Aksoy Laboratory- EPH Yale University, School of Medicine

Tsetse flies rely on their obligate symbiont Wigglesworthia glossinidia to fulfill their nutritional needs. The complete genome sequence of Wigglesworthia is now available. Article, Sequence Data Commentary by Brendan Wren, Commentary at SciDevNet, Washington Post Article

		Each Tsetse Fly Harbors approximately 100 million cells of Wigglesworthia in its bacteriome (Bac). (A) shows the V-shaped white bacteriome structure in anterior midgut filled with blood, (B) a section through the bacteriome with bacteria seen lying free in the cytoplasm of the bacteriocyte.

Wigglesworthia phylogeny Characterization of Wigglesworthia from the four species groups of tsetse: fusca, morsitans, palpalis and austeni, has shown that they form a distinct lineage. The evolutionary relationship of the different tsetse species has been independently determined based on observed variation in the Internal Transcribed Spacer (ITS-2) regions of rDNA. This analysis has shown similarity between the phylogeny of genus Glossina and the phylogeny of their bacterial symbionts, implying that a tsetse ancestor had been infected with a bacterium some 50-80 million years ago, and from this ancestral pair species of tsetse and their associated Wigglesworthia strains radiated without horizontal transfer events between species.

Wigglesworthia Genome The genome size of Wigglesworthia had been estimated to range from 705 to 770-kb based on Pulsed-Field Gel Electrophoresis analysis. Its completely sequenced genome is 697,724 bp in Wigglesworthia brevipalpis -about one-sixth of that of the related free-living Escherichia coli (4.6 Mb). The completely annotated sequence of Wigglesworthia genome has revealed the presence of 621 predicted coding sequences (CDSs) with an average length of 988 bp. It has been possible to assign biological roles to 522 (84%) of these putative proteins, while 95 proteins (16%) matched hypothetical proteins of unknown function. Comparative analysis of the CDSs indicate that the Wigglesworthia genome contains a subset of the genes of free-living bacteria, such as the enteric E. coli and Salmonella typhimurium, further supporting that it shares an ancestor with them.

Role of flagella functions in Wigglesworthia Wigglesworthia is transmitted to the intrauterine larva through the mother’s milk gland secretions. It is not known, whether whole bacteriocytes or Wigglesworthia cells are transferred from the mother to her larva. Wigglesworthia genome does not apparently encode for a secretion system that would mediate uptake and entry into the eukaryotic larval cells. However, it has retained the machinery for the synthesis of a complete flagellar apparatus, including the basal body, hook, filament, filament cap regions, and the integral membrane proteins required for motility functions, motA and motB. While retention of genes associated with the flagellar operons is suggestive of a functional role, neither flagellum nor motility has been observed in Wigglesworthia in adult bacteriocytes. It is possible that the expression of a functional flagellum at certain life stages might facilitate the transmission of Wigglesworthia cells to the intrauterine progeny via milk-secretions. It is also possible that the flagellar structure in Wigglesworthia may function as a Type III-secretion system to export putative proteins to enable entry into the host larval or pupal gut cells destined to be bacteriocytes.

Wigglesworthia and parasitic pathogens In comparison with the obligates, parasitic Rickettsia has little capability for biosynthesis of amino acids, cofactors, or nucleic acids, but has significantly more genes that encode for products in DNA metabolism and transport-related functions. Similar to the free-living enterics and intracellular parasites which rely on their complex and flexible surface structures as protection from host defense mechanisms and environmental changes, Wigglesworthia genome encodes for enzymes involved in the LPS and peptidoglycan biosynthesis, products integral to its Gram negative cell wall structure. The retention of membrane capabilities by Wigglesworthia may reflect the biology of this symbiosis, where this organism may need protection from the host environment and defenses while in transit to the intrauterine progeny.

Wigglesworthia compared with the obligate mutualist Buchnera Although the obligate mutualists Buchnera and Wigglesworthia share apparent functional and evolutionary similarities in regards to their symbiotic associations with their insect hosts, their genetic blueprints are quite different. Wigglesworthia shares only 69% of its CDSs with Buchnera, and these mostly represent the indispensable house keeping genes. In comparison with Buchnera, a greater proportion of the Wigglesworthia genome is committed to the synthesis of products involved in cellular processes, cell structure, fatty acid metabolism, and especially, biosynthesis of cofactors. In contrast, a greater percentage of the Buchnera genome encodes for the biosynthesis of amino acids.


Role of Wigglesworthia in tsetse–vitamin biosynthesis
Supplementing the eukaryotic diet with metabolic products is thought to play a central role in the functional basis of the obligate mutualists. The single diet of tsetse—vertebrate blood—is known to be low in vitamins, and coupled with data from dietary supplementation experiments of antibiotic-fed (symbiont-free) tsetse flies, a putative role in vitamin metabolism has been assigned to its symbionts. Analysis of the CDSs indiate that this genome has retained 62 genes involved in the biosynthesis of cofactors, prosthetic groups and carriers, and has the potential to synthesize biotin, thiazole, lipoic acid, FAD (riboflavin, B2), folate, pantothenate, thiamine (B1), pyridoxine (B6), protoheme, and nicotinamide.