Extracellular vesicles from mesenchymal stem cells of dental pulp and adipose tissue display distinct transcriptomic characteristics suggestive of potential therapeutic targets.

Objective: Mesenchymal stem cells (MSCs) are isolated from various human tissues and used for therapy, in which beneficial effects are attributed mainly to mesenchymal stem cell-derived extracellular vesicles (MSC-EVs). Whereas MSCs of diverse tissue types share cardinal stem cell features, it is becoming evident that MSCs of each tissue type possess unique properties as well. For designing efficient stem cellbased therapies, it is crucial to understand the unique properties associated with MSCs and MSC-EVs of each tissue type. Such unique properties can be analyzed through transcriptomic approaches using comprehensive gene expression databases and sophisticated analytical tools. Here, we comparatively studied the transcriptomes in MSC-EVs of dental pulp and adipose tissue. Additionally, the transcriptomes of MSC-EVs were compared with the cellular transcriptomes of MSCs for the same tissue types. Methods: MSCs were cultured from human dental pulp and adipose tissue specimens. Conditioned culture media were collected to prepare MSC-EVs, from which RNAs were isolated and subjected to next-generation sequencing for transcriptomic analysis. Gene expression signatures in MSC-EVs of each tissue type were investigated using gene set analysis. Results: MSC-EVs obtained from dental pulp-derived MSCs showed distinct transcriptomic signatures of neurogenesis and neural retina development while MSC-EVs of adipose tissue-derived MSCs showed signatures of mitochondrial activity and skeletal system development. The transcriptomes of MSC-EVs resembled the cellular transcriptomes of MSCs, and the genes associated with neurogenesis were highly expressed in both MSCs and MSC-EVs of dental pulp. Adipose tissue-derived MSCs and MSC-EVs highly expressed genes associated with angiogenesis, hair growth, and dermal matrices. Conclusion: The clear and distinct signatures of neurogenesis and neural retina development in dental pulp-derived MSC-EVs imply neurodegenerative disorders and retinal diseases as putative therapeutic targets. In contrast, the transcripts in adipose tissue-derived MSC-EVs could be useful in rejuvenating the skin and musculoskeletal system. Further insights into MSC-EVs of divergent tissue types may expand the list of potential therapeutic targets.

Feminizing Wolbachia endosymbiont disrupts maternal sex chromosome inheritance in a butterfly species.

Wolbachia is a maternally inherited ubiquitous endosymbiotic bacterium of arthropods that displays a diverse repertoire of host reproductive manipulations. For the first time, we demonstrate that Wolbachia manipulates sex chromosome inheritance in a sexually reproducing insect. Eurema mandarina butterfly females on Tanegashima Island, Japan, are infected with the wFem Wolbachia strain and produce all-female offspring, while antibiotic treatment results in male offspring. Fluorescence in situ hybridization (FISH) revealed that wFem-positive and wFem-negative females have Z0 and WZ sex chromosome sets, respectively, demonstrating the predicted absence of the W chromosome in wFem-infected lineages. Genomic quantitative polymerase chain reaction (qPCR) analysis showed that wFem-positive females lay only Z0 eggs that carry a paternal Z, whereas females from lineages that are naturally wFem-negative lay both WZ and ZZ eggs. In contrast, antibiotic treatment of adult wFem females resulted in the production of Z0 and ZZ eggs, suggesting that this Wolbachia strain can disrupt the maternal inheritance of Z chromosomes. Moreover, most male offspring produced by antibiotic-treated wFem females had a ZZ karyotype, implying reduced survival of Z0 individuals in the absence of feminizing effects of Wolbachia. Antibiotic treatment of wFem-infected larvae induced male-specific splicing of the doublesex (dsx) gene transcript, causing an intersex phenotype. Thus, the absence of the female-determining W chromosome in Z0 individuals is functionally compensated by Wolbachia-mediated conversion of sex determination. We discuss how Wolbachia may manipulate the host chromosome inheritance and that Wolbachia may have acquired this coordinated dual mode of reproductive manipulation first by the evolution of female-determining function and then cytoplasmically induced disruption of sex chromosome inheritance.

Insect Sex Determination Manipulated by Their Endosymbionts: Incidences, Mechanisms and Implications.

