Comparative transcriptomic evidence of physiological changes and potential relationships in vertebrates under different dormancy states.

Dormancy represents a fascinating adaptive strategy for organisms to survive in unforgiving environments. After a period of dormancy, organisms often exhibit exceptional resilience. This period is typically divided into hibernation and aestivation based on seasonal patterns. However, the mechanisms by which organisms adapt to their environments during dormancy, as well as the potential relationships between different states of dormancy, deserve further exploration. Here, we selected Perccottus glenii and Protopterus annectens as the primary subjects to study hibernation and aestivation, respectively. Based on histological and transcriptomic analysis of multiple organs, we discovered that dormancy involved a coordinated functional response across organs. Enrichment analyses revealed noteworthy disparities between the two dormant species in their responses to extreme temperatures. Notably, similarities in gene expression patterns pertaining to energy metabolism, neural activity, and biosynthesis were noted during hibernation, suggesting a potential correlation between hibernation and aestivation. To further explore the relationship between these two phenomena, we analyzed other dormancy-capable species using data from publicly available databases. This comparative analysis revealed that most orthologous genes involved in metabolism, cell proliferation, and neural function exhibited consistent expression patterns during dormancy, indicating that the observed similarity between hibernation and aestivation may be attributable to convergent evolution. In conclusion, this study enhances our comprehension of the dormancy phenomenon and offers new insights into the molecular mechanisms underpinning vertebrate dormancy.

Dramatic genome-wide reprogramming of mRNA in hypometabolic muscle.

In response to seasonal droughts, the green striped burrowing frog Cyclorana alboguttata enters a reversible hypometabolic state called aestivation where heart rate and oxygen consumption can be reduced despite warm (>25C°) ambient temperatures. With a view to understanding molecular mechanisms we profiled aestivating versus control gastrocnemius muscle using mRNA sequencing. This indicated an extensive metabolic reprogramming, with nearly a quarter of the entire transcriptome (3996 of 16,960 mRNA) exhibiting a nominal >2-fold change. Consistent with a physiological adaptation to spare carbohydrate reserves, carbohydrate catabolism was systemically downregulated. A 630-fold downregulation of ENO3 encoding the enolase enzyme was most striking. The 590 frog orthologs of mRNA encoding the mitoproteome were, viewed as a population, significantly downregulated during aestivation, although not to the same extent as mRNA encoding carbohydrate catabolism. Prominent examples include members of the TCA cycle (IDH2), electron transport chain (NDUFA6), the ATP synthase complex (ATP5F1B) and ADP/ATP intracellular transport (SLC25A4). Moreover, mRNA derived from the mt genome itself (e.g. mt-ND1) were also downregulated. Most prominent among the upregulated mRNA are those encoding aspects of regulated proteolysis including the proteosome (e.g. PSME4L), peptidases (USP25), atrogins (FBXO32) and ubiquitination (VCP). Finally, we note the ∼5-fold upregulation of the mRNA EIFG3 that encodes part of the EIF4F complex. This possesses global control of protein synthesis. Given protein synthesis is repressed in aestivating frogs this indicates the skeletal musculature is poised for accelerated translation of mRNA upon emergence, supporting a strategy to rapidly restore function when the summer rains come.

Canagliflozin regulates metabolic reprogramming in diabetic kidney disease by inducing fasting-like and aestivation-like metabolic patterns.

