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

Pascal Herren, Alison M Dunn, Nicolai V Meyling, Carlotta Savio, Helen Hesketh

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Pascal Herren, Alison M Dunn, Nicolai V Meyling, Carlotta Savio, Helen Hesketh. Effect of CO2 Concentrations on Entomopathogen Fitness and Insect-Pathogen Interactions. Microbial ecology, 2024, 87 (1), pp.34. 10.1007/s00248-024-02347-6. hal-05156677

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

Pascal Herren<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> ID · Alison M. Dunn<fnref n="1" /> ID · Nicolai V. Meyling<fnref n="2" /> ID · Carlotta Savio<fnref n="4" />,<fnref n="5" /> ID · Helen Hesketh<fnref n="3" /> ID

Received: 30 October 2023 / Accepted: 14 January 2024

© The Author(s) 2024

## 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** Host-pathogen Interactions · *Tenebrio molitor* · *Bacillus thuringiensis* · *Metarhizium brunneum* · Insect Culture · Biocontrol

## Introduction

CO₂ (carbon dioxide) has the potential to affect host-pathogen interactions if either the host, pathogen, or both are affected by changes in CO₂ concentrations. Numerous insect species are constantly exposed to CO₂ concentrations above the atmospheric level, which is currently recorded as approximately 420 ppm (parts per million) [1]. Elevated CO₂ 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 CO₂ concentration in soil air (inside soil pores), for example, is typically higher than the atmospheric CO₂ concentration due to decreased gas exchange [4], hence soil-dwelling insect species are exposed to elevated CO₂ concentrations in their environment. Furthermore, it is known that CO₂ can accumulate in colonies of social insects reaching up to 60,000 ppm in leaf-cutting ant colonies [5], and 92,000 ppm

<figure><img src="image_2.png" /></figure>

<footnote>Helen Hesketh
hhesketh@ceh.ac.uk</footnote>

<footnote n="1">¹ Faculty of Biological Sciences, University of Leeds,
Leeds LS2 9JT, UK</footnote>

<footnote n="2">² Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg 1871, Denmark</footnote>

<footnote n="3">³ UK Centre for Ecology & Hydrology, Maclean Building,
Benson Lane, Crowmarsh Gifford, Wallingford,
Oxfordshire OX10 8BB, UK</footnote>

<footnote n="4">⁴ Université Paris-Saclay, INRAE, Micalis Institute, Domaine de Vilvert, Jouy-en-Josas, AgroParisTech 78350, France</footnote>

<footnote n="5">⁵ Department of Plant Sciences, Wageningen University,
Wageningen 6708 PB, The Netherlands</footnote>
in termite mounds [6]. Insects that are mass-reared for food and feed purposes can also be exposed to elevated CO₂ concentrations because they are typically kept at high densities in closed systems [7], which facilitates the accumulation of CO₂ [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 CO₂ per kg of body mass per day or approximately 1,000 g CO₂ per kg body mass gain [2]. Despite the utilisation of appropriate ventilations, CO₂ is still likely to accumulate in production facilities of *T. molitor* [8, 11]; for example, in a closed experimental *T. molitor* rearing, CO₂ concentrations reached up to 6,000 ppm [12]. The maximum permitted CO₂ 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 CO₂ 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 CO₂ concentrations inside stored grains can exceed atmospheric CO₂ concentration [18] and when there is microbial or insect activity, CO₂ 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 CO₂ concentrations affect host-pathogen interactions in both mass-reared and wild insects [25]. Improving our understanding of the effects of CO₂ on entomopathogens and their interactions with insect hosts will help to guide decisions of whether CO₂ should be considered a relevant factor to include for insect-pathogen interaction experiments and in the design of insect mass rearing facilities.

CO₂ is known to affect entomopathogenic organisms; for example, *Pseudoxylaria* spp., an entomopathogenic fungus infecting termites (*Odontotermes obesus*), showed reduced growth when exposed to elevated CO₂ 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 CO₂ compared to 350 ppm CO₂ [26]. CO₂ 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 CO₂ concentrations [28] and *Candida albicans*, a fungal pathogen of humans, switches from the monocellular to the more virulent filamentous growth at elevated CO₂ concentrations [27]. Nevertheless, the impact of CO₂ on the virulence of entomopathogenic organisms that can infect economically important insects remains unknown.

