Acute and chronic effects of Titanium dioxide (TiO2) PM1 on honey bee gut microbiota under laboratory conditions

Abstract

Apis mellifera is an important provider of ecosystem services, and during flight and foraging behaviour is exposed to environmental pollutants including airborne particulate matter (PM). While exposure to insecticides, antibiotics, and herbicides may compromise bee health through alterations of the gut microbial community, no data are available on the impacts of PM on the bee microbiota. Here we tested the effects of ultrapure Titanium dioxide (TiO2) submicrometric PM (i.e., PM1, less than 1 µm in diameter) on the gut microbiota of adult bees. TiO2 PM1 is widely used as a filler and whitening agent in a range of manufactured objects, and ultrapure TiO2 PM1 is also a common food additive, even if it has been classified by the International Agency for Research on Cancer (IARC) as a possible human carcinogen in Group 2B. Due to its ubiquitous use, honey bees may be severely exposed to TiO2 ingestion through contaminated honey and pollen. Here, we demonstrated that acute and chronic oral administration of ultrapure TiO2 PM1 to adult bees alters the bee microbial community; therefore, airborne PM may represent a further risk factor for the honey bee health, promoting sublethal effects against the gut microbiota.

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
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Acute and chronic eects
of Titanium dioxide (TiO2) PM1
on honey bee gut microbiota
under laboratory conditions
G. Papa1, G. Di Prisco2,3,4, G. Spini5, E. Puglisi5* & I. Negri1
Apis mellifera is an important provider of ecosystem services, and during ight and foraging behaviour
is exposed to environmental pollutants including airborne particulate matter (PM). While exposure
to insecticides, antibiotics, and herbicides may compromise bee health through alterations of the
gut microbial community, no data are available on the impacts of PM on the bee microbiota. Here we
tested the eects of ultrapure Titanium dioxide (TiO2) submicrometric PM (i.e., PM1, less than 1 µm
in diameter) on the gut microbiota of adult bees. TiO2 P M 1 is widely used as a ller and whitening
agent in a range of manufactured objects, and ultrapure TiO2 P M 1 is also a common food additive,
even if it has been classied by the International Agency for Research on Cancer (IARC) as a possible
human carcinogen in Group 2B. Due to its ubiquitous use, honey bees may be severely exposed to TiO2
ingestion through contaminated honey and pollen. Here, we demonstrated that acute and chronic
oral administration of ultrapure TiO2 P M 1 to adult bees alters the bee microbial community; therefore,
airborne PM may represent a further risk factor for the honey bee health, promoting sublethal eects
against the gut microbiota.
Honey bees (Apis mellifera Linnaeus) are important providers of ecosystem services, both regulating, through
pollination of a wide range of crops and uncultivated plants, and provisioning, for the delivery of honey, pollen,
propolis and other products to humans. Moreover, the honey bee is an important bioindicator of environmental
contamination, and both the insect and its products are used for the detection of environmental pollutants1–5.
In the last decade, one of the major problems plaguing honey bees is a phenomenon named Colony Col-
lapse Disorder (CCD) which causes loss of colonies worldwide6–8. e multifactorial origin of CCD is widely
acknowledged, and environmental stressors such as pesticides, heavy metals, or airborne particulate matter (PM)
pollutants may play a key role in driving the bees’ decline9.
While the eects of pesticides and heavy metals on bees are widely recognised, till now very few data are
available on the eects of PM on bees’ health. ese studies include lethal and sublethal eects (e.g., behaviour,
gene expression, and cellular alterations) of the exposure to lead and cadmium oxides and TiO2 nanoparticles10–13.
