Published papers

Neocaridina, a crustacean, exhibits distinctive tail morphogenesis during molting, making it a potential model for post-embryonic morphogenesis. After the first molting, two significant changes occur in the tail: the uropod branches are cleared and the telson undergoes convergent elongation. Cross-sectional analysis showed that uropod and telson branching begins immediately after hatching as cuticle branching. The surface structure of the developing tail suggested that telson elongation is achieved through anisotropic cuticle furrow extension during molting, linked to epithelial cell shape changes occurring post-hatching. An in vivo live imaging system with UV-LED resin revealed gradual telson contraction before molting. Additionally, a draft genome of Neocaridina denticulata was provided. This research enhances understanding of arthropod morphogenesis through molting and lays groundwork for further developmental and cytological studies in Neocaridina.

Abstract: Spider silk is an archetypal biopolymer material with extreme tensile properties arising from its complex hierarchical assembly. While recent advances in sequencing have yielded abundant insights, relatively little is known concerning post-translational modifications (PTMs) in spider silk. Here, we probe the PTM landscape of dragline silk from the Jorō spider (Trichonephila clavata) using a combination of mass spectroscopy and solid-state nuclear magnetic resonance (NMR). The results reveal a wide array of potential modifications, including hydroxyproline, phosphorylation, and dityrosine cross-links, encompassing the different spidroin constituents. Notably, the MaSp3 repetitive region displayed numerous PTMs, whereas MaSp1 and MaSp2 variants showed distinct phosphorylation patterns in its terminal domains. The N-terminal domain (NTD) phosphorylation sites were found predominantly at the dimer interface, suggesting a modulatory function with respect to its pH-driven dimerization function, a hypothesis supported by studies using phosphomimetic NTD mutants. Possible roles of phosphoserine in limiting β-sheet formation, and hydroxyproline in disrupting β-turns are also discussed. Impact statement: Spider silk is an archetypal biomaterial that can outperform our most sophisticated artificial fibers. The secret to its mechanical properties lies in its complex hierarchical structure—encompassing the nano- to macroscales—that forms through a process of molecular self-assembly of the constituent spidroin proteins. While recent advances in "biomateriomics” have given us tremendous insights into the sequence–function relationships that determine spider silk behavior, the picture is still far from complete. One area that has received little attention is posttranslational modifications (PTMs). PTMs are ubiquitous biological phenomena that are crucial for providing dynamic control of the proteome, and effectively expand the structural and functional design space of proteins beyond that provided by the canonical amino acids. Here, we undertook a comprehensive analysis of PTMs from spider dragline silk fiber, which revealed numerous potential sites for a wide array of modifications. The results provide a fascinating window into additional layers of complexity underlying the mechanical behavior of spider silk, and suggest further avenues for creating novel, dynamically tunable, bioinspired materials.

Spider mites are a group of arachnids belonging to Acari (mites and ticks), family Tetranychidae, known to produce nanoscale silk fibers characterized by a high Young's modulus. The silk fibroin gene of spider mites has been computationally predicted through genomic analysis of Tetranychus urticae Koch, but it has yet to be confirmed by proteomic evidence. In this work, we sequenced and assembled the transcriptome from two genera of spider mites, Tetranychus kanzawai Kishida and Panonychus citri (McGregor), and combined it with silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multiomics analysis. As a result, two fibroins were identified, which were different genes than those previously predicted by computational methods. The amino acid composition and secondary structure suggest similarity to aciniform or cylindrical spidroins of spider silk, which partly mirrors their mechanical properties, exhibiting a high Young's modulus. The availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes that sometimes emerge in multiple lineages in a convergent manner and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks. Significance: Here we sequenced and assembled the transcriptome from two genera of spider mites, T. kanzawai and P. citri, and combined it with silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multiomics analysis. Spider mite silk is especially characterized by its extremely fine nano-scale diameter and high Young's modulus, even exceeding those of spider silks. The availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes, which independently evolved in mites, insects, and spiders but yet show sequence convergence, and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks.

Studies examining long-term longitudinal metabolomic data and their reliability in large-scale populations are limited. Therefore, we aimed to evaluate the reliability of repeated measurements of plasma metabolites in a prospective cohort setting and to explore intra-individual concentration changes at three time points over a 6-year period. The study participants included 2999 individuals (1317 men and 1682 women) from the Tsuruoka Metabolomics Cohort Study, who participated in all three surveys—at baseline, 3 years, and 6 years. In each survey, 94 plasma metabolites were quantified for each individual and quality control (QC) sample. The coefficients of variation of QC, intraclass correlation coefficients, and change rates of QC were calculated for each metabolite, and their reliability was classified into three categories: excellent, fair to good, and poor. Seventy-six percent (71/94) of metabolites were classified as fair to good or better. Of the 39 metabolites grouped as excellent, 29 (74%) in men and 26 (67%) in women showed significant intra-individual changes over 6 years. Overall, our study demonstrated a high degree of reliability for repeated metabolome measurements. Many highly reliable metabolites showed significant changes over the 6-year period, suggesting that repeated longitudinal metabolome measurements are useful for epidemiological studies.

