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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

Germfree
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
Germfree
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
GoldenGateAssembly:BGMtoolkitformodular,multipartassembly
with
functions?
InitiallyengineeredinE.coli,andtransferredtoBGMspeciesusingthepMMB67EHorigin
Pec ncooperative
structure
Promoters+RBS
CP18
CP18lacO
CP4
CP4lacO
CP44
CP32
CP6
CP7
CP12b
CP25
CP25lacO
CP25tetO
PA1
PA1lacO
PA1tetO
PA2
PA3

LacIq
Lac
LacUV5
LambdaPR
T5lacO
T7
T7tetO
T7lacO
recN
araBAD
Ptac
EM7
PPddc

ExampleofBGMpartsforGoldenGateassembly RED TYPE =


role in digesting
G.polymers
apicola genes
Terminators
plant
from
MarkersandOrigins
CodingSequences
for diges ng
ingested
pollen
rpoC
AmpRpMMB67EH
Reporters
Enzymes
pec
n
or
using
EOUdouble
KanRpMMB67EH
GFP
4HPAhydroxylase
ilvGEDA
SpecRpMMB67EH
mRuby2
(LDOPAproduction)
resul
ngcellulases
sugars
M13rrnD
secreted
Venus
BBa_B0015
mTurquoise2
Homeostasis
T7
E2crimson
T7RNApolymerase
NanoLucluciferase
RNAinterference
Repressors
GFPdsRNA
LacI
tubulinalpha1dsRNA
TetR
TyrosinehydroxylasedsRNA
trpR
DOPAdecarboxylasedsRNA
cI
TyrosinedecarboxylasedsRNA
Rop
TyraminebetahydroxylasedsRNA
AraC
dCas9
Bacterialgenedisruption
LtrBgroupIIintron
LtrA

SpecRp15A

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

6
7

3
DNAparts

4
1

AmpRp15A

and
pectinases
KanRp15A

4
Expression
vector

Could be energy source for bees

GoldenGate
AssemblyusingBsaI

P. Engel et al. 2012 PNAS

Leeetal.2015ACSSynthBiol.;Mutaliketal.2013.NatMethods;Jensen&Hammer1990AEM

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

4
seconds

10 Minute Gap between Trials

Examples of responses to aversive training

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, Fishers 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

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