Fukatsu, Takema

ABSTRACT 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.

ABSTRACTA gammaproteobacterial facultative symbiont of the genusRickettsiellawas recently identified in the pea aphid,Acyrthosiphon pisum. Infection with this symbiont altered the color of the aphid body from red to green, potentially affecting the host's ecological characteristics, such as attractiveness to different natural enemies. In European populations ofA. pisum, the majority ofRickettsiella-infected aphids also harbor another facultative symbiont, of the genusHamiltonella. We investigated thisRickettsiellasymbiont for its interactions with the coinfectingHamiltonellasymbiont, its phenotypic effects onA. pisumwith and withoutHamiltonellacoinfection, and its infection prevalence inA. pisumpopulations. Histological analyses revealed that coinfectingRickettsiellaandHamiltonellaexhibited overlapping localizations in secondary bacteriocytes, sheath cells, and hemolymph, whileRickettsiella-specific localization was found in oenocytes.Rickettsiellainfections consistently altered hosts' body color from red to green, where the greenish hue was affected by both host and symbiont genotypes.Rickettsiella-Hamiltonellacoinfections also changed red aphids to green; this greenish hue tended to be enhanced byHamiltonellacoinfection. With different host genotypes,Rickettsiellainfection exhibited either weakly beneficial or nearly neutral effects on host fitness, whereasHamiltonellainfection andRickettsiella-Hamiltonellacoinfection had negative effects. Despite considerable frequencies ofRickettsiellainfection in European and North AmericanA. pisumpopulations, noRickettsiellainfection was detected among 1,093 insects collected from 14 sites in Japan. On the basis of these results, we discuss possible mechanisms for the interaction ofRickettsiellawith other facultative symbionts, their effects on their hosts' phenotypes, and their persistence in natural host populations. We propose the designation “CandidatusRickettsiella viridis” for the symbiont.

ABSTRACT “ Candidatus Midichloria mitochondrii” is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus . Bacteria phylogenetically related to “ Ca . Midichloria mitochondrii” (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales . This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family “ Candidatus Midichloriaceae.” In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

ABSTRACT Here we investigated the bacterial endosymbionts of weevils of the genus Curculio . From all four species of Curculio weevils examined, a novel group of bacterial gene sequences were consistently identified. Molecular phylogenetic analyses demonstrated that the sequences formed a distinct clade in the Gammaproteobacteria , which was not related to previously known groups of weevil endosymbionts such as Nardonella spp. and Sodalis -allied symbionts. In situ hybridization revealed that the bacterium was intracellularly harbored in a bacteriome associated with larval midgut. In adult females, the bacterium was localized in the germalia at the tip of each overiole, suggesting vertical transmission via ovarial passage. Diagnostic PCR surveys detected high prevalence of the bacterial infection in natural host populations. Electron microscopy identified the reduced cell wall of the bacterial cells, and the bacterial genes exhibited AT-biased nucleotide composition and accelerated molecular evolution, which are suggestive of a long-lasting endosymbiotic association. On the basis of these results, we conclude that the novel endocellular bacteria represent the primary symbiont of Curculio weevils and proposed the designation “ Candidatus Curculioniphilus buchneri.” In addition to “ Ca. Curculioniphilus,” we identified Sodalis -allied gammaproteobacterial endosymbionts from the chestnut weevil, Curculio sikkimensis , which exhibited partial infection frequencies in host insect populations and neither AT-biased nucleotide composition nor accelerated molecular evolution. We suggest that such Sodalis -allied secondary symbionts in weevils might provide a potential source for symbiont replacements, as has occurred in an ancestor of Sitophilus grain weevils.