Effect of Quorum Sensing Inducers and Inhibitors on Cytoplasmic Incompatibility Induced by Wolbachia (Rickettsiales: Anaplasmataceae) in American Serpentine Leafminer (Diptera: Agromyzidae): Potential Tool for the Incompatible Insect Technique.

Agricultural crops around the world are attacked by approximately 3,000-10,000 species of pest insect. There is increasing interest in resolving this problem using environmentally friendly approaches. Wolbachia (Hertig), an insect endosymbiont, can modulate host reproduction and offspring sex through cytoplasmic incompatibility (CI). The incompatible insect technique (IIT) based on CI-Wolbachia is a promising biological control method. Previous studies have reported an association between CI and Wolbachia density, which may involve a quorum sensing (QS) mechanism. In this study, we investigated the effect of manipulating QS in Wolbachia using several chemicals including 3O-C12-HSL; C2HSL; spermidine (QS inducers), 4-phenylbutanoyl; and 4-NPO (QS inhibitors) on American serpentine leafminer (Liriomyza trifolii [Burgess]), an agricultural pest. The results showed that inducing QS with 3O-C12-HSL decreased the proportion of hatched eggs and increased Wolbachia density, whereas QS inhibition with 4-phenylbutanoyl had the opposite effects. Thus, manipulating QS in Wolbachia can alter cell density and the proportion of hatched eggs in the host L. trifolii, thereby reducing the number of insect progeny. These findings provide evidence supporting the potential efficacy of the IIT based on CI-Wolbachia for the environmentally friendly control of insect pest populations.

Unpredicted impacts of insect endosymbionts on interactions between soil organisms, plants and aphids.

Ecologically significant symbiotic associations are frequently studied in isolation, but such studies of two-way interactions cannot always predict the responses of organisms in a community setting. To explore this issue, we adopt a community approach to examine the role of plant-microbial and insect-microbial symbioses in modulating a plant-herbivore interaction. Potato plants were grown under glass in controlled conditions and subjected to feeding from the potato aphid Macrosiphum euphorbiae. By comparing plant growth in sterile, uncultivated and cultivated soils and the performance of M. euphorbiae clones with and without the facultative endosymbiont Hamiltonella defensa, we provide evidence for complex indirect interactions between insect- and plant-microbial systems. Plant biomass responded positively to the live soil treatments, on average increasing by 15% relative to sterile soil, while aphid feeding produced shifts (increases in stem biomass and reductions in stolon biomass) in plant resource allocation irrespective of soil treatment. Aphid fecundity also responded to soil treatment with aphids on sterile soil exhibiting higher fecundities than those in the uncultivated treatment. The relative allocation of biomass to roots was reduced in the presence of aphids harbouring H. defensa compared with plants inoculated with H. defensa-free aphids and aphid-free control plants. This study provides evidence for the potential of plant and insect symbionts to shift the dynamics of plant-herbivore interactions.

Genetic Modification of Sodalis Species by DNA Transduction.

Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing, and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram-negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms.IMPORTANCE A large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherent nature of these associations, insect endosymbionts cannot be usually isolated in pure culture or genetically manipulated. Here we use a broad-host-range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad-host-range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

Conjugal DNA Transfer in Sodalis glossinidius, a Maternally Inherited Symbiont of Tsetse Flies.

