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Abstract Background The Japanese cedar (Cryptomeria japonica D. Don) is one of the most important Japanese forest trees, occupying approximately 44% of artificial forests and planted in East Asia, the Azores Archipelago, and certain islands in the Indian Ocean. Although the huge genome of the species (ca. 9 Gbp) with abundant repeat elements may have represented an obstacle for genetic analysis, this species is easily propagated by cutting, flowered by gibberellic acid, transformed by Agrobacterium, and edited by CRISPR/Cas9. These characteristics of C. japonica recommend it as a model conifer species for which reference genome sequences are necessary. Results Herein, we report the first chromosome-level assembly of C. japonica (2n = 22) using third-generation selfed progeny (estimated homozygosity rate = 0.96). Young leaf tissue was used to extract high molecular weight DNA (> 50 kb) for HiFi PacBio long-read sequencing and to construct an Hi-C/Omni-C library for Illumina short-read sequencing. The 29× and 26× genome coverage of HiFi and Illumina reads, respectively, for de novo assembly yielded 2,651 contigs (9.1 Gbp, N50 contig size 12.0 Mbp). Hi-C analysis mapped 97% of the nucleotides on 11 chromosomes. The assembly was verified through comparison with a consensus linkage map comprising 7,781 markers. BUSCO analysis identified ∼ 91% conserved genes. Conclusions Annotations of genes and comparisons of repeat elements with other Cupressaceae and Pinaceae species provide a fundamental resource for conifer research.

Abstract Limb reduction has occurred multiple times in tetrapod history. Among ratites, wing reductions range from mild vestigialization to complete loss, with emus (Dromaius novaehollandiae) serving as a model for studying the genetic mechanisms behind limb reduction. Here, we explore the developmental mechanisms underlying wing reduction in emu. Our analyses reveal that immobilization resulting from the absence of distal muscles contributes to skeletal shortening, fusion and left-right intraindividual variation. Expression analysis and single cell-RNA sequencing identify muscle progenitors displaying a dual lateral plate mesodermal and myogenic signature. These cells aggregate at the proximal region of wing buds and undergo cell death. We propose that this cell death, linked to the lack of distal muscle masses, underlines the morphological features and variability in skeletal elements due to reduced mechanical loading. Our results demonstrate that differential mobility during embryonic development may drive morphological diversification in vestigial structures.

Abstract The unculturable nature of intracellular obligate symbionts presents a significant challenge for elucidating gene functionality, necessitating the development of gene manipulation techniques. One of the best-studied obligate symbioses is that between aphids and the bacterial endosymbiont Buchnera aphidicola. Given the extensive genome reduction observed in Buchnera, the remaining genes are crucial for understanding the host-symbiont relationship, but a lack of tools for manipulating gene function in the endosymbiont has significantly impeded the exploration of the molecular mechanisms underlying this mutualism. In this study, we introduced a novel gene manipulation technique employing synthetic single-stranded peptide nucleic acids (PNAs). We targeted the critical Buchnera groEL using specially designed antisense PNAs conjugated to an arginine-rich cell-penetrating peptide (CPP). Within 24 h of PNA administration via microinjection, we observed a significant reduction in groEL expression and Buchnera cell count. Notably, the interference of groEL led to profound morphological malformations in Buchnera, indicative of impaired cellular integrity. The gene knockdown technique developed in this study, involving the microinjection of CPP-conjugated antisense PNAs, provides a potent approach for in vivo gene manipulation of unculturable intracellular symbionts, offering valuable insights into their biology and interactions with hosts.

Orchids and arbuscular mycorrhiza (AM) plants evolved independently and have different structures and fungal partners, but they both facilitate nutrient uptake. Orchid mycorrhiza (OM) supports orchid seed germination, but unlike AM, its role in disease resistance of mature plants is largely unknown. Here, we examined whether OM induces systemic disease resistance against a necrotrophic pathogen in a similar fashion to AM. We investigated the priming effect of mycorrhizal fungi inoculation on resistance of a terrestrial orchid, Bletilla striata, to soft rot caused by Dickeya fangzhongdai. We found that root colonization by a compatible OM fungus primed B. striata seedlings and induced systemic resistance against the infection. Transcriptome analysis showed that priming was mediated by the downregulation of jasmonate and ethylene pathways and that these pathways are upregulated once infection occurs. Comparison with the reported transcriptome of AM fungus–colonized rice leaves revealed similar mechanisms in B. striata and in rice. These findings highlight a novel aspect of commonality between OM and AM plants in terms of induced systemic resistance.

Abstract Understanding the genetic basis for adapting to thermal environments is important due to serious effects of global warming on ectothermic species. Various genes associated with thermal adaptation in lizards have been identified mainly focusing on changes in gene expression or the detection of positively selected genes using coding regions. Only a few comprehensive genome‐wide analyses have included noncoding regions. This study aimed to identify evolutionarily conserved and accelerated genomic regions using whole genomes of eight Anolis lizard species that have repeatedly adapted to similar thermal environments in multiple lineages. Evolutionarily conserved genomic regions were extracted as regions with overall sequence conservation (regions with fewer base substitutions) across all lineages compared with the neutral model. Genomic regions that underwent accelerated evolution in the lineage of interest were identified as those with more base substitutions in the target branch than in the entire background branch. Conserved elements across all branches were relatively abundant in “intergenic” genomic regions among noncoding regions. Accelerated regions (ARs) of each lineage contained a significantly greater proportion of noncoding RNA genes than the entire multiple alignment. Common genes containing ARs within 5 kb of their vicinity in lineages with similar thermal habitats were identified. Many genes associated with circadian rhythms and behavior were found in hot‐open and cool‐shaded habitat lineages. These genes might play a role in contributing to thermal adaptation and assist future studies examining the function of genes involved in thermal adaptation via genome editing.

