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Glyphosate perturbs the gut microbiota of honey bees
Erick V. S. Motta
a,1
, Kasie Raymann
a,2
, and Nancy A. Moran
a,1
a
Department of Integrative Biology, University of Texas at Austin, Austin, TX 78712
Edited by Margaret J. McFall-Ngai, University of Hawaii at Manoa, Honolulu, HI, and approved August 21, 2018 (received for review March 6, 2018)
Glyphosate, the primary herbicide used globally for weed control,
targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)
enzyme in the shikimate pathway found in plants and some
microorganisms. Thus, glyphosate may affect bacterial symbionts
of animals living near agricultural sites, including pollinators such
as bees. The honey bee gut microbiota is dominated by eight
bacterial species that promote weight gain and reduce pathogen
susceptibility. The gene encoding EPSPS is present in almost all
sequenced genomes of bee gut bacteria, indicating that they are
potentially susceptible to glyphosate. We demonstrated that the
relative and absolute abundances of dominant gut microbiota
species are decreased in bees exposed to glyphosate at concen-
trations documented in the environment. Glyphosate exposure of
young workers increased mortality of bees subsequently exposed
to the opportunistic pathogen Serratia marcescens. Members of
the bee gut microbiota varied in susceptibility to glyphosate,
largely corresponding to whether they possessed an EPSPS of class
I (sensitive to glyphosate) or class II (insensitive to glyphosate).
This basis for differences in sensitivity was confirmed using
in vitro experiments in which the EPSPS gene from bee gut bacte-
ria was cloned into Escherichia coli. All strains of the core bee gut
species, Snodgrassella alvi, encode a sensitive class I EPSPS, and
reduction in S. alvi levels was a consistent experimental result.
However, some S. alvi strains appear to possess an alternative
mechanism of glyphosate resistance. Thus, exposure of bees to
glyphosate can perturb their beneficial gut microbiota, potentially
affecting bee health and their effectiveness as pollinators.
honey bees
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microbiome
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glyphosate
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Snodgrassella alvi
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Serratia
The broad-spectrum herbicide glyphosate [N-(phosphono-
methyl)glycine] has long been the primary weed management
system, and its use is growing in connection with crops geneti-
cally engineered to be resistant to glyphosate (1, 2). Its mecha-
nism of action, inhibition of 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS), an enzyme in the shikimate pathway, prevents
the biosynthesis of aromatic amino acids and other secondary
metabolites in plants and some microorganisms (3). EPSPS
catalyzes the reaction between phosphoenolpyruvate (PEP) and
shikimate 3-phosphate (S3P) (4), and glyphosate is a competitive
inhibitor that blocks the PEP-binding site (5). EPSPS enzymes
from different organisms vary in molecular weight (46–178 kDa)
and sequence homology (6) and form two phylogenetic clusters
that differ in tolerance to glyphosate. Class I enzymes are sen-
sitive to glyphosate and are present in all plants and in some
bacteria, such as Escherichia coli (4); class II enzymes are only
found in some bacteria, such as Staphylococcus aureus, and can
tolerate high concentrations of glyphosate (7, 8).
Animals lack the shikimate pathway, which is why glyphosate is
considered one of the least toxic pesticides used in agriculture (9).
However, some evidence suggests that glyphosate affects non-
target organisms, for example, changing the behavior of honey
bees (10), reducing reproduction of soil-dwelling earthworms (11),
and affecting the growth of microalgae and aquatic bacteria (12).
Glyphosate is also associated with changes in plant endophytic
and rhizosphere microbiomes (2) and with disturbances of gut
microbiota of animals living near agricultural sites (13).
Honey bees and bumble bees are major pollinators of flow-
ering plants, including many crops. When foraging, they can be
exposed to a variety of xenobiotics, such as glyphosate. This
herbicide is known to affect the growth of microorganisms (13–
15), and the health of bees is intrinsically related to their distinct
gut microbial community (16, 17). The honey bee gut microbiota
is dominated by eight bacterial species: Lactobacillus spp. Firm-
4, Lactobacillus spp. Firm-5 (phylum Firmicutes), Bifidobacte-
rium spp. (phylum Actinobacteria), Snodgrassella alvi,Gilliamella
apicola,Frischella perrara,Bartonella apis, and Alpha 2.1 (phylum
Proteobacteria) (18). Each of these species exhibits strain di-
versity corresponding to differences in metabolic capabilities and
tolerances to xenobiotics (19, 20). Newly emerged workers (NEWs)
are nearly free of gut bacteria and acquire their normal microbial
community orally through social interactions with other workers
during the first few days after emergence (21). Bees deprived of
their normal microbiota show reduced weight gain and altered
metabolism (22), increased pathogen susceptibility (17), and in-
creased mortality within hives (23).