In polluted environments, airborne PM is known to stuck to the body of the bee and can be also ingested
through contaminated pollen and honey that represent the food sources of the bee colony3,5,14. If ingested, dusts
can come into contact with the gut microbiome lining the intestinal epithelium posing a hazard to the bacte-
rial community. Recent evidence suggests that environmental stressors can indirectly compromise bee health
through gut microbiota disruption15. e honey bee harbors a simple and distinct gut community, thought to
be the result of a long-lasting evolutionary relationship15. While no evidence has been reported until now on the
impacts of PM on the bee health, alterations of the gut microbial community composition were demonstrated
in bees exposed to antibiotics16, glyphosate17, insecticides18,19 and sublethal doses of cadmium and selenite20.
e gut of worker bees is dominated by nine clusters of bacterial species comprising between 95% and 99.9%
of total diversity in almost all individuals, based on investigations carried out on 16S rDNA21–23 and on total
DNA metagenomics of intestinal samples24. e two omnipresent Gram-negative species are Snodgrassella alvi
OPEN
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  
          
 *email: edoardo.puglisi@unicatt.it
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and Gilliamella apicola, both members of Phylum Proteobacteria25. Among Gram-positive bacteria, two groups
of species in the Firmicutes phylum are ubiquitous and abundant, referred to as the Lactobacillus Firm-4 and
Lactobacillus Firm-5 clades26. Although oen less abundant, the cluster of the species Bidobacterium asteroides27
is also found in most adult worker bees. Less numerous and even less prevalent are the Proteobacteria species
Frischella perrara28, Bartonella apis29, Parasaccharibacter apium23 and a group of Gluconobacteria species des-
ignated Alpha2.1. ese species have narrow niches in the intestines of bees (e.g., F. perrara) or are generalists,
i.e., they are also found in the hive environment (for example, P. apium, Lactobacillus kunkeei and the Alpha2.1
group), which may explain their relatively lower frequency in bee gut detections. While other bacteria may occa-
sionally be present, these nine species groups represent bacterial lineages that appear to be specically adapted
to life alongside their hosts, bees. is gut microbiota organization is well described by Kwong & Moran30.
e newly hatched larvae are free of bacteria that start colonizing the gut thanks to interactions with worker
bees and the hive environment31,32. During the metamorphosis of the larvae to pupae and nally, into adult bees,
the lining of the intestine is renewed: the newly emerged adult bees have very few bacteria in the intestine and
are readily colonized by the typical intestinal microbial community33.
Titanium dioxide (TiO2) is a naturally occurring metal oxide. As rutile, TiO2 is not rare in nature and may
concentrate in the heavy fraction of sediments. Synthetic TiO2 in form of sub-micrometric PM, i.e., PM1, less
than 1µm in diameter, is widely used as a ller and whitening agent in a range of manufactured objects, such
as plastics, paints, paper, printing inks, textiles, catalysts, oor and roong materials, and vehicles components.
TiO2 is also a common ingredient in cosmetics, pharmaceuticals, sunscreen and as a food additive. While TiO2
used for non-food applications has surface coatings of alumina and silica, to reduce photoactivity, and organic
surface treatments, conferring hydrophobic properties, TiO2 as a food additive is ultrapure, and PM size ranges
from 400µm to 30 nm34,35.
e potential ecological and human health impact of exposure to TiO2 is of growing concern. Indeed, inhala-
tion and intra-tracheally administration of PM of TiO2, including nano-meter sized particles, induces lung cancer
in rats36. TiO2 has indeed been classied by the International Agency for Research on Cancer (IARC) as a pos-
sible human carcinogen in Group 2B (IARC, 2010). New evidence suggests that oral exposure to nanosized TiO2
promotes chronic intestinal inammation and carcinogenesis in rats35, as well as dysbiosis of gut microbiota37.
Trace element analyses already demonstrated that Titanium can be detected in honey, pollen and bees, espe-
cially in high density urban and residential/suburban areas38–41. Titanium can be only found in combined form
and the most widespread chemical form both from natural and anthropogenic sources is TiO2. erefore, honey
bees may be severely exposed to TiO2 ingestion through contaminated honey and pollen, and in severely polluted
areas, PM1 of TiO2 can be detected in pollen grains and honey (Negri, personal communication).
e toxicity of TiO2 PM1at nano-scale has been already studied in honey bee with histological and immuno-
histochemical approaches12 shedding light on the negative eects at high doses. However, in-depth studies on the
eects on the gut environment of the TiO2 ingested by bees are still lacking, and no studies on specic impacts
on the bee’s gut microbiota are available. Here we wanted to ll this gap by studying the eect of TiO2 on honey
bees gut microbiota in the experimental context of controlled oral administration, providing evidence that inges-
tion of PM1 of TiO2 can alter the bee microbial community. Sub-lethal doses were employed, while acute and
chronic exposures were assessed at 24 and 96h post-emergency for acute and chronic experiments respectively.
