Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.

Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.

Liquid-liquid phase separation promotes animal desiccation tolerance.

Proteinaceous liquid-liquid phase separation (LLPS) occurs when a polypeptide coalesces into a dense phase to form a liquid droplet (i.e., condensate) in aqueous solution. In vivo, functional protein-based condensates are often referred to as membraneless organelles (MLOs), which have roles in cellular processes ranging from stress responses to regulation of gene expression. Late embryogenesis abundant (LEA) proteins containing seed maturation protein domains (SMP; PF04927) have been linked to storage tolerance of orthodox seeds. The mechanism by which anhydrobiotic longevity is improved is unknown. Interestingly, the brine shrimp Artemia franciscana is the only animal known to express such a protein (AfrLEA6) in its anhydrobiotic embryos. Ectopic expression of AfrLEA6 (AWM11684) in insect cells improves their desiccation tolerance and a fraction of the protein is sequestered into MLOs, while aqueous AfrLEA6 raises the viscosity of the cytoplasm. LLPS of AfrLEA6 is driven by the SMP domain, while the size of formed MLOs is regulated by a domain predicted to engage in protein binding. AfrLEA6 condensates formed in vitro selectively incorporate target proteins based on their surface charge, while cytoplasmic MLOs formed in AfrLEA6-transfected insect cells behave like stress granules. We suggest that AfrLEA6 promotes desiccation tolerance by engaging in two distinct molecular mechanisms: by raising cytoplasmic viscosity at even modest levels of water loss to promote cell integrity during drying and by forming condensates that may act as protective compartments for desiccation-sensitive proteins. Identifying and understanding the molecular mechanisms that govern anhydrobiosis will lead to significant advancements in preserving biological samples.

Differential expression profiling of heat stressed tardigrades reveals major shift in the transcriptome.

Tardigrades are renowned for their extreme stress tolerance, which includes the ability to endure complete desiccation, high levels of radiation and very low sub-zero temperatures. Nevertheless, tardigrades appear to be vulnerable to high temperatures and thus the potential effects of global warming. Here, we provide the first analysis of transcriptome data obtained from heat stressed specimens of the eutardigrade Ramazzottius varieornatus, with the aim of providing new insights into the molecular processes affected by high temperatures. Specifically, we compare RNA-seq datasets obtained from active, heat-exposed (35 °C) tardigrades to that of active controls kept at 5 °C. Our data reveal a surprising shift in transcription, involving 9634 differentially expressed transcripts, corresponding to >35% of the transcriptome. The latter data are in striking contrast to the hitherto observed constitutive expression underlying tardigrade extreme stress tolerance and entrance into the latent state of life, known as cryptobiosis. Thus, when examining the molecular response, heat-stress appears to be more stressful for R. varieornatus than extreme conditions, such as desiccation or freezing. A gene ontology analysis reveals that the heat stress response involves a change in transcription and presumably translation, including an adjustment of metabolism, and, putatively, preparation for encystment and subsequent diapause. Among the differentially expressed transcripts we find heat-shock proteins as well as the eutardigrade specific proteins (CAHS, SAHS, MAHS, RvLEAM, and Dsup). The latter proteins thus seem to contribute to a general stress response, and may not be directly related to cryptobiosis.

Interstellar space biology via Project Starlight.

Our ability to explore the cosmos by direct contact has been limited to a small number of lunar and interplanetary missions. However, the NASA Starlight program points a path forward to send small, relativistic spacecraft far outside our solar system via standoff directed-energy propulsion. These miniaturized spacecraft are capable of robotic exploration but can also transport seeds and organisms, marking a profound change in our ability to both characterize and expand the reach of known life. Here we explore the biological and technological challenges of interstellar space biology, focusing on radiation-tolerant microorganisms capable of cryptobiosis. Additionally, we discuss planetary protection concerns and other ethical considerations of sending life to the stars.

Tardigrada: An Emerging Animal Model to Study the Endoplasmic Reticulum Stress Response to Environmental Extremes.

