Abstract:

Objective: This study aims to clarify the composition of gut fungi and bacteria of Agrilus mali in natural population in different developmental stages and different environmental conditions, and infer the possible effects on growth, development and physiological changes of the hosts. Method: The intact intestines of the adult (A) and larva (L) of Agrilus mali collected from wild fruit forest (W) and cultivated orchard (C) were dissected. The ITS2 sequence (fungi) and V3-V4 variant region (bacteria) of 16S rRNA were determined by Illumina MiSeq technology. The number of operating taxonomic units (OTU) of gut symbiotic microorganisms was counted, the species abundance, composition and alpha diversity were analyzed, and PICRUSt2 was used to predict function of OTUs. Result: A total of 177 028 ITS2 sequences of intestinal fungi and 253 712 16S rRNA high-quality sequences of bacteria were obtained from the gut microorganisms of A. mali. After cluster analysis, 285 (fungi) and 1 470 (bacteria) OTUs were obtained, respectively. For fungi, 5 phyla, 22 classes, 50 orders, 92 families, and 122 genera were identified; and for bacteria, totally 40 phyla, 103 classes, 231 orders, 364 families, and 594 genera were identified. There were 11 unique genera of fungi in CA, 28 in CL, 37 in WA, 12 in WL, and there were 2 unique genera of bacteria in CA, 223 in CL, 47 in WA, 68 in WL. A total of 7 genera of fungal communities and 21 genera of bacterial communities were determined as the core taxa. In addition, for intestinal fungal community, both the species diversity and richness of adults were greater than those of larvae, however for the bacterial community, species diversity in adults was greater than that in larvae, but the richness in the former was lower than in the later. Also, both the species diversity and richness of gut microorganism community of the larvae were much greater in cultivated than those in wild. Furthermore, a total of 74 fungal metabolic pathways and 426 bacterial metabolic pathways were annotated through MetaCyc Pathway. A total of 868 fungal metabolic pathways and 2 188 bacterial metabolic pathways were annotated through KEGG. Conclusion: There are differences in species and abundance of gut fungi and bacteria communities of A. mali at different developmental stages and in different environments. The species diversity and abundance of gut fungi in adults are greater than those in larvae. However, the intestinal bacteria in adults have lower abundance than that in larvae, suggesting that fungi play a significant role during developing process of adults while bacteria play a significant role during developing process of larvae. Additionally, although the species and abundance of intestinal microorganisms are quite difference, their functions are almost the same, indicating that a fixed group or symbiotic bacteria play a role in the gut of A. mali, which may be the result of long-term co-evolution between A. mali and its host.

Key words: Agrilus mali, gut microorganisms, high throughput sequencing, diversity, PICRUSt2 function prediction