Trehalose Consumption Research Articles

Trehalose consumption ameliorates pathogenesis in an inducible mouse model of the Fragile X-associated tremor/ataxia syndrome

ABSTRACT Trehalose is a naturally occurring sugar found in various food and pharmaceutical preparations with the ability to enhance cellular proteostasis and reduce the formation of toxic intracellular protein aggregates, making it a promising therapeutic candidate for various neurodegenerative disorders. Objectives Here, we explored the effectiveness of nutritional trehalose supplementation in ameliorating symptoms in a mouse model of Fragile X-associated tremor/ataxia syndrome (FXTAS), an incurable late onset manifestation of moderately expanded trinucleotide CGG repeat expansion mutations in the 5’ untranslated region of the fragile X messenger ribonucleoprotein 1 gene (FMR1). Methods An inducible mouse model of FXTAS expressing 90 CGG repeats in the brain had been previously developed, which faithfully captures hallmarks of the disorder, the formation of intracellular inclusions, and the disturbance of motor function. Taking advantage of the inducible nature of the model, we investigated the therapeutic potential of orally administered trehalose under two regimens, modelling disease prevention and disease treatment. Results and Discussion Trehalose's effectiveness in combating protein aggregation is frequently attributed to its ability to induce autophagy. Accordingly, trehalose supplementation under the prevention regimen ameliorated the formation of intranuclear inclusions and improved the motor deficiencies resulting from the induced expression of 90 CGG repeats, but it failed to reverse the existing nuclear pathology as a treatment strategy. Given the favorable safety profile of trehalose, it is promising to further explore the potential of this agent for early stage FXTAS.

Dietary Trehalose as a Bioactive Nutrient

Trehalose is a naturally occurring, non-reducing disaccharide comprising two covalently-linked glucose molecules. It possesses unique physiochemical properties, which account for multiple biological roles in a variety of prokaryotic and eukaryotic organisms. In the past few decades, intensive research on trehalose has uncovered its functions, and extended its uses as a sweetener and stabilizer in the food, medical, pharmaceutical, and cosmetic industries. Further, increased dietary trehalose consumption has sparked research on how trehalose affects the gut microbiome. In addition to its role as a dietary sugar, trehalose has gained attention for its ability to modulate glucose homeostasis, and potentially as a therapeutic agent for diabetes. This review discusses the bioactive effects of dietary trehalose, highlighting its promise in future industrial and scientific contributions.

Chronic Consumption of Trehalose Reduces Lifespan in Drosophila Model.

Trehalose has been widely used as a kind of food additives. But in recent years, with several new studies of trehalose, some harmful effects had also been found. Drosophila melanogaster was used as a model organism to explore whether trehalose affects the lifespan. The results showed that high concentrations of trehalose could significantly shorten the lifespan of female flies by 12.5%, when compared to controls.

Study protocol for a pilot randomised controlled trial evaluating the effectiveness of oral trehalose on inflammatory factors, oxidative stress, nutritional and clinical status in traumatic head injury patients receiving enteral nutrition

IntroductionIn traumatic brain injury (TBI) patients, inflammatory processes and oxidative stress have been linked to the development of neurodegenerative diseases, disability, increased rate of muscle catabolism, malnutrition, hospital stay and...

Combined induction of mTOR-dependent and mTOR-independent pathways of autophagy activation as an experimental therapy for Alzheimer's disease-like pathology in a mouse model

Alzheimer's disease (AD) is associated with amyloid-β (Aβ) accumulation that might be hindered by autophagy. There are two ways to induce autophagy: through mTOR-dependent and mTOR-independent pathways (here, by means of rapamycin and trehalose, respectively). The aim of this study was to evaluate the contribution of these pathways and their combination to the treatment of experimental AD. Mice were injected bilaterally intracerebroventricularly with an Aβ fragment (25–35) to set up an AD model. Treatment with rapamycin (10 mg/kg, every other day), trehalose consumption with drinking water (2 mg/mL, ad libitum), or their combination started 2 days after the surgery and lasted for 2 weeks. Open-field, plus-maze, and passive avoidance tests were used for behavioral phenotyping. Neuronal density, Aβ accumulation, and the expression of autophagy marker LC3-II and neuroinflammatory marker IBA1 were measured in the frontal cortex and hippocampus. mRNA levels of autophagy genes (Atg8, Becn1, and Park2) were assessed in the hippocampus. Trehalose but not rapamycin caused pronounced prolonged autophagy induction and transcriptional activation of autophagy genes. Both drugs effectively prevented Aβ deposition and microglia activation. Autophagy inhibitor 3-methyladenine significantly attenuated autophagy activation and disturbed the effect of the inducers on Aβ load. The inducers substantially reversed behavioral and neuronal deficits in Aβ-injected mice. In many cases, the best outcomes were achieved with the combined treatment. Thus, trehalose alone or combined autophagy activation by the two inducers may be a promising treatment approach to AD-like neurodegeneration. Some aspects of interaction between mTOR-dependent and mTOR-independent pathways of autophagy are discussed.

