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

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. Supplementary Information The online version contains supplementary material available at 10.1007/s00248-024-02347-6.


Introduction
CO 2 (carbon dioxide) has the potential to affect hostpathogen interactions if either the host, pathogen, or both are affected by changes in CO 2 concentrations.Numerous insect species are constantly exposed to CO 2 concentrations above the atmospheric level, which is currently recorded as approximately 420 ppm (parts per million) [1].Elevated CO 2 concentrations can be a result of the respiration of insects [2,3] or a product of increased microbial activity and subsequent accumulation in enclosed areas [4].The CO 2 concentration in soil air (inside soil pores), for example, is typically higher than the atmospheric CO 2 concentration due to decreased gas exchange [4], hence soil-dwelling insect species are exposed to elevated CO 2 concentrations in their environment.Furthermore, it is known that CO 2 can accumulate in colonies of social insects reaching up to 60,000 ppm in leaf-cutting ant colonies [5], and 92,000 ppm in termite mounds [6].Insects that are mass-reared for food and feed purposes can also be exposed to elevated CO 2 concentrations because they are typically kept at high densities in closed systems [7], which facilitates the accumulation of CO 2 [8].
The yellow mealworm, Tenebrio molitor, is an insect species that is increasingly being mass-reared to produce proteins and fats to feed livestock and for use in aquaculture [9,10].Respiration of T. molitor larvae produces approximately 60 g CO 2 per kg of body mass per day or approximately 1,000 g CO 2 per kg body mass gain [2].Despite the utilisation of appropriate ventilations, CO 2 is still likely to accumulate in production facilities of T. molitor [8,11]; for example, in a closed experimental T. molitor rearing, CO 2 concentrations reached up to 6,000 ppm [12].The maximum permitted CO 2 concentrations in production facilities are regulated by law in most countries to ensure the health and safety of employees [13].For example, the long-term (8 h) exposure limit of CO 2 concentration in the workplace is 5,000 ppm in many countries including the UK [14], the US [15], and countries belonging to the EU [16], which is more than tenfold higher than atmospheric concentrations.
Besides the use of T. molitor to produce feed, the yellow mealworm is also a global pest of stored grains and grain by-products [17].The CO 2 concentrations inside stored grains can exceed atmospheric CO 2 concentration [18] and when there is microbial or insect activity, CO 2 concentrations may increase even further [19,20].Various organisms (entomopathogens) such as bacteria, fungi, protists, nematodes, and viruses can infect T. molitor [21,22].Some of these entomopathogens are used as biological control agents against T. molitor in stored grains [23,24] but at the same time, entomopathogens can also cause lethal or sublethal diseases in insects mass-reared for food and feed leading to economic losses in production systems [21].Currently, there is a dearth of knowledge on how CO 2 concentrations affect host-pathogen interactions in both mass-reared and wild insects [25].Improving our understanding of the effects of CO 2 on entomopathogens and their interactions with insect hosts will help to guide decisions of whether CO 2 should be considered a relevant factor to include for insect-pathogen interaction experiments and in the design of insect mass rearing facilities.
CO 2 is known to affect entomopathogenic organisms; for example, Pseudoxylaria spp., an entomopathogenic fungus infecting termites (Odontotermes obesus), showed reduced growth when exposed to elevated CO 2 concentrations [6].Furthermore, the number of conidia produced by different strains of the entomopathogenic fungal species Metarhizium anisopliae, Isaria farinosa, and Beauveria bassiana were generally decreased at 1,000 ppm CO 2 compared to 350 ppm CO 2 [26].CO 2 has also been found to affect the virulence of pathogenic organisms of humans [27]; in the human-pathogenic bacterium Bacillus cereus, for example, the expression of virulence genes was higher at elevated CO 2 concentrations [28] and Candida albicans, a fungal pathogen of humans, switches from the monocellular to the more virulent filamentous growth at elevated CO 2 concentrations [27].Nevertheless, the impact of CO 2 on the virulence of entomopathogenic organisms that can infect economically important insects remains unknown.
In this study, we examined the effects of CO 2 on two entomopathogens, the bacterium Bacillus thuringiensis, and the fungus Metarhizium brunneum, which both naturally infect T. molitor [21,22].We used in vitro experiments and full-factorial bioassays to study interactions between CO 2 , insects, and pathogens.The pathogens were selected because both B. thuringiensis and M. brunneum can be found in stored grains [29][30][31]; grain products are both an important habitat of T. molitor and often used to feed T. molitor larvae in production systems [11].Species of the genus Metarhizium are facultative entomopathogens, as these fungi can also colonize the rhizosphere of plants or live as saprotrophs [32,33].The impact of CO 2 on fungal germination and growth in the external insect host environment is therefore highly relevant.On the other hand, B. thuringiensis is thought to only multiply inside the insect host while the environment (external to the insect host) constitutes a transition compartment for the spores and crystals without reproduction [34].Therefore, the effects of CO 2 on the viability and virulence of spores and crystals in the environment (e.g., soil or stored grains) are relevant to evaluate.The aims of this study were to assess the effects of elevated CO 2 (4,500 ± 500 ppm) on: (1) the in vitro germination of conidia and mycelial growth of M. brunneum, (2) the in vitro viability and persistence of B. thuringiensis spores, and (3) the in vivo interactions between M. brunneum or B. thuringiensis and the larvae of T. molitor.

