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.
References
Al Naggar Y, Dabour K, Masry S, Sadek A, Naiem E Giesy JP (2020)
Sublethal effects of chronic exposure to CdO or PbO nanoparticles or
their binary mixture on the honey bee (Apis mellifera L.).
Environ Sci Pollut Res, 27(16), 19004-19015.
https://doi.org/10.1007/s11356-018-3314-2
Ami EB, Yuval B, Jurkevitch E (2010) Manipulation of the microbiota of
mass-reared Mediterranean fruit flies Ceratitis capitata(Diptera: Tephritidae) improves sterile male sexual performance. ISME J,
4(1), 28-37. https://doi.org/10.1038/ismej.2009.82
Anders S, Pyl PT, Huber W (2015) HTSeq—a Python framework to work with
high-throughput sequencing data. Bioinformatics, 31(2), 166-169.
https://doi.org/10.1093/bioinformatics/btu638
Anderson MJ (2008) A new method for non‐parametric multivariate analysis
of variance. Austral Ecol, 26(1), 32-46.
https://doi.org/10.1111/j.1442-9993.2001.01070.pp.x
Apprill A, McNally S, Parsons R, Weber L (2015) Minor revision to V4
region SSU rRNA 806R gene primer greatly increases detection of SAR11
bacterioplankton. Aquat Microb Ecol, 75(2), 129-137.
https://doi.org/10.3354/ame01753
Aufauvre J, Misme-Aucouturier B, Viguès B, Texier C, Delbac F, Blot N
(2014) Transcriptome analyses of the honeybee response to Nosema
ceranae and insecticides. PLoS One, 9(3), e91686.
https://doi.org/10.1371/journal.pone.0091686
Bassene H, Niang EHA, Fenollar F, Doucoure S, Faye O, Raoult D, Sokhna
C, Mediannikov O (2020) Role of plants in the transmission ofAsaia sp., which potentially inhibit the Plasmodium sporogenic
cycle in Anopheles mosquitoes. Sci Rep, 10(1), 1-10.
https://doi.org/10.1038/s41598-020-64163-5
Bebane PS, Hunt BJ, Pegoraro M, Jones AC, Marshall H, Rosato E, Mallon
EB (2019) The effects of the neonicotinoid imidacloprid on gene
expression and DNA methylation in the buff-tailed bumblebee Bombus
terrestris . Proc Royal Soc B, 286(1905), 20190718.
https://doi.org/10.1098/rspb.2019.0718
Beyers DW, Rice JA, Clements WH, Henry CJ (1999) Estimating
physiological cost of chemical exposure: integrating energetics and
stress to quantify toxic effects in fish. Can J Fish Aquat Sci, 56(5),
814-822. https://doi.org/10.1139/f99-006
Bindea G, Galon J, Mlecnik B (2013) CluePedia Cytoscape plugin: pathway
insights using integrated experimental and in silico data.
Bioinformatics, 29(5), 661-663.
https://doi.org/10.1093/bioinformatics/btt019
Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A,
Fridman WH, Pagès F, Trajanoski Z, Galon J (2009) ClueGO: a Cytoscape
plug-in to decipher functionally grouped gene ontology and pathway
annotation networks. Bioinformatics, 25(8), 1091-1093.
https://doi.org/10.1093/bioinformatics/btp101
Bisanz JE(2018) qiime2R: Importing QIIME2 artifacts and associated data
into R sessions. https://github.com/jbisanz/qiime2R.
Bokulich NA, Kaehler BD, Rideout JR, Dillon M, Bolyen E, Knight R,
Huttley GA, Caporaso JG (2018) Optimizing taxonomic classification of
marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier
plugin. Microbiome, 6(1), 1-17.
https://doi.org/10.1186/s40168-018-0470-z
Bolger AM, Lohse M, Usadel B (2014). Trimmomatic: a flexible trimmer for
Illumina sequence data. Bioinformatics, 30(15), 2114-2120.
https://doi.org/10.1093/bioinformatics/btu170
Bolyen E, Rideout JR, Dillon MR et al (2019) Reproducible, interactive,
scalable and extensible microbiome data science using QIIME 2. Nat
Biotechnol, 37, 852–857.
