Small, smaller, smallest: the origins and evolution of ancient dual symbioses in a Phloem-feeding insect

Abstract

Many insects rely on bacterial symbionts with tiny genomes specialized for provisioning nutrients lacking in host diets. Xylem sap and phloem sap are both deficient as insect diets, but differ dramatically in nutrient content, potentially affecting symbiont genome evolution. For sap-feeding insects, sequenced symbiont genomes are available only for phloem-feeding examples from the suborder Sternorrhyncha and xylem-feeding examples from the suborder Auchenorrhyncha, confounding comparisons. We sequenced genomes of the obligate symbionts, Sulcia muelleri and Nasuia deltocephalinicola, of the phloem-feeding pest insect, Macrosteles quadrilineatus (Auchenorrhyncha: Cicadellidae). Our results reveal that Nasuia-ALF has the smallest bacterial genome yet sequenced (112 kb), and that the Sulcia-ALF genome (190 kb) is smaller than that of Sulcia in other insect lineages. Together, these symbionts retain the capability to synthesize the 10 essential amino acids, as observed for several symbiont pairs from xylem-feeding Auchenorrhyncha. Nasuia retains genes enabling synthesis of two amino acids, DNA replication, transcription, and translation. Both symbionts have lost genes underlying ATP synthesis through oxidative phosphorylation, possibly as a consequence of the enriched sugar content of phloem. Shared genomic features, including reassignment of the UGA codon from Stop to tryptophan, and phylogenetic results suggest that Nasuia-ALF is most closely related to Zinderia, the betaproteobacterial symbiont of spittlebugs. Thus, Nasuia/Zinderia and Sulcia likely represent ancient associates that have co-resided in hosts since the divergence of leafhoppers and spittlebugs >200 Ma, and possibly since the origin of the Auchenorrhyncha, >260 Ma.

Keywords: Nasuia deltocephalinicola; Sulcia muelleri; gene loss; genome evolution; leafhopper; nutritional symbioses.

Fig. 1.—

Schematic summary of the evolution of symbiotic associations in major lineages of Auchenorrhyncha, with emphasis on the Cicadomorpha (cicadas, spittlebugs, leafhoppers, and treehoppers). The focal symbionts, Nasuia and Sulcia from Macrosteles quadrilineatus (Cicadellidae: Deltocephalinae), are demarcated with a blue and red arrow, respectively. Phylogenies show evolutionary events in symbioses based on molecular phylogenetic studies (Moran et al. 2003, 2005; Takiya et al. 2006; Urban and Cryan 2012; Noda et al. 2012; Bressan and Mulligan 2013; Hou et al. 2013; Koga et al. 2013). Events are color-coded as follows: Host insect phylogeny (from Cryan and Urban 2012) gray background; Sulcia, red; betaproteobacterial symbiont lineage (BetaSymb = Zinderia + Nasuia + Vidania), blue; symbiont loss, black dashed line; and symbiont replacement, green. Identified symbiont names are given at the tips. This schematic is not a full account of host relationships and symbiont associations, as many species and genera remain to be explored, and some inferred losses may reflect incomplete knowledge. Sulcia is hypothesized to have been associated with the Auchenorrhyncha since its emergence 260–280 Ma (see time scale and corresponding blue box; Moran et al. 2005). Although further evidence is required to test the hypothesis that the Auchenorrhyncha share a common betaproteobacterial symbiont (BetaSymb) that was acquired early in its diversification, this potential relationship is shown here with a blue dashed line. The hypothesis that the Cicadomorpha do share the closely related BetaSymb (Zinderia + Nasuia), as evidenced by our genomic and phylogenetic results, is shown in a solid blue line (as is Vidania found throughout the Fulgoroidea based on Urban and Cryan 2012).

Fig. 2.—

Fluorescence in situ hybridization of the bacteriome of Macrosteles quadrilineatus (Cicadellidae: Deltocephalinae), housing Sulcia muelleri (green; Bacteroidetes), and Nasuia deltocephalinicola (red; Betaproteobacteria). Blue fluorescence shows host nuclear DNA. (a) Localization of the bacteriome along the anterolateral abdominal segments. (b) The dissected bacteriome comprising two distinct bacteriocyte types that house Sulcia-ALF and Nasuia-ALF. White scale bars equal 100 μm (a) and 50 μm. (b) Note.—Red autofluorescence of the host thoracic plates.

Fig. 3.—

Genome map of the co-primary symbionts of Macrosteles quadrilineatus: Sulcia-ALF (left) and Nasuia-ALF (right). Genomes are scaled relative to nucleotide count. The outermost track (blue) shows genes coded on the plus strand, whereas the adjacent tracks (teal) show genes coded on the minus strand. The other tracks show RNA genes, including the rRNA operons (purple), ncRNA (red), and tRNA (orange). The two graphs show GC skew (outermost: green = values above genome average, purple = below genome average) and GC content (innermost: gray = values above genome average, black = below genome average). Plots were created with DNAPlotter (Carver et al. 2009).

Fig. 4.—

Synteny plot showing gene order conservation and gene loss between Sulcia-ALF (Macrosteles quadrilineatus) and Sulcia-GWSS (Homalodisca coagulata). Axes are in the number of nucleotides. Lettered breaks correspond to gene maps on the right hand side. Plots a–c show specific gene loss between Sulcia-GWSS (top) and Sulcia-ALF (bottom). Conserved orthologs are colored blue connected with gray shading, whereas genes that are lost in Sulcia-ALF are shown in red.

Fig. 5.—

Genomic content of the co-primary symbionts from the leafhoppers, Macrosteles quadrilineatus (Sulcia-ALF and Nasuia-ALF) and Homalodisca coagulata (Sulcia-GWSS and Baumannia); and, the spittlebug, Clastoptera arizonana (Sulcia-CARI & Zinderia). The major pathways include DNA replication, DNA repair, translation and processing, protein stability, and energy metabolism. Colored boxes indicate genes that are present, and white boxes indicate gene absence. Striped boxes indicate genes that are present, but possibly pseudogenized, or with uncertain function.

Fig. 6.—

Relationships between Nasuia deltocephalinicola and other Betaproteobacteria. Phylogenetic tree was inferred with maximum likelihood criteria for 107 taxa for 42 proteins (approx. 10,000 amino acid columns). Circles at nodes indicate bootstrap support values: black = >75, gray = 55–75, and white = <55. See supplementary table S1, Supplementary Material online, for complete taxon sampling, and supplementary figure S2, Supplementary Material online, for full phylogeny.