Methods
All insect rearing and experiments took place in two separate 50-litre LEEC Culture Safe CO 2 incubators adjacent to each other, one used for low [450 ppm (± 50 ppm)] CO 2 , and one used for high [4,500 ppm (± 500 ppm)] CO 2 concentrations (see Supplementary methods).The low CO 2 concentration corresponds approximately with ambient CO 2 concentration, whereas the choice of the high CO 2 concentration was based on maximum permitted concentrations for human safe working [13][14][15][16] and data from experimental setups [12].To allow for maximum gas exchange in the Petri dishes in which the microorganisms were grown, the lids of all Petri dishes (unless otherwise stated) were 1 3 elevated by adding 2 cm wide plastic strips between the lids and the lower dish.Metarhizium brunneum isolate KVL12-30 (culture collection of the Department of Plant and Environmental Sciences, University of Copenhagen, Denmark) and Bacillus thuringiensis serovar morrisoni tenebrionis 4AA1 (Bacillus genetic stock center, Ohio State University, USA) were used in experiments.The in vitro and the in vivo experiments were performed on three and two independent occasions, respectively.

Germination and Growth of M. brunneum
The germination of M. brunneum conidia was assessed by adding 100 μl of 10 6 conidia/ml (see Supplementary methods) on each of three replicate (per condition and time point) 10 ml SDAY/4 (16.25 g Sabouraud dextrose agar, 2.5 g yeast extract, and 11.25 g agar in 1 l dH 2 O) Petri dishes.The suspensions were spread using a Drigalski spatula and the Petri dishes were incubated at either low or high CO 2 for 6, 8, 10, 12, 14, 18, or 24 h.Thereafter, 100 conidia were counted at three different locations on each Petri dish (300 conidia per Petri dish) and the numbers of germinated and un-germinated conidia were noted.A conidium was considered as germinated when it had a germ tube at least as long as the smallest diameter of the conidium.
The colony growth rates of M. brunneum at different CO 2 concentrations were assessed by adding 2 μl of 10 6 conidia/ ml on the centre of each of ten replicate 30 ml SDAY/4 Petri dishes and subsequent incubation at either low or high CO 2 .The area of each colony was measured using a digital calliper on two perpendicular diameters, every second day for eight days, starting two days after the preparation of the Petri dishes.The average of the two diameters per colony was used as one data point for calculating the growth rate (mm/day) between days two and eight.Petri dishes that dried out before the end of the experiment were excluded from the analysis.

