Intracellularity, extracellularity, and squeezing in the symbiotic organ underpin nurturing and functioning of bacterial symbiont in leaf beetles.

Cassidine leaf beetles are associated with genome-reduced symbiotic bacteria Stammera involved in pectin digestion. Stammera cells appear to be harbored in paired symbiotic organs located at the foregut-midgut junction either intracellularly or extracellularly, whereas the symbiont is extracellular in the ovary-accessory glands of adult females and during caplet transmission in eggs. However, using fluorescence and electron microscopy, an intracellular symbiotic configuration of Stammera was observed in Notosacantha species. Detailed inspection of other cassidine species revealed fragmented cell membrane and cytoplasm of the symbiotic organs, wherein Stammera cells are in an intermediate status between intracellularity and extracellularity. We also identified a mitochondria-rich region adjacent to the symbiont-filled region and well-developed muscle fibers surrounding the whole symbiotic organ. Based on these observations, we discuss why the Stammera genome has been reduced so drastically and how symbiont-derived pectinases are produced and supplied to the host's alimentary tract for plant cell wall digestion.

Self-stabilization mechanism encoded by a bacterial toxin facilitates reproductive parasitism.

A wide variety of maternally transmitted endosymbionts in insects are associated with reproductive parasitism, whereby they interfere with host reproduction to increase the ratio of infected females and spread within populations.1,2 Recent successes in identifying bacterial factors responsible for reproductive parasitism3,4,5,6,7 as well as further omics approaches8,9,10,11,12 have highlighted the common appearance of deubiquitinase domains, although their biological roles-in particular, how they link to distinct manipulative phenotypes-remain poorly defined. Spiroplasma poulsonii is a helical and motile bacterial endosymbiont of Drosophila,13,14 which selectively kills male progeny with a male-killing toxin Spaid (S. poulsonii androcidin), which encodes an ovarian tumor (OTU) deubiquitinase domain.6 Artificial expression of Spaid in flies reproduces male-killing-associated pathologies that include abnormal apoptosis and neural defects during embryogenesis6,15,16,17,18,19; moreover, it highly accumulates on the dosage-compensated male X chromosome,20 congruent with cellular defects such as the DNA damage/chromatin bridge breakage specifically induced upon that chromosome.6,21,22,23 Here, I show that without the function of OTU, Spaid is polyubiquitinated and degraded through the host ubiquitin-proteasome pathway, leading to the attenuation of male-killing activity as shown previously.6 Furthermore, I find that Spaid utilizes its OTU domain to deubiquitinate itself in an intermolecular manner. Collectively, the deubiquitinase domain of Spaid serves as a self-stabilization mechanism to facilitate male killing in flies, optimizing a molecular strategy of endosymbionts that enables the efficient manipulation of the host at a low energetic cost.

Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality.

Some Wolbachia endosymbionts induce male killing, whereby male offspring of infected females are killed during development; however, the origin and diversity of the underlying mechanisms remain unclear. In this study, we identified a 76 kbp prophage region specific to male-killing Wolbachia hosted by the moth Homona magnanima. The prophage encoded a homolog of the male-killing gene oscar in Ostrinia moths and the wmk gene that induces various toxicities in Drosophila melanogaster. Upon overexpressing these genes in D. melanogaster, wmk-1 and wmk-3 killed all males and most females, whereas Hm-oscar, wmk-2, and wmk-4 had no impact on insect survival. Strikingly, co-expression of tandemly arrayed wmk-3 and wmk-4 killed 90% of males and restored 70% of females, suggesting their conjugated functions for male-specific lethality. While the male-killing gene in the native host remains unknown, our findings highlight the role of bacteriophages in male-killing evolution and differences in male-killing mechanisms among insects.

Mechanisms Underpinning Morphogenesis of a Symbiotic Organ Specialized for Hosting an Indispensable Microbial Symbiont in Stinkbugs.