In this study, we investigated the effects of glyphosate expo-
sure on the size and composition of the honey bee gut micro-
biome. We found the microbiome was affected by glyphosate
exposure during and after gut colonization, and that glyphosate
exposure during early gut colonization increased mortality of
bees exposed to an opportunistic pathogen. Additionally, bee gut
bacteria differ in glyphosate susceptibility. We explored the
molecular mechanisms of this variability in glyphosate tolerance
by expressing the EPSPS of bee gut symbionts in E. coli. Some
bee gut bacteria tolerate glyphosate by virtue of a class II EPSPS,
but a few strains with susceptible class I EPSPS depend on other,
Significance
Increased mortality of honey bee colonies has been attributed
to several factors but is not fully understood. The herbicide
glyphosate is expected to be innocuous to animals, including
bees, because it targets an enzyme only found in plants and
microorganisms. However, bees rely on a specialized gut
microbiota that benefits growth and provides defense against
pathogens. Most bee gut bacteria contain the enzyme targeted
by glyphosate, but vary in whether they possess susceptible
versions and, correspondingly, in tolerance to glyphosate. Ex-
posing bees to glyphosate alters the bee gut community and
increases susceptibility to infection by opportunistic patho-
gens. Understanding how glyphosate impacts bee gut symbi-
onts and bee health will help elucidate a possible role of this
chemical in colony decline.
Author contribu tions: E.V.S.M. and N.A.M. des igned research; E.V.S.M. an d K.R. per-
formed research; E.V.S.M., K.R., and N.A.M. analyzed data; and E.V.S.M. , K.R., and
N.A.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This open access article is distributed under Creative Commons Attribution-NonCommercial-
NoDeriv atives Licen se 4.0 (CC BY-NC- ND).
Data deposition: All new sequence data are available on NCBI BioProject (accession nos.
PRJNA432210 and PRJNA480015).
1
To whom corresp ondence may be addressed . Email: erickvsm@utex as.edu or nancy.
moran@austin.utexas.edu.
2
Present address: Department of Biology, University of North Carolina at Greensboro,
Greensboro, NC 27403.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1803880115/-/DCSupplemental.
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yet unknown, mechanisms for tolerance. Overall, our results
show that glyphosate exposure can perturb the gut microbiota of
honey bees, and that compositional shifts typically favor species
tolerant to glyphosate and disfavor sensitive species.
Results and Discussion
Glyphosate Perturbs the Honey Bee Gut Bacterial Community. Hun-
dreds of adult worker bees were collected from a single hive,
treated with either 5 mg/L glyphosate (G-5), 10 mg/L glyphosate
(G-10) or sterile sucrose syrup (control) for 5 d, and returned to
their original hive. Bees were marked on the thorax with paint to
make them distinguishable in the hive. Glyphosate concentra-
tions were chosen to mimic environmental levels, which typically
range between 1.4 and 7.6 mg/L (24), and may be encountered by
bees foraging at flowering weeds. To determine the effects of
glyphosate on the size and composition of the gut microbiome,
15 bees were sampled from each group before reintroduction to
the hive (day 0) and postreintroduction (day 3), and relative and
absolute abundances of gut bacteria were assessed using deep
amplicon sequencing of the V4 region of the bacterial 16S rRNA
gene and quantitative PCR (qPCR).
At day 0, glyphosate exposure had little effect on the bee gut
microbiome size, but the absolute and relative abundances of the
core species, S. alvi, were significantly lower in the G-10 group (Fig.
1andSI Appendix,Fig.S1). The effects of glyphosate exposure on
thebeegutmicrobiomeweremoreprominentatday3,aftertreated
bees were returned to the hive. The total number of gut bacteria
decreased for both treatment groups, relative to control, but this
drop was significant only for the G-5 group, which also exhibited
more severe compositional shifts (Fig. 1). The absolute abundances
of four dominant gut bacteria, S. alvi,Bifidobacterium,Lactobacillus
Firm-4 and Firm-5 were decreased (Fig. 1), and the relative abun-
dance of G. apicola increasedintheG-5group(SI Appendix,Fig.
S1). Surprisingly, only Lactobacillus Firm-5 decreased in absolute
abundance in the G-10 group (Fig. 1). This experiment was repeated
using bees from a different hive and season, and similar trends were
observed (SI Appendix,Fig.S2). As in the first experiment, signifi-
cant reductions in abundancewereobservedforS. alvi in bees
treated with glyphosate (SI Appendix,Fig.S2).
The relative lack of effects of the G-10 treatment on the
microbiota composition at day 3 posttreatment is unexplained, but
may reflect other effects of glyphosate on bees. Our recapture
method fails to sample bees that died or abandoned the hive.
