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Aphids harbor proteobacterial endosymbionts such as Buchnera aphidicola housed in specialized bacteriocytes derived from host cells. The endosymbiont Buchnera supplies essential amino acids such as arginine to the host cells and, in turn, obtains sugars needed for its survival from the hemolymph. The mechanism of sugar supply in aphid bacteriocytes has been rarely studied. It also remains unclear how Buchnera acquires its carbon source. The hemolymph sugars in Acyrthosiphon pisum are composed of the disaccharide trehalose containing two glucose molecules. Here, we report for the first time that trehalose is transported and used as a potential carbon source by Buchnera across the bacteriocyte plasma membrane via trehalose transporters. The current study characterized the bacteriocyte trehalose transporter Ap_ST11 (LOC100159441) using the Xenopus oocyte expression system. The Ap_ST11 transporter was found to be proton-dependent with a K

Glass transition behavior of carnosine (β-alanyl-L-histidine) and its impact on the storage stability of freeze-dried lactic acid bacteria (LAB) were investigated. Glass transition temperature of anhydrous carnosine was determined to be 99 oC. From the water sorption isotherm and glass transition temperature curve, the water activity where glass transition occurs at 25 oC (typical room temperature) was determined to be 0.28. These values for carnosine were higher than for sucrose, but lower than for trehalose. Effect of carnosine on the viability of freeze-dried LAB (Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus paracasei) before and after storage at 37 oC for 4 weeks was compared with that of other antioxidants (cysteine, glutathione, and ascorbic acid). Carnosine and cysteine strongly ameliorated reductions in cell viability after the storage. Carnosine showed the highest number of viable cells for L. reuteri and L. paracasei. The stabilizing mechanism of carnosine is thought to be attributed to water substitution, glass transition, antioxidation, and optimum pH.

African chironomid (

Significance Anhydrobiosis is a reversible ametabolic state that occurs in response to severe desiccation. The largest anhydrobiotic animal known is the larva of the African chironomid Polypedilum vanderplanki. Here, we investigated how the metabolism of larvae changes during the desiccation–rehydration cycle and how simple biochemical processes determine viability of the chironomid. Major findings suggest that, in addition to its known anhydroprotectant role, trehalose acts as a major source of energy for rehydration. Citrate and adenosine monophosphate, accumulated in the dry state, allow rapid resumption of metabolism during the recovery phase. Finally, metabolic waste is stored as stable or nontoxic compounds such as allantoin, xanthurenic acid, or ophthalmic acid that may also act as antioxidants., Some organisms have evolved a survival strategy to withstand severe dehydration in an ametabolic state, called anhydrobiosis. The only known example of anhydrobiosis among insects is observed in larvae of the chironomid Polypedilum vanderplanki. Recent studies have led to a better understanding of the molecular mechanisms underlying anhydrobiosis and the action of specific protective proteins. However, gene regulation alone cannot explain the rapid biochemical reactions and independent metabolic changes that are expected to sustain anhydrobiosis. For this reason, we conducted a comprehensive comparative metabolome–transcriptome analysis in the larvae. We showed that anhydrobiotic larvae adopt a unique metabolic strategy to cope with complete desiccation and, in particular, to allow recovery after rehydration. We argue that trehalose, previously known for its anhydroprotective properties, plays additional vital roles, providing both the principal source of energy and also the restoration of antioxidant potential via the pentose phosphate pathway during the early stages of rehydration. Thus, larval viability might be directly dependent on the total amount of carbohydrate (glycogen and trehalose). Furthermore, in the anhydrobiotic state, energy is stored as accumulated citrate and adenosine monophosphate, allowing rapid reactivation of the citric acid cycle and mitochondrial activity immediately after rehydration, before glycolysis is fully functional. Other specific adaptations to desiccation include potential antioxidants (e.g., ophthalmic acid) and measures to avoid the accumulation of toxic waste metabolites by converting these to stable and inert counterparts (e.g., xanthurenic acid and allantoin). Finally, we confirmed that these metabolic adaptations correlate with unique organization and expression of the corresponding enzyme genes.

