Exploring the hemolymph microbiome: an emerging lever for health management in industrial Tenebrio molitor rearing

The farming of edible insects represents a promising alternative strategy for the sustainable production of proteinrich food and feed with a low environmental footprint. Rearing substrates strongly influence insect performance and play a key role in shaping insect- associated microbiota, which in turn can affect host physiology and immune competence. While gut microbiota has been extensively investigated, the microbiota associated with insect hemolymph and its potential role in immune responses remain largely unexplored. The HeMiTool project (Hemolymph Microbiome of insects: a promising Tool to develop innovative strategies to control pests and protect beneficials) aims to fill this knowledge gap by providing an in-depth characterisation of hemolymph microbiome–immune system dynamics in mature larvae of Tenebrio molitor L. Larvae were continuously reared on different agro-industrial byproducts under controlled conditions, allowing a robust comparison of diet-related effects. Hemolymph and gut microbial communities were analysed using a combined culture- dependent and culture-independent approach, including high-throughput 16S rRNA gene sequencing and quantitative PCR. In parallel, key chemical and biochemical parameters of the hemolymph were assessed, and some immune-related traits were investigated. Methodological optimisation of hemolymph extraction and processing ensured the reliability of downstream microbiological and immunological analyses. Preliminary results indicate that both hemolymph and gut-associated microbial communities are influenced by the rearing substrate, supporting the existence of a previously overlooked, dietresponsive hemolymph microbiome compartment. Observed trends in hemolymph- associated parameters suggest possible interactions between nutrition and host physiological status, although further investigation is required to clarify these relationships. Overall, this study provides first insights into the hemolymph microbiome of an edible insect species and highlights its relevance as an emerging component of insect biology. These findings provide a foundation for future diet-based strategies aimed at improving robustness and health stability in mass-reared edible insects. This work was funded by the PRIN 2022 PNRR project HeMiTool (Grant no. P20228WWB7), European Union – Next Generation EU, Mission 4 Component 1.