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.

The industrial rearing of the yellow mealworm (Tenebrio molitor) for feed and food purposes on agricultural by-products may expose larvae and adults to entomopathogens used as biocontrol agents in crop production. Bacterial spores/toxins or fungal conidia from species such as Bacillus thuringiensis or Metarhizium brunneum could affect the survival and growth of insects. Therefore, the aim of this study was to investigate the potential benefits of a wheat bran diet supplemented with probiotic bacteria and dried egg white on larval development and survival and its effects on the gut microbiome composition. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactiplantibacillus plantarum WJB, were added to wheat bran feed with and without dried egg white, as an additional protein source, directly from neonate larval hatching until reaching a body mass of 20 mg. Subsequently, larvae from the various diets were exposed for 72 h to B. thuringiensis, M. brunneum, or their combination. Larval survival and growth were recorded for 14 days, and the bacterial microbiota composition was analyzed using 16S rDNA sequencing prior to pathogen exposure and on days 3 and 11 after inoculation with the pathogens. The results showed increased survival for T. molitor larvae reared on feed supplemented with P. pentosaceus in the case of co-infection. Larval growth was also impacted in the co-infection treatment. No significant impact of egg white or of P. pentosaceus on larval growth was recorded, while the addition of Lb. plantarum resulted in a minor increase in individual mass gain compared with infected larvae without the latter probiotic. On day 14, B. thuringiensis was no longer detected and the overall bacterial community composition of the larvae was similar in all treatments. On the other hand, the relative operational taxonomic unit (OTU) abundance was dependent on day, diet, and probiotic. Interestingly, P. pentosaceus was present throughout the experiments, while Lb. plantarum was not found at a detectable level, although its transient presence slightly improved larval performance. Overall, this study confirms the potential benefits of some probiotics during the development of T. molitor while underlining the complexity of the relationship between the host and its microbiome.

The yellow mealworm (Tenebrio molitor) is a promising insect species for mass-rearing for the production of feed and food. In mass-production systems, insects may be exposed to abiotic stressors such as heat stress as well as potentially lethal pathogens. To ensure mass-reared T. molitor populations are healthy and productive there is a need to understand both the risks, and potential benefits of heat stress, on the fitness of insects and their susceptibility to pathogens. In this study, we investigated the effects of a short (2 h) or a long (14 h) heat stress (38 degrees C) exposure on the susceptibility and the immune responses of T. molitor larvae exposed to a fungal pathogen (Metarhizium brunneum). Larvae were exposed to the pathogen either immediately or five days after the heat stress treatments. The development of heat stressed larvae and their offspring was also assessed. A short heat stress immediately before exposure to M. brunneum increased the survival probability of T. molitor larvae, which correlated with increased antibacterial activity in the hemolymph. The exposure of larvae to short, or long heat stresses five days before pathogen exposure did not affect their survival, despite a temporary lowered body mass gain of heat stressed larvae. However, heat stressed larvae showed decreased hemocyte concentrations when exposed to M. brunneum. We also found an increased body weight in larval offspring of females that had been exposed to a short heat stress as larvae themselves. These findings demonstrate the importance of understanding the effects of heat stress in the long-term. The beneficial effects of heat stress on pathogen susceptibility in T. molitor and the negative effects on body mass gain are only transient, whereas negative effects on immune response (hemocyte concentrations) persist over an extended period.

The yellow mealworm (Tenebrio molitor L.; Coleoptera: Tenebrionidae) has been proposed during the last decade as a suitable insect species for feed and food production. The inclusion of by- and side-products of cereal and vegetable production in the feed of yellow mealworm increases the probability of exposure to Bacillus thuringiensis svmorrisoni biovar tenebrionis (Btt), an entomopathogen used in the biological control of coleopteran pest species. The lack of studies on effects of Btt spores and toxin on T. molitor in mass-rearing systems led us to explore its effect on survival and growth of larvae and its persistence in larvae and in frass. Larvae of different body weight (3, 5, 10 and 40 mg) were exposed by free feeding for 72 hours to a range of Btt spore - crystal concentrations spiked to wheat bran (WB). Susceptibility to the entomopathogen was concentration-dependent and linked to the larval body weight, larvae of higher weight being less susceptible. Growth and feed conversion ratio of survivors showed significant impact for low-weight larvae while less or no effect was recorded for larvae having higher weight. Btt was still recovered from larvae and frass 14 days after feeding with non-contaminated WB, indicating a certain level of persistence in the tested conditions, on alive 40-mg larvae, implying a potential risk in T. molitor mass rearing.

Numerous insect species and their associated microbial pathogens are exposed to elevated CO2 concentrations in both artificial and natural environments. However, the impacts of elevated CO2 on the fitness of these pathogens and the susceptibility of insects to pathogen infections are not well understood. The yellow mealworm, Tenebrio molitor, is commonly produced for food and feed purposes in mass-rearing systems, which increases risk of pathogen infections. Additionally, entomopathogens are used to control T. molitor, which is also a pest of stored grains. It is therefore important to understand how elevated CO2 may affect both the pathogen directly and impact on host-pathogen interactions. We demonstrate that elevated CO2 concentrations reduced the viability and persistence of the spores of the bacterial pathogen Bacillus thuringiensis. In contrast, conidia of the fungal pathogen Metarhizium brunneum germinated faster under elevated CO2. Pre-exposure of the two pathogens to elevated CO2 prior to host infection did not affect the survival probability of T. molitor larvae. However, larvae reared at elevated CO2 concentrations were less susceptible to both pathogens compared to larvae reared at ambient CO2 concentrations. Our findings indicate that whilst elevated CO2 concentrations may be beneficial in reducing host susceptibility in mass-rearing systems, they may potentially reduce the efficacy of the tested entomopathogens when used as biological control agents of T. molitor larvae. We conclude that CO2 concentrations should be carefully selected and monitored as an additional environmental factor in laboratory experiments investigating insect-pathogen interactions.