We investigated experimentally whether a short peptide, PvLEA-22, which consists of two tandem repeats of an 11-mer motif of Group 3 late embryogenesis abundant proteins, has a chaperone-like function for denatured proteins. Lysozyme was selected as a target protein. Turbidity measurements indicated that the peptide suppresses the heat-induced aggregation of lysozyme when added at a molar ratio of PvLEA-22/lysozyme40. Circular dichroism and differential scanning calorimetry measurements confirmed that the lysozyme was denatured on heating but spontaneously refolded on subsequent cooling in the presence of the peptide. As a result, up to 80% of the native catalytic activity of lysozyme was preserved. Similar chaperone-like activity was also observed for a peptide with the same amino acid composition as PvLEA-22 but whose sequence is scrambled. To elucidate the underlying mechanism of the chaperone function of these peptides, we performed coarse-grained molecular dynamics simulations. This revealed that a denatured lysozyme molecule is shielded by several peptide molecules in aqueous solution, which acts as a physical barrier, reducing the opportunities for collision between denatured proteins. An important finding was that a peptide bound to the denatured protein is very rapidly replaced by another; due to such rapid exchange, peptide-protein contact time is very short, that is, on the order of ∼200 ns. Therefore, the peptide does not constrain the behavior of the denatured protein, which can refold freely.

Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a ‘sandbox’ for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin–calcineurin–NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin–calmodulin kinase–CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

Larvae of the African midge Polypedilum vanderplanki (Diptera: Chironomidae) show a form of extreme desiccation tolerance known as anhydrobiosis. The cell line Pv11 was recently established from the species, and these cells can also survive under desiccated conditions, and proliferate normally after rehydration. Here we report the identification of a new promoter, 121, which has strong constitutive transcriptional activity in Pv11 cells and promotes effective expression of exogenous genes. Using a luciferase reporter assay, this strong transcriptional activity was shown to be conserved in cell lines from various insect species, including S2 (Drosophila melanogaster, Diptera), SaPe-4 (Sarcophaga peregrina, Diptera), Sf9 (Spodoptera frugiperda, Lepidoptera) and Tc81 (Tribolium castaneum, Coleoptera) cells. In conjunction with an appropriate selection maker gene, the 121 promoter was able to confer zeocin resistance on SaPe-4 cells and allowed the establishment of stable SaPe-4 cell lines expressing the fluorescent protein AcGFP1; this is the first report of heterologous gene expression in this cell line. These results show the 121 promoter to be a versatile tool for exogenous gene expression in a wide range of insect cell lines, particularly useful to those from non-model insect species.

We tested whether a short model peptide derived from a group 3 late embryogenesis abundant (G3LEA) protein is able to maintain the fluorescence activity of a red fluorescent protein, mKate2, in the dry state. The fluorescence intensity of mKate2 alone decreased gradually through repeated dehydration-rehydration treatments. However, in the presence of the LEA model peptide, the peak intensity was maintained almost perfectly during such stress treatments, which implies that the three dimensional structure of the active site of mKate2 was protected even under severe desiccation conditions. For comparison, similar experiments were performed with other additives such as a native G3LEA protein, trehalose and BSA, all of whose protective abilities were lower than that of the LEA model peptide., Highlights • We prepared a 22-mer model peptide of a group-3 LEA protein. • The fluorescent peak of a red fluorescent protein was almost lost on drying. • The model peptide suppressed such desiccation-induced damage. • This indicates that the 3D structure of the fluorophore was protected. • The peptide exhibited the highest protective effect among the reagents tested.

Simple Summary Anhydrobiosis is a unique ametabolic state that is characterized by extreme body water loss due to desiccation, generally over 95%. The sleeping chironomid, Polypedilum vanderplanki, is an anhydrobiotic insect inhabiting semi-arid regions in Africa. Pv11 is an anhydrobiotic insect cell line established from the P. vanderplanki larvae, and the cell line also displays the extreme desiccation tolerance. This unique feature could be useful for developing a dry preservation technique. However, effective expression systems for protein production have not been available due to a limited number of experimental tools in Pv11 cells. To solve this problem, we identified a minimal promoter from the genome sequence of P. vanderplanki because promoters designed for model organisms are not available in Pv11 cells, although minimal promoters are commonly used for molecular biology experiments and biotechnology fields. The identification of the minimal promoter allowed us to develop an effectively inducible expression system in Pv11 cells. By using the system, several proteins including a functional protein with complex structures were successfully expressed in the cells. The successful establishment of the inducible system on Pv11 cells could contribute to bio-industrial protein production. Abstract The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin – calcineurin – NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin – calmodulin Kinase – CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose–induced Ca2+ surge to the anhydrobiosis and the temporal different roles of each signaling pathway.

