Figure 7: Gene ontology terms of A) the 30 most significantly upregulated and B) downregulated genes in the DEP treatment colored by category and sorted by -log10FDR.
Discussion
In this study, we found that oral exposure to diesel exhaust particles (DEPs) changes the gut microbiome and gene expression of bumblebee workers, while DEP exposure via air did not. Brake dust, the second pollutant we tested via oral exposure, did not induce changes in the gut microbiome or gene expression in the bumblebee workers.
While the composition of the microbial gut community in control, solvent control, and brake dust exposure treatment was similar, we detected major shifts in the DEP treatment. This raises several interesting questions: 1) How do DEPs affect the bacteria to induce changes in the gut microbiome composition? 2) Which components in diesel exhaust are responsible for the observed changes? Our hypothesis is that PAHs could be the component of DEP affecting bacteria directly. DEPs contain different PAHs, a class of organic compounds well-known to be toxic, mutagenic, and genotoxic to various life forms (Patel et al. 2020, Sun et al. 2021). Also, shifts in the microbial gut community due to PAH exposure have been reported in different animals, such as fish, sea cucumbers, or potworms (Enchytraeidae) (DeBofsky et al. 2020, DeBofsky 2021, Ding et al. 2020, Quintanilla-Mena et al. 2021, Zhao et al. 2019). Therefore, we suspect PAHs to be the leading cause of changes in the bumblebee gut microbiome in our study. However, the large amount of elemental carbon in DEPs, may itself provide another explanation. The DEPs may function like activated carbon with its large surface-area-to-volume ratio and may adsorb microbes that are then discharged by excretion (Naka et al. 2001, Rivera-Utrilla et al. 2001, Wichmann 2007). Even though activated carbon has no direct negative impact, constant adsorption and discharge might disrupt the bacterial community resulting in the compositional and quantitative changes similar to those observed in our study.
The bacterium Snodgrassella , one of the dominant core bacteria in undisturbed gut microbiomes of bumblebees (Hammer et al. 2021), is nearly absent after the DEP exposure. Snodgrassella , together with Gilliamella , forms a biofilm coating the inner wall of the ileum (Hammer et al. 2021, Martinson et al. 2012). Both, host and symbionts could profit from this biofilm formation as it prevents bacteria from washout and enables the formation of a syntrophic network (Kwong et al. 2014, Powell et al. 2016, Zhang et al. 2022). Additionally, the biofilm could protect the host against gut parasites, such as C. bombi, who need to attach to the gut wall to persist (Koch et al. 2019, Näpflin & Schmid-Hempel 2018). However, the mutualistic relationship between the microbes seems to be disrupted by DEP exposition, as Snodgrassella abundance is extremely diminished. In contrast, Gilliamella increases in relative abundance after DEP exposure. This indicates that Gilliamella may be able to form a biofilm independently from Snodgrassella . A relatively simple explanation for the higher relative abundance ofGilliamella might be that the reduction of Snodgrassellaleaves Gilliamella as the only dominant bacterium in the gut and therefore Gilliamella might thrive better or fill the void.Snodgrassella seems especially prone to pollutants, as Rothman et al. (2020) already reported a decrease in its relative abundance after exposure of bees to copper, selenate, or glyphosate. Additionally, we found an unknown bacterium from the family Neisseriaceae, the same family to which also Snodgrassella belongs, having a lower relative abundance after DEP exposure. If this is a consistent result, it might indicate a general susceptibility of this family to DEPs.
The higher abundance of Asaia in the DEP treatment was driven by two samples, in which Asaia dominates the bacterial community with relative abundances of 99 % and 67 %, respectively. Asaiais a flower-associated acetic acid bacterium, which is commonly found in the gut of members of different insect orders, such as Hemiptera, Diptera, and Hymenoptera (Bassene et al. 2020, Crotti et al. 2009, Kautz et al. 2013). It can dominate the gut microbiome of Anophelesmosquitos, which is why it is considered a potential tool in malaria control (Capone et al. 2013, Favia et al. 2008). While there have been reports of Asaia in bumblebees, the dominance of Asaia in some of the DEP samples is rather uncommon (Bosmans 2018). DEPs might disrupt the natural microbiome community opening the door for opportunistic bacteria such as Asaia (Favia et al. 2007). Even though we kept the bumblebees in this experiment indoors throughout their lives, Asaia bacteria may derive from pollen fed to the bumblebees before the start of the experiment.
We detected an interesting pattern in the genus Lactobacillus , one of the core gut bacteria of bumblebees (Hammer et al. 2021). While the species L. bombicola , a bumblebee-associated bacterium, has a lower abundance after DEP exposure, the abundance of the honeybee-associated L. apis increases. Again, the disruption of the original microbiome caused by DEPs might explain that foreign bacteria can establish themselves in the microbiome. As the pollen fed to the bumblebees before the experiment was collected by honeybees, it could be the source of L. apis .
The DEP-induced changes in the gut microbiome may affect bumblebee health, as core bacteria could prevent infections by parasites. The abundance of Gilliamella , Lactobacillus andSnodgrassella is negatively correlated with the parasitesCrithidia and Nosema , while non-core bacteria are more abundant in infected bumblebees (Cariveau et al. 2014, Koch et al. 2012, Koch & Schmid-Hempel 2012, Mockler et al. 2018). The biofilm formation of Snodgrassella and Gilliamella may form a physical barrier to the trypanosome C. bombi which needs to attach to the ileum wall to persist (Koch et al. 2019, Näpflin & Schmid-Hempel 2018). The disruption of this biofilm and the higher abundance of non-core bacteria, such as Asaia , may increase the parasite susceptibility of bumblebees exposed to DEPs.
