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.     

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.     

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.     

Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster

When ectotherms are exposed to low temperatures, they enter a cold‐induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill‐coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using nuclear magnetic resonance (NMR) spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold‐induced perturbations. The metabolites of cold‐hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations.     

Cold acclimation allows Drosophila flies to maintain mitochondrial functioning under cold stress

Environmental stress generally disturbs cellular homeostasis. Researchers have hypothesized that chilling injury is linked to a shortage of ATP. However, previous studies conducted on insects exposed to nonfreezing low temperatures presented conflicting results. In this study, we investigated the mitochondrial bioenergetics of Drosophila melanogaster flies exposed to chronic cold stress (4 °C). We assessed mitochondrial oxygen consumption while monitoring the rate of ATP synthesis at various times (0, 1, 2, and 3 days) during prolonged cold stress and at two assay temperatures (25 and 4 °C). We compared organelle responses between cold-susceptible and cold-acclimated phenotypes. Continuous exposure to low temperature provoked temporal declines in the rates of mitochondrial respiration and ATP synthesis. Respiratory control ratios (RCRs) suggested that mitochondria were not critically uncoupled. Nevertheless, after 3 days of continuous cold stress, a sharp decline in the mitochondrial ATP synthesis rate was observed in control flies when they were assayed at low temperature. This change was associated with reduced survival capacity in control flies. In contrast, cold-acclimated flies exhibited high survival and maintained higher rates of mitochondrial ATP synthesis and coupling (i.e., higher RCRs). Adaptive changes due to cold acclimation observed in the whole organism were thus manifested in isolated mitochondria. Our observations suggest that cold tolerance is linked to the ability to maintain bioenergetics capacity under cold stress.     

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.     

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.     

Effects of acclimation temperature on membrane phospholipids in the flesh fly Sarcophaga similis

Cold acclimation is a well‐known strategy for enhancing cold tolerance in ectotherms including insects. Nevertheless, information on the physiological mechanisms underpinning this phenomenon is still limited. Biological membrane integrity is critical for insects to perform at low temperatures, and an advantage is conferred on those insects that can adjust the composition of their membrane phospholipids. Such changes contribute to homeoviscous adaptation, a process that allows membranes to maintain a liquid–crystalline (fluid) state at low temperatures. Here we investigated phospholipids in the flesh fly Sarcophaga similis acclimated to various temperatures. Significant differences were observed in the composition of their fatty acyl chains: flies acclimated to low temperatures showed a higher proportion of palmitic and oleic acids at the expense of palmitoleic acid. Other fatty acids (stearic, linoleic, linolenic, arachidonic, eicosapentaenoic acids) were not significantly changed. The degree of unsaturation decreased in cold‐acclimated flies, but the difference was quite small. The weighted average chain length and number of double bonds were unchanged among flies acclimated to different temperatures. As temperatures decreased, the percentage of phosphatidylethanolamine increased to twice that of phosphatidylcholine. We discuss the role of these phospholipid changes in cold acclimation.     

Dietary sugars affect cold tolerance of Drosophila melanogaster

In spite of the extensive knowledge of the biology and the genetics of Drosophila melanogaster, the mechanisms by which this fly builds up cold tolerance remain poorly understood. Recent studies have reported that acclimation-mediated acquisition of cold tolerance is associated with moderate accumulation of sugars in drosophilids. However, it is not known whether there is a genuine causative link between cold tolerance and body sugar accumulation in Drosophila flies. We thus tested whether increasing body sugars levels, via dietary enrichment, will promote the cold tolerance of D. melanogaster adults. We gradually augmented the concentration of four different sugars (sucrose, fructose, glucose and trehalose) in rearing diets and tested the basal cold tolerance (acute and chronic). Using SIM-GC/MS approach, we verified whether feeding of larvae and adults on sugar-enriched diets was associated with increasing body sugars. We also tested whether development, body mass, fat stores, metabolites composition and metabolic pathways were altered by these dietary manipulations. The data confirm an effective incorporation of all sugars. Contrary to the expectation, cold tolerance was negatively affected by exogenous sugars, especially when supplemented at high concentrations. Rearing on high-sugar doses induced system-wide metabolic alteration associated with carbohydrate metabolism imbalance, a developmental delay and a fresh mass reduction. Our data show that high dietary sugars create a metabolic imbalance and negatively affect cold tolerance. This study provides an intriguing connection between nutritional conditions and thermal trait. It also underlines that careful attention should be given to dietary factors when studying thermal traits.     

Expression of Ixodes scapularis antifreeze glycoprotein enhances cold tolerance in Drosophila melanogaster

Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies.     

Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance

In Drosophila melanogaster (D. melanogaster), neurosecretory insulin-like peptide-producing cells (IPCs), analogous to mammalian pancreatic β cells are involved in glucose homeostasis. Extending those findings, we have developed in the adult fly an oral glucose tolerance test and demonstrated that IPCs indeed are responsible for executing an acute glucose clearance response. To further develop D. melanogaster as a relevant system for studying age-associated metabolic disorders, we set out to determine the impact of adult-specific partial ablation of IPCs (IPC knockdown) on insulin-like peptide (ILP) action, metabolic outcomes and longevity. Interestingly, while IPC knockdown flies are hyperglycemic and glucose intolerant, these flies remain insulin sensitive as measured by peripheral glucose disposal upon insulin injection and serine phosphorylation of a key insulin-signaling molecule, Akt. Significant increases in stored glycogen and triglyceride levels as well as an elevated level of circulating lipid measured in adult IPC knockdown flies suggest profound modulation in energy metabolism. Additional physiological outcomes measured in those flies include increased resistance to starvation and impaired female fecundity. Finally, increased life span and decreased mortality rates measured in IPC knockdown flies demonstrate that it is possible to modulate ILP action in adult flies to achieve life span extension without insulin resistance. Taken together, we have established and validated an invertebrate genetic system to further investigate insulin action, metabolic homeostasis and regulation of aging regulated by adult IPCs.Key words: Drosophila melanogaster, insulin-producing cells (IPCs), drosophila insulin-like peptides (DILPs), type 2 diabetes, oral glucose tolerance test (OGTT), insulin sensitivity, energy metabolism, life span     

Effects of acclimation temperature on thermal tolerance and membrane phospholipid composition in the fruit fly Drosophila melanogaster

Adaptative responses of ectothermic organisms to thermal variation typically involve the reorganization of membrane glycerophospholipids (GPLs) to maintain membrane function. We investigated how acclimation at 15, 20 and 25 degrees C during preimaginal development influences the thermal tolerance and the composition of membrane GPLs in adult Drosophila melanogaster. Long-term cold survival was significantly improved by low acclimation temperature. After 60 h at 0 degrees C, more than 80% of the 15 degrees C-acclimated flies survived while none of the 25 degrees C-acclimated flies survived. Cold shock tolerance (1h at subzero temperatures) was also slightly better in the cold acclimated flies. LT50 shifted down by ca 1.5 degrees C in 15 degrees C-acclimated flies in comparison to those acclimated at 25 degrees C. In contrast, heat tolerance was not influenced by acclimation temperature. Low temperature acclimation was associated with the increase in proportion of ethanolamine (from 52.7% to 58.5% in 25 degrees C-acclimated versus 15 degrees C-acclimated flies, respectively) at the expense of choline in GPLs. Relatively small, but statistically significant changes in lipid molecular composition were observed with decreasing acclimation temperature. In particular, the proportions of glycerophosphoethanolamines with linoleic acid (18:2) at the sn-2 position increased. No overall change in the degree of fatty acid unsaturation was observed. Thus, cold tolerance but not heat tolerance was influenced by preimaginal acclimation temperature and correlated with the changes in GPL composition in membranes of adult D. melanogaster.     

Metabolic regulatory network alterations in response to acute cold stress and ginsenoside intervention

Acute stress may trigger systemic biochemical and physiological changes in living organisms, leading to a rapid loss of homeostasis, which can be gradually reinstated by self-regulatory mechanisms and/or drug intervention strategy. However, such a sophisticated metabolic regulatory process has so far been poorly understood, especially from a holistic view. Urinary metabolite profiling of Sprague-Dawley rats exposed to cold temperature (-10 degrees C) for 2 h using GC/MS in conjunction with modern multivariate statistical techniques revealed drastic biochemical changes as evidenced by fluctuations of urinary metabolites and demonstrated the protective effect of total ginsenosides (TGs) in ginseng extracts on stressed rats. The metabonomics approach enables us to visualize significant alterations in metabolite expression patterns as a result of stress-induced metabolic responses and post-stress compensation, and drug intervention. Several major metabolic pathways including catecholamines, glucocorticoids, the tricarboxylic acid (TCA) cycle, tryptophan (nicotinate), and gut microbiota metabolites were identified to be involved in metabolic regulation and compensation required to restore homeostasis.     

Partial ablation of adult Drosophilainsulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance

In Drosophila melanogaster (D. melanogaster), neurosecretory insulin-like peptide-producing cells (IPCs), analogous to mammalian pancreatic b cells are involved in glucose homeostasis. Extending those findings, we have developed in the adult fly an oral glucose tolerance test and demonstrated that IPCs indeed are responsible for executing an acute glucose clearance response. To further develop D. melanogaster as a relevant system for studying age-associated metabolic disorders, we set out to determine the impact of adult-specific partial ablation of IPCs (IPC knockdown) on insulin-like peptide (ILP) action, metabolic outcomes and longevity. Interestingly, while IPC knockdown flies are hyperglycemic and glucose intolerant, these flies remain insulin sensitive as measured by peripheral glucose disposal upon insulin injection and serine phosphorylation of a key insulin-signaling molecule, Akt. Significant increases in stored glycogen and triglyceride levels as well as an elevated level of circulating lipid measured in adult IPC knockdown flies suggest profound modulation in energy metabolism. Additional physiological outcomes measured in those flies include increased resistance to starvation and impaired female fecundity. Finally, increased life span and decreased mortality rates measured in IPC knockdown flies demonstrate that it is possible to modulate ILP action in adult flies to achieve life span extension without insulin resistance. Taken together, we have established and validated an invertebrate genetic system to further investigate insulin action, metabolic homeostasis and regulation of aging regulated by adult IPCs.     

A proteomic analysis of cold stress responses in rice seedlings

Using proteomic analysis, an investigation aimed at a better understanding of the molecular adaptation mechanisms of cold stress was carried out in rice (Oryza sativa). The seedlings were exposed to a progressively low temperature stress treatment from normal temperature to 15, 10, and 5 degrees C. Proteins were extracted from the leaves collected from both control and stressed seedlings. By fractionation, approximately 1700 protein spots were separated and visualized on CBB-stained 2-D gels. Sixty protein spots were found to be up-regulated in responding to the progressively low temperature stress and displayed different dynamic patterns. As an initial work, 41 of these proteins were identified using MALDI-TOF MS or ESI/MS/MS. These cold responsive proteins, besides two proteins of unknown function, include four factors of protein biosynthesis, four molecular chaperones, two proteases, and eight enzymes involved in biosynthesis of cell wall components, seven antioxidative/detoxifying enzymes, and proteins linked to energy pathway, as well as a protein involved in signal transduction. The functional proteomes illuminate the facts, at least in plant cell, that protein quality control mediated by chaperones and proteases and enhancement of cell wall components play important roles in tolerance to cold stress. Using TargetP program, the subcellular localization of the identified proteins was analyzed. Proteins (43.9%) were predicted to be located in the chloroplasts, implying that chloroplast proteome is virtually subjective to cold stress. The physiological implications, revealed from the experimental data, are discussed in context of a complex metabolic network in plant cells responsive to cold stress.     

Cold shock and heat shock: a comparison of the protection generated by brief pretreatment at less severe temperatures

Abstract Brief exposure to low (0^oC) or high (40^oC) temperature elicits a protective response that prevents injury when the flesh fly, Sarcophaga crassipalpis Macquart, is subjected to more severe cold (-10^oC) or heat (45^oC). Both the low and high temperature responses were found in all developmental stages of the fly, but were most pronounced in the pupal and pharate adult stages. The protective responses generated by brief exposure to 0 or 40^oC appear similar in that both result in a rapid acquisition of cold or heat tolerance and a loss of protection after the flies are returned to 25^oC. The protection generated by chilling is obvious within 10 min of exposure to 0^oC while a 30 min exposure to 40^oC is required to induce the high temperature protection. High temperature protects against cold shock injury within a narrow range (around 36^oC) but we have no evidence that low temperature can protect against heat injury. We previously demonstrated that the rapid increase in cold tolerance correlates with concomitant increases in glycerol concentration, but in this study we found no significant elevation in glycerol in heat-shocked flies. Thus the physiological and biochemical bases for the rapid responses to cold and heat appear to be different.     

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.     

When nonshivering thermogenesis equals maximum metabolic rate: thermal acclimation and phenotypic plasticity of fossorial Spalacopus cyanus (Rodentia)

Many small mammals inhabiting fluctuating and cold environments display enhanced capacity for seasonal changes in nonshivering thermogenesis (NST) and thermoregulatory maximum metabolic rate (MMR). However, it is not known how this plasticity remains in a mammal that rarely experiences extreme thermal fluctuations. In order to answer this question, we determined body mass (m(b)), basal metabolic rate (BMR), NST, MMR, and minimum thermal conductance (C) on a Chilean fossorial caviomorph (Spalacopus cyanus) from a coastal population, acclimated to cold (15 degrees C) and warm (30 degrees C) conditions. NST was measured as the maximum response of metabolic rate (NST(max)) after injection of norepinephrine (NE) in thermoneutrality minus BMR. Maximum metabolic rate was assessed in animals exposed to enhanced heat-loss atmosphere (He-O2) connected with an open-flow respirometer. Body mass and metabolic variables increased significantly after cold acclimation with respect to warm acclimation but to a low extent (BMR, 26%; NST, 10%; and MMR, 12%). However, aerobic scope (MMR/BMR), calculated shivering thermogenesis (ST), and C did not change with acclimation regime. Our data suggest that physiological plasticity of S. cyanus is relatively low, which is in accordance with a fossorial mode of life. Although little is known about MMR and NST in fossorial mammals, S. cyanus has remarkably high NST; low MMR; and surprisingly, a nil capacity of ST when compared with other rodents.