Members of the genus Arsenophonus comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an Arsenophonus endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. We propose provisional nomenclature for this bacterium in the genus Arsenophonus as "Candidatus Arsenophonus arthropodicus." Phylogenetic analyses indicate that "Candidatus Arsenophonus arthropodicus" is closely related to the Arsenophonus endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of Arsenophonus in insects. To this end, we describe methods for the culture of "Candidatus Arsenophonus arthropodicus" in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.Many members of the class Insecta maintain mutualistic associations with one or more specialized symbiotic bacteria (2). Bacteria that participate in these associations are classified either as primary (P) or secondary (S) endosymbionts, because they often coexist in a single insect host. The P-endosymbionts are predicted to be ancient in origin because their phylogenies are concordant with those of their host insects over a substantial period of evolutionary time, indicating long-term coevolution. On the other hand, the S-endosymbionts are predicted to be recent in origin because their phylogenies show little or no concordance with their insect hosts, indicating recent acquisition.While the ancient P-endosymbionts are known to have defined mutualistic functions in their insect hosts, the role of the S-endosymbionts is not yet well understood. From an evolutionary standpoint it seems likely that S-endosymbionts have beneficial (mutualistic) roles in their insect hosts because they are maintained predominantly through a maternal (vertical) transmission strategy. Several recent studies have provided experimental evidence for a number of beneficial effects conferred by the S-endosymbionts of aphids, which recently received new nomenclature (20). These benefits include host plant specialization (17, 27), increased resistance to hymenopteran parasitoids (21, 22), and increased tolerance to heat stress (5, 19). In addition, there is evidence indicating that S-endosymbionts can provide some level of functional compensation for the loss of P-endosymbionts in a laboratory population of aphids (16). While these studies are both exciting and encouraging, the ability to perform experimentation in these systems would be greatly enhanced with the opportunity to genetically manipulate S-endosymbionts. The application of recombinant DNA technology would permit functional analysis of individual genes in the endosymbiont genomes, providing a platform to e...

Sodalis glossinidius, a maternally transmitted endosymbiont of tsetse flies, maintains two phylogenetically distinct type-III secretion systems encoded by chromosomal symbiosis regions designated SSR-1 and SSR-2. Although both symbiosis regions are closely related to extant pathogenicity islands with similar gene inventories, SSR-2 has undergone novel degenerative adaptations in the transition to mutualism. Notably, SSR-2 lacks homologs of genes found in SSR-1 that encode secreted effector proteins known to facilitate the host cell cytoskeletal rearrangements necessary for bacterial entry and uptake into eukaryotic cells. Also, as a result of relaxed selection, SSR-2 has undergone inactivation of genes encoding components of the type-III secretion system needle substructure. In the current study, we used quantitative PCR to determine the expression profiles of ysaV (SSR-1) and invA (SSR-2) transcripts when S. glossinidius infects an insect cell line, and we used an invasion assay to characterize the phenotype of an S. glossinidius mutant that lacks the ability to produce an OrgA protein that is required for function of the SSR-2 secretome. Whereas SSR-1 is required for bacterial invasion of host cells and ysaV is expressed when bacteria contact host cells, SSR-2 is required for bacterial proliferation after entry, and invA is only expressed in the intracellular stage of infection. These results demonstrate that degenerative genetic adaptations in SSR-2 have promoted functional diversification of the Sodalis SSR-2 type-III secretion system.

Extrachromosomal element pSOG3 (52,162 nucleotides) in the genome of Sodalis glossinidius contains redundant phage-related gene pairs, indicating that it may have been formed by the fusion of two ancestral phage genomes followed by gene degradation. We suggest that pSOG3 is a prophage that has undergone genome degeneration accompanying host adaptation to symbiosis.