Microbial mutualists are pivotal for insect adaptation, which often entails the evolution of elaborate organs for symbiosis. Addressing what mechanisms underpin the development of such organs is of evolutionary interest. Here, we investigated the stinkbug Plautia stali, whose posterior midgut is transformed into a specialized symbiotic organ. Despite being a simple tube in newborns, it developed numerous crypts in four rows, whose inner cavity hosts a specific bacterial symbiont, during the 1st to 2nd nymphal instar stages. Visualization of dividing cells revealed that active cell proliferation was coincident with the crypt formation, although spatial patterns of the proliferating cells did not reflect the crypt arrangement. Visualization of visceral muscles in the midgut, consisting of circular muscles and longitudinal muscles, uncovered that, strikingly, circular muscles exhibited a characteristic arrangement running between the crypts specifically in the symbiotic organ. Even in the early 1st instar stage, when no crypts were seen, two rows of epithelial areas delineated by bifurcated circular muscles were identified. In the 2nd instar stage, crossing muscle fibers appeared and connected the adjacent circular muscles, whereby the midgut epithelium was divided into four rows of crypt-to-be areas. The crypt formation proceeded even in aposymbiotic nymphs, revealing the autonomous nature of the crypt development. We propose a mechanistic model of crypt formation wherein the spatial arrangement of muscle fibers and the proliferation of epithelial cells underpin the formation of crypts as midgut evaginations. IMPORTANCE Diverse organisms are associated with microbial mutualists, in which specialized host organs often develop for retaining the microbial partners. In light of the origin of evolutionary novelties, it is important to understand what mechanisms underpin the elaborate morphogenesis of such symbiotic organs, which must have been shaped through interactions with the microbial symbionts. Using the stinkbug Plautia stali as a model, we demonstrated that visceral muscular patterning and proliferation of intestinal epithelial cells during the early nymphal stages are involved in the formation of numerous symbiont-harboring crypts arranged in four rows in the posterior midgut to constitute the symbiotic organ. Strikingly, the crypt formation occurred normally even in symbiont-free nymphs, revealing that the crypt development proceeds autonomously. These findings suggest that the crypt formation is deeply implemented into the normal development of P. stali, which must reflect the considerably ancient evolutionary origin of the midgut symbiotic organ in stinkbugs.

A male-killing gene encoded by a symbiotic virus of Drosophila.

In most eukaryotes, biparentally inherited nuclear genomes and maternally inherited cytoplasmic genomes have different evolutionary interests. Strongly female-biased sex ratios that are repeatedly observed in various arthropods often result from the male-specific lethality (male-killing) induced by maternally inherited symbiotic bacteria such as Spiroplasma and Wolbachia. However, despite some plausible case reports wherein viruses are raised as male-killers, it is not well understood how viruses, having much smaller genomes than bacteria, are capable of inducing male-killing. Here we show that a maternally inherited double-stranded RNA (dsRNA) virus belonging to the family Partitiviridae (designated DbMKPV1) induces male-killing in Drosophila. DbMKPV1 localizes in the cytoplasm and possesses only four genes, i.e., one gene in each of the four genomic segments (dsRNA1-dsRNA4), in contrast to ca. 1000 or more genes possessed by Spiroplasma or Wolbachia. We also show that a protein (designated PVMKp1; 330 amino acids in size), encoded by a gene on the dsRNA4 segment, is necessary and sufficient for inducing male-killing. Our results imply that male-killing genes can be easily acquired by symbiotic viruses through reassortment and that symbiotic viruses are hidden players in arthropod evolution. We anticipate that host-manipulating genes possessed by symbiotic viruses can be utilized for controlling arthropods.

Perplexing dynamics of Wolbachia proteins for cytoplasmic incompatibility.

The mechanism of symbiont-induced cytoplasmic incompatibility (CI) has been a long-standing mystery. A new study on Wolbachia's Cif proteins in PLOS Biology provides supportive evidence for the "Host modification model," although the alternative "Toxin-antidote model" is still in the running.

Male-killing toxin in a bacterial symbiont of Drosophila.

Several lineages of symbiotic bacteria in insects selfishly manipulate host reproduction to spread in a population 1 , often by distorting host sex ratios. Spiroplasma poulsonii2,3 is a helical and motile, Gram-positive symbiotic bacterium that resides in a wide range of Drosophila species 4 . A notable feature of S. poulsonii is male killing, whereby the sons of infected female hosts are selectively killed during development1,2. Although male killing caused by S. poulsonii has been studied since the 1950s, its underlying mechanism is unknown. Here we identify an S. poulsonii protein, designated Spaid, whose expression induces male killing. Overexpression of Spaid in D. melanogaster kills males but not females, and induces massive apoptosis and neural defects, recapitulating the pathology observed in S. poulsonii-infected male embryos5-11. Our data suggest that Spaid targets the dosage compensation machinery on the male X chromosome to mediate its effects. Spaid contains ankyrin repeats and a deubiquitinase domain, which are required for its subcellular localization and activity. Moreover, we found a laboratory mutant strain of S. poulsonii with reduced male-killing ability and a large deletion in the spaid locus. Our study has uncovered a bacterial protein that affects host cellular machinery in a sex-specific way, which is likely to be the long-searched-for factor responsible for S. poulsonii-induced male killing.

Common and unique strategies of male killing evolved in two distinct Drosophila symbionts.