Tardigrada (also known as "water bears") are hydrophilous microinvertebrates with a bilaterally symmetrical body and four pairs of legs usually terminating with claws. Water bears are quite complex animals and range from 50 to 1200 µm in length. Their body is divided into a head segment and four trunk segments, each bearing a pair of legs. They inhabit almost all terrestrial and aquatic environments, from the ocean depths to highest mountains ranges. However, one of their best known and unusual features is their capability for cryptobiosis. In this state tardigrades are able to survive extremely low and high temperatures and atmospheric pressures, complete lack of water, high doses of radiation, high concentrations of toxins and even a cosmic vacuum. The cellular mechanisms enabling cryptobiosis are poorly understood, although it appears the synthesis of certain types of molecules (sugars and proteins) enable the prevention of cellular damage at different levels. The endoplasmic reticulum (ER) is a morphologically and functionally diverse organelle able to integrate multiple extracellular and internal signals and generate adaptive cellular responses. However, the ER morphology and activity in the case of tardigrades has been studied rarely and in the context of oogenesis, functioning of the digestive system, and in the role and function of storage cells. Thus, there are no direct studies on the contribution of the ER in the ability of this organism to cope with environmental stress during cryptobiosis. Nevertheless, it is highly probable that the ER has a crucial role in this uncommon process. Since water bears are easy to handle laboratory animals, they may represent an ideal model organism to uncover the important role of the ER in the cell response to extreme environmental stress conditions.

New insights into survival strategies of tardigrades.

Life is set within a narrow frame of physicochemical factors, yet, some species have adapted to conditions far beyond these constraints. Nature appears to have evolved two principal strategies for living organisms to cope with hostile conditions. One way is to remain active, retaining metabolism through adaptations that enable the organism to match the physiological requirements of environmental change. The other is to enter a state of dormancy with metabolic suppression. One form of metabolic suppression, known as cryptobiosis, is a widespread state across life kingdoms, in which metabolism comes to a reversible standstill. Among animals, nematodes, rotifers and tardigrades, comprise species that have the ability to enter cryptobiosis at all stages of their life cycle. Tardigrades are microscopic cosmopolitan metazoans found in permanent and temporal aquatic environments. They are renowned for their ability to tolerate extreme stress and are particularly resistant after having entered a cryptobiotic state known as a "tun". As new molecular tools allow for a more detailed investigation into their enigmatic adaptations, tardigrades are gaining increasing attention. In this graphical review, we provide an outline of survival strategies found among tardigrades and we summarize current knowledge of the adaptive mechanisms that underlie their unique tolerance to extreme or changing environments.

Role of Intrinsic Disorder in Animal Desiccation Tolerance.

This review compares the molecular strategies employed by anhydrobiotic invertebrates to survive extreme water stress. Intrinsically disordered proteins (IDPs) play a central role in desiccation tolerance in all species investigated. Various hypotheses about the functions of anhydrobiosis-related intrinsically disordered (ARID) proteins, including late embryogenesis abundant (LEA) and tardigrade-specific intrinsically disordered proteins, are evaluated by broad sequence characterization. A surprisingly wide range in sequence characteristics, including hydropathy and the frequency and distribution of charges, is discovered. Interestingly, two clusters of similar proteins are found that potentially correlate with distinct functions. This may indicate two broad groups of ARID proteins, composed of one group that folds into functional conformations during desiccation and a second group that potentially displays functions in the hydrated state. A broad range of physiochemical properties suggest that folding may be induced by factors such as hydration level, molecular crowding, and interactions with binding partners. This plasticity may be required to fine-tune the ARID-proteome response at different hydration levels during desiccation. Furthermore, the sequence properties of some LEA proteins share qualities with IDPs known to undergo liquid-liquid phase separations during environmental challenges.

Hit pause: Developmental arrest in annual killifishes and their close relatives.