Abstract Aims Schizophrenia is a chronic relapsing psychiatric disorder that is characterized by many symptoms and has a high heritability. There were studies showing that the phospholipid abnormalities in subjects with schizophrenia (Front Biosci, S3, 2011, 153; Schizophr Bull, 48, 2022, 1125; Sci Rep, 7, 2017, 6; Anal Bioanal Chem, 400, 2011, 1933). Disturbances in prefrontal cortex phospholipid and fatty acid composition have been reported in subjects with schizophrenia (Sci Rep, 7, 2017, 6; Anal Bioanal Chem, 400, 2011, 1933; Schizophr Res, 215, 2020, 493; J Psychiatr Res, 47, 2013, 636; Int J Mol Sci, 22, 2021). For exploring the signaling pathways contributing to the lipid changes in previous study (Sci Rep, 7, 2017, 6), we performed two types of transcriptome analyses in subjects with schizophrenia: an unbiased transcriptome analysis solely based on RNA‐seq data and a correlation analysis between levels of gene expression and lipids. Methods RNA‐Seq analysis was performed in the postmortem prefrontal cortex from 10 subjects with schizophrenia and 5 controls. Correlation analysis between the transcriptome and lipidome from 9 subjects, which are the same samples in the previous lipidomics study (Sci Rep, 7, 2017, 6). Results Extraction of differentially expressed genes (DEGs) and further sequence and functional group analysis revealed changes in gene expression levels in phosphoinositide 3‐kinase (PI3K)‐Akt signaling and the complement system. In addition, a correlation analysis clarified alterations in ether lipid metabolism pathway, which is not found as DEGs in transcriptome analysis alone. Conclusions This study provided results of the integrated analysis of the schizophrenia‐associated transcriptome and lipidome within the PFC and revealed that lipid‐correlated alterations in the transcriptome are enriched in specific pathways including ether lipid metabolism pathway.

Gene expression patterns differ in different tissues, and the expression pattern of genes in the mammalian testis is known to be extremely variable in different species. To clarify how the testis transcriptomic pattern has evolved in particular species, we examined the evolution of the adult testis transcriptome in Theria using 10 species: two marsupials (opossum and Tasmanian devil), six eutherian (placental) mammals (human, chimpanzee, bonobo, gorilla, rhesus macaque, and mouse), and two outgroup species (platypus and chicken). We show that 22 testis-expressed genes are marsupial-specific, suggesting their acquisition in the stem lineage of marsupials after the divergence from eutherians. Despite the time length of the eutherian stem lineage being similar to that of the marsupial lineage, acquisition of testis-expressed genes was not found in the stem lineage of eutherians; rather, their expression patterns differed by species, suggesting rapid gene evolution in the eutherian ancestors. Fifteen testis-expressed genes are therian-specific, and for three of these genes, the evolutionary tempo is markedly faster in eutherians than in marsupials. Our phylogenetic analysis of Rho GTPase-activating protein 28 (ARHGAP28) suggests the adaptive evolution of this gene in the eutherians, probably together with the expression pattern differentiation.

For understanding the evolutionary mechanism of sexually selected exaggerated traits, it is essential to uncover its molecular basis. By using broad-horned flour beetle that has male-specific exaggerated structures (mandibular horn, head horn and gena enlargement), we investigated the transcriptomic and functional characters of sex-biased genes. Comparative transcriptome of male vs. female prepupal heads elucidated 673 sex-biased genes. Counter-intuitively, majority of them were female-biased (584 genes), and GO enrichment analysis showed cell-adhesion molecules were frequently female-biased. This pattern motivated us to hypothesize that female-biased transcripts (i.e. the transcripts diminished in males) may play a role in outgrowth formation. Potentially, female-biased genes may act as suppressors of weapon structure. In order to test the functionality of female-biased genes, we performed RNAi-mediated functional screening for top 20 female-biased genes and 3 genes in the most enriched GO term (cell-cell adhesion, fat1/2/3, fat4 and dachsous). Knockdown of one transcription factor, zinc finger protein 608 (zfp608) resulted in the formation of male-like gena in females, supporting the outgrowth suppression function of this gene. Similarly, knockdown of fat4 induced rudimental, abnormal mandibular horn in female. fat1/2/3RNAi, fat4RNAi and dachsousRNAi males exhibited thick and/or short mandibular horns and legs. These cell adhesion molecules are known to regulate tissue growth direction and known to be involved in the weapon formation in Scarabaeoidea beetles. Functional evidence in phylogenetically distant broad-horned flour beetle suggest that cell adhesion genes are repeatedly deployed in the acquisition of outgrowth. In conclusion, this study clarified the overlooked functions of female-biased genes in weapon development.

Abstract The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.

Abstract The snow crab, Chionoecetes opilio, is a giant deep-sea brachyuran. While several decapod crustaceans generally continue to molt and grow throughout their lifetime, the snow crab has a fixed number of molts. Adolescent males continue to molt proportionately to their previous size until the terminal molt at which time an allometric increase in chela size occurs and an alteration of behavioral activities occurs, ensuring breeding success. In this study, we investigated the circulating concentrations of methyl farnesoate (an innate juvenile hormone in decapods) (MF) before or after the terminal molt in males. We then conducted eyestalk RNAseq to obtain molecular insight into the regulation of physiological changes after the terminal molt. Our analyses revealed an increase in MF titers after the terminal molt. This MF surge may be caused by suppression of the genes that encode MF-degrading enzymes and mandibular organ-inhibiting hormone that negatively regulates MF biosynthesis. Moreover, our data suggests that behavioral changes after the terminal molt may be driven by the activation of biogenic amine-related pathways. These results are important not only for elucidating the physiological functions of MFs in decapod crustaceans, which are still largely unknown, but also for understanding the reproductive biology of the snow crab.

