Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR.

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.

Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel.

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.

Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades.

Tardigrades are small aquatic animals known for the tolerant ability against various extreme stresses. Recent studies identified several tardigrade-unique proteins as protective factors of biomolecules from extreme stresses. Due to the limitation of the technique available in tardigrades, the function of these protective molecules has largely been studied utilizing the systems of in vitro and the heterologous expression in other organisms. Although RNAi is feasible in tardigrades, their effects are variable and not always sufficient. To analyze the functions of the tardigrade protective proteins, in vivo genetic manipulations have been desired. In this study, we used a tardigrade Hypsibius exemplaris as a model whose genome is available, and developed the delivery method of Cas9 ribonucleoproteins (RNPs) to adult tardigrade cells. Cas9 RNPs containing two kinds of crRNAs were injected to the body cavity of adult tardigrades and subjected to the subsequent electroporation to facilitate the incorporation of RNPs to the cells. Using this delivery method, we detected the deletion of the intervening region between two crRNAs from the genome. Intriguingly, all examined joining sites exhibited no incorporation of insertions/deletions (indels), suggesting that no-indel end-joining is dominant repair system in this tardigrade. We also detected similar removal of the intervening region even in the tardigrades injected with Cas9 RNPs without electroporation and in this case the no-indel end-joining is detected in still dominant but not all examined joining sites. This study provides the development of the delivery method of Cas9 RNPs to tardigrade cells and our data also suggested that simultaneous application of more than two crRNAs/gRNAs are recommended to disrupt the target gene by CRISPR/Cas9 system to avoid scarless repair in the tardigrade.

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 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.

Mblk-1 Transcription Factor Family: Its Roles in Various Animals and Regulation by NOL4 Splice Variants in Mammals.

Transcription factors play critical roles in regulation of neural development and functions. A transcription factor Mblk-1 was previously reported from a screen for factors possibly important for the higher brain functions of the honeybee. This review first summarizes how Mblk-1 was identified, and then provides an overview of the studies of Mblk-1 and their homologs. Mblk-1 family proteins are found broadly in animals and are shown to affect transcription activities. Studies have revealed that the mammalian homologs can interact with several cofactors and together regulate transcription. Interestingly, a recent study using the mouse homologs, Mlr1 and Mlr2, showed that one of their cofactor proteins, NOL4, have several splice variants with different effects on the transactivation activities of Mlr proteins. These findings suggest that there is an additional layer of the regulation of Mblk-1 family proteins by cofactor splice variants and provide novel insights into our current understanding of the roles of the conserved transcription factor family.

Acute phase response in amputated tail stumps and neural tissue-preferential expression in tail bud embryos of the Xenopus neuronal pentraxin I gene.

Regeneration of lost organs involves complex processes, including host defense from infection and rebuilding of lost tissues. We previously reported that Xenopus neuronal pentraxin I (xNP1) is expressed preferentially in regenerating Xenopus laevis tadpole tails. To evaluate xNP1 function in tail regeneration, and also in tail development, we analyzed xNP1 expression in tailbud embryos and regenerating/healing tails following tail amputation in the 'regeneration' period, as well as in the 'refractory' period, when tadpoles lose their tail regenerative ability. Within 10 h after tail amputation, xNP1 was induced at the amputation site regardless of the tail regenerative ability, suggesting that xNP1 functions in acute phase responses. xNP1 was widely expressed in regenerating tails, but not in the tail buds of tailbud embryos, suggesting its possible role in the immune response/healing after an injury. xNP1 expression was also observed in neural tissues/primordia in tailbud embryos and in the spinal cord in regenerating/healing tails in both periods, implying its possible roles in neural development or function. Moreover, during the first 48 h after amputation, xNP1 expression was sustained at the spinal cord of tails in the 'regeneration' period tadpoles, but not in the 'refractory' period tadpoles, suggesting that xNP1 expression at the spinal cord correlates with regeneration. Our findings suggest that xNP1 is involved in both acute phase responses and neural development/functions, which is unique compared to mammalian pentraxins whose family members are specialized in either acute phase responses or neural functions.

