Effect of CO2 Concentrations on Entomopathogen Fitness and Insect-Pathogen Interactions

Abstract

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.

Keywords: Bacillus thuringiensis; Biocontrol; Host-pathogen Interactions; Insect Culture; Metarhizium brunneum; Tenebrio molitor.

Fig. 1

Schematic representation of the experimental design. A Larvae reared at either low or high CO₂ for 18 days were exposed to B. thuringiensis previously exposed to either low or high CO₂ for two days or to water as a control. B Larvae reared at either low or high CO₂ for 18 days were exposed to M. brunneum grown at low or high CO₂ for 14 days. The lids of the Petri dishes were elevated by adding a 2 cm wide plastic strip between the lower dish and the lid. A, B Each arrow represents one treatment (12 treatments in total, n = 5, 30 larvae per cup, two experimental repetitions). The survival, feed intake and weight of the larvae were assessed every second day for a period of 14 days after pathogen exposure. Figure created with BioRender (www.biorender.com)

Fig. 2

Three-parameter log-logistic models for germination of M. brunneum conidia over time (hours) at either low (e = 9.58 (confidence limits = 9.49 and 9.66); b = -12.74; d = 99.83) or high (e = 7.77 (confidence limits = 7.66 and 7.87); b = -9.83; d = 99.90) CO₂ concentrations. The shaded areas represent the 95% confidence intervals

Fig. 3

Survival of T. molitor larvae reared at either low (blue) or high (red) CO₂ concentrations after exposure to pathogens for a period of 14 days. A Cumulative survival probability of larvae exposed to either low or high CO₂ without (dotted survival curves) and with B. thuringiensis exposure (continuous survival curves). B Cumulative survival probabilities of larvae exposed to either low or high CO₂ without (dotted survival curves) and with M. brunneum exposure (continuous survival curves). A, B Different letters to the right of the survival curves indicate statistical differences among treatments at p < 0.05. The shaded areas represent the 95% confidence intervals. Hazard ratios and p-values of fixed and random effects of the mixed effects cox proportional hazards models are displayed in Table 2. Figure created with GraphPad Prism version 9.3.1

Fig. 4

Feed intake per 30 larvae during exposure (two days) to the pathogens. A Feed intake during exposure to B. thuringiensis in Exp. (experiment) 1 and 2: control (no exposure to B. thuringiensis), lowCO₂-larv (larvae exposed to low CO₂), highCO₂-larv (larvae exposed to high CO₂), lowCO₂-Bt (B. thuringiensis exposed to low CO₂), highCO₂-Bt (B. thuringiensis exposed to high CO₂). B Feed intake during exposure to M. brunneum in Exp. (experiment) 1 and 2: control (no exposure to M. brunneum), lowCO₂-larv (larvae exposed to low CO₂), highCO₂-larv (larvae exposed to high CO₂), lowCO₂-Mb (M. brunneum grown at low CO₂), highCO₂-Mb (M. brunneum grown at high CO₂). A, B Boxplots show median, interquartile range, and minimum and maximum. Different letters above boxplots indicate statistical differences among treatments at p < 0.05 for each experiment separately. Degrees of freedom, F-values and p-values of the two-way ANOVAs are displayed in Table 3. Figure created with GraphPad Prism version 9.3.1

Fig. 5

Weight gain per larva (mg) during 14 days after exposure to the pathogens. A Weight gain after exposure to B. thuringiensis in Exp. (experiment) 1 and 2: control (no exposure to B. thuringiensis), lowCO₂-larv (larvae exposed to low CO₂), highCO₂-larv (larvae exposed to high CO₂), lowCO₂-Bt (B. thuringiensis exposed to low CO₂), highCO₂-Bt (B. thuringiensis exposed to high CO₂). B Weight gain after exposure to M. brunneum in Exp. (experiment) 1 and 2: control (no exposure to M. brunneum), lowCO₂-larv (larvae exposed to low CO₂), highCO₂-larv (larvae exposed to high CO₂), lowCO₂-Mb (M. brunneum grown at low CO₂), highCO₂-Mb (M. brunneum grown at high CO₂). A, B Boxplots show median, interquartile range, and minimum and maximum. Different letters above boxplots indicate statistical differences among treatments at p < 0.05 for each experiment separately. Degrees of freedom, F-values and p-values of the two-way ANOVAs are displayed in Table 3. Figure created with GraphPad Prism version 9.3.1