Abstract Background Detecting genomic variants and their accumulation processes during species diversification and adaptive radiation is important for understanding the molecular and genetic basis of evolution. Anolis lizards in the West Indies are good models for studying evolutionary mechanisms because of the repeated evolution of their morphology and the ecology. We performed de novo genome assembly of six Cuban Anolis lizards with different ecomorphs and thermal habitats (Anolis isolepis, Anolis allisoni, Anolis porcatus, Anolis allogus, Anolis homolechis, and Anolis sagrei). We carried out a comparative analysis of these genome assemblies to investigate the genetic changes that occurred during their diversification. Results We reconstructed novel draft genomes with relatively long scaffolds and high gene completeness, with the scaffold N50 ranging from 5.56 to 39.79 Mb and vertebrate Benchmarking Universal Single-Copy Orthologs completeness ranging from 77.5% to 86.9%. Comparing the repeat element compositions and landscapes revealed differences in the accumulation process between Cuban trunk-crown and trunk-ground species and separate expansions of several families of LINE in each Cuban trunk-ground species. Duplicated gene analysis suggested that the proportional differences in duplicated gene numbers among Cuban Anolis lizards may be associated with differences in their habitat ranges. Additionally, Pairwise Sequentially Markovian Coalescent analysis suggested that the effective population sizes of each species may have been affected by Cuba’s geohistory. Conclusions We provide draft genomes of six Cuban Anolis lizards and detected species and lineage-specific transposon accumulation and gene copy number changes that may be involved in adaptive evolution. The change processes in the past effective population size was also estimated, and the factors involved were inferred. These results provide new insights into the genetic basis of Anolis lizard diversification and are expected to serve as a stepping stone for the further elucidation of their diversification mechanisms.

Abstract Background In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), adventitious shoots can be induced simply by placing internodal segments on phytohormone-free culture medium. The shoots form locally on the epidermis of the apical region of the segments, but not the basal region. Levels of endogenous auxin and cytokinin transiently increase in the segments after 1 week of culture. Results Here, we conducted RNA-seq analysis to compare gene expression patterns in apical and basal regions of segments before culture and after 1 week of culture for adventitious shoot formation. The results revealed 8987 differentially expressed genes in a de novo assembly of 76,684 genes. Among them, 276 genes were upregulated in the apical region after 1 week of culture relative to before culture and the basal region after 1 week of culture. These genes include 18 phytohormone-response genes and shoot-formation-related genes. Validation of the gene expression by quantitative real-time PCR assay confirmed that the expression patterns were similar to those of the RNA-seq data. Conclusions The transcriptome data show that expression of cytokinin biosynthesis genes is induced along with the acquisition of cellular pluripotency and the initiation of cell division by wounding in the apical region of internodal segments, that trigger adventitious shoot formation without callusing.

Abstract Termite castes express specialized phenotypes for their own tasks and are a good example of insect polyphenism. To understand the comprehensive gene expression profiles during caste differentiation, RNA-seq analysis based on the genome data was performed during the worker, presoldier, and nymphoid molts in Reticulitermes speratus. In this species, artificial induction methods for each molt have already been established, and the time scale has been clarified. Three different periods (before the gut purge (GP), during the GP, and after the molt) were discriminated in each molt, and two body parts (head and other body regions) were separately sampled. The results revealed that many differentially expressed genes (head: 2884, body: 2579) were identified in each molt. Based on the independent real-time quantitative PCR analysis, we confirmed the different expression patterns of seven out of eight genes in the presoldier molt. Based on the GO and KEGG enrichment analyses, the expressions of genes related to juvenile hormone titer changes (e.g., JH acid methyltransferase), nutrition status (e.g., Acyl-CoA Delta desaturase), and cell proliferation (e.g., insulin receptor), were shown to specifically fluctuate in each molt. These differences may have a crucial impact on caste differentiation. These data are important resources for future termite sociogenomics.

Abstract Aphids have evolved bacteriocytes or symbiotic host cells that harbor the obligate mutualistic bacterium Buchnera aphidicola. Because of the large cell size (approximately 100 μm in diameter) of bacteriocytes and their pivotal role in nutritional symbiosis, researchers have considered that these cells are highly polyploid and assumed that bacteriocyte polyploidy may be essential for the symbiotic relationship between the aphid and the bacterium. However, little is known about the ploidy levels and dynamics of aphid bacteriocytes. Here, we quantitatively analyzed the ploidy levels in the bacteriocytes of the pea-aphid Acyrthosiphon pisum. Image-based fluorometry revealed the hyper polyploidy of the bacteriocytes ranging from 16- to 256-ploidy throughout the lifecycle. Bacteriocytes of adult parthenogenetic viviparous females were ranged between 64 and 128C DNA levels, while those of sexual morphs (oviparous females and males) were comprised of 64C, and 32–64C cells, respectively. During post-embryonic development of viviparous females, the ploidy level of bacteriocytes increased substantially, from 16 to 32C at birth to 128–256C in actively reproducing adults. These results suggest that the ploidy levels are dynamically regulated among phenotypes and during development. Our comprehensive and quantitative data provides a foundation for future studies to understand the functional roles and biological significance of the polyploidy of insect bacteriocytes.

Excess boron (B) is toxic to plants and thereby causes DNA damage and cell death in root meristems. However, the underlying mechanisms which link boron and DNA damage remain unclear. It has been reported that the rpt5a-6 mutant of the 26S proteasome is sensitive to excess boron, resulting in more frequent cell death in root meristem and reduced root elongation. In this study, we showed that a reduction in root growth in the rpt5a mutant in the presence of high boron levels is repressed by a mutation in NAC domain containing transcription factor NAC103, a substrate of the proteasome, which functions in the unfolded protein response pathway. The mutation in NAC103 alleviated excess-B-induced DNA damage and cell death in root meristems of the rpt5a mutant. Superoxide (O2−) staining with nitroblue tetrazolium revealed that boron stress causes O2− accumulation in root tips, which was higher in the rpt5a-6 mutant, whereas the accumulation was lower in the rpt5a-6 nac103-3 double mutant. Our work demonstrates the overall involvement of NAC103 in maintaining healthy root meristem under excess boron conditions in the absence of RPT5A proteasome subunit.

