Microbial symbioses in treehoppers: Multipartite masterpieces or muddled mess?

Multipartite microbial symbioses in tropical treehoppers (Membracidae) appear to be intriguing examples of holometabolome complexity. Treehopper symbioses were first examined by Agnes Rau, a student of Paul Buchner, in her dissertation in 1943. Rau found Brazilian treehoppers hosted up to 6 species of bacteriome symbiont, which Buchner highlighted as remarkable. U Using genomics, metatranscriptomics, pathway analysis, and FISH we revisited these insects, collecting ~47 species, dissecting and sequencing the bacteriomes to understand the nature of these symbioses. We assembled metagenomes and metatranscriptomes and characterized gene expression repertoires. Results revealed 8 symbionts that were abundant in Brazilian, but not U.S., treehoppers. The obligate symbiont Sulcia was universally present, but its gene expression varied depending on other symbionts. The symbiont Nasuia, contributing only a few amino acids, was sometimes absent and replaced by a yeast-like Ophiocordyceps symbiont. Divergent Arsenophonus-like and Sodalis-like symbionts were nearly universally present at close to 1:1 abundance, suggesting possible essential functions. A Brenneria-like symbiont and a new Bombella sp. occurred in half of these samples including U.S. treehoppers. Follow-up PCR surveys of ant trophobionts and honeydew showed Bombella sp. may be transferred although its phylogeny suggested co-evolution with membracids. Gene ontology enrichment suggested a possible role in branched-chain amino acid and mo-molybdoperin biosynthesis, iron homeostasis, or defense functions. Together, these data suggest a rich pattern of symbiont gain, loss, and metabolic complementarity. It remains unclear what factors facilitate the high symbiont biodiversity in neotropical treehoppers or whether trophobiosis with ants and bees plays a role.