Results
SEM–EDX results. Scanning electron microscope (SEM) coupled with X-ray (EDX) analyses were used to
assess the morphology, chemical composition and size of TiO2 particles delivered to the bees.
SEM analyses showed that the TiO2 stock powder had a sub-micrometre size < 1µm (between 800 and
200nm) and an ellipsoidal/spherical shape (Fig.1A). Moreover, EDX analyses and compositional mapping
conrm the purity of TiO2 rutile (Fig.1B–E).
SEM–EDX analyses demonstrated the absence of TiO2 in haemolymph collected from the acute, chronic and
control samples (FigureS1A,B). In the haemolymph, EDX spectra (FigureS1B) showed the presence of many
elements e.g., Mg, Ca, Na, S, P, K, Cl42.
SEM observation performed on chronic and control gut highlighted the presence of many lanceolate crystals
likely due to precipitation of salts. EDX analysis conrmed their chemical composition as K2SO4 (Fig.2A–D).
In the rectum of treated bees, TiO2 was found both associated (Fig.2E,F) and not associated (Fig.2G–L) with
K2SO4 crystals.
Bacterial diversity in the studied honey bees. e gut bacterial diversity was assessed by means of
Illumina HTS of bacterial 16S amplicons covering the V3-V4 regions. A total of 537,641 sequences were pro-
duced, ltered and downscaled to 216,000 (i.e., 12,000 per each sample) aer elimination of homopolymers,
sequences not aligning to the target region, chimaeras, non-bacterial sequences and rarefaction to the least
populated sample. Aer this downscaling, one out of 18 samples (a replicate of the 100X acute test) was elimi-
nated because it had a lower number of sequences. e resulting Good’s index of coverage of the rareed samples
was 94.7 ± 1.2%, indicating that the vast majority of bees gut bacterial diversity was covered by the sequencing.
e structure of honey bee gut bacterial community was investigated at OTUs level, testing with a CCA model
if the treatment (control vs chronic vs acute) and the dose had signicant eects on the bacterial gut communities
of the studied bees. Results (Fig.3) indicated that the bees from the acute and the chronic experiments, being
sampled at dierent life stages, hosted very dierent gut microbiota. Within each exposure time, it was found
that samples were clearly grouped among doses for chronic exposure, but not for the acute. Similar outcomes
were obtained by Principal Component Analyses (FigureS2).
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Figure1. Control sample of TiO2 rutile powder and elemental mapping. (A) SEM image showing the
morphology and size and (B) EDX spectrum showing the purity of the TiO2 powder. (C) SEM-BSE image and
the (D) titanium and (E) oxygen element maps.
Figure2. Elemental mapping of the rectum in control (A–D) and chronical samples (E–L). (A,E,G,K) SEM-
BSE images; (B–D) element analysis highlighting the presence of K2SO4 crystals in the control sample; (F)
titanium dioxide associated with K2SO4 crystals; (H–L) titanium dioxide not associated with K2SO4 crystals.