Abstract Background Despite the morphological diversity of animals, their basic anatomical patterns—the body plans in each animal phylum—have remained highly conserved over hundreds of millions of evolutionary years. This is attributed to conservation of the body plan-establishing developmental period (the phylotypic period) in each lineage. However, the evolutionary mechanism behind this phylotypic period conservation remains under debate. A variety of hypotheses based on the concept of modern synthesis have been proposed, such as negative selection in the phylotypic period through its vulnerability to embryonic lethality. Here we tested a new hypothesis that the phylotypic period is developmentally stable; it has less potential to produce phenotypic variations than the other stages, and this has most likely led to the evolutionary conservation of body plans. Results By analyzing the embryos of inbred Japanese medaka embryos raised under the same laboratory conditions and measuring the whole embryonic transcriptome as a phenotype, we found that the phylotypic period has greater developmental stability than other stages. Comparison of phenotypic differences between two wild medaka populations indicated that the phylotypic period and its genes in this period remained less variational, even after environmental and mutational modifications accumulated during intraspecies evolution. Genes with stable expression levels were enriched with those involved in cell-cell signalling and morphological specification such as Wnt and Hox, implying possible involvement in body plan development of these genes. Conclusions This study demonstrated the correspondence between the developmental stage with low potential to produce phenotypic variations and that with low diversity in micro- and macroevolution, namely the phylotypic period. Whereas modern synthesis explains evolution as a process of shaping of phenotypic variations caused by mutations, our results highlight the possibility that phenotypic variations are readily limited by the intrinsic nature of organisms, namely developmental stability, thus biasing evolutionary outcomes.

Summary: Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid, Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286 bp and 853,149 bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera–Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.

Abstract The sex determination gene doublesex (dsx) encodes a transcription factor with two domains, oligomerization domain 1 (OD1) and OD2, and is present throughout insects. Sex-specific Dsx splicing isoforms regulate the transcription of target genes and trigger sex differentiation in all Holometabola examined to date. However, in some hemimetabolous insects, dsx is not spliced sexually and its sequence is less conserved. Here, to elucidate evolutionary changes in dsx in domain organisation and regulation in termites, we searched genome and/or transcriptome databases for the dsx OD1 and OD2 in seven termite species and their sister group (Cryptocercus woodroaches). Molecular phylogenetic and synteny analyses identified OD1 sequences of termites and C. punctulatus that clustered with dsx of Holometabola and regarded them as dsx orthologues. The Cryptocercus dsx orthologue containing OD2 was spliced sexually, as previously shown in other insects. However, OD2 was not found in all termite dsx orthologues. These orthologues were encoded by a single exon in three termites for which genome information is available; they were not alternatively spliced but transcribed in a male-specific manner in two examined species. Evolution of dsx regulation from sex-specific splicing to male-specific transcription may have occurred at an early stage of social evolution in termites.

The organization of social insect colonies requires sophisticated mechanisms to regulate caste composition according to colony demands. In termites, the soldier caste is responsible for the inhibition of soldier differentiation, but the mechanism underlying the regulation of soldier differentiation is still unclear. In this study, we performed transcriptome analyses to identify genes expressed in workers that fluctuated in the presence of soldiers in the subterranean termite Reticulitermes speratus. First, soldier differentiation was artificially induced via juvenile hormone (JH) application, and the inhibitory effects of soldier differentiation on soldier presence were evaluated. Second, transcriptomes were prepared from workers with or without soldiers under JH treatment, and expression analyses were performed to identify differentially expressed genes (DEGs) for each treatment. The expression levels of several DEGs were verified by quantitative real-time PCR. The results indicated that only a small number of DEGs were upregulated by the presence of soldiers. A homology search of DEGs and gene ontology (GO) analysis of the DEGs showed that some genes were responsible for the regulation of hormone levels, social interaction, and response to xenobiotic substances, suggesting that they could be involved in developmental arrest and pheromonal regulation in workers. Moreover, GO analysis indicated that the expression of many genes, including those involved in hormone metabolic processes, fluctuated with JH application. Suppression of soldier differentiation in the presence of soldiers could be accomplished by the expression of a large number of genes required for soldier differentiation.

Abstract Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

Liverworts have a unique oil body organelle unrelated to lipid stores found in other eukaryotes. Here the authors show that oil body formation is analogous to that of cell plates, relying on periodic redirection of the secretory pathway and a syntaxin-1 homolog, and that oil bodies contribute to defence against herbivory.

AbstractThe enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiaeSchizosaccharomyces pombeSHOTA

Abstract Objective Due to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used to produce a pollen-free cultivar. In order to reduce the time and cost needed for production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica to rapidly identify pollen-free mutants. Results We developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. LAMP markers were developed for field stations, where PCR machines are unavailable. The LAMP method only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be read by the naked eye. Because the causative mutations were deletions, we developed two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers. These assays can be visualized using capillary or agarose gel electrophoresis.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5’-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5’-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

Morphotypes of arbuscular mycorrhizal (AM) symbiosis, Arum, Paris, and Intermediate types, are mainly determined by host plant lineages. It was reported that the phytohormone gibberellin (GA) inhibits the establishment of Arum-type AM symbiosis in legume plants. In contrast, we previously reported that GA promotes the establishment of Paris-type AM symbiosis in Eustoma grandiflorum, while suppressing Arum-type AM symbiosis in a legume model plant, Lotus japonicus. This raises a hitherto unexplored possibility that GA-mediated transcriptional reprogramming during AM symbiosis is different among plant lineages as the AM morphotypes are distinct. Here, our comparative transcriptomics revealed that several symbiosis-related genes were commonly upregulated upon AM fungal colonization in L. japonicus (Arum-type), Daucus carota (Intermediate-type), and E. grandiflorum (Paris-type). Despite of the similarities, the fungal colonization levels and the expression of symbiosis-related genes were suppressed in L. japonicus and D. carota but were promoted in E. grandiflorum in the presence of GA. Moreover, exogenous GA inhibited the expression of genes involved in biosynthetic process of the pre-symbiotic signal component, strigolactone, which resulted in the reduction of its endogenous accumulation in L. japonicus and E. grandiflorum. Additionally, differential regulation of genes involved in sugar metabolism suggested that disaccharides metabolized in AM roots would be different between L. japonicus and D. carota/E. grandiflorum. Therefore, this study uncovered the conserved transcriptional responses during mycorrhization regardless of the distinct AM morphotype. Meanwhile, we also found diverse responses to GA among phylogenetically distant AM host plants.