In this study, we examined the effects of CO₂ 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 CO₂, insects, and pathogens. The pathogens were selected because both *B. thuringiensis* and *M. brunneum* can be found in stored grains [29–31]; 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 CO₂ 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 CO₂ 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 CO₂ (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 CO₂ incubators adjacent to each other, one used for low [450 ppm (± 50 ppm)] CO₂, and one used for high [4,500 ppm (± 500 ppm)] CO₂ concentrations (see Supplementary methods). The low CO₂ concentration corresponds approximately with ambient CO₂ concentration, whereas the choice of the high CO₂ concentration was based on maximum permitted concentrations for human safe working [13–16] 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
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 10⁶ 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 dH₂O) Petri dishes. The suspensions were spread using a Drigalski spatula and the Petri dishes were incubated at either low or high CO₂ 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 CO₂ concentrations were assessed by adding 2 µl of 10⁶ conidia/ml on the centre of each of ten replicate 30 ml SDAY/4 Petri dishes and subsequent incubation at either low or high CO₂. 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 10³ 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 dH₂O) Petri dishes. The suspensions were spread using a Drigalski spatula and the Petri dishes were incubated at either low or high CO₂. At both CO₂ concentrations, 100 µl of sterile dH₂O 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 6×10⁵ spores/ml (see Supplementary methods) were added and the Petri dishes containing the coverslips were incubated at either low or high CO₂. Additionally, 100 µl of sterile dH₂O 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 CO₂ concentrations for 18 days. *Bacillus thuringiensis* spores and crystals mixed in diet were exposed to either low or high CO₂ concentrations for two days. *Metarhizium brunneum* was grown at either low or high CO₂ concentrations for 14 days. The pathogens (100 µl of 4×10⁹ spores/ml per 100 mg diet for *B. thuringiensis* and 100 µl of 10⁸ 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 CO₂; 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
<figure><img src="image_3.png" /><figcaption>**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</figcaption></figure>

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 ANOVA's (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].

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)

The effect of CO₂ on *M. brunneum* conidia germination was described using a three-parameter log-logistic model ($y = \frac{d}{(1+e^{(b(\ln(x)-\ln(i))})}$; 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 CO₂ concentrations were compared using the compParm function [37]. *Metarhizium brunneum* growth rates at different CO₂ concentrations were analysed using a one-way ANOVA. Experimental repetitions were combined, as no interactive effect of repetition and CO₂ 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 CO₂ 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.
<figure><img src="image_4.png" /><figcaption>**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</figcaption></figure>

<table><caption>**Table 1** *Metarhizium brunneum* colony growth rate, *Bacillus thuringiensis* spore viability and persistence at either low or high CO₂ concentrations.<fnref n="1" /></caption><thead><tr><th></th><th><i>M. brunneum</i> colony growth rate (mm/d ± SEM)<fnref n="2" /></th><th><i>B. thuringiensis</i> spore viability (cfu/ml ± SEM)<fnref n="3" /></th><th><i>B. thuringiensis</i> spore persistence (cfu/ml ± SEM)<fnref n="4" /></th></tr></thead><tbody><tr><td>Low CO₂</td><td>3.67 ± 0.04<sup>a</sup></td><td>762.33 ± 61.11<sup>a</sup></td><td>650.62 ± 98.07<sup>a</sup></td></tr><tr><td>High CO₂</td><td>3.73 ± 0.81<sup>a</sup></td><td>682.00 ± 58.76<sup>b</sup></td><td>213.58 ± 43.11<sup>b</sup></td></tr></tbody></table>

Mixed effects Cox proportional hazards models were used to analyse the survival of the larvae in the in vivo assays (fixed effects: pathogen exposure, CO₂ exposure of larvae, CO₂ 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 CO₂ 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 CO₂ were found in previous three-way ANOVAs.

