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Testing microbial pest control products in bees, a comparative study on different bee species and their interaction with two representative microorganisms

Environmental Sciences Europe volume 36, Article number: 169 (2024) Cite this article

Abstract

Background

The evaluation of the impact of pesticides on non-target species, like bees, is a crucial factor in registration procedures. Therefore, standardized test procedures have been developed on OECD level assessing the effects of chemicals on honey bees or bumble bees. Unfortunately, these protocols cannot directly be adapted for testing products that contain microorganisms. Interest in the use of microorganisms has increased in recent years due to their specificity to target species while not harming non-target organisms. This study aimed to evaluate optimal conditions to assess the effects of microbial plant protection products on bee species according to currently available test protocols. Some of the most commonly used microorganisms for plant protection, Bacillus thuringiensis subspecies aizawai (B. t. a. ABTS 1857) and Beauveria bassiana (B. b. ATCC 74040) were tested on Apis mellifera, Bombus terrestris, and Osmia bicornis at different temperatures (18, 26, 33 °C) under laboratory conditions.

Results

Exposure to the product containing B. t. a. ABTS 1857 resulted in higher mortality compared to B. b. ATCC 74040 in all tested bee species. A temperature-dependent effect towards higher mortality at higher temperatures of 26 °C or 33 °C was observed in O. bicornis exposed to both microorganisms. A. mellifera showed variable responses, but for B. terrestris there was mostly no effect of temperature when exposed to microorganisms in high concentrations. However, temperature affected longevity of bee species in the non-exposed control group. A. mellifera mortality increased with decreasing temperatures, while B. terrestris and O. bicornis mortality increased with increasing temperatures. A test duration of 15 or 20 days was found to be suitable for testing these microorganisms.

Conclusion

In conclusion, 26 °C should be considered the worst-case scenario for testing B. bassiana on all tested bee species. For testing B. thuringiensis, a temperature of 33 °C is recommended for A. mellifera, whereas B. terrestris and O. bicornis should be tested at 26 °C.

Background

The use of chemical plant protection products (PPPs), including insecticides, fungicides, and herbicides, recently raised several concerns because of their potential side effects on pollinators and other non-target insects as well as development of resistance in the target organisms. Public reports and policy makers drive the trend towards the use of biological plant protection products as eco-friendly alternatives, for example, by the commitment of the European Green Deal in 2019 (COM/2019/640) [14]. Biological PPPs use beneficial insects and microorganisms as natural antagonists of target pest species or natural substances for plant protection. This approach differ from the use of synthetic chemical products that are currently employed. Recent studies evaluated the use of natural antagonists such as entomopathogenic microorganisms as alternatives for chemical insecticides, which include bacteria, fungi, viruses and protozoa [7, 15, 20, 49]. The main proposed advantages of the chosen microorganisms are their potential specificity to their target species and their fast environmental degradation.

The Gram-positive entomopathogenic bacterium Bacillus thuringiensis (Firmicutes, Bacillaceae) and the entomopathogenic fungus Beauveria bassiana (Ascomycota, Cordycipitaceae) are the most often studied microorganisms and have long use history. Both cover a broad spectrum of orders of host species due to the existence of different subspecies and strains [19, 29, 40, 50]. It has been proven that effects on non-target species from exposure to a particular strain or subspecies of either B. thuringiensis or B. bassiana cannot be extrapolated to another strain or subspecies [10, 50].

Bacillus thuringiensis (B. t.) is a ubiquitous, spore forming bacterium that produces parasporal crystal (cry-) proteins or delta-endotoxins during its stationary phase [5, 38]. These proteins are thermostable and toxic to many insects [9, 19]. The toxins act in the digestive tract of the host. They enter the intestine membrane, bind to special receptors causing receptor destruction, and finally lead to sepsis [27, 39]. In addition to the direct lethal effects of the toxins, epithelial alterations can lead to sub-lethal effects due to a higher risk of haemocoel invasion by other microorganisms [11]. B. t. strains are presumed to be specific to their target host family, but effects of different formulations and/or strains on non-target species of other families have been recently demonstrated [3, 15, 30, 45]. The observed effects were alterations of the midgut microbiome of adult honey bees [42], an increased developmental time or decreased adult emergence rates of Drosophila species [3].

