). The overwintering females, however, switch from SMR to much higher metabolic rate during flight, either seeking for optimal microhabitat within the shelter or in response to disturbances by air current or predator attack. These behaviors result in fast oxidation of lipid reserves and, therefore, the autumn load of energy reserves may actually limit winter survival under specific circumstances. Next, we show that the level of females' cold hardiness is physiologically set relatively weak for overwintering in open field, above-ground habitats, but is ecologically entirely sufficient for overwintering in most underground spaces. The characteristics of suitable overwintering shelters are: no or limited risk of contact with ice crystals, no or limited air movements, winter temperatures relatively stable between +2 and + 6 °C, winter minimum does not drop below -4 °C for longer than one week, or below -8 °C for longer than 1 day.

Study of interactions between carboxylated core shell magnetic nanoparticles and polymyxin B by capillary electrophoresis with inductively coupled plasma mass spectrometry.

In this work, interactions of carboxylated core shell magnetic nanoparticles with polymyxin B sulfate were studied by connecting capillary electrophoresis with inductively coupled plasma mass spectrometry. The interaction was probed by affinity mode of capillary electrophoresis with 25 mM phosphate buffer at physiological pH. 54Fe, 56Fe, 57Fe, 34S, and 12C isotopes were used to monitor the migration of an electroosmotic flow marker and the interaction of the nanoparticles with polymyxin B. The analysis of interaction data showed two distinct interaction regions, one with low polymyxin B concentration, the second with high polymyxin B concentration. These regions differed in the strength of the interaction, 1.49 × 107 M-1 and 1.60 × 104 M-1, and in the stoichiometry of 0.7 and 3.5, respectively. These differences can be explained by the decrease of electrostatic repulsion between nanoparticles caused by polymyxin B. This is also in agreement with the nanoparticles peak shapes: sharp for low polymyxin B concentrations and broad for high polymyxin B concentrations.

Fat body disintegration after freezing stress is a consequence rather than a cause of freezing injury in larvae of Drosophila melanogaster.

Extracellular freezing of insect body water may cause lethal injury either by direct mechanical stress exerted by growing ice crystals on cells and tissues or, indirectly, by deleterious physico-chemical effects linked to freeze-induced cell dehydration. Here we present results showing that the macroscopic damage (cell ruptures, tissue disintegration) to fat body of Drosophila melanogaster is not directly caused by mechanical forces linked to growth of ice crystals but rather represents a secondary consequence of other primary freeze injuries occurring at subcellular or microscopic levels. Larvae of D. melanogaster were acclimated to produce variants ranging from freeze susceptible to freeze tolerant. Then, larvae were exposed to supercooling and freezing stresses at different subzero temperatures. The larval survival and macroscopic damage to fat body tissue was scored in 1632 larvae exposed to cold stress. In most cases, fat body damage was not evident immediately following cold stress but developed later. This suggests that the fat body disintegration is a consequence rather than a cause of cold injury. Analysis of fat body membrane phospholipids revealed that increased freeze tolerance was associated with increased relative proportion of phosphatidylethanolamines (PEs) at the expense of phosphatidylcholines (PCs). The PE/PC ratio increased from 1.08 in freeze-susceptible larvae to 2.10 in freeze-tolerant larvae. The potential effects of changing PE/PC ratio on phospholipid bilayer stability upon supercooling and freezing stress are discussed.

Supercooling and freezing as eco-physiological alternatives rather than mutually exclusive strategies: A case study in Pyrrhocoris apterus.

Overwintering insects are categorized either as freeze tolerant or freeze avoiding (supercooling) based on their ability or inability, respectively, to tolerate the formation of ice in their body. The freeze tolerant insects set their supercooling point (SCP) higher for winter to stimulate freezing at higher temperatures, while freeze avoiding insects survive winter in a supercooled state by depressing their SCP. Some supercooling insects, however, were found to survive in frozen state when freezing occurred through inoculation by external ice at mild subzero temperatures. Here, we assessed the potential relevance of inoculative freezing and freeze tolerance strategy in an insect that was so far considered as a classical example of a 'supercooler', the linden bug (Pyrrhocoris apterus). Microclimatic conditions of the overwintering microhabitat of P. apterus (leaf litter layer with buffered temperature fluctuations, mild sub-zero extremes, high humidity, and presence of ice) present a potentially high risk of inoculative freezing. We found that P. apterus is highly susceptible to inoculation by external ice. The temperature at which inoculative freezing occurred (above -3°C) was much higher compared to SCP (-16 °C to -20 °C in winter). The insects were inoculated through body openings and across cuticle and were able to survive after freezing. There was, however, a distinct critical ice fraction, corresponding to 38.7-42.8% of total body water, beyond which survival rapidly decreased to zero. We found that P. apterus adaptively reduces the actual ice fraction below critical ice fraction for winter season. Since many insect species overwinter in habitats similar to that of P. apterus, the ability to tolerate freezing after inoculation by external ice crystals could be much more common among 'supercooling' insects than it is currently appreciated.

Thermal analysis of ice and glass transitions in insects that do and do not survive freezing.

Some insects rely on the strategy of freeze tolerance for winter survival. During freezing, extracellular body water transitions from the liquid to the solid phase and cells undergo freeze-induced dehydration. Here, we present results of a thermal analysis (from differential scanning calorimetry) of ice fraction dynamics during gradual cooling after inoculative freezing in variously acclimated larvae of two drosophilid flies, Drosophila melanogaster and Chymomyza costata Although the species and variants ranged broadly between 0 and close to 100% survival of freezing, there were relatively small differences in ice fraction dynamics. For instance, the maximum ice fraction (IFmax) ranged between 67.9% and 77.7% total body water (TBW). Chymomyza costata larvae showed statistically significant phenotypic shifts in parameters of ice fraction dynamics (melting point and IFmax) upon entry into diapause, cold acclimation and feeding on a proline-augmented diet. These differences were mostly driven by colligative effects of accumulated proline (ranging between 6 and 487 mmol kg-1 TBW) and other metabolites. Our data suggest that these colligative effects per se do not represent a sufficient mechanistic explanation for high freeze tolerance observed in diapausing, cold-acclimated C. costata larvae. Instead, we hypothesize that accumulated proline exerts its protective role via a combination of mechanisms. Specifically, we found a tight association between proline-induced stimulation of glass transition in partially frozen body liquids (vitrification) and survival of cryopreservation in liquid nitrogen.

True lab-in-a-syringe technology for bioassays.

In our work, we introduced a novel concept of the lab-in-a-syringe tests. We solved the problem of detection in already published LIS tests by putting all the reaction and detection pads directly into the syringe barrel. We also used more layers to make the results visible for users. Two detection layouts: (i) with using rounded pads-based detection, and (ii) with using rectangular detection pads, were studied. As the proof of concept, we studied the determination of Ni(II) using dimethylglyoxime as the reagent and blocking of the interference of Fe(II). The calibrations for Ni(II) at the optimal conditions has excellent R2 of 0.998 with production costs of 0.2 USD per one test.

Reprint of: Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth, Cydia pomonella.

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.

Accumulation of free amino acids during exposure to drought in three springtail species.

Springtails are closely related to insects, but they differ from these with respect to water balance, in particular because springtails are small and have high integumental permeability to water. Here we report a series of experiments addressing the dynamics of osmoregulation, water content and accumulation of free amino acids (FAAs) in three springtail species during exposure to a gradually increasing environmental desiccation simulating conditions in drought exposed soil. Folsomia candida and Protaphorura fimata (both living in the deeper soil layers; euedaphic species) were active throughout the 3week exposure, with the developing drought regime ending at -3.56MPa (the soil water activity at the permanent wilting point of plants is -1.5MPa) and remained hyperosmotic (having an body fluid osmolality higher than the corresponding environment) to their surrounding air. Sinella curviseta (living in upper soil/litter layers; hemiedaphic species) also survived this exposure, but remained hypoosmotic throughout (i.e. with lower osmolality than the environment). The body content of most FAAs increased in response to drought in all three species. Alanine, proline and arginine were the most significantly upregulated FAAs. By combining our results with data in the literature, we could account for 82% of the observed osmolality at -3.56MPa in F. candida and 92% in P. fimata. The osmolality of S. curviseta was only slightly increased under drought, but here FAAs were considerably more important as osmolytes than in the two other species. We propose that FAAs probably have general importance in drought tolerance of springtails.

Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth, Cydia pomonella.

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.

Physiology of cold tolerance in the bark beetle, Pityogenes chalcographus and its overwintering in spruce stands.

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.

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).

Physiological and biochemical responses to cold and drought in the rock-dwelling pulmonate snail, Chondrina avenacea.

The pulmonate snail Chondrina avenacea lives on exposed rock walls where it experiences drastic daily and seasonal fluctuations of abiotic conditions and food availability. We found that tolerance to dry conditions was maintained at a very high level throughout the year and was mainly based on the snails' ability to promptly enter into estivation (quiescence) whenever they experienced drying out of their environment. Snails rapidly suppressed their metabolism and minimized their water loss using discontinuous gas exchange pattern. The metabolic suppression probably included periods of tissue hypoxia and anaerobism as indicated by accumulation of typical end products of anaerobic metabolism: lactate, alanine and succinate. Though the drought-induced metabolic suppression was sufficient to stimulate moderate increase of supercooling capacity, the seasonally highest levels of supercooling capacity and the highest tolerance to subzero temperatures were tightly linked to hibernation (diapause). Hibernating snails did not survive freezing of their body fluids and instead relied on supercooling strategy which allowed them to survive when air temperatures dropped to as low as -21 °C. No accumulation of low-molecular weight compounds (potential cryoprotectants) was detected in hibernating snails except for small amounts of the end products of anaerobic metabolism.

Long-term cold acclimation extends survival time at 0°C and modifies the metabolomic profiles of the larvae of the fruit fly Drosophila melanogaster.

BACKGROUND: Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately -5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum. PRINCIPAL FINDINGS: We found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt(50)) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt(50) (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines. CONCLUSION: Third-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring.

Seasonal changes of free amino acids and thermal hysteresis in overwintering heteropteran insect, Pyrrhocoris apterus.

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.