Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations

Fluctuating temperatures are a predominant feature of the natural environment but their effects on ectotherm physiology are not well-understood. The warm periods of fluctuating thermal regimes (FTRs) provide opportunities for repair leading to increased survival, but there are also indications of negative effects of warm exposure. In this study, we examined respiration and oxidative stress in adult Alphitobius diaperinus exposed to FTRs and to constant low temperatures. We hypothesized that cold exposure will cause oxidative stress and that FTRs would reduce the amount of chill injuries, via activation of the antioxidant system. We measured V˙CO2, activities of super oxide dismutase (SOD), amounts of total (GSHt) and oxidized glutathione (GSSG) during cold and warm periods of FTRs. Increased severity of cold exposure caused a decrease in the glutathione pool. SOD levels increased during the recovery period in the more severe FTR. The antioxidant response was sufficient to counter the reactive oxygen species production, as the GSH:GSSG ratio increased. We conclude that cold stress causes oxidative damage in these beetles, and that a warm recovery period activates the antioxidant system allowing repair of cold-induced damage, leading to the increased survival previously noted in beetles exposed to fluctuating versus constant temperatures.     

Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes

Environmental stress deleteriously affects every aspect of an ectotherm's biological function. Frequent exposure of terrestrial insects to temperature variation has thus led to the evolution of protective biochemical and physiological mechanisms. However, the physiological mechanisms underlying the positive impact of fluctuating thermal regimes (FTRs) on the fitness and survival of cold-exposed insects have not been studied. We have thus investigated the metabolic changes in adults of the beetle Alphitobius diaperinus in order to determine whether FTRs trigger the initiation of a metabolic response involving synthesis of protective compounds, such as free amino acids (FAAs) and polyols. The metabolic profile was analyzed during constant fluctuating thermal regimes (the beetles had daily pulses at higher temperatures that enabled them to recover) and compared with constant cold exposure and untreated controls. The increase of several essential amino acids (Lys, Iso, Leu, Phe and Trp) in cold-exposed beetles supports the conclusion that it results from the breakdown of proteins. Some FAAs have been shown to have cryoprotective properties in insects, but the relationship between FAAs, cold tolerance and survival has not yet been well defined. Instead of considering FAAs only as a part of the osmo- and cryoprotective arsenal, they should also be regarded as main factors involved in the multiple regulatory pathways activated during cold acclimation. Under FTRs, polyol accumulation probably contributes to the increased duration of survival in A. diaperinus.     

Insect cold tolerance and repair of chill-injury at fluctuating thermal regimes: role of ion homeostasis

Adults of the bug Pyrrhocoris apterus and the beetle Alphitobius diaperinus developed chill-injury slower and survived longer when they were exposed to fluctuating thermal regimes (FTRs, where periods of low temperature were alternated with periods of higher temperature on a daily basis) rather than to constant low temperatures. The extracellular (haemolymph) concentrations of potassium ions increased with significantly higher rates in the insects exposed to constant low temperatures than in those exposed to FTRs. The concentrations of magnesium and sodium ions were maintained relatively constant or decreased slightly in both thermal regimes. The loss of body water and the increase of haemolymph osmolality contributed to, but could not fully explain, the ion concentration changes, which probably resulted also from impairing the function of an active metabolic component (ion pump) at low temperatures. This explanation was supported by observing (in P. apterus) the return toward normal [K+] during the warm "recovery" period of the FTR. Collectively, the paper stresses the importance of considering the temperature fluctuations in the experimental studies on insect cold tolerance and suggests that the positive effect of the FTR on cold tolerance may consist, at least partially, in allowing the primary ion pumping systems to re-establish the ion gradients across cell membranes and epithelia during the recovery periods at a higher temperature.     

