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.

Cytoplasmic incompatibility in the semivoltine longicorn beetle Acalolepta fraudatrix (Coleoptera: Cerambycidae) double infected with Wolbachia.

Wolbachia are obligatory endosymbiotic α-proteobacteria found in many arthropods. They are maternally inherited, and can induce reproductive alterations in the hosts. Despite considerable recent progress in studies on the associations between Wolbachia and various taxonomic groups of insects, none of the researches have revealed the effects of Wolbachia on longicorn beetles as the host insect. Acalolepta fraudatrix is a forest longicorn beetle that is distributed in East Asia. In this study, the relationship between Wolbachia and A. fraudatrix was investigated. Out of two populations of A. fraudatrix screened for Wolbachia using the genes ftsZ, wsp, and 16S rRNA, only one of the populations showed detection of all three genes indicating the presence of Wolbachia. Electron microscopy and fluorescent in situ hybridization also confirmed that the A. fraudatrix population was infected with Wolbachia. Sequencing the wsp genes derived from single insects revealed that two strains of Wolbachia coexisted in the insects based on the detection of two different sequences of the wsp gene. We designated these strains as wFra1 and wFra2. The bacterial titers of wFra1 were nearly 2-fold and 3-fold higher than wFra2 in the testes and ovaries, respectively. The two strains of Wolbachia in the insects were completely eliminated by rearing the insects on artificial diets containing 1% concentration of tetracycline for 1 generation. Reciprocal crosses between Wolbachia-infected and Wolbachia-uninfected A. fraudatrix demonstrated that only eggs produced by the crosses between Wolbachia-infected males and Wolbachia-uninfected females did not hatch, indicating that Wolbachia infecting A. fraudatrix causes cytoplasmic incompatibility in the host insect. This is the first report showing the effect of Wolbachia on reproductive function in a longicorn beetle, A. fraudatrix.

Small genome symbiont underlies cuticle hardness in beetles.

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.

A Novel, Extremely Elongated, and Endocellular Bacterial Symbiont Supports Cuticle Formation of a Grain Pest Beetle.

The saw-toothed grain beetle, Oryzaephilus surinamensis (Silvanidae), is a cosmopolitan stored-product pest. Early studies on O. surinamensis in the 1930s described the presence of peculiar bacteriomes harboring endosymbiotic bacteria in the abdomen. Since then, however, the microbiological nature of the symbiont has been elusive. Here we investigated the endosymbiotic system of O. surinamensis in detail. In the abdomen of adults, pupae, and larvae, four oval bacteriomes were consistently identified, whose cytoplasm was full of extremely elongated tubular bacterial cells several micrometers wide and several hundred micrometers long. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont was the most closely related to the endosymbiont of a grain pest beetle, Rhyzopertha dominica (Bostrichidae). The symbiont was detected in developing embryos, corroborating vertical symbiont transmission through host generations. The symbiont gene showed AT-biased nucleotide composition and accelerated molecular evolution, plausibly reflecting degenerative evolution of the symbiont genome. When the symbiont infection was experimentally removed, the aposymbiotic insects grew and reproduced normally, but exhibited a slightly but significantly more reddish cuticle and lighter body mass. These results indicate that the symbiont of O. surinamensis is not essential for the host's growth and reproduction but contributes to the host's cuticle formation. Symbiont genome sequencing and detailed comparison of fitness parameters between symbiotic and aposymbiotic insects under various environmental conditions will provide further insights into the symbiont's biological roles for the stored-product pest.IMPORTANCE Some beetles notorious as stored-product pests possess well-developed symbiotic organs called bacteriomes for harboring specific symbiotic bacteria, although their biological roles have been poorly understood. Here we report a peculiar endosymbiotic system of a grain pest beetle, Oryzaephilus surinamensis, in which four oval bacteriomes in the abdomen are full of extremely elongated tubular bacterial cells. Experimental symbiont elimination did not hinder the host's growth and reproduction, but resulted in emergence of reddish beetles, uncovering the symbiont's involvement in host's cuticle formation. We speculate that the extremely elongated symbiont cell morphology might be due to the degenerative symbiont genome deficient in bacterial cell division and/or cell wall formation, which highlights an evolutionary consequence of intimate host-symbiont coevolution.

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.

Infection Density Dynamics of the Citrus Greening Bacterium "Candidatus Liberibacter asiaticus" in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants.