## Results

First, the effects of CO₂ on different pathogen traits outside of the host were tested. The time to 50% germination of *M. brunneum* conidia was significantly lower at high CO₂ compared to low CO₂ (t = 26.07; p < 0.001). At both CO₂ levels, the germination of conidia was > 99% after 24 h (Fig. 2).

*Metarhizium brunneum* colony growth rate was not affected by CO₂ (Table 1). In contrast, *B. thuringiensis* spores incubated at high CO₂ showed significantly lower viability than spores incubated at low CO₂ (Table 1). Similarly, *B. thuringiensis* spore persistence was significantly decreased at high compared to low CO₂ 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 CO₂ (Fig. 1). We tested the larval density in the two CO₂ 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 CO₂ was indeed not affected by CO₂ (p = 0.311, χ<fnref n="2" /> = 1.026, d.f. = 1,6). Likewise, CO₂ did not affect the weight of the larvae at the start of the experiment (p = 0.387, χ<fnref n="2" /> = 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 CO₂ 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 CO₂ resulting in approximately 12 and 8% higher survival after 14 days, respectively. There was no effect of CO₂ on survival of control larvae (p = 0.771, Fig. 3A, B). Moreover, exposure of the pathogens to different CO₂ concentrations before exposure of the larvae did not affect the virulence of *B. thuringiensis* or *M. brunneum* (Table 2).

The effect of CO₂ 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 CO₂ 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). CO₂ 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 CO₂ 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,

<footnote n="1">¹Means (± SEM) followed by different letters within a column indicate significant differences among the treatments. SEM, standard error of the mean; cfu, colony forming units</footnote>

<footnote n="2">²p = 0.305, F = 1.077, d.f. = 1,43</footnote>

<footnote n="3">³p < 0.001, χ² = 16.971, d.f. = 1,58</footnote>

<footnote n="4">⁴p < 0.001, χ² = 13.419, d.f. = 1,52</footnote>
**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

<figure><img src="image_5.png" /><figcaption>**Table 2** Results of mixed effects cox proportional hazards models to analyse survival of *T. molitor* larvae<fnref n="1" />,<fnref n="2" /></figcaption></figure>

<table><thead><tr><th></th><th>HR ± SE</th><th>p</th></tr></thead><tbody><tr><td><strong>Bacillus thuringiensis (Bt)</strong></td><td></td><td></td></tr><tr><td>Exposure of larvae to Bt</td><td>11.505 ± 0.233</td><td><strong>&lt; 0.001</strong></td></tr><tr><td>Exposure of larvae to CO₂</td><td>0.693 ± 0.088</td><td><strong>&lt; 0.001</strong></td></tr><tr><td>Exposure of Bt to CO₂</td><td>0.992 ± 0.089</td><td>0.930</td></tr><tr><td>Cup</td><td>-</td><td>0.963</td></tr><tr><td>Experimental repetition</td><td>-</td><td>0.969</td></tr><tr><td><strong>Metarhizium brunneum (Mb)</strong></td><td></td><td></td></tr><tr><td>Exposure of larvae to Mb</td><td>31.352 ± 0.242</td><td><strong>&lt; 0.001</strong></td></tr><tr><td>Exposure of larvae to CO₂</td><td>0.816 ± 0.091</td><td><strong>0.025</strong></td></tr><tr><td>Exposure of Mb to CO₂</td><td>0.954 ± 0.093</td><td>0.620</td></tr><tr><td>Cup</td><td>-</td><td><strong>0.011</strong></td></tr><tr><td>Experimental repetition</td><td>-</td><td><strong>&lt; 0.001</strong></td></tr></tbody></table>

weight gain was not affected by exposure of either the larvae or *M. brunneum* to different CO₂ concentrations (Fig. 5B; Table 3).