Uncovering the benefits of fluctuating thermal regimes on cold tolerance of drosophila flies by combined metabolomic and lipidomic approach

When exposed to constant low temperatures (CLTs), insects often suffer from cumulative physiological injuries that can severely compromise their fitness and survival. Yet, mortality can be considerably lowered when the cold stress period is interrupted by periodic warm interruption(s), referred to as fluctuating thermal regimes, FTRs. In this study, we have shown that FTRs strongly promoted cold tolerance of Drosophila melanogaster adults. We then assessed whether this marked phenotypic shift was associated with detectable physiological changes, such as synthesis of cryoprotectants and/or membrane remodeling. To test these hypotheses, we conducted two different time-series Omics analyzes in adult flies submitted to CLTs vs. FTRs: metabolomics (GC/MS) and lipidomics (LC/ESI/MS) targeting membrane phospholipids. We observed increasing levels in several polyhydric alcohols (arabitol, erythritol, sorbitol, mannitol, glycerol), sugars (fructose, mannose) and amino acids (serine, alanine, glutamine) in flies under CLT. Prolonged exposure to low temperature was also associated with a marked deviation of metabolic homeostasis and warm interruptions as short as 2 h were sufficient to periodically return the metabolic system to functionality. Lipidomics revealed an increased relative proportion of phosphatidylethanolamines and a shortening of fatty acyl chains in flies exposed to cold, likely to compensate for the ordering effect of low temperature on membranes. We found a remarkable correspondence in the time-course of changes between the metabolic and phospholipids networks, both suggesting a fast homeostatic regeneration during warm intervals under FTRs. In consequence, we suggest that periodic opportunities to restore system-wide homeostasis contribute to promote cold tolerance under FTRs.     

Proteomic profiling of a parasitic wasp exposed to constant and fluctuating cold exposure

When insects are exposed to fluctuating thermal regimes (FTRs) (i.e., cold exposure alternating with periodic short pulses to high temperature), in contrast to constant low temperature (CLT), mortality due to accumulation of chill injuries is markedly reduced. To investigate the physiological processes behind the positive impact of FTR, based on a holistic approach, two-dimensional electrophoresis (2-DE) analysis were performed with the parasitic wasp Aphidius colemani. Parasitoid proteomes revealed 369 well-distinguishable protein spots, where the overall response to cold exposure was clearly specific to treatments (CLT versus FTR). The reduced mortality under FTR was associated with up-regulation of several proteins playing key roles in energy metabolism (glycolysis, TCA cycle, synthesis and conversion of ATP), protein chaperoning (Hsp70/Hsp90), and protein degradation (proteasome). Our results also support the idea that cytoskeleton components, particularly actin arrangement, could play a role in the higher survival rates of insects under FTR.     

The impact of fluctuating thermal regimes on the survival of a cold-exposed parasitic wasp, Aphidius colemani

Abstract.  The impact of fluctuating thermal regimes on the cold tolerance of the parasitoid Aphidius colemani at the mummy stage is examined. The hypothesis is tested that, if a cold period is interrupted by a return to a higher temperature for a short time, a physiological recovery is possible and may lead to higher survival. Mummies are exposed to a constant temperature of 4 °C and, when periodic sudden transfers to 20 °C for 2 h are applied, survival of immature parasitoids is markedly improved, proportionally to the warming frequency. The time lag before emergence diminishes with the duration of cold exposure and with warming frequency. The sex ratio of emergent adults after cold exposure indicates that males may be more susceptible than females to cold-injury. It is suggested that the transfer to 20 °C allows a re-activation of the normal metabolism, leading to repair and recovery of any injuries caused by prolonged chilling. The study underlines the importance of cyclic temperature changes on insect survival.     

A model for the time–temperature–mortality relationship in the chill-susceptible beetle, Alphitobius diaperinus, exposed to fluctuating thermal regimes

Exposing insects to a fluctuating thermal regime (FTR) compared with constant low temperature (CLT) significantly reduces cold-induced mortality. The beneficial effects of FTR result from physiological repair during warming intervals. The duration and the temperature experienced during the recovery period are supposed to strongly impact the resulting cold survival; however, disentangling the effects of both recovery variables had not been broadly investigated. In this study, we investigate cold tolerance (lethal time, Lt₅₀) of the polyphagous beetle Alphitobius diaperinus. We examined adult survival under various CLTs (0, 5, 10 and 15 °C), and under 20 different FTR conditions, where the 0 °C exposure alternated with various recovery temperatures (Rt) (5, 10, 15 and 20 °C) combined with various recovery durations (Rds) (0.5, 1, 2, 3 and 4 h). Under CLTs, Lt₅₀ increased with temperature until no mortality occurred above the upper limit of cold injury zone (ULCIZ). Under FTRs, Lt₅₀ increased with both Rt and Rd. The magnitude of the survival gain was clearly boosted when Rt was above the ULCIZ (at 20 °C). Based on a data matrix of lethal times with multiple Rt×Rd combinations, a predictive model showed that cold survival increased exponentially with Rt and Rd. This model was subsequently validated with additional survival tests. We suggest that increasing recovery durations associated with optimal recovery temperatures eventually leads to a progressive chilling compensation.     