AIMS/HYPOTHESIS: Sodium-glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) are antihyperglycaemic drugs that protect the kidneys of individuals with type 2 diabetes mellitus. However, the underlying mechanisms mediating the renal benefits of SGLT2i are not fully understood. Considering the fuel switches that occur during therapeutic SGLT2 inhibition, we hypothesised that SGLT2i induce fasting-like and aestivation-like metabolic patterns, both of which contribute to the regulation of metabolic reprogramming in diabetic kidney disease (DKD). METHODS: Untargeted and targeted metabolomics assays were performed on plasma samples from participants with type 2 diabetes and kidney disease (n=35, 11 women) receiving canagliflozin (CANA) 100 mg/day at baseline and 12 week follow-up. Next, a systematic snapshot of the effect of CANA on key metabolites and pathways in the kidney was obtained using db/db mice. Moreover, the effects of glycine supplementation in db/db mice and human proximal tubular epithelial cells (human kidney-2 [HK-2]) cells were studied. RESULTS: Treatment of DKD patients with CANA for 12 weeks significantly reduced HbA1c from a median (interquartile range 25-75%) of 49.0 (44.0-57.0) mmol/mol (7.9%, [7.10-9.20%]) to 42.2 (39.7-47.7) mmol/mol (6.8%, [6.40-7.70%]), and reduced urinary albumin/creatinine ratio from 67.8 (45.9-159.0) mg/mmol to 47.0 (26.0-93.6) mg/mmol. The untargeted metabolomics assay showed downregulated glycolysis and upregulated fatty acid oxidation. The targeted metabolomics assay revealed significant upregulation of glycine. The kidneys of db/db mice undergo significant metabolic reprogramming, with changes in sugar, lipid and amino acid metabolism; CANA regulated the metabolic reprogramming in the kidneys of db/db mice. In particular, the pathways for glycine, serine and threonine metabolism, as well as the metabolite of glycine, were significantly upregulated in CANA-treated kidneys. Glycine supplementation ameliorated renal lesions in db/db mice by inhibiting food intake, improving insulin sensitivity and reducing blood glucose levels. Glycine supplementation improved apoptosis of human proximal tubule cells via the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. CONCLUSIONS/INTERPRETATION: In conclusion, our study shows that CANA ameliorates DKD by inducing fasting-like and aestivation-like metabolic patterns. Furthermore, DKD was ameliorated by glycine supplementation, and the beneficial effects of glycine were probably due to the activation of the AMPK/mTOR pathway.

Aestivation in Nature: Physiological Strategies and Evolutionary Adaptations in Hypometabolic States.

Aestivation is considered to be one of the "purest" hypometabolic states in nature, as it involves aerobic dormancy that can be induced and sustained without complex factors. Animals that undergo aestivation to protect themselves from environmental stressors such as high temperatures, droughts, and food shortages. However, this shift in body metabolism presents new challenges for survival, including oxidative stress upon awakening from aestivation, accumulation of toxic metabolites, changes in energy sources, adjustments to immune status, muscle atrophy due to prolonged immobility, and degeneration of internal organs due to prolonged food deprivation. In this review, we summarize the physiological and metabolic strategies, key regulatory factors, and networks utilized by aestivating animals to address the aforementioned components of aestivation. Furthermore, we present a comprehensive overview of the advancements made in aestivation research across major species, including amphibians, fish, reptiles, annelids, mollusks, and echinoderms, categorized according to their respective evolutionary positions. This approach offers a distinct perspective for comparative analysis, facilitating an understanding of the shared traits and unique features of aestivation across different groups of organisms.

Terrestrial Zen master: Transcriptomics in long-term aestivation and arousal of Mediterranean earthworms.

Earthworms have a crucial role in the maintenance of the biotic and abiotic soil properties, which is important for the biodiversity and productivity of terrestrial ecosystems, especially in the current scenario of climate change. Aestivation is a form of dormancy witnessed in organisms living in deserts or semiarid environments such as the ones found in the central part of the Iberian Peninsula. This work employs next-generation sequencing techniques to explore the changes in gene expression of different aestivation times (1 month and 1 year) as well as changes in gene expression upon arousal. Not surprisingly, the more the aestivation persisted the higher levels of gene downregulation were observed. Conversely, upon arousal, a quick recovery of the levels of gene expression were noted, comparable to the control. Transcriptional changes related to immune responses coming predominantly from abiotic stressors in aestivating earthworms and from biotic stressors in aroused earthworms triggered regulation of the cell fate via apoptosis. Long-term aestivation seemed to be enabled by remodeling of the extracellular matrix, activity of DNA repair mechanisms, and inhibitory neurotransmitters, which could also play a role in lifespan increase. Arousal from 1-month aestivation was on the other hand, characterized by regulation of the cell division cycle. Since aestivation is considered as an unfavorable metabolic state, aroused earthworms probably go through a damage removal process and a subsequent reparation process. This study provides the first transcriptomic investigation done on earthworms in such long aestivation times as well as arousal demonstrating the resilience and adaptability of Carpetania matritensis.