https://doi.org/10.1038/s41587-019-0209-9
Bosmans L, Pozo MI, Verreth C, Crauwels S, Wilberts L, Sobhy IS, Wäckers
F, Jacquemyn H, Lievens B (2018) Habitat-specific variation in gut
microbial communities and pathogen prevalence in bumblebee queens
(Bombus terrestris ). PLoS One, 13(10), e0204612.
https://doi.org/10.1371/journal.pone.0204612
Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP
(2016) DADA2: High-resolution sample inference from Illumina amplicon
data. Nat Methods, 13(7), 581-583.
https://doi.org/10.1038/nmeth.3869
Calow P (1991) Physiological costs of combating chemical toxicants:
ecological implications. Comp Biochem Physiol C Toxicol Pharmacol,
100(1-2), 3-6. https://doi.org/10.1016/0742-8413(91)90110-f
Cameron SA, Sadd BM (2020) Global trends in bumble bee health. Annu Rev
Entomol, 65, 209-232.
https://doi.org/10.1146/annurev-ento-011118-111847
Capone A, Ricci I, Damiani C et al (2013) Interactions betweenAsaia , Plasmodium and Anopheles : new insights into
mosquito symbiosis and implications in malaria symbiotic control.
Parasites Vectors, 6(1), 1-13.
https://doi.org/10.1186/1756-3305-6-182
Cardoso P, Barton PS, Birkhofer K et al (2020) Scientists’ warning to
humanity on insect extinctions. Biol Conserv, 242, 108426.
https://doi.org/10.1016/j.biocon.2020.108426
Cariveau DP, Elijah Powell J, Koch H, Winfree R, Moran NA (2014)
Variation in gut microbial communities and its association with pathogen
infection in wild bumble bees (Bombus ). ISME J, 8(12), 2369-2379.
https://doi.org/10.1038/ismej.2014.68
Chen J, Guo Y, Huang S, Zhan H, Zhang M, Wang J, Shu Y (2021)
Integration of transcriptome and proteome reveals molecular mechanisms
underlying stress responses of the cutworm, Spodoptera litura ,
exposed to different levels of lead (Pb). Chemosphere, 283, 131205.
https://doi.org/10.1016/j.chemosphere.2021.131205
Christen V, Schirrmann M, Frey JE, Fent K (2018) Global transcriptomic
effects of environmentally relevant concentrations of the neonicotinoids
clothianidin, imidacloprid, and thiamethoxam in the brain of honey bees
(Apis mellifera ). Environ Sci Technol, 52(13), 7534-7544.
https://doi.org/10.1021/acs.est.8b01801
Colgan TJ, Fletcher IK, Arce AN, Gill RJ, Ramos Rodrigues A, Stolle E,
Chittka L, Wurm Y (2019) Caste‐and pesticide‐specific effects of
neonicotinoid pesticide exposure on gene expression in bumblebees. Mol
Ecol, 28(8), 1964-1974. https://doi.org/10.1111/mec.15047
Crotti E, Damiani C, Pajoro M et al (2009) Asaia , a versatile
acetic acid bacterial symbiont, capable of cross‐colonizing insects of
phylogenetically distant genera and orders. Environ Microbiol, 11(12),
3252-3264. https://doi.org/10.1111/j.1462-2920.2009.02048.x
Daisley BA, Chmiel JA, Pitek AP, Thompson GJ, Reid G (2020) Missing
microbes in bees: how systematic depletion of key symbionts erodes
immunity. Trends Microbiol, 28(12), 1010-1021.
https://doi.org/10.1016/j.tim.2020.06.006
David JP, Coissac E, Melodelima C, Poupardin R, Riaz MA, Chandor-Proust
A, Reynaud S (2010) Transcriptome response to pollutants and
insecticides in the dengue vector Aedes aegypti using next-generation
sequencing technology. BMC Genom, 11(1), 1-12.
https://doi.org/10.1186/1471-2164-11-216
DeBofsky A, Xie Y, Grimard C, Alcaraz AJ, Brinkmann M, Hecker M, Giesy
JP (2020) Differential responses of gut microbiota of male and female
fathead minnow (Pimephales promelas ) to a short-term
environmentally-relevant, aqueous exposure to benzo [a] pyrene.