Since bees exposed to glyphosate may exhibit impaired spatial
processing, compromising their return to hives (10, 24), bees in the
G-10 group that consumed more glyphosate-laced sugar syrup
before reintroduction to the hive may have been less likely to
return to the hive after foraging. Since fewer than 20% of bees
Lactobacillus Firm-5 Lactobacillus Firm-4 Gilliamella apicola Snodgrassella alvi Bifidobacterium Frischella perrara Bartonella apis
Alpha 2.1 Other Lactobacillus Fructobacillus Klebsiella Serratia Escherichia
0.0
2.4e+6
4.8e+6
7.2e+6
9.6e+6
1.2e+7
**
0.0
8.0e+6
1.6e+7
2.4e+7
3.2e+7
4.0e+7
*
0.0
1.6e+6
3.2e+6
4.8e+6
6.4e+6
8.0e+6
0.0
2.0e+6
4.0e+6
6.0e+6
8.0e+6
1.0e+7
*
*
0.0
2.0e+6
4.0e+6
6.0e+6
8.0e+6
1.0e+7 *
0.0
1.6e+6
3.2e+6
4.8e+6
6.4e+6
8.0e+6
0.0
1.2e+6
2.4e+6
3.6e+6
4.8e+6
6.0e+6
1.35e+8
0.0
2.25e+7
4.50e+7
6.75e+7
9.00e+7
0.0
2.25e+7
4.50e+7
6.75e+7
9.00e+7
0.0
2.25e+7
4.50e+7
6.75e+7
9.00e+7
Day 3Day 0 Day 3Day 0
Relative abundance Absolute abundance
Control
5 mg/L
Glyphosate
10 mg/L
Glyphosate
16S rDNA copies16S rDNA copies
A
B
Gilliamella apicola
Snodgrassella alvi Bifidobacterium Frischella perrara Bartonella apis
Lactobacillus Firm-5 Lactobacillus Firm-4
0%
25%
50%
75%
100%
0%
25%
50%
75%
100%
0%
25%
50%
75%
100%
0.0
1.8e+7
3.6e+7
5.4e+7
7.2e+7
9.0e+7
Total bacteria
**
1.
35
e+
8
Day 0 Day 3
C
G-5
G-10
C
G-5
G-10
Day 0 Day 3
C
G-5
G-10
C
G-5
G-10
Day 0 Day 3
C
G-5
G-10
C
G-5
G-10
Day 0 Day 3
C
G-5
G-10
C
G-5
G-10
Day 0 Day 3
C
G-5
G-10
C
G-5
G-10
Day 0 Day 3
C
G-5
G-10
C
G-5
G-10
Day 0 Day 3
C
G-5
G-10
C
G-5
G-10
Day 0 Day 3
C
G-5
G-10
C
G-5
G-10
Fig. 1. Changes in gut microbiota composition following glyphosate exposure of honey bees with established gut communities. (A) Stacked column graph
showing the relative and absolute abundances of gut bacterial species in control bees and bees treated with 5 mg/L or 10 mg/L glyphosate at
posttreatment days 0 and 3. Each column represents one bee. (B) Boxplots of bacterial 16S rDNA copies for control (C) and glyphosate-treated (G-5 and G-10)
bees at posttreatment days 0 and 3 (n=15 for each group and time point). Box-and-whisker plots show high, low, and median values, with lower and upper
edges of each box denoting first and third quartiles, respectively. *P<0.05 and **P<0.01, Wilcoxon rank sum test followed by Bonferroni correction.
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reintroduced to the hive were recovered, recovered bees may not
represent the total effect of glyphosate on treatment groups.
Glyphosate Affects Early Gut Bacterial Colonization. Glyphosate ar-
rests bacterial growth without directly killing the cells, so we
hypothesized that it would have a greater effect on actively di-
viding cells present during early gut colonization. To test this,
NEWs, which are nearly free of gut bacteria (21), were simul-
taneously exposed to an inoculum consisting of their normal
microbial community and to glyphosate. This simultaneous ex-
posure is relevant to field situations, since glyphosate has been
detected in hives and honey samples (25, 26), indicating that
honey bee foragers can transport residues of this herbicide to the
colony and contaminate other bees, including NEWs, and food
resources. Also, glyphosate is a stable, water-soluble chemical
that can persist in the environment for long periods (10).
Assessment of gut microbiomes, as described in the previous
section, identified all eight core gut taxa in both control and
treatment groups (Fig. 2A), showing that glyphosate does not
eliminate colonization by any core member. Average total bacte-
rial abundance was slightly lower in glyphosate-treated bees, but
this was not statistically significant (Fig. 2B). S. alvi was the most
strongly affected member of the gut microbiota and decreased in
both absolute and relative abundance, while Lactobacillus Firm-4
increased in relative abundance (Fig. 2 C–Eand SI Appendix,Fig.
S3). Based on relative abundance, gut community compositions of
glyphosate-treated bees differed from those of controls (principal
coordinate analysis of weighted UniFrac) (27), permanova test
with 9,999 permuations; P=0.0078, pseudo-F statistic =6.66)
(Fig. 2F). Thus, glyphosate exposure during early development of
the gut community can interfere with normal colonization by al-
tering the abundance of beneficial bacterial species.
