Evolutionary dynamics of type VI secretion systems in fruit fly-associated Enterobacter

Naima Bel Mokhtar^1,2Panagiota Stathopoulou¹Elias Asimakis¹Antonios Augustinos³Julieta Salgueiro⁴Malini Alleck⁵Preeaduth Sookar⁵ÓSCAR DEMBILIO⁶Diego F Segura⁷George Tsiamis^1*

• ¹Department of Sustainable Agriculture, University of Patras, Patras, Greece
• ²Panepistemio Patron, Patras, Greece
• ³Department of Plant Protection Patras, Institute of Industrial and Forage Crops, Hellenic Agricultural Organization ‘DIMITRA’, Patras, Greece
• ⁴Centro de Recursos Naturales Renovables de la Zona Semiárida, CERZOS, Universidad Nacional del Sur, Bahía Blanca, Argentina
• ⁵Agro-Industry and Food Security Division, Ministry of Agro-Industry, Food Security Blue Economy & Fisheries, Agricultural Services, Mauritius, Mauritius
• ⁶Empresa de Transformación Agraria S.A., S.M.E., M.P. (TRAGSA), Paterna, Spain
• ⁷Facultad de Ciencias Agronómicas y Veterinarias, Universidad del Salvador, Buenos Aires, Argentina

Species in the genus Enterobacter are widely distributed and occupy diverse ecological niches. Although many species within this genus have been extensively isolated and characterized, their symbiotic associations with Tephritidae fruit flies remain understudied, particularly through comparative genomic analyses. To address this gap, we conducted a whole-genome comparative analysis of thirteen Enterobacter strains isolated from the most economically significant fruit fly species: Anastrepha fraterculus, Bactrocera dorsalis, Bactrocera zonata, Ceratitis capitata, and Zeugodacus cucurbitae. The results revealed that different fruit flies harbor distinct Enterobacter species, with Enterobacter hormaechei being the most prevalent across hosts. Notably, distinct E. hormaechei subspecies were associated with specific hosts, suggesting a potential host-driven adaptation and coevolution. Pangenome analysis highlighted a dynamic genetic structure among these strains, with significant differences in the core, shell, and species-specific gene composition. The high proportion of metabolism-related genes in the core genome suggests a conserved role in essential biological functions, whereas the enrichment of mobile genetic elements (prophages and transposons) and cell motility genes within the shell and species-specific genomes highlights the genomic plasticity and potential host-specific adaptations. Three distinct subtypes of T6SS (type VI secretion systems) gene clusters, T6SS_C1, T6SS_C2, and T6SS_C3, were detected across Enterobacter strains. T6SS_C1 and T6SS_C2 were identified in most Enterobacter strains, whereas T6SS_C3 cluster was restricted to a single isolate. Although these clusters contained thirteen core T6SS genes, they were characterized by different gene synteny and effector/immunity gene content, suggesting that different Enterobacter strains may utilize distinct mechanisms for interbacterial interactions, host manipulation, and environmental adaptation. Overall, our findings reveal the genetic basis of the symbiosis between Enterobacter species and fruit flies, shedding light on their evolutionary dynamics, diversity of T6SS, and functional traits. These results open new avenues for developing microbiome-based strategies for pest management, including the targeted manipulation of microbial communities to enhance sterile insect technique (SIT) outcomes.