Viability and Persistence of B. thuringiensis
The in vitro viability of B. thuringiensis spores was assessed by adding 100 μl of 10 3 spores/ml (see Supplementary methods) to each of ten replicate 10 ml LB-Agar (lysogeny broth agar; 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g bacteriological agar in 1 l dH 2 O) Petri dishes.The suspensions were spread using a Drigalski spatula and the Petri dishes were incubated at either low or high CO 2 .At both CO 2 concentrations, 100 μl of sterile dH 2 O was spread on each of three replicate 10 ml LB-Agar Petri dishes as controls (in the case of contamination this would be apparent on these Petri dishes).The numbers of colonies per Petri dish were counted after 24 h to calculate cfu/ml (colony forming units/ml; see Supplementary methods).
To measure in vitro persistence of B. thuringiensis spores, the method of Wood et al. [35] was adapted.Nine replicate autoclaved glass coverslips (22 × 22 mm) were placed inside an empty sterile Petri dish (three coverslips per Petri dish).On each coverslip, 100 μl of 6 × 10 5 spores/ ml (see Supplementary methods) were added and the Petri dishes containing the coverslips were incubated at either low or high CO 2 .Additionally, 100 μl of sterile dH 2 O was added on a separate coverslip in each Petri dish as a control (in the case of contamination this would be apparent on these coverslips).After two days the coverslips were transferred individually to 50 ml Falcon tubes containing 15 ml PBS (phosphate buffered saline) with Triton X-100 (0.1% v/v) and the tubes were put on an orbital shaker at 200 rpm at 25 °C for 15 min.Thereafter, 10 μl of the resulting suspensions were pipetted onto LB-Agar plates.By tilting the Petri dish on one side, the diluted suspensions ran down the media forming straight lines (three technical replicates on different Petri dishes were prepared).The average of the three technical replicates was used as one data point to calculate cfu/ml.

In Vivo Bioassays
Tenebrio molitor larvae were reared at either low or high CO 2 concentrations for 18 days.Bacillus thuringiensis spores and crystals mixed in diet were exposed to either low or high CO 2 concentrations for two days.Metarhizium brunneum was grown at either low or high CO 2 concentrations for 14 days.The pathogens (100 μl of 4 × 10 9 spores/ml per 100 mg diet for B. thuringiensis and 100 μl of 10 8 conidia/ ml per 100 mg diet for M. brunneum) were mixed into the larval diet [wheat bran (96% w/w) and dried egg white (4% w/w)] as described in the Supplementary methods.The larvae were exposed to lethal concentrations (previously determined in pre-experimental bioassays) of each pathogen separately in a full-factorial bioassay (n = 5, 30 larvae per cup).Furthermore, two groups of unexposed larvae (one at low and one at high CO 2 ; n = 5, 30 larvae per cup) were prepared as control treatments (Fig. 1).The larvae per cup were weighed as a group the day before, and on the day of exposure to the pathogens.Two days after the start of the exposure to the pathogens, the larvae were transferred to fresh cups.The larvae and the remaining diet in each cup were separated from frass by using a sieve (0.5 mm) 2, 4, 6, 8, 10, 12 and 14 days after exposure.Larval mortality was also assessed on the same days after exposure and dead larvae were removed.The remaining diet and live larvae were weighed individually and new diet (0.6 × weight of live larvae) and water agar (1% w/v; 0.6 × weight of live The effect of CO 2 on M. brunneum conidia germination was described using a three-parameter log-logistic model ( y = d (1+e (b(ln(x)−ln(i)) ) ; where y = germinated conidia (%), i = inflection point (i.e., hours to 50% germination), b = slope, d = upper limit, x = time in hours) using the drc package [37].The times to 50% germination at different CO 2 concentrations were compared using the compParm function [37].Metarhizium brunneum growth rates at different CO 2 concentrations were analysed using a one-way ANOVA.Experimental repetitions were combined, as no interactive effect of repetition and CO 2 was found in a previous two-way ANOVA.Bacillus thuringiensis spore persistence, spore viability, and density of larvae before the start of the in vivo assays at different CO 2 concentrations were compared by implementing generalized linear mixed models with a negative binomial error distribution (used for over dispersed count data) using the lme4 package [38] with experimental repetition included as a random effect.larvae) were added on the same days as larval mortality was assessed (the value 0.6 was established in a pre-experimental bioassay to ensure that the larvae did not starve between feeding time points).The larvae from one cup treated with B. thuringiensis in the second experimental repetition were excluded from analysis because the cup was tipped over during the experiment.

