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The effects of the microbiome

on the behaviors of bees
Nancy A. Moran
The University of Texas at Austin

outline
• Bees and their gut microbiota
• Experimental approaches for studying bee
gut microbiota

• Bees as a behavioral model
• Example experiments on effects of
microbiota on bee behavior

• Issues with using bee microbiota as a model

Apis mellifera (western honey bee)
Large social colonies, dominated by female workers
Most important agricultural pollinator, >$15 billion annually
Problems with colony health in last decade
Distinctive gut microbiota
2015 White House Report

The bee gut microbiota (BGM)

~ 9 bacterial species
form dense,
spatially organized
communities
In hindguts of adult
worker honeybees.

Bartonella apis
Parasacchar apium
Frischella perrara
Snodgrassella alvi
Gilliamella apicola
Bifidobacterium sp.
Lactobacillus Firm4
Lactobacillus Firm5
Other

Crop and Midgut:
few microbes
Ileum

Rectum

Hindgut:
99% of gut microbes
Kwong & Moran Nature Rev Microbiol 2016

Gut microbiota across the life span of the honey bee worker
Stable community of ~109 cells
by day 5 of adulthood

# bacteria per gut

Few bacteria, erratic composition
in larvae and very young adults

Adult microbiota sometimes disrupted
by varied Enterobacteriaceae including insect pathogens
Kwong & Moran Nat Rev Microbiol 2016

Gut Microbiota: Honeybees v Humans
Similarities
 Dominated by specialized coevolved bacterial species that do not
occur in the outside environment or in food, require low oxygen
conditions
 Socially transmitted during early life, mostly within family groups
 High levels of strain diversity within symbiont species
 Mixture of Gram negative and Gram positive bacteria
 Occur in nutrient poor distal gut; can utilize complex plant
polymers
 Dysbiosis (old hosts or following antibiotics) characterized by
Enterobacteriaceae, such as Serratia, Klebsiella, Hafnia, etc.
 Host immune system modulates community composition
 History of antibiotic treatment; presence of resistance in many
strains
Differences
 Only ~9 species comprise >95% of microbiota in healthy bees
versus hundreds of species in human microbiota
 All species can be grown axenically in the laboratory
 Obviously differences in host biology development and immune

Team
Members
Strategy
for
studying
bee
gutgut
symbionts
within
hosts
Strategy for studying defined bee
communities
within
hosts

Moran

Moran

Ellington

Alper

community Davies
Monitor
Barrick
size, composition and
activity within hosts

– bacterial genome evolution, bee gut microbiome

Germ‐free
Inoculate with
Ellington

T7
RNAP,
nucleic acid circuits, protease evolution
Hosts
known strains
(=Newly Emerged
Feed in engineering, promoter development
Alper
– metabolic
Adult Bees)
sucrose
solution or onevolution, genetic stability
Barrick

experimental
Apis mellifera
sterilized
Bombus impatiens
pollen genetics,
Davies
– microbial

on hosts,
Monitor effects
antimicrobial
development
ability to colonize different hosts

Producing germ free honey bees for microbiome experiments
Larvae develop in outdoor hives

Stages of pupae

Pupae are pulled from frames in lab

Pupae in lab dish: kept at 35oC

Gut lining
&
contents
shed

Initially
germ free

Protocols for
in vitro rearing
of bee larvae

http://blogs.ifas.ufl.edu/entnemdept/categor
y/change-category/

Bumble bees: whole colonies can be grown in lab
last numerous months, available commercially

Laboratory Colony of Bombus impatiens

Experimental
colonization
by S. alvi
Microbiome
Mind Control
Natural social transmission in hive
4 days post exposure

Honey bee gut
microbiota (BGM)

Lab-inoculated with S. alvi wkB2
4 days post exposure

Neuropeptide
product

Stability, robustness, safety

Brain

Control honey bee
and hive behavior
W Kwong

Circuits of Orthogonal
Proteases (COPS)

S. alvi
(rRNA FISH)

insect DNA

Broad-host-range
synthetic control
episome (Arkmid)

Engineered function

Ileum cross section: experimental coinfection with S. alvi and G. apicola

S. alvi

G. apicola

bee cell nuclei

bee cytoplasm

M. Steele, UT Austin

Team
Members
Strategy
for
studying
bee
gut symbionts
within
hosts
Strategy for studying conventionalized
gut versus
‘germ-free’
bees

