Transcriptional Sensitivity of Southern Ocean Plankton Communities
to Changes in Temperature and Micronutrient Availability
Andrew Allen
Erin Bertrand
John McCrow
Hong Zheng
Ahmed Moustafa
Jeff McQuaid
SIO and JCVI
David Hutchins
Kai Xu
Nathan Walworth
USC
Deborah Bronk
Rachel Sipler
Jenna Spackeen
VIMS
Photo: Jeff McQuaid
Mak Saito
Dawn Moran
Abigail Noble
WHOI
Change and Projected Change in the Ross Sea
Sea Surface Temperature:
• Slight negative SST trend over past ~20-30 years; expected to continue 2-3 decades
(Mayewski et al 2009, Comiso et al 2000, Lebedev 2007)
• Then expected to warm substantially by the end of the next century (Bracegirdle
and Stephenson, 2012); specific projections difficult (Smith et al 2014)
Iron:
• Relative magnitude of sources still unclear
• Shelf sediments, dust, sea ice
• Result: change is likely but difficult to predict- direction and magnitude
(Smith et al 2012, 2014)
Change is coming: understand diversity and transcriptional
baselines, response of existing communities
Photos: Jeff McQuaid, Dawn Moran
Ice
Jan
18S rRNA analysis of Spring and
Summer water column and sea ice
McMurdo Sound communities
Nov
Alveolata
Haptophyta
Cryptophyta
Chlorophyta
10%
1%
0.1%
Opisthokonta
Diatoms
Dawn Moran, Jeff McQuaid, Greg Wanger
Saito Lab, WHOI
Ahmed Moustafa, JCVI, AUC Egypt
Ice
Jan
CFB
Nov
16S rRNA analysis
of Spring and
Summer water
column McMurdo
Sound
communities
Alphaproteobacteria
2 µm
Sea Ice diatom and bacteria
(Greg Wanger)
Gammaproteobacteria
Saito Lab, WHOI
Ahmed Moustafa, JCVI, AUC Egypt
Question: What is the transcriptional response of
McMurdo Sound phytoplankton and bacterial
communities to changes in temperature, iron, and
vitamin B12 availability?
Approach: Manipulative experiments, late summer
McMurdo Sound of the Ross Sea, Jan 2013
Photos: Erin Bertrand, Jeff McQuaid
McMurdo Sound of the Ross Sea, late austral summer 2012/13
Ross Island
MODIS
16 Jan 2013: Collect 1000 L trace metal clean
seawater; return via helicopter to McMurdo
Station, Crary Lab
Three types of manipulative experiments
• Short term batch- manipulate Fe, B12
• Semicontinuous dilution- manipulate temperature, Fe
• Continuous flow- “ecostats”- manipulate temperature, Fe, CO2
Fe and B12 Dynamics in the Ross Sea
2 of 4 previous bottle experiments in the Ross Sea
showed B12 limitation of phytoplankton growth
• secondary to Fe limitation
Function of initial community composition?
• Bacterial abundance
• Phytoplankton community
MetE MetH
Summer
Spring
MetE
Bertrand et al 2007, 2011
Cyanobacteria
MetE
MetE
MetH
B12
MetH
MetH
Eukaryotic
phytoplankton
MetH
Heterotrophic bacteria
and archaea
Photodegradation
Short term bioassays: B12 and iron
Characterize transcriptional response of initial community to micronutrient additions at 0°C
• 1 nM FeCl3, 200 pM B12
Control
+B12
Incubate 0°C, 24h
• RNA samples
• Nitrate, bicarbonate
uptake (Bronk Lab)
• Chla measure
After 96h, Chla measure
+Fe
Harvest trace metal clean
seawater, return to Crary
Lab
Photos: Jen Erxleben
+B12Fe
Fill 2.7 L bottles
and supplement
Bertrand et al in prep
1.6
0h
24 h
-1
Chl a (g L )
1.4
96 h
*
*
*
1.2
1.0
0.8
Chlorophyll a at 96h:
community was independently
B12 and Fe limited
0.6
0.4
0.2
Cont +Fe +B12 +B12Fe
0.0012
0.0010
*
0.0008
*
0.0006
0.0004
At 24h: Nitrate uptake rate increased in +Fe
treatments
No change in chlorophyll a or primary
productivity
0.0002
0.0000
0.20
-1 -1
HCO3 (mol C L h )
-1 -1
NO3 (mol N L h )
0.0
0.0014
Sequence community RNA after 24 hours
rRNA removal to enrich mRNA
target bacteria and phytoplankton
0.15
0.10
0.05
0.00
t=0
Cont +Fe +B12 +B12Fe
Bertrand et al in prep
Metatranscriptome analysis pipeline