Stable endosymbiotic relationships between cnidarian animals and dinoflagellate algae are vital for sustaining coral reef ecosystems. Recent studies have shown that elevated seawater temperatures can cause the collapse of their endosymbiosis, known as ‘bleaching’, and result in mass mortality. However, the molecular interplay between temperature responses and symbiotic states still remains unclear. To identify candidate genes relevant to the symbiotic stability, we performed transcriptomic analyses under multiple conditions using the symbiotic and apo-symbiotic (symbiont free) Exaiptasia diaphana, an emerging model sea anemone. Gene expression patterns showed that large parts of differentially expressed genes in response to heat stress were specific to the symbiotic state, suggesting that the host sea anemone could react to environmental changes in a symbiotic state-dependent manner. Comparative analysis of expression profiles under multiple conditions highlighted candidate genes potentially important in the symbiotic state transition under heat-induced bleaching. Many of these genes were functionally associated with carbohydrate and protein metabolisms in lysosomes. Symbiont algal genes differentially expressed in hospite encode proteins related to heat shock response, calcium signaling, organellar protein transport, and sugar metabolism. Our data suggest that heat stress alters gene expression in both the hosts and symbionts. In particular, heat stress may affect the lysosome-mediated degradation and transportation of substrates such as carbohydrates through the symbiosome (phagosome-derived organelle harboring symbiont) membrane, which potentially might attenuate the stability of symbiosis and lead to bleaching-associated symbiotic state transition.

Abstract One of the most striking examples of phenotypic plasticity is the different phenotypes (i.e., castes) within a same nest of social insects. Castes usually derive from a single genotype initially by receiving social cues among individuals during development. Specific gene expression changes may be involved in caste differentiation, and thus, the regulatory mechanism of these changes should be clarified in order to understand social maintenance and evolution. The damp‐wood termite Zootermopsis nevadensis is one of the most important model termite species, due to not only the availability of genomic, transcriptomic, and epigenomic information but also evidence that soldier‐ and worker‐destined individuals can be identified in natural conditions. Given that the nutritional intakes via social interactions are crucial for caste differentiation in this species, there is a possibility that transcriptomic changes are influenced by the nutritional difference among these individuals. Here, whole body RNA‐seq analysis of 3rd‐instar larvae with biological replications and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted. We found the drastic expression differences during caste developments between soldier‐ and worker‐destined individuals. The results indicated that there are several key signaling pathways responsible for caste formations, which are involved in developments and social interactions. Particularly, the nutritional sensitive signaling was upregulated in soldier‐destined individuals, while some metabolic pathways were identified in worker‐destined individuals. These bioinformatic data obtained should be utilized to examine the molecular mechanisms of caste determination in social insects.

Root-knot nematodes (RKNs) are among the most devastating pests in agriculture. Solanum torvum Sw. (Turkey berry) has been used as a rootstock for eggplant (aubergine) cultivation because of its resistance to RKNs, including Meloidogyne incognita and M. arenaria. We previously found that a pathotype of M. arenaria, A2-J, is able to infect and propagate in S. torvum. In vitro infection assays showed that S. torvum induced the accumulation of brown pigments during avirulent pathotype A2-O infection, but not during virulent A2-J infection. This experimental system is advantageous because resistant and susceptible responses can be distinguished within a few days, and because a single plant genome can yield information about both resistant and susceptible responses. Comparative RNA-sequencing analysis of S. torvum inoculated with A2-J and A2-O at early stages of infection was used to parse the specific resistance and susceptible responses. Infection with A2-J did not induce statistically significant changes in gene expression within one day post-inoculation (DPI), but afterward, A2-J specifically induced the expression of chalcone synthase, spermidine synthase, and genes related to cell wall modification and transmembrane transport. Infection with A2-O rapidly induced the expression of genes encoding class III peroxidases, sesquiterpene synthases, and fatty acid desaturases at 1 DPI, followed by genes involved in defense, hormone signaling, and the biosynthesis of lignin at 3 DPI. Both isolates induced the expression of suberin biosynthetic genes, which may be triggered by wounding during nematode infection. Histochemical analysis revealed that A2-O, but not A2-J, induced lignin accumulation at the root tip, suggesting that physical reinforcement of cell walls with lignin is an important defense response against nematodes. The S. torvum-RKN system can provide a molecular basis for understanding plant-nematode interactions.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

The extent of cellular heterogeneity involved in neuronal regeneration after spinal cord injury (SCI) remains unclear. Therefore, we established stress-responsive transgenic zebrafish embryos with SCI. As a result, we found an SCI-induced cell population, termed SCI stress-responsive regenerating cells (SrRCs), essential for neuronal regeneration post-SCI. SrRCs were mostly composed of subtypes of radial glia (RGs-SrRCs) and neuron stem/progenitor cells (NSPCs-SrRCs) that are able to differentiate into neurons, and they formed a bridge across the lesion and connected with neighbouring undamaged motor neurons post-SCI. Compared to SrRCs at the caudal side of the SCI site (caudal-SrRCs), rostral-SrRCs participated more actively in neuronal regeneration. After RNA-seq analysis, we discovered that caveolin 1 (cav1) was significantly upregulated in rostral-SrRCs and that cav1 was responsible for the axonal regrowth and regenerative capability of rostral-SrRCs. Collectively, we define a specific SCI-induced cell population, SrRCs, involved in neuronal regeneration, demonstrate that rostral-SrRCs exhibit higher neuronal differentiation capability and prove that cav1 is predominantly expressed in rostral-SrRCs, playing a major role in neuronal regeneration after SCI.

