Search for genes gained by horizontal gene transfer in an entomopoxvirus, with special reference to the analysis of the transfer of an ABC transporter gene.

Although it is generally believed that large DNA viruses capture genes by horizontal gene transfer (HGT), the detailed manner of such transfer has not been fully elucidated. Here, we searched for genes in the coleopteran entomopoxvirus (EV) Anomala cuprea entomopoxvirus (ACEV) that might have been gained by ACEV by HGT. We classified the potential source organisms for HGT into three categories: the host A. cuprea; other organisms, including viruses unrelated to EVs; and organisms with uncertain host attribution. Of the open reading frames (ORFs) of the ACEV genome, 2.1 % were suggested to have been gained from the host by ACEV or its recent ancestor via HGT; 8.7 % were possibly from organisms other than the host, and 3.7 % were possibly from the third category of organisms via HGT. The analysis showed that ACEV contains some interesting ORFs obtained by HGT, including a large ATP-binding cassette protein (ABC transporter) ORF and a tenascin ORF (IDs ACV025 and ACV123, respectively). We then performed a detailed analysis of the HGT of the ACEV large ABC transporter ORF-the largest of the ACEV ORFs. mRNA sequences obtained by RNA-seq from fat bodies-sites of ACEV replication-and midgut tissues-sites of initial infection-of the virus's host A. cuprea larvae were subjected to BLAST analysis. One type of ABC transporter ORF from the fat bodies and two types from the midgut tissues, one of which was identical to that in the fat bodies, had the greatest identity to the ABC transporter ORF of ACEV. The two types from the host had high levels of identity to each other (approximately 95 % nucleotide sequence identity), strongly suggesting that the host ABC transporter group consisting of the two types was the origin of ACV025. We then determined the sequence (12,381 bp) containing a full-length gene of the A. cuprea ABC transporter. It turned out to be a transcription template for the abovementioned mRNA found in both tissues. In addition, we determined a large part (ca. 6.9 kb) of the template sequence for the mRNA found only in the midgut tissues. The results showed that the ACEV ABC transporter ORF is missing parts corresponding to introns of the host ABC transporter genes, indicating that the ORF was likely acquired by HGT in the form of mRNA. The presence of definite duplicated sequences adjacent to the ACEV ABC transporter genes-a sign of LINE-1 retrotransposon-mediated HGT-was not observed. An approximately 2-month ACV025 transcription experiment suggested that the transporter sequence is presumed to be continuously functional. The amino acid sequence of ACV025 suggests that its product might function in the regulation of phosphatide in the host-cell membranes.

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.

Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

Genetic variations and microbiome of the poultry red mite Dermanyssus gallinae.

The poultry red mite Dermanyssus gallinae poses a significant threat to the health of hens and poultry production. A comprehensive understanding of D. gallinae is necessary to develop sustainable and efficacious control methods. Here we examined 144 D. gallinae collected from 18 poultry farms throughout the Japanese Archipelago for their genetic variations based on mitochondrial cytochrome c oxidase subunit I (COI) sequences, and microbiome variations based on amplicon sequencing of the 16S ribosomal RNA gene. According to COI sequencing, the Japanese samples were categorized into three haplogroups, which did not reflect the geographical distribution. Microbiome analyses found that the major bacteria associated with D. gallinae were Bartonella, Cardinium, Wolbachia, and Tsukamurella, with Bartonella being most predominant. Among 144 individual mites, all possessed one of the two major types of Bartonella (Bartonella sp. A), while 140 mites possessed the other type (Bartonella sp. B). The presence of the two strains of Bartonella was also confirmed by a single copy gene, rpoB. The presence of Bartonella in laid eggs suggested transovarial vertical transmission. Given that obligate blood-feeding arthropods generally require a supply of B vitamins from symbiotic bacteria, Bartonella may play an important role in mite survival. Rickettsiella, a major symbiont in European D. gallinae populations, and suggested to be an important symbiont by genomic data, was rarely found in Japanese populations. Cardinium detected from D. gallinae fell into a major clade found widely in arthropods, whereas Wolbachia detected in Japanese D. gallinae appear to be a new lineage, located at the base of Wolbachia phylogeny. Of the mitochondrial phylogeny, infection patterns of Cardinium and Wolbachia were strongly correlated, possibly suggesting one or both of the symbionts induce reproductive manipulations and increase spread in the host populations.

