Double trouble? Quantifying the risk from co-exposure to multiple pathogens in Tenebrio molitor at different CO₂ concentrations

The insect mass-rearing industry to produce feed and food is expanding rapidly. Insects in production frequently encounter multiple pathogens and environmental stressors simultaneously, which can lead to significant economic losses. Our understanding of the interactions between different stressors remains limited, and existing methods primarily focus on determining overall patterns of additivity, synergism, or antagonism. However, the interactions between different stressors may exhibit more intricate response patterns, such as time or dose dependency. With the expanding industry of insect production, it becomes vital to conduct comprehensive risk assessment of diseases, using approaches that can detect both lethal and sublethal effects. Here, we assessed the risk of co-exposure to a fungal (Metarhizium brunneum) and a bacterial (Bacillus thuringiensis) pathogen in the yellow mealworm (Tenebrio molitor) at ambient and elevated carbon dioxide (CO₂) concentrations. We assessed total larval biomass per treatment group, survival, and individual weight gain 14 and 20 days after pathogen exposure. To analyse the data, we used a mixture toxicity (MIXTox) model, which identifies dose ratio or dose level dependency in addition to overall antagonism or synergism. The interactions between the two pathogens were mostly antagonistic or additive at both CO₂ concentrations and time points, indicating that the observed effects during co-exposure did not exceed the expected combined effects of the individual exposure. We did not find evidence that the interactions between the pathogens substantially change at elevated CO₂. The antagonistic interactions measured in the bioassays are likely to be indirect via the insect host, as we did not detect direct inhibition between the two pathogens in in vitro experiments. Here we show that using the MIXTox model is a powerful tool to assess the effects of co-exposure to pathogens and quantify risk of disease in mass-reared insects.