Typically, captive honey bees do not defecate, and dead bac-
terial cells and the released DNA accumulate in the gut (23).
Thus, we also analyzed changes in bacterial abundance after
glyphosate exposure by extracting both DNA and RNA from the
guts of treatment and control bees in a second colonization ex-
periment. We included a positive control group, in which bees
were exposed to tylosin, an antibiotic used in beekeeping. This
antibiotic treatment was expected to perturb the microbiota, but
the decrease was significant only for RNA samples (SI Appendix,
Fig. S4). Glyphosate exposure resulted in nonsignificant decreases
in total bacteria for both DNA and RNA assays. We also checked
changes in absolute abundance for three core bacterial species,
S. alvi,Lactobacillus Firm-4, and Lactobacillus Firm-5. Tylosin
treatment resulted in reductions for 16S rRNA copies (SI Ap-
pendix,Fig.S4). Effects of glyphosate treatment were specific to
S. alvi, which was the only assayed species showing significant
reductions in absolute abundance, observed for both DNA and
RNA assays (SI Appendix, Fig. S4). This experiment suggests that
measures based on DNA are partly obscured by DNA from dead
bacterial cells, although this effect does not entirely mask shifts in
bacterial abundance.
Glyphosate Exposure Makes Young Worker Bees More Susceptible to
Serratia.To determine whether glyphosate-induced perturbation
of microbiota colonization affects host health, we measured the
susceptibility of glyphosate-treated bees to an opportunistic
bacterial pathogen. NEWs were exposed to glyphosate in the
stage of acquiring their normal microbial community. After 5
d of treatment, bees were challenged with Serratia marcescens
kz19, an opportunistic pathogen commonly detected at very low
frequencies in the bee gut (28, 29).
For bees lacking gut microbiota, Serratia challenge resulted in
increased mortality relative to that observed for bees with a con-
ventional gut microbiota, regardless of glyphosate exposure (Fig.
2Gand SI Appendix,Fig.S5). For bees with a conventional gut
microbiota, glyphosate treatment resulted in increased mortality
after Serratia challenge. To determine whether this increased
mortality was attributable to the effects of glyphosate on the gut
microbiota or to direct effects of glyphosate on bees, we included
control groups not challenged with Serratia.Inbeesexposedto
glyphosate, but not challenged with Serratia, survival rates were
not significantly affected by glyphosate and were much higher than
in the Serratia-challenged groups (Fig. 2G), demonstrating that a
direct effect of glyphosate on bees is not the basis of the high
mortality of glyphosate-exposed, pathogen-challenged bees.
Our results show that glyphosate reduces the protective effect
of the gut microbiota against opportunistic pathogens and that S.
alvi is the bacterial species most negatively affected by glyphosate
exposure. By itself, S. alvi appears to give some protection, but
not as much as the whole gut microbiota (SI Appendix, Fig. S6).
S. alvi forms a biofim on the wall of the gut ileum (18, 21, 30),
which may function as a mechanical barrier against pathogen
invasion. Some S. alvi strains encode type VI secretion systems
(31), which could contribute to colonization resistance through
contact-dependent inhibition of Serratia. Furthermore, host ex-
pression of antimicrobial peptides is upregulated after S. alvi
colonization (32), which could increase resistance to infection by
pathogens. Besides a direct protective effect, S. alvi may be
critical in enabling the full microbiota to assemble, thus enabling
greater protection.
Relative
abundance
Glyphosate
Control
PCoA - Weighted Unifrac
PC1 (72.01%)
PC2 (11.63%)
ate
A
CEB
Absolute
abundance
Lactobacillus Firm-5 Lactobacillus Firm-4 Gilliamella apicola Snodgrassella alvi
Bifidobacterium Frischella perrara Bartonella apis Alpha 2.1
0.06
0.04
0.02
0.00
-0.02
-0.04
-0.06 -0.1 0.0 0.1
F
D
C G
0
9
18
27
36
45
***
CG
C G
0
5
10
15
20
25
**
16S rDNA copies
% abundance
% abundance
16S rDNA copies
etasohpylGlortnoC
0
25%
50%
75%
100%
0.0
3.0e+7
6.0e+7
9.0e+7
1.2e+8
S. alvi Firm-4S. alvi
Day 0
Serratia challenge
Percent survival
***
G
012345678
0
20
40
60
80
100
Days
CG
0.0
3.0e+7
6.0e+7
9.0e+7
1.2e+8 Tot al
0.0
6.0e+6
1.2e+7
1.8e+7
2.4e+7
**
G
MF+Ser
MF+Gly+Ser
MF
MF+Gly
GH
GH+Gly
GH+Ser
GH+Gly+Se
r
***
Day 0 Day 0 Day 0
Fig. 2. Changes in gut microbiota composition following glyphosate expo-
sure of young honey bees and susceptibility to Serratia infection. (A)Stacked
column graph showing the relative and absolute abundances of gut bacterial
species in control and glyphosate-treated bees. Each column represents one
bee. (B–E) Boxplots of total bacterial 16S rDNA copies and of absolute and
relative abundances of two gut bacterial species for control (n=14) and
glyphosate-treated (n=11) bees. **P<0.01, and ***P<0.001, Wilcoxon rank
sum test followed by Bonferroni correction. (F) Principal coordinate analysis of
gut community composition using weighted UniFrac (permanova test with
9,999 permuations; P=0.0078, pseudo-F statistic =6.66). (G)Thepercent
survival of age-controlled bees after Serratia kz19 exposure, shown as a
Kaplan–Meier survival c urve. ***P<0.001, coxph model implemented in the
“survival”package in R. GH, gut homogenate-exposed bees; Gly, glyphosate
treatment; MF, microbiota-free bees; Ser, Serratia challenge.