The entomopathogenic fungus Beauveria bassiana (B. b.) is used in greenhouses and field for the protection of vegetables. Together all B. b. subspecies and strains cover a broad spectrum of hosts of almost all insect orders, acari and the bee-specific ectoparasitic mite Varroa destructor  [13, 47, 48]. B. bassiana has a high temperature dependency with an optimal temperature of about 25 °C in the laboratory [16, 17]. The fungi act on the host´s cuticle through penetration by conidial hyphae followed by an invasion of the internal tissue. The host’s innate immune system will be overcame, leading to host’s death. By the production of new conidia, which are spread in the environment, new hosts can be infected [29, 50].

In registration procedures, a risk assessment for each product used for plant protection has to be conducted, based on the provided studies before approval. So far, standardized test procedures are available at OECD level for testing chemicals. The OECD 213 and the OECD 245 guidelines specify the conduction of acute and chronic oral toxicity tests of chemical PPPs on adult honey bees under laboratory conditions [31, 32]. OECD 247 furthermore provides procedures to assess acute oral toxicity to B. terrestris [33]. Guidelines for toxicity tests on Osmia species are being developed. Recently, Azpiazu et al. [2] described a first methodological approach to perform toxicity tests in the laboratory using Osmia species. In contrast to chemical PPPs, biological PPPs contain live and dynamic (micro) organisms and thus there is a need for modifications in these guidelines, including test duration, given that most microorganisms require incubation in their hosts until they can cause infectivity or pathogenicity. Up to now, no standardized test procedures are available for testing microorganisms on bees and developing protocols to assess microbial product effects on Apis and non-Apis bees is mandatory [4].

The current study aimed to (1) compare the response of different model bee species: Apis mellifera, Bombus terrestris and Osmia bicornis to exposure to several microorganisms; (2) to evaluate the required modifications of the available OECD-guidelines for testing of microbial plant protection products; and (3) to define optimal test conditions for both, bees and microorganisms.

Methods

Experimental setup and treatment conditions

The plant protection products Naturalis® containing Beauveria bassiana strain ATCC 74040 (2.3 × 107 spores/ml) and FlorBac™ containing Bacillus thuringiensis sub. aizawai strain ABTS 1857 (15,000 IU/mg) were tested. Naturalis® is used against the glasshouse whitefly (Trialeurodes vaporariorum) and FlorBac™ against various species of the order Lepidoptera, both in vegetable cultivation.

The maximal treatment concentration (100%) was set according to the maximum field recommended concentration (MFRC) of product label instructions and the substances were tested in dilution series with 100%, 50%, and 10% of the MFRC. If the 100% acute exposure assay did not show any effects on longevity, the following acute exposure dilution was not tested because no effects are expected. The MFRC for Naturalis® is defined as 2 L (product)/ha in 1500 L/ha water, which corresponds to a concentration of 1330 mg/kg, and for FlorBac™ it is defined as 1.5 kg (product)/ha in 900 L/ha water, which corresponds to a concentration of 1317 mg/kg. Thus, 100% concentrations were set to the calculated MFRC with corresponding dilution series (Table 1).

Table 1 Overview of test concentrations used in the feeding assay
Full size table

Treatment solutions were prepared daily in a 2 M sucrose solution. Sucrose solution was produced using distilled water. The product FlorBac™ had to be pre-dissolved in 2 mL of distilled water as it is a powder. Naturalis® was directly dissolved in the sucrose solution. Test series were conducted in chronic as well as in acute exposure assays (for details see respective paragraphs for every species and Supplementary Table S1). Negative control group was feed with 2 M sucrose solution only. The chronic and acute assays were performed simultaneously and thus the same negative control group was used for both assays per species and temperature.

Test organisms were kept in stainless steel cages (10 × 8.5 × 5.5 cm) with a glass pane under constant climatic conditions in darkness except during observation (OECD 213, 245). Each cage had two holes at the top to enable the placement of a feeder and handling.

The assay was repeated at 18 °C, 26 °C, and 33 °C with a relative humidity of 65%. Mortality and food consumption were recorded daily. The study was conducted from October 2021 to February 2022.

