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.