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Dierences among samples were also reected by α-diversity analyses on the total number of OTUs identied
in each treatment, which highlighted a clear trend: bees exposed to both chronic and acute TiO2 PM displayed
a higher diversity as compared to their relative controls. e dierence was signicant according to LSD test
between the chronic control and the two acute treatments (Fig.4).
e distinct bacterial community structures induced by TiO2 PM exposure were also highlighted by hierar-
chical clustering of sequences classied at the genus level (Fig.5): in agreement with the CCA results, chronic
and acute experiments were grouped in two main separate clusters sharing less than 70% of similarity. e two
controls for each treatment representing adults sampled at two dierent life stages were also grouped separately
one from the other. Gilliamella was the dominant genus in the acute experiments (24h), with relative percentages
reaching more than 80% of total abundance in a number of acute treatments at both 10X and 100X, followed by
Lactobacillus and Acetobacter. A very dierent composition was identied in 96h bees of the chronic exposure
experiment (Fig.5): here the dominant genus was Lactobacillus, which decreased in the TiO2 exposed bees. e
latter was however enriched in Bidobacterium, reaching relative concentration between 5 and 12% in a number
of chronically exposed bees. A particular feature was detected in two treated replicates at 10X and 100X, with
Acetobacter covering the vast majority (i.e., > 98%) of the observed diversity at the genus level.
Distinctive features in the gut microbiota of honey‑bees acutely exposed to TiO2. Aer den-
ing that the two bee groups from the acute and the chronic exposure experiments had very dierent gut bacte-
rial compositions (Figs.3 and 5), separate analyses were carried out on the two groups, focusing on the relative
presence of the most abundant OTUs classied at the species level.
In the acute exposure experiments, G.apicola was the most abundant species with an average of ca 70% of the
total bacterial community, followed by Lactobacillus apis, S. alvi, Lactobacillus kimbladii and Acetobacter tropicalis
(Fig.6a). e clustering did not show clear discrimination between control and treated bees, but a Metastats
model on the same OTUs revealed a number of signicant dierences (Fig.6b). Specically, a signicant reduc-
tion with increasing acute doses of TiO2 was found for L. kimbladii. e same trend was detected for L. apis and
S. alvi, but with no statistical signicance.
Distinctive features in the gut microbiota of honey‑bees chronically exposed to TiO2. A
stronger dierentiation between control and TiO2 exposed bees was found in the chronic experiments. Here, the
composition at the species level was very dierent, reecting the age dierences between this and the previous
group of bees. G. apicola was still present, but with much lower relative abundances (< 2%). On the contrary,
a strong increase in abundances was found for B. apis, L. apis, L. kimbladii, Commensalibacter intestini, and
Bartonella spp. (Fig.7a). Much higher was also the number of species displaying signicant dierences between
treatments and controls (Fig.7b), thus highlighting signicant eects of chronic TiO2 exposure on the honey
bees gut microbiota. Specically, L. apis, Lactobacillus melliventris and Bartonella spp. were signicantly reduced
by the exposure to TiO2, in most cases with a dose dependent eect. On the contrary, Bombella intestini was
found to be signicantly enriched only in the X100 treatment.
Figure3. Hypothesis-driven canonical correspondence analysis (CCA) of honey bees gut microbiota testing
the signicance of the treatment and dose eects on the abundances of all analysed OTUs. Samples are labelled
according to the 6 groups studied.
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Discussion
In the present study, we explored the potential eects of acute and chronic oral administration of pure TiO2 rutile
on honey bee gut microbiota. e particles used were PM1; ranging from 800 to 200nm as characterized by
SEM–EDX analysis. In a preliminary assay, we fed newly emerged worker bees with four dierent concentrations
of TiO2 in 1M sucrose solution, including 100ng/µL and 10ng/µL as test doses, aer the rejection of the two
higher doses 104ng/µL and 103ng/µL. Newly emerged worker bees were orally exposed to the selected doses of
TiO2 in an acute (single treatment, sampling at 24h) and a chronic application (treatments repeated every 24h,
sampling at 96h). In both assays, we obtained no mortality for the entire trials. is is partly in contrast with
similar experiments on the honey bee, that however applied higher doses than the one tested here: TiO2 resulted
highly toxic at 1000ng/µL12, while in another study the LC50 at 96h resulted in 5.9ng/µL13. Nevertheless, TiO2
showed high mortality at a very high dose of 2400ng/µL) in cutworm (Spodoptera litura)43.