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, "Candidatus Liberibacter asiaticus," and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of "Ca. Liberibacter asiaticus" in field populations of D. citri with experiments using field-collected insects to address how "Ca. Liberibacter asiaticus" infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from "Ca. Liberibacter asiaticus"-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were "Ca. Liberibacter asiaticus" positive. The infections were systemic across head-thorax and abdomen, ranging from 10(3) to 10(7) bacteria per insect. In spring, the infection densities were low in March, at ∼ 10(3) bacteria per insect, increasing up to 10(6) to 10(7) bacteria per insect in April and May, and decreasing to 10(5) to 10(6) bacteria per insect in late May, whereas the infection densities were constantly ∼ 10(6) to 10(7) bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with "Ca. Liberibacter asiaticus" infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected "Ca. Liberibacter asiaticus"-infected insects suggested that (i) "Ca. Liberibacter asiaticus"-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼ 10(6) bacteria per insect) of "Ca. Liberibacter asiaticus" density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits "Ca. Liberibacter asiaticus" to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.

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.

Spiroplasma as a model insect endosymbiont.

Members of the genus Spiroplasma are actively motile and helical bacteria of the class Mollicutes, which are associated with a variety of arthropods and plants. Some spiroplasmas cause female-biased sex ratios of their host insects as a result of selective death of the male offspring during embryogenesis. Several strains of male-killing spiroplasmas have been successfully transfected into Drosophila melanogaster by haemolymph injection and maintained in laboratory fly stocks. Spiroplasma-Drosophila endosymbiosis represents an ideal model system for analysing the molecular mechanisms underlying host-symbiont interactions. The infection dynamics exhibited by the symbiont within the host, the effects of external and environmental factors on the symbiotic association and symbiont interactions with the host's immune system have been investigated using this system. Comparisons between a male-killing Spiroplasma strain and its non-male-killing variant revealed that, in addition to different male-killing abilities, they also differed in infection dynamics and resistance to host innate immunity. It is currently unclear whether these different phenotypes are interconnected to each other. However, if so, such pleiotropy could facilitate our understanding of the genetic and molecular mechanisms of the endosymbiotic system.

Longicorn beetle that vectors pinewood nematode carries many Wolbachia genes on an autosome.

Monochamus alternatus is the longicorn beetle notorious as a vector of the pinewood nematode that causes the pine wilt disease. When two populations of M. alternatus were subjected to diagnostic polymerase chain reaction (PCR) detection of four Wolbachia genes, only the ftsZ gene was detected from one of the populations. The Wolbachia ftsZ gene persisted even after larvae were fed with a tetracycline-containing diet for six weeks. The inheritance of the ftsZ gene was not maternal but biparental, exhibiting a typical Mendelian pattern. The ftsZ gene titres in homozygotic ftsZ(+) insects were nearly twice as high as those in heterozygotic ftsZ(+) insects. Exhaustive PCR surveys revealed that 31 and 30 of 214 Wolbachia genes examined were detected from the two insect populations, respectively. Many of these Wolbachia genes contained stop codon(s) and/or frame shift(s). Fluorescent in situ hybridization confirmed the location of the Wolbachia genes on an autosome. On the basis of these results, we conclude that a large Wolbachia genomic region has been transferred to and located on an autosome of M. alternatus. The discovery of massive gene transfer from Wolbachia to M. alternatus would provide further insights into the evolution and fate of laterally transferred endosymbiont genes in multicellular host organisms.

High and low temperatures differently affect infection density and vertical transmission of male-killing Spiroplasma symbionts in Drosophila hosts.

We investigated the vertical transmission, reproductive phenotype, and infection density of a male-killing Spiroplasma symbiont in two Drosophila species under physiological high and low temperatures through successive host generations. In both the native host Drosophila nebulosa and the nonnative host Drosophila melanogaster, the symbiont infection and the male-killing phenotype were stably maintained at 25 degrees C, rapidly lost at 18 degrees C, and gradually lost at 28 degrees C. In the nonnative host, both the high and low temperatures significantly suppressed the infection density of the spiroplasma. In the native host, by contrast, the low temperature suppressed the infection density of the spiroplasma whereas the high temperature had little effect on the infection density. These results suggested that the low temperature suppresses both the infection density and the vertical transmission of the spiroplasma whereas the high temperature suppresses the vertical transmission preferentially. The spiroplasma density was consistently higher in the native host than in the nonnative host, suggesting that the host genotype may affect the infection density of the symbiont. The temperature- and genotype-dependent instability of the symbiont infection highlights a complex genotype-by-genotype-by-environment interaction and may be relevant to the low infection frequencies of the male-killing spiroplasmas in natural Drosophila populations.

Spiroplasma infection causes either early or late male killing in Drosophila, depending on maternal host age.