Rhizospheric microbiomes differ between active and dormant plants due to changes in root activity and exudate production, especially under environmental stress. In arid regions, native plants such as Potaninia mongolica Maxim enter dormancy to survive harsh conditions. However, rhizospheric microbial and chemical differences between active and dormant states of plants remain poorly described. This study investigated rhizospheric microbial communities and soil chemical changes in the case of active and dormant P.mongolica plants. Rhizospheric soil samples were collected, and soil texture and chemical variables were analyzed. High-throughput sequencing targeting the 16S rRNA and ITS regions was conducted to profile bacterial and fungal communities, respectively. Results showed that the dominant fungal phyla were Ascomycota and Basidiomycota, while Proteobacteria and Actinobacteria were the dominant bacterial phyla in both plant states. Although bacterial diversity did not differ significantly between active and dormant plants (p > 0.05, Welch’s t-test), fungal diversity was significantly different. Among soil chemical variables, total nitrogen was notably elevated in the rhizosphere of dormant plants (mean = 7.93; SD = 5.91). These findings reveal differences in fungal community structure and nitrogen levels in the rhizosphere between active and dormant plant states. Understanding these interactions contributes to our knowledge of desert plant microbiome dynamics and may inform the use of microbial indicators or amendments to support vegetation restoration in arid environments.

Tardigrades possess the ability to enter an almost completely dehydrated state, anhydrobiosis. The CAHS (cytosolic abundant heat-soluble) protein family has been identified as one of the anhydrobiosis-related proteins. In particular, CAHS3 protein from an anhydrobiotic tardigrade, Ramazzottius varieornatus, shows heat-solubility and reversible condensation and is one of the most highly expressed among the CAHS paralogs. A recently developed tardigrade-specific vector showed tissue-specific expression of RvCAHS3 most pronounced in the epidermis in vivo, contrary to the idea that anhydrobiotic genes are uniformly expressed in all tardigrade cells. In this study, we investigated the regulation of RvCAHS3 gene expression through in vivo expression experiments using tardigrade vectors with a series of truncated upstream regions coupled with in silico analysis to identify the anhydrobiosis-related genes that are expressed under the same regulatory system as RvCAHS3. As a result, the 300–350 bp region upstream of RvCAHS3 is critical for regulating gene expression in tardigrade vector experiments, and three motifs conserved between two species of anhydrobiotic tardigrades were identified within a 500 bp region directly upstream of RvCAHS3 start codon. These motifs, which have also been identified upstream of other CAHS genes, could be associated with the regulatory system of anhydrobiosis-related genes in tardigrades.

Background: In gonochoristic animals, the sex determination pathway induces different morphological and behavioral features that can be observed between sexes, a condition known as sexual dimorphism. While many components of this sex differentiation cascade show high levels of diversity, factors such as the Doublesex-Mab-3-Related Transcription factor (DMRT) are widely conserved across animal taxa. Species of the phylum Tardigrada exhibit remarkable diversity in morphology and behavior between sexes, suggesting a pathway regulating this dimorphism. Despite the wealth of genomic and zoological knowledge accumulated in recent studies, the sexual differences in tardigrades genomes have not been identified. In the present study, we focused on the gonochoristic species Paramacrobiotus metropolitanus and employed omics analyses to unravel the molecular basis of sexual dimorphism. Results: Transcriptome analysis between sex-identified specimens revealed numerous differentially expressed genes, of which approximately 2,000 male-biased genes were focused on 29 non-male-specific genomic loci. From these regions, we identified two Macrobiotidae family specific DMRT paralogs, which were significantly upregulated in males and lacked sex specific splicing variants. Furthermore, phylogenetic analysis indicated all tardigrade genomes lack the doublesex ortholog, suggesting doublesex emerged after the divergence of Tardigrada. In contrast to sex-specific expression, no evidence of genomic differences between the sexes was found. We also identified several anhydrobiosis genes that exhibit sex-biased expression, suggesting a possible mechanism for protection of sex-specific tissues against extreme stress. Conclusions: This study provides a comprehensive analysis for analyzing the genetic differences between sexes in tardigrades. The existence of male-biased, but not male-specific, genomic loci and identification of the family specific male-biased DMRT subfamily provides the foundation for understanding the sex determination cascade. In addition, sex-biased expression of several tardigrade-specific genes which are involved their stress tolerance suggests a potential role in protecting sex-specific tissue and gametes.