Killifishes survive and persist in extreme environments by exploiting both aquatic and terrestrial habitats for egg deposition, and by adjusting the length of development to match availability of water to support larval growth and maturation. Annual killifishes persist in ephemeral bodies of water through the production of drought-tolerant embryos. Survival of the environmental stresses associated with their highly variable and seasonal habitat is supported by their ability to enter into at least two states of metabolic and developmental dormancy, diapause or quiescence. There are three stages of diapause in annual killifishes, one occurring prior to gastrulation, one about midway through development, and one in late pre-hatching embryos. Quiescence may occur at any developmental stage. In addition, delayed hatching is known to occur in close relatives of the annual killifishes, and may be superficially confused with pre-hatching diapause. These types of developmental delay are induced by different cues and serve different purposes in the life history of the species. Thus, it is likely that the molecular mechanisms that induce dormancy and support survival are unique in each case. It is imperative that we properly define these forms of developmental dormancy in our studies in order to put our results into the proper ecological and evolutionary context. Here the unique characteristics of these distinct categories of developmental delay are reviewed. Developmental Dynamics 246:858-866, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Recovery and reproduction of an Antarctic tardigrade retrieved from a moss sample frozen for over 30 years.

Long-term survival has been one of the most studied of the extraordinary physiological characteristics of cryptobiosis in micrometazoans such as nematodes, tardigrades and rotifers. In the available studies of long-term survival of micrometazoans, instances of survival have been the primary observation, and recovery conditions of animals or subsequent reproduction are generally not reported. We therefore documented recovery conditions and reproduction immediately following revival of tardigrades retrieved from a frozen moss sample collected in Antarctica in 1983 and stored at -20 °C for 30.5 years. We recorded recovery of two individuals and development of a separate egg of the Antarctic tardigrade, Acutuncus antarcticus, providing the longest records of survival for tardigrades as animals or eggs. One of the two resuscitated individuals and the hatchling successfully reproduced repeatedly after their recovery from long-term cryptobiosis. This considerable extension of the known length of long-term survival of tardigrades recorded in our study is interpreted as being associated with the minimum oxidative damage likely to have resulted from storage under stable frozen conditions. The long recovery times of the revived tardigrades observed is suggestive of the requirement for repair of damage accrued over 30 years of cryptobiosis. Further more detailed studies will improve understanding of mechanisms and conditions underlying the long-term survival of cryptobiotic organisms.

Identification of differentially expressed water-insoluble proteins in the encystment process of Colpoda cucullus by two-dimensional electrophoresis and LC-MS/MS analysis.

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; ß-tubulin) was enhanced.

Fluorescence shadow imaging of Hypsibius exemplaris reveals morphological differences between sucrose- and CaCl2-induced osmobiotes.

Tardigrades are renowned for their ability to survive a wide array of environmental stressors. In particular, tardigrades can curl in on themselves while losing a significant proportion of their internal water content to form a structure referred to as a tun. In surviving varying conditions, tardigrades undergo distinct morphological transformations that could indicate different mechanisms of stress sensing and tolerance specific to the stress condition. Methods to effectively distinguish between morphological transformations, including between tuns induced by different stress conditions, are lacking. Herein, an approach for discriminating between tardigrade morphological states is developed and utilized to compare sucrose- and CaCl2-induced tuns, using the model species Hypsibius exemplaris. A novel approach of shadow imaging with confocal laser scanning microscopy enabled production of three-dimensional renderings of Hys. exemplaris in various physiological states resulting in volume measurements. Combining these measurements with qualitative morphological analysis using scanning electron microscopy revealed that sucrose- and CaCl2-induced tuns have distinct morphologies, including differences in the amount of water expelled during tun formation. Further, varying the concentration of the applied stressor did not affect the amount of water lost, pointing towards water expulsion by Hys. exemplaris being a controlled process that is adapted to the specific stressors.

The Evolution of Temperature and Desiccation-Related Protein Families in Tardigrada Reveals a Complex Acquisition of Extremotolerance.