Statistical Analysis
Differences were considered as significant at p < 0.05 and data was only subjected to one-, two-or three-way ANO-VAs (analysis of variances) when normality (QQ-plots) and homogeneity of variances (Levene test, p > 0.05) assumptions were satisfied.Tukey's HSD (Honestly Significant Difference) tests were used to separate the means.All statistical analyses were performed using R v. 4.1.0[36].

Results
First, the effects of CO 2 on different pathogen traits outside of the host were tested.The time to 50% germination of M. brunneum conidia was significantly lower at high CO 2 compared to low CO 2 (t = 26.07;p < 0.001).At both CO 2 levels, the germination of conidia was > 99% after 24 h (Fig. 2).
Metarhizium brunneum colony growth rate was not affected by CO 2 (Table 1).In contrast, B. thuringiensis spores incubated at high CO 2 showed significantly lower viability than spores incubated at low CO 2 (Table 1).Similarly, B. thuringiensis spore persistence was significantly decreased at high compared to low CO 2 concentration (Table 1).
To investigate host-pathogen interactions, full-factorial bioassays were performed in which the pathogens and the host were exposed to either low or high CO 2 (Fig. 1).We tested the larval density in the two CO 2 conditions before the start of the experiments to ensure that it did not affect our results.The larval density in the rearing containers at low and high CO 2 was indeed not affected by CO 2 (p = 0.311, χ2 = 1.026, d.f.= 1,6).Likewise, CO 2 did not affect the weight of the larvae at the start of the experiment (p = 0.387, χ2 = 0.748, d.f.= 1,97).The germination rates of M. brunneum conidia were > 99% in all treatments and experimental repetitions.Larvae reared at high CO 2 were significantly less susceptible (i.e., less likely to die) to B. thuringiensis (Fig. 3A; Table 2) and M. brunneum (Fig. 3B; Table 2) than larvae reared at low CO 2 resulting in approximately 12 and 8% higher survival after 14 days, respectively.There was no effect of CO 2 on survival of control larvae (p = 0.771, Fig. 3A, B).Moreover, exposure of the pathogens to different CO 2 concentrations before exposure of the larvae did not affect the virulence of B. thuringiensis or M. brunneum (Table 2).
The effect of CO 2 concentration and pathogen exposure on feed intake was measured during pathogen exposure.The feed intake per larva was reduced by B. thuringiensis exposure (Fig. 4A), but CO 2 did not affect feed intake in either the control or B. thuringiensis exposed larvae (Fig. 4A; Table 3).Similarly, feed intake was reduced by M. brunneum exposure in the second iteration of the experiment, and in certain treatments of the first iteration (Fig. 4B).CO 2 did not affect the feed intake during M. brunneum or control exposure (Fig. 4B; Table 3).
Exposure of larvae to B. thuringiensis significantly reduced weight gain of the larvae over the course (14 days) of the experiments (Fig. 5A).However, weight gain was not affected by exposure of either the larvae or B. thuringiensis to different CO 2 concentrations (Fig. 5A; Table 3).Exposure of larvae to M. brunneum did not affect the weight gain over the course of the experiment except for one treatment in the second iteration of the experiment (Fig. 5B).Furthermore, Mixed effects cox proportional hazards models were used to analyse the survival of the larvae in the in vivo assays (fixed effects: pathogen exposure, CO 2 exposure of larvae, CO 2 exposure of pathogens; random effects: experimental repetition, cup) using the coxme package [39].Only significant fixed effects were retained in the final models and pairwise comparisons of treatments were performed using Tukey contrasts with single-step adjustment for multiple testing using the multcomp package [40].The effect of CO 2 on larval weight at the start of the experiment was analysed using a generalized linear mixed model with a gamma error distribution using the lme4 package [38] with experimental repetition included as a random effect.Weight gain per larva for the duration of the experiments and feed intake during exposure data were analysed separately for both experimental repetitions (Exp. 1 and 2) using two-way ANOVAs because interactive effects of experimental repetition and exposure of larvae or pathogens to CO 2 were found in previous three-way ANOVAs.Means (± SEM) followed by different letters within a column indicate significant differences among the treatments.SEM, standard error of the mean; cfu, colony forming units 2 p = 0.305, F = 1.077, d.f.= 1,43  3).

