Research Interests: Engineering, Genetics, Entomology, Chemistry, Animal Behavior, and 15 moreComputational Biology, Fluorescence Microscopy, Biology, Developmental neuroscience, Medicine, Gene expression, Behavioral Neuroscience, Biological Sciences, Bees, Brain, Anatomy and Physiology, Animals, Cells, Honeybee, and Arrestin
Figure 1. Graphical experimental scheme of the desiccation assay. In total, 29 or 30 tardigrades were dropped onto the filter paper and placed in the sealed desiccation chamber. The humidity in the chamber was controlled by the presence... more
Figure 1. Graphical experimental scheme of the desiccation assay. In total, 29 or 30 tardigrades were dropped onto the filter paper and placed in the sealed desiccation chamber. The humidity in the chamber was controlled by the presence of saturated salt solution or water, which had no contact with the animals.
Supplementary Text, Figures, and Tables
Amino acid sequences of <i>Paramacrobiotus</i> sp. TYO trehalase genes.
Table S2. Gene loss in stress-response regulatory pathways in tardigrades (in Hara et al., 2021)
Amino acid sequences of <i>Paramacrobiotus</i> sp. TYO TPS-TPP genes.
Dataset S6. The Bayesian tree of the TPP gene in newick format (in Hara et al., 2021)
A nucleotide sequence of <i>Paramacrobiotus</i> sp. TYO 18S rRNA
Coding nucleotide sequences of the genome-based gene set of a trehalose-producing tardigrade <i>Paramacrobiotus sp.</i> TYO.
Amino acid sequence of TPS-TPP genes of <i>Botryllus schlosseri</i> modeled by on-demand gene prediction. Genome assembly was obtained from <i>Botryllus schlosseri</i> genome project... more
Amino acid sequence of TPS-TPP genes of <i>Botryllus schlosseri</i> modeled by on-demand gene prediction. Genome assembly was obtained from <i>Botryllus schlosseri</i> genome project (http://botryllus.stanford.edu/botryllusgenome/).
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Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various... more
Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins t...
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts.... more
Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts. In particular, exposure to space radiation represents a serious potential long-term threat to the health of astronauts because the amount of radiation exposure accumulates during their time in space. Therefore, health risks associated with exposure to space radiation are an important topic in space travel, and characterizing space radiation in detail is essential for improving the safety of space missions. In the first part of this review, we provide an overview of the space radiation environment and briefly present current and future endeavors that monitor different space radiation environments. We then present research evaluating adverse biological effects caused by exposure to various space radiation environments and how these can be reduced. We...
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Reproductive strategy is an important aspect of biological diversity. In tardigrades, several reproductive modes, including sexual reproduction, are known. However, tardigrade mating behavior has been observed only rarely in most species,... more
Reproductive strategy is an important aspect of biological diversity. In tardigrades, several reproductive modes, including sexual reproduction, are known. However, tardigrade mating behavior has been observed only rarely in most species, and in some cases, especially in the freely ovipositing eutardigrades, remains entirely unknown. In the present study, we cultured two sexually reproducing tardigrade species that lay eggs freely, Paramacrobiotus sp. TYO strain and Macrobiotus shonaicus, to investigate and compare their courtship, mating, and chromosome morphology. Mating behavior was observed and recorded in both species. The entire mating sequence, including courtship, was categorized into five discrete steps common to two species, as follows: [1] Tracking: the male tracks and orientates toward the female; [2] Touching: the male makes contact with the cloaca of the female; [3] Standstill: the female ceases movement until male ejaculation is complete; [4] Ejaculation: the male curls its caudal end and ejaculates into the cloaca from close range; [5] Contraction: the female contracts its ventral side after ejaculation to capture spermatozoa deposited in the external environment in close proximity to the cloaca. Some notable differences between the two species were observed in the steps 3–4. First, oviposition was observed at 40 min in Paramacrobiotus sp. TYO strain, and a few days after mating in M. shonaicus, respectively. Comparisons of chromosome morphology before and after mating indicated that oocytes are arrested at metaphase I in both species. Spermatozoa attach to the interior of the chorion of laid eggs.