Winter warming facilitates range expansion: cold tolerance of the butterfly<Emphasis Type="Italic"> Atalopedes campestris</Emphasis>

Our ability to predict ecological and evolutionary responses to climate change requires an understanding of the mechanistic links between climate and range limits. The warming trend over the past half-century has generated numerous opportunities to develop much-needed case studies of these links. Species that are only limited by climatic factors are likely to shift range quickly during periods of warming. Such species directly impact recipient communities and indicate trends that will become more widespread. Because minimum temperature (T (min)) is rising at twice the rate of maximum temperature, species with this range-limiting factor may be especially responsive to global warming. In this study, I test the hypothesis that rising T (min) has directly affected the range of a skipper butterfly. Atalopedes campestris has moved northward rapidly this century, recently colonizing eastern Washington where January T (min) has risen 3 degrees C in 50 years. The results show that: 1. A. campestris' range lies completely within the -4 degrees C January average minimum isotherm, and that recently colonized areas were below this threshold earlier this century. 2. In acute cold stress experiments, -4 to -7 degrees C proved to be a critical thermal limit: median supercooling point was -6.3 degrees C, and minimum lethal temperature (LT50 with 12-h exposure) was -5.7 degrees C. 3. In chronic cold stress experiments, survivorship declined sharply in diurnally fluctuating thermal regimes typical of the current range edge. High mortality occurred under constant 0 degrees C conditions as well as in fluctuating regimes, implying that thermal insulation from snow would not protect A. campestris. 4. There was no evidence of evolution in cold tolerance at the range margin, despite strong selection. Thus, winter warming was apparently a prerequisite for the range expansion. Characteristics of this species that seem to be associated with its rapid response are that it is an opportunistic species, it is not habitat or dispersal limited, and it is constrained by T (min).     

In situ development of sublethal stress in Escherichia coli: effects on enumeration.

Development of sublethal stress in Escherichia coli exposed in situ to estuarine waters was examined during various seasons. An electrochemical detection technique was utilized to derive a stress index based upon the difference between a predicted electrochemical response time in Trypticase soy broth or EC medium at 44.5 degrees C estimated from a standard curve for unstressed cells and an observed response time for cells exposed to seawater. This stress index was related to recovery efficiencies of seawater-exposed cells, using a variety of standard and resuscitative enumeration procedures. Stress was further studied by determination of the adenylate energy charge. Sublethal stress as measured by the electrochemical detection method was an inverse function of water temperature, with maximum stress occurring after exposure to temperatures below 10 degrees C. Total adenylates and ATP decreased dramatically at low temperatures, although energy charge remained relatively constant under various environmental conditions. Decreases in E. coli ATP suggest that ATP may not be an adequate measure of biomass for in situ stressed cells. Discrepancies in enumeration efficiency were most pronounced at temperatures below 10 degrees C. Resuscitative procedures for solid-media techniques increased the recovery of stressed cells under cold water conditions but were not as effective as the standard most-probable-number procedure.     

