Isolation of Hermetia illucens larvae core gut microbiota by two different cultivation strategies

Hermetia illucens larvae (black soldier fly larvae, BSFL) convert efficiently organic waste to high quality biomass. To gain knowledge on the specific functions of gut microbes in this process it is a prerequisite to culture members of the core gut microbiota. Two different cultivation strategies were applied here for this purpose, a dilution-to-extinction cultivation and direct plating using six different media to culture aerobic heterotrophic bacteria. A total of 341 isolates were obtained by the dilution-to-extinction cultivation and 138 isolates by direct plating from guts of BSFL reared on chicken feed. Bacterial isolates were phylogenetically identified at the genus level by 16S rRNA gene sequencing (phylotyping) and differentiated at the strain level by genomic fingerprinting (genotyping). The main proportion of isolates was assigned to Proteobacteria, Firmicutes (Bacilli), and Actinobacteria. Predominant genera discussed in literature as member of a potential BSFL core gut microbiota, Providencia, Proteus, Morganella, Enterococcus, Bacillus, and members of the family Enterobacteriaceae, were isolated. A high intra-phylotype diversity was obtained by genomic fingerprinting which was especially enhanced by the dilution-to-extinction cultivation. This study showed that the application of different cultivation strategies including a dilution-to-extinction cultivation helps to culture a higher diversity of the BSFL gut microbiota and that genomic fingerprinting gives a better picture on the genetic diversity of cultured bacteria which cannot be covered by a 16S rRNA gene sequence based identification alone.

Automated summary

Hermetia illucens larvae, also known as black soldier fly larvae (BSFL), are efficient in converting organic waste into high-quality biomass. This study explored the role of gut microbes in this process by culturing members of the core gut microbiota. Two cultivation strategies were employed, dilution-to-extinction cultivation and direct plating, resulting in the isolation of a diverse range of bacterial strains. Phylogenetic analysis identified predominant genera, such as Providencia, Proteus, Morganella, Enterococcus, Bacillus, and members of the family Enterobacteriaceae, which have been previously associated with the BSFL gut microbiota. Genomic fingerprinting provided insights into the genetic diversity of the cultured bacteria that could not be obtained solely through 16S rRNA gene sequencing.