Moran

Ellington

Alper

community
Monitor
Conventionalized
gut Davies
Barrick
microbiota,
indistinguishable
size,
composition
and
from
thatwithin
of hive
bees
activity
hosts

Inoculate with
Moran
– bacterial
genome evolution, bee gut microbiome
homogenized
hind gutswith
of
Germ‐free
Inoculate
Ellington
– T7 RNAP,
nucleic acid circuits, protease evolution
hive bees
Hosts
known
strains

(=Newly Emerged
Alper
– metabolic engineering, promoter development
Adult Bees)

Barrick
– experimental evolution, genetic stability
Apis mellifera
Compare phenotypes of germ-free

Bombus impatiens
on hosts,
Monitor effects
Davies
– microbial genetics, bees
antimicrobial
development
and bees with conventionalized
ability to colonize different hosts

microbiota

Designer BGM Communities
BGM species can be inoculated
into uncolonized pupae
Bee pupae

No Exposure or
non-BGM Exposure

+BGM
Exposure

<105 >108
Bacteria per adult gut

BGM communities can be
passaged and replicated by
co-housing bees

Experiments on the
Bee Gut Microbiota:
Genomics,
Genetics,
Metabolism,
Antibiotic resistance,
Host gene expression,
Effects on pathogens,

Some G. apicola
strains from Apis
degrade
pollen vectors
cell wall
Pipeline
for production
ofcan
BGM
Arkmid
Bee
Microbiota:
coevolved
community
components,
and can use resul
ng sugars
Golden Gate Assembly: BGM toolkit for modular, multipart assembly
with
functions?
Initially engineered in E. coli, and transferred to BGM species using  the pMMB67EH origin
Pec ncooperative
structure
Promoters + RBS
CP18
CP18‐lacO
CP4
CP4‐lacO
CP44
CP32
CP6
CP7
CP12b
CP25
CP25‐lacO
CP25‐tetO
PA1
PA1‐lacO
PA1‐tetO
PA2
PA3

LacIq
Lac
LacUV5
Lambda PR
T5‐lacO
T7
T7‐tetO
T7‐lacO
recN
araBAD
Ptac
EM7
PPddc

Example of BGM parts for Golden Gate assembly  RED TYPE =
role in digesting
G.polymers
apicola genes
Terminators
plant
from
Markers and Origins
Coding Sequences
for diges ng
ingested
pollen
rpoC
AmpR‐pMMB67EH
Reporters
Enzymes
pec
n
or
using
EOU‐double
KanR‐pMMB67EH
GFP
4‐HPA hydroxylase
ilvGEDA
SpecR‐pMMB67EH
mRuby2
(L‐DOPA production)
resul
ngcellulases
sugars
M13‐rrnD
secreted
Venus
BBa_B0015
mTurquoise2
Homeostasis
T7
E2‐crimson
T7 RNA polymerase
NanoLuc luciferase
RNA interference 
Repressors
GFP dsRNA
LacI
tubulin alpha‐1 dsRNA
TetR
Tyrosine hydroxylase dsRNA
trpR
DOPA decarboxylase dsRNA
cI
Tyrosine decarboxylase dsRNA
Rop
Tyramine beta hydroxylase dsRNA
AraC
dCas9
Bacterial gene disruption
LtrB group II intron
LtrA

SpecR‐p15A

fermentation of
released
Strains with pec nase
genessugars

degrade pec n in culture.

resulting short
chain fatty acids
Apparent HGT from
2
3plant pathogens
available
to host

8

6
7

3
DNA parts

4
1

AmpR‐p15A

and
pectinases
KanR‐p15A

5

2

1

4
Expression
vector

Could be energy source for bees

Golden Gate
Assembly using BsaI

8

5

7

6

P. Engel et al. 2012 PNAS

Lee et al. 2015 ACS Synth Biol. ; Mutalik et al. 2013. Nat Methods; Jensen & Hammer 1990 AEM 

Genetic engineering of Bee Gut Microbiota bacterial species
conjugative plasmids used to introduce genes into BGM
species
pMMB67EH expressing GFP in
Snodgrassella. alvi wkB2 Gilliamella apicola wkB7

Broad host range
plasmid vector
expressing cargo
genes

Visualizing engineered BGM species in live and dead
hosts

Bartonella apis
expressing
mRuby in dissected
ileum
Serratia N10A28
(bee-derived)
expressing E2Crimson in guts of
live bees.