Survival of Bactrocera latifrons (Diptera: Tephritidae) adults under constant and fluctuating low temperatures

Bactrocera latifrons (Hendel) is believed to have originated in Southeast Asia but has invaded Hawaii and most recently East Africa. This insect has also been recorded on Okinawa Island, the far south of Kyushu Island, Japan. To assess the overwintering ability of B. latifrons adults, survival at constant temperatures (8, 10, 12, 14, 15 °C) and under fluctuating thermal regimes (FTRs) was investigated. At 14 or 15 °C, more than 30 % of females survived for 90 days. Time required to kill 95 % of B. latifrons at 8 °C was estimated to be 13 days; at 10 °C, 29 days; and at 12 °C, 38 days for females, and 8, 17, and 24 days at the same above temperatures, respectively, for males, suggesting low cold tolerance of this species. The results show that females survive cold temperatures better than males. Under an FTR of 11 °C (22 h)/20 °C (2 h) (average 11.8 °C) survival of females drastically increased compared to that at a constant temperature of 12 °C, whereas the survival of males increased slightly. Survival under FTRs indicates that adult B. latifrons may not overwinter in the north of Tanegashima Island, located 30 km south of Kyushu Island, Japan.     

Thermal biology of the alien ground beetle <Emphasis Type="Italic">Merizodus soledadinus</Emphasis> introduced to the Kerguelen Islands

Thermal tolerance is one of the major determinants of successful establishment and spread of invasive aliens. Merizodus soledadinus (Coleoptera, Carabidae) was accidentally introduced to Kerguelen from the Falkland Islands in 1913. On Kerguelen, the climate is cooler than the Falklands Islands but has been getting warmer since the 1990s, in synchrony with the rapid expansion of M. soledadinus. We aimed to investigate the thermal sensitivity in adults of M. soledadinus and hypothesised that climate warming has assisted the colonisation process of M. soledadinus. We examined (1) survival of constant low temperatures and at fluctuating thermal regimes, (2) the critical thermal limits (CT_min and CT_max) of acclimated individuals (4, 8 and 16°C), (3) the metabolic rates of acclimated adults at temperatures from 0 to 16°C. The FTRs moderately increased the duration of survival compared to constant cold exposure. M. soledadinus exhibited an activity window ranged from −5.5 ± 0.3 to 38 ± 0.5°C. The Q ₁₀ after acclimation to temperatures ranging from 0 to 16°C was 2.49. Our work shows that this species is only moderately cold tolerant with little thermal plasticity. The CT_min of M. soledadinus are close to the low temperatures experienced in winter on Kerguelen Islands, but the CT_max are well above summer conditions, suggesting that this species has abundant scope to deal with current climate change.     

Stress intensity and fitness in the parasitoid Aphidius ervi (Hymenoptera: Braconidae): temperature below the development threshold combined with a fluctuating thermal regime is a must

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Evaluation of the physiological status of the Pacific sardine, <Emphasis Type="Italic">Sardinops sagax caeruleus</Emphasis>, acclimated to different thermal regimes based on selected blood parameters

Despite the widespread recognition of the importance of temperature in the population dynamics and distribution of Pacific sardine (Sardinops sagax caeruleus), few studies have examined the species’ physiological response to temperature under controlled conditions. The effect of fluctuating thermal regimes on blood plasma cortisol, glucose, aspartate aminotransferase (ALT), alanine aminotransferase (AST) and red blood cell (RBC) counts of Pacific sardine was evaluated to monitor physiological response. Sardines from the southern subpopulation that inhabits waters off Baja California, Mexico, were collected during fall 2008. Acclimation to four fluctuating temperature regimes (two symmetric and two asymmetric) and a constant, optimal temperature occurred over 20 d. Symmetric and asymmetric regimes were designed to simulate regional summer (18–23°C) and winter (13–18°C) temperature ranges within 24 h. In the optimal regime (OR), sardines were acclimated to 18°C. In symmetric regimes, sardines were exposed for 7 h to the high and low temperatures, while in asymmetric regimes exposure to the high temperature was shorter (4 vs.10 h). Blood sampling took place during the low and high temperature exposure, and we tested for differences in blood parameters compared to OR. Plasma cortisol and glucose concentration of sardines from summer regimes were only significantly higher at 23°C. AST activities were significantly higher than OR during the high and low temperatures of both summer treatments. No differences were found in individuals exposed to the winter regimes. ALT activities were significantly higher than OR only during the high and low temperature of the symmetric summer regime. RBC were significantly higher than OR for both summer regimes. Sardines from southern subpopulation exhibit a negative physiological response to high temperatures, yet they can acclimate to the lower (≤18°C) temperatures typical of the California Current System. Temperatures ≥23°C may be considered a detrimental sublethal temperature, particularly for long exposure periods.     