Background: A striking aspect of evolution is that it often converges on similar trajectories. Evolutionary convergence can occur in deep time or over short time scales, and is associated with the imposition of similar selective pressures. Repeated convergent events provide a framework to infer the genetic basis of adaptive traits. The current study examines the genetic basis of secondary web loss within web-building spiders (Araneoidea). Specifically, we use a lineage of spiders in the genus Tetragnatha (Tetragnathidae) that has diverged into two clades associated with the relatively recent (5 mya) colonization of, and subsequent adaptive radiation within, the Hawaiian Islands. One clade has adopted a cursorial lifestyle, and the other has retained the ancestral behavior of capturing prey with sticky orb webs. We explore how these behavioral phenotypes are reflected in the morphology of the spinning apparatus and internal silk glands, and the expression of silk genes. Several sister families to the Tetragnathidae have undergone similar web loss, so we also ask whether convergent patterns of selection can be detected in these lineages. Results: The cursorial clade has lost spigots associated with the sticky spiral of the orb web. This appears to have been accompanied by loss of silk glands themselves. We generated phylogenies of silk proteins (spidroins), which showed that the transcriptomes of cursorial Tetragnatha contain all major spidroins except for flagelliform. We also found an uncharacterized spidroin that has higher expression in cursorial species. We found evidence for convergent selection acting on this spidroin, as well as genes involved in protein metabolism, in the cursorial Tetragnatha and divergent cursorial lineages in the families Malkaridae and Mimetidae. Conclusions: Our results provide strong evidence that independent web loss events and the associated adoption of a cursorial lifestyle are based on similar genetic mechanisms. Many genes we identified as having evolved convergently are associated with protein synthesis, degradation, and processing, which are processes that play important roles in silk production. This study demonstrates, in the case of independent evolution of web loss, that similar selective pressures act on many of the same genes to produce the same phenotypes and behaviors.

The Tardigrada are a clade with a disputed and complex taxonomy. The three traditional tardigrade classes are the Heterotardigrada, Eutardigrada and the dubious, monotypic Mesotardigrada. A recent molecular study by Guil et al (Zoologica Scripta, 48, 2019, 120) suggested that the Apochela, previously considered an order of Eutardigrada, should become a new class, Apotardigrada, with the Parachela becoming THE new Eutardigrada. This new diagnosis was presented alongside compelling morphological evidence. Here, we test the assumptions of the molecular evidence for Apotardigrada by analysing tardigrade 18S and 28S data under four separate conditions, along with new BUSCO data, across both Bayesian- and maximum-likelihood phylogenetic approaches. We found variable conclusions regarding the status of Apotardigrada could be drawn by comparing the ribosomal RNA and BUSCO results, and caution against using branch length data to determine taxonomic hierarchy.

Secretory-abundant heat-soluble (SAHS) proteins are unique heat-soluble proteins of Tardigrada and are believed to play an essential role in anhydrobiosis, a latent state of life induced by desiccation. To investigate the dynamic properties, molecular dynamics (MD) simulations of a SAHS protein, RvSAHS1, were performed in solution and under dehydrating conditions. For comparison purposes, MD simulations of a human liver-type fatty-acid binding protein (LFABP) were performed in solution. Furthermore, high-speed atomic force microscopy observations were conducted to ascertain the results of the MD simulations. Three properties of RvSAHS1 were found as follows. (1) The entrance region of RvSAHS1 is more flexible and can be more extensive in solutions compared with that of a human LFABP because there is no salt bridge between the βD and βE strands. (2) The intrinsically disordered domain in the N-terminal region significantly fluctuates and can form an amphiphilic α-helix. (3) The size of the entrance region gets smaller along with dehydration, keeping the β-barrel structure. Overall, the obtained results provide atomic-level dynamics of SAHS proteins.