Epigenetic DNA base methylation plays important roles in gene expression regulation. We here describe a gene expression regulation network consisting of many DNA methyltransferases each frequently changing its target sequence-specificity. Our object Helicobacter pylori, a bacterium responsible for most incidence of stomach cancer, carries a large and variable repertoire of sequence-specific DNA methyltransferases. By creating a dozen of single-gene knockout strains for the methyltransferases, we revealed that they form a network controlling methylome, transcriptome and adaptive phenotype sets. The methyltransferases interact with each other in a hierarchical way, sometimes regulated positively by one methyltransferase but negatively with another. Motility, oxidative stress tolerance and DNA damage repair are likewise regulated by multiple methyltransferases. Their regulation sometimes involves translation start and stop codons suggesting coupling of methylation, transcription and translation. The methyltransferases frequently change their sequence-specificity through gene conversion of their target recognition domain and switch their target sets to remodel the network. The emerging picture of a metamorphosing gene regulation network, or firework, consisting of epigenetic systems ever-changing their specificity in search for adaptation, provides a new paradigm in understanding global gene regulation and adaptive evolution.

Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and brain-transcriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing, and biting/striking occurred only during intervals between mouth-locking. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pair showed a strong gene expression correlation, whereas those in D20 fighting pairs showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in a grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in brain-transcriptomic synchronization (PIBS) suggested that this synchronization provides a physiological basis for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 group found genes enriched for ion transport, synaptic function, and learning and memory. Brain-transcriptomic synchronization could be a general phenomenon and may provide a new cornerstone with which to investigate coordinating and sustaining social interactions between two interacting partners of vertebrates.

Diverse animals, including insects, harbor microbial symbionts within their gut, body cavity, or cells. The subsocial parastrachiid stinkbug Parastrachia japonensis is well-known for its peculiar ecological and behavioral traits, including its prolonged non-feeding diapause period and maternal care of eggs/nymphs in an underground nest. P. japonensis harbors a specific bacterial symbiont within the gut cavity extracellularly, which is vertically inherited through maternal excretion of symbiont-containing white mucus. Thus far, biological roles of the symbiont in the host lifecycle has been little understood. Here we sequenced the genome of the uncultivable gut symbiont “Candidatus Benitsuchiphilus tojoi.” The symbiont has an 804 kb circular chromosome encoding 606 proteins and a 14.5 kb plasmid encoding 13 proteins. Phylogenetic analysis indicated that the bacterium is closely related to other obligate insect symbionts belonging to the Gammaproteobacteria, including Buchnera of aphids and Blochmannia of ants, and the most closely related to Ishikawaella, an extracellular gut symbiont of plataspid stinkbugs. These data suggested that the symbiont genome has evolved like highly reduced gamma-proteobacterial symbiont genomes reported from a variety of insects. The presence of genes involved in biosynthesis pathways for amino acids, vitamins, and cofactors in the genome implicated the symbiont as a nutritional mutualist, supplementing essential nutrients to the host. Interestingly, the symbiont’s plasmid encoded genes for thiamine and carotenoid synthesis pathways, suggesting the possibility of additional functions of the symbiont for protecting the host against oxidative stress and DNA damage. Finally, possible involvement of the symbiont in uric acid metabolism during diapause is discussed.

The harlequin ladybird beetle, Harmonia axyridis, has remarkable phenotypic diversity, with over 200 colour patterns. Here, Ando et al. show that this patterning is regulated by the transcription factor gene pannier and has diversified by repeated inversions and cis-regulatory modifications of pannier.

Autophagy is an essential system for degrading and recycling cellular components for survival during starvation conditions. Under sucrose starvation, application of a papain protease inhibitor E-64d to the Arabidopsis root and tobacco BY-2 cells induced the accumulation of vesicles, labeled with a fluorescent membrane marker FM4-64. The E-64d–induced vesicle accumulation was reduced in the mutant defective in autophagy-related genes ATG2, ATG5, and ATG7, suggesting autophagy is involved in the formation of these vesicles. To clarify the formation of these vesicles in detail, we monitored time-dependent changes of tonoplast, and vesicle accumulation in sucrose-starved cells. We found that these vesicles were derived from the tonoplast and produced by microautophagic process. The tonoplast proteins were excluded from the vesicles, suggesting that the vesicles are generated from specific membrane domains. Concanamycin A treatment in GFP-ATG8a transgenic plants showed that not all FM4-64–labeled vesicles, which were derived from the tonoplast, contained the ATG8a-containing structure. These results suggest that ATG8a may not always be necessary for microautophagy.

Abstract Background Mycorrhizal symbiosis is one of the most fundamental types of mutualistic plant-microbe interaction. Among the many classes of mycorrhizae, the arbuscular mycorrhizae have the most general symbiotic style and the longest history. However, the genomes of arbuscular mycorrhizal (AM) fungi are not well characterized due to difficulties in cultivation and genetic analysis. In this study, we sequenced the genome of the AM fungus Rhizophagus clarus HR1, compared the sequence with the genome sequence of the model species R. irregularis, and checked for missing genes that encode enzymes in metabolic pathways related to their obligate biotrophy. Results In the genome of R. clarus, we confirmed the absence of cytosolic fatty acid synthase (FAS), whereas all mitochondrial FAS components were present. A KEGG pathway map identified the absence of genes encoding enzymes for several other metabolic pathways in the two AM fungi, including thiamine biosynthesis and the conversion of vitamin B6 derivatives. We also found that a large proportion of the genes encoding glucose-producing polysaccharide hydrolases, that are present even in ectomycorrhizal fungi, also appear to be absent in AM fungi. Conclusions In this study, we found several new genes that are absent from the genomes of AM fungi in addition to the genes previously identified as missing. Missing genes for enzymes in primary metabolic pathways imply that AM fungi may have a higher dependency on host plants than other biotrophic fungi. These missing metabolic pathways provide a genetic basis to explore the physiological characteristics and auxotrophy of AM fungi.