The sex-determining systems of arthropods are surprisingly diverse. Some species have male or female heterogametic sex chromosomes while other species do not have sex chromosomes. Most species are diploids but some species, including wasps, ants, thrips and mites, are haplodiploids (n in males; 2n in females). Many of the sexual aberrations, such as sexual mosaics, sex-specific lethality and conversion of sexuality, can be explained by developmental defects including double fertilization of a binucleate egg, loss of a sex chromosome or perturbation of sex-determining gene expression, which occur accidentally or are induced by certain environmental conditions. However, recent studies have revealed that such sexual aberrations can be caused by various groups of vertically-transmitted endosymbiotic microbes such as bacteria of the genera Wolbachia, Rickettsia, Arsenophonus, Spiroplasma and Cardinium, as well as microsporidian protists. In this review, we first summarize the accumulated data on endosymbiont-induced sexual aberrations, and then discuss how such endosymbionts affect the developmental system of their hosts and what kinds of ecological and evolutionary effects these endosymbionts have on their host populations.

Transfection of feminizing Wolbachia endosymbionts of the butterfly, Eurema hecabe, into the cell culture and various immature stages of the silkmoth, Bombyx mori.

Wolbachia are maternally inherited endosymbiotic bacteria of invertebrates that can manipulate the reproductive systems of their arthropod hosts in a variety of ways. To establish a useful model system for investigating the mechanism of Wolbachia-induced host feminization, we conducted the following series of experiments: (1) feminizing Wolbachia of the butterfly, Eurema hecabe, were transferred into cell cultures of the silkmoth, Bombyx mori, and (2) the transfected Wolbachia in cell cultures were inoculated into B. mori at four immature stages. Wolbachia were successfully transfected into the cell cultures and stably maintained for more than 1 year (>30 passages). However, none of the inoculated insects produced mature oocytes that were Wolbachia-positive. This finding was consistent with the fact that Wolbachia was not detected in individuals in subsequent generations. In contrast, Wolbachia were detected at relatively high frequencies (60-80% of individuals) in the somatic tissues of inoculated insects. Real-time quantitative polymerase chain reaction revealed that the Wolbachia densities in the cultured cells were approximately tenfold higher than those in the native host E. hecabe. Among B. mori individuals inoculated at various developmental stages, those inoculated at early stages exhibited higher Wolbachia densities at the adult stage. The Wolbachia densities in individuals inoculated at the second-instar stage were comparable to those in intact E. hecabe. These results suggest that infection and/or proliferation of Wolbachia in germline cells are actively hindered by regulation in B. mori but feasible in somatic cells and that the Wolbachia densities in somatic tissues are regulated by the living host insects.

A natural population of the butterfly Eurema hecabe with Wolbachia-induced female-biased sex ratio not by feminization.

In butterflies, the adult sex ratio observed in the field is usually male-biased, although the sex ratio of their progeny is 1:1. This is due to the higher motility and larger behavioral range of males than females. As expected, the sex ratio of Eurema hecabe butterflies collected at 6 localities throughout Japan was male-biased. However, in Tsukuba, located in the central part of Japan, the sex ratio was found to be biased toward females. Their progeny reared in the laboratory also exhibited a female-biased sex ratio. A single strain of Wolbachia is considered to be the cause of the sex-ratio distortion, because antibiotic treatment reversed the sex ratio to 1:1, and only a single nucleotide sequence of wsp, a highly variable Wolbachia gene, was detected by molecular analysis. Cytogenetic analysis excluded the possibility of feminization as the underlying mechanism. In addition, when the wild-caught females that had already mated in nature were treated with antibiotics before oviposition, egg-hatch rates were extremely low, suggesting that the same Wolbachia strain also caused cytoplasmic incompatibility. Our findings suggest the possibility that a single strain of Wolbachia induces 2 distinct reproductive manipulations in the same host.

Unexpected mechanism of symbiont-induced reversal of insect sex: feminizing Wolbachia continuously acts on the butterfly Eurema hecabe during larval development.

When the butterfly Eurema hecabe is infected with two different strains (wHecCI2 and wHecFem2) of the bacterial endosymbiont Wolbachia, genetic males are transformed into functional females, resulting in production of all-female broods. In an attempt to understand how and when the Wolbachia endosymbiont feminizes genetically male insects, larval insects were fed an antibiotic-containing diet beginning at different developmental stages until pupation. When the adult insects emerged, strikingly, many of them exhibited sexually intermediate traits in their wings, reproductive organs, and genitalia. The expression of intersexual phenotypes was strong in the insects treated from first instar, moderate in the insects treated from third instar, and weak in the insects treated from fourth instar. The insects treated from early larval instar grew and pupated normally but frequently failed to emerge and died in the pupal case. The dead insects in the pupal case contained lower densities of the feminizing Wolbachia endosymbiont than the successfully emerged insects, although none of them were completely cured of the symbiont infection. These results suggest the following: (i) the antibiotic treatment suppressed the population of feminizing Wolbachia endosymbionts; (ii) the suppression probably resulted in attenuated feminizing activity of the symbiont, leading to expression of intersexual host traits; (iii) many of the insects suffered pupal mortality, possibly due to either intersexual defects or Wolbachia-mediated addiction; and hence (iv) the feminizing Wolbachia endosymbiont continuously acts on the host insects during larval development for expression of female phenotypes under a male genotype. Our finding may prompt reconsideration of the notion that Wolbachia-induced reproductive manipulations are already complete before the early embryonic stage and provide insights into the mechanism underlying the symbiont-induced reversal of insect sex.