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Unlike mammals,Xenopus laevistadpoles possess high ability to regenerate their lost organs. In amphibians, the main source of regenerated tissues is lineage-restricted tissue stem cells, but the mechanisms underlying induction,... more
Unlike mammals,Xenopus laevistadpoles possess high ability to regenerate their lost organs. In amphibians, the main source of regenerated tissues is lineage-restricted tissue stem cells, but the mechanisms underlying induction, maintenance and differentiation of these stem/progenitor cells in the regenerating organs are poorly understood. We previously reported thatinterleukin-11(il-11) is highly expressed in the proliferating cells of regeneratingXenopustadpole tails. Here, we show thatil-11knockdown (KD) shortens the regenerated tail length, and the phenotype is rescued by forced-il-11-expression in the KD tadpoles. Moreover, marker genes for undifferentiated notochord, muscle, and sensory neurons are downregulated in the KD tadpoles, and the forced-il-11-expression in intact tadpole tails induces expression of these marker genes. Our findings demonstrate thatil-11is necessary for organ regeneration, and suggest that IL-11 plays a key role in the induction and maintenance of undif...
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In insect brains, the mushroom bodies (MBs, a higher center) comprise intrinsic neurons, termed Kenyon cells (KCs). We previously showed that the honeybee (Apis mellifera L.) MBs comprise four types of KCs, in addition to the previously... more
In insect brains, the mushroom bodies (MBs, a higher center) comprise intrinsic neurons, termed Kenyon cells (KCs). We previously showed that the honeybee (Apis mellifera L.) MBs comprise four types of KCs, in addition to the previously known three types of KCs: class I large-type KCs (lKCs), class I small-type KCs (sKCs) and class II KCs, novel class I 'middle-type' KCs (mKCs), which are characterized by the preferential expression of a gene, termed mKast. Although mKast was originally discovered during the search for genes whose expression is enriched in the optic lobes (OLs) in the worker brain, subsequent analysis revealed that the gene is expressed in an mKC-preferential manner in the MBs. To gain more insights into the function of mKast in the honeybee brain, we here performed expression analysis of mKast and immunohistochemistry of the mKast protein. Prominent mKast expression was first detected in the brain after the P7 pupal stage. In addition, mKast was expressed a...
Research Interests: Biochemistry, Evolutionary Biology, Genetics, Neuroscience, Developmental Biology, and 15 moreBiology, Ecology, Immunohistochemistry, Medicine, Gene expression, Multidisciplinary, In Situ Hybridization, Brain, Animals, Gene, PLoS one, Ol, Mushroom Bodies, Al, and reverse transcriptase polymerase chain reaction
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ABSTRACTTardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution.... more
ABSTRACTTardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Ecdysozoa by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Similar to bdelloid rotifers, nematodes and other animals of the water film, limno-terrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated l...
Research Interests: Evolutionary Biology, Biology, Extremophiles, Genome Size, Horizontal Gene Transfer, and 15 moreBiological Sciences, DNA, Phylogeny, Animals, Desiccation, Genome, Reproducibility of Results, Genomic Library, Base Sequence, Proteome, Cryptobiosis, Gene Expression Regulation, Extremophile, Gene expression profiling, and Medical and Health Sciences
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Research Interests: Engineering, Genetics, Entomology, Chemistry, Animal Behavior, and 15 moreComputational Biology, Fluorescence Microscopy, Biology, Developmental neuroscience, Medicine, Gene expression, Behavioral Neuroscience, Biological Sciences, Bees, Brain, Anatomy and Physiology, Animals, Cells, Honeybee, and Arrestin
Trehalose is a versatile non-reducing sugar. In some animal groups possessing its intrinsic production machinery, it is used as a potent protectant against environmental stresses, as well as blood sugar. However, the trehalose... more
Trehalose is a versatile non-reducing sugar. In some animal groups possessing its intrinsic production machinery, it is used as a potent protectant against environmental stresses, as well as blood sugar. However, the trehalose biosynthesis genes remain unidentified in the large majority of metazoan phyla, including vertebrates. To uncover the evolutionary history of trehalose production machinery in metazoans, we scrutinized the available genome resources and identified bifunctional trehalose-6-phosphate synthase-trehalose-6-phosphate phosphatase (TPS–TPP) genes in various taxa. The scan included our newly sequenced genome assembly of a desiccation-tolerant tardigrade Paramacrobiotus sp. TYO, revealing that this species retains TPS–TPP genes activated upon desiccation. Phylogenetic analyses identified a monophyletic group of the many of the metazoan TPS–TPP genes, namely ‘pan-metazoan’ genes, that were acquired in the early ancestors of metazoans. Furthermore, coordination of our re...