Secretory-abundant heat-soluble (SAHS) proteins, which constitute a protein family unique to tardigrades, are thought to be essential for anhydrobiosis. Our previous study has revealed that one of the SAHS proteins of Ramazzottius varieornatus (RvSAHS1) has a more flexible entrance than a mammalian fatty-acid-binding protein, which has a crystal structure similar to that of RvSAHS1. Recently, SAHS paralogs that are expressed abundantly and specifically in the early embryos of this tardigrade and Hypsibius exemplaris have been identified. Comparing these amino-acid sequences with that of RvSAHS1, we have found characteristic differences as I113F and D146T. In this study, we investigate I113F and D146T mutants' properties of RvSAHS1 using molecular dynamics simulations and compare the structures and fluctuations of their entrances with those of the wild type. The two mutants exhibit different properties at the entrance of the β-barrel structure. The I113F mutant tends to close the entrance more than the wild type due to the enhanced hydrophobic network inside the cavity. The D146T mutant, in contrast to the I113F mutant, tends to open the entrance. The mechanism by which this mutation opens the entrance is also discussed. Even though only a single mutation located far from the entrance is added to the wild type, there is a clear difference in the tendency to open and close the β-barrel entrance. It indicates that the entrance properties of the SAHS protein are sensitive to the amino-acid sequence.

Protein-based materials are considered versatile biomaterials, and their biodegradability is an advantage for sustainable development. Bagworm produces strong silk for use in unique situations throughout its life stages. Rigorous molecular analyses of Eumeta variegata suggested that the particular mechanical properties of its silk are due to the coexistence of poly-A and GA motifs. However, little molecular information on closely related species is available, and it is not understood how these properties were acquired evolutionarily or whether the motif combination is a conserved trait in other bagworms. Here, we performed a transcriptome analysis of two other bagworm species (Canephora pungelerii and Bambalina sp.) belonging to the family Psychidae to elucidate the relationship between the fibroin gene and silk properties. The obtained transcriptome assemblies and tensile tests indicated that the motif combination and silk properties were conserved among the bagworms. Furthermore, our analysis showed that C. pungelerii produces extraordinarily strong silk (breaking strength of 1.4 GPa) and indicated that the cause may be the C. pungelerii -specific balance of crystalline/amorphous regions in the H-fibroin repetitive domain. This particular H-fibroin architecture may have been evolutionarily acquired to produce strong thread to maintain bag stability during the relatively long development period of Canephora species relative to other bagworms.

Trichonephila clavipes (Linnaeus, 1767) is known as a golden silk orb-weaver and belongs to the family Araneidae. T. clavipes is one of the few spider species whose genome has been reported and model organism for a molecular biology. Here, we present the complete mitochondrial genome sequence (mtDNA) of T. clavipes. The sequence was obtained using a long-read Nanopore technology and corrected with an Illumina technology. The circular genome is 14,902 bp in length, and the AT content was 77.21%. The T. clavipes mitochondrial genome contains 13 protein-coding genes (PCGs), 22 tRNA genes, and 2 rRNA genes. The majority of PCGs were found on the heavy strain.

Polyrhachis lamellidens is a temporary socially parasitic ant whose new queen utilizes other ant species in the early stages of colony foundation. Field observations and rearing experiments suggest that Camponotus japonicus is a host species of P. lamellidens. It is presumed that Camponotus obscuripes is also a host of P. lamellidens by rearing experiments and field observations; however, there are no records of P. lamellidens workers or brood coexisting in C. obscuripes colonies in field observations, and there is no clear evidence that C. obscuripes is a natural host of P. lamellidens. We conducted detailed field observations, behavioral tests, and rearing experiments to show that C. obscuripes is a host of temporary social parasites. We found colonies with P. lamellidens queens, workers, and larvae intermixed with C. obscuripes workers in the field. Behavioral tests showed that workers of both species in mixed colonies did not attack each other and maintained nestmate recognition ability, which suggests a collaborative nestmate relationship. Furthermore, a rearing experiment confirmed social parasitism by P. lamellidens among C. obscuripes by producing a mixed brood-producing colony. These are the first field and laboratory records of temporary social parasitism involving P. lamellidens and C. obscuripes.

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.

When extracting DNA of invertebrates for long-read sequencing, not only enough quantity and size of the DNA but, depending on the species, elimination of contamination of endosymbiotic Wolbachia genome also has to be achieved. These requirements become troublesome, especially in small-sized species with a limited number of individuals available for the experiment. In this chapter, using tiny parasitoid wasps (Reclinervellus nielseni) parasitizing spiders as hosts, we developed a method of eliminating the Wolbachia genomes by means of an antibiotic administration to adult wasps via honey solution. Twenty days of rifampicin treatment since their emergence from cocoons resulted in a significant decrease in the Wolbachia genomes while keeping good DNA conditions for nanopore sequencing. An adequate quantity of DNA was then gained by pooling several individuals. The method could be applied to other insects or invertebrates that can be maintained by laboratory feeding with liquid food.

