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.
Eukaryotic cells acquired novel organelles during evolution through mechanisms that remain largely obscure. The existence of the unique oil body compartment is a synapomorphy of liverworts that ...represents lineage-specific acquisition of this organelle during evolution, although its origin, biogenesis, and physiological function are yet unknown. We find that two paralogous syntaxin-1 homologs in the liverwort Marchantia polymorpha are distinctly targeted to forming cell plates and the oil body, suggesting that these structures share some developmental similarity. Oil body formation is regulated by an ERF/AP2-type transcription factor and loss of the oil body increases M. polymorpha herbivory. These findings highlight a common strategy for the acquisition of organelles with distinct functions in plants, via periodical redirection of the secretory pathway depending on cellular phase transition.
Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their ...inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle’s cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella’s sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host’s cuticle formation and hardening. Notably, Nardonella’s tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host’s aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host’s final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host–symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.
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.
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.
Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal ...immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug
, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont,
Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.
Differentiation of the non-reproductive caste is a unique feature of eusocial insects. Apoptosis in oocytes plays a major role in constraining the reproductivity of the eusocial insects including ...bees, ants, and termites. However, the regulation of reproductive constraint in non-reproductives of primitively eusocial insects other than hymenopterans and blattodeans is almost unknown. Here, we investigated the soldier sterility in a hemipteran insect, the social aphid Ceratovacuna japonica. We compared the gonads of soldiers, that are completely sterile, with those of reproductives in their viviparous development. We found that soldiers possess a pair of ovaries and the same number of germaria as reproductives, but soldiers’ ovarioles were small and lacking gastrulating embryos. Unlike in most model social insects, the staining of cleaved Caspase-3 showed apoptosis in the maternal nutritive cells, rather in the oocyte, of soldier ovaries. In addition, the ubiquitous C. japonica vasa1 and piwi2a expression indicates the developmental failure of embryos in soldier ovaries. The absence of posterior nos1, an insect posterior determinant, indicates deficient posterior patterning in soldier ovarioles. Our findings suggest a different mode of reproductive constraint, which regulates both oogenesis and embryogenesis in a viviparous insect ovary. This is the first report of the reproductive constraint in a viviparous social insect at the molecular level.
Schematic illustration of cell death and development in the ovarioles of reproductive and soldier aphids. Ovarioles came from first-instar reproductives and soldiers. Signals of markers are indicated by the colour keys shown at the bottom right of each column in the illustration. Colour intensities of markers correspond to their expression levels. The anterior region of the ovariole is to the left. The staging of embryos in reproductive ovarioles followed that of the pea aphid described by Miura et al. (2003). In the reproductive ovarioles, signals of Casp-3 and PI are not detectable. Cjvas1 and Cjpiwi2a are expressed in the germarium, oocytes, and PGCs. Cjnos1 is expressed in the germarium, oocytes, embryos, and PGCs, with posterior localization in the embryo. In soldier ovarioles, Casp-3 signals are detected in the nurse cells of the germarium and follicle cells of the embryo. PI is universally detected in the ovarioles. Cjvas1 and Cjpiwi2a are ubiquitously expressed in the germarium, oocytes, and embryos. Cjnos1 is expressed in the germarium and oocyte but is not detectable in the embryos. Display omitted
•Viviparous reproductive constraint was investigated in primitively eusocial aphids.•Regulation of ovariole number does not contribute to soldier aphid sterility.•Soldier reproduction is constrained by apoptosis in maternal nutritive cells.•Necrosis results in degeneration of oocytes and embryos in soldier aphid ovaries.•Embryos in soldier ovaries ectopically express germline genes.
The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional ...regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.
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•ALaP-seq to profile PML body-associated regions•PML bodies frequently associate with Yp locus to regulate Y-linked genes•PML bodies exclude DNMT3A to maintain hypo-methylated state of associated promoters
Kurihara et al. determined genetic loci associated with PML bodies in mouse ESCs by developing APEX-mediated chromatin labeling and purification (ALaP). PML bodies frequently associate with the short arm of the Y chromosome to regulate clustered Y-linked genes by maintaining hypo-methylated DNA through excluding DNMT3A.
Termites have colonized many habitats and are among the most abundant animals in tropical ecosystems, which they modify considerably through their actions. The timing of their rise in abundance and ...of the dispersal events that gave rise to modern termite lineages is not well understood. To shed light on termite origins and diversification, we sequenced the mitochondrial genome of 48 termite species and combined them with 18 previously sequenced termite mitochondrial genomes for phylogenetic and molecular clock analyses using multiple fossil calibrations. The 66 genomes represent most major clades of termites. Unlike previous phylogenetic studies based on fewer molecular data, our phylogenetic tree is fully resolved for the lower termites. The phylogenetic positions of Macrotermitinae and Apicotermitinae are also resolved as the basal groups in the higher termites, but in the crown termitid groups, including Termitinae + Syntermitinae + Nasutitermitinae + Cubitermitinae, the position of some nodes remains uncertain. Our molecular clock tree indicates that the lineages leading to termites and Cryptocercus roaches diverged 170 Ma (153-196 Ma 95% confidence interval CI), that modern Termitidae arose 54 Ma (46-66 Ma 95% CI), and that the crown termitid group arose 40 Ma (35-49 Ma 95% CI). This indicates that the distribution of basal termite clades was influenced by the final stages of the breakup of Pangaea. Our inference of ancestral geographic ranges shows that the Termitidae, which includes more than 75% of extant termite species, most likely originated in Africa or Asia, and acquired their pantropical distribution after a series of dispersal and subsequent diversification events.
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.
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.
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.
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