Tardigrada is an ecdysozoan lineage famed for its resilience. Tardigrades can tolerate high doses of radiation, low-oxygen environments, desiccation, and both high and low temperatures under a dormant state called "anhydrobiosis", which is a reversible halt of metabolism upon almost complete desiccation. A large amount of research has focused on the genetic pathways related to these capabilities, and a number of genes have been identified and linked to the extremotolerant response of tardigrades. However, the history of these genes is unclear, and the origins and history of extremotolerant genes within Tardigrada remain a mystery. Here, we generate the first phylogenies of six separate protein families linked with desiccation and radiation tolerance in Tardigrada: cytosolic abundant heat-soluble protein, mitochondrial abundant heat-soluble protein, secretory abundant heat-soluble protein, meiotic recombination 11 homolog, and the newly discovered Echiniscus testudo abundant heat-soluble proteins (alpha and beta). The high number of independent gene duplications found amongst the six gene families studied suggests that tardigrades have a complex history with numerous independent adaptations to cope with aridity within the limnoterrestrial environment. Our results suggest that tardigrades likely transitioned from a marine environment to a limnoterrestrial environment only twice, once in stem Eutardigrada and once in Heterotardigrada, which explains the unique adaptations to anhydrobiosis present in both classes.

The major inducible small heat shock protein HSP20-3 in the tardigrade Ramazzottius varieornatus forms filament-like structures and is an active chaperone.

The tardigrade Ramazzottius varieornatus has remarkable resilience to a range of environmental stresses. In this study, we have characterised two members of the small heat shock protein (sHSP) family in R. varieornatus, HSP20-3 and HSP20-6. These are the most highly upregulated sHSPs in response to a 24 h heat shock at 35 0C of adult tardigrades with HSP20-3 being one of the most highly upregulated gene in the whole transcriptome. Both R. varieornatus sHSPs and the human sHSP, CRYAB (HSPB5), were produced recombinantly for comparative structure-function studies. HSP20-3 exhibited a superior chaperone activity than human CRYAB in a heat-induced protein aggregation assay. Both tardigrade sHSPs also formed larger oligomers than CRYAB as assessed by size exclusion chromatography and transmission electron microscopy of negatively stained samples. Whilst both HSP20-3 and HSP20-6 formed particles that were variable in size and larger than the particles formed by CRYAB, only HSP20-3 formed filament-like structures. The particles and filament-like structures formed by HSP20-3 appear inter-related as the filament-like structures often had particles located at their ends. Sequence analyses identified two unique features; an insertion in the middle region of the N-terminal domain (NTD) and preceding the critical-sequence identified in CRYAB, as well as a repeated QNTN-motif located in the C-terminal domain of HSP20-3. The NTD insertion is expected to affect protein-protein interactions and subunit oligomerisation. Removal of the repeated QNTN-motif abolished HSP20-3 chaperone activity and also affected the assembly of the filament-like structures. We discuss the potential contribution of HSP20-3 to protein condensate formation.

New insights into osmobiosis and chemobiosis in tardigrades.

Tardigrades are renowned for their ability to enter the extremotolerant state of latent life known as cryptobiosis. While it is widely accepted that cryptobiosis can be induced by freezing (cryobiosis) and by desiccation (anhydrobiosis), the latter involving formation of a so-called tun, the exact mechanisms underlying the state-as well as the significance of other cryptobiosis inducing factors-remain ambiguous. Here, we focus on osmotic and chemical stress tolerance in the marine tidal tardigrade Echiniscoides sigismundi. We show that E. sigismundi enters the tun state following exposure to saturated seawater and upon exposure to locality seawater containing the mitochondrial uncoupler DNP. The latter experiments provide evidence of osmobiosis and chemobiosis, i.e., cryptobiosis induced by high levels of osmolytes and toxicants, respectively. A small decrease in survival was observed following simultaneous exposure to DNP and saturated seawater indicating that the tardigrades may not be entirely ametabolic while in the osmobiotic tun. The tardigrades easily handle exposure to ultrapure water, but hypo-osmotic shock impairs tun formation and when exposed to ultrapure water the tardigrades do not tolerate DNP, indicating that tolerance towards dilute solutions involves energy-consuming processes. We discuss our data in relation to earlier and more contemporary studies on cryptobiosis and we argue that osmobiosis should be defined as a state of cryptobiosis induced by high external osmotic pressure. Our investigation supports the hypothesis that the mechanisms underlying osmobiosis and anhydrobiosis are overlapping and that osmobiosis likely represents the evolutionary forerunner of cryptobiosis forms that involve body water deprivation.

