Microbial associates of the elm leaf beetle: uncovering the absence of resident bacteria and the influence of fungi on insect performance.

Microbial symbionts play crucial roles in the biology of many insects. While bacteria have been the primary focus of research on insect-microbe symbiosis, recent studies suggest that fungal symbionts may be just as important. The elm leaf beetle (ELB, Xanthogaleruca luteola ) is a serious pest species of field elm ( Ulmus minor ). Using culture-dependent and independent methods, we investigated the abundance and species richness of bacteria and fungi throughout various ELB life stages and generations, while concurrently analyzing microbial communities on elm leaves. No persistent bacterial community was found to be associated with the ELB or elm leaves. By contrast, fungi were persistently present in the beetle's feeding life stages and on elm leaves. Fungal community sequencing revealed a predominance of the genera Penicillium and Aspergillus in insects and on leaves. Culture-dependent surveys showed a high prevalence of two fungal colony morphotypes closely related to Penicillium lanosocoeruleum and Aspergillus flavus . Among these, the Penicillium morphotype was significantly more abundant on feeding-damaged compared with intact leaves, suggesting that the fungus thrives in the presence of the ELB. We assessed whether the detected prevalent fungal morphotypes influenced ELB's performance by rearing insects on (i) surface-sterilized leaves, (ii) leaves inoculated with Penicillium spores, and (iii) leaves inoculated with Aspergillus spores. Insects feeding on Penicillium -inoculated leaves gained more biomass and tended to lay larger egg clutches than those consuming surface-sterilized leaves or Aspergillus -inoculated leaves. Our results demonstrate that the ELB does not harbor resident bacteria and that it might benefit from associating with Penicillium fungi.IMPORTANCEOur study provides insights into the still understudied role of microbial symbionts in the biology of the elm leaf beetle (ELB), a major pest of elms. Contrary to expectations, we found no persistent bacterial symbionts associated with the ELB or elm leaves. Our research thus contributes to the growing body of knowledge that not all insects rely on bacterial symbionts. While no persistent bacterial symbionts were detectable in the ELB and elm leaf samples, our analyses revealed the persistent presence of fungi, particularly Penicillium and Aspergillus on both elm leaves and in the feeding ELB stages. Moreover, when ELB were fed with fungus-treated elm leaves, we detected a potentially beneficial effect of Penicillium on the ELB's development and fecundity. Our results highlight the significance of fungal symbionts in the biology of this insect.