The sleeping chironomidPolypedilum vanderplankiis capable of anhydrobiosis, a striking example of adaptation to extreme desiccation. Tolerance to complete desiccation in this species is associated with the emergence of multiple paralogs of protective genes. One of the gene families highly expressed under anhydrobiosis and involved in this process are protein-L-isoaspartate (D-aspartate) O-methyltransferases (PIMTs). Recently, a closely related anhydrobiotic midge from Malawi,P. pembai, showing the ability to tolerate complete desiccation similar to that ofP. vanderplanki, but experiences more frequent desiccation-rehydration cycles due to differences in ecology, was discovered. Here, we sequenced and assembled the genome ofP. pembaiand performed a population genomics analysis of several populations ofP. vanderplankiand a population ofP. pembai.We observe positive selection and radical changes in the genetic architecture of thePIMTlocus between the two species, including multiple duplication events in theP. pembailineage. In particular,PIMT-4, the most highly expressed of thesePIMTs, is present in six copies in theP. pembai; these copies differ in expression profiles, suggesting possible sub- or neofunctionalization. The nucleotide diversity (π) of the genomic region carrying these new genes is decreased inP. pembai, but not in the orthologous region carrying the ancestral gene inP. vanderplanki, providing evidence for a selective sweep associated with post-duplication adaptation in the former. Overall, our results suggest an extensive recent and likely ongoing, adaptation of the mechanisms of anhydrobiosis.

Larvae of the sleeping chironomid Polypedilum vanderplanki are known for their extraordinary ability to survive complete desiccation in an ametabolic state called “anhydrobiosis”. The unique feature of P. vanderplanki genome is the presence of expanded gene clusters associated with anhydrobiosis. While several such clusters represent orthologues of known genes, there is a distinct set of genes unique for P. vanderplanki. These include Lea-Island-Located (LIL) genes with no known orthologues except two of LEA genes of P. vanderplanki, PvLea1 and PvLea3. However, PvLIL proteins lack typical features of LEA such as the state of intrinsic disorder, hydrophilicity and characteristic LEA_4 motif. They possess four to five transmembrane domains each and we confirmed membrane targeting for three PvLILs. Conserved amino acids in PvLIL are located in transmembrane domains or nearby. PvLEA1 and PvLEA3 proteins are chimeras combining LEA-like parts and transmembrane domains, shared with PvLIL proteins. We have found that PvLil genes are highly upregulated during anhydrobiosis induction both in larvae of P. vanderplanki and P. vanderplanki-derived cultured cell line, Pv11. Thus, PvLil are a new intriguing group of genes that are likely to be associated with anhydrobiosis due to their common origin with some LEA genes and their induction during anhydrobiosis.

Anhydrobiosis, an adaptive ability to withstand complete desiccation, is in insects limited to a single species: the nonbiting midge Polypedilum vanderplanki (the sleeping chironomid). Evolution of anhydrobiosis in a single representative of a large genus is associated with drastic changes in genome structure, including the emergence of new multimember gene families directly involved in desiccation tolerance. Among them, Late Embryogenesis Abundant (LEA) proteins, which protect other proteins from aggregation caused by desiccation, are believed to originate via horizontal gene transfer from a bacterial donor. To obtain new insights on the biological background of the expanded 27-member LEA protein group in P. vanderplanki, we investigated the expression of corresponding genes in a P. vanderplanki-derived cell line, capable of anhydrobiosis, in a normal state and during induction of desiccation tolerance. We found that all LEA proteins genes identified in P. vanderplanki’s genome except PvLea16 and PvLea17 are also expressed in Pv11 cells. Their expression was elevated in response to anhydrobiosis-inducing trehalose treatment. Expression patterns of PvLea genes were well preserved in Pv11 cells in comparison to P. vanderplanki’s larvae both in the control group and during the anhydrobiosis cycle. We also investigated localization of LEA proteins in Pv11 cells and Sf9 cells and found a different level of conservation in intracellular localization of the protein expressed in mammalian and insect cells.