Unfortunately, the studies by Özkan etal.13 and Ferrara etal.12 do not provide a specic characterization of
the size, morphology and purity of TiO2 dust delivered to the bees. In our research, TiO2 dust were carefully
characterised by SEM/EDX, demonstrating the absence of contaminants or coatings and the dimensions greater
than 200nm. Such size might also explain the absence of TiO2 PM in the haemolymph since particles might not
be able to cross the barrier of the gut epithelium. However, a more in-depth study involving the analysis of the
gut epithelium should be carried out to exclude the presence of TiO2 PM from the epithelial cells or cytological
abnormalities.
An indirect impact of TiO2 on haemolymph has been thoroughly investigated in other organisms. In Mytilus
galloprovincialis, TiO2 has been shown to aect several immune parameters in both circulating haemocytes and
haemolymph serum, resulting in immunomodulation44–46. In larvae of Galleria mellonella (Lepidoptera: Pyrali-
dae), exposure with dietary TiO2 nanoparticles (NPs) has dose-dependent toxic eects and can enhance the
stress-resistant capacity with a signicant increase in the total protein amount and content of malondialdehyde
(MDA) and glutathione S-transferase activity at 100, 500 and 1000 ppm47. In our experiments, SEM–EDX reveals
the typical haemolymph elements spectrum (Mg, Ca, Na, S, P, K, Cl) of the honey bee48.
e analyses of bacterial community composition allowed by HTS of 16S amplicons clearly show that the
acute and chronic exposed bees populations had hosted very dierent bacterial gut populations (Figs.5, 6 and
7). is is expected since analyses were carried out aer 1 dpe (day post-emergence) for the acute experiment
and at 4 dpe for the chronic experiment. e β-diversity analyses here carried out by means of unconstrained
(PCA and hierarchical clustering, Fig.5 and FigureS2) and constrained (CCA, Fig.3) analyses showed a clear
distinction in bacterial community structure between the chronic and the acute experiments, and in the chronic
experiments among controls and treated bees. ese results are in line with several studies on other chemical
stressors16–20, thus conrming that also PM modulates the honey bees gut bacterial community.
Figure4. Number of observed OTUs (S) for the chronic and acute treatments and their respective controls.
Signicant dierences are highlighted with dierent letters according to Tukey’s HSD test for comparison of
means.
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e results obtained in the unexposed controls are quite in agreement with previous evidence and showed
the typical community of adult worker bee microbiota that is dominated by Lactobacillus, Bidobacterium,
Gilliamella, Snodgrassella. e presence of Bartonella and Commensalibacter in 4 dpe bees from the chronic
experiment is in agreement with the characteristic gut microbial community of the so-called “winter bees”, i.e.,
the last generations of workers characterised by overwintering individuals with an extended lifespan to ensure
colony survival until spring49. e variability occurring in control bees is in agreement with other studies, where
it has been demonstrated that, while many of the phylotypes are consistently present in adult worker bees, their
relative abundance can vary across individuals49,50, and representative of these two genera can be found also in
early stages of development20,51.
In the treated populations, the two tested doses of TiO2 had signicant impacts on the gut bacterial communi-
ties, with more dierences between exposed and control bees in the chronic as compared to the acute exposure.
As far as we know, this is the rst study where the eects of TiO2 particles where specically studied on bees gut
microbiota, but they can be compared with a number of studies that previously assessed the detrimental impacts
of airborne PM on the bees physiology12,13. Regarding α-diversity indexes, we found a dose-dependent increase
in diversity, (Fig.3), while the opposite was found for the neonicotinoid insecticide iacloprid18, polystyrene
particles at µm levels52, and antibiotics16. It must be highlighted that in these cited works a signicant mortal-
ity was detected, whereas in our study sub-lethal doses were tested and no mortality registered. It can thus be
speculated that the microbiota responded to the sub-lethal stressors by increasing the diversity, as postulated by
the ecological theory of the intermediate disturbance hypothesis rstly proposed by Connell in 197853.