Discussion
In this study, elevated CO 2 concentrations were found to decrease the viability and persistence of B. thuringiensis spores in vitro, whilst decreasing the duration of conidial germination of M. brunneum.Interestingly, exposure of the pathogens to different CO 2 concentrations before infection did not affect the virulence of these entomopathogens toward T. molitor larvae, but larvae reared at elevated CO 2 were less susceptible (i.e., less likely to die) to the pathogens than larvae reared at ambient CO 2 .These findings are important because T. molitor larvae are often exposed to CO 2 concentrations above ambient conditions [8,11,18].
Here we show that CO 2 levels affect the susceptibility of T. molitor to entomopathogens, which has implications for both mass-rearing of mealworms for food and feed purposes,  the growth rates of different M. anisopliae strains are either positively or negatively affected by elevated CO 2 (650 and 1,000 ppm) [26].We, in contrast, did not find an effect of CO 2 at industrially relevant concentrations on the growth rate of M. brunneum in vitro.This is, to our knowledge, the first study that measures the direct effects of elevated environmental CO 2 on the persistence and viability of a bacterial entomopathogen.However, it is known from other species that CO 2 can reduce bacterial growth [44].We found that the persistence of B. thuringiensis spores was almost three times lower at elevated CO 2 .Surprisingly, there was no effect of exposure of B. thuringiensis to elevated CO 2 on the subsequent virulence in the insect host.This could be because the crystals of B. thuringiensis that are essential for the infection process might not be affected by CO 2 .Moreover, we speculate that the spores kept at elevated CO 2 could have been only temporarily inactivated (dormant) and might be reactivated in the host.It has been shown for other species of the Bacillus genus that suboptimal thermal and pH conditions during incubation can increase the time to germination of spores [45].
Interestingly, we could not detect any sublethal effects of elevated CO 2 on the larvae.In contrast, in a study by Li et al. [12], T. molitor larvae reared in a closed system had a lower weight gain compared to larvae reared in an open system, which was argued to be due to higher CO 2 concentrations in the closed system [12].However, these differences could and for biocontrol of this insect species.In addition to our main findings, we also found that CO 2 did not affect the feed intake of the larvae during exposure to the pathogens and overall, did not affect the individual weight gain of the larvae.Investigating sub-lethal effects such as these is crucial, especially for the production of insects because a reduction in weight gain leads to economic losses, as the overall mass of insects produced is reduced.
It is challenging to put our study in context with other studies on CO 2 because the few other studies that have been published investigating the effects of CO 2 on insectpathogen interactions either use lower (< 1,000 ppm) or significantly higher (> 50,000 ppm) CO 2 concentrations than in this present study.To our knowledge, this is the first study to measure the effect of industrially relevant CO 2 concentrations for the mass-rearing of T. molitor and other reared insect species.Elevated CO 2 concentrations have been suggested to act as a cue promoting the germination of an entomophthoralean fungus (Entomophaga maimaiga) as CO 2 concentrations might be elevated near the insect cuticle [41].This increased germination of fungal conidia is in accordance with our study.However, decreased germination and mycelial growth of a hypocrealean fungus (B.bassiana) were reported as a result of a very high CO 2 concentration (400,000 ppm) [42].Similarly, 50,000 ppm CO 2 decreased the mycelial growth and sporulation of M. brunneum, Aspergillus sp., and B. bassiana in vitro [43].Moreover, it was proposed (without statistical analyses) that understanding the reliability of biocontrol of storage pests.
To ensure meaningful conclusions, we suggest it is crucial to consider CO 2 effects (i.e., through monitoring and using pertinent CO 2 concentrations) when studying any insect pathogen systems that are likely to be exposed to elevated CO 2 in their natural or artificially maintained environments.
also have been due to other factors such as different relative humidity or different concentrations of other gases in the two systems.It is important to note that elevated CO 2 concentrations may be more detrimental to insects when the relative humidity is low, because elevated CO 2 forces the insects to keep their spiracles open, which can result in water loss [46].
Our study supports prior findings by Borisade & Magan [26] who exposed desert locusts (Schistocerca gregaria) and house crickets (Acheta domesticus) to elevated CO 2 concentrations (1,000 ppm).The authors suggested that S. gregaria and A. domesticus kept at elevated CO 2 showed increased survival and lethal times, respectively, when exposed to B. bassiana, although this was not statistically validated [26].In contrast to these findings, the survival of red flour beetles (Tribolium castaneum) exposed to B. bassiana was significantly decreased at very high CO 2 concentrations (440,000 ppm) [42].Due to our experimental design, we can disentangle the effects of CO 2 on the interactions between the pathogens and T. molitor, demonstrating that previous exposure of the pathogens to elevated CO 2 did not affect the virulence of the pathogens, but that rearing the larvae at elevated CO 2 decreases the susceptibility of the larvae to the pathogens.One possible explanation is that CO 2 may affect the insect immune response.For example, in Drosophila melanogaster the production of antimicrobial peptides was inhibited by CO 2 (130,000 ppm) correlating with increased susceptibility to bacterial infections [47].Moreover, in T. castaneum CO 2 increased the production of benzoquinones [48] (a quinone that is also produced by T. molitor [49]), which inhibit B. bassiana [50].The mechanism underlying the decreased susceptibility of T. molitor to pathogens at elevated CO 2 concentrations remains to be investigated.Moreover, it would be beneficial for the production of T. molitor and other mass-reared insect species to investigate the CO 2 concentrations T. molitor is evolutionarily adapted to in order to optimise rearing conditions.Tenebrio molitor might be adapted to elevated CO 2 concentrations whereas other species may be adapted to different CO 2 concentrations.
Here, we demonstrate that CO 2 directly affects a bacterial and a fungal entomopathogen in vitro and their in vivo interactions with an insect host.Based on these results, we conclude that the tested elevated CO 2 concentration (4,500 ± 500 ppm) in T. molitor mass-rearing systems is beneficial for larvae exposed to the tested pathogens by increasing larval survival.Furthermore, we did not find any sublethal effects of CO 2 on T. molitor larvae that would affect the overall productivity of the mass-rearing system.For biocontrol of T. molitor, our results indicate that the efficacies of the two tested entomopathogens may be lowered at elevated CO 2 concentrations, which has implications for

