Isolation and identification of culturable gut microbiota in the larval stage of lesser mealworm (Alphitobius diaperinus)

The highly prevalent pest Alphitobius diaperinus (Coleoptera: Tenebrionidae) causes significant structural damage in poultry farms. Despite previous investigations on its carriage of pathogenic microorganisms, our understanding of its microbiome remains limited. This study aimed to analyze the diversity of culturable gut microbiota in A. diaperinus obtained from laboratory breeding. Fifteen seventh instar larvae underwent a 24-hour starvation period, followed by surface disinfection. Dissected midguts were homogenized and plated on nutrient agar (NA), brain heart infusion agar (BHI), and Bacillus cereus agar (BC). The cultured isolates were subjected to gram staining, phylogenetic analysis, biochemical property evaluation, and metabolic activity assessment. Bacterial counts were higher in BHI (2.51x10⁵ CFU/gut) than in NA (2.25x10⁵ CFU/gut), possibly due to nutrient richness. NA exhibited a dominant colony morphology of gram-negative bacilli, while BHI displayed additional distinct colonies of gram-positive cocci. Surprisingly, yeast-like colonies were observed on BC plates. Based on 16S rRNA gene sequences, eight bacterial isolates were identified as Enterobacter sp., and two as Staphylococcus sp. Using RNA gene ITS region sequences, two yeast isolates were identified as Debaryomyces sp. and Hyphopichia sp. A preliminary species-level identification of bacteria (Enterobacter cloacae, Staphylococcus gallinarum, and Staphylococcus succinus) was achieved using API systems and complementary biochemical tests. Discrepancies between phylogenetic analysis and phenotypic data suggest the potential existence of new species or subspecies. Further comprehensive studies are required to confirm this hypothesis.