Sample collection
200-2000 mL
filtered onto
0.2 um cartridge
filters
RNA extraction
Library prep
Sequencing
and cleanup plus
rRNA removal
NuGen cDNA
synthesis and
Truseq library
preparation
Paired end Illumina
Hiseq 2000
Targets Bacteria, Archaea, Eukaryotic nuclear and organelle mRNA
Read Filtering
Filtering of primers,
adapters, rRNA;
quality trimming
Assembly
Combined assembly
by merging in
stages
(CLC de novo)
ORF Calling
Prediction of ORFs
on assembled
contigs and further
filtering of rRNA
PhyloDB:
• 25 million peptide sequences
• Does not rely on blast hit for functional annotation
• MMETSP transcriptome sequencing included
PhyloDB Annotation
(1)Taxonomy via best blast hit
(2) functional domains
(PFams/TIGRFams), KEGG,
KOG, GO, EC, pathway,
transporter and
transmembrane domains,
organelle assignment
Sequencing and Assembly Summary
Pooled libraries
Raw
Trimmed
Mapped
rRNA
Assigned
Eukaryotes
Bacteria
Archaea
Virus
Unknown phylogeny
% rRNA, % mapped
100
Percent of trimmed reads
441,425,648
414,698,752
299,185,770
21,624,539
171,783,433
141,024,961
28,424,075
214,737
954,416
1,165,244
ORFS
194,173
136,144
56,031
245
1096
657
Assigned Reads:
82% eukaryotic
16% bacterial
% mapped
% rRNA
% spike
80
1.0
0.8
60
0.6
40
0.4
20
0.2
0
0.0
Co
n
tA
Co
n
tB
Co
n
tC
F
eA
F
eB
C
2 A 12 B 12 C Fe A Fe B Fe C
Fe
2
B1
B
B
2
2
B1
B1
B1
% spike 1
Reads
Community contributions to mRNA pool
Eukaryotes
Bacteria and Archaea
Metazoa
Control
+Fe
+B12
Fungi
+B12Fe
Other Eukayotes
A.
Control
+Fe
+B12
Archaea
Other Bacteria
B.
+B12Fe
Betaproteobacteria
Haptophyta
Gammaproteobacteria
Cryptophyta
Alphaproteobacteria
Streptophyta
Firmicutes
Chlorophyta
Cyanobacteria
Dinophyta
Bacteroidetes/Chlorobi
Ciliophora
Actinobacteria
Other Stramenopiles
0
Pelagophyte
10
20
30
40
50
% of reads assigned to bacteria and archaea
Centric Diatom
Pennate Diatom
0
10
20
30
40
50
% of reads assigned to eukaryotes
Error bars: 1SD, triplicates
60
Transcriptome differences reflect
changes in gene expression levels
within the community, NOT
community composition changes
60
Community contributions to mRNA pool
Eukaryotes
Diatoms
Metazoa
Control
+Fe
+B12
Fungi
+B12Fe
Other Eukayotes
A.
other centric
Control
+Fe
+B12
Coscinodiscophyceae
+B12Fe
C.
Thalassiosira
Haptophyta
Cryptophyta
Chaetoceros
Streptophyta
other pennate
Chlorophyta
Pseudo-nitzchia
Dinophyta
Fragilariopsis
Ciliophora
Other Stramenopiles
0
Pelagophyte
10
20
30
40
50
% of diatom reads assigned
Centric Diatom
Pennate Diatom
0
10
20
30
40
50
% of reads assigned to eukaryotes
Error bars: 1SD, triplicates
60
Fragilariopsis- dominated diatom
community, no significant
differences: changes reflect
transcriptome responses to
micronutrients, not community
composition changes
60
Transcriptional changes induced by micronutrient additions
Bacteroidetes/Chlorobi
Cont vs Fe
B12 vs B12 Fe
Cont vs B12
Bacteroidetes/Chlorobi
Fe vs B12Fe
Gammaproteobacteria
Gammaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Dinophyta
Dinophyta
Centric diatoms
Centric diatoms
Pennate diatoms
Pennate diatoms
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Percentage of differentially expressed
ORFs (FDR< 0.05)
0.0
0.5
1.0
1.5
2.0
2.5
Percentage of reads mapped to diff.
expressed ORFs (FDR< 0.05)
Differentially expressed: edgeR pairwise comparisons of triplicate treatments, FDR < 0.05
B12 exhibits strong influence on short term transcriptional response across all
three most abundant groups per kingdom
fmol CBA1 per g total protein
Hypothesized
B12- starvation
biomarkers from
culture work:
fmol MetE per g total protein
Diatom responses to changes in B12 availability
4
High B12
MetE
Low B12
3
2
1