A new continuous cell line from the pest insect, Anomala cuprea (Coleoptera; Scarabaeidae): emergence of contractile cells.

Insect contractile cells frequently appear at an early phase of cell culture, but in most cases, they disappear before a continuous cell line is established, so the cell line ceases to contract. Continuous contractile insect cell lines are currently available from only one species each of Hymenoptera and Diptera. Here, we obtained a new cell line that contracted long after being established as a continuous cell line. The cell line contracted for a short period at an early phase of insect cell culture before a continuous cell line was established, but then did not contract again for several years. After this cell line entered the continuous growth phase, it produced spontaneously contractile tissues for about 4 mo but stopped contracting again. This is the first instance of a cell line that contracted after its establishment as a non-contractile continuous cell line. It is unclear whether the contractile cells survive or die after contraction ceases at an early phase of cell culture, and our results indicate that potential contractile cells survive for years after they stop to contract. The cells of this line sometimes produced complex contractile structures, such as sheet-like tissues. Only a few continuous cell lines have been derived from scarabaeid beetles. The new continuous cell line was derived from the culture of the fat bodies of the scarab beetle Anomala cuprea, which is a pest in the agriculture and forestry of Japan. The population doubling time of the new cell line was 2.5 d and thus it grows very rapidly among coleopteran continuous cell lines. Our new cell line will facilitate research on the physiology and pathology of Coleoptera, including scarab beetles, and may also contribute to research on invertebrate muscles.

Spatial distribution of orally administered viral fusolin protein in the insect midgut and possible synergism between fusolin and digestive proteases to disrupt the midgut peritrophic matrix.

Oral inoculation of entomopoxvirus spindles, microstructures composed of fusolin protein, causes disruption of the peritrophic matrix (PM), a physical barrier against microbe infection, in the insect midgut. Although the atomic structure of fusolin has been determined, little has been directly elucidated of the mechanism of disruption of the PM. In the present study, we first performed an immunohistochemical examination to determine whether fusolin acts on the PM directly or indirectly in the midgut of Bombyx mori larvae that were inoculated with spindles of Anomala cuprea entomopoxvirus. This revealed that the PM, rather than the midgut cells, was the attachment site for fusolin. Fusolin broadly attached to the PM from the anterior to the posterior region, both to its ectoperitrophic and endoperitrophic surfaces and within the PM. These results likely explain why the whole of the PM is rapidly disintegrated. Second, we administered protease inhibitors mixed with spindles and observed decreased midgut protease activity and reduced disruption of the PM. This suggests that midgut protease(s) is also positively involved in PM disruption. Based on the present results, we propose an overall mechanism for the disruption of the PM by administration of fusolin.

Maternally transmitted non-bacterial male killer in Drosophila biauraria.

A maternally inherited, all-female trait is widely found among arthropods, which is caused by bacterial endosymbionts such as Wolbachia, Rickettsia, Spiroplasma and Cardinium We discovered a single female of Drosophila biauraria, collected from Tomakomai, Hokkaido, Japan, that produced all-female offspring. This all-female trait was maternally inherited in the iso-female line (SP12F) by backcrossing with males of a normal line (SP11-20) with a 1 : 1 sex ratio derived from the same population. The all-female trait was not affected by tetracycline treatment performed for two consecutive generations. However, the microinjection of filter-sterilized homogenate of SP12F females into SP11-20 females established all-female matrilines. Our data suggest the role of transmissible agents, most likely viruses, but not bacteria or protists, as the possible cause of the all-female phenotype, which is likely to be achieved by killing of male embryos because egg hatch rates of SP12F were nearly half those of SP11-20. This is the first report in Diptera to demonstrate a maternally inherited virus-like element as the cause of the male-killing phenotype in D. biauraria.

Misdirection of dosage compensation underlies bidirectional sex-specific death in Wolbachia-infected Ostrinia scapulalis.