Concerning the changes in species abundances, we found that in the acute test only one species, L. kimbladii,
was signicantly reduced by the highest dose of TiO2 applied (Fig.6). Firstly described in 2014 by Olofsson54
and colleagues, this species was more recently proposed as a possible probiotic55,56. Interestingly, L. kimbladii
was instead found at higher abundances in the 96h bees from the chronic exposure, with values higher than
the control in the two treated theses (Fig.7). is change may point to an adaptation in time of the microbial
community, with a probiotic species becoming more abundant to counteract the chronic eects of a chemical
stressor, as previously shown for Bidobacterium species in bees exposed to the insecticide Nitenpyram19.
Figure5. Hierarchical clustering of sequences classied at the genus level for both chronic and acute
treatments. Bars of dierent colours indicated the relative percentage of the genera identied in the honey bees
gut. Only genera participating with > 1% in at least one sample are shown, while taxa with lower participations
were added to the “other” group. Similar samples were clustered using the average linkage algorithm.
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While in the acute experiment only L. kimbladii was signicantly inhibited by the TiO2 particles (Fig.6), in the
chronic exposure three species were inhibited: L. apis, L. melliventris and Bartonella spp. (Fig.7). L. apis is one of
the most studied components of the bees gut microbiome, whose functions as probiotics were demonstrated by
processes such as the attenuation of immune dysregulation57 and the inhibition of Paenibacillus larvae and other
pathogens58; the induction of resistance towards bacterial infection was also demonstrated for L. melliventris,
which was accordingly proposed as a bee probiotic59. Finally, the reduction of species belonging to Bartonella
was also found in bees parasitized by Varroa,60, but this genus was also found to be increased aer exposure to
glyphosate17. e only taxon whose relative presence was signicantly increased by chronic TiO2 exposure was
B. intestinii, an acetic acid bacterium (AAB) rstly isolated in honey bees in 2017 by Yun and colleagues61. is
species, together with other AAB, may play a role in the regulation of the innate immune system homeostasis62
and may thus represent an adaptation of the gut bacterial community to counteract the stressors sub-lethal eects.
Titanium oxide nanoparticles are known to exert a toxic eect on several bacteria, with proposed modes of
action related to both chemical and physical interactions with the cells envelopes63, and alterations in human
gut bacterial communities are oen found including detrimental eects, as recently reviewed by Lams etal.,.
(2020)64. Here we provide for the rst time modulation of the honey bees gut microbiota induced by both acute
and chronic exposure to TiO2 PM1, with stronger eects in the latter case. e eects we report are related to
doses that are probably higher than the ones that can be found under eld conditions and can be considered
sub-lethal since no mortality was observed among all treated bees, and an increase in bacterial diversity was
even found. On the other side, some negative eects related to the decrease in the relative percentages of some
benecial lactobacilli were however observed, and for this reason the role of airborne particulate as a further risk
factor for Apis mellifera health in addition to other chemical stressors should be further explored.
Figure6. (a) Hierarchical clustering of sequences of OTUs participating with > 1% in at least one sample
for the acute exposure experiment. Rare OTUs with lower participation were added to the “other” group; (b)
metastats model showing dierences among treatments for the most abundant OTUs. Signicant dierences are
highlighted with dierent letters according to Tukey’s HSD test for comparison of means.
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Materials and methods
Honey bees. Experiments were conducted during October 2018 with Apis mellifera ligustica colonies main-
tained in the experimental apiary of the University of Napoli “Federico II”, Department of Agricultural Sciences.
Brood frames with capped cells from two colonies were selected and kept in a climatic chamber at 36°C and
60% relative humidity for approximately 16h. Newly emerged workers were randomly selected and used for the
bioassays.
Preparation of TiO2 feeding solution. Titanium dioxide rutile (TiO2) powder was acquired from 2B
Minerals S.r.l. (Modena, Italy). Suspensions were prepared by dissolving 0.5g TiO2 in 50 mL distilled H2O,
vortexed for ~ 20s and sonicated for 15min to increase dispersion and ensure the maximum distribution of
particles in water13. A 1M sucrose solution was then prepared with TiO2 suspensions.
