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outline
Bees and their gut microbiota
Experimental approaches for studying bee
gut microbiota
~ 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
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
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
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
Stages of pupae
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/
Experimental
colonization
by S. alvi
Microbiome
Mind Control
Natural social transmission in hive
4 days post exposure
Neuropeptide
product
Brain
Circuits of Orthogonal
Proteases (COPS)
S. alvi
(rRNA FISH)
insect DNA
Broad-host-range
synthetic control
episome (Arkmid)
Engineered function
S. alvi
G. apicola
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
No Exposure or
non-BGM Exposure
+BGM
Exposure
<105 >108
Bacteria per adult gut
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
SpecRp15A
fermentation of
released
Strains with pec nase
genessugars
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
GoldenGate
AssemblyusingBsaI
Leeetal.2015ACSSynthBiol.;Mutaliketal.2013.NatMethods;Jensen&Hammer1990AEM
Bartonella apis
expressing
mRuby in dissected
ileum
Serratia N10A28
(bee-derived)
expressing E2Crimson in guts of
live bees.
aaRS
Trophallaxis
Dopamine
antagonist
diminishes
ability to
associate
a scent with
punishment
Feeding L-DOPA
to bees results in
linear increase in
titres of dopamine
in hemolymph.
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
dopamine
BGM Serratia
N10A28-L-DOPA
Ddc
Bee host
L-DOPA
Slide 24
1
35
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
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)