Chemosphere, 252, 126461.
https://doi.org/10.1016/j.chemosphere.2020.126461
DeBofsky A, Xie Y, Challis JK, Jain N, Brinkmann M, Jones PD, Giesy JP
(2021) Responses of juvenile fathead minnow (Pimephales promelas )
gut microbiome to a chronic dietary exposure of benzo [a] pyrene.
Environ Poll, 278, 116821.
https://doi.org/10.1016/j.envpol.2021.116821
DeGrandi-Hoffman G, Corby-Harris V, DeJong EW, Chambers M, Hidalgo G
(2017) Honey bee gut microbial communities are robust to the fungicide
Pristine® consumed in pollen. Apidologie, 48(3), 340-352.
https://doi.org/10.1007/s13592-016-0478-y
DeGruttola AK, Low D, Mizoguchi A, Mizoguchi E (2016) Current
understanding of dysbiosis in disease in human and animal models.
Inflamm Bowel Dis, 22(5), 1137-1150.
https://doi.org/10.1097/MIB.0000000000000750
Desneux N, Decourtye A, Delpuech JM (2007) The sublethal effects of
pesticides on beneficial arthropods. Annu Rev Entomol, 52(1), 81-106.
https://doi.org/10.1146/annurev.ento.52.110405.091440
Díaz S, Fargione J, Chapin III FS, Tilman D (2006) Biodiversity loss
threatens human well-being. PLoS Biol, 4(8), e277.
https://doi.org/10.1371/journal.pbio.0040277
Ding J, Zhu D, Wang HT, Lassen SB, Chen QL, Li G, Lv M, Zhu YG (2020)
Dysbiosis in the gut microbiota of soil fauna explains the toxicity of
tire tread particles. Environ Sci Technol, 54(12), 7450-7460.
https://doi.org/10.1021/acs.est.0c00917
Dinno A (2017) dunn.test: Dunn’s Test of Multiple Comparisons Using Rank
Sums. R package version 1.3.5,
https://CRAN.R-project.org/package=dunn.test
Dirzo R, Young HS, Galetti M, Ceballos G, Isaac NJ, Collen B (2014)
Defaunation in the Anthropocene. Science, 345(6195), 401-406.
https://doi.org/10.1126/science.1251817
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P,
Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq
aligner. Bioinformatics, 29(1), 15-21.
https://doi.org/10.1093/bioinformatics/bts635
Douglas AE (2015) Multiorganismal insects: diversity and function of
resident microorganisms. Annu Rev Entomol, 60, 17.
https://doi.org/10.1146%2Fannurev-ento-010814-020822
Engel P, Kwong WK, McFrederick Q et al (2016) The bee microbiome: impact
on bee health and model for evolution and ecology of host-microbe
interactions. MBio, 7(2), e02164-15.
https://doi.org/10.1128/mBio.02164-15
Engel P, Moran NA (2013) The gut microbiota of insects–diversity in
structure and function. FEMS Microbiol Rev, 37(5), 699-735.
https://doi.org/10.1111/1574-6976.12025
Ernst F, Shetty S, Borman T, Lahti L (2022a) mia: Microbiome analysis. R
package version 1.5.17, https://github.com/microbiome/mia.
Ernst F, Borman T, Lahti L (2022b) miaViz: Microbiome Analysis Plotting
and Visualization. R package version 1.6.0.
Favia G, Ricci I, Damiani C et al (2007) Bacteria of the genusAsaia stably associate with Anopheles stephensi , an Asian
malarial mosquito vector. Proc Nat Acad Sci U.S.A., 104(21), 9047-9051.
https://doi.org/10.1073/pnas.0610451104
Favia G, Ricci I, Marzorati M, Negri I, Alma A, Sacchi L, Bandi lC,
Daffonchio D (2008) Bacteria of the Genus Asaia : A Potential
Paratransgenic Weapon Against Malaria. In: Aksoy S (eds) Transgenesis
and the Management of Vector-Borne Disease. Advances in Experimental
Medicine and Biology, vol 627. Springer, New York, NY.
https://doi.org/10.1007/978-0-387-78225-6_4
Feldhaar H, Otti O (2020) Pollutants and their interaction with diseases
of social Hymenoptera. Insects, 11(3), 153.
https://doi.org/10.3390/insects11030153
Fernandes AD, Macklaim JM, Linn TG, Reid G, Gloor GB (2013) ANOVA-like
differential expression (ALDEx) analysis for mixed population RNA-Seq.