Test organisms

Apis mellifera

The day before exposure worker winter honey bees were collected from three colonies at the Julius Kuehn-Institute (Braunschweig). Winter bees might be exposed to biological PPPs during their foraging flights in early spring including the blooming of several fruit trees like cherry and apple. Healthy colonies comprised at least 10,000–12,000 bees and a one-year-old fertile queen (Buckfast). All colonies had low levels of Varroa infestation. Where the natural mite fall was lower than one mite per day, treatment with formic acid against Varroa destructor was conducted during summer at least 10 weeks before starting the experiment. Bees were caged in groups of 10 individuals per colony (OECD 213, 245) [31, 32]. This grouping was conducted because winter bees were used and otherwise a random pool would have led to additional stress for the bees. Honey bees were kept for one day in the incubator for acclimatization, fed with 2 M sucrose solution only (OECD 213, 245). Feeding was performed using a modified 1 mL plastic syringe with removed tips and was changed daily (OECD 213, 245). Each treatment consisted of six replicates, respectively, of two cages per colony.

In the acute exposure assay, bees were starved for 2 h before the initiation of the test (OECD 213). 200 µl of treatment solutions were provided for a maximum of 6 h, resulting in an average individual exposure of 20 µl/bee (OECD 213). After full consumption of the treatment solution, feeder was removed and replaced by a new feeder containing sucrose solution only (OECD 213). Furthermore, bees were fed ad libitum with sucrose solution only. OECD 213 recommends a test duration of 48 h or 96 h if mortality rises to more than 10% after the first 24 h. Here, the test duration was extended to 15 days to include life history traits and infection time of microorganisms.

Treatment solutions were replaced daily in the chronic exposure assay and exposure period lasted for 10 days (OECD 245). Observation period was extended to another five days and total time including the exposure period was thus 15 days. The extension was performed to include life history traits and infection time of microorganisms.

Bombus terrestris

Six bumble bee colonies were obtained from a commercial supplier (Biobest Group NV, Belgium), and consisted of one queen and approximately 25–30 workers. The colonies were delivered 12 days before the start of the experiment in a plastic box with a surrounding cardboard and were fed by a pollen–sucrose mix (Supplementary Table S2 for pollen information). All colonies were placed in an incubator at 26 °C and 65% relative humidity (OECD 247) [33].

Adult bumble bee workers were caged in groups of five individuals when colonies raised a size of 60–80 workers. OECD 247 recommends a single housing of bumble bee workers to prevent hierarchy fights and to enable controlled feeding because bumble bees do not share food. However, test duration of OECD 247 is maximum 96 h. Preliminary experiments showed, that when bumble bees were kept for a longer test duration, a better performance of bumble bee survival was ensured in a cohort of five. The maximum number of five individuals further prevents hierarchy fights. Bumble bee workers were weighted beforehand and were homogenously distributed by weight to treatment groups (OECD 247). This minimized the potential effect of size polymorphism of bumble bee workers. Treatment groups consisted of five replicates. Caging was performed one day before the start of the exposure for acclimatization of bumble bees, similar to the honey bee assay. Bumble bees were fed with a modified 5-mL plastic syringe with removed tips. Furthermore, two additional holes were added to the sites of the syringe because bumble bees cannot consume headlong.

Bumble bees were exposed to 5 mL treatment solutions for 24 h in the acute exposure assay, which is in contrast to the 4 h exposure period of 40 µl of treatment solution recommended in OECD 247. The exposure period was adjusted because of the cohort caging and to ensure that all bumble bees were successfully exposed as bumble bees perform no trophallaxis. Exposure scenario in chronic assay was the same as in the honey bee chronic exposure assay. The observation period lasted for 20 days, including the chronic exposure period, in both bumble bee exposure assays (acute and chronic). The extended observation period was chosen because control mortality was low until day 15 and thus it was not possible to test for treatment-related effects in some cases.