White arrows: B. apis cells
Red arrows:
autofluorescent pollen
grains

Example: Engineering L-DOPA production into bee gut bacteria

aaRS

L-DOPA oxidized to melanin
In engineered E. coli on left

Homeostasis circuitry should allow regulated production

Bees as a behavioral model



Extensive literature on bee behaviors including learning
assays, motility, aggression, sociality, etc
Neurotransmitters and many hormones largely conserved
across worms, insects, mammals
Many behavioral effects documented in bees
Learning, social behavior, aggressivity, gustatory
responses, activity level, buzzing response …

Trophallaxis

Sucrose Sensitivity: Proboscis
extension response

Bees fed dopamine
learn to associate a
color with punishment
faster than controls

Agarwal et al. (2011)

Dopamine
antagonist
diminishes
ability to
associate
a scent with
punishment

Vergoz et al. (2007)

Feeding L-DOPA
to bees results in
linear increase in
titres of dopamine
in hemolymph.

Woodring and Harris (1999)

Previous results on effects of L-DOPA & dopamine in bees

Aversive Training:
Bees learn to associate
scent with punishment

Sting Extension
Observation Interval

Scent
Presented 1

Bee in harness
Nonanol = training odor
Punishment = 7V @ 60Hz for 2 s
Each trial = 1 min (trial plus rests)
Repeat after 10 minutes
Score for Sting Extension Response

Training
Interval

Rest
2

3

4
seconds

5

10 Minute Gap between Trials

Examples of responses to aversive training

1

Example study: Does inoculation with gut symbiont
overproducing L-DOPA improve bee learning?
Previous studies & feeding trials: More dopamine improves aversive learning in bees.
Experiment:
Inoculate bees with BGM Serratia N10 A28 producing either L-DOPA or Rcrimson
(control). Bees with L-DOPA strain expected to have more dopamine in brains

dopamine

BGM Serratia
N10A28-L-DOPA

Ddc
Bee host

L-DOPA

Prediction: Bees with L-DOPAproducing symbiont will learn
faster and remember better.

Slide 24
1

Nancy Moran, 10/30/2016

Does inoculation with gut symbiont
overproducing L-DOPA improve learning?

3-5 days colonization
TESTING
8 replicate groups of
5 bees per condition
6 trials
1 h later:
Bees responding at
any trial tested for
recall

35

*p<0.05, Fisher’s Exact

Sting Extension Response

1d old bees
Fed Serratia N10A28
-E2-Crimson control
-pBTK587 (L-DOPA)

Responses of Trained
Bees Tested 1h later

30

*

*

25

20

68%
38%

15

10

5

0
pBTK587
L-DOPA

* p<0.05, Kruskal-Wallis

E2c
Control

Responding

Not Responding
Preliminary results:
L-DOPA symbiont improves learning and memory

Ongoing studies of engineered BGM and
behavior

Hunger,
proboscis extension

Aversive learning

Aggression, stinging,
cohort alert
ADH2, KivD

HpaB, HpaC

ATF1/ATF2

delivery via
universal protein
secretion system

Insulin

L-DOPA

Isopentyl acetate

(hormone)

(neurotransmitter)

(alarm pheromone)

Animal Welfare in the context of research on bees
As an invertebrate, currently not covered under IACUC policies or any laws.
Nonetheless, we strive to avoid procedures that might result in prolonged
suffering of bees.
At the end of experiments, bees are killed by freezing; they die within 2
minutes.
Many researchers study bees as a domestic, agriculturally important species.

Challenges encountered working with bees as models
Honey bees have complex social lives within large colonies. Studying them
in the lab environment, outside the context of the colony, is highly artificial.
Have not yet been able to establish germ-free colonies: typical colonies
have >30,000 workers and are difficult to raise indoors.
Numerous sources of variation need to be controlled: bee behavior varies
genetically and with age.
Despite homologies in endocrine systems, immune systems, and nervous
systems, obviously, many aspects of human biology do not apply to bees.

Thanks to the Entire Bee Team
Andy Ellington
Jeffrey Barrick
Sean Leonard
Eli Powell
Margaret Steele
Waldan Kwong
Philipp Engel
Aaron Saleen
Bryan Davies
Shaunak Kar
Jiri Perutka
Dennis Mishler
Kim Hammond