In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada.

Water is essential for life, but anhydrobiotic tardigrades can survive almost complete dehydration. Anhydrobiosis has been a biological enigma for more than a century with respect to how organisms sustain life without water, but the few choices of genetic toolkits available in tardigrade research have been a challenging circumstance. Here, we report the development of an in vivo expression system for tardigrades. This transient transgenic technique is based on a plasmid vector (TardiVec) with promoters that originated from an anhydrobiotic tardigrade Ramazzottius varieornatus. It enables the introduction of GFP-fused proteins and genetically encoded indicators such as the Ca2+ indicator GCaMP into tardigrade cells; consequently, the dynamics of proteins and cells in tardigrades may be observed by fluorescence live imaging. This system is applicable for several tardigrades in the class Eutardigrada: the promoters of anhydrobiosis-related genes showed tissue-specific expression in this work. Surprisingly, promoters functioned similarly between multiple species, even for species with different modes of expression of anhydrobiosis-related genes, such as Hypsibius exemplaris, in which these genes are highly induced upon facing desiccation, and Thulinius ruffoi, which lacks anhydrobiotic capability. These results suggest that the highly dynamic expression changes in desiccation-induced species are regulated in trans. Tissue-specific expression of tardigrade-unique unstructured proteins also suggests differing anhydrobiosis machinery depending on the cell types. We believe that tardigrade transgenic technology opens up various experimental possibilities in tardigrade research, especially to explore anhydrobiosis mechanisms.

Nondomain biopolymers: Flexible molecular strategies to acquire biological functions.

A long-standing assumption in molecular biology posits that the conservation of protein and nucleic acid sequences emphasizes the functional significance of biomolecules. These conserved sequences fold into distinct secondary and tertiary structures, enable highly specific molecular interactions, and regulate complex yet organized molecular processes within living cells. However, recent evidence suggests that biomolecules can also function through primary sequence regions that lack conservation across species or gene families. These regions typically do not form rigid structures, and their inherent flexibility is critical for their functional roles. This review examines the emerging roles and molecular mechanisms of "nondomain biomolecules," whose functions are not easily predicted due to the absence of conserved functional domains. We propose the hypothesis that both domain- and nondomain-type molecules work together to enable flexible and efficient molecular processes within the highly crowded intracellular environment.

Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk.

Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining high-quality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders. We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.

Transcriptome analysis of the tardigrade Hypsibius exemplaris exposed to the DNA-damaging agent bleomycin.

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 anti-oxidative 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.

Composition of Minor Ampullate Silk Makes Its Properties Different from Those of Major Ampullate Silk.

Spider's minor ampullate silk, or MI-silk, exhibits distinct mechanical properties and water resistance compared to its major ampullate counterpart (MA-silk). The principal protein constituent of MI-silk is known as minor ampullate spidroin, or MiSp, and while its sequence has been deciphered and is thought to underlie the differences in properties with MA-silk, the composition of MI-silk and the relationship between its composition and properties remain elusive. In this study, we set out to investigate the mechanical properties, water resistance, and proteome of MA-silk and MI-silk from Araneus ventricosus and Trichonephila clavata. We also synthesized artificial fibers from major ampullate spidroin, MaSp1 and 2, and MiSp to compare their properties. Our proteomic analysis reveals that the MI-silk of both araneids is composed of MiSp, MaSp1, and spidroin constituting elements (SpiCEs). The absence of MaSp2 in the MI-silk proteome and the comparison of the water resistance of artificial fibers suggest that the presence of MaSp2 is the reason for the disparity in water resistance between MI-silk and MA-silk.

Time-series transcriptomic screening of factors contributing to the cross-tolerance to UV radiation and anhydrobiosis in tardigrades.

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.

Multiomics study of a heterotardigrade, Echinisicus testudo, suggests the possibility of convergent evolution of abundant heat-soluble proteins in Tardigrada.

BACKGROUND: Many limno-terrestrial tardigrades can enter an ametabolic state, known as anhydrobiosis, upon desiccation, in which the animals can withstand extreme environments. Through genomics studies, molecular components of anhydrobiosis are beginning to be elucidated, such as the expansion of oxidative stress response genes, loss of stress signaling pathways, and gain of tardigrade-specific heat-soluble protein families designated CAHS and SAHS. However, to date, studies have predominantly investigated the class Eutardigrada, and molecular mechanisms in the remaining class, Heterotardigrada, still remains elusive. To address this gap in the research, we report a multiomics study of the heterotardigrade Echiniscus testudo, one of the most desiccation-tolerant species which is not yet culturable in laboratory conditions. RESULTS: In order to elucidate the molecular basis of anhydrobiosis in E. testudo, we employed a multi-omics strategy encompassing genome sequencing, differential transcriptomics, and proteomics. Using ultra-low input library sequencing protocol from a single specimen, we sequenced and assembled the 153.7 Mbp genome annotated using RNA-Seq data. None of the previously identified tardigrade-specific abundant heat-soluble genes was conserved, while the loss and expansion of existing pathways were partly shared. Furthermore, we identified two families novel abundant heat-soluble proteins, which we named E. testudo Abundant Heat Soluble (EtAHS), that are predicted to contain large stretches of disordered regions. Likewise the AHS families in eutardigrada, EtAHS shows structural changes from random coil to alphahelix as the water content was decreased in vitro. These characteristics of EtAHS proteins are analogous to those of CAHS in eutardigrades, while there is no conservation at the sequence level. CONCLUSIONS: Our results suggest that Heterotardigrada have partly shared but distinct anhydrobiosis machinery compared with Eutardigrada, possibly due to convergent evolution within Tardigrada. (276/350).