PloS One, 8(7), e67019.
https://doi.org/10.1371/journal.pone.0067019
Fox J, Weisberg S (2019) An R Companion to Applied Regression, Third
edition. Sage, Thousand Oaks CA.
https://socialsciences.mcmaster.ca/jfox/Books/Companion/.
Gao J, Jin SS, He Y, Luo JH, Xu CQ, Wu YY, Hou CS, Wang Q, Diao QY
(2020) Physiological analysis and transcriptome analysis of Asian honey
bee (Apis cerana cerana ) in response to sublethal neonicotinoid
imidacloprid. Insects, 11(11), 753.
https://doi.org/10.3390/insects11110753
Gizaw G, Kim Y, Moon K, Choi JB, Kim YH, Park JK (2020) Effect of
environmental heavy metals on the expression of detoxification-related
genes in honey bee Apis mellifera . Apidologie, 51(4), 664-674.
https://doi.org/10.1007/s13592-020-00751-8
Gieré R, Querol X (2010) Solid particulate matter in the atmosphere.
Elements, 6(4), 215-222.
https://doi.org/10.2113/gselements.6.4.215
Greim H (2019) Diesel engine emissions: are they no longer tolerable?.
Arch Toxicol, 93(9), 2483-2490.
https://doi.org/10.1007/s00204-019-02531-5
Hamilton G A, Hartnett HE (2013) Soot black carbon concentration and
isotopic composition in soils from an arid urban ecosystem. Org Geochem,
59, 87-94. https://doi.org/10.1016/j.orggeochem.2013.04.003
Hammer TJ, Le E, Martin AN, Moran NA (2021) The gut microbiota of
bumblebees. Insectes Soc, 68(4), 287-301.
https://doi.org/10.1007/s00040-021-00837-1
Harrison JF (2009). Tracheal system. In Encyclopedia of insects (pp.
1011-1015). Academic Press.
https://doi.org/10.1016/B978-0-12-374144-8.00265-4
Harrison RM, Jones AM, Gietl J, Yin J, Green DC (2012) Estimation of the
contributions of brake dust, tire wear, and resuspension to nonexhaust
traffic particles derived from atmospheric measurements. Environ Sci
Technol, 46(12), 6523-6529. https://doi.org/10.1021/es300894r
Hartig F (2022) DHARMa: Residual Diagnostics for Hierarchical
(Multi-Level / Mixed) Regression Models. R package version 0.4.6,
https://CRAN.R-project.org/package=DHARMa
Hladun KR, Di N, Liu TX, Trumble JT (2016) Metal contaminant
accumulation in the hive: Consequences for whole‐colony health and brood
production in the honey bee (Apis mellifera L.). Environ Toxicol
Chem, 35(2), 322-329. https://doi.org/10.1002/etc.3273
Holzinger A, Mair MM, Lücker D, Seidenath D, Opel T, Langhof N, Otti O,
Feldhaar H (2022) Comparison of fitness effects in the earthwormEisenia fetida after exposure to single or multiple anthropogenic
pollutants. Sci Total Environ, 156387.
https://doi.org/10.1016/j.scitotenv.2022.156387
Hothorn T, Bretz F, Westfall P (2008) Simultaneous inference in general
parametric models. Biom J, 50(3), 346-363.
https://doi.org/10.1002/bimj.200810425
Iijima A, Sato K, Yano K, Tago H, Kato M, Kimura H, Furuta N (2007)
Particle size and composition distribution analysis of automotive brake
abrasion dusts for the evaluation of antimony sources of airborne
particulate matter. Atmospheric Environ, 41(23), 4908-4919.
https://doi.org/10.1016/j.atmosenv.2007.02.005
Kassambara A (2020) ggpubr: ’ggplot2’ Based Publication Ready Plots. R
package version 0.4.0, https://CRAN.R-project.org/package=ggpubr
Kautz S, Rubin BE, Moreau CS (2013) Bacterial infections across the
ants: frequency and prevalence of Wolbachia , Spiroplasma ,
and Asaia . Psyche, 2013.