Preliminary investigation of effects of copper on a terrestrial population of the antarctic rotifer Philodina sp.

Terrestrial microinvertebrates in Antarctica are potentially exposed to contaminants due to the concentration of human activity on ice-free areas of the continent. As such, knowledge of the response of Antarctic microinvertebrates to contaminants is important to determine the extent of anthropogenic impacts. Antarctic Philodina sp. were extracted from soils and mosses at Casey station, East Antarctica and exposed to aqueous Cu for 96 h. The Philodina sp. was sensitive to excess Cu, with concentrations of 36 µg L-1 Cu (48 h) and 24 µg L-1 Cu (96 h) inhibiting activity by 50%. This is the first study to be published describing the ecotoxicologically derived sensitivity of a rotifer from a terrestrial population to metals, and an Antarctic rotifer to contaminants. It is also the first study to utilise bdelloid rotifer cryptobiosis (chemobiosis) as a sublethal ecotoxicological endpoint. This preliminary investigation highlights the need for further research into the responses of terrestrial Antarctic microinvertebrates to contaminants.

Examples of Extreme Survival: Tardigrade Genomics and Molecular Anhydrobiology.

Tardigrades are ubiquitous meiofauna that are especially renowned for their exceptional extremotolerance to various adverse environments, including pressure, temperature, and even ionizing radiation. This is achieved through a reversible halt of metabolism triggered by desiccation, a phenomenon called anhydrobiosis. Recent establishment of genome resources for two tardigrades, Hypsibius exemplaris and Ramazzottius varieornatus, accelerated research to uncover the molecular mechanisms behind anhydrobiosis, leading to the discovery of many tardigrade-unique proteins. This review focuses on the history, methods, discoveries, and current state and challenges regarding tardigrade genomics, with an emphasis on molecular anhydrobiology. Remaining questions and future perspectives regarding prospective approaches to fully elucidate the molecular machinery of this complex phenomenon are discussed.

Functional and Conformational Plasticity of an Animal Group 1 LEA Protein.

Group 1 (Dur-19, PF00477, LEA_5) Late Embryogenesis Abundant (LEA) proteins are present in organisms from all three domains of life, Archaea, Bacteria, and Eukarya. Surprisingly, Artemia is the only genus known to include animals that express group 1 LEA proteins in their desiccation-tolerant life-history stages. Bioinformatics analysis of circular dichroism data indicates that the group 1 LEA protein AfLEA1 is surprisingly ordered in the hydrated state and undergoes during desiccation one of the most pronounced disorder-to-order transitions described for LEA proteins from A. franciscana. The secondary structure in the hydrated state is dominated by random coils (42%) and ß-sheets (35%) but converts to predominately α-helices (85%) when desiccated. Interestingly, AfLEA1 interacts with other proteins and nucleic acids, and RNA promotes liquid-liquid phase separation (LLPS) of the protein from the solvent during dehydration in vitro. Furthermore, AfLEA1 protects the enzyme lactate dehydrogenase (LDH) during desiccation but does not aid in restoring LDH activity after desiccation-induced inactivation. Ectopically expressed in D. melanogaster Kc167 cells, AfLEA1 localizes predominantly to the cytosol and increases the cytosolic viscosity during desiccation compared to untransfected control cells. Furthermore, the protein formed small biomolecular condensates in the cytoplasm of about 38% of Kc167 cells. These findings provide additional evidence for the hypothesis that the formation of biomolecular condensates to promote water stress tolerance during anhydrobiosis may be a shared feature across several groups of LEA proteins that display LLPS behaviors.