AMPK activity is required for the induction of anhydrobiosis in a tardigrade Hypsibius exemplaris, and its potential up-regulator is PP2A.

The anhydrobiotic tardigrade, Hypsibius exemplaris, was previously considered to require de novo gene expression and protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity for successful anhydrobiosis. These indicate that H. exemplaris has signal transduction systems responding to desiccation stress, with the involvement of phosphorylation events. To this end, we carried out time-series phosphoproteomics of H. exemplaris exposed to mild desiccation stress and detected 48 phosphoproteins with significant differential regulations. Among them, immediate and successive reduction of phosphorylation levels of AMP-activated protein kinase (AMPK) was observed. The subsequent chemical genetic approach showed that AMPK was activated during the preconditioning stage for anhydrobiosis, and inhibition of its activity impaired successful anhydrobiosis. As PP2A is known to dephosphorylate AMPK in other organisms, we suggested that decreased phosphorylation levels of AMPK upon mild desiccation stress were caused by dephosphorylation by PP2A. Accordingly, phosphoproteomics of animals pre-treated with the PP1/PP2A inhibitor cantharidic acid (CA) lacked the decrease in phosphorylation levels of AMPK. These observations suggest that AMPK activity is required for successful anhydrobiosis in H. exemplaris, and its phosphorylation state is possibly regulated by PP2A.

Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus.

Tardigrada, 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 Panarthropoda 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. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. 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 levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda.

Comparison of the transcriptomes of two tardigrades with different hatching coordination.

BACKGROUND: Tardigrades are microscopic organisms, famous for their tolerance against extreme environments. The establishment of rearing systems of multiple species has allowed for comparison of tardigrade physiology, in particular in embryogenesis. Interestingly, in-lab cultures of limnic species showed smaller variation in hatching timing than terrestrial species, suggesting a hatching regulation mechanism acquired by adaptation to their habitat. RESULTS: To this end, we screened for coordinated gene expression during the development of two species of tardigrades, Hypsibius exemplaris and Ramazzottius varieornatus, and observed induction of the arthropod molting pathway. Exposure of ecdysteroids and juvenile hormone analog affected egg hatching but not embryonic development in only the limnic H. exemplaris. CONCLUSION: These observations suggest a hatching regulation mechanism by the molting pathway in H. exemplaris.

Patterns of population structure and complex haplotype sharing among field isolates of the green alga Chlamydomonas reinhardtii.

The nature of population structure in microbial eukaryotes has long been debated. Competing models have argued that microbial species are either ubiquitous, with high dispersal and low rates of speciation, or that for many species gene flow between populations is limited, resulting in evolutionary histories similar to those of macroorganisms. However, population genomic approaches have seldom been applied to this question. Here, we analyse whole-genome resequencing data for all 36 confirmed field isolates of the green alga Chlamydomonas reinhardtii. At a continental scale, we report evidence for putative allopatric divergence, between both North American and Japanese isolates, and two highly differentiated lineages within N. America. Conversely, at a local scale within the most densely sampled lineage, we find little evidence for either spatial or temporal structure. Taken together with evidence for ongoing admixture between the two N. American lineages, this lack of structure supports a role for substantial dispersal in C. reinhardtii and implies that between-lineage differentiation may be maintained by reproductive isolation and/or local adaptation. Our results therefore support a role for allopatric divergence in microbial eukaryotes, while also indicating that species may be ubiquitous at local scales. Despite the high genetic diversity observed within the most well-sampled lineage, we find that pairs of isolates share on average ~9% of their genomes in long haplotypes, even when isolates were sampled decades apart and from different locations. This proportion is several orders of magnitude higher than the Wright-Fisher expectation, raising many further questions concerning the evolutionary genetics of C. reinhardtii and microbial eukaryotes generally.

Nanopore sequencing: Review of potential applications in functional genomics.

Molecular biology has been led by various measurement technologies, and increased throughput has developed omics analysis. The development of massively parallel sequencing technology has enabled access to fundamental molecular data and revealed genomic and transcriptomic signatures. Nanopore sequencers have driven such evolution to the next stage. Oxford Nanopore Technologies Inc. provides a new type of single molecule sequencer using protein nanopore that realizes direct sequencing without DNA synthesizing or amplification. This nanopore sequencer can sequence an ultra-long read limited by the input nucleotide length, or can determine DNA/RNA modifications. Recently, many fields such as medicine, epidemiology, ecology, and education have benefited from this technology. In this review, we explain the features and functions of the nanopore sequencer, introduce various situations where it has been used as a critical technology, and expected future applications.