Simple Summary We found that incubation with a solution containing ~50 mM neodymium (one of the rare-earth elements, REE) induces a rapid of active metabolism in mammalian cells. We have named this state REEbernation and found that the process involves a rapid replacement of calcium with neodymium in membranes and organelles of a cell, allowing it to maintain its shape and membrane integrity under extreme conditions, including vacuum. Furthermore, phosphate exchange is blocked because of non-dissolvable neodymium salts formation, which “discharged” the cell. We also showed that REEbernation is characterized by instant shutting down RNA transcriptional activity in the cells, providing an intriguing opportunity to study a snapshot of gene expression at a given time point. Finally, we found that the REEbernation state is reversible, and we could restore the metabolism and proliferation capacity of the cells. The REEbernation provides a new method to reversibly place a cell into “on-hold” mode, opening opportunities to develop protocols for biological samples fixation with a minimum effect on the omics profile for biomedical needs. Abstract Until now, the ability to reversibly halt cellular processes has been limited to cryopreservation and several forms of anabiosis observed in living organisms. In this paper we show that incubation of living cells with a solution containing ~50 mM neodymium induces a rapid shutdown of intracellular organelle movement and all other evidence of active metabolism. We have named this state REEbernation (derived from the terms REE (rare earth elements) and hibernation) and found that the process involves a rapid replacement of calcium with neodymium in membranes and organelles of a cell, allowing it to maintain its shape and membrane integrity under extreme conditions, such as low pressure. Furthermore, phosphate exchange is blocked as a result of non-dissolvable neodymium salts formation, which “discharged” the cell. We further showed that REEbernation is characterized by an immediate cessation of transcriptional activity in observed cells, providing an intriguing opportunity to study a snapshot of gene expression at a given time point. Finally, we found that the REEbernation state is reversible, and we could restore the metabolism and proliferation capacity of the cells. The REEbernation, in addition to being an attractive model to further investigate the basic mechanisms of cell metabolism control, also provides a new method to reversibly place a cell into “on-hold” mode, opening opportunities to develop protocols for biological samples fixation with a minimum effect on the omics profile for biomedical needs.

The sleeping chironomid (Polypedilum vanderplanki Hinton) lives on temporary rock pools in the semi-arid tropical regions of Africa. Its larvae are able to survive the dry season in a completely desiccated ametabolic state known as anhydrobiosis. So far, P. vanderplanki was the only species among all insects showing demonstrated anhydrobiotic ability. Here, we show that a new related species originating from Malawi, Polypedilum pembai sp.n., is also anhydrobiotic and that its desiccation tolerance mechanism is probably similar to what is observed in P. vanderplanki. The new species, P. pembai sp.n., is described with special attention to the common and different morphological features, compared with P. vanderplanki. Phylogenetic analysis showed that both species are closely related, suggesting that anhydrobiosis evolved only once in their common ancestor about 49 Ma somewhere in Africa, before the divergence of two species, one in the sub-Saharan area and another in southeastern Africa.

Trehalose is a promising natural cryoprotectant, but its cryoprotective effect is limited due to difficulties in transmembrane transport. Thus, expressing the trehalose transporter TRET1 on various mammalian cells may yield more trehalose applications. In this study, we ran comparative cryopreservation experiments between the TRET1-expressing CHO-K1 cells (CHO-TRET1) and the CHO-K1 cells transfected with an empty vector (CHO-vector). The experiments involve freezing under various trehalose concentrations in an extracellular medium. The freeze-thawing viabilities of CHO-TRET1 cells are higher than those of CHO-vector cells for most freezing conditions. This result differs from control experiments with a transmembrane type cryoprotectant, dimethyl sulfoxide (Me2SO), which had similar viabilities in each condition for both cell types. We conclude that the trehalose loaded into the cells with TRET1 significantly improves the cryoprotective effect. The higher viabilities occurred when the extracellular trehalose concentration exceeded 200 mM, with 250-500 mM being optimal, and a cooling rate below 30 K/min, with 5-20 K/min being optimal.

Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic Polypedilum vanderplanki embryo endure complete desiccation because of their ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells preserved an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase, preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including pre-incubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pvll-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pvll-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation-tolerant related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

L-isoaspartyl O-methyltransferases (PIMTs) are known mostly because of their protein chain reparation activity in plant seeds, but also in many other organisms, from bacteria to mammals, including human. The main function of PIMT is recognition and methylation of spontaneously isomerized aspartate residue (Asp) in the amino acid sequences of cellular proteins, and thus preventing their conformation shifts and subsequent inactivation or loss of particular functions. The correlation between PIMT activity and accumulation of isomerized aspartate residues is well studied and used as a marker of isoAsp presence, whereas regulation pathways of PIMT genes are not clear yet. In our study, we propose a possible model for multiple paralogous PIMT gene regulation in the genome of an anhydrobiotic insect Polypedilum vanderplanki. Analysis of promoter regions revealed that PIMT genes possess enriched motif nearby +1 site, associated with GO term heat-shock polytene chromosome puffing. Poly(ADP-ribose) polymerase (PARP) is involved in this process of chromatin loosening and subsequent transcription activation of related genes. This result implied that expression of PIMT genes in P. vanderplanki could be regulated by chromatin modification.

Polipedilum vanderplanki is the most complex known organism able to survive body desiccation via entering a state of suspended metabolism called anhydrobiosis. This unique ability is based on the specific molecular machinery involving a synthesis of non-reducing sugar trehalose and a variety of protective proteins. Genes encoding these protective proteins are extensively duplicated in the P. vanderplanki genome and become hugely upregulated in response to desiccation. Some of these highly expressed genes encode substitutions of amino acids crucial for the function of corresponding proteins. An intriguing group of protective proteins in P. vanderplanki are thioredoxins (TRX). These antioxidant proteins are important for P. vanderplanki anhydrobiosis since desiccation is tightly related to the elevated production of free radicals and oxidative damage. The TRX set is unprecedentedly expanded in the P. vanderplanki genome up to 25 TRX genes. Genomes of congeneric midge Polipedilum nubifer, Apis mellifera, Drosophila melanogaster, and Anopheles gambiae encode only 3–7 TRX genes. Moreover, all three P. vanderplanki thioredoxin genes most expressed at 24 h of P. vanderplanki larva desiccation encode for proteins missing the typical CxxC motif.

Chinese hamster ovary cells (CHO–K1 cells) in which the trehalose transporter (TRET1) is expressed can have greater cryoprotection than ordinary CHO–K1 cells. This study examines the uptake characteristics of trehalose into cells via TRET1 and determines the influence of intracellular trehalose on the freeze–thaw viabilities. In our experiments, the intracellular trehalose concentration is controlled by the extracellular trehalose concentration and the immersion time in a freezing solution. In this freezing solution, both kinds of CHO–K1 cells are independently dispersed with various amount of trehalose, and then put into the CO2 incubator for 0–6 h. After a set immersion time, the cell-suspended sample is cooled to 193 K, stored for 1 week, then quickly thawed at 310 K and its viability measured. The uptake amount of intracellular trehalose is measured before freezing. We find an upper limit for the uptake amount of trehalose when the extracellular trehalose concentration is about 400 mM, at which the freeze–thaw viability is the highest. When the extracellular trehalose concentration exceeds 400 mM, shorter immersion times are needed to obtain the maximum freeze–thaw viability. Also, longer immersion weakens the cells. Our analyses indicate that when the extracellular trehalose-concentration is less than 400 mM, the trehalose uptake occurs more slowly with less dehydration, resulting in less stress on the cell. When the extracellular trehalose concentration exceeds the saturation level, the cell is stressed by the excess dehydration due to the remaining osmotic pressure, with apoptosis occurring before freezing.