Transcriptomic analysis provides insights into the immune responses and nutrition in Ostrinia furnacalis larvae parasitized by Macrocentrus cingulum.

Macrocentrus cingulum is a principal endoparasite of Ostrinia furnacalis larvae. M. cingulum larvae repress host immune responses for survival and ingest host nutrients for development until emerging. However, most investigations focused on the mechanisms of how wasps repress the host immunity, the triggered immune responses and nutrient status altered by wasps in host are neglected. In this study, we found that parasitized O. furnacalis larvae activated fast recognition responses and produced some effectors such as lysozyme and antimicrobial peptides, along with more consumption of trehalose, glucose, and even lipid to defend against the invading M. cingulum. However, the expression of peroxidase 6 and superoxide dismutase 2 (SOD 2) was upregulated, and the messenger RNA (mRNA) levels of cellular immunity-related genes such as thioester-containing protein 2 (TEP 2) and hemocytin were also reduced, suggesting that some immune responses were selectively shut down by wasp parasitization. Taken together, all the results indicated that parasitized O. furnacalis larvae selectively activate the immune recognition response, and upregulate effector genes, but suppress ROS reaction and cellular immunity, and invest more energy to fuel certain immune responses to defend against the wasp invading. This study provides useful information for further identifying key components of the nutrition and innate immune repertoire which may shape host-parasitoid coevolutionary dynamics.

Improving SIRT1 by trehalose supplementation reduces oxidative stress, inflammation, and histopathological scores in the kidney of aged rats.

The aging process leads to progressive loss of kidney function. Sirtuin1 (SIRT1) exerts renoprotective effects by conferring resistance to cellular stresses. Trehalose potentially displayed various beneficial effects to promote health span. In this study, we investigated the effects of trehalose on renal SIRT1 and kidney function in senescent rats. Trehalose (2% w/v) was administrated in drinking water for 1month to male aged rats (24months). Then, the level of SIRT1 mRNA and protein, malondialdehyde, total antioxidant capacity, tumor necrosis factor α as well as parameters related to the function and histology of the kidneys were evaluated. Trehalose supplementation increased the level of SIRT1, whereas alleviated the level of oxidative stress, inflammation, and histopathology scores in senescent tissues. However, trehalose administration did not alter kidney function indices in old rats. Collectively, these findings suggested that trehalose was an effective intervention to ameliorate some aspects of age-associated injury in the old kidneys. PRACTICAL APPLICATIONS: Aging is associated with impairment in renal structure and function. Trehalose is a natural disaccharide, which is widely distributed in many organisms. The consumption of trehalose as a dietary supplement is increasing worldwide. This study showed that trehalose administration to aged rats had renoprotective effects through reducing oxidative stress and inflammation, which was mediated by SIRT1. Our results provide useful information for individuals using this sugar as a supplement.

Bacterial α-diglucoside metabolism: perspectives and potential for biotechnology and biomedicine.