Osmia bicornis

Cocoons of female red mason bees were delivered from a commercial supplier (WAB-Mauerbienenzucht, Konstanz, Germany) and kept at 2.5 °C until emergence. Altogether, 1,200 cocoons were placed in plastic boxes (13 × 18 × 6 cm) in groups of 25 for emergence. Plastic boxes were spiked on the top and the site with several holes for ventilation. The temperature was increased over four days from 10 °C at day one, to 15 °C for another day and finally to 25 °C for two days at a constant relative humidity of 65% in darkness to induce emergence. Emerged mason bees were placed in groups of five individuals in the stainless steel cages. Five replicates were used per treatment. Mason bees were fed with 2 M sucrose solution or exposed to treatment solution via 5-mL plastic syringes. Adjustment to the plastic syringe was done similarly as for bumble bees to ensure feeding. Additionally, mason bees were fed with a 50:50 pollen mixture. Pollen mixture was prepared from honey bee collected pollen pellets, purchased from an organic beekeeper (Andreas Bock, Ökologische Imkerei, Mertingen, Germany, Supplementary Table S2 for detailed pollen information), and 2 M sucrose solution. This additional feeding was performed because newly emerged females were used, and thus the provision of pollen as a protein source ensures high vitality during the early developmental stages.

Acute exposure lasted for 24 h to ensure a successful exposure because mason bees do not share food via trophallaxis. Chronic exposure was performed similar to the honey bee and bumble bee chronic exposure assay. Observation period was set to 15 days similar to the honey bee assay. Methods described in Azpiazu et al. [2] were not adopted because the experiment described in this study was performed beforehand in 2022.

Statistical analysis

Statistical analysis was performed in R (version 4.2.2) using the user interface RStudio (2022.12.0). Mortality analysis was performed using the survival (version 3.5–8) [43], survminer (version 0.4.9) [24], coxme (version 2.2–20) [44] and emmeans (version 1.10.1) [26] packages. Kaplan–Meier estimators were calculated for graphical visualization of data. Survival data were analyzed by a Cox proportional hazard mixed effect model using the coxme function. Data were analyzed first species-wise and for a more detailed analysis, data were further split by species and exposure scenario. PPP treatment interacting with temperature were set as explanatory variables including cage number as a random effect: survival ~ PPP * temperature + (1|cage). Pairwise comparisons were performed afterwards using the emmean function with an integrated False Discovery Rate (FDR) method to correct for multiple testing. Furthermore, an additional model taking the concentration as a covariate into account was performed. Since the experiment considered several factors, like temperature, bee species, microorganism species, and concentrations, it was decided to focus on the major questions rather than to discuss the concentrations themselves. The results of all tested concentrations and the comparison can be found in the Supplementary.

Finally, a Cox proportional hazard model was calculated to estimate differences between different treatment groups and species. Plant protection product, temperature and species were set in hazard models as explanatory variables. The untreated A. mellifera control group was chosen as reference group for the comparison as A. mellifera is reported to be prevailing model organism in several approaches of the risk assessment [23]. The control group tested at 26 °C was chosen for the comparison of acute exposure scenarios, and control group tested at 33 °C for the chronic exposure scenario according to the test conditions described in OECD 213 and 245, respectively.

Differences in food consumption were compared via an ANOVA analysis and significant differences were determined using a Tukey HSD test.

Results

Survival probability was highest in A. mellifera at 33 °C compared to 26 °C and 18 °C (both: coxme, p < 0.05) when tested without any additional treatment (Figs. 1–2). In contrast, in B. terrestris and O. bicornis assay, temperature of 33 °C led to a significant lower survival probability compared to 26 °C and 18 °C (both: coxme, p < 0.05) (Figs. 1–2).

Fig. 1
figure 1

Survival probability under chronic exposure to 100% of maximum field recommended concentration of product. The figure shows the effect of treatment substances on worker longevity of A Apis mellifera, B Bombus terrestris, and C Osmia bicornis with 95% confidence interval. The products contain either B. t. a. ABTS 1857 or B. b. ATCC 74040. Sample sizes were N = 6 cages/treatment, n = 10 bees/cage (A. mellifera); N = 5 cages/treatment, n = 5 bees/cage (B. terrestris) and N = 5 cages/treatment, n = 5 bees/cage (O. bicornis). Different letters indicate significant differences (coxme: survival ~ treatment (PPP) * temperature + (1|cage), p < 0.05). Comparison was performed among panels within each species