Symbiont-induced male-killing phenotypes have been found in a variety of insects. Conventionally, these phenotypes have been divided into two categories according to the timing of action: early male killing at embryonic stages and late male killing at late larval stages. In Drosophila species, endosymbiotic bacteria of the genus Spiroplasma have been known to cause early male killing. Here, we report that a spiroplasma strain normally causing early male killing also induces late male killing depending on the maternal host age: male-specific mortality of larvae and pupae was more frequently observed in the offspring of young females. As the lowest spiroplasma density and occasional male production were also associated with newly emerged females, we proposed the density-dependent hypothesis for the expression of early and late male-killing phenotypes. Our finding suggested that (1) early and late male-killing phenotypes can be caused by the same symbiont and probably by the same mechanism; (2) late male killing may occur as an attenuated expression of early male killing; (3) expression of early and late male-killing phenotypes may be dependent on the symbiont density, and thus, could potentially be affected by the host immunity and regulation; and (4) early male killing and late male killing could be alternative strategies adopted by microbial reproductive manipulators.

Prevalence of a non-male-killing spiroplasma in natural populations of Drosophila hydei.

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.

Tissue-specific infection dynamics of male-killing and nonmale-killing spiroplasmas in Drosophila melanogaster.

The male-killing spiroplasma strain NSRO causes an extremely female-biased sex ratio of the host, Drosophila melanogaster, as a result of selective death of male offspring during embryogenesis. The spiroplasma strain NSRO-A, a variant of NSRO, does not cause such symptoms. In an attempt to gain insights into the mechanism underlying the symbiont-induced reproductive phenotype, infection densities of the spiroplasmas in different tissues were monitored during host aging using a quantitative PCR technique. The density dynamics in the hemolymph were reminiscent of those in the whole body, whereas the density dynamics in the fat body, intestine and ovary were not. These results suggest that the majority of the spiroplasmas colonize and proliferate in the hemolymph of the host. In the hemolymph and whole body, the infection densities of NSRO were generally higher than those of NSRO-A, which may be related to the different reproductive phenotypes caused by the spiroplasmas.

Asymmetrical interactions between Wolbachia and Spiroplasma endosymbionts coexisting in the same insect host.

We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.

Functional analysis of unique class II insertion sequence IS1071.

Various xenobiotic-degrading genes on many catabolic plasmids are often flanked by two copies of an insertion sequence, IS1071. This 3.2-kb IS element has long (110-bp) terminal inverted repeats (IRs) and a transposase gene that are phylogenetically related to those of the class II transposons. However, the transposition mechanism of IS1071 has remained unclear. Our study revealed that IS1071 was only able to transpose at high frequencies in two environmental beta-proteobacterial strains, Comamonas testosteroni and Delftia acidovorans, and not in any of the bacteria examined which belong to the alpha- and gamma-proteobacteria. IS1071 was found to have the functional features of the class II transposons in that (i) the final product of the IS1071 transposition was a cointegrate of its donor and target DNA molecules connected by two directly repeated copies of IS1071, one at each junction; (ii) a 5-bp duplication of the target sequence was observed at the insertion site; and (iii) a tnpA mutation of IS1071 was efficiently complemented by supplying the wild-type tnpA gene in trans. Deletion analysis of the IS1071 IR sequences indicated that nearly the entire region of the IRs was required for its transposition, suggesting that the interaction between the transposase and IRs of IS1071 might be different from that of the other well-characterized class II transposons.

Population dynamics of male-killing and non-male-killing spiroplasmas in Drosophila melanogaster.

The endosymbiotic bacteria Spiroplasma spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 x 10(8) dnaA copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 x 10(9) dnaA copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 x 10(-5) for NSRO and 3 x 10(-4) for NSRO-A.

Hidden from the host: Spiroplasma bacteria infecting Drosophila do not cause an immune response, but are suppressed by ectopic immune activation.

Insects and other arthropods have an effective innate immune system that can clear infections with bacteria and other microorganisms. Despite this ability, one group of bacteria, the spiroplasmas, survive unharmed within the haemolymph of a wide range of arthropod hosts. We investigated the interaction between one member of this clade, a relative of Spiroplasma poulsonii, and the immune system of its Drosophila host. Expression of antimicrobial genes in spiroplasma-infected flies did not differ from wild-type controls either in the naturally infected state, nor after septic shock. We therefore concluded that spiroplasma infection did not induce an immune response in its host, but that this absence of response was unlikely to be because the bacterium inhibited response. Further experiments revealed immune reactions induced ectopically did reduce parasite titre. We therefore conclude that this bacterium has a novel form of interaction with its host, being hidden from the host immune system, but potentially suppressible by it.