Comparison of Sexual Reproductive Behaviors in Two Species of Macrobiotidae (Tardigrada: Eutardigrada).

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.

Molecular palaeontology illuminates the evolution of ecdysozoan vision.

Colour vision is known to have arisen only twice-once in Vertebrata and once within the Ecdysozoa, in Arthropoda. However, the evolutionary history of ecdysozoan vision is unclear. At the molecular level, visual pigments, composed of a chromophore and a protein belonging to the opsin family, have different spectral sensitivities and these mediate colour vision. At the morphological level, ecdysozoan vision is conveyed by eyes of variable levels of complexity; from the simple ocelli observed in the velvet worms (phylum Onychophora) to the marvellously complex eyes of insects, spiders, and crustaceans. Here, we explore the evolution of ecdysozoan vision at both the molecular and morphological level; combining analysis of a large-scale opsin dataset that includes previously unknown ecdysozoan opsins with morphological analyses of key Cambrian fossils with preserved eye structures. We found that while several non-arthropod ecdysozoan lineages have multiple opsins, arthropod multi-opsin vision evolved through a series of gene duplications that were fixed in a period of 35-71 million years (Ma) along the stem arthropod lineage. Our integrative study of the fossil and molecular record of vision indicates that fossils with more complex eyes were likely to have possessed a larger complement of opsin genes.

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the "Ohanami Project", to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

eRP arrangement: a strategy for assembled genomic contig rearrangement based on replication profiling in bacteria.

BACKGROUND: The reduced cost of sequencing has made de novo sequencing and the assembly of draft microbial genomes feasible in any ordinary biology lab. However, the process of finishing and completing the genome remains labor-intensive and computationally challenging in some cases, such as in the study of complete genome sequences, genomic rearrangements, long-range syntenic relationships, and structural variations. METHODS: Here, we show a contig reordering strategy based on experimental replication profiling (eRP) to recapitulate the bacterial genome structure within draft genomes. During the exponential growth phase, the majority of bacteria show a global genomic copy number gradient that is enriched near the replication origin and gradually declines toward the terminus. Therefore, if genome sequencing is performed with appropriate timing, the short-read coverage reflects this copy number gradient, providing information about the contig positions relative to the replication origin and terminus. RESULTS: We therefore investigated the appropriate timing for genomic DNA sampling and developed an algorithm for the reordering of the contigs based on eRP. As a result, this strategy successfully recapitulates the genomic structure of various structural mutants with draft genome sequencing. CONCLUSIONS: Our strategy was successful for contig rearrangement with intracellular DNA replication behavior mechanisms and can be applied to almost all bacteria because the DNA replication system is highly conserved. Therefore, eRP makes it possible to understand genomic structural information and long-range syntenic relationships using a draft genome that is based on short reads.

De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions.

BACKGROUND: The phytoflagellated protozoan, Euglena gracilis, has been proposed as an attractive feedstock for the accumulation of valuable compounds such as ß-1,3-glucan, also known as paramylon, and wax esters. The production of wax esters proceeds under anaerobic conditions, designated as wax ester fermentation. In spite of the importance and usefulness of Euglena, the genome and transcriptome data are currently unavailable, though another research group has recently published E.gracilis transcriptome study during our submission. We herein performed an RNA-Seq analysis to provide a comprehensive sequence resource and some insights into the regulation of genes including wax ester metabolism by comparative transcriptome analysis of E.gracilis under aerobic and anaerobic conditions. RESULTS: The E.gracilis transcriptome analysis was performed using the Illumina platform and yielded 90.3 million reads after the filtering steps. A total of 49,826 components were assembled and identified as a reference sequence of E.gracilis, of which 26,479 sequences were considered to be potentially expressed (having FPKM value of greater than 1). Approximately half of all components were estimated to be regulated in a trans-splicing manner, with the addition of protruding spliced leader sequences. Nearly 40 % of 26,479 sequences were annotated by similarity to Swiss-Prot database using the BLASTX program. A total of 2080 transcripts were identified as differentially expressed genes (DEGs) in response to anaerobic treatment for 24 h. A comprehensive pathway enrichment analysis using the KEGG pathway revealed that the majority of DEGs were involved in photosynthesis, nucleotide metabolism, oxidative phosphorylation, fatty acid metabolism. We successfully identified a candidate gene set of paramylon and wax esters, including novel ß-1,3-glucan and wax ester synthases. A comparative expression analysis of aerobic- and anaerobic-treated E.gracilis cells indicated that gene expression changes in these components were not extensive or dynamic during the anaerobic treatment. CONCLUSION: The RNA-Seq analysis provided comprehensive transcriptome information on E.gracilis for the first time, and this information will advance our understanding of this unique organism. The comprehensive analysis indicated that paramylon and wax ester metabolic pathways are regulated at post-transcriptional rather than the transcriptional level in response to anaerobic conditions.

Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes.

BACKGROUND: The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes. RESULTS: We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58× coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons. CONCLUSIONS: PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of "finished grade" because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.