The transcriptome analysis revealed significant changes in gene expression after oral exposure of bumblebees to a sublethal dose of DEPs. In total, 165 genes were upregulated, and 159 genes were downregulated. GO enrichment analysis and network analysis indicate that these changes could be related to a general stress response against pollutants. While upregulated GO terms involve many metabolic and catabolic processes, downregulated GO terms include metabolic and biosynthetic processes. DEP exposure might deplete stored reserves causing the observed changes as a consequence of higher energetic costs. Changes in metabolism seem to be a typical reaction to pollutants in insects which seems reasonable as they often interfere with biochemical processes. Transcriptional changes in bumblebees and honeybees exposed to sublethal doses of neonicotinoids are mainly linked to metabolic processes (Bebane 2019, Colgan 2019, Gao et al. 2020, Shi et al. 2017). Exposure to heavy metals or PAHs induces similar changes in spiders, mosquitos, moths, and fireflies (Chen et al. 2021, David et al. 2010, Li et al. 2016, Zhang et al. 2019, Zhang et al. 2020). Even though the changes differ in detail, certain processes seem commonly involved in the response to pollutants. Consistent with our findings, exposure to insecticides or PAHs affects mitochondrial functioning, an important part of the insect energy metabolism (Colgan et al. 2019, Zhang et al. 2019, Zhang et al. 2020). This supports the idea of increased energy demand caused by pollutants (Beyers et al. 1999, Calow 1991). We also observed an upregulation of signal transduction in our study, similar to observations in honeybees and fireflies exposed to Imidacloprid and the PAH benzo(a)pyrene, respectively (Gao et al. 2020, Zhang et al. 2019, 2020). Typically, chemical stressors, such as PAHs, insecticides, and heavy metals, affect genes associated with detoxification processes and drug metabolism (Chen et al. 2021, David et al. 2010, Gizaw et al. 2020, Zhang et al. 2019). However, in our study, we did not find any differentially expressed detoxification-related genes. Possibly the number of PAHs attached to the DEPs was not enough to trigger a reaction that would lead to a measurable increase in detoxification. Overall, the observed changes in gene expression after oral DEP exposure of bumblebees resemble a general stress response to pollutants.
In contrast to oral exposure, we did not find any effect on gene expression after exposure of bumblebees to DEPs via the air. To cause changes, DEPs need to enter the tracheal system or attach to sensory organs, such as the antennae. The exposure of bumblebees for three minutes per day may not have been enough to affect them. Particles on the antennae may have been removed quickly by cleaning behavior and the spiracles seem to be an effective protective barrier against the uptake of particles into the tracheae (Harrison 2009, Schönitzer 1986). Thus, our results should be taken with care because probably only very few particles entered the tracheal system of the bumblebees.
Unlike DEPs, oral exposure to brake dust particles did not affect the gut microbial community nor the gene expression of the bumblebees. However, some concerns remain about the experimental procedure. For one, we did not use brake dust from a real braking scenario, but rather artificially milled brake pads. Dust derived from them may have different physicochemical properties. Milled brake dust particles have a much higher mean particle size than DEPs (10 µm vs. 0.01 µm). As we defined treatment concentration per weight, these different physical properties lead to big differences in the particle counts of the treatment solutions, i.e. solutions with brake dust contained far fewer particles than those with DEPs. Moreover, large brake dust particles tend to sink to the bottom of the feeding syringes which might have reduced the particle uptake. While brake dust seems not to affect the bumblebees, further studies are needed to address the indicated limitations of the present study.
Taken together, the results from our microbiome and transcriptome analysis indicate potential consequences for insect health, here shown in bumblebees, after oral DEP exposure. Gut dysbiosis may increase the susceptibility of bumblebees to pathogens, while a general stress response may lower available energetic resources. To evaluate these hypotheses further studies should investigate the combined effect of DEP exposure and other stressors, such as parasites, limited food availability, or abiotic factors. Bumblebees may be able to compensate for facing one stressor but will eventually be overstrained by multiple stressors. Additionally, whole colony experiments would add to the evaluation of DEPs as a potential contributor to insect losses, as effects may be small on the individual level but accumulate on the colony level.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The microbiome and RNA-Seq sequencing data were deposited at NCBI’s Sequence Read Archive (SRA) under Bioproject numbers PRJNA907197 (16S microbiome sequencing) and PRJNA907822 (transcriptome sequencing), respectively.
Author contributions
DS, AW, OO, and HF conceived the idea, designed the experiment, and wrote the manuscript. AM, TH, TO, NL, and DB produced and analyzed the particulate matter. DS, MR, and AW carried out the experiment. DS and AW performed the data analysis. DS, AW, OO, and HF interpreted the results. All authors read and approved of the final manuscript.
Funding
This project was funded by the Bavarian State Ministry of the Environment and Consumer Protection as part of the project network BayOekotox.
Acknowledgements
We thank Sara Pölloth, Simon Bitz, Frederic Hüftlein, and Helena Hartmann for helping with the lab work, and Michaela Hochholzer and Andrea Kirpal (Keylab Genomics and Bioinformatics) for preparing the NGS libraries.
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