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Fig. 2
figure 2

Survival probability under acute exposure to 100% of maximum field recommended concentration of product. The figure shows the effect of treatment substances on worker longevity of A Apis mellifera, B Bombus terrestris, and C. Osmia bicornis with 95% confidence interval. The products contain either B. t. a. ABTS 1857 or B. b. ATCC 74040. Sample sizes were N = 6 cages/treatment, n = 10 bees/cage (A. mellifera); N = 5 cages/treatment, n = 5 bees/cage (B. terrestris) and N = 5 cages/treatment, n = 5 bees/cage (O. bicornis). Different letters indicate significant differences (coxme: survival ~ treatment (PPP) * temperature + (1|cage), p < 0.05). Comparison was performed among panels within each species

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In general, the exposure to the product containing B. t. a. ABTS 1857 or B. b. ATCC 74040 led in all tested bee species exposed to 100% or 50% concentration to a significantly higher mortality rate (coxme, p < 0.0001) regardless the exposure scenario (acute or chronic) or the temperature conditions compared to the control group (Figs. 1–2, Supplementary Figures S1, S2). The exposure to 10% of B. b. ATCC 74040 had no effect on survival in A. mellifera (coxme, p = 0.0847) (Supplementary Figures S3, S4). The successful exposure to B. b. ATCC 74040 was additionally verified in a germination assay, and non-treated control groups were absent of any B. b. ATCC 74040 hyphae, which confirmed no cross-contamination between groups (more details see Supplementary).

Considering both factors (temperature and treatment), there were no temperature-dependent differences in B. terrestris mortality exposed to both microorganisms under chronic exposure (coxme, p > 0.05) and exposed to B. b. ATCC 74040 under acute exposure (coxme, p > 0.05) (Figs. 1, 2). Temperature affected B. terrestris mortality when exposed acute to B. t. a. ABTS 1857 and reared at 26 °C compared to a rearing at 33 °C (coxme, p < 0.04) (Fig. 2). When lower concentrations were tested, temperature effects were observed between B. terrestris mortality exposed to both microorganisms (Supplementary Figures S1–S4). Rearing temperatures of 26 °C and 33 °C led to higher mortality in O. bicornis when exposed to both products containing the microorganisms at all concentrations (coxme, p < 0.05) (Figs. 1, 2, Supplementary Figures S1–S4).

The sensitivity comparison among species showed that O. bicornis was more sensitive to 33 °C compared to the reference (cox, p < 0.001) (Fig. 3E); whereas sensitivity was lower in B. terrestris at 18 °C and 26 °C and A. mellifera at 33 °C compared to the reference (cox, p < 0.04) (Fig. 3E). Sensitivity of A. mellifera was lower at higher temperatures of 33 °C under acute exposure to B. b. ATCC 74040 compared to reference (cox, p < 0.001), whereas sensitivity of O. bicornis was higher at higher temperatures of 33 °C to both microorganisms compared to reference (cox, p < 0.001) (Fig. 3A, C). In all tested bee species, the sensitivity was higher at higher temperatures under chronic exposure to the product with B. t. a. ABTS 1857 compared to the reference (cox, p < 0.001) (Fig. 3B). Irrespective of the temperature, a significant increase in hazard ratio was observed for all chronic exposure scenarios of to both microorganisms (cox, p < 0.001), except for A. mellifera reared at 33 °C showing variable response (Fig. 3, Supplementary Figures S5, S6). Overall, in the most cases mortality trends showed that sensitivity of O. bicornis increased with higher temperatures, whereas sensitivity of A. mellifera decreased with higher temperatures (Fig. 3).

Fig. 3
figure 3

Sensitivity comparison. The figure shows a comparison of the hazard ratio of tested species (Apis mellifera, Bombus terrestris and Osmia bicornis) at different temperatures compared to the OECD reference group. For acute exposure the Apis mellifera 26 °C non-treated group (A, C) was set as reference group, and for chronic exposure Apis mellifera 33 °C (B, D). A, B show the hazard ratio of bees treated with B. t. a. ABTS 1857; C, D the comparison under B. b. ATCC 74040 exposure; E shows the comparison of untreated control groups only at different temperatures with A. mellifera reared at 26 °C as reference group. The products contain either 100% of maximum recommended concentration of B. t. a. ABTS 1857 or B. b. ATCC 74040. Missing values resulted from no mortality within these groups and no calculable hazard ratio. Stars indicate significant differences to the respective reference group (Cox proportional hazard test, * p < 0.05, ** p < 0.01, *** p < 0.001). F represents photographs of fungal hyphae growing on bees’ tongue resulting from a germination assay of B. b. ATCC 74040 exposed bees

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The consumption of sucrose solution was measured during the whole exposure duration and calculated individually for all tested bee species and treatment concentrations (Supplementary Figure S7). The average consumption of A. mellifera worker bees was 40 µl per bee per day, whereas B. terrestris workers consumed on average 231 µl and O. bicornis 48 µl per bee per day. There were neither significant differences in the consumption rate at the different temperatures nor under the tested treatment concentrations (ANOVA, p > 0.05). Although there are obvious variances within the consumption rate per bee, there was no time-dependent trend in food consumption.