The impact of elevated aestivation temperatures on the behaviour of bogong moths (Agrotis infusa).

Bogong moths are an iconic Australian insect. They migrate annually in spring from low elevation locations in southern Australia to the Australian Alps where they aestivate during summer. As summer ends they make their return journey to the breeding grounds where they mate, lay eggs, and die. Given the moth's extreme behaviour in seeking out cool alpine habitat and with the knowledge that average temperatures at their aestivation sites are rising because of climate change, we first asked whether increased temperatures affect bogong moth activity during aestivation. We found that moth behaviour patterns changed from showing peaks at dawn and dusk with supressed activity during the day at cooler temperatures to near-constant activity at all times of day at 15 °C. Second, we asked whether moth mass changes after aestivating at different temperatures for a week due to dehydration or consumption of body energy reserves. We found that moth wet mass loss increased with increasing temperature, but found no difference in dry mass among temperature treatments. Overall, our results suggest that bogong moth aestivation behaviour changes with temperature and that it may be lost at around 15 °C. The impact of warming on the likelihood of individuals to complete their aestivation in the field should be investigated as a matter of priority to better understand the impact of climate change on the Australian alpine ecosystem.

Morpho-functional changes of lungfish Protopterus dolloi skin in the shift from freshwater to aestivating conditions.

African dipnoi (Protopterus sp.) are obligate air-breathing fish that, during dry season, may experience a period of dormancy named aestivation. Aestivation is characterized by complete reliance on pulmonary breathing, general decrease of metabolism and down-regulation of respiratory and cardiovascular functions. To date, little is known about morpho-functional rearrangements induced by aestivation in the skin of African lungfishes. Our study aims to identify, in the skin of P. dolloi, structural modifications and stress-induced molecules in response to short-term (6 days) and long-term (40 days) aestivation. Light microscopy showed that short-term aestivation induces major reorganization, with narrowing of epidermal layers and decrease of mucous cells; prolonged aestivation is characterized by regenerative processes and re-thickening of epidermal layers. Immunofluorescence reveals that aestivation correlates with an increased oxidative stress and changes of Heat Shock Proteins expression, suggesting a protective role for these chaperons. Our findings revealed that lungfish skin undergoes remarkable morphological and biochemical readjustments in response to stressful conditions associated with aestivation.

Cardiac microRNA expression profile in response to estivation.

Couch's spadefoot toad (Scaphiopus couchii) spends most of the year underground in a hypometabolic state known as estivation. During this time, they overcome significant dehydration and lack of food through many mechanisms including employing metabolic rate depression (MRD), increasing urea concentration, switching to lipid oxidation as the primary energy source, and decreasing their breathing and heart rate. MicroRNA (miRNA) are known to regulate translation by targeting messenger RNA (mRNA) for degradation or temporary storage, with several studies having reported that miRNA is differentially expressed during MRD, including estivation. Thus, we hypothesized that miRNA would be involved in gene regulation during estivation in S. couchii heart. Next-generation sequencing and bioinformatic analyses were used to assess changes in miRNA expression in response to two-month estivation and to predict the downstream effects of this expression. KEGG and GO analyses indicated that ribosome and cardiac muscle contraction are among the pathways predicted to be upregulated, whereas cell signaling and fatty acid metabolism were predicted to be downregulated. Together these results suggest that miRNAs contribute to the regulation of gene expression related to cardiac muscle physiology and energy metabolism during estivation.

Estivation and Postestivation Development of Hemlock Woolly Adelgid (Adelges tsugae) (Hemiptera: Adelgidae) at Different Temperatures.

