The close association between the olive fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) and bacteria has been known for more than a century. Recently, the presence of a host-specific, hereditary, unculturable symbiotic bacterium, designated ‘Candidatus Erwinia dacicola’, has been described inside the cephalic organ of the fly, called the oesophageal bulb. In the present study, the 16S rRNA gene sequence variability of ‘Ca. E. dacicola’ was examined within and between 26 Italian olive fly populations sampled across areas where olive trees occur in the wild and areas where cultivated olive trees have been introduced through history. The bacterial contents of the oesophageal bulbs of 314 olive flies were analysed and a minimum of 781 bp of the 16S rRNA gene was sequenced. The corresponding host fly genotype was assessed by sequencing a 776 bp portion of the mitochondrial genome. Two ‘Ca. E. dacicola’ haplotypes were found (htA and htB), one being slightly more prevalent than the other (57 %). The two haplotypes did not co-exist in the same individuals, as confirmed by cloning. Interestingly, the olive fly populations of the two main Italian islands, Sicily and Sardinia, appeared to be represented exclusively by the htB and htA haplotypes, respectively, while peninsular populations showed both bacterial haplotypes in different proportions. No significant correlation emerged between the two symbiont haplotypes and the 16 host fly haplotypes observed, suggesting evidence for a mixed model of vertical and horizontal transmission of the symbiont during the fly life cycle.
In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and ‘the rest’ of the present family Halobacteriaceae.
Pathogenic scuticociliates, which are common in the haemolymph or tissues of maricultured animals and often cause serious diseases, are a species-rich assemblage with mostly unresolved systematic relationships, especially in some less-studied groups. In the present study, we sequenced the small-subunit rRNA gene of six species of scuticociliates, Uronemella parafilificum, Metanophrys sinensis, Parauronema longum, Cohnilembus verminus, Porpostoma notata and Ancistrum crassum, the last two of which have not been studied previously using molecular analyses. Phylogenetic trees were constructed using Bayesian inference, maximum-likelihood and maximum-parsimony methods to assess the inter- and intra-generic relationships of scuticociliates. Results revealed the following: 1) Porpostoma did not cluster with the Philasteridae, Cohnilembidae or any other family of the order Philasterida; 2) sequences of Uronemella parafilificum and Uronemella filificum showed a difference of 1.02 % (15 nt sites), revealing a close relationship between them; 3) the approximately unbiased test rejected monophyly of both Metanophrys and Parauronema, indicating that the terminal position of the anterior end of the paroral membrane and the structure of membranelle 1 are unreliable characters for distinction of genera in this group of scuticociliates; 4) Ancistrum crassum grouped with Boveria subcylindrica, showing a close phylogenetic relationship between the orders Thigmotrichida and Pleuronematida; and 5) Parauronema longum, Cyclidium plouneouri and Cyclidium porcatum should be removed from their currently assigned genera.