國枝 武和

Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.

Tardigrades can survive hostile environments such as desiccation by adopting a state of anhydrobiosis. Numerous tardigrade species have been described thus far, and recent genome and transcriptome analyses revealed that several distinct strategies were employed to cope with harsh environments depending on the evolutionary lineages. Detailed analyses at the cellular and subcellular levels are essential to complete these data. In this work, we analyzed a tardigrade species that can withstand rapid dehydration, Ramazzottius varieornatus. Surprisingly, we noted an absence of the anhydrobiotic-specific extracellular structure previously described for the Hypsibius exemplaris species. Both Ramazzottius varieornatus and Hypsibius exemplaris belong to the same evolutionary class of Eutardigrada. Nevertheless, our observations reveal discrepancies in the anhydrobiotic structures correlated with the variation in the anhydrobiotic mechanisms.

Among hymenopteran insects, aculeate species such as bees, ants, and wasps have enlarged and morphologically elaborate mushroom bodies (MBs), a higher-order brain center in the insect, implying their relationship with the advanced behavioral traits of aculeate species. The molecular bases leading to the acquisition of complicated MB functions, however, remains unclear. We previously reported the constitutive and MB-preferential expression of an ecdysone-signaling related transcription factor, Mblk-1/E93, in the honey bee brain. Here, we searched for target genes of Mblk-1 in the worker honey bee MBs using chromatin immunoprecipitation sequence analyses and found that Mblk-1 targets several genes involved in synaptic plasticity, learning, and memory abilities. We also demonstrated that Mblk-1 expression is self-regulated via Mblk-1-binding sites, which are located upstream of Mblk-1. Furthermore, we showed that the number of the Mblk-1-binding motif located upstream of Mblk-1 homologs increased associated with evolution of hymenopteran insects. Our findings suggest that Mblk-1, which has been focused on as a developmental gene transiently induced by ecdysone, has acquired a novel expression pattern to play a role in synaptic plasticity in honey bee MBs, raising a possibility that molecular evolution of Mblk-1 may have partly contributed to the elaboration of MB function in insects.

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 that reversibly form condensates upon dehydration-like stress. Through a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed “TFE-Dependent ReversiblY condensing Proteins (T-DRYPs).” Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeleton-like proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and could contribute to the exceptional physical stability in a dehydrated state.

The genus Paramacrobiotus was erected in 2009 from the genus Macrobiotus, and 43 Paramacrobiotus species have been described to date. Although the first genome sequence in the genus was reported for the TYO strain of Paramacrobiotus sp., which is a dioecious species and has five bivalent chromosomes, its precise taxonomic identification remained undetermined. Here, we report its morphology, confirming the presence of a microplacoid, cuticular bulge on the inner side of legs I–III, and granulation on the inner side of legs IV under both light and electron microscopy, and smooth areoles on the egg shell, indicating that it differs from other described species. In addition, the previously described karyotype 2n=10 of this strain is clearly distinct from other species of the genus Paramacrobiotus, supporting the hypothesis that the strain represents a new species. Molecular analyses for the small and large ribosomal subunit (18S rDNA, 28S rDNA), the internal transcribed spacer 2 (ITS-2) and cytochrome C oxidase subunit I (COI) were also performed. The TYO strain is most similar in the analysed nuclear markers to Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa & Roszkowska, 2020 and Paramacrobiotus sp. strain MG.002 (p-distances in 18S rDNA: 0.53%, 28S rDNA: 0.98–1.12%, and ITS-2: 9.9%), which corroborates with the overall morphological similarity between these taxa. Despite the close relationship between the TYO strain and P. experimentalis, the genetic species delimitation based on molecular analysis indicates that the TYO strain indeed is a distinct species. Therefore, this tardigrade is described here as Paramacrobiotus metropolitanus sp. nov.

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 results with the previous horizontal gene transfer studies illuminated that the two tardigrade lineages, nematodes and bdelloid rotifers, all of which include desiccation-tolerant species, independently acquired the TPS-TPP homologues via horizontal transfer accompanied with loss of the 'pan-metazoan' genes. Our results indicate that the parallel evolution of trehalose synthesis via recurrent loss and horizontal transfer of the biosynthesis genes resulted in the acquisition and/or augmentation of anhydrobiotic lives in animals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In the honey bee, the mushroom bodies (MBs), a higher-order center in insect brain, comprise interneurons termed Kenyon cells (KCs). We previously reported that Mblk-1, which encodes a transcription factor involved in ecdysteroid-signaling, is expressed preferentially in the large-type KCs (lKCs) in the pupal and adult worker brain and that phosphorylation by the Ras/MAPK pathway enhances the transcriptional activity of Mblk-1 in vitro. In the present study, we performed immunoblotting and immunofluorescence studies using affinity-purified anti-Mblk-1 and anti-phosphorylated Mblk-1 antibodies to analyze the distribution and phosphorylation of Mblk-1 in the brains of pupal and adult workers. Mblk-1 was preferentially expressed in the lKCs in both pupal and adult worker brains. In contrast, some Mblk-1 was phosphorylated almost exclusively in the pupal stages, and phosphorylated Mblk-1 was preferentially expressed in the MB neuroblasts and lKCs in pupal brains. Immunofluorescence studies revealed that both Mblk-1 and phosphorylated Mblk-1 are located in both the cytoplasm and nuclei of the lKC somata in the pupal and adult worker brains. These findings suggest that Mblk-1 plays a role in the lKCs in both pupal and adult stages and that phosphorylated Mblk-1 has pupal stage-specific functions in the MB neuroblasts and lKCs in the honey bee brain.

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 especially consider the deleterious effects on cellular DNA and how cells activate DNA repair mechanisms. The latest technologies being developed, e.g., a fluorescent ubiquitination-based cell cycle indicator, to measure real-time cell cycle progression and DNA damage caused by exposure to ultraviolet radiation are presented. Progress in examining the combined effects of microgravity and radiation to animals and plants are summarized, and our current understanding of the relationship between psychological stress and radiation is presented. Finally, we provide details about protective agents and the study of organisms that are highly resistant to radiation and how their biological mechanisms may aid developing novel technologies that alleviate biological damage caused by radiation. Future research that furthers our understanding of the effects of space radiation on human health will facilitate risk-mitigating strategies to enable long-term space and planetary exploration.

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.