Preliminary palatability tests on bees were carried out with the following dilutions: 104ng/µL, 103ng/µL,
100ng/µL, 10ng/µL. e last two concentrations were chosen for the experiments following rejection of the
more concentrated solutions by the bees.
Chronic and acute exposure. Two groups of 30 newly eclosed bees were randomly collected from the
combs. e bees were fed adlibitum with 1 µL of 10ng/µL and 100ng/µL TiO2 solutions (1M sucrose), respec-
tively (hereinaer CH10- and CH100-bees), and then placed into plastic cages containing 1.5mL of the same
solution to which they were previously fed. Control bees consisted of 30 individuals fed adlibitum with an
untreated sucrose solution (1M).
e solution was changed every 24h. During the experiments, all caged bees were kept in a climatic chamber
at 36°C and 60% relative humidity. Aer 96h the bees were anesthetized with CO2 for ~ 30s and the whole gut
dissected as previously described65. Guts were stored in absolute ethanol and immediately refrigerated at -80°C
for the subsequent microbiological analysis. Experiments were conducted in triplicate (i.e., 3 replicates each
made by three guts pooled together). Bees survival was recorded each day.
e experimental design of acute exposure was the same as in chronic exposure except for the duration of
the experiment that in acute exposure lasted only 24h instead of 96, and bees were fed only once with 10ng/µL
or 100ng/µL TiO2 solutions (hereinaer AC10- and AC100-bees).
Figure7. (a) Hierarchical clustering of sequences of OTUs participating with > 1% in at least one sample for
the chronic exposure experiment. Rare OTUs with lower participation were added to the “other” group; (b)
metastats model showing dierences among treatments for the most abundant OTUs. Signicant dierences are
highlighted with dierent letters according to Tukey’s HSD test for comparison of means.
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TiO2 detection in the gut. To assess morphology, average size and chemical purity of TiO2 powder, few
µgrams were mounted onto stubs and analysed through a Scanning Electron Microscope (SEM) provided with
X-ray spectroscopy (EDX) (Zeiss Gemini SEM 500—Bruker Quanta X-Flash 61|31). Secondary Electrons (SE),
BackScattered Electrons (BSE) images, and EDX point analyses, were acquired as previously described3,14.
e presence of TiO2 in the haemolymph and gut of chronic bees was investigated by SEM–EDX. Haemo-
lymph from 4 randomly selected CH10- and CH100-bees plus 4 randomly selected control bees was sampled
from the dorsal aorta, following the method standardized by Garrido and colleagues66. e bee gut (rectum with
excrements) from CH10- and CH100-bees plus control bees (n = 4 randomly selected, for each concentration
and control) were mounted onto stub and dried in a sterilized oven at 20°C for 40min. Samples were carbon
coated and analyzed with SEM/EDX.
Microbiota analysis. DNA extraction. DNA was extracted from gut samples collected for microbiological
analyses. For each dose—acute and chronical—n. 3 groups of three guts were analysed. From these samples, the
total microbial DNA was extracted using the Fast DNA SPIN Kit for Soil (MP Biomedicals, USA) with the fol-
lowing modications: each sample was homogenized in the FastPrep for 40s at speed setting of 6.5 twice, keep-
ing it in ice between the two homogenisation steps, while the nal centrifugation was carried out at 14,000×g for
15min, and the nal resuspension of the Binding Matrix was carried out in 50 µL of nuclease-free water.
e DNA extractions were checked with electrophoresis on a 1% agarose gel, and then quantied using a
QuBit uorometer (Invitrogen, United Kingdom).