## Discussion

In this study, elevated CO₂ 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 CO₂ concentrations before infection did not affect the virulence of these entomopathogens toward *T. molitor* larvae, but larvae reared at elevated CO₂ were less susceptible (i.e., less likely to die) to the pathogens than larvae reared at ambient CO₂. These findings are important because *T. molitor* larvae are often exposed to CO₂ concentrations above ambient conditions [8, 11, 18]. Here we show that CO₂ levels affect the susceptibility of *T. molitor* to entomopathogens, which has implications for both mass-rearing of mealworms for food and feed purposes,

<footnote n="1">¹HR ± SE (hazard ratio ± standard error) and *p*-values of fixed effects: Exposure of larvae to *Bt*, Exposure of larvae to CO₂, Exposure of *Bt* to CO₂, Exposure of larvae to *Mb*, Exposure of *Mb* to CO₂; and *p*-values of random effects: Cup and Experimental repetition</footnote>

<footnote n="2">²Bold *p*-values denote statistical significance at *p* < 0.05</footnote>
<figure><img src="image_6.png" /><figcaption>**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</figcaption></figure>

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

<figure><img src="image_7.png" /><figcaption>**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</figcaption></figure>

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
<table><caption>**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<fnref n="1" />,<fnref n="2" /></caption><thead><tr><th rowspan="2"></th><th colspan="3">Feed intake during exposure</th><th colspan="3">Individual weight gain</th></tr><tr><th>d.f.</th><th>F</th><th>p</th><th>d.f.</th><th>F</th><th>p</th></tr></thead><tbody><tr><td colspan="7"><strong>B. thuringiensis</strong>, Experiment 1</td></tr><tr><td>Bt</td><td>2,24</td><td>105.01</td><td><strong>&lt;0.001</strong></td><td>2,24</td><td>94.34</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Larv-CO<sub>2</sub></td><td>1,24</td><td>0.17</td><td>0.684</td><td>1,24</td><td>0.19</td><td>0.668</td></tr><tr><td>Bt * Larv-CO<sub>2</sub></td><td>2,24</td><td>1.07</td><td>0.358</td><td>2,24</td><td>0.09</td><td>0.917</td></tr><tr><td colspan="7"><strong>Bt</strong> * Larv-CO<sub>2</sub> Experiment 2</td></tr><tr><td>Bt</td><td>2,23</td><td>471.13</td><td><strong>&lt;0.001</strong></td><td>2,23</td><td>48.22</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Larv-CO<sub>2</sub></td><td>1,23</td><td>0.01</td><td>0.945</td><td>1,23</td><td>0.50</td><td>0.488</td></tr><tr><td>Bt * Larv-CO<sub>2</sub></td><td>2,23</td><td>1.30</td><td>0.29</td><td>2,23</td><td>0.58</td><td>0.570</td></tr><tr><td colspan="7"><strong>M. brunneum</strong>, Experiment 1</td></tr><tr><td>Mb</td><td>2,24</td><td>16.24</td><td><strong>&lt;0.001</strong></td><td>2,24</td><td>3.96</td><td><strong>0.033</strong></td></tr><tr><td>Larv-CO<sub>2</sub></td><td>1,24</td><td>3.24</td><td>0.084</td><td>1,24</td><td>0.04</td><td>0.837</td></tr><tr><td>Mb * Larv-CO<sub>2</sub></td><td>2,24</td><td>1.69</td><td>0.207</td><td>2,24</td><td>0.18</td><td>0.838</td></tr><tr><td colspan="7"><strong>Mb</strong> * Larv-CO<sub>2</sub> Experiment 2</td></tr><tr><td>Mb</td><td>2,24</td><td>87.37</td><td><strong>&lt;0.001</strong></td><td>2,23</td><td>8.54</td><td><strong>0.002</strong></td></tr><tr><td>Larv-CO<sub>2</sub></td><td>1,24</td><td>0.58</td><td>0.452</td><td>1,23</td><td>2.03</td><td>0.167</td></tr><tr><td>Mb * Larv-CO<sub>2</sub></td><td>2,24</td><td>1.64</td><td>0.216</td><td>2,23</td><td>1.10</td><td>0.350</td></tr></tbody></table>

