Evolutionary and metabolic consequences of symbiont addition and replacement in Brazilian treehoppers

Interaction between eukaryotic hosts and their microbial symbionts are ubiquitous and complex. The endosymbioses observed within Brazilian treehoppers are of key interest because of the unprecedented diversity that has been observed in their multipartite bacteriome-bound community of microbes. Using a combination of metagenomics, comparative genomic, phylogenomic, transcriptomics, and metatranscriptomics methods, our study provides preliminary evidence of these endosymbionts’ identity, location, and functions in the host. This symbiotic community includes well-studied primary symbionts Sulcia and Nasuia. However, our results confirmed that Nasuia is missing from some species of Brazilian treehopper. Additional symbionts include Arsenophonus, Sodalis, Rickettsia, Bombella, and Wolbachia. We also detected fragments of Asaia, Brenneria, and a yeast-like symbiont (YLS). Phylogenetic analysis indicated that Arsenophonus, Sodalis, and Wolbachia appeared to be both vertically and horizontally transferred, whereas Sulcia and Nasuia, are characteristically maintained through strict vertical transmission. Analysis also indicated long branching monophylies for Brenneria, Rickettsia, and the YLS, which may suggest vertical transmission. Finally, our analysis revealed treehopper-associated Bombella-like symbiont which was phylogenetically distinct from the Asaia symbiont identified in two samples. Pseudogene analysis provided insight that Arsenophonus, Sodalis, and Wolbachia appear to be undergoing rapid pseudogenization of their genomes, implying that these symbionts may be in the early stages of genome degeneration following the theory of Muller’s Ratchet. Pathway analysis indicated that Arsenophonus provides pathways for a wide range of amino acid, cofactor, vitamin, and sugar biosynthesis, making it a decent candidate for the replacement of Nasuia. These results are supported by gene expression (DEG) analysis which showed upregulation of genes for functions that could support the host or co-symbionts. Pathway analysis of Sodalis revealed an incompetent genome that does not contribute complete pathways for any amino acid, cofactor, vitamin, or sugar biosynthesis, while maintaining pathways related to the catabolism of amino acids and sugars. This potentially implies that Sodalis has already lost the majority of genes related to these life-sustaining processes or is acting as a selfish symbiont or parasite within the host. This selfish symbiont/parasite hypothesis is further supported by DEG enrichment analysis which identified 9 different upregulated gene ontology terms all involving different types of ion sequestering. Finally, Wolbachia appears to have some host beneficial, and some potentially host detrimental traits. Pathway tools determined that in some hosts, Wolbachia uniquely biosynthesizes four B-12 like cobamide compounds which it may synthesize for itself and/or it may play a role in supplementing the host’s nutrient poor diet. However, DEG analysis also found an upregulated gene related to cytoplasmic incompatibility, which could indicate that Wolbachia has some ability to manipulate reproduction of the host treehopper. The future directions for this study include long read sequencing for assembly challenges, confirming symbiont transmission mode through microscopy, and sequencing the treehopper genome to analyze horizontal gene transfers.