Hemlock woolly adelgid (Adelges tsugae) is the most important pest of hemlocks in the eastern United States, where it completes three generations a year. We investigated the impact of temperature (8, 12, 16, and 20°C) on the estivation and postaestivation stages of the A. tsugae sistens generation. Temperature significantly impacted development and survival of this generation. The highest mortality occurred at the coolest temperature (8°C). Adelges tsugae developed rapidly as the temperature increased and optimum temperatures for development ranged between 17 and 22°C for the different instars. The estimated lower temperature threshold was 0°C for second instar nymphs and 3 -5°C for the other instars and the preoviposition period. Estivating first-instar sistentes resumed development (as evidenced by segments becoming visible) after 40-100 d at the constant temperatures (fastest at 16°C) then required only 105 degree-days (DD) for 50% of the individuals to molt. Subsequent instars developed rapidly (another 470 DD total to reach adult), and oviposition began at ~623 DD from the time the first instars resumed development. This study provides valuable data required to develop an annual phenology model for A. tsugae which will assist in timing monitoring and control treatments.

Isotopic evidence that aestivation allows malaria mosquitoes to persist through the dry season in the Sahel.

Data suggest that the malaria vector mosquito Anopheles coluzzii persists during the dry season in the Sahel through a dormancy mechanism known as aestivation; however, the contribution of aestivation compared with alternative strategies such as migration is unknown. Here we marked larval Anopheles mosquitoes in two Sahelian villages in Mali using deuterium (2H) to assess the contribution of aestivation to persistence of mosquitoes through the seven-month dry season. After an initial enrichment period, 33% of An. coluzzii mosquitoes were strongly marked. Seven months following enrichment, multiple analysis methods supported the ongoing presence of marked mosquitoes, compatible with the prediction that the fraction of marked mosquitoes should remain stable throughout the dry season if local aestivation is occurring. The results suggest that aestivation is a major persistence mechanism of An. coluzzii in the Sahel, contributing at least 20% of the adults at the onset of rains. This persistence strategy could influence mosquito control and malaria elimination campaigns.

Pro- and anti-apoptotic microRNAs are differentially regulated during estivation in Xenopus laevis.

Xenopus laevis, the African clawed frog, undergoes seasonal estivation to survive periods of drought when its lake-bed habitats dry up. The frog can lose ∼30% of its total body water, leading to conditions of impaired blood flow and ischemia which risk cellular survival under these harsh conditions. MicroRNAs are short, noncoding, single-stranded RNAs 21-24 nt long that have been widely implicated in hypometabolic responses, and serve functions including apoptosis survival. The levels of three pro-apoptotic and four anti-apoptotic miRNAs were measured in liver and skeletal muscle of estivating X. laevis, and bioinformatic analysis was performed to verify potential mRNA targets of these miRNAs. Members of pro-apoptotic miRNAs miR-15a, miR-16, and miR-101 showed upregulation as a result of dehydration stress, while anti-apoptotic miRNAs miR-19b, miR-21, miR-92a, and miR-155 showed differential regulation between the two tissues. Together, these miRNAs act in a more diverse fashion than arbitrarily pro- or anti-apoptotic, and encompass functions ranging from the inhibition of cell proliferation through cell cycle arrest to the prevention of skeletal muscle atrophy.

Genome-wide comparative analysis of DNAJ genes and their co-expression patterns with HSP70s in aestivation of the sea cucumber Apostichopus japonicus.

DNAJ proteins function as co-chaperones of HSP70 and play key roles in cell physiology to promote protein folding and degradation, especially under environmental stress. Based on our previous study on HSP70, a systematic study of DNAJ was performed in sea cucumber Apostichopus japonicus using the transcriptomic and genomic data, identifying 43 AjDNAJ genes, including six AjDNAJA genes, eight AjDNAJB genes, and 29 AjDNAJC genes. Slight expansion and conserved genomic structure were observed using the phylogenetic and syntenic analysis. Differential period-specific and tissue-specific expression patterns of AjDNAJs were observed between adult and juvenile individuals during aestivation. Strong tissue-specific expression correlations between AjDNAJ and AjHSP70 genes were found, indicating that the involvements of AjHSP70IVAs in the aestivation of sea cucumbers were regulated by AjDNAJs. Several key genes with significant expression correlations, such as AjDNAJB4L and AjHSP70IVAs, were suggested to function together under heat stress. Together, these findings provide early insight into the involvement of AjDNAJs in the aestivation and their roles as co-chaperones of AjHSP70s.

