Comparative Genomics of the Dual-Obligate Symbionts from the Treehopper, Entylia carinata (Hemiptera: Membracidae), Provide Insight into the Origins and Evolution of an Ancient Symbiosis

Abstract

Insect species in the Auchenorrhyncha suborder (Hemiptera) maintain ancient obligate symbioses with bacteria that provide essential amino acids (EAAs) deficient in their plant-sap diets. Molecular studies have revealed that two complementary symbiont lineages, "Candidatus Sulcia muelleri" and a betaproteobacterium ("Ca. Zinderia insecticola" in spittlebugs [Cercopoidea] and "Ca. Nasuia deltocephalinicola" in leafhoppers [Cicadellidae]) may have persisted in the suborder since its origin ∼300 Ma. However, investigation of how this pair has co-evolved on a genomic level is limited to only a few host lineages. We sequenced the complete genomes of Sulcia and a betaproteobacterium from the treehopper, Entylia carinata (Membracidae: ENCA), as the first representative from this species-rich group. It also offers the opportunity to compare symbiont evolution across a major insect group, the Membracoidea (leafhoppers + treehoppers). Genomic analyses show that the betaproteobacteria in ENCA is a member of the Nasuia lineage. Both symbionts have larger genomes (Sulcia = 218 kb and Nasuia = 144 kb) than related lineages in Deltocephalinae leafhoppers, retaining genes involved in basic cellular functions and information processing. Nasuia-ENCA further exhibits few unique gene losses, suggesting that its parent lineage in the common ancestor to the Membracoidea was already highly reduced. Sulcia-ENCA has lost the abilities to synthesize menaquinone cofactor and to complete the synthesis of the branched-chain EAAs. Both capabilities are conserved in other Sulcia lineages sequenced from across the Auchenorrhyncha. Finally, metagenomic sequencing recovered the partial genome of an Arsenophonus symbiont, although it infects only 20% of individuals indicating a facultative role.

Keywords: aminoacyl tRNA synthetases; bacteria; facultative symbiont; gene loss; genome evolution; nutritional symbioses.

Fig. 1.

—Entylia carinata (Membracidae) host and fluorescence in situ hybridization of bacteriome organs containing the bacterial symbionts, “Ca. Sulcia muelleri” (red; Bacteroidetes) and “Ca. Nasuia koganicola” (green; Betaproteobacteria). DNA is counterstained with DAPI (blue) primarily showing host DNA. (A) Image showing E. carinata adult. (B) Lateral habitus of E. carinata nymph abdomen and (C) showing bacteriocytes containing bacteria (white arrows illustrate Nasuia containing bacteriocytes). White scale bars represent 200 µm (B) and 100 µm (C). Note: Green autofluorescence observed in abdominal cavity (without arrows) for Image (C). Photograph of E. carinata adult (A) provided by Kyle Kittelberger.

Fig. 2.

—The relationship between percent G + C nucleotide content and genome size of the major co-primary bacterial symbiont lineages in the Auchenorrhyncha (Hemiptera). Symbionts are color-coded by their lineage of origin (discussed in text; see labels, e.g., Hodgkinia = grey). Inset graph shows that G + C content is significantly correlated with genome size (Phylogenetic Generalized Lease Squares [PGLS]: correlation = −0.98 and P-value = <0.001) in sequenced Sulcia lineages. Sulcia phylogeny reconstructed with 16S rRNA using ML criteria in RAxML (Stamatakis 2014) and PGLS performed in R with nlme and ape (Paradis et al. 2004; Pinheiro et al. 2017). Representative host insects are abbreviated as follows: ALF = Macrosteles quadrilineatus, PUNC = M. quadripunctulatus, and ENCA = Entylia carinata.

Fig. 3.

—Maximum likelihood phylogeny of Nasuia and other Betaproteobacteria based on 42 protein-coding genes for 108 taxa (104 ingroup taxa + 4 outgroup taxa). Relevant bootstrap support values are shown at each inter-node. Host lineages/names in Cicadomorpha are given beneath the branches. Representative host insects are abbreviated as follows: ALF = Macrosteles quadrilineatus, PUNC = M. quadripunctulatus, and ENCA = Entylia carinata.

Fig. 4.

—Summary of the lineage-specific gene losses (excluding hypothetical genes) between obligate symbionts from the treehopper Entylia carinata (Sulcia- and Nasuia-ENCA) and leafhopper Macrosteles spp. (Sulcia- and Nauia-ALF & PUNC). The hypothetical establishment date of obligate symbionts in the common ancestor of the Auchenorrhyncha and the hypothetical diversification date of leafhoppers and treehoppers are based on previous studies (Shcherbakov 2002; Moran et al. 2005; Cryan and Svenson 2010; Koga et al. 2013). Evolutionary events of Sulcia and Nasuia are colored in red and green, respectively. Pseudogenes are shaded in gray.

Fig. 5.

—(A) Gene retention and losses involved in DNA replication and repair, transcription, translation and energy metabolisms in obligate symbionts from the treehopper Entylia carinata (Sul- and Nas-ENCA), leafhopper Macrosteles quadrilineatus (Sul- and Nas-ALF), and spittlebug Clastoptera arizonana (Sul- and Zind-CARI). (B) Retention of genes with the same predicted function in both Nasuia–ENCA and Sulcia-ENCA (genes shaded red) relative to their related lineages in the ALF leafhopper. Conserved orthologs are shaded blue and unique genes lost involved in other cellular functions are shaded white. (B) Note: tRNA genes omitted.

Fig. 6.

—Predicted branched-chain amino acid pathways in Sulcia-ENCA. The missing ilvE (branched-chain EAA transaminase) gene is shaded in gray. Substrates required for pathway completion and predicted to be imported by the host are shown in red box. Metabolites synthesized in Sulcia-ENCA are shown in blue box.