Abstract Females of social Hymenoptera only mate at the beginning of their adult lives and produce offspring until their death. In most ant species, queens live for over a decade, indicating that ant queens can store large numbers of spermatozoa throughout their long lives. To reveal the prolonged sperm storage mechanisms, we identified enriched genes in the sperm-storage organ (spermatheca) relative to those in body samples in Crematogaster osakensis queens using the RNA-sequencing method. The genes encoding antioxidant enzymes, proteases, and extracellular matrix-related genes, and novel genes that have no similar sequences in the public databases were identified. We also performed differential expression analyses between the virgin and mated spermathecae or between the spermathecae at 1-week and 1-year after mating, to identify genes altered by the mating status or by the sperm storage period, respectively. Gene Ontology enrichment analyses suggested that antioxidant function is enhanced in the spermatheca at 1-week after mating compared with the virgin spermatheca and the spermatheca at 1-year after mating. In situ hybridization analyses of 128 selected contigs revealed that 12 contigs were particular to the spermatheca. These genes have never been reported in the reproductive organs of insect females, suggesting specialized roles in ant spermatheca.

Aquaculture industries are under threat from noxious red tides, but harm can be mitigated by precautions such as early harvesting and restricting fish feeding to just before the outbreak of a red tide. Therefore, accurate techniques for forecasting red-tide outbreaks are strongly needed. Omics analyses have the potential to expand our understanding of the eco-physiology of these organisms at the molecular level, and to facilitate identification of molecular markers for forecasting their population dynamics and occurrence of damages to fisheries. Red tides of marine raphidophytes, especially Chattonella species, often extensively harm aquaculture industries in regions with a temperate climate around the world. A red tide of Chattonella tends to develop just after an input of nutrients along the coast. Chattonella displays diurnal vertical migration regulated by a weak blue light, so it photosynthesizes in the surface layer during the daytime and takes up nutrients in the bottom layer during the nighttime. Superoxide produced by Chattonella cells is a strong candidate for the cause of its toxicity to bacteria and fishes. Here we conducted mRNA-seq of Chattonella antiqua to identify genes with functions closely related to the dynamics of the noxious red tide, such as photosynthesis, photoreception, nutrient uptake, and superoxide production. The genes related to photosynthetic pigment biosynthesis and nutrient uptake had high similarity with those of model organisms of plants and algae and other red-tide microalgae. We identified orthologous genes of photoreceptors such as aureochrome (newly five genes), the cryptochrome/photolyase (CRY/PHR) family (6-4PHR, plant CRY or cyclobutane pyrimidine dimer [CPD] Class III, CPD Class II, and CRY-DASH), and phytochrome (four genes), which regulate various physiological processes such as flagellar motion and cell cycle in model organisms. Six orthologous genes of NADPH oxidase, which produces superoxide on the cell membrane, were found and divided into two types: one with 5–6 transmembrane domains and another with 11 transmembrane domains. The present study should open the way for analyzing the eco-physiological features of marine raphidophytes at the molecular level.

Many scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex differentiation gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.

Mammalian hibernators store fat extensively in white adipose tissues (WATs) during pre-hibernation period (Pre-HIB) to prepare for hibernation. However, the molecular mechanisms underlying the pre-hibernation remodeling of WAT have not been fully elucidated. Syrian hamsters, a food-storing hibernator, can hibernate when exposed to a winter-like short day photoperiod and cold ambient temperature (SD-Cold). Animals subjected to prolonged SD-Cold had smaller white adipocytes and beige-like cells within subcutaneous inguinal WAT (iWAT). Time-course analysis of gene expression with RNA-sequencing and quantitative PCR demonstrated that the mRNA expression of not only genes involved in lipid catabolism (lipolysis and beta-oxidation) but also lipid anabolism (lipogenesis and lipid desaturation) was simultaneously up-regulated prior to hibernation onset in the animals. The enhanced capacity of both lipid catabolism and lipid anabolism during hibernation period (HIB) is striking contrast to previous observations in fat-storing hibernators that only enhance catabolism during HIB. The mRNA expression of mTORC1 and PPAR signaling molecules increased, and pharmacological activation of PPARs indeed up-regulated lipid metabolism genes in iWAT explants from Syrian hamsters. These results suggest that the Syrian hamster rewires lipid metabolisms while preparing for hibernation to effectively utilize body fat and synthesize it from food intake during HIB.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-02005-4

Beetle horns are attractive models for studying the evolution of novel traits, as they display diverse shapes, sizes, and numbers among closely related species within the family Scarabaeidae. Horns radiated prolifically and independently in two distant subfamilies of scarabs, the dung beetles (Scarabaeinae), and the rhinoceros beetles (Dynastinae). However, current knowledge of the mechanisms underlying horn diversification remains limited to a single genus of dung beetles, Onthophagus. Here we unveil 11 horn formation genes in a rhinoceros beetle, Trypoxylus dichotomus. These 11 genes are mostly categorized as larval head- and appendage-patterning genes that also are involved in Onthophagus horn formation, suggesting the same suite of genes was recruited in each lineage during horn evolution. Although our RNAi analyses reveal interesting differences in the functions of a few of these genes, the overwhelming conclusion is that both head and thoracic horns develop similarly in Trypoxylus and Onthophagus, originating in the same developmental regions and deploying similar portions of appendage patterning networks during their growth. Our findings highlight deep parallels in the development of rhinoceros and dung beetle horns, suggesting either that both horn types arose in the common ancestor of all scarabs, a surprising reconstruction of horn evolution that would mean the majority of scarab species (~35,000) actively repress horn growth, or that parallel origins of these extravagant structures resulted from repeated co-option of the same underlying developmental processes.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.