Diversity in Expression Biases of Lineage-Specific Genes During Development and Anhydrobiosis Among Tardigrade Species.

Lineage-specific genes can contribute to the emergence and evolution of novel traits and adaptations. Tardigrades are animals that have adapted to tolerate extreme conditions by undergoing a form of cryptobiosis called anhydrobiosis, a physical transformation to an inactive desiccated state. While studies to understand the genetics underlying the interspecies diversity in anhydrobiotic transitions have identified tardigrade-specific genes and family expansions involved in this process, the contributions of species-specific genes to the variation in tardigrade development and cryptobiosis are less clear. We used previously published transcriptomes throughout development and anhydrobiosis (5 embryonic stages, 7 juvenile stages, active adults, and tun adults) to assess the transcriptional biases of different classes of genes between 2 tardigrade species, Hypsibius exemplaris and Ramazzottius varieornatus. We also used the transcriptomes of 2 other tardigrades, Echiniscoides sigismundi and Richtersius coronifer, and data from 3 non-tardigrade species (Adenita vaga, Drosophila melanogaster, and Caenorhabditis elegans) to help identify lineage-specific genes. We found that lineage-specific genes have generally low and narrow expression but are enriched among biased genes in different stages of development depending on the species. Biased genes tend to be specific to early and late development, but there is little overlap in functional enrichment of biased genes between species. Gene expansions in the 2 tardigrades also involve families with different functions despite homologous genes being expressed during anhydrobiosis in both species. Our results demonstrate the interspecific variation in transcriptional contributions and biases of lineage-specific genes during development and anhydrobiosis in 2 tardigrades.

Tolerance to Anhydrobiotic Conditions Among Two Coexisting Tardigrade Species Differing in Life Strategies.

Water availability is one of the most important factors for terrestrial life. Terrestrial habitats may periodically become dry, which can be overcome by an organism's capability to undergo anhydrobiosis. In animals, this phenomenon has been reported for invertebrates, with tardigrades being the best-known. However, different tardigrade species appear to significantly differ in their anhydrobiotic abilities. While several studies have addressed this issue, established experimental protocols for tardigrade dehydration differ both within and among species, leading to ambiguous results. Therefore, we apply unified conditions to estimate intra-and interspecies differences in anhydrobiosis ability reflected by the return to active life. We analysed Milnesium inceptum and Ramazzottius subanomalus representing predatory and herbivorous species, respectively, and often co-occur in the same habitat. The results indicated that the carnivorous Mil. inceptum displays better anhydrobiosis survivability than the herbivorous Ram. subanomalus. This tendency to some degree coincides with the time of "waking up" since Mil. inceptum showed first movements and full activity of any first individual later than Ram. subanomalus. The movements of all individuals were however observed to be faster for Mil. inceptum. Differences between the experimental groups varying in anhydrobiosis length were also observed: the longer tun state duration, the more time was necessary to return to activity.

Third-Stage Dispersal Juveniles of Bursaphelenchus xylophilus Can Resist Low-Temperature Stress by Entering Cryptobiosis.

Nematodes can enter cryptobiosis by dehydration as an adaptation to low-temperature environments and recover from cryptobiosis by rehydration after environmental improvement. In this work, the survival of Bursaphelenchusxylophilus third-stage dispersal juveniles was studied in response to low-temperature treatment. The average survival rates were 1.7% after -80 °C treatment for 30 d and 82.2% after -20 °C treatment for 30 d. The changes of water content and inorganic salt ions that occur in pine trees during winter gradually alter the osmotic pressure in the liquid environment to dehydrate B. xylophilus juveniles, resulting in improved survival after low-temperature treatment. The survival rate at -20 °C improved to 92.1% when the juveniles entered cryptobiosis by osmotic regulation. The results of this study demonstrate that B. xylophilus third-stage dispersal juveniles can resist low-temperature stress through cryptobiosis, providing the theoretical basis for the identification of areas potentially vulnerable to B. xylophilus in the mid-temperature and cold temperature zones of China.