One of the major damaging factors for living organisms experiencing water insufficiency is oxidative stress. Loss of water causes a dramatic increase in the production of reactive oxygen species (ROS). Thus, the ability for some organisms to survive almost complete desiccation (called anhydrobiosis) is tightly related to the ability to overcome extraordinary oxidative stress. The most complex anhydrobiotic organism known is the larva of the chironomid Polypedilum vanderplanki. Its antioxidant system shows remarkable features, such as an expansion of antioxidant genes, their overexpression, as well as the absence or low expression of enzymes required for the synthesis of ascorbate and glutathione and their antioxidant function. In this chapter, we summarize existing data about the antioxidant system of this insect, which is able to cope with substantial oxidative damage, even in an intracellular environment that is severely disturbed due to water loss.

Anhydrobiotic larvae of an African chironomid Polypedilum vanderplanki are known to be highly resistant to various abiotic stress factors, including ultraviolet radiation. The comparison of survival rates after different doses of UV irradiation between P. vanderplanki larvae and closely related non-anhydrobiotic Polypedilum nubifer larvae showed strongly enhanced resistance of P. vanderplanki to UV irradiation, especially in completely desiccated state. Plasmid-based assay showed an evidence of contribution of LEA4 protein to the protection of the larvae’s DNA against UV damage.

Water is essential for living organisms. Terrestrial organisms are incessantly exposed to the stress of losing water, desiccation stress. Avoiding the mortality caused by desiccation stress, many organisms acquired molecular mechanisms to tolerate desiccation. Larvae of the African midge, Polypedilum vanderplanki, and its embryonic cell line Pv11 tolerate desiccation stress by entering an ametabolic state, anhydrobiosis, and return to active life after rehydration. The genes related to desiccation tolerance have been comprehensively analyzed, but transcriptional regulatory mechanisms to induce these genes after desiccation or rehydration remain unclear. Here, we comprehensively analyzed the gene regulatory network in Pv11 cells and compared it with that of Drosophila melanogaster, a desiccation sensitive species. We demonstrated that nuclear transcription factor Y subunit gamma-like, which is important for drought stress tolerance in plants, and its transcriptional regulation of downstream positive feedback loops have a pivotal role in regulating various anhydrobiosis-related genes. This study provides an initial insight into the systemic mechanism of desiccation tolerance.

The larvae of the African midge, Polypedilum vanderplanki, can enter an ametabolic state called anhydrobiosis to conquer fatal desiccation stress. The Pv11 cell line, derived from embryos of the midge, shows desiccation tolerance by pretreatment with trehalose before desiccation; they can resume proliferation after rehydration. To address the underlying molecular mechanisms, we desiccated Pv11 cells after pretreatment with the medium containing trehalose and induced proliferation by rehydration. We collected the cells at each before and after desiccation and rehydration step and performed CAGE-seq of mRNA of those cells. By analysing differentially expressed genes (DEGs) among the results of CAGE-seq, we detected 384 DEGs after trehalose treatment and 14 DEGs after rehydration. Hierarchical clustering of the identified DEGs indicated that rehydration returns their expression pattern to that in the control culture state. DEGs involved in various stress responses, detoxification of harmful chemicals, and regulation of oxidoreduction were upregulated by trehalose treatment. DEGs for rehydration supported that DNA repair is one of the potential mechanisms involves recovery. This study provided initial insight into the molecular mechanisms underlying the extreme desiccation tolerance of Pv11 cells with a potential for proliferation following rehydration.

Significance Anhydrobiosis is an ametabolic state found in several organisms that can survive extreme desiccation. It is of practical interest because its application to other systems might allow room temperature preservation of cells, tissues, or organs in the dry state. The insect Polypedilum vanderplanki is the most complex animal that can enter anhydrobiosis. Proteins responsible for desiccation tolerance in P. vanderplanki are relatively well studied, but little is known about mechanisms underlying their induction during desiccation. Here, we show that the heat shock transcription factor regulatory network was coopted during the evolution of P. vanderplanki to activate many known desiccation-protective genes, including genes encoding late embryogenesis abundant (LEA) proteins., Polypedilum vanderplanki is a striking and unique example of an insect that can survive almost complete desiccation. Its genome and a set of dehydration–rehydration transcriptomes, together with the genome of Polypedilum nubifer (a congeneric desiccation-sensitive midge), were recently released. Here, using published and newly generated datasets reflecting detailed transcriptome changes during anhydrobiosis, as well as a developmental series, we show that the TCTAGAA DNA motif, which closely resembles the binding motif of the Drosophila melanogaster heat shock transcription activator (Hsf), is significantly enriched in the promoter regions of desiccation-induced genes in P. vanderplanki, such as genes encoding late embryogenesis abundant (LEA) proteins, thioredoxins, or trehalose metabolism-related genes, but not in P. nubifer. Unlike P. nubifer, P. vanderplanki has double TCTAGAA sites upstream of the Hsf gene itself, which is probably responsible for the stronger activation of Hsf in P. vanderplanki during desiccation compared with P. nubifer. To confirm the role of Hsf in desiccation-induced gene activation, we used the Pv11 cell line, derived from P. vanderplanki embryo. After preincubation with trehalose, Pv11 cells can enter anhydrobiosis and survive desiccation. We showed that Hsf knockdown suppresses trehalose-induced activation of multiple predicted Hsf targets (including P. vanderplanki-specific LEA protein genes) and reduces the desiccation survival rate of Pv11 cells fivefold. Thus, cooption of the heat shock regulatory system has been an important evolutionary mechanism for adaptation to desiccation in P. vanderplanki.