https://doi.org/10.1155/2013/936341
Kim KH, Kabir E, Kabir S (2015) A review on the human health impact of
airborne particulate matter. Environ Int, 74, 136-143.
https://doi.org/10.1016/j.envint.2014.10.005
Koch H, Cisarovsky G, Schmid‐Hempel P (2012) Ecological effects on gut
bacterial communities in wild bumblebee colonies. J Anim Ecol, 81(6),
1202-1210. https://doi.org/10.1111/j.1365-2656.2012.02004.x
Koch H, Schmid-Hempel P (2011a) Bacterial communities in central
European bumblebees: low diversity and high specificity. Microb Ecol,
62(1), 121-133. https://doi.org/10.1007/s00248-011-9854-3
Koch H, Schmid-Hempel P (2011b) Socially transmitted gut microbiota
protect bumble bees against an intestinal parasite. Proc Nat Acad Sci
U.S.A., 108(48), 19288-19292.
https://doi.org/10.1073/pnas.1110474108
Koch H, Schmid‐Hempel P (2012) Gut microbiota instead of host genotype
drive the specificity in the interaction of a natural host‐parasite
system. Ecol Lett, 15(10), 1095-1103.
https://doi.org/10.1111/j.1461-0248.2012.01831.x
Koch H, Woodward J, Langat MK, Brown MJ, Stevenson PC (2019) Flagellum
removal by a nectar metabolite inhibits infectivity of a bumblebee
parasite. Curr Biol, 29(20), 3494-3500.
https://doi.org/10.1016/j.cub.2019.08.037
Kolde R (2019) pheatmap: Pretty Heatmaps. R package version 1.0.12,
https://CRAN.R-project.org/package=pheatmap
Kwong WK, Moran NA (2016) Gut microbial communities of social bees. Nat
Rev Microbiol, 14(6), 374-384.
https://doi.org/10.1038/nrmicro.2016.43
Levy M, Kolodziejczyk AA, Thaiss CA, Elinav E (2017) Dysbiosis and the
immune system. Nat Rev Immunol, 17(4), 219-232.
https://doi.org/10.1038/nri.2017.7
Li CC, Wang Y, Li GY, Yun YL, Dai YJ, Chen J, Peng Y (2016)
Transcriptome profiling analysis of wolf spider Pardosa
pseudoannulata (Araneae: Lycosidae) after cadmium exposure. Int J Mol
Sci, 17(12), 2033. https://doi.org/10.3390/ijms17122033
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change
and dispersion for RNA-seq data with DESeq2. Genome Biol, 15(12), 1-21.
https://doi.org/10.1186/s13059-014-0550-8
Łukowski A, Popek R, Jagiełło R, Mąderek E, Karolewski P (2018)
Particulate matter on two Prunus spp. decreases survival and performance
of the folivorous beetle Gonioctena quinquepunctata . Environ Sci
Pollut Res, 25(17), 16629-16639.
https://doi.org/10.1007/s11356-018-1842-4
Martin M (2011) Cutadapt removes adapter sequences from high-throughput
sequencing reads. EMBnet J, 17(1), 10-12.
https://doi.org/10.14806/ej.17.1.200
Martino C, Morton JT, Marotz CA, Thompson LR, Tripathi A, Knight R,
Zengler K (2019) A novel sparse compositional technique reveals
microbial perturbations. mSystems, 4(1), e00016-19.
https://doi.org/10.1128/mSystems.00016-19
Martinson VG, Danforth BN, Minckley RL, Rueppell O, Tingek S, Moran NA
(2011) A simple and distinctive microbiota associated with honey bees
and bumble bees. Mol Ecol, 20(3), 619-628.