Naturally occurring single and double infection with Wolbachia strains in the butterfly Eurema hecabe: transmission efficiencies and population density dynamics of each Wolbachia strain.

Wolbachia belonging to Alphaproteobacteria are transovarially transmitted bacteria responsible for reproductive alterations in a wide range of arthropods. In natural populations of the butterfly Eurema hecabe, there are two different types of Wolbachia-infected individuals. Individuals singly infected with Wolbachia strain wHecCI exhibit strong cytoplasmic incompatibility, whereas those doubly infected with wHecCI and wHecFem exhibit feminization. Here, we examined the infection frequencies and population densities of each Wolbachia strain in different host tissues (ovary, testis, fat body, midgut, Malpighian tubule and leg), and the cost of infection in offspring produced by single-infected and double-infected mothers of E. hecabe. The vertical transmission rate of wHecCI was nearly 100%, and that of wHecFem was c. 80%. The wHecCI densities were 10(3)-10(4)-fold higher than the wHecFem densities. In most tissues, the wHecCI densities were significantly higher in offspring of single-infected mothers than in offspring of double-infected mothers. In offspring of double-infected mothers, however, the wHecCI densities were not affected by the presence of wHecFem, suggesting a lack of interaction between the wHecCI and wHecFem densities. The offspring development time was dependent on the infection status of the mothers. These results imply that the maternal infection status affects the Wolbachia densities and fitness of offspring.

Molecular phylogeography of two sibling species of Eurema butterflies.

The common yellow butterfly Eurema hecabe is widely distributed in East Asia, and is one of the most burdensome species for taxonomists due to the numerous geographic and seasonal wing colour patterns. Moreover, within this species, individuals with a yellow wing fringe that occur in temperate regions of Japan (Y type) proved to be biologically different from others that occur widely in subtropical regions of Japan and all over East Asia (B type). To unveil the genetic variation within and between the two types, a total of 50 butterflies collected at 18 geographic localities in East Asia were examined for nucleotide sequence variation of three mitochondrial regions: cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and NADH dehydrogenase subunit 5 (ND5). In addition, they were also examined for infection status with the endosymbiotic bacteria Wolbachia. The three mitochondrial sequences consistently showed that (i) Y type and B type were highly divergent, (ii) nucleotide variation within B type was very small although sampled from a geographically wide range, and (iii) a weak association existed between mitochondrial DNA haplotypes and Wolbachia infection status.

Genetic structure of sibling butterfly species affected by Wolbachia infection sweep: evolutionary and biogeographical implications.

It was recently recognized that in Japan, the common yellow butterfly, Eurema hecabe, consists of two sibling species, which have been unnamed yet and tentatively called yellow (Y) type and brown (B) type. We investigated the diversity of nuclear and mitochondrial genes in Japanese populations of Y type and B type of E. hecabe. The phylogeny based on nuclear genes agreed with the distinction between Y type and B type, which had been also supported by a wide array of biological data. However, the phylogeny based on mitochondrial genes did not reflect the distinction. PCR survey of Wolbachia revealed that B-type populations were all infected while Y-type populations contained both infected and uninfected individuals. A single genotype of Wolbachia, which was inferred to be a CI-inducing strain from their wsp gene sequence, was prevalent in these populations. Notably, the mitochondrial phylogeny was in perfect agreement with the pattern of Wolbachia infection, suggesting that the Wolbachia infection had affected the mitochondrial genetic structure of the host insects. Probably, the Wolbachia strain and the associated mitochondrial genomes have been occasionally introduced from B-type populations to Y-type populations through migration and subsequent interspecific hybridization, and CI-driven population sweep has been spreading the Wolbachia strain and the particular mitochondrial haplotypes, which originated from B-type populations, into Y-type populations. On the basis of these results together with the geological and biogeographical knowledge of the Japanese Archipelago, we proposed an evolutionary hypothesis on the invasion and spread of Wolbachia infection in B-type and Y-type of E. hecabe.