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The Bee Gut Contains Bacterial Species with both Sensitive and
Insensitive Types of EPSPS. Bacterial EPSPS exists as two main
types, corresponding to two phylogenetic clusters, that differ in
sensitivity to glyphosate: Class I is naturally sensitive, whereas
class II is insensitive (8). To identify the EPSPS types present in
the bee gut microbiota, a phylogenetic tree was constructed using
the EPSPS protein of bacterial strains isolated from honey bee
and bumble bee guts and of other representative organisms (Fig.
3). EPSPS sequences from S. alvi,G. apicola,F. perrara,Bifido-
bacterium, and Apibacter adventoris (phylum Bacteroidetes) (33)
clustered with those from other organisms containing a class I
EPSPS, and thus these bacteria are predicted to be sensitive to
glyphosate. In contrast, sequences from B. apis and Lactobacillus
Firm-4 clustered with other bacteria containing a class II EPSPS,
as did Parasaccharibacter apium (Alpha 2.2), a bacterium com-
monly detected in honey bee larvae and hives, but rare in the
guts of workers (34), and Paenibacillus larvae, the agent of
American foulbrood in honey bee larvae (35); these bacteria are
predicted to be unaffected by glyphosate exposure. Lactobacillus
Firm-5 strains for which genomes are available lack the gene
encoding EPSPS and were excluded from our analysis.
Bee Gut Bacteria Vary in Glyphosate Sensitivity at the Species and
Strain Levels. Several bee gut-associated bacterial strains isolated
from honey bees and bumble bees were cultured in vitro in the
presence or absence of a high dose of glyphosate. Most S. alvi
and G. apicola strains tested, which contain a class I EPSPS,
either do not grow or have a delay in growth when cultured in the
presence of glyphosate; no such effect is observed for strains
containing a class II EPSPS, Lactobacillus Firm-4 and B. apis
(Fig. 3 and SI Appendix, Fig. S7). However, S. alvi strains wkB2
and wkB298, despite containing a class I EPSPS, grow as well in
the presence of glyphosate as in its absence, with no initial delay
in growth. We looked for potential single-site mutations in the
EPSPS active site of these strains, which is known to confer
tolerance to glyphosate (36), but no mutations were observed,
indicating that the resistance in these S. alvi strains results from
other mechanisms.
A previous study of the genes required by S. alvi to live in the
bee hindgut showed that the aromatic amino acid biosynthetic
pathway is required for growth in this niche (30), which is con-
sistent with low aromatic amino acid concentrations in the
hindgut (37). Thus, bee gut bacterial strains having a glyphosate-
susceptible EPSPS are predicted to drop in abundance following
exposure, as observed for S. alvi (Fig. 1 and SI Appendix, Fig. S2)
and Bifidobacterium (Fig. 1) in the hive experiments. Lactoba-
cillus Firm-4, which encodes a class II EPSPS, and Firm-5, which
does not contain the target enzyme of glyphosate, also had their
abundances reduced in the hive experiment (Fig. 1), which was
not expected. This may be explained by the fact that these strains
lack the aromatic amino acid biosynthetic pathway (18), relying
on uptake of aromatic amino acids released by other bacterial
species, such as S. alvi, in the bee gut environment. The increase
in G. apicola relative abundance (SI Appendix, Fig. S1) was
unpredicted, but was also observed in a previous study on mi-
crobial community responses to antibiotic perturbation (23).
Glyphosate Resistance Is Independent of EPSPS Class in Some Bee Gut
Strains. To understand the mechanism that prevents some bee
gut bacterial strains from growing in the presence of glyphosate,
we complemented E. coli ΔaroA with aroA genes cloned from bee
gut bacterial strains as well as with the E. coli K12 aroA,whichis
known to be sensitive to glyphosate. E. coli ΔaroA cannot syn-
thesize aromatic amino acids and does not grow in minimal media,
but grows normally when transformed with an arabinose-inducible
plasmid carrying the intact E. coli aroA gene (Fig. 4).