Discussion

This study showed how test conditions like ambient temperature can affect longevity and sensitivity of the three tested model bee species (A. mellifera, B. terrestris, and O. bicornis) when exposed to products containing the microorganisms B. t. a. ABTS 1857 and B. b. ATCC 74040. Consequently, the study revealed that when conducting acute and chronic laboratory feeding assays, using biological plant protection products with microorganisms, temperature conditions related to bee-specific host response cannot be neglected.

In contrast to A. mellifera and B. terrestris, there have been no studies assessing the effects of microorganisms on O. bicornis or other mason bees [7, 15]. This study provided a first comparative insight into the effects of microorganisms on Osmia species. Chronic exposure to both tested microorganisms led to a decreasing survival probability in all tested bee species, which was further reflected in an increased hazard ratio among species. Previous studies have repeatedly shown a high mortality rate of B. terrestris when exposed to microorganisms [6, 8, 18, 22, 25] and a dose-dependent effect on A. mellifera mortality [41]. However, acute exposure scenarios in A. mellifera may not result in adverse effects on longevity at lower concentrations [28]. When testing microorganisms, it is not uncommon to obtain contradictory results due to the use of different strains and subspecies, and therefore, caution should be taken when interpreting those [15]. Our findings showed that both tested microorganisms reduced survival probability of O. bicornis under both exposure scenarios as well. Therefore, the laboratory results indicated the necessity of further testing such products under field conditions to evaluate realistic risks to bees. Additionally, a sterilized treatment group was tested to assess potential effects of the formulation or existing metabolites in the product. The results showed that the inactivated treatment group did not affect the survival of exposed bees and indicate that the observed effects may relate to the microorganisms or its metabolites (see Supplementary material) [1].

Risk assessment authorities evaluate the PPPs before permission depending on submitted studies which are mostly conducted due to developed guidelines or guidance at OECD level. However, currently available OECD guidelines  [31,32,33] are developed for testing of chemicals and not suitable or need to be adapted for testing biological PPPs containing microorganisms. For example, the guidelines recommend different temperatures for acute and chronic exposure scenarios to bee species. The test temperatures are chosen to preserve the bees. In contrast, microorganisms may have a different optimal temperature range for their development and a compromise regarding the temperature condition should be made when setting up laboratory assays to test these microorganisms to bees. Except for B. terrestris, the results indicated an additional temperature-dependent effect when bees are exposed to microorganisms. The additional temperature effect may resulted from the temperature preference of the bees on the one hand and from the preferences of the microorganisms on the other hand.

A. mellifera keep their brood nests at constant temperatures of about 33–35 °C with a relative humidity of 65–70%, which explains the beneficial impact of the higher temperature of 33 °C on A. mellifera longevity alone or in combination with the exposure to the microorganisms. In contrast, the hive temperature can vary depending on the ambient temperature and can range between 20 °C and 35 °C. The temperature in the honey super or on the edge combs have different temperature compared to the brood nest [34]. In B. terrestris colonies the ambient temperature is on average at 20 °C and rising temperatures towards 30 °C will result in stress response like increased worker fanning activity [46]. This corresponds to the high mortality rate in the untreated control group at 33 °C (Fig. 1B). The solitary bee O. bicornis was the most sensitive to increasing temperatures within this study (in acute and chronic treatments), resulting in high mortality rates within the control groups at high temperatures of 33 °C compared to rearing at 18 °C. O. bicornis prefers ambient temperatures of 18–24 °C for their nest sites and avoid temperatures higher than 28 °C [35]. An explanation might be that higher temperatures can lead to physiological changes in bees such as loss in body weight [35].

