Shifting microbiomes complement life stage transitions and diet of the bird parasite Philornis downsi from the Galapagos Islands.

Domestication disconnects an animal from its natural environment and diet, imposing changes in the attendant microbial community. We examine these changes in Philornis downsi (Muscidae), an invasive parasitic fly of land birds in the Galapagos Islands. Using a 16S rDNA profiling approach we studied the microbiome of larvae and adults of wild and laboratory-reared populations. These populations diverged in their microbiomes, significantly more so in larval than in adult flies. In field-collected second-instar larvae, Klebsiella (70.3%) was the most abundant taxon, while in the laboratory Ignatzschineria and Providencia made up 89.2% of the community. In adults, Gilliamella and Dysgonomonas were key members of the core microbiome of field-derived females and males but had no or very low representation in the laboratory. Adult flies harbour sex-specific microbial consortia in their gut, as male core microbiomes were significantly dominated by Klebsiella. Thus, P. downsi microbiomes are dynamic and shift correspondingly with life cycle and diet. Sex-specific foraging behaviour of adult flies and nest conditions, which are absent in the laboratory, may contribute to shaping distinct larval, and adult male and female microbiomes. We discuss these findings in the context of microbe-host co-evolution and the implications for control measures.

Host-specific associations affect the microbiome of Philornis downsi, an introduced parasite to the Galápagos Islands.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.

Azospirillum brasilense does not affect population structure of specific rhizobacterial communities of inoculated maize (Zea mays).

Positive response of plant species to plant growth-promoting rhizobacteria have led to an increased interest in their use as bacterial inoculants. However, the introduction of exogenous bacteria into natural ecosystems may perturb bacterial populations within the microbial community and lead to the disruption of indigenous populations performing key functional roles. In this study the effect of Azospirillum brasilense inoculation on maize (Zea mays) rhizosphere Actinobacteria, Bacteroidetes, alpha-Proteobacteria, Pseudomonas and Bdellovibrio spp. was assessed using a polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) approach in conjunction with group-specific primers. The DGGE fingerprints analysis revealed that the introduction of A. brasilense did not alter or disrupt the microbial system at the group-specific level. However, some communities such as the alpha-Proteobacteria and Bdellovibrio were influenced by plant age while the other bacterial groups remained unaffected. Based on these as well as previous data, it can be inferred that inoculation with A. brasilense does not perturb the natural bacterial populations investigated.

Inoculation with the plant-growth-promoting rhizobacterium Azospirillum brasilense causes little disturbance in the rhizosphere and rhizoplane of maize (Zea mays).

Inoculation with Azospirillum brasilense exerts beneficial effects on plant growth and crop yields. In this study, a comparative analysis of maize (Zea mays) root inoculated or not inoculated with A. brasilense strains was performed in two soils. Colonization dynamics of the rhizobacteria were tracked in various root compartments using 16S rRNA-targeted probes and 4',6'diamidino-2-phenylindole staining, and the structure of bacterial populations in the same samples was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction products of the 16S rRNA gene. Based on whole cell hybridization, a large fraction of the bacterial community was found to be active in both the rhizoplane-endorhizosphere and rhizosphere soil compartments, in both soil types. A DGGE fingerprint analysis revealed that plant inoculation with A. brasilense had no effect on the structural composition of the bacterial communities, which were also found to be very similar at the root tip and at zones of root branching. However, rhizobacterial populations were strongly influenced by plant age, and their complexity decreased in the rhizoplane-endorhizosphere in comparison to rhizosphere soil. A clone library generated from rhizosphere DNA revealed a highly diverse community of soil and rhizosphere bacteria, including an indigenous Azospirillum-like organism. A large proportion of these clones was only distantly related to known species.

Poly beta-hydroxybutyrate depolymerase (PhaZ) in Azospirillum brasilense and characterization of a phaZ mutant.

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-hydroxybutyrate (PHB) under sub-optimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments. PHB depolymerase (PhaZ) is an essential enzyme in PHB degradation. The phaZ gene was identified in Azospirillum brasilense, cloned, sequenced, and shown to be located on the chromosome. Insertion of a kanamycin-resistant cassette within phaZ of A. brasilense resulted in a phaZ mutant that was unable to degrade PHB; however, carbon source utilization was similar in both the wild-type and the mutant strain. The ability of the wild-type to endure starvation conditions, ultraviolet irradiation, heat, and osmotic shock, and to grow in the presence of hydrogen peroxide was higher than that of the mutant strain. By contrast, the ability of the phaZ mutant strain to endure desiccation was higher than that of the wild-type strain. No differences between the strains were seen in their ability to endure sonication, or to survive in carrier materials used for soil inoculants. In addition, motility was the same between the two strains, whereas cell aggregation and exopolysaccharide production were higher in the wild-type than in the phaZ mutant strain.

Involvement of the reserve material poly-beta-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization.

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.

Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant.

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(beta-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (beta-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.

Involvement of outer-membrane proteins in the aggregation of Azospirillum brasilense.

A bioassay was developed to investigate biological factors involved in the aggregation of Azospirillum brasilense strain Cd. Cells were grown for 24 h under aggregation-inducing and non-aggregation-inducing conditions (high and low C:N, respectively) and sonicated for 20 s. The cells were washed by centrifugation and resuspended in potassium phosphate buffer containing the two types of sonication extract. A greater extent of aggregation and higher flocculation were observed after 2-3 h incubation in the presence of sonicates from cells grown at high C:N (H-cells) compared to cells grown at low C:N. Flocculation did not occur after incubation of these cells in phosphate buffer. Boiled or proteinase K-treated sonicates originating from H-cells had lower aggregation-inducing capacity. After fractionation of the crude sonicate, both the outer-membrane protein (OMP) and the total membrane (mostly OMP) fractions possessed relatively high aggregation specific activities. The aggregation-inducing capacity of the OMP fraction strongly correlated with its protein concentration in the bioassay. Treatment of this fraction with proteinase K also decreased its aggregation-inducing activity. These findings suggest that OMPs are involved in the aggregation process of cells of A. brasilense.

Aggregation in Azospirillum brasilense: effects of chemical and physical factors and involvement of extracellular components.

A medium for consistent induction of aggregation of Azospirillum brasilense cells was developed and used to study the effects of chemical and physical factors as well as extracellular components involved in this phenomenon. Growth of A. brasilense strain Cd in a high C:N medium using fructose and ammonium chloride as C and N sources, respectively, resulted in flocculation visible to the naked eye after 24 h. No cell aggregates were formed after 72 h growth in low C:N medium. Aggregating cells, but not cells grown under low C:N, accumulated high amounts of poly-beta-hydroxybutyrate and the cell envelope contained a well-defined electron-dense layer outside the outer membrane. Suspending the aggregates in 0.2 or 0.5 M urea was the only treatment effective for disrupting aggregates. The concentration of exopolysaccharide produced by four different strains of A. brasilense, differing in their capacity to aggregate, strongly correlated with the extent of aggregation. Electrophoretic protein profiles from different fractions of aggregating and non-aggregating cells were compared. Differences were observed in the pattern of low-molecular-mass proteins and in the polar flagellin that has previously been proposed to be involved in adhesion processes. However, a mutant lacking both lateral and polar flagella showed the strongest aggregation. The involvement of polysaccharides and/or proteins in aggregation of A. brasilense is discussed.