Background: Tardigrades are microscopic animals that are capable of tolerating extreme environments by entering a desiccated state of suspended animation known as anhydrobiosis. While antioxidative stress proteins, antiapoptotic pathways and tardigrade-specific intrinsically disordered proteins have been implicated in the anhydrobiotic machinery, conservation of these mechanisms is not universal within the phylum Tardigrada, suggesting the existence of overlooked components. Results: Here, we show that a novel Mn-dependent peroxidase is an important factor in tardigrade anhydrobiosis. Through time-series transcriptome analysis of Ramazzottius varieornatus specimens exposed to ultraviolet light and comparison with anhydrobiosis entry, we first identified several novel gene families without similarity to existing sequences that are induced rapidly after stress exposure. Among these, a single gene family with multiple orthologs that is highly conserved within the phylum Tardigrada and enhances oxidative stress tolerance when expressed in human cells was identified. Crystallographic study of this protein suggested Zn or Mn binding at the active site, and we further confirmed that this protein has Mn-dependent peroxidase activity in vitro. Conclusions: Our results demonstrated novel mechanisms for coping with oxidative stress that may be a fundamental mechanism of anhydrobiosis in tardigrades. Furthermore, localization of these sets of proteins mainly in the Golgi apparatus suggests an indispensable role of the Golgi stress response in desiccation tolerance.

Tardigrades are microscopic animals that are renowned for their capabilities of tolerating near-complete desiccation by entering an ametabolic state called anhydrobiosis. However, many species also show high tolerance against radiation in the active state as well, suggesting cross-tolerance via the anhydrobiosis mechanism. Previous studies utilized indirect DNA damaging agents to identify core components of the cross-tolerance machinery in species with high anhydrobiosis capacities. However, it was difficult to distinguish whether transcriptomic changes were specific to DNA damage or mutual with anhydrobiosis. To this end, we performed transcriptome analysis on bleomycin-exposed Hypsibius exemplaris. We observed induction of several tardigrade-specific gene families, including a previously identified novel antioxidative stress family, which may be a core component of the cross-tolerance mechanism. We also identified enrichment of the tryptophan metabolism pathway, for which metabolomic analysis suggested engagement of this pathway in stress tolerance. These results provide several candidates for the core component of cross-tolerance, as well as possible anhydrobiosis machinery.

We describe two new species of leptocheliid tanaidaceans, Chondrochelia sublitoralis sp. nov. and Neoleptochelia japonica sp. nov., from Japan. Chondrochelia individuals were collected from 113 to 185 m depth in the Kumano Sea. Chondrochelia sublitoralis sp. nov. resembles Chondrochelia africana, Chondrochelia corsica, Chondrochelia taitungensis, and Chondrochelia tanykeraia in having females with slender chelipeds but differs from them in the length ratio of antennular articles and the setal pattern on maxillipeds, cheliped, pereopods 4–6, and pleopods. Males differ from Chondrochelia durbanensis, for which information on females is lacking, in having pereopod-1 propodus lacking ventrodistal spiniform setae and a uniarticulate uropodal exopod. Chondrochelia sublitoralis sp. nov. varies intraspecifically in the number of female antennular articles, five in larger and four in smaller females. Neoleptochelia japonica sp. nov., collected from the coast of Okinawa Island, differ from Neoleptochelia javaensis (previously the sole species in Neoleptochelia) in the setal pattern on antennae, maxillipeds, cheliped, and pleopods in females; and that on the cheliped in males. Male pereopods 1–3 show a unique subchelate condition that we speculate may function for clasping during copulation. Several females in both species had the second thoracomere demarcated on the carapace, whereas others did not. Cytochrome c oxidase subunit I (COI) gene sequences determined for C. sublitoralis sp. nov. and N. japonica sp. nov. showed intraspecific K2P distances of 0–0.4% and 0–0.5%, respectively. We inferred the phylogenetic positions of the two species in Leptocheliidae based on COI data.

Genome sequencing of small species, such as those of meinofauna, can be challenging due to the extremely low input of genomic DNA. While nanopore sequencing is a promising technology for genome assembly due to its limitless long reads, recommended input of 1 μg for the Ligation Sequencing Kit often precludes the use of this technology. Here, I detail an unbiased droplet-based multiple displacement amplification of picogram order of DNA to realize nanopore sequencing with ultralow input of genomic DNA. For this purpose, a microfluidic chip of 10X Genomics Chromium Controller is utilized. With this method, over 10 μg of unbiased amplicons around 10 kbp in length can be obtained from as low as 50 μg of input DNA, which is enough for the construction of multiple sequencing libraries, or for the size selection of longer DNA fragments.