In a competitive microbial environment, nutrient acquisition is a major contributor to the survival of any individual bacterial species, and the ability to access uncommon energy sources can provide a fitness advantage. One set of soluble carbohydrates that have attracted increased attention for use in biotechnology and biomedicine is the α-diglucosides. Maltose is the most well-studied member of this class; however, the remaining four less common α-diglucosides (trehalose, kojibiose, nigerose, and isomaltose) are increasingly used in processed food and fermented beverages. The consumption of trehalose has recently been shown to be a contributing factor in gut microbiome disease as certain pathogens are using α-diglucosides to outcompete native gut flora. Kojibiose and nigerose have also been examined as potential prebiotics and alternative sweeteners for a variety of foods. Compared to the study of maltose metabolism, our understanding of the synthesis and degradation of uncommon α-diglucosides is lacking, and several fundamental questions remain unanswered, particularly with regard to the regulation of bacterial metabolism for α-diglucosides. Therefore, this minireview attempts to provide a focused analysis of uncommon α-diglucoside metabolism in bacteria and suggests some future directions for this research area that could potentially accelerate biotechnology and biomedicine developments. KEY POINTS: • α-diglucosides are increasingly important but understudied bacterial metabolites. • Kinetically superior α-diglucoside enzymes require few amino acid substitutions. • In vivo studies are required to realize the biotechnology potential of α-diglucosides.

Is there a causal relationship between trehalose consumption and Clostridioides difficile infection?

Trehalose metabolism appears to play a role in the pathogenicity of some microbes. It has been claimed that trehalose consumption may be a risk factor for Clostridioides difficile infection (CDI), but the evidence for a causal link is contentious. Epidemic ribotypes of C. difficile harbour mutations or have acquired extra genes that mean these strains can utilize lower concentrations of bioavailable trehalose, providing a competitive metabolic advantage in some CDI animal models. By contrast, evidence has emerged to show that trehalose-induced microbiota changes can help protect/reduce CDI in other models. In addition, C. difficile trehalose metabolic variants are widespread among epidemic and nonepidemic ribotypes alike, and the occurrence of these trehalose variants was not associated with increase disease severity or mortality. Currently, there is no proven causal association between the incidence or severity of human CDI and the presence of trehalose metabolism variants. Furthermore, microbial metabolism reduces trehalose bioavailability, potentially removing this competitive advantage for C. difficile trehalose metabolism variants. Taken together, trehalose consumed as part of a normal diet has no increased risk of CDI.

Daily consumption of one teaspoon of trehalose can help maintain glucose homeostasis: a double-blind, randomized controlled trial conducted in healthy volunteers

BackgroundTrehalose is a natural disaccharide that is widely distributed. A previous study has shown that daily consumption of 10 g of trehalose improves glucose tolerance in individuals with signs of metabolic syndrome. In the present study, we determined whether a lower dose (3.3 g/day) of trehalose improves glucose tolerance in healthy Japanese volunteers.MethodsThis was a randomized, double-blind, placebo-controlled study of healthy Japanese participants (n = 50). Each consumed 3.3 g of trehalose (n = 25) or sucrose (n = 25) daily for 78 days. Their body compositions were assessed following 0, 4, 8, and 12 weeks; and serum biochemical parameters were assayed and oral 75-g glucose tolerance tests were performed at baseline and after 12 weeks.ResultsThere were similar changes in body composition and serum biochemistry consistent with established seasonal variations in both groups, but there were no differences in any of these parameters between the two groups. However, whereas after 12 weeks of sucrose consumption, the plasma glucose concentration 2 h after a 75-g glucose load was significantly higher than the fasting concentration, after 12 weeks of trehalose consumption the fasting and 2-h plasma glucose concentrations were similar. Furthermore, an analysis of the participants with relatively high postprandial blood glucose showed that the plasma glucose concentration 2 h after a 75-g glucose load was significantly lower in the trehalose group than in the sucrose group.ConclusionsOur findings suggest that trehalose helps lower postprandial blood glucose in healthy humans with higher postprandial glucose levels within the normal range, and may therefore contribute to the prevention of pathologies that are predisposed to by postprandial hyperglycemia,, even if the daily intake of trehalose is only 3.3 g, an amount that is easily incorporated into a meal.Trial registrationUMIN, UMIN000033536. Registered 27 July 2018.

Lactotrehalose, an Analog of Trehalose, Increases Energy Metabolism Without Promoting Clostridioides difficile Infection in Mice