Splicing variants of NOL4 differentially regulate the transcription activity of Mlr1 and Mlr2 in cultured cells.

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'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.

Sexual dimorphism in the tardigrade Paramacrobiotus metropolitanus transcriptome.

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.

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

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

A comparative ultrastructure study of the tardigrade Ramazzottius varieornatus in the hydrated state, after desiccation and during the process of rehydration.

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.

Proteomic analysis of the royal jelly and characterization of the functions of its derivation glands in the honeybee.

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.

Expression analysis of XPhyH-like during development and tail regeneration in Xenopus tadpoles: possible role of XPhyH-like expressing immune cells in impaired tail regenerative ability.

Xenopus tadpoles have high regenerative ability of amputated tails except during the 'refractory period', 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 'non-self' 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 'non-self'.

Identification of kakusei, a nuclear non-coding RNA, as an immediate early gene from the honeybee, and its application for neuroethological study.

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.

Description of a model tardigrade Paramacrobiotus metropolitanus sp. nov. (Eutardigrada) from Japan with a summary of its life history, reproduction and genomics.

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 IIII, 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: 0.981.12%, 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.

Mblk-1/E93, an ecdysone related-transcription factor, targets synaptic plasticity-related genes in the honey bee mushroom bodies.

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.

Loss of genes for DNA recombination and repair in the reductive genome evolution of thioautotrophic symbionts of Calyptogena clams.

BACKGROUND: Two Calyptogena clam intracellular obligate symbionts, Ca. Vesicomyosocius okutanii (Vok; C. okutanii symbiont) and Ca. Ruthia magnifica (Rma; C. magnifica symbiont), have small genomes (1.02 and 1.16 Mb, respectively) with low G+C contents (31.6% and 34.0%, respectively) and are thought to be in an ongoing stage of reductive genome evolution (RGE). They lack recA and some genes for DNA repair, including mutY. The loss of recA and mutY is thought to contribute to the stabilization of their genome architectures and GC bias, respectively. To understand how these genes were lost from the symbiont genomes, we surveyed these genes in the genomes from 10 other Calyptogena clam symbionts using the polymerase chain reaction (PCR). RESULTS: Phylogenetic trees reconstructed using concatenated 16S and 23S rRNA gene sequences showed that the symbionts formed two clades, clade I (symbionts of C. kawamurai, C. laubieri, C. kilmeri, C. okutanii and C. soyoae) and clade II (those of C. pacifica, C. fausta, C. nautilei, C. stearnsii, C. magnifica, C. fossajaponica and C. phaseoliformis). recA was detected by PCR with consensus primers for recA in the symbiont of C. phaseoliformis. A detailed homology search revealed a remnant recA in the Rma genome. Using PCR with a newly designed primer set, intact recA or its remnant was detected in clade II symbionts. In clade I symbionts, the recA coding region was found to be mostly deleted.In the Rma genome, a pseudogene of mutY was found. Using PCR with newly designed primer sets, mutY was not found in clade I symbionts but was found in clade II symbionts. The G+C content of 16S and 23S rRNA genes in symbionts lacking mutY was significantly lower than in those with mutY. CONCLUSIONS: The extant Calyptogena clam symbionts in clade II were shown to have recA and mutY or their remnants, while those in clade I did not. The present results indicate that the extant symbionts are losing these genes in RGE, and that the loss of mutY contributed to the GC bias of the genomes during their evolution.

Parallel evolution of trehalose production machinery in anhydrobiotic animals via recurrent gene loss and horizontal transfer.

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.

Publisher Correction: Developmental stage-specific distribution and phosphorylation of Mblk-1, a transcription factor involved in ecdysteroid-signaling in the honey bee brain.

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