Male killing is a selfish reproductive manipulation caused by symbiotic bacteria, where male offspring of infected hosts are selectively killed. The underlying mechanisms and the process of their evolution are of great interest not only in terms of fundamental biology, but also their potential applications. The two bacterial Drosophila symbionts, Wolbachia and Spiroplasma, have independently evolved male-killing ability. This raises the question whether the underlying mechanisms share some similarities or are specific to each bacterial species. Here, we analyse pathogenic phenotypes of D. bifasciata infected with its natural male-killing Wolbachia strain and compare them with those of D. melanogaster infected with male-killing Spiroplasma We show that male progeny infected with the Wolbachia strain die during embryogenesis with abnormal apoptosis. Interestingly, male-killing Wolbachia infection induces DNA damage and segregation defects in the dosage-compensated chromosome in male embryos, which are reminiscent of the phenotypes caused by male-killing Spiroplasma in D. melanogaster By contrast, host neural development seems to proceed normally unlike male-killing Spiroplasma infection. Our results demonstrate that the dosage-compensated chromosome is a common target of two distinct male killers, yet Spiroplasma uniquely evolved the ability to damage neural tissue of male embryos.

Male-killing symbiont damages host's dosage-compensated sex chromosome to induce embryonic apoptosis.

Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner.

Male-killing Spiroplasma induces sex-specific cell death via host apoptotic pathway.

Some symbiotic bacteria cause remarkable reproductive phenotypes like cytoplasmic incompatibility and male-killing in their host insects. Molecular and cellular mechanisms underlying these symbiont-induced reproductive pathologies are of great interest but poorly understood. In this study, Drosophila melanogaster and its native Spiroplasma symbiont strain MSRO were investigated as to how the host's molecular, cellular and morphogenetic pathways are involved in the symbiont-induced male-killing during embryogenesis. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, anti-cleaved-Caspase-3 antibody staining, and apoptosis-deficient mutant analysis unequivocally demonstrated that the host's apoptotic pathway is involved in Spiroplasma-induced male-specific embryonic cell death. Double-staining with TUNEL and an antibody recognizing epidermal marker showed that embryonic epithelium is the main target of Spiroplasma-induced male-specific apoptosis. Immunostaining with antibodies against markers of differentiated and precursor neural cells visualized severe neural defects specifically in Spiroplasma-infected male embryos as reported in previous studies. However, few TUNEL signals were detected in the degenerate nervous tissues of male embryos, and the Spiroplasma-induced neural defects in male embryos were not suppressed in an apoptosis-deficient host mutant. These results suggest the possibility that the apoptosis-dependent epidermal cell death and the apoptosis-independent neural malformation may represent different mechanisms underlying the Spiroplasma-induced male-killing. Despite the male-specific progressive embryonic abnormality, Spiroplasma titers remained almost constant throughout the observed stages of embryonic development and across male and female embryos. Strikingly, a few Spiroplasma-infected embryos exhibited gynandromorphism, wherein apoptotic cell death was restricted to male cells. These observations suggest that neither quantity nor proliferation of Spiroplasma cells but some Spiroplasma-derived factor(s) may be responsible for the expression of the male-killing phenotype.

Atypical cadherins Dachsous and Fat control dynamics of noncentrosomal microtubules in planar cell polarity.

How global organ asymmetry and individual cell polarity are connected to each other is a central question in studying planar cell polarity (PCP). In the Drosophila wing, which develops PCP along its proximal-distal (P-D) axis, we previously proposed that the core PCP mediator Frizzled redistributes distally in a microtubule (MT)-dependent manner. Here, we performed organ-wide analysis of MT dynamics by introducing quantitative in vivo imaging. We observed MTs aligning along the P-D axis at the onset of redistribution and a small but significant excess of + ends-distal MTs in the proximal region of the wing. This characteristic alignment and asymmetry of MT growth was controlled by atypical cadherins Dachsous (Ds) and Fat (Ft). Furthermore, the action of Ft was mediated in part by PAR-1. All these data support the idea that the active reorientation of MT growth adjusts cell polarity along the organ axis.

Wing-to-Leg homeosis by spineless causes apoptosis regulated by Fish-lips, a novel leucine-rich repeat transmembrane protein.

Growth, patterning, and apoptosis are mutually interactive during development. For example, cells that select an abnormal fate in a developing field are frequently removed by apoptosis. An important issue in this process that needs to be resolved is the mechanism used by cells to discern their correct fate from an abnormal fate. In order to examine this issue, we developed an animal model that expresses the dioxin receptor homolog Spineless (Ss) ectopically in the Drosophila wing. The presence of mosaic clones ectopically expressing ss results in a local transformation of organ identity, homeosis, from wing into a leg or antenna. The cells with misspecified fates subsequently activate c-Jun N-terminal kinase to undergo apoptosis in an autonomous or nonautonomous manner depending on their position within the wing, suggesting that a cell-cell interaction is, at least in some cases, involved in the detection of misspecified cells. Similar position dependence is commonly observed when various homeotic genes controlling the body segments are ectopically expressed. The autonomous and nonautonomous apoptosis caused by ss is regulated by a novel leucine-rich repeat family transmembrane protein, Fish-lips (Fili) that interacts with surrounding normal cells. These data support a mechanism in which the lack of some membrane proteins helps to recognize the presence of different cell types and direct these cells to an apoptotic fate in order to exclude them from the normal developing field.