Polypedilum vanderplanki is a striking and unique example of an insect that can survive almost complete desiccation. Its genome and a set of dehydration-rehydration transcriptomes, together with the genome of Polypedilum nubifer (a congeneric desiccation-sensitive midge), were recently released. Here, using published and newly generated datasets reflecting detailed transcriptome changes during anhydrobiosis, as well as a developmental series, we show that the TCTAGAA DNA motif, which closely resembles the binding motif of the Drosophila melanogaster heat shock transcription activator (Hsf), is significantly enriched in the promoter regions of desiccation-induced genes in P. vanderplanki, such as genes encoding late embryogenesis abundant (LEA) proteins, thioredoxins, or trehalose metabolism-related genes, but not in P. nubifer Unlike P. nubifer, P. vanderplanki has double TCTAGAA sites upstream of the Hsf gene itself, which is probably responsible for the stronger activation of Hsf in P. vanderplanki during desiccation compared with P. nubifer To confirm the role of Hsf in desiccation-induced gene activation, we used the Pv11 cell line, derived from P. vanderplanki embryo. After preincubation with trehalose, Pv11 cells can enter anhydrobiosis and survive desiccation. We showed that Hsf knockdown suppresses trehalose-induced activation of multiple predicted Hsf targets (including P. vanderplanki-specific LEA protein genes) and reduces the desiccation survival rate of Pv11 cells fivefold. Thus, cooption of the heat shock regulatory system has been an important evolutionary mechanism for adaptation to desiccation in P. vanderplanki.

One of the major damaging factors for living organisms experiencing water insufficiency is oxidative stress. Loss of water causes a dramatic increase in the production of reactive oxygen species (ROS). Thus, the ability for some organisms to survive almost complete desiccation (called anhydrobiosis) is tightly related to the ability to overcome extraordinary oxidative stress. The most complex anhydrobiotic organism known is the larva of the chironomid Polypedilum vanderplanki. Its antioxidant system shows remarkable features, such as an expansion of antioxidant genes, their overexpression, as well as the absence or low expression of enzymes required for the synthesis of ascorbate and glutathione and their antioxidant function. In this chapter, we summarize existing data about the antioxidant system of this insect, which is able to cope with substantial oxidative damage, even in an intracellular environment that is severely disturbed due to water loss.

The anhydrobiosis is a unique state notable for a complete lack of detectable metabolic activity and an ability to withstand extreme stresses. It is induced, as its name suggests, by severe water loss, extending up to complete desiccation. Despite of severe stress, organisms entering anhydrobiosis normally retain their viability. This phenomena is ensured by a variety of protective mechanisms, including expression of protective proteins. The latter include Heat shock proteins (Hsp), which have long been recognized for their importance in a wide range of stress protection mechanisms. This chapter summarizes the theory and available experimental data, both suggesting the importance of Hsp in invertebrate anhydrobiosis. However, most of experimental data are the results of expression studies. We show that they are insufficient to make robust conclusions on the role of Hsp in anhydrobiosis. To date, only two robust evidences based on loss of-function-experiments are available, leaving for the future research the complete elucidation of Hsp function in anhydrobiosis.