How to resist soil desiccation: Transcriptional changes in a Mediterranean earthworm during aestivation.

Earthworms have a central role in ministering the terrestrial ecosystems and are proving to have an important role in modulating the effects climate change has on soil. Aestivation is a form of dormancy employed by the organisms living in deserts and arid environments, when confronted with prolonged periods of drought. Understanding global metabolic adjustments required for withstanding the harsh conditions of the ever more severe Iberian drought, we performed a global transcriptomic exploration of the endogeic earthworm Carpetania matritensis during aestivation. There were a total of 6352 differentially expressed transcripts in the aestivating group, with 65% being downregulated. Based on GO and KEGG enrichment analyses, downregulated genes seem to be indicative of an overall metabolic depression during aestivation. Indeed we noted a reduction of protein turnover and macromolecule metabolism coupled with suppression of genes involved in digestion. Upregulated genes, namely antioxidant genes and DNA repair genes showed clear signs of abiotic stress caused by ROS generation. Abiotic stress led to transcriptomic changes of genes involved in immune response, mostly affecting the NF-kb signaling pathway as well as changes in apoptotic genes indicating the necessity of investigating these processes in a tissue specific manner. Lastly we uncovered a possible mechanism for water retention by nitrogenous waste accumulation. This study provides the first ever transcriptomic investigation done on aestivating earthworms and as such serves as a general framework for investigation on other earthworm species and other soil invertebrates, which is becoming increasingly important with the current scenario of climate change.

Seasonal cellular stress phenomena and phenotypic plasticity in land snail Helix lucorum populations from different altitudes.

Temperature, a major abiotic environmental factor, regulates various physiological functions in land snails and therefore determines their biogeographical distribution. Thus, species with different distributions may present different thermal tolerance limits. Additionally, the intense reactivation of snail metabolic rate upon arousal from hibernation or estivation may provoke stress. Land snails, Helix lucorum, display a wide altitudinal distribution resulting in populations being exposed to different seasonal temperature variations. The aim of the present study was to investigate the expression of heat shock proteins (Hsps), mitogen activated protein kinases (MAPKs) and proteins that are related to apoptosis (Bcl-2, ubiquitin), that have 'cytoprotective' roles and are also considered to be reliable indicators of stress because of their crucial role in maintaining cellular homeostasis. These proteins were assessed in H. lucorum individuals from two different populations, one at Axios (sea level, 0 m) and the other at Kokkinopilos (Olympus, 1250 m), as well as after mutual population exchanges, in order to find out whether the different responses of these stress-related proteins depend solely on the environmental temperature. The results showed seasonally altered levels in all studied proteins in the hepatopancreas and foot of snails, both among different populations and between the same populations exposed to varying altitudes. However, individuals of the same population in their native habitat or acclimatized to a different habitat showed a relatively similar pattern of expression, supporting the induction of the specific proteins according to the life history of each species.

Clustering genomic organization of sea cucumber miRNAs impacts their evolution and expression.

Echinoderms are marine deuterostomes with fascinating adaptation features such as aestivation and organ regeneration. However, post-transcriptional gene regulation by microRNAs (miRNAs) underlying these features are largely unexplored. Here, using homology-based and de novo approaches supported by expression data, we provided a comprehensive annotation of miRNA genes in the sea cucumber Apostichopus japonicus. By linkage and phylogenic analyses, we characterized miRNA genomic organization, evolutionary history and expression regulation. The results showed that sea cucumbers evolved a large number of new miRNAs, which tended to form polycistronic clusters via tandem duplication that had been especially active in the echinoderms. Most new miRNAs were weakly expressed, but miRNA clustering increased the expression level of clustered new miRNAs. The most abundantly expressed new miRNAs were organized in a single tandem cluster (cluster n2), which was activated during aestivation and intestine regeneration. Overall, our analyses suggest that clustering of miRNAs is important for their evolutionary origin, expression control, and functional cooperation.