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Genomic DNA stores all genetic information and is indispensable for maintenance of normal cellular activity and propagation. Radiation causes severe DNA lesions, including double-strand breaks, and leads to genome instability and even... more
Genomic DNA stores all genetic information and is indispensable for maintenance of normal cellular activity and propagation. Radiation causes severe DNA lesions, including double-strand breaks, and leads to genome instability and even lethality. Regardless of the toxicity of radiation, some organisms exhibit extraordinary tolerance against radiation. These organisms are supposed to possess special mechanisms to mitigate radiation-induced DNA damages. Extensive study using radiotolerant bacteria suggested that effective protection of proteins and enhanced DNA repair system play important roles in tolerability against high-dose radiation. Recent studies using an extremotolerant animal, the tardigrade, provides new evidence that a tardigrade-unique DNA-associating protein, termed Dsup, suppresses the occurrence of DNA breaks by radiation in human-cultured cells. In this review, we provide a brief summary of the current knowledge on extremely radiotolerant animals, and present novel ins...
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Organ regenerative ability depends on the animal species and the developmental stage. The molecular bases for variable organ regenerative ability, however, remain unknown. Previous studies have identified genes preferentially expressed in... more
Organ regenerative ability depends on the animal species and the developmental stage. The molecular bases for variable organ regenerative ability, however, remain unknown. Previous studies have identified genes preferentially expressed in the blastema tissues in various animals, but transcriptome analysis of the isolated proliferating blastema cells has not yet been reported. In the present study, we used RNA-sequencing analysis to analyze the gene expression profile of isolated proliferating blastema cells of regenerating Xenopus laevis tadpole tails. We used flow cytometry to isolate proliferating cells, and non-proliferating blastema cells, from regenerating tadpole tails as well as proliferating tail bud cells from tail bud embryos, the latter two of which were used as control cells, based on their DNA content. Among the 28 candidate genes identified by RNA-sequencing analysis, quantitative reverse transcription-polymerase chain reaction identified 10 genes whose expression was ...
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Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the... more
Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial l...
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The honeybee is a social insect that exhibits various social behaviors. To elucidate the neural basis of honeybee behavior, we detected neural activity in freely-moving honeybee workers using an immediate early gene (IEG) that is... more
The honeybee is a social insect that exhibits various social behaviors. To elucidate the neural basis of honeybee behavior, we detected neural activity in freely-moving honeybee workers using an immediate early gene (IEG) that is expressed in a neural activity-dependent manner. In European honeybees (Apis mellifera), we identified a novel nuclear non-coding RNA, termed kakusei, as the first insect IEG, and revealed the neural activity pattern in foragers. In addition, we isolated a homologue of kakusei, termed Acks, from the Japanese honeybee (Apis cerana), and detected active neurons in workers fighting with the giant hornet.