https://doi.org/10.1111/j.1365-294X.2010.04959.x
Martinson VG, Moy J, Moran N A (2012) Establishment of characteristic
gut bacteria during development of the honeybee worker. Appl Environ
Microbiol, 78(8), 2830-2840. https://doi.org/10.1128/AEM.07810-11
Miličić M, Popov S, Branco VV, Cardoso P (2021) Insect threats and
conservation through the lens of global experts. Conserv Lett, 14(4),
e12814. https://doi.org/10.1111/conl.12814
Mockler BK, Kwong WK, Moran NA, Koch H (2018) Microbiome structure
influences infection by the parasite Crithidia bombi in bumble
bees. Appl Environ Microbiol, 84(7), e02335-17.
https://doi.org/10.1128/AEM.02335-17
Motta EV, Raymann K, Moran NA (2018) Glyphosate perturbs the gut
microbiota of honey bees. Proc Nat Acad Sci U.S.A., 115(41),
10305-10310. https://doi.org/10.1073/pnas.1803880115
Näpflin K, Schmid-Hempel P (2018) High gut microbiota diversity provides
lower resistance against infection by an intestinal parasite in
bumblebees. Am Nat, 192(2), 131-141.
https://doi.org/10.1086/698013
Naka K, Watarai S, Inoue K, Kodama Y, Oguma K, Yasuda T, Kodama H (2001)
Adsorption effect of activated charcoal on enterohemorrhagicEscherichia coli . J Vet Med Sci, 63(3), 281-285.
https://doi.org/10.1292/jvms.63.281
Ndakidemi B, Mtei K, Ndakidemi PA (2016) Impacts of synthetic and
botanical pesticides on beneficial insects. Agric Sci, 7(06), 364.
http://dx.doi.org/10.4236/as.2016.76038
Negri I, Mavris C, Di Prisco G, Caprio E, Pellecchia M (2015) Honey bees
(Apis mellifera , L.) as active samplers of airborne particulate
matter. PLoS One, 10(7), e0132491.
https://doi.org/10.1371/journal.pone.0132491
Noriega JA, Hortal J, Azcárate FM et al (2018) Research trends in
ecosystem services provided by insects. Basic Appl Ecol, 26, 8-23.
https://doi.org/10.1016/j.baae.2017.09.006
Patel A B, Shaikh S, Jain KR, Desai C, Madamwar D (2020) Polycyclic
aromatic hydrocarbons: sources, toxicity, and remediation approaches.
Front Microbiol, 11, 562813.
https://doi.org/10.3389/fmicb.2020.562813
Powell E, Ratnayeke N, Moran NA (2016) Strain diversity and host
specificity in a specialized gut symbiont of honeybees and bumblebees.
Mol Ecol, 25(18), 4461-4471. https://doi.org/10.1111/mec.13787
Prat O, Degli-Esposti D (2019) New challenges: Omics technologies in
ecotoxicology. In Ecotoxicology (pp. 181-208). Elsevier.
https://doi.org/10.1016/B978-1-78548-314-1.50006-7
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J,
Glöckner FO (2012) The SILVA ribosomal RNA gene database project:
improved data processing and web-based tools. Nucleic Acids Res, 41(D1),
D590-D596. https://doi.org/10.1093/nar/gks1219
Quintanilla-Mena M, Vega-Arreguin J, Río-García D, Patiño-Suárez V,
Peraza-Echeverria S, Puch-Hau C (2021) The effect of benzo [a]
pyrene on the gut microbiota of Nile tilapia (Oreochromis
niloticus ). Appl Microbiol Biotechnol, 105(20), 7935-7947.
https://doi.org/10.1007/s00253-021-11592-5
Raymann K, Shaffer Z, Moran NA (2017) Antibiotic exposure perturbs the
gut microbiota and elevates mortality in honeybees. PLoS Biol, 15(3),
e2001861. https://doi.org/10.1371/journal.pbio.2001861
R Core Team (2022) R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria. URL
https://www.R-project.org/.
Rivera‐Utrilla J, Bautista‐Toledo I, Ferro‐García MA, Moreno‐Castilla C
(2001) Activated carbon surface modifications by adsorption of bacteria
and their effect on aqueous lead adsorption. J Chem Technol Biotechnol,
76(12), 1209-1215. https://doi.org/10.1002/jctb.506
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package
for differential expression analysis of digital gene expression data.