Transformants carrying the aroA gene from S. alvi,G. apicola,
and B. apis were able to grow in minimal media at a similar rate to
the transformant carrying the aroA gene from E. coli (Fig. 4). The
addition of 10 mM glyphosate to the media resulted in a delay in
growth of ∼48–72 h for transformants carrying the aroA gene from
all S. alvi and G. apicola strains tested (Fig. 4). This is expected if
glyphosate binds to a susceptible EPSPS, blocking the shikimate
pathway and preventing bacterial growth until the concentration
of PEP or EPSPS exceeds that of glyphosate, allowing the trans-
formants to resume growth. On the other hand, the transformant
carrying the aroA gene from B. apis did not exhibit the growth
delay in the presence of glyphosate (Fig. 4), as predicted since this
aroA version encodes an insensitive class II EPSPS. Moreover, the
addition of increased concentrations of arabinose in the media or
reduction in glyphosate concentration sped up the growth of all
transformant strains (SI Appendix, Fig. S8), which corroborates
the reversible mechanism of EPSPS inhibition by glyphosate.
Although S. alvi strains wkB2 and wkB298 were resistant to
glyphosate (Fig. 3 and SI Appendix, Fig. S7), their aroA versions
were sensitive to glyphosate (Fig. 4). Potentially, some bee gut
microbes may have evolved alternative glyphosate resistance
mechanisms due to a history of exposure, similar to the re-
sistance observed for the antibiotic tetracycline used in bee-
keeping (38). Therefore, we overexpressed, in WT E. coli, certain
genes encoding transporters from some bee gut bacteria,
including wkB2 and wkB298 strains, that could be involved in
Snodgrassella alvi wkB298
Snodgrassella alvi Gris1-6
Gilliamella apicola wkB30
Gilliamella apicola wkB72
Snodgrssella alvi wkB2
Lactobacillus Firm-4 BI-2.5
Lactobacillus mellis Hon2
Gilliamella apicola App2-1
Gilliamella apicola Bim3-2
Bifidobacterium sp. wkB344
Gilliamella apicola wkB1
Snodgrassella alvi Occ4-2
Snodgrassella alvi wkB9
Bifidobacterium sp. Bin2
Bartonella apis PEB0150
Frischella perrara PEB0191
Uncultured microbe AM79
Bartonella apis BBC0178
Lactobacillus Firm-4 LV8-1
Snodgrassella alvi Fer1-2
Lactobacillus Firm-4 BI-1.1
Zea mays CAA44974
Pseudomonas putida
Snodgrassella alvi wkB273
Gilliamella apicola App4-10
Gilliamella apicola M6-3G
Nicotiana tabacum P23981
Bifidobacterium sp. wkB338
Escherichia coli K-12
Snodgrassella alvi Nev4-2
Oryza sativa AAL07437
Pseudomonas fluorescens G2
Apibacter adventoris wkB180
Snodgrassella alvi WF3-3
Snodgrassella alvi wkB332
Parasaccharibacter apium MRS3-2
Streptococcus pneumoniae
Bartonela apis wkB233A
Gilliamella apicola wkB308
Bacillus subtilis subsp. subtilis
Halovibrio variabilis
Petunia hybrida P11043
Paenibacillus larvae
Apibacter adventoris wkB301
Bartonella apis PEB0122
Bartonella apis PEB0149
Apibacter adventoris wkB309
Staphylococcus aureus subsp. aureus
Snodgrassella alvi App6-4
Gilliamella apicola wkB112
Bartonella apis BBC0122
Arabidopsis thaliana P05466
Ochrobactrum anthropi
Snodgrassella alvi wkB339
Parasaccharibacter apium G7-7-3C
Gilliamella apicola Nev6-6
Lactobacillus sp. Bim4-9
BV < 70 BV 70-90 BV > 90 0.4
Class I EPSPS
Class II EPSPS
us
24h 36h 48h
InsectaGro MRS
24h 36h 48h
01
OD treated/untreated
Fig. 3. Maximum-likelihood phylogeny based on amino acid sequences of
EPSPS (PhyML 3.1, LG model +Gamma4, 100 bootstrap replicates). Bee gut
bacterial strains, other bacteria, and some plant species are represented in
the phylogeny. The heatmap represents the growth of some bee bacterial
strains in the presence/absence of 10 mM glyphosate at three time points
(24, 36, and 48 h). Glyphosate was dissolved in the culture media (InsectaGro
or MRS broth, based on bacterial preferences). A value of 1 indicates that
growth is the same in the presence or absence of glyphosate.
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glyphosate resistance: yhhS, which encodes a membrane trans-
porter conferring glyphosate tolerance when overexpressed in
E. coli (39), and tetC, which encodes an efflux pump that pro-
vides tetracycline resistance to S. alvi wkB2 (38). However, these
transporters were not able to reverse the delay in E. coli growth
caused by glyphosate (SI Appendix, Fig. S9). As the glyphosate
tolerance exhibited by some S. alvi strains does not appear to be
due to a resistant EPSPS or to transport by YhhS or TetC, these
strains are likely to employ a novel mechanism of glyphosate
resistance. Future studies might identify this mechanism and
determine the evolutionary origin of resistance.
