Kaur, S.; Sunny, J. S.; Stinson, S. A.; Rondinelli, M. A.; Alexander, J.; Vint, A.; Doyle, R. T.; Hug, L. A.; diCenzo, G. C.

Beetles (order Coleoptera) are a diverse insect group whose associated microbial communities remain understudied despite their ecological, commercial, and biotechnological importance. Larvae of the darkling beetle species Tenebrio molitor (mealworms) and Zophobas atratus (superworms) have gained attention for their reported ability to ingest and possibly degrade plastics such as polyethylene and polystyrene. This degradation is believed to be mediated by enzymes from their gut microbiota, yet the underlying microbial mechanisms remain unclear. Using deep shotgun metagenomics, we generated comprehensive gut metagenomes for mealworms and superworms, recovering 53 and 100 high-quality metagenome-assembled genomes (MAGs), respectively. We found that the gut microbiomes of both insect species are dominated by bacteria from the phyla Bacillota and Pseudomonadota, with superworms trending towards greater bacterial diversity than mealworms. Comparing gut bacterial communities of insects fed polyethylene or polystyrene versus controls revealed no clear plastic ingestion effects beyond those attributable to starvation. On the other hand, insect cohort contributed substantially to variation in bacterial community composition, explaining 23% and 33% of the total variation in mealworms and superworms, respectively. Finally, we computationally identified numerous secreted proteins with sequence similarity to enzymes previously implicated in polyethylene and polystyrene degradation, and we provide support for dye decolorizing peroxidase (DyP)-type peroxidases as being the enzyme class mostly likely to initiate plastic oxidation in the digestive tracts of mealworms and superworms. Overall, this study provides high-quality metagenomes and MAGs for mealworms and superworms, revealing substantial under-explored microbial biodiversity, and highlighting DyP-type peroxidases as promising targets for future plastic biodegradation studies.