Insight into the transmission biology and species-specific functional capabilities of tsetse (Diptera: glossinidae) obligate symbiont Wigglesworthia

Abstract

Ancient endosymbionts have been associated with extreme genome structural stability with little differentiation in gene inventory between sister species. Tsetse flies (Diptera: Glossinidae) harbor an obligate endosymbiont, Wigglesworthia, which has coevolved with the Glossina radiation. We report on the ~720-kb Wigglesworthia genome and its associated plasmid from Glossina morsitans morsitans and compare them to those of the symbiont from Glossina brevipalpis. While there was overall high synteny between the two genomes, a large inversion was noted. Furthermore, symbiont transcriptional analyses demonstrated host tissue and development-specific gene expression supporting robust transcriptional regulation in Wigglesworthia, an unprecedented observation in other obligate mutualist endosymbionts. Expression and immunohistochemistry confirmed the role of flagella during the vertical transmission process from mother to intrauterine progeny. The expression of nutrient provisioning genes (thiC and hemH) suggests that Wigglesworthia may function in dietary supplementation tailored toward host development. Furthermore, despite extensive conservation, unique genes were identified within both symbiont genomes that may result in distinct metabolomes impacting host physiology. One of these differences involves the chorismate, phenylalanine, and folate biosynthetic pathways, which are uniquely present in Wigglesworthia morsitans. Interestingly, African trypanosomes are auxotrophs for phenylalanine and folate and salvage both exogenously. It is possible that W. morsitans contributes to the higher parasite susceptibility of its host species.

Importance: Genomic stasis has historically been associated with obligate endosymbionts and their sister species. Here we characterize the Wigglesworthia genome of the tsetse fly species Glossina morsitans and compare it to its sister genome within G. brevipalpis. The similarity and variation between the genomes enabled specific hypotheses regarding functional biology. Expression analyses indicate significant levels of transcriptional regulation and support development- and tissue-specific functional roles for the symbiosis previously not observed in obligate mutualist symbionts. Retention of the genetically expensive flagella within these small genomes was demonstrated to be significant in symbiont transmission and tailored to the unique tsetse fly reproductive biology. Distinctions in metabolomes were also observed. We speculate an additional role for Wigglesworthia symbiosis where infections with pathogenic trypanosomes may depend upon symbiont species-specific metabolic products and thus influence the vector competence traits of different tsetse fly host species.

FIG 1

Localization of Wigglesworthia within the tsetse fly. Wigglesworthia resides within the bacteriome organ (top left) and is found free in the cytoplasm of specialized cells known as bacteriocytes (top right). (Bottom) Wigglesworthia is present extracellularly in the milk gland tissue. (Bottom left) Fluorescence in situ hybridization (FISH) staining for Wigglesworthia. (Bottom right) Schematic drawing based on FISH results shown on the left. DAPI staining indicates nuclei in bacteriocytes and milk gland tubule cells. Pink fluorescent rhodamine staining shows Wigglesworthia within bacteriocytes and in milk gland lumen.

FIG 2

Linearized comparison of the genomes of WGB and WGM. (A) Genome alignment as represented from Mauve. Each colored block represents a locally collinear block (LCB) of DNA that has not undergone rearrangement within its boundaries. Bar height indicates average nucleotide similarity within a region. The green LCB is inverted, as indicated by the relative reverse orientation of the block in each genome. (B) Annotation of the WGM genome. Each gene is shown as a trapezoid with the straight side representing the start codon. Genes above the line are on the positive strand, while genes below the line are on the negative strand. The shaded area indicates the inverted region relative to the WGB genome. Genes are labeled and color coded according to the different functional categories assigned.

FIG 3

Comparative analyses of the WGM and WGB genomes. (A) The Wigglesworthia pangenome consists of a core 599 CDSs with an additional 21 and 19 unique CDSs within the WGM and WGB genomes, respectively. (B) Distribution of unique CDSs present per functional category.

FIG 4

Wigglesworthia flagella are utilized during maternal transmission. (A) Normalized qRT-PCR-based gene expression results for the fliC and motA genes in the bacteriome (BAC), different stages of intrauterine larvae (L1, L2, and L3), newly deposited pupae (Pu-early), late pupae (Pu-late), and carcasses of mothers that carry the corresponding intrauterine larvae (Mom-L1, Mom-L2, and Mom-L3). Asterisks indicate statistically significant differences between the bacteriome and various developmental stages. ***, P < 0.0001; **, P < 0.001; *, P < 0.05. (B) Images of WGM FliC-specific antibody staining on cross sections of the tsetse fly common milk duct (column A), duct within the larval gut (column B), larval bacteriome (column C), and adult bacteriome (column D). Row 1 represents DAPI staining, row 2 represents FliC antibody staining, and row 3 represents the merged images of rows 1 and 2.

FIG 5

qRT-PCR of hemH, groEL, and thiC expression during tsetse fly development. Gene expression results are shown for hemH (A), groEL (B), and thiC (C) in the adult bacteriome (BAC), different stages of intrauterine larvae (L1, L2, and L3), newly deposited pupae (Pu-early), late pupae (Pu-late), and carcasses of mothers that carry the corresponding intrauterine larvae (Mom-L1, Mom-L2, and Mom-L3). All data were normalized to the ribosomal gene rpsC Asterisks indicate statistically significant differences between the bacteriome and different developmental stages. ***, P < 0.0001; *, P < 0.05.

FIG 6

Summary of dN/dS ratio calculations relative to genome position. Genes putatively influenced by purifying selection are represented by blue diamonds. Genes within 2 standard deviations of the mean dN/dS ratios are represented by red squares. Genes with >2 and >3 standard deviations from the mean dN/dS ratio are represented by yellow triangles and green circles, respectively.