Stable associations between insects and bacterial species are widespread in nature. This is the case for many economically important insects, such as tsetse flies. Tsetse flies are the vectors of Trypanosoma brucei, the etiological agent of African trypanosomiasis-a zoonotic disease that incurs a high socioeconomic cost in regions of endemicity. Populations of tsetse flies are often infected with the bacterium Sodalis glossinidius Following infection, S. glossinidius establishes a chronic, stable association characterized by vertical (maternal) and horizontal (paternal) modes of transmission. Due to the stable nature of this association, S. glossinidius has been long sought as a means for the implementation of anti-Trypanosoma paratransgenesis in tsetse flies. However, the lack of tools for the genetic modification of S. glossinidius has hindered progress in this area. Here, we establish that S. glossinidius is amenable to DNA uptake by conjugation. We show that conjugation can be used as a DNA delivery method to conduct forward and reverse genetic experiments in this bacterium. This study serves as an important step in the development of genetic tools for S. glossinidius The methods highlighted here should guide the implementation of genetics for the study of the tsetse-Sodalis association and the evaluation of S. glossinidius-based tsetse fly paratransgenesis strategies.IMPORTANCE Tsetse flies are the insect vectors of T. brucei, the causative agent of African sleeping sickness-a zoonotic disease that inflicts a substantial economic cost on a broad region of sub-Saharan Africa. Notably, tsetse flies can be infected with the bacterium S. glossinidius to establish an asymptomatic chronic infection. This infection can be inherited by future generations of tsetse flies, allowing S. glossinidius to spread and persist within populations. To this effect, S. glossinidius has been considered a potential expression platform to create flies which reduce T. brucei stasis and lower overall parasite transmission to humans and animals. However, the efficient genetic manipulation of S. glossinidius has remained a technical challenge due to its complex growth requirements and uncharacterized physiology. Here, we exploit a natural mechanism of DNA transfer among bacteria and develop an efficient technique to genetically manipulate S. glossinidius for future studies in reducing trypanosome transmission.

Genome evolution in an ancient bacteria-ant symbiosis: parallel gene loss among Blochmannia spanning the origin of the ant tribe Camponotini.

Stable associations between bacterial endosymbionts and insect hosts provide opportunities to explore genome evolution in the context of established mutualisms and assess the roles of selection and genetic drift across host lineages and habitats. Blochmannia, obligate endosymbionts of ants of the tribe Camponotini, have coevolved with their ant hosts for ∼40 MY. To investigate early events in Blochmannia genome evolution across this ant host tribe, we sequenced Blochmannia from two divergent host lineages, Colobopsis obliquus and Polyrhachis turneri, and compared them with four published genomes from Blochmannia of Camponotus sensu stricto. Reconstructed gene content of the last common ancestor (LCA) of these six Blochmannia genomes is reduced (690 protein coding genes), consistent with rapid gene loss soon after establishment of the symbiosis. Differential gene loss among Blochmannia lineages has affected cellular functions and metabolic pathways, including DNA replication and repair, vitamin biosynthesis and membrane proteins. Blochmannia of P. turneri (i.e., B. turneri) encodes an intact DnaA chromosomal replication initiation protein, demonstrating that loss of dnaA was not essential for establishment of the symbiosis. Based on gene content, B. obliquus and B. turneri are unable to provision hosts with riboflavin. Of the six sequenced Blochmannia, B. obliquus is the earliest diverging lineage (i.e., the sister group of other Blochmannia sampled) and encodes the fewest protein-coding genes and the most pseudogenes. We identified 55 genes involved in parallel gene loss, including glutamine synthetase, which may participate in nitrogen recycling. Pathways for biosynthesis of coenzyme A, terpenoids and riboflavin were lost in multiple lineages, suggesting relaxed selection on the pathway after inactivation of one component. Analysis of Illumina read datasets did not detect evidence of plasmids encoding missing functions, nor the presence of coresident symbionts other than Wolbachia. Although gene order is strictly conserved in four Blochmannia of Camponotus sensu stricto, comparisons with deeply divergent lineages revealed inversions in eight genomic regions, indicating ongoing recombination despite ancestral loss of recA. In sum, the addition of two Blochmannia genomes of divergent host lineages enables reconstruction of early events in evolution of this symbiosis and suggests that Blochmannia lineages may experience distinct, host-associated selective pressures. Understanding how evolutionary forces shape genome reduction in this system may help to clarify forces driving gene loss in other bacteria, including intracellular pathogens.