and for biocontrol of this insect species. In addition to our main findings, we also found that CO₂ 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 CO₂ because the few other studies that have been published investigating the effects of CO₂ on insect-pathogen interactions either use lower (<1,000 ppm) or significantly higher (>50,000 ppm) CO₂ concentrations than in this present study. To our knowledge, this is the first study to measure the effect of industrially relevant CO₂ concentrations for the mass-rearing of *T. molitor* and other reared insect species. Elevated CO₂ concentrations have been suggested to act as a cue promoting the germination of an entomophthoralean fungus (*Entomophaga maimaiga*) as CO₂ 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 CO₂ concentration (400,000 ppm) [42]. Similarly, 50,000 ppm CO₂ 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

the growth rates of different *M. anisopliae* strains are either positively or negatively affected by elevated CO₂ (650 and 1,000 ppm) [26]. We, in contrast, did not find an effect of CO₂ 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 CO₂ on the persistence and viability of a bacterial entomopathogen. However, it is known from other species that CO₂ can reduce bacterial growth [44]. We found that the persistence of *B. thuringiensis* spores was almost three times lower at elevated CO₂. Surprisingly, there was no effect of exposure of *B. thuringiensis* to elevated CO₂ 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 CO₂. Moreover, we speculate that the spores kept at elevated CO₂ 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 CO₂ 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 CO₂ concentrations in the closed system [12]. However, these differences could

<footnote n="1">¹Abbreviations: *Bt*: *B. thuringiensis* treatment (either no *Bt*, *Bt* exposed to low CO₂, or *Bt* exposed to high CO₂); *Mb*: *M. brunneum* treatment (either no *Mb*, *Mb* exposed to low CO₂, or *Mb* exposed to high CO₂); *Larv-CO₂*: exposure of larvae to either low or high CO₂</footnote>

<footnote n="2">²Bold *p*-values denote statistical significance at *p* < 0.05</footnote>
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 CO₂ concentrations may be more detrimental to insects when the relative humidity is low, because elevated CO₂ 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 CO₂ concentrations (1,000 ppm). The authors suggested that *S. gregaria* and *A. domesticus* kept at elevated CO₂ 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 CO₂ concentrations (440,000 ppm) [42]. Due to our experimental design, we can disentangle the effects of CO₂ on the interactions between the pathogens and *T. molitor*, demonstrating that previous exposure of the pathogens to elevated CO₂ did not affect the virulence of the pathogens, but that rearing the larvae at elevated CO₂ decreases the susceptibility of the larvae to the pathogens. One possible explanation is that CO₂ may affect the insect immune response. For example, in *Drosophila melanogaster* the production of antimicrobial peptides was inhibited by CO₂ (130,000 ppm) correlating with increased susceptibility to bacterial infections [47]. Moreover, in *T. castaneum* CO₂ 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 CO₂ 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 CO₂ concentrations *T. molitor* is evolutionarily adapted to in order to optimise rearing conditions. *Tenebrio molitor* might be adapted to elevated CO₂ concentrations whereas other species may be adapted to different CO₂ concentrations.

Here, we demonstrate that CO₂ 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 CO₂ 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 CO₂ 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 efficiencies of the two tested entomopathogens may be lowered at elevated CO₂ concentrations, which has implications for

understanding the reliability of biocontrol of storage pests. To ensure meaningful conclusions, we suggest it is crucial to consider CO₂ effects (i.e., through monitoring and using pertinent CO₂ concentrations) when studying any insect pathogen systems that are likely to be exposed to elevated CO₂ in their natural or artificially maintained environments.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00248-024-02347-6.

**Acknowledgements** We would like to thank Ŷnsect for providing the initial *T. molitor* population. Furthermore, we would like to thank Alan Bloomfield and Daisy Fairchild for their technical assistance.

**Author Contributions** All authors contributed to the study conception and design. P.H. performed the experimental work, data collection, and analysis. P.H. wrote the first draft of the manuscript. N.V.M., A.M.D., C.S., and H.H. critiqued the manuscript for intellectual content. All authors read and approved the final manuscript.

**Funding** This work was done within the project 'Insect Doctors' which has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 859850.

**Data Availability** The datasets generated for the current study are available from the corresponding author on reasonable request.

## Declarations

**Competing Interests** The authors declare no competing interests.

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