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Mlr1 (Mblk-1-related protein-1) and Mlr2 are mouse homologs of transcription factor Mblk-1 (Mushroom body large-type Kenyon cell-specific protein-1), which we originally identified from the honeybee brain. In the present study, aiming at... more
Mlr1 (Mblk-1-related protein-1) and Mlr2 are mouse homologs of transcription factor Mblk-1 (Mushroom body large-type Kenyon cell-specific protein-1), which we originally identified from the honeybee brain. In the present study, aiming at identifying coregulator(s) of Mlr1 and Mlr2 from the mouse brain, we used yeast two-hybrid screening of mouse brain cDNA library to search for interaction partners of Mlr 1 and Mlr2, respectively. We identified nucleolar protein 4 (NOL4) splicing variants as major interaction partners for both Mlr1 and Mlr2. Among the three murine NOL4 splicing variants, we further characterized NOL4-S, which lacks an N-terminal part of NOL4-L, and NOL4-SΔ, which lacks nuclear localization signal (NLS)-containing domain of NOL4-S. A GST pull-down assay revealed that Mlr1 interacts with both NOL4-S and NOL4-SΔ, whereas Mlr2 interacts with NOL4-S, but not with NOL4-SΔ. These results indicate that the NLS-containing domain of NO4-S Is necessary for in vitro binding with Mlr2, but not for that with Mlr1. Furthermore, a luciferase assay using Schneider&amp;amp;amp;amp;amp;amp;amp;amp;#39;s Line 2 cells revealed that transactivation activity of Mlr1 was significantly suppressed by both NOL4-S and NOL4-SΔ, with almost complete suppression by NOL4-SΔ. In contrast, transactivation activity of Mlr2 was significantly suppressed by NOL4-S but rather activated by NOL4-SΔ. Our findings suggest that transactivation activities of Mlr1 and Mlr2 are differentially regulated by splicing variants of NOL4, which are expressed in a tissue-selective manner.
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To identify candidate royal jelly (RJ) proteins that might affect the physiologic status of honeybee colony members, we used shotgun proteomics to comprehensively identify the RJ proteome as well as proteomes of the hypopharyngeal gland... more
To identify candidate royal jelly (RJ) proteins that might affect the physiologic status of honeybee colony members, we used shotgun proteomics to comprehensively identify the RJ proteome as well as proteomes of the hypopharyngeal gland (HpG), postcerebral gland (PcG), and thoracic gland (TG), from which RJ proteins are assumed to be derived. We identified a total of 38 nonredundant RJ proteins, including 22 putative secretory proteins and Insulin-like growth factor-binding protein complex acid labile subunit. Among them, 9 proteins were newly identified from RJ. Comparison of the RJ proteome with the HpG, PcG, and TG proteomes revealed that 17 of the 22 putative secretory RJ proteins were derived from some of these glands, suggesting that the RJ proteome is a cocktail of proteins from these three glands. Furthermore, pathway analysis suggested that the HpG proteome represents the molecular basis of the extremely high protein-synthesizing ability, whereas the PcG proteome suggests that the PcG functions as a reservoir for the volatile compounds and a primer pheromone. Finally, to further characterize the possible total RJ proteome, we identified putative secretory proteins in the proteomes of these three glands. This will be useful for predicting novel RJ protein components in future studies.
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Adenosine deaminase-related growth factors (ADGF), also known as CECR1 in vertebrates, are a novel family of growth factors with sequence similarity to classical cellular adenosine deaminase. Although genes for ADGF/CECR1 have been... more
Adenosine deaminase-related growth factors (ADGF), also known as CECR1 in vertebrates, are a novel family of growth factors with sequence similarity to classical cellular adenosine deaminase. Although genes for ADGF/CECR1 have been identified in both invertebrates as well as vertebrates, their in vivo functions in vertebrates remain unknown. We isolated cDNA clones for two cerc 1s from Xenopus laevis. Both recombinant Xenopus CECR1s exhibited adenosine deaminase and growth factor activity, and the adenosine deaminase activity was found to be indispensable for growth factor activity. The Xenopus cerc 1s are expressed in the somites, pronephros, eyes, cement gland, neural tube, and neural floor plate of the embryos. Knock-down of these two genes using morpholino oligonucleotides caused a reduction in the body size and abnormalities of the body axis in the Xenopus embryos, accompanied by selective changes in the expression of developmental marker genes. Injection of adenosine, agonists for adenosine/P1 receptors, or adenosine deaminase inhibitor into late gastrula archenteron embryos resulted in developmental defects similar to those caused by morpholino oligonucleotide injection. These results show, for the first time, the involvement of CECR1s via the adenosine/P1 receptors in vertebrate embryogenesis via regulation of extracellular adenosine concentrations.