ABSTRACT Members of the genus Rickettsiella are bacterial pathogens of insects and other arthropods. Recently, a novel facultative endosymbiont, “Candidatus Rickettsiella viridis,” was described in the pea aphid Acyrthosiphon pisum, whose infection causes a striking host phenotype: red and green genetic color morphs exist in aphid populations, and upon infection with the symbiont, red aphids become green due to increased production of green polycyclic quinone pigments. Here we determined the complete genome sequence of the symbiont. The 1.6-Mb circular genome, harboring some 1,400 protein-coding genes, was similar to the genome of entomopathogenic Rickettsiella grylli (1.6 Mb) but was smaller than the genomes of phylogenetically allied human pathogens Coxiella burnetii (2.0 Mb) and Legionella pneumophila (3.4 Mb). The symbiont’s metabolic pathways exhibited little complementarity to those of the coexisting primary symbiont Buchnera aphidicola, reflecting the facultative nature of the symbiont. The symbiont genome harbored neither polyketide synthase genes nor the evolutionarily allied fatty acid synthase genes that are suspected to catalyze the polycyclic quinone synthesis, indicating that the green pigments are produced not by the symbiont but by the host aphid. The symbiont genome retained many type IV secretion system genes and presumable effector protein genes, whose homologues in L. pneumophila were reported to modulate a variety of the host's cellular processes for facilitating infection and virulence. These results suggest the possibility that the symbiont is involved in the green pigment production by affecting the host’s metabolism using the secretion machineries for delivering the effector molecules into the host cells. IMPORTANCE Insect body color is relevant to a variety of biological aspects such as species recognition, sexual selection, mimicry, aposematism, and crypsis. Hence, the bacterial endosymbiont “Candidatus Rickettsiella viridis,” which alters aphid body color from red to green, is of ecological interest, given that different predators preferentially exploit either red- or green-colored aphids. Here we determined the complete 1.6-Mb genome of the symbiont and uncovered that, although the red-green color transition was ascribed to upregulated production of green polycyclic quinone pigments, the symbiont genome harbored few genes involved in the polycyclic quinone biosynthesis. Meanwhile, the symbiont genome contained type IV secretion system genes and presumable effector protein genes, whose homologues modulate eukaryotic cellular processes for facilitating infection and virulence in the pathogen Legionella pneumophila. We propose the hypothesis that the symbiont may upregulate the host’s production of polycyclic quinone pigments via cooption of secretion machineries and effector molecules for pathogenicity.

Speciation is a continuous process and analysis of species pairs at different stages of divergence provides insight into how it unfolds. Previous genomic studies on young species pairs have revealed peaks of divergence and heterogeneous genomic differentiation. Yet less known is how localised peaks of differentiation progress to genome-wide divergence during the later stages of speciation in the presence of persistent gene flow. Spanning the speciation continuum, stickleback species pairs are ideal for investigating how genomic divergence builds up during speciation. However, attention has largely focused on young postglacial species pairs, with little knowledge of the genomic signatures of divergence and introgression in older stickleback systems. The Japanese stickleback species pair, composed of the Pacific Ocean three-spined stickleback (Gasterosteus aculeatus) and the Japan Sea stickleback (G. nipponicus), which co-occur in the Japanese islands, is at a late stage of speciation. Divergence likely started well before the end of the last glacial period and crosses between Japan Sea females and Pacific Ocean males result in hybrid male sterility. Here we use coalescent analyses and Approximate Bayesian Computation to show that the two species split approximately 0.68-1 million years ago but that they have continued to exchange genes at a low rate throughout divergence. Population genomic data revealed that, despite gene flow, a high level of genomic differentiation is maintained across the majority of the genome. However, we identified multiple, small regions of introgression, occurring mainly in areas of low recombination rate. Our results demonstrate that a high level of genome-wide divergence can establish in the face of persistent introgression and that gene flow can be localized to small genomic regions at the later stages of speciation with gene flow.

A working knowledge of the proximate factors intrinsic to sterile caste differentiation is necessary to understand the evolution of eusocial insects. Genomic and transcriptomic analyses in social hymenopteran insects have resulted in the hypothesis that sterile castes are generated by the novel function of co-opted or recruited universal gene networks found in solitary ancestors. However, transcriptome analysis during caste differentiation has not been tested in termites, and evolutionary processes associated with acquiring the caste are still unknown. Termites possess the soldier caste, which is regarded as the first acquired permanently sterile caste in the taxon. In this study, we performed a comparative transcriptome analysis in termite heads during 3 molting processes, i.e., worker, presoldier and soldier molts, under natural conditions in an incipient colony of the damp-wood termite Zootermopsis nevadensis. Although similar expression patterns were observed during each molting process, more than 50 genes were shown to be highly expressed before the presoldier (intermediate stage of soldier) molt. We then performed RNA interference (RNAi) of the candidate 13 genes, including transcription factors and uncharacterized protein genes, during presoldier differentiation induced by juvenile hormone (JH) analog treatment. Presoldiers induced after RNAi of two genes related to TGFβ (Transforming growth factor beta) signaling were extremely unusual and possessed soldier-like phenotypes. These individuals also displayed aggressive behaviors similar to natural soldiers when confronted with Formica ants as hypothetical enemies. These presoldiers never molted into the next instar, presumably due to the decreased expression levels of the molting hormone (20-hydroxyecdysone; 20E) signaling genes. These results suggest that TGFβ signaling was acquired for the novel function of regulating between JH and 20E signaling during soldier differentiation in termites.

Local regulation of synaptic efficacy is thought to be important for proper networking of neurons and memory formation. Dysregulation of global translation influences long-term memory in mice, but the relevance of the regulation specific for local translation by RNA granules remains elusive. Here, we demonstrate roles of RNG105/caprin1 in long-term memory formation. RNG105 deletion in mice impaired synaptic strength and structural plasticity in hippocampal neurons. Furthermore, RNG105-deficient mice displayed unprecedentedly severe defects in long-term memory formation in spatial and contextual learning tasks. Genome-wide profiling of mRNA distribution in the hippocampus revealed an underlying mechanism: RNG105 deficiency impaired the asymmetric somato-dendritic localization of mRNAs. Particularly, RNG105 deficiency reduced the dendritic localization of mRNAs encoding regulators of AMPAR surface expression, which was consistent with attenuated homeostatic AMPAR scaling in dendrites and reduced synaptic strength. Thus, RNG105 has an essential role, as a key regulator of dendritic mRNA localization, in long-term memory formation.

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.