mTOR Signaling in Metabolic Stress Adaptation.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular homeostasis that integrates environmental and nutrient signals to control cell growth and survival. Over the past two decades, extensive studies of mTOR have implicated the importance of this protein complex in regulating a broad range of metabolic functions, as well as its role in the progression of various human diseases. Recently, mTOR has emerged as a key signaling molecule in regulating animal entry into a hypometabolic state as a survival strategy in response to environmental stress. Here, we review current knowledge of the role that mTOR plays in contributing to natural hypometabolic states such as hibernation, estivation, hypoxia/anoxia tolerance, and dauer diapause. Studies across a diverse range of animal species reveal that mTOR exhibits unique regulatory patterns in an environmental stressor-dependent manner. We discuss how key signaling proteins within the mTOR signaling pathways are regulated in different animal models of stress, and describe how each of these regulations uniquely contribute to promoting animal survival in a hypometabolic state.

The amino acid and lipophilic profiles of Chilo partellus (Swinhoe) larvae fluctuate with diapause.

The Chilo partellus (Crambidae: Lepidoptera) larvae undergoes both hibernation and estivation in India. Although, much has been done on reproductive physiological aspects, little is known about biochemical changes happening during hibernation and estivation in C. partellus. Thus, we mapped changes in amino acid and lipophilic profiles of C. partellus larvae while undergoing hibernation and estivation using high-performance liquid chromatography and gas chromatography mass spectroscopy. The studies revealed higher amounts of amino acids namely, serine, glycine, histidine, arginine, proline, tyrosine, and methionine in estivation, while lower in hibernation as compared with nondiapause larvae of C. partellus. Furthermore, the amounts of aspartic acid, glutamic acid, and alanine in hibernation, and threonine, valine, isoleucine, phenylalanine, and leucine in estivation were on par with nondiapause larvae. The lipophilic compounds namely, linoleic acid, stearic acid, eicosanoic acid, and n-pentadecanol were lower in hibernation than estivation and nondiapause larvae of C. partellus. Palmitoleic acid and methyl 3-methoxytetradecanoate contents were higher in hibernation than estivation and nondiapause, while myristic acid and lathosterol contents were higher in estivation than hibernation and nondiapause larvae of C. partellus. Cholesterol content was higher, while squalene and gamma-ergostenol were lower in hibernation and estivation as compared with nondiapause larvae of C. partellus. These findings suggest that certain amino acids may be constituents of heat-shock proteins and help C. partellus during estivation. However, the lipophilic compounds could be helpful in maintaining development during hibernation and estivation in C. partellus.

Aestivation motifs explain hypertension and muscle mass loss in mice with psoriatic skin barrier defect.

AIM: Recent evidence suggests that arterial hypertension could be alternatively explained as a physiological adaptation response to water shortage, termed aestivation, which relies on complex multi-organ metabolic adjustments to prevent dehydration. Here, we tested the hypothesis that chronic water loss across diseased skin leads to similar adaptive water conservation responses as observed in experimental renal failure or high salt diet. METHODS: We studied mice with keratinocyte-specific overexpression of IL-17A which develop severe psoriasis-like skin disease. We measured transepidermal water loss and solute and water excretion in the urine. We quantified glomerular filtration rate (GFR) by intravital microscopy, and energy and nitrogen pathways by metabolomics. We measured skin blood flow and transepidermal water loss (TEWL) in conjunction with renal resistive indices and arterial blood pressure. RESULTS: Psoriatic animals lost large amounts of water across their defective cutaneous epithelial barrier. Metabolic adaptive water conservation included mobilization of nitrogen and energy from muscle to increase organic osmolyte production, solute-driven maximal anti-diuresis at normal GFR, increased metanephrine and angiotensin 2 levels, and cutaneous vasoconstriction to limit TEWL. Heat exposure led to cutaneous vasodilation and blood pressure normalization without parallel changes in renal resistive index, albeit at the expense of further increased TEWL. CONCLUSION: Severe cutaneous water loss predisposes psoriatic mice to lethal dehydration. In response to this dehydration stress, the mice activate aestivation-like water conservation motifs to maintain their body hydration status. The circulatory water conservation response explains their arterial hypertension. The nitrogen-dependency of the metabolic water conservation response explains their catabolic muscle wasting.