Biotechnology

ABSTRACT Bacterial endosymbionts of the pine bark adelgid, Pineus strobi (Insecta: Hemiptera: Adelgidae), were investigated using transmission electron microscopy, 16S and 23S rRNA-based phylogeny, and fluorescence in situ hybridization. Two morphologically different symbionts affiliated with the Gammaproteobacteria were present in distinct bacteriocytes. One of them (“ Candidatus Annandia pinicola”) is most closely related to an endosymbiont of Adelges tsugae , suggesting that they originate from a lineage already present in ancient adelgids before the hosts diversified into the two major clades, Adelges and Pineus . The other P. strobi symbiont (“ Candidatus Hartigia pinicola”) represents a novel symbiont lineage in members of the Adelgidae. Our findings lend further support for a complex evolutionary history of the association of adelgids with a phylogenetically diverse set of bacterial symbionts.

ABSTRACTThe tick-borne bacterium “CandidatusNeoehrlichia mikurensis” has recently been recognized as a human pathogen. Together withBorrelia afzelii, it is one of the most common pathogens found in the tickIxodes ricinus. Here, we compared the epidemiologies of “Ca. Neoehrlichia mikurensis” andB. afzeliiby longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole (Myodes glareolus), using real-time PCR for detection and quantification. The prevalences of “Ca. Neoehrlichia mikurensis” andB. afzeliiwere determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of “Ca. Neoehrlichia mikurensis” increased significantly during the sampling season. The clearance rate of “Ca. Neoehrlichia mikurensis” was significantly higher than that ofB. afzelii. We found a high frequency of double infections; 46% of all samples infected with “Ca. Neoehrlichia mikurensis” also had a coinfection withB. afzelii. The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.

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“CandidatusCardinium hertigii” (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. InCulicoides(Diptera: Ceratopogonidae) biting midges, the prevalence of “CandidatusCardinium” infection was documented as moderate, with no detectable sex bias. We therefore investigated whether “CandidatusCardinium” affects important fitness parameters, such as survival and body size, inCulicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account “CandidatusCardinium” infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with “CandidatusCardinium” infection data. The findings revealed no difference in survival ofCulicoidesinfected with “CandidatusCardinium” and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for “CandidatusCardinium”-infected versus uninfected midges. In aggregate, these findings support our conclusion that “CandidatusCardinium” does not have an overt effect on the survival and size of adultC. imicolamidges. “CandidatusCardinium” may affect immature stages or may alter adult reproductive performance.

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.