Trehalose is a disaccharide that might be used in the treatment of cardiometabolic diseases. However, trehalose consumption promotes the expansion of Clostridioides difficile ribotypes that metabolize trehalose via trehalose-6-phosphate hydrolase. Furthermore, brush border and renal trehalases can reduce the efficacy of trehalose by cleaving it into monosaccharides. We investigated whether a trehalase-resistant analogue of trehalose (lactotrehalose) has the same metabolic effects of trehalose without expanding C difficile. We performed studies with HEK293 and Caco2 cells, primary hepatocytes from mice, and human intestinal organoids. Glucose transporters were overexpressed in HEK293 cells, and glucose tra2nsport was quantified. Primary hepatocytes were cultured with or without trehalose or lactotrehalose, and gene expression patterns were analyzed. C57B6/J mice were given oral antibiotics and trehalose or lactotrehalose in drinking water, or only water (control), followed by gavage with the virulent C difficile ribotype 027 (CD027); fecal samples were analyzed for toxins A (ToxA) or B (ToxB) by enzyme-linked immunosorbent assay. Other mice were given trehalose or lactotrehalose in drinking water for 2 days before placement on a chow or 60% fructose diet for 10 days. Liver tissues were collected and analyzed by histologic, serum biochemical, RNA sequencing, autophagic flux, and thermogenesis analyses. We quantified portal trehalose and lactotrehalose bioavailability by gas chromatography mass spectrometry. Fecal microbiomes were analyzed by 16S ribosomal RNA sequencing and principal component analyses. Lactotrehalose and trehalose each blocked glucose transport in HEK293 cells and induced a gene expression pattern associated with fasting in primary hepatocytes. Compared with mice on the chow diet, mice on the high-fructose diet had increased circulating cholesterol, higher ratios of liver weight-to-body weight, hepatic lipid accumulation (steatosis), and liver gene expression patterns of carbohydrate-responsive de novo lipogenesis. Mice given lactotrehalose while on the high-fructose diet did not develop any of these features and had increased whole-body caloric expenditure compared with mice given trehalose or water and fed a high-fructose diet. Livers from mice given lactotrehalose had increased transcription of genes that regulate mitochondrial energy metabolism compared with liver from mice given trehalose or controls. Lactotrehalose was bioavailable in venous and portal circulation and fecal samples. Lactotrehalose reduced fecal markers of microbial branched-chain amino acid biosynthesis and increased expression of microbial genes that regulate insulin signaling. In mice given antibiotics followed by CD027, neither lactotrehalose nor trehalose increased levels of the bacteria or its toxin in stool-in fact, trehalose reduced the abundance of CD027 in stool. Lactotrehalose and trehalose reduced markers of inflammation in rectal tissue after CD027 infection. Lactotrehalose is a trehalase-resistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fasting-mimetics for the treatment of diabetes and nonalcoholic fatty liver disease.

Metabolic constraints drive self-organization of specialized cell groups.

How phenotypically distinct states in isogenic cell populations appear and stably co-exist remains unresolved. We find that within a mature, clonal yeast colony developing in low glucose, cells arrange into metabolically disparate cell groups. Using this system, we model and experimentally identify metabolic constraints sufficient to drive such self-assembly. Beginning in a uniformly gluconeogenic state, cells exhibiting a contrary, high pentose phosphate pathway activity state, spontaneously appear and proliferate, in a spatially constrained manner. Gluconeogenic cells in the colony produce and provide a resource, which we identify as trehalose. Above threshold concentrations of external trehalose, cells switch to the new metabolic state and proliferate. A self-organized system establishes, where cells in this new state are sustained by trehalose consumption, which thereby restrains other cells in the trehalose producing, gluconeogenic state. Our work suggests simple physico-chemical principles that determine how isogenic cells spontaneously self-organize into structured assemblies in complimentary, specialized states.

Clostridium difficile trehalose metabolism variants are common and not associated with adverse patient outcomes when variably present in the same lineage.