The yellow mealworm, Tenebrio molitor , is currently one of the most important insect species produced for livestock feed and human consumption. High-density rearing conditions make the risk of disease and infections by parasitic symbionts a challenge in the mass production of these insects. However, certain symbionts are beneficial and should be favoured in order to promote healthy insect populations. Knowledge of parasitic symbionts and their management is essential for the insect rearing industry and its associated research. Here we review the documented microbial infectious agents, invertebrate parasites, and beneficial symbionts occurring in T. molitor . Furthermore, we discuss detection, prevention, and treatment methods for disease management in T. molitor production systems to inform future management and decision making in T. molitor rearing.

The mass production of insects is rapidly expanding globally, supporting multiple industrial needs. However, parasite infections in insect mass-production systems can lower productivity and can lead to devastating losses. High rearing densities and artificial environmental conditions in mass-rearing facilities affect the insect hosts as well as their parasites. Environmental conditions such as temperature, gases, light, vibration, and ionizing radiation can affect productivity in insect mass-production facilities by altering insect development and susceptibility to parasites. This review explores the recent literature on environment-host-parasite interactions with a specific focus on mass-reared insect species. Understanding these complex interactions offers opportunities to optimise environmental conditions for the prevention of infectious diseases in mass-reared insects.

We examined the contents of nutritional importance, i.e., amino acids, fatty acids and minerals of different developmental stages of drones of two honey bee subspecies, namely Apis mellifera carnica and A. m. mellifera. The results revealed that, in general, individual amino acid amounts and therefore the total protein increased along with the developmental stages of the drones. No statistically significant differences were found between the same developmental stages of the two subspecies. The reverse, i.e., a decrease with developmental stage occurred in relation to the fatty acid composition. Most of the minerals were higher at advanced developmental stages. Overall, the high protein content (31.4–43.4%), small amount of fat (9.5–11.5%) and abundance of minerals such asiron and zinc, make drones a suitable nutritional resource. Even though nutrient content, especially protein, was higher in the pupae than the prepupae, we propose prepupae also as a commercial product based on their higher biomass production. Provided standard production protocols maintaining hygiene and safety will be adhered to, we propose that drone honey bees can be utilized as human food or animal feed.

Drone brood of Apis mellifera is often removed from the beehive to control the honeybee parasite Varroa destructor. Instead of discarding the drone brood, it could rather be used as a new food source for human nutrition. However, studies on microbiological hazards caused by edible insects are rare, especially in the case of drone brood. In this survey, microbial total viable cell counts and the most common foodborne bacteria were assessed in raw drone brood. Samples were taken from 24 beehives from four apiaries in Switzerland. The drone brood combs were harvested either by the beekeepers with their personal equipment or by the researchers with sterile equipment. No difference in the total viable cell counts was found between these two methods. All samples were free of Salmonella. Viable counts of Bacillus cereus, coagulase-positive staphylococci, Enterobacteriaceae and Escherichia coli were all below the detection limits of the recommended ISO reference methods. However, Listeria monocytogenes was detected in eight samples (all <10 cfu/g), which has not been reported in edible insects until now.

Dead sporulating female fly cadavers infected by the house fly-pathogenic fungus Entomophthora muscae are attractive to healthy male flies, which by their physical inspection may mechanically trigger spore release and by their movement create whirlwind airflows that covers them in infectious conidia. The fungal artillery of E. muscae protrudes outward from the fly cadaver, and consists of a plethora of micrometric stalks that each uses a liquid-based turgor pressure build-up to eject a jet of protoplasm and the initially attached spore. The biophysical processes that regulate the release and range of spores, however, are unknown. To study the physics of ejection, we design a biomimetic 'soft cannon' that consists of a millimetric elastomeric barrel filled with fluid and plugged with a projectile. We precisely control the maximum pressure leading up to the ejection, and study the cannon efficiency as a function of its geometry and wall elasticity. In particular, we predict that ejection velocity decreases with spore size. The calculated flight trajectories under aerodynamic drag predict that the minimum spore size required to traverse a quiescent layer of a few millimetres around the fly cadaver is approximately 10 µm. This corroborates with the natural size of E. muscae conidia (approx. 27 µm) being large enough to traverse the boundary layer but small enough (less than 40 µm) to be lifted by air currents. Based on this understanding, we show how the fungal spores are able to reach a new host.

Entomophthoralean fungi are insect pathogenic fungi and are characterized by their active discharge of infective conidia that infect insects. Our aim was to study the effects of temperature on the discharge and to characterize the variation in the associated temporal pattern of a newly discovered Pandora species with focus on peak location and shape of the discharge. Mycelia were incubated at various temperatures in darkness, and conidial discharge was measured over time. We used a novel modification of a statistical model (pavpop), that simultaneously estimates phase and amplitude effects, into a setting of generalized linear models. This model is used to test hypotheses of peak location and discharge of conidia. The statistical analysis showed that high temperature leads to an early and fast decreasing peak, whereas there were no significant differences in total number of discharged conidia. Using the proposed model we also quantified the biological variation in the timing of the peak location at a fixed temperature.