S. alvi Strains May Vary in Sensitivity to Glyphosate in Vivo. Our
phylogenetic analysis and cloning experiments demonstrated
that, despite displaying variable susceptibility to glyphosate
in vitro, all S. alvi strains possess a glyphosate-sensitive class I
EPSPS. Therefore, we investigated whether this variation in
susceptibility occurs in vivo. NEWs were monoinoculated with
two different S. alvi strains: wkB2, which grows in the presence of
high concentrations of glyphosate, or wkB339, which exhibits a
delay in growth in the presence of high concentrations of
glyphosate (Fig. 5A). Bees were hand fed bacterial suspensions
to inoculate with a control number of S. alvi cells, exposed to
glyphosate for 3 d, and sampled at days 1 and 3 during exposure.
Both S. alvi wkB2 and S. alvi wkB339 increased in abundance
between days 1 and 3 in unexposed bees, consistent with previous
findings that S. alvi can colonize guts of monoinoculated bees
(30, 40). Based on qPCR estimates of S. alvi abundance on day 3,
glyphosate exposure had a negative effect on growth of both
strains [two-way analysis of variance (ANOVA), treatment ef-
fect, P<0.0001]. S. alvi wkB339 was more affected by glyphosate
exposure, based on significant interaction between strain and
treatment (two-way ANOVA, P<0.0204). Correspondingly, the
absolute abundance of the glyphosate-sensitive strain, wkB339,
was significantly lower in glyphosate-treated bees compared with
controls (Tukey’s test, P<0.001) or wkB2-treated bees (Tukey’s
test, P<0.001) (Fig. 5 Band C). Potentially, strain differences in
glyphosate sensitivity may contribute to the observed variation in
the overall decrease in S. alvi abundance when bees with their
native gut microbiota are exposed to glyphosate.
Conclusion
As in many animals, honey bees rely on their gut microbial
community for a variety of functions, including food processing
(25, 26), regulation of immune system (33, 34), and defense
against pathogens (17, 27). Perturbations of this system have the
potential to lead to negative consequences for host fitness. We
found that glyphosate affects the bee gut microbiota composition
and that bacterial species and strains within this community vary
in susceptibility to glyphosate. Recent experimental and observa-
tional studies have provided evidence that dysbiosis affecting the
bee gut can increase susceptibility to pathogen invasion (23, 41,
42). Our results also suggest that establishment of a normal mi-
crobial community is crucial for protection against opportunistic
pathogens of honey bees. Furthermore, our results highlight one
potential mechanism by which glyphosate affects bee health.
While some species in the bee gut can tolerate high concen-
trations of glyphosate due to the presence of a class II EPSPS
enzyme, others are sensitive due to the presence of a class I
EPSPS. A consistent effect of glyphosate on the bee gut micro-
biota was a negative impact on growth of S. alvi, which possesses a
sensitive EPSPS. However, some strains of S. alvi may tolerate
glyphosate through an as yet unknown mechanism. Since bee gut
symbionts affect bee development, nutrition, and defense against
natural enemies, perturbations of these gut communities may be a
factor making bees more susceptible to environmental stressors
including poor nutrition and pathogens.
Methods
More details are provided in SI Appendix.
Effects of Glyphosate on the Honey Bee Gut Microbiome. Adult workers with
established gut communities were collected from a hive at University of
Texas, Austin (UT Austin), marked on the thorax with paint, fed glyphosate (5
or 10 mg/L) or sterile sucrose syrup for 5 d, and returned to the same hive.
Fifteen bees from each group were sampled before and 3 d after reintro-
duction to the hive. This experiment was repeated using bees from a different
hive and different year. DNA was extracted from dissected guts and used as
template for qPCR analyses. DNA samples from the first experiment were
submitted for Illumina sequencing at the Genomic Sequencing and Analysis
Facility (GSAF) at UT Austin. Illumina sequence reads were processed using
QIIME 1.9.1 (43).
Effects of Glyphosate on Early Gut Colonization and Susceptibility to Serratia
Infection. Hundreds of late-stage pupae were removed from brood frames
and allowed to emerge under sterile conditions in laboratory. (Experiment A)
NEWs were exposed to bee gut homogenate for 5 d, then hand fed 1 mM
glyphosate or sterile sugar syrup on 2 alternate days. Fifteen bees from each
group were sampled 2 d after the last hand feeding. DNA was extracted from
dissected guts, used as template for qPCR analyses, and submitted for Illumina
sequencing at the GSAF, UT Austin. (Experiment B) NEWs were exposed to a
bee gut homogenate or sterile sucrose syrup. Each group was divided into
two subgroups and treated with 0.1 mM glyphosate or sterile sucrose syrup
for 5 d. After that, half of the subgroups was exposed to the opportunistic
pathogen S. marcescens kz19, whereas the other half was used as controls.
