Disruption of ATP homeostasis during chronic cold stress and recovery in the chill susceptible beetle (Alphitobius diaperinus)

This study examined the impact of fluctuating thermal regimes (FTRs) on cold tolerance of the polyphagous beetle Alphitobius diaperinus. Daily pulses of elevated temperatures can provide breaks in chronic cold stress, potentially allowing for physiological recovery and improving survival. Perturbations in central metabolism appear to be a common physiological response in insects exposed to low temperatures. It has been suggested that energy supplies, which may be depleted during cold exposure, can be regenerated during the warming pulses of FTRs. This study tested the assumption that chronic cold stress may induce ATP depletion and that recovery during FTR warming pulses may allow re-establishment of ATP supplies. In this study, A. diaperinus were exposed to cold stress under different thermal regimes (constant or fluctuating). The results did not confirm the aforementioned assumption. No cold-induced ATP depletion was observed. The lowest ATP levels were repeatedly detected in the untreated controls. The data show that homoeostasis of ATP is lost when adults A. diaperinus are exposed to cold stress, whatever thermal regime (constant or fluctuating). ATP accumulation may be viewed as a symptom of a production/consumption imbalance under cold stress conditions. Periodic short (2-h) warming pulses clearly improved cold survival. Cellular homeostasis, however, probably requires a longer recovery period to be fully restored.     

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.     

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.     

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.     

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.     

Does fluctuating thermal regime trigger free amino acid production in the parasitic wasp Aphidius colemani (Hymenoptera: Aphidiinae)?

When stressful cold-exposure is interrupted by short warm intervals, physiological recovery is possible, and this improves markedly the survival of insects. Fluctuating thermal regime (FTR) may act as a cue triggering the initiation of a metabolic response involving synthesis of cryoprotective compounds, such as free amino acids (FAA). Since specific changes in FAA levels can provide a good indication of the overall response of an organism to stressful conditions, we investigated temporal changes in FAA body contents of the parasitoid Aphidius colemani Viereck during exposure to FTR (4 degrees C: 20 degrees C for 22 h: 2 h per day) versus constant low temperature (4 degrees C). Physiological response during cold-exposure was clearly dissimilar between thermal treatments. Under constant cold-exposure FAA pool increased, whereas it decreased with cold-exposure duration in FTR. No single FAA accumulation could explain the higher survival under FTR. We propose that instead of considering FAA as a part of cryoprotective arsenal, FAA accumulation should rather be regarded as a symptom of a cold-induced physiological response. This is much less manifest under FTR, as the warm intervals likely allow a periodic reactivation of normal metabolic activities and a recovery of developmental processes.     

Changes in free amino acids in Alphitobius diaperinus (Coleoptera: Tenebrionidae) during thermal and food stress

Temporal changes in free amino acid pools are examined in starved and cold-exposed (10 degrees C) beetles (Alphitobius diaperinus) (control beetles; 4, 7, 14, 28 and 56 days of cold-exposure). The range of individual responses and the effect of gender on survival and free amino acid levels are evaluated. Females survived significantly longer (Lt50 and Lt90) than males under stressing conditions. Pro, Gln, Ala, Arg and Thr are the major components of the free amino acid pool in the whole body of adult beetles. Multivariate analysis indicates that the duration of exposure explains most of the observed variation in amino acid levels, while sex effect is not significant. Moreover, amino acid levels fluctuate strongly within each sampled date, revealing a high inter-individual heterogeneity. However, this heterogeneity decreases after four weeks of cold-exposure and starvation. The increase in Ala level and the concomitant decrease of Pro after four weeks suggest that Pro might be an important fuel for metabolism when fat reserves are reduced. We conclude that changes in free amino acid pools are due to a combination of reduced individual heterogeneity, cold-acclimation and amino acid degradation for energetic purposes.     

Effect of food shortage and temperature on oxygen consumption in the lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

Abstract.  Temperature and food availability are limiting factors for the establishment of tropical insects in temperate countries. In the alien pest beetle, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), starvation and temperature have a significant impact on metabolic rate with oxygen consumption ranging from 0.5 µmol/g fresh mass (FM)/h at 12 °C to 3.4 µmol/g FM/h at 24 °C. At 12 °C, oxygen consumption decreased continuously during an entire period of starvation. However, at 16, 20 and 24 °C, beetles display a marked hyperactivity that leads to an increase in the oxygen consumption level during the first week of starvation, followed by a steep decrease until the end of the starvation period. Oxygen consumption either does not decline in fed beetles (observed at higher temperatures) or declines at a much shallower rate than in starved beetles (observed at cooler temperatures). During the first week of refeeding, Oxygen consumption rose steeply at 16, 20 and 24 °C before levelling off to the initial value ( t ₀). At 12 °C, no compensation process was observed during recovery. This study reveals that an important threshold in the biology of A. diaperinus lies between 12 and 16 °C, leading to the onset of reduced locomotor activity and the promotion of survival to the detriment of reproduction. This 'sit and wait' behaviour is proposed as an adaptive strategy (i.e. inactivity and lower oxygen consumption coupled with low energetic requirements and high recovery abilities). Such behaviour and the observed hyperactivity were rarely described in insects before the present study. Together, the previous and present results suggest that A. diaperinus populations are likely maintained in temperate regions by immigration from warmer situations.     

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.     

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.     

Long‐term after‐effects of cold exposure in adult Alphitobius diaperinus (Tenebrionidae): the need to link survival ability with subsequent reproductive success

1. The effects of cold acclimation and cold exposure on the survival and reproductive capabilities of Alphitobius diaperinus (Tenebrionidae) adult beetles are examined. 2. First, the impact of temperature on survival duration was assessed by placing beetles in a range of cool temperature treatments. Second, the importance of acclimation duration was assessed. Third, the impact of thermal stress on subsequent reproductive ability was examined for beetles that had no previous cold exposure, and for beetles that had been subjected to previous cold exposure (i.e. acclimated) at various conditions, including fluctuating temperatures. 3. In all groups, the number of recorded survivors was strongly impacted by recovery period duration (i.e. 2 vs. 10 days). Survival of non‐acclimated and 3‐day acclimated beetles, expressed as lethal time for 50% of the samples, was reduced significantly when the insects were re‐assessed for survival at 10 days after being returned to optimal growth conditions (7.9 ± 0.4 vs. 5.1 ± 0.6 days and 8.8 ± 0.5 vs. 6.8 ± 0.6 days, respectively). 4. Insects that had been subject to cold acclimation expressed better subsequent reproduction success than non‐acclimated beetles. This beneficial impact increased when the acclimation period was prolonged, but some longer acclimation periods had no significant impact on survival. 5. Our results indicate that cold exposure has the capacity to irreversibly damage the reproductive system and that insect survival depends on the duration of the recovery period. Both the survival ability and subsequent reproductive output have to be examined to objectively determine insect cold resistance.     

Evaluating (13)C enrichment data of free amino acids for precise metabolic flux analysis

Metabolic flux analysis using (13)C enrichment data of intracellular free amino acids (FAAs) can improve the time resolution of flux estimation compared to analysis of proteinogenic amino acid data owing to the faster turnover times of FAAs. The nature of the (13)C enrichment dynamics of FAAs remains obscure, however, especially with regard to its dependence on culture conditions, even though an understanding of dynamic behavior is important for precise metabolic flux estimation. In this study, we analyzed the (13)C enrichment dynamics of free and proteinogenic amino acids in a series of continuous culture experiments with Escherichia coli. The results indicated that the effect of protein degradation on the (13)C enrichment of FAAs was negligible under cellular growth conditions. Furthermore, they showed that the time scale necessary for (13)C enrichment dynamics of FAAs to reach a steady state depends on culture conditions such as oxygen uptake rate, which was likely due to different pool sizes of intracellular metabolites. The results demonstrate the importance of analyzing (13)C enrichment dynamics for the precise estimation of metabolic fluxes using FAA data.     

Limited plasticity of low temperature tolerance in an Australian cantharid beetle Chauliognathus lugubris

Spatial and/or taxonomic bias in thermal tolerance and plasticity data can severely impact projections of climate change responses and limit the understanding of the evolution of thermal performance curves. Thus, further data from under‐represented groups and geographical locations are important for synthesizing and predicting the physiological responses of insects to climate variability. For example, the magnitude of rapid cold‐hardening (RCH) and seasonal acclimatization of low temperature tolerance are typically poorly documented for nondipteran species from the southern Hemisphere. Moreover, few studies assess RCH responses under different acclimation regimes. To address this paucity of data, the low temperature survival, RCH and acclimation ability of Chauliognathus lugubris (F.) are assessed from an adult aggregation collected in Armidale, New South Wales, Australia. Beetles are acclimated to either 27 or 20 °C for 1 week and then tested for their ability to survive cold shock or rapidly cold‐harden. There is no effect of acclimation on low temperature survival (mean survival range at ?5.4 °C for 2 h: 4–52% in 27 and 20 °C acclimation groups). In addition, beetles show no significant improvement in survival after acute thermal pretreatments. In conclusion, these data suggest a generally poor acclimation potential of low temperature survival and no RCH responses in adult Australian cantharid beetles, which is accordance with what might be expected given the microclimate experienced, their ability for behavioural regulation and the life history of the species.     

Comparison of two acetylcholinesterase gene cDNAs of the lesser mealworm, Alphitobius diaperinus, in insecticide susceptible and resistant strains

Two cDNAs encoding different acetylcholinesterase (AChE) genes (AdAce1 and AdAce2) were sequenced and analyzed from the lesser mealworm, Alphitobius diaperinus. Both AdAce1 and AdAce2 were highly similar (95 and 93% amino acid identity, respectively) with the Ace genes of Tribolium castaneum. Both AdAce1 and AdAce2 have the conserved residues characteristic of AChE (catalytic triad, intra-disulfide bonds, and so on). Partial cDNA sequences of the Alphitobius Ace genes were compared between two tetrachlorvinphos resistant (Kennebec and Waycross) and one susceptible strain of beetles. Several single nucleotide polymorphisms (SNPs) were detected, but only one non-synonymous mutation was found (A271S in AdAce2). No SNPs were exclusively found in the resistant strains, the A271S mutation does not correspond to any mutations previously reported to alter sensitivity of AChE to organophosphates or carbamates, and the A271S was found only as a heterozygote in one individual from one of the resistant A. diaperinus strains. This suggests that tetrachlorvinphos resistance in the Kennebec and Waycross strains of A. diaperinus is not due to mutations in either AChE gene. The sequences of AdAce1 and AdAce2 provide new information about the evolution of these important genes in insects.     

The effect of low ambient temperature on the febrile responses of rats to semi-purified human endogenous pyrogen

The febrile responses of Sprague-Dawley rats to semi-purified human endogenous pyrogen were studied at a thermoneutral ambient temperature (26 degrees C) and in the cold (3 degrees C). It was found that while rats developed typical monophasic febrile responses at thermoneutrality, febrile responses were absent in the cold-exposed rats. Experiments were conducted to determine whether this lack of febrile responses in cold-exposed rats was due to an inability of these animals to generate or retain heat in the cold. Thermogenesis and vasoconstriction were stimulated in cold-exposed rats by selectively cooling the hypothalamus, using chronically implanted thermodes. It was shown that, using this stimulus, metabolic rate could be increased by more than 50 percent and body temperature could be driven up at a rate of 5 degrees C/hour in rats exposed to the cold. Therefore, it was concluded that the lack of febrile responses of cold-exposed rats to pyrogen is in no way due to a physical or physiological inability to retain heat. Instead, it appears that in some manner cold exposure suppresses the sensitivity or responsiveness of the rat to pyrogenic stimuli.     

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.     

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.     

Increased nonshivering thermogenesis, brown fat cytochrome-c oxidase activity, GDP binding, and uncoupling protein mRNA levels after short daily cold exposure of Phodopus sungorus

In their natural environment, burrowing rodents experience rather fluctuating ambient temperatures and are acutely cold exposed only for short periods outside their burrows. The effect of short daily cold exposure on basal metabolic rate, nonshivering thermogenesis, brown fat thermogenesis, and uncoupling protein mRNA was studied in the Djungarian hamster, Phodopus sungorus. They were kept at 23 degrees C and exposed to 5 degrees C daily either for one 4-h period or twice for 2 h (in 12-h intervals). At the same time control hamsters were kept continuously either at thermoneutrality (23 degrees C) or at 5 degrees C. Two 2-h cold exposures daily were sufficient to increase basal metabolic rate and nonshivering thermogenesis to the same level as continuous cold exposure, whereas one 4-h cold period per day did not result in a significant increase of both parameters. Brown fat thermogenesis (as measured by cytochrome-c oxidase activity and GDP binding to the mitochondrial uncoupling protein) increased to the same extent by both treatments with short daily cold exposure. However, this increase was less than in the chronically cold-exposed hamsters. A similar result was found for uncoupling protein mRNA: both short-term cold-exposed hamsters increased uncoupling protein mRNA levels to a similar extent, but less than after chronic cold treatment. It is concluded that short daily cold exposures are sufficient to cause adaptive increases of the capacity of metabolic heat production as well as brown fat thermogenic properties.