Phylogenetic trees based on the InvA and 16S rDNA sequences from SZPE, S. glossinidius, and selected plant and animal pathogens. For the InvA tree, only clades supported by bootstrap values greater than 60% are shown as resolved. Accession numbers of InvA sequences are as follows: SZPE (), S. glossinidius (), Yersinis pestis (NC001972), S. enterica (), S. flexneri (NC002698), P. syringae (), Ralstonia solanacearum (), Chlamydia pneumoniae (NC000922), Erwinia amylovora (), Rhizobium sp. NGR234 (), Xanthomonas campestris (). The invA homolog from Bordetella bronchiseptica was obtained from a dataset produced by the Bordetella bronchiseptica Sequencing Group at the Sanger Center, and can be obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/bb/.

Effect of divalent cations and temperature on invA, spaPQ, and fusA gene expression in SZPE. To calculate relative transcript numbers, standard curves for each gene were generated by serially diluting RNA templates with the highest transcript numbers for each gene. “Fold-difference” values were determined by comparing transcript numbers in each sample to a control sample, defined within each experiment as the sample with the lowest number of transcripts for a given gene. At high concentrations of Ca²⁺ or Mg²⁺ (≥200 μM), but not Cu²⁺, invA and spaPQ transcription is repressed. Transcription of the housekeeping gene, fusA, is not affected by any of the metal cations tested. At a reduced assay temperature (15°C), inv/spa expression is completely repressed regardless of Ca²⁺ availability.

Quantitative RT-PCR analysis of gene expression in weevil bacteriomes at different developmental stages. Expression of invA, spaPQ, and fusA was analyzed for three individual cohorts of reproductively synchronized animals at 3-day intervals through the course of their development into adulthood. To calculate relative transcript numbers, standard curves were generated for each gene by serially diluting RNA templates with the highest transcript numbers for each gene. “Fold-difference” values were determined by comparing transcript numbers in each sample to a control sample, defined within each cohort as the sample with the lowest number of transcripts for a given gene. Peak levels of the invA and spaPQ transcripts are detected in weevils undertaking pupation and metamorphosis, whereas levels of the housekeeping gene (fusA) transcripts remain constant throughout weevil development.