Evolution of multi-partner symbiotic systems in the Cerataphidini tribe: genome reduction of Buchnera and frequent turnover of companion symbionts

Many insect species rely on multiple microorganisms to fulfill their essential nutritional requirements. In aphids, dual-symbiotic systems involving the ancient obligate symbiont Buchnera aphidicola and a more recently acquired companion symbiont, such as Serratia symbiotica, have been well-documented in Lachninae and Chaitophorinae subfamilies. Recently, a novel Buchnera-Arsenophonus dual-symbiosis was discovered in Ceratovacuna japonica (Hormaphidinae). This study expands the knowledge of bacterial symbionts in the Hormaphidinae subfamily, particularly focusing on Ceratovacuna nekoashi and Pseudoregma panicola, to elucidate the evolutionary trajectory of dual-symbiosis within this lineage. Our 16S ribosomal RNA gene amplicon sequencing analysis revealed that Cerataphidini species consistently harbor companion symbionts alongside Buchnera, whereas Hormaphidini and Nipponaphidini species harbor only Buchnera. Notably, Arsenophonus in C. nekoashi and C. japonica have distinct phylogenetic origins, and P. panicola has acquired Symbiopectobacterium instead of Arsenophonus. Microscopic analyses demonstrated that these companion symbionts are maternally transmitted and occupy distinct cell types from those harboring Buchnera. Genome sequencing revealed extreme reduction in Buchnera genomes to [~]0.4 Mbp in both C. nekoashi and P. panicola, comparable to C. japonica but significantly smaller than Buchnera genomes of [~]0.6 Mbp in mono-symbioses. The companion symbionts possess complete riboflavin biosynthesis pathways lost in Buchnera. Our findings suggest that Buchnera genome reduction and companion symbiont acquisition occurred in the common ancestor of Cerataphidini, followed by multiple companion symbiont replacements, revealing a dynamic evolutionary pattern of multi-partner symbioses.