Bioinformatics, 26(1), 139-140.
https://doi.org/10.1093/bioinformatics/btp616
Rönkkö T, Timonen H (2019) Overview of sources and characteristics of
nanoparticles in urban traffic-influenced areas. J Alzheimers Dis,
72(1), 15-28. https://doi.org/10.3233/jad-190170
Rothman JA, Russell KA, Leger L, McFrederick QS, Graystock P (2020) The
direct and indirect effects of environmental toxicants on the health of
bumblebees and their microbiomes. Proc Royal Soc B, 287(1937), 20200980.
https://doi.org/10.1098/rspb.2020.0980
Rothman, JA, Leger L, Graystock P, Russell K, McFrederick QS (2019) The
bumble bee microbiome increases survival of bees exposed to selenate
toxicity. Environ Microbiol, 21(9), 3417-3429.
https://doi.org/10.1111/1462-2920.14641
Sánchez-Bayo F, Wyckhuys KA (2019) Worldwide decline of the entomofauna:
A review of its drivers. Biol Conserv, 232, 8-27.
https://doi.org/10.1016/j.biocon.2019.01.020
Schirmer K, Fischer BB, Madureira DJ, Pillai S (2010) Transcriptomics in
ecotoxicology. Anal Bioanal Chem, 397(3), 917-923.
https://doi.org/10.1007/s00216-010-3662-3
Schönitzer K (1986) Quantitative aspects of antenna grooming in bees
(Apoidea: Hymenoptera). Ethology, 73(1), 29-42.
https://doi.org/10.1111/j.1439-0310.1986.tb00997.x
Seidenath D, Holzinger A, Kemnitz K, Langhof N, Lücker D, Opel T, Otti
O, Feldhaar H (2021) Individual vs. combined short-term effects of soil
pollutants on colony founding in a common ant species. Front Insect Sci,
13. https://doi.org/10.3389/finsc.2021.761881
Shi TF, Wang YF, Liu F, Qi L, Yu LS (2017) Sublethal effects of the
neonicotinoid insecticide thiamethoxam on the transcriptome of the honey
bees (Hymenoptera: Apidae). J Econ Entomol, 110(6), 2283-2289.
https://doi.org/10.1093/jee/tox262
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N,
Schwikowski B, Ideker T (2003) Cytoscape: a software environment for
integrated models of biomolecular interaction networks. Genome Res,
13(11), 2498–2504. https://doi.org/10.1101/gr.1239303
Shreiner AB, Kao JY, Young VB (2015) The gut microbiome in health and in
disease. Curr Opin Gastroenterol, 31(1), 69.
https://doi.org/10.1097%2FMOG.0000000000000139
Straub L, Strobl V, Neumann P (2020) The need for an evolutionary
approach to ecotoxicology. Nat Ecol Evol, 4(7), 895-895.
https://doi.org/10.1038/s41559-020-1194-6
Subramanian A, Tamayo P, Mootha KM et al (2005) Gene set enrichment
analysis: a knowledge-based approach for interpreting genome-wide
expression profiles. Proc Natl Acad Sci U.S.A., 102(43), 15545-15550.
https://doi.org/10.1073/pnas.0506580102
Sun K, Song Y, He F, Jing M, Tang J, Liu R (2021) A review of human and
animals exposure to polycyclic aromatic hydrocarbons: Health risk and
adverse effects, photo-induced toxicity and regulating effect of
microplastics. Sci Total Environ, 773, 145403.
https://doi.org/10.1016/j.scitotenv.2021.145403
Theis KR, Dheilly NM, Klassen JL et al (2016) Getting the hologenome
concept right: an eco-evolutionary framework for hosts and their
microbiomes. mSystems, 1(2), e00028-16.
https://doi.org/10.1128/mSystems.00028-16
Thorpe A, Harrison RM (2008) Sources and properties of non-exhaust
particulate matter from road traffic: a review. Sci Total Environ,
400(1-3), 270-282. https://doi.org/10.1016/j.scitotenv.2008.06.007
Turner S, Pryer KM, Miao VP, Palmer JD (1999) Investigating deep
phylogenetic relationships among cyanobacteria and plastids by small
subunit rRNA sequence analysis. J Eukaryot Microbiol, 46(4), 327-338.
https://doi.org/10.1111/j.1550-7408.1999.tb04612.x
Valavanidis A, Fiotakis K, Vlachogianni T (2008) Airborne particulate
matter and human health: toxicological assessment and importance of size
and composition of particles for oxidative damage and carcinogenic
mechanisms. J Environ Sci Health C: Toxicol, 26(4), 339-362.
https://doi.org/10.1080/10590500802494538
Wiaterek C (2012) Reibbeläge. In: Breuer, B., Bill, K.H. (Eds.),
Bremsenhandbuch. ATZ/MTZFachbuch.