Endosymbiotic bacteria of the genus Wolbachia often manipulate the reproductive system of their hosts to propagate themselves in host populations. Ostrinia scapulalis moths infected with Wolbachia (wSca) produce female-only progeny (sex chromosomes: ZW), whereas females cured of the infection by antibiotic treatment produce male-only progeny (ZZ). The occurrence of female- and male-only progeny has been attributed to the specific death of the opposite sex during embryonic and larval development. In this bidirectional sex-specific lethality, embryos destined to die express a phenotypic sex opposite to their genotypic sex. On the basis of these findings, we suggested that wSca carries a genetic factor that feminizes the male host, the W chromosome of the host has lost its feminizing function, and discordance between the genotypic and phenotypic sexes underlies this sex-specific death. In the present study, we examined whether the failure of dosage compensation was responsible for this sex-specific mortality. Quantitative PCRs showed that Z-linked gene expression levels in embryos destined to die were not properly dosage compensated; they were approximately two-fold higher in the male progeny of wSca-infected females and approximately two-fold lower in the female progeny of infected-and-cured females. These results support our hypothesis that misdirection of dosage compensation underlies the sex-specific death.

A short, high-temperature treatment of host larvae to analyze Wolbachia-host interactions in the moth Ostrinia scapulalis.

Maternally inherited endosymbiotic bacteria of the genus Wolbachia cause various reproductive alterations in their hosts. Wolbachia induces male-specific death during embryonic and larval stages in the moth Ostrinia scapulalis. To investigate how the density of Wolbachia affects their performance in the host, we attempted to reduce its density using a short, high-temperature treatment of the host at the larval stage. Individuals cured of infection as well as sexual mosaics, which harbor Wolbachia, were obtained by this method in the next generation. The sex of uninfected offspring was exclusively male, similar to that of the offspring of larvae treated with antibiotics. A strong correlation was found between Wolbachia density in female moths and the sex ratio of their progeny. These results suggest that a short, high-temperature treatment at the larval stage reduced the density of Wolbachia in the adult stage, and, hence, inhibited interference with the host's development in the next generation. Since the direct effects of the heat treatment on Wolbachia were transient, this method may be useful for specifying the critical time for interference by Wolbachia in host development.

A male-killing Wolbachia carries a feminizing factor and is associated with degradation of the sex-determining system of its host.

Endosymbiotic bacteria of the genus Wolbachia induce diverse reproductive alterations in their insect hosts. Wolbachia (wSca) infecting the moth Ostrinia scapulalis causes unusual male killing, in which males (genotype: ZZ) selectively die during embryonic and larval development, whereas females (genotype: ZW), in turn, selectively die when cured of infection. To gain insight into the interaction between wSca and the host, we analysed phenotypic and genetic sexes of the embryos and larvae of normal, wSca-infected, and infected-and-cured O. scapulalis by diagnosing the sex-specifically spliced transcripts of Osdsx-a homologue of the sex-determining gene doublesex-and sex chromatin in interphase nuclei, respectively. It was observed that the female-type Osdsx was expressed in the infected male (ZZ) progenies destined to die, whereas the male-type Osdsx was expressed in the cured female (ZW) progenies destined to die. These findings suggest that (i) wSca, a male killer, carries a genetic factor that feminizes the male host, (ii) the sex-determining system of the host is degraded, and (iii) a mismatch between the genetic and phenotypic sexes underlies the sex-specific death.

Expression of a doublesex homologue is altered in sexual mosaics of Ostrinia scapulalis moths infected with Wolbachia.

A homologue of the sex-determining gene doublesex, Osdsx, was identified in the adzuki bean borer Ostrinia scapulalis. Three isoforms of the Osdsx transcript (Osdsx(M), Osdsx(FL) and Osdsx(FS)) differing in length were found. Osdsx(M) was specifically found in males, and contained an 852-bp ORF encoding a protein of 284 amino acids. Osdsx(FL) and Osdsx(FS) were found in females, and had the same 813-bp ORF encoding a protein of 271 amino acids. The Osdsx gene was inferred to have six exons and five introns. The variation in the transcript could be explained by the alternative splicing of Osdsx: Osdsx(M) was formed by the splicing of exons 1, 2, 5 and 6, Osdsx(FS) by the splicing of exons 1-4 and 6, and Osdsx(FL) by the splicing of exons 1-6. RT-PCR analysis indicated that Osdsx was transcribed in a sex-specific manner in all somatic tissues examined, regardless of developmental stage. In Wolbachia-induced sexual mosaics of O. scapulalis, which are genetically male, the female-specific isoform of Osdsx (Osdsx(FL)) was shown to be expressed in addition to the male-specific isoform (Osdsx(M)). This finding provides the first evidence that Wolbachia manipulates the sex of its host by interfering either with the sex-specific splicing of Osdsx itself or with another upstream sex determination process.