No glyphosate
020 40 60 80 100
0.0
0.1
0.2
0.3
0.4
0.5
E. coli aroA (OD600)
Time (h)
K12
wkB2
wkB298
wkB339
Nev4-2
wkB112
wkB233A
aroA gene:
10 mM glyphosate
020 40 60 80 100
0.0
0.1
0.2
0.3
0.4
0.5
Time (h)
E. coli aroA (OD600)
Fig. 4. Growth curves of E. coli ΔaroA BW25113 expressing the aroA gene
from different bee-associated bacterial strains (B. apis in red, E. coli in black,
G. apicola in blue, and S. alvi in green) cultured in minimal media in the
presence or absence of 10 mM glyphosate.
102
103
104
105
106
107
108
109
1010
CCGG
wkB339wkB2
Log10 16S rDNA copies
S. alvi wkB2 S. alvi wkB339
B
A
Control
Glyphosate
0 1020304050
0.0
0.1
0.2
0.3
0.4
0.5
0.6
OD600
Time (h)
0 1020304050
0.0
0.1
0.2
0.3
0.4
0.5
0.6
OD600
Time (h)
***
***
Two-way ANOVA summary
C
Source SS df MS F p
Strain 1.55 1 1.55 5.87 0.0227
Treatment 7.24 1 7.24 27.44 <0.0001
Interaction 1.61 1 1.61 6.1 0.0204
Error 6.86 26 0.26
Tot al 17 .2 6 29
Fig. 5. Variation in S. alvi strain sensitivity to glyphosate. (A) Growth curves
of S. alvi wkB2 and wkB339 cultured in InsectaGro media in the presence or
absence of 10 mM glyphosate. Experiment was performed in triplicate, and
each data point represents the average optical density (600 nm, with SD
bars). (B) Boxplots of S. alvi wkB2 and wkB339 abundances in bees exposed
or not to 0.1 mM glyphosate for 3 d estimated by qPCR. Box-and-whisker
plots show high, low, and median values, with lower and upper edges of
each box denoting first and third quartiles, respectively. ***P<0.001, two-
way ANOVA with Tukey’s correction for multiple comparisons. (C) Two-way
ANOVA for effects of S. alvi strain and glyphosate treatment.
Motta et al. PNAS Latest Articles
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5of6
APPLIED BIOLOGICAL
SCIENCES
Bees were exposed to similar amounts of glyphosate (∼1.7 μg) in experi-
ments A and B.
S. alvi Colonization During Glyphosate Exposure. NEWs were hand fed 5 μL
sucrose syrup containing ∼10
5
cells of S. alvi wkB2 or wkB339 or sterile sucrose
syrup as control. Each group was divided into two subgroups and treated with
0.1 mM glyphosate or sterile sucrose syrup for 3 d immediately following
bacterial exposure. Eight bees were sampled from each subgroup at days 1
and 3, and DNA was extracted from dissected guts. S. alvi-specific primers (44)
were used to amplify total copies of 16S rDNA of each sample by qPCR.
In Vitro Experiments with Bee Gut Bacterial Strains. Honey bee and bumble
bee gut bacterial strains (SI Appendix, Table S1) were cultured in InsectaGro
or MRS broth in the presence or absence of 10 mM glyphosate in a 96-well
plate and incubated in a plate reader at 35 °C and 5% CO
2
for 48 h. Optical
density was measured at 600 nm every 6 h. Experiments were performed in
triplicate.
Plasmid Construction and Transformation. The aroA,yhhS,andtetC genes
from various bacterial strains were PCR amplified and cloned into the
arabinose-inducible pBAD30 vector (45) by Gibson assembly (46) and then
used to transform E. coli strain BW25113 or a derivative lacking the aroA gene
by electroporation. Primer sequences are listed in SI Appendix, Table S2.
Growth Rate Analysis of Transformed E. coli.Transformed E. coli cells were
cultured in duplicate in 24-well plates containing M9 minimal medium (47)
with appropriate antibiotics, varying concentrations of glyphosate, and
varying concentrations of arabinose. The plates were incubated in a plate
reader at 37 °C for 24–96 h. Optical density was measured at 600 nm
every hour.
ACKNOWLEDGMENTS. We thank Eli Powell and Zack Shaffer for technical
assistance, Erik Quandt for cloning advice, Waldan Kwong and Hauke Koch
for providing bee gut bacterial strains, Margaret Steele for constructive
comments, Kim Hammond for maintaining hives, Jeffrey Barrick for pro-
viding E. coli ΔaroA BW25113, and the UT Austin GSAF for sequencing ser-
vices. This work was supported by Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior, Brazil (13578-13-8 to E.V.S.M.), the USDA National
Institute of Food and Agriculture (2017-67012-26088 to K.R. and 2018-67013-
27540 to N.A.M.), and the National Institutes of Health (R01GM108477-02
to N.A.M.).
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