Viewig + Teuber Verlag, Wiesbaden, Germany.
Wichmann HE (2007) Diesel exhaust particles. Inhal Toxicol, 19(sup1),
241-244. https://doi.org/10.1080/08958370701498075
Wickham H (2016) ggplot2: Elegant Graphics for Data Analysis. New York,
NY: Springer. https://doi.org/10.1007/978-3-319-24277-4_9
Wickham H, Girlich M (2022) tidyr: Tidy Messy Data. R package version
1.2.1, https://CRAN.R-project.org/package=tidyr
Yang Y, Ma S, Yan Z, Liu F, Diao Q, Dai P (2019) Effects of three common
pesticides on survival, food consumption and midgut bacterial
communities of adult workers Apis cerana and Apis
mellifera . Environ Pollut, 249, 860-867.
https://doi.org/10.1016/j.envpol.2019.03.077
Yilmaz P, Parfrey LW, Yarza P, Gerken J, Pruesse E, Quast C, Schweer T,
Peplies J, Ludwig W, Glöckner FO (2014) The SILVA and “all-species
living tree project (LTP)” taxonomic frameworks. Nucleic Acids Res,
42(D1), D643-D648. https://doi.org/10.1093/nar/gkt1209
Zereini F, Wiseman CLS (2010) Urban Airborne Particulate Matter.
Springer, Berlin, Germany
https://doi.org/10.1007/978-3-642-12278-1
Zhang W, Chen W, Li Z, Ma L, Yu J, Wang H, Liu Z, Xu B (2018)
Identification and characterization of three new cytochrome P450 genes
and the use of RNA interference to evaluate their roles in antioxidant
defense in Apis cerana cerana Fabricius. Front Physiol, 9, 1608.
https://doi.org/10.3389/fphys.2018.01608
Zhang QL, Guo J, Deng XY, Wang F, Chen JY, Lin LB (2019) Comparative
transcriptomic analysis provides insights into the response to the benzo
(a) pyrene stress in aquatic firefly (Luciola leii ). Sci Total
Environ, 661, 226-234.
https://doi.org/10.1016/j.scitotenv.2019.01.156
Zhang ZJ, Zheng H (2022) Bumblebees with the socially transmitted
microbiome: A novel model organism for gut microbiota research. Insect
Sci, 29, 958–976 https://doi.org/10.1111/1744-7917.13040
Zhang QL, Jiang YH, Dong ZX, Li HW, Lin LB (2021) Exposure to benzo
[a] pyrene triggers distinct patterns of microRNA transcriptional
profiles in aquatic firefly Aquatica wuhana (Coleoptera:
Lampyridae). J Hazard Mater, 401, 123409.
https://doi.org/10.1016/j.jhazmat.2020.123409
Zhao Y, Liu H, Wang Q, Li B, Zhang H, Pi Y (2019) The effects of benzo
[a] pyrene on the composition of gut microbiota and the gut health
of the juvenile sea cucumber Apostichopus japonicus Selenka. Fish
Shellfish Immunol, 93, 369-379.
https://doi.org/10.1016/j.fsi.2019.07.073
Zilber-Rosenberg I, Rosenberg E (2008) Role of microorganisms in the
evolution of animals and plants: the hologenome theory of evolution.
FEMS Microbiol Rev, 32(5), 723-735.
https://doi.org/10.1111/j.1574-6976.2008.00123.x
Zöllner C (2019) Einsatz optischer und analytischer Methoden zur
Bewertung des Betriebsverhaltens von Partikelfiltersystemen für die
Anwendung im Verkehr. In: Brüggemann D (eds.) Thermodynamik: Energie -
Umwelt – Technik, Band 34., ISBN: 978-3-8325-5032-5, Logos, Berlin
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