Fig. 1
Fig. 1 Schematic representation of the experimental design.A Larvae reared at either low or high CO 2 for 18 days were exposed to B. thuringiensis previously exposed to either low or high CO 2 for two days or to water as a control.B Larvae reared at either low or high CO 2 for 18 days were exposed to M. brunneum grown at low or high CO 2 for 14 days.The lids of the Petri dishes were elevated by adding a

Table 1
Metarhizium brunneum colony growth rate, Bacillus thuringiensis spore viability and persistence at either low or high CO 2 concentrations.

Table 2
Results Exposure of larvae to Bt, Exposure of larvae to CO 2 , Exposure of Bt to CO 2 , Exposure of larvae to Mb, Exposure of Mb to CO 2 ; and p-values of random effects: Cup and Experimental repetition 2 Bold p-values denote statistical significance at p < 0.05 of mixed effects cox proportional hazards models to analyse survival of T. molitor larvae 1,2 Bacillus thuringiensis (Bt) HR ± SE p Exposure of larvae to Bt 11.505 ± 0.233 < 0.001 Exposure of larvae to CO 2 0.693 ± 0.088 < 0.001 Exposure of Bt to CO 2 0.992 ± 0.1 HR ± SE (hazard ratio ± standard error) and p-values of fixed effects:

Table 3
Results of two-way ANOVAs to analyse feed intake of T. molitor larvae during the two days of pathogen exposure and individual weight gain of larvae during 14 days of individual experimental repetitions 1,2 Bt: B. thuringiensis treatment (either no Bt, Bt exposed to low CO 2 , or Bt exposed to high CO 2 ); Mb: M. brunneum treatment (either no Mb, Mb exposed to low CO 2 , or Mb exposed to high CO 2 ); Larv-CO 2 : exposure of larvae to either low or high CO 2 1 Abbreviations: 2 Bold p-values denote statistical significance at p < 0.05 1 3