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We previously demonstrated that the gene for a member of the humoral C-type lectin family is transiently expressed in the regenerating legs of the American cockroach [Arai et al., Insect Biochem. Mol. Biol. 28, 987-994 (1998)]. To... more
We previously demonstrated that the gene for a member of the humoral C-type lectin family is transiently expressed in the regenerating legs of the American cockroach [Arai et al., Insect Biochem. Mol. Biol. 28, 987-994 (1998)]. To identify candidate lectin(s) involved in tail regeneration in the Xenopus laevis tadpole, we isolated a 35-kDa Ca(2+)-dependent lectin (XCL-1) from adult Xenopus serum and cloned its cDNA. Although XCL-1 gene expression was not induced in the regenerating tails, we isolated a cDNA for an XCL-1-related protein (XCL-2) by reverse transcription-polymerase chain reaction. In contrast to the XCL-1 gene, XCL-2 gene expression was significantly increased in the regenerating tails, suggesting its role in tail regeneration. Although both XCL-1 and XCL-2 belong to a recently identified Xenopus lectin family (X-lectins), XCL-1 and XCL-2 exhibit distinct developmental gene expression from two other known X-lectin members, both of which are expressed principally in the embryonic stage, whereas the XCL-1 and XCL-2 genes are predominantly expressed in the adult and middle/late tadpole stages, respectively, suggesting multiple functions of X-lectin family members. Thus, the presence of multifunctional Ca(2+)-dependent lectin family and the induction of the member gene in regenerating organs are conserved among insects and vertebrates.
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To date, there are only few reports of immediate early genes (IEGs) available in insects. Aiming at identifying a conserved IEG in insects, we characterized an Egr homolog of the honeybee (AmEgr: Apis mellifera Egr). AmEgr was transiently... more
To date, there are only few reports of immediate early genes (IEGs) available in insects. Aiming at identifying a conserved IEG in insects, we characterized an Egr homolog of the honeybee (AmEgr: Apis mellifera Egr). AmEgr was transiently induced in whole worker brains after seizure induction. In situ hybridization for AmEgr indicated that neural activity of a certain mushroom body (a higher brain center) neuron subtype, which is the same as that we previously identified using another non-coding IEG, termed kakusei, is more enhanced in forager brains. These findings suggest that Egr can be utilized as an IEG in insects.
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Xenopus tadpoles have high regenerative ability of amputated tails except during the &amp;amp;amp;amp;amp;amp;amp;amp;#39;refractory period&amp;amp;amp;amp;amp;amp;amp;amp;#39;, when the ability is transiently lost. We previously... more
Xenopus tadpoles have high regenerative ability of amputated tails except during the &amp;amp;amp;amp;amp;amp;amp;amp;#39;refractory period&amp;amp;amp;amp;amp;amp;amp;amp;#39;, when the ability is transiently lost. We previously demonstrated that distinct immune responses occur in tail stumps between the refractory and pre/post-refractory regeneration periods. Furthermore, treatment with an immunosuppressant, FK506, restores the tail regenerative ability during the refractory period. Based on these findings, we previously proposed that autoreactive immune cells infiltrate the tail stumps to attack blastema cells as &amp;amp;amp;amp;amp;amp;amp;amp;#39;non-self&amp;amp;amp;amp;amp;amp;amp;amp;#39; during the refractory period, resulting in the impaired regenerative ability. The immune cells that attack the blastema cells, however, remained unclear. Here we screened for genes whose expression in the tail stumps was altered by FK506 treatment during the refractory period and identified a Xenopus homolog of phytanoyl-CoA dioxygenase (PhyH)-like. XPhyH-like expression transiently increased in tail stumps after amputation during the refractory period, and was reduced by FK506 treatment. XPhyH-like expression in the whole tadpole body specifically increased during the refractory period and was enriched in the blood cell fraction. These findings suggest that XPhyH-like is expressed in autoreactive immune cells that are distributed in the whole body during the refractory period and transiently infiltrate the tail stumps to attack the blastema cells as &amp;amp;amp;amp;amp;amp;amp;amp;#39;non-self&amp;amp;amp;amp;amp;amp;amp;amp;#39;.