DNA amplication. e V3-V4 region of the bacterial 16S rRNA gene (between 480 and 490bp) was amplied
by PCR using the universal primers 343f. (5′-TAC GGR AGG CAG CAG-3′), and 802r (5′-TACNVGGG TWT CTA
ATC C-3′)67. A two-step PCR protocol was implemented in order to reduce the possibility of generating non-
specic primer annealing, as detailed in Berry etal.68. e PCR reaction mix comprised of 20.5 µL of MegaMix
(Microzone Limited, United Kingdom), 1.25 μL of each primer (10μM), and 2 μL (1ng/μL concentration) of
DNA template. ermal cycling conditions were as follows: Step 1: an initial denaturation at 94°C for 5min,
followed by 25 cycles at 94°C for 30s, 50°C for 30s, 72°C for 30s, followed by a nal extension at 72°C for
10min. Step 2: initial hold at 95°C for 5min, followed by 10 cycles of 95°C for 30s, 50°C for 30s, and 30°C
for 30s; then, a nal extension at 72°C for 10min. At the second step, each sample was amplied using a dedi-
cated forward primer with a 9- base extension at the 5′ end, which acts as a tag, in order to make simultaneous
analyses of all samples in a single sequencing run possible e DNA amplications were checked with electro-
phoresis on a 1% agarose gel, and then quantied using a QuBit uorometer (Invitrogen, United Kingdom). PCR
products generated from the second step were multiplexed as a single pool using equivalent molecular weights
(20ng). e pool was then puried using the solid phase reversible immobilization (SPRI) method with Agen-
court AMPure XP kit (REF A63880, Beckman Coulter, Milan, Italy), then sequenced by Fasteris S.A. (Geneva,
Switzerland). e TruSeq DNA sample preparation kit (REF 15026486, Illumina Inc, San Diego, CA) was used
for amplicon library preparation, whereas the sequencing was carried out with the MiSeq Illumina instrument
(Illumina Inc., San Diego, CA) generating 300bp paired-end reads.
Sequence data preparation and statistical analyses. High-throughput sequencing data ltering,
multiplexing and preparation for concomitant statistical analyses were carried out as previously detailed69. In
summary, paired-reads were assembled to reconstruct the full V3-V4 amplicons using the “pandaseq” script70
with a maximum of 2 allowed mismatches and at least 30bp of overlap between the read pairs. was then carried
out with the Fastx-toolkit was then employed for samples demultiplexing (http://hanno nlab.cshl.edu/fastx _toolk
it/).Mothur v.1.32.171 was applied in order to remove sequences with large homopolymers (≥ 10), sequences that
did not align within the targeted V3-V4 region, chimeric sequences72 and sequences not classied as bacterial
aer alignment against the Mothur version of the RDP training data set. Mothur and R (http://www.R-proje
ct.org/) were employed to analyze the resulting high-quality sequences following the operational taxonomic
unit (OTU) and the taxonomy-based approach. For the OTU approach, sequences were rst aligned against the
SILVA reference aligned database for bacteria73 using the NAST algorithm and a kmer approach74,75, and then
clustered at the 3% distance using the average linkage algorithm. OTUs having a sum of their abundances across
all samples of than 0.1% of the total were grouped into a single “rare OTUs” group. For taxonomy based analyses,
sequences were classied into taxa using an amended version of the Greengenes database76.
Mothur and R were also employed for statistical analyses on OTU and taxonomy matrixes using hierarchical
clustering with the average linkage algorithm at dierent taxonomic levels, Principal component analysis (PCA)
for unconstrained samples grouping, Canonical correspondence analyses (CCA) to assess the signicance of
dierent treatments on the analysed diversity. Features that were signicantly dierent between treatments were
identied with Metastats76.
Sequence data were submitted to the National Centre for Biotechnology Information Sequence Read Archive
(BioProject accession number PRJNA693145).
Received: 29 November 2020; Accepted: 22 February 2021
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Acknowledgements
GP was partially supported by the Doctoral School on the Agro-Food System (Agrisystem) of the Università
Cattolica del Sacro Cuore (Italy). We thank Tiziano Catelani and Paolo Gentile for advices on the SEM-EDX
analyses and Francesco Pennacchio for the permission to use the Entomology laboratory at the Federico II
University of Naples (Italy).
Author contributions
G.P., I.N., conceived and designed the experiments; G.P., G.P.D., G.S. performed the experiments; G.P., I.N., E.P.,
analysed data G.P., G.D.P., I.N., E.P. wrote the article. All authors reviewed the manuscript.
Competing interests
e authors declare no competing interests.
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