In contrast, the optimum temperature for a B. bassiana infection is between 23 °C and 28 °C and 60–70% relative humidity [50]. Peng et al. [36] showed that B. bassiana was effective to honey bees at an optimal temperature of 25 °C. Rearing temperatures of 18 °C and 26 °C led in A. mellifera to an additional effect on mortality when exposed to B. bassiana chronically (Fig. 1A). Furthermore, higher temperatures of 33 °C can inhibit an infection with B. bassiana effectively [12, 36], which was shown by the lowest mortality of A. mellifera at the 33 °C group (Fig. 1A). B. bassiana treatment further caused a reduced survival probability at 18 °C compared to 28 °C in a bee-vectoring experiment with B. terrestris [21]. An infection with B. thuringiensis works best at temperatures of 32 °C compared to lower temperatures in the target species Ostrinia nubilalis [37]. Taking the temperature into account when testing microorganisms to bees is thus mandatory. An ambient temperature of 26 °C would preserve all tested bees on the one hand and enables to evaluate the effects of tested fungi on the other hand. When testing bacteria, which are most effective at higher temperatures such as B. t. a. ABTS 1857, a test temperature of 33 °C is recommended for testing A. mellifera, while 26 °C is recommended for testing B. terrestris and O. bicornis.

The sufficient length of the test duration is another critical point, which should be assigned correctly when conducting laboratory assays [15]. The OECD guidelines recommend a test duration of ten days for chronic exposure scenarios (OECD 245) or up to 96 h for acute exposure scenarios (OECD 213, 247). However, infection and reproduction capacity of microorganisms can last from minutes to days and thus test duration should be adapted [4, 15]. Our results showed, that a study duration of 15 days for A. mellifera and O. bicornis and of 20 days for B. terrestris might be sufficient to assess the effects of the tested microorganisms. In contrast to OECD 247, B. terrestris should be housed in small groups of five individuals since the test duration is extended to several days.

Conclusion

For non-infected honey bees a low temperature of 18 °C resulted in increased mortality. Fungal infections with B. b. ATCC 74040 led to increased mortality for honey bees at 18 °C. In contrast, a high temperature of 33 °C resulted in a higher control mortality for B. terrestris and O. bicornis compared to other temperatures, but low mortality in honey bees under B. b. ATCC 74040 treatment. Comparing these obvious trade-offs among bees at low and high temperatures, temperature conditions are to be adjusted in laboratory assays when testing different bee species. A temperature of 26 °C might be considered as the worst-case scenario for testing fungi effects on all tested bee species, as this temperature might be beneficial for both, the survival probability of the three model bee species and the infection and germination probability of the tested microorganism. When B. thuringiensis is tested, 33 °C can be considered the worst-case scenario for honey bees, but 26 °C for testing B. terrestris or O. bicornis.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

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Acknowledgements

We want to thank Finja Mense and Michelle Röthig for their assistance in germination assay and Michel Metje for providing the photographs of Beauveria infected bees.

Funding

Open Access funding enabled and organized by Projekt DEAL. We acknowledge the financial support by the Federal Ministry of Food and Agriculture for funding initial research in this field.

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Authors and Affiliations

  1. Institute for Bee Protection, Julius Kühn‑Institute (JKI) – Federal Research Centre for Cultivated Plants, Messeweg 11-12, 38104, Braunschweig, Germany

    Karoline Wueppenhorst, Kevin Nack, Silvio Erler, Jens Pistorius & Abdulrahim T. Alkassab

  2. Zoological Institute, Technische Universität Braunschweig, Mendelssohnstraße 4, 38106, Braunschweig, Germany

    Karoline Wueppenhorst & Silvio Erler

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  1. Karoline Wueppenhorst
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  5. Abdulrahim T. Alkassab
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Contributions

A.T.A. conceived the study question, K.N. performed the experiment, K.W., K.N. analyzed the data, K.W., S.E., A.T.A. interpreted the data, K.W. was major contributor in writing the manuscript, J.P. obtained study funding. All authors read, revised and approved the final manuscript.

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Correspondence to Karoline Wueppenhorst.

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Wueppenhorst, K., Nack, K., Erler, S. et al. Testing microbial pest control products in bees, a comparative study on different bee species and their interaction with two representative microorganisms. Environ Sci Eur 36, 169 (2024). https://doi.org/10.1186/s12302-024-00994-7

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