Diurnal variation around an optimum and near‐critically high temperature does not alter the performance of an ectothermic aquatic grazer

The growing threat of global climate change has led to a profusion of studies examining the effects of warming on biota. Despite the potential importance of natural variability such as diurnal temperature fluctuations, most experimental studies on warming are conducted under stable temperatures. Here, we investigated whether the responses of an aquatic invertebrate grazer (Lymnaea stagnalis) to an increased average temperature differ when the thermal regime is either constant or fluctuates diurnally. Using thermal response curves for several life‐history and immune defense traits, we first identified the optimum and near‐critically high temperatures that Lymnaea potentially experience during summer heat waves. We then exposed individuals that originated from three different populations to these two temperatures under constant or fluctuating thermal conditions. After 7 days, we assessed growth, reproduction, and two immune parameters (phenoloxidase‐like activity and antibacterial activity of hemolymph) from each individual. Exposure to the near‐critically high temperature led to increased growth rates and decreased antibacterial activity of hemolymph compared to the optimum temperature, whilst temperature fluctuations had no effect on these traits. The results indicate that the temperature level per se, rather than the variability in temperature was the main driver altering trait responses in our study species. Forecasting responses in temperature‐related responses remains challenging, due to system‐specific properties that can include intraspecific variation. However, our study indicates that experiments examining the effects of warming using constant temperatures can give similar predictions as studies with fluctuating thermal dynamics, and may thus be useful indicators of responses in nature.     

Cyclic CO(2) emissions during the high temperature pulse of fluctuating thermal regime in eye-pigmented pupae of Megachile rotundata

Megachile rotundata (Hymenoptera: Megachilidae), the primary pollinator used in alfalfa seed production, may need to be exposed to low-temperature storage to slow the insects' development to better match spring emergence with the alfalfa bloom. It has been demonstrated that using a fluctuating thermal regime (FTR) improves the tolerance of pupae to low temperatures. Carbon dioxide emission rates were compared between four different FTRs, all with a base temperature of 6 °C and a daily high-temperature pulse. Four different high-temperature pulses were examined, 15 or 25 °C for 2 h and 20 °C for 1 or 2 h. A subset of pupae at the FTR base temperature of 6 °C exhibited continuous gas exchange and, once ramped to 20 or 25 °C, shifted to cyclic gas exchange. As temperatures were ramped down from the high-temperature pulse to 6 °C, the pupae reverted to continuous gas exchange. The following conclusions about the effect of FTR on the CO₂ emissions of M. rotundata pupae exposed to low-temperature storage during the spring incubation were reached: 1) the high temperature component of the FTR was the best predictor of respiratory pattern; 2) neither pupal body mass nor days in FTR significantly affected which respiratory pattern was expressed during FTRs; 3) cyclic gas exchange was induced only in pupae exposed to temperatures greater than 15 °C during the FTR high temperature pulse; and 4) a two hour pulse at 25 °C doubled the number of CO₂ peaks observed during the FTR pulse as compared to a two hour pulse at 20 °C.     

Fatty acid composition and extreme temperature tolerance following exposure to fluctuating temperatures in a soil arthropod

Ectotherms commonly adjust their lipid composition to ambient temperature to counteract detrimental thermal effects on lipid fluidity. However, the extent of lipid remodeling and the associated fitness consequences under continuous temperature fluctuations are not well-described. The objective of this study was to investigate the effect of repeated temperature fluctuations on fatty acid composition and thermal tolerance. We exposed the springtail Orchesella cincta to two constant temperatures of 5 and 20 °C, and a continuously fluctuating treatment between 5 and 20 °C every 2 days. Fatty acid composition differed significantly between constant low and high temperatures. As expected, animals were most cold tolerant in the low temperature treatment, while heat tolerance was highest under high temperature. Under fluctuating temperatures, fatty acid composition changed with temperature initially, but later in the experiment fatty acid composition stabilized and closely resembled that found under constant warm temperatures. Consistent with this, heat tolerance in the fluctuating temperature treatment was comparable to the constant warm treatment. Cold tolerance in the fluctuating temperature treatment was intermediate compared to animals acclimated to constant cold or warmth, despite the fact that fatty acid composition was adjusted to warm conditions. This unexpected finding suggests that in animals acclimated to fluctuating temperatures an additional underlying mechanism is involved in the cold shock response. Other aspects of homeoviscous adaptation may protect animals during extreme cold. This paper forms a next step to fully understand the functioning of ectotherms in more thermally variable environments.     

Effects of a fluctuating temperature regime experienced by Daphnia during diel vertical migration on Daphnia life history parameters

Many freshwater zooplankton species perform a diel vertical migration (DVM) and spend the day within the lower, colder hypolimnion of stratified lakes. Trade-offs that arise from this migration have already attracted much attention and the cold temperature in the hypolimnion is thought to be the main cost of this behaviour. In this study we additionally looked at the extra costs daphnids have from being exposed to a fluctuating temperature regime (cold during the day and warm during the night) which is less well studied until today. In our experiment Daphnia hyalina Leydig and Daphnia magna Straus either spent 24 h in constant warm water (19 °C), 24 h in constant cold water (12 °C), or spent 12 h in warm and 12 h in cold water in an alternating way (fluctuating temperature regime). We expected the values of the life history parameters of Daphnia in the fluctuating temperature regime to be exactly halfway between the values of the life history parameters in the warm and cold treatments because the daphnids spent exactly half of the time in warm water, and half of the time in cold water. Concordant with earlier studies our results showed that age at first reproduction and egg development time were reduced at higher temperatures. In the fluctuating temperature regime the values of both parameters were exactly halfway between the values at permanently warm and cold temperature regimes. In contrast, somatic growth was higher at higher temperatures but was lower in the fluctuating temperature regime than expected from the mean somatic growth rate. This suggests that a fluctuating temperature regime experienced by migrating daphnids in stratified lakes involves additional costs for the daphnids.     

Phenotypic plasticity is not affected by experimental evolution in constant,predictable or unpredictable fluctuating thermal environments

The selective past of populations is presumed to affect the levels of phenotypic plasticity. Experimental evolution at constant temperatures is generally expected to lead to a decreased level of plasticity due to presumed costs associated with phenotypic plasticity when not needed. In this study, we investigated the effect of experimental evolution in constant, predictable and unpredictable daily fluctuating temperature regimes on the levels of phenotype plasticity in several life history and stress resistance traits in Drosophila simulans. Contrary to the expectation, evolution in the different regimes did not affect the levels of plasticity in any of the traits investigated even though the populations from the different thermal regimes had evolved different stress resistance and fitness trait means. Although costs associated with phenotypic plasticity are known, our results suggest that the maintenance of phenotypic plasticity might come at low and negligible costs, and thus, the potential of phenotypic plasticity to evolve in populations exposed to different environmental conditions might be limited.     

Assay conditions in laboratory experiments: is the use of constant rather than fluctuating temperatures justified when investigating temperature-induced plasticity?

Temperature is an important selective agent in nature. Consequently, temperature-induced plasticity which may help buffering detrimental effects of temperature variation has received considerable attention over recent decades. Laboratory studies have almost exclusively used constant temperatures, while in nature, temperature typically shows pronounced daily fluctuations. Using a factorial design with constant versus fluctuating temperatures and a higher versus a lower mean temperature, we here investigate in the butterfly Lycaena tityrus whether the use of constant temperatures is justified. Fluctuating compared to constant temperatures caused shorter development times, increased heat but decreased cold stress resistance, decreased heat-shock protein expression, and increased immunocompetence. Thus, overall, fluctuating temperatures were more beneficial to the butterflies compared to constant ones. However, despite substantial variation across temperature regimes, the ranking of trait values among treatments remained largely unaffected (e.g. lower constant as well as fluctuating temperatures caused increased pupal mass). Thus, we tentatively conclude that there is no general reason for concern about using constant temperatures in studies investigating phenotypic plasticity, which seem to comprise a fair proxy. However, substantial differences in mean values as well as interactive effects suggest that one needs to be cautious. We further demonstrate negative effects of high temperatures on butterfly immune function, which seem to result from a trade-off between the latter and the heat shock response.