BackgroundClostridium difficile ribotype-027, ribotype-078, and ribotype-017 are virulent and epidemic lineages. Trehalose metabolism variants in these ribotypes, combined with increased human trehalose consumption, have been hypothesised to have contributed to their emergence and virulence.Methods5232 previously whole-genome sequenced C. difficile isolates were analysed. Clinical isolates were used to investigate the impact of trehalose metabolism variants on mortality. Import data were used to estimate changes in dietary trehalose. Ribotype-027 virulence was investigated in a clinically reflective gut model.FindingsTrehalose metabolism variants found in ribotype-027 and ribotype-017 were widely distributed throughout C. difficile clade-2 and clade-4 in 24/29 (83%) and 10/11 (91%) of sequence types (STs), respectively. The four-gene trehalose metabolism cluster described in ribotype-078 was common in genomes from all five clinically-important C. difficile clades (40/167 [24%] STs).The four-gene cluster was variably present in 208 ribotype-015 infections (98 [47%]); 27/208 (13%) of these patients died within 30-days of diagnosis. Adjusting for age, sex, and infecting ST, there was no association between 30-day all-cause mortality and the four-gene cluster (OR 0.36 [95%CI 0.09–1.34, p = 0.13]).Synthetic trehalose imports in the USA, UK, Germany and the EU were < 1 g/capita/year during 2000–2006, and < 9 g/capita/year 2007–2012, compared with dietary trehalose from natural sources of ~100 g/capita/year.Trehalose supplementation did not increase ribotype-027 virulence in a clinically-validated gut model.InterpretationTrehalose metabolism variants are common in C. difficile. Increases in total dietary trehalose during the early-mid 2000s C. difficile epidemic were likely relatively minimal. Alternative explanations are required to explain why ribotype-027, ribotype-078 and ribotype-017 have been successful.FundingNational Institute for Health Research. Gut model experiments only: Hayashibara Co. Ltd.

Multiple toxicity studies of trehalose in mice by intragastric administration

In the present study, aberration, body weight, food consumption, haematology, organ coefficients, and both gross and microscopic appearance of some histiocytes were compared between the test and control groups. A sperm abnormality test, bone marrow cell micronucleus test, and a haematology study were conducted at levels of 1.25g/kg, 2.5g/kg, and 5g/kg of trehalose. In both the sperm abnormality test and bone marrow cell micronucleus test, statistically significant differences were observed between the positive control and treatment groups (P<0.05), while no statistical difference was observed among the negative control, high dose, moderate dose and low dose groups (P>0.05). In the haematology study, there was no significant difference found from the controls at P>0.05. The results obtained in the present study could support the conclusion that consumption of trehalose has no adverse effects for humans.

Behaviour of dehydrated baker's yeast during reduction reactions in a biphasic medium.

The behaviour of dry baker's yeast ( Saccharomyces cerevisiae type II, Sigma) used as biocatalyst without preliminary growth for the synthesis of 2-heptanol from 2-heptanone in a biphasic system is presented. Cells undergo intracellular trehalose consumption with a stoichiometric ethanol production during the first 15 h of the process. This metabolism is then replaced by acetate accumulation. These reactions are disconnected from the biocatalytic reaction and do not provide reduced cofactors. 2-Heptanone is metabolised by two pathways. The first leads to 2-heptanol (molar yield close to 55%, enantioselectivity higher than 99%, with a slight decrease at the end of the process) and the second corresponds to material incorporation into the biomass. This latter phenomenon is assumed to provide the biocatalyst with the reduced cofactors needed for the reduction process. Overall, the process yielded ca. 1.4 g/l 2-heptanol in 50 h reaction, which is close to that observed with fresh cells previously grown for 15 h.

Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies

This paper contains a review of the history, natural occurrence, human consumption, metabolism, manufacture, and the results of eight standardized animal safety studies using trehalose. Trehalose (α,α-trehalose) is a naturally occurring sugar containing two d-glucose units in an α,α-1,1 linkage. Trehalose functions in many organisms as an energy source or a protectant against the effects of freezing or dehydration. It also possesses physical and/or chemical properties that are different than other sugars, which may make trehalose an attractive ingredient in food, health and beauty and pharmaceutical products. Data are presented supporting safe human consumption of trehalose in doses up to 50 g, and the physiologic ability of humans to digest it. No consistent treatment-related, dose-dependent adverse effects were observed in any of the eight safety studies performed at doses up to 10% of the diets. On the basis of these toxicity studies, human studies in which doses of trehalose were administered to various populations, and consumption of trehalose in commercial products in Japan, it is concluded that trehalose is safe for use as an ingredient in consumer products when used in accordance with current Good Manufacturing Practices.

Intracellular accumulation of trehalose during streptomycin formation by Streptomyces griseus.

When washed mycelium of Streptomyces griseus HUT 6037 was incubated in 0.5% NaCl solution containing 14C-gl cosamine with shaking at 28°C, the activity of the mycelium to incorporate radioactivity into the cell wall decreased rapidly, while that into streptomycin increased. During this physiological change, UDP-Af-acetylmuramyl-pentapeptide, UDP-N-acetylglucosamine, glutamic acid and trehalose accumulated in the mycelium. The latter two substances accumulated much more than the former two. When the time courses of the activities of incorporation of 14C-glucosamine into the four substances, mucopeptide and streptomycin were examined, a decrease in the activity into peptidoglycan led to an increase into streptomycin, glutamic acid and trehalose. In a pH-stat batch culture with a defined medium, trehalose was accumulated in the cell before glucose was consumed. However, after glucose was consumed, the consumption of trehalose began. Streptomycin production continued until intracellular trehalose completely disappeared in spite of the lack of glucose in the culture medium.

13C nuclear magnetic resonance study of trehalose mobilization in yeast spores.

Using high-resolution 13C nuclear magnetic resonance, we examined the mobilization of endogenous trehalose in suspensions of yeast asci. Sporulation of yeast cells in [1-13C]acetate resulted in incorporation of label into the C-3 and C-4 positions of trehalose within the asci. During germination of these asci with [1-13C]glucose, the consumption of both endogenous trehalose and exogenous glucose were followed simultaneously by 13C nuclear magnetic resonance, as was the formation of glycerol and ethanol, their glycolytic and products. Time courses for carbohydrate consumption indicated that trehalose, although it decreased to 25% of its initial value upon germination, was not preferentially catabolized and did not provide the primary energy supply for germination with glucose. The ratio of trehalose to glucose catabolized was 0.09. Exogenous glucose levels appeared to regulate trehalose mobilization since trehalose was only consumed when sufficiently high levels (more than 2 mM) of glucose were present. Upon glucose depletion newly synthesized [1-13C]trehalose was observed. Nuclear magnetic resonance spectra of extracts confirmed the trehalose peak assignments and showed products of [1-13C]glucose catabolism. In addition by quantitating trehalose consumption and 2-deoxyglucose incorporation in dormant yeast asci, we found that 3.8 +/- 0.l4 molecules of 2-deoxyglucose were incorporated for each trehalose molecule consumed. Trehalose can therefore function as a carbohydrate source for ATP formation during dormancy.

The effect of controlled atmospheres on carbohydrate metabolism in the tissue of Ephestia cautella (walker) pupae

Ephestia cautella pupae, 0–24 hr old, were exposed for 24 hr to a controlled atmosphere, either high in carbon dioxide or low in oxygen at 26°C. Tissue levels of glycogen, trehalose, glucose, α-glycerophosphate, glycerol, lactate, alanine, ATP, ADP, AMP, citrate and malate were determined in exposed pupae. Hypercarbia tended to increase the consumption of glycogen but had no clear effect on trehalose consumption. Glucose levels were similar to insect exposed to normal and to hypercarbic atmospheres but significantly higher in insects exposed to hypoxia. α-Glycerphosphate, glycerol, lactate and alanine levels were high in 0–24 hr old pupae but fell after 24 hr exposure to normal of hypoxic atmospheres. The levels of these metabolites fell less when nitrogen was replaced by carbon dioxide. Hypercarbia reduced the total amount of adenine nucleotides and the energy charge. However, 1% oxygen in nitrogen markedly affected the ATP level. Citrate was reduced in hypercarbia and in hypoxia, whereas malate was reduced by hypercarbia but increased in hypoxia.

Neurosecretory cells in insect brain and production of hypoglycaemic hormone.

INSECTS have a hyperglycaemic hormone, produced by neurosecretory cells of the corpus cardiacum (CC). This hormone stimulates the production of the major blood carbohydrate trehalose from glycogen stored in fat body cells1. No hypotrehalosaemic hormone has so far been reported and the level of blood trehalose has been thought to depend on: the secretory activity of the CC neurosecretory cells controlled by nerves from the brain2,3; the relationship between production and consumption of trehalose, which in many insects, including flies, serves as fuel for flight. Also, in vitro experiments indicate that medium containing trehalose can inhibit the synthesis or liberation of trehalose from fat body cells, and it has been suggested that such feedback control is an important factor in the natural regulation of trehalose formation4,5. It was therefore surprising to find that seven decapitated 6-d-old blowflies, Calliphora erythrocephala, not only remained alive for 30 h, during which locomotion and heart beat continued, but that their blood trehalose level had increased from 21.9 g l−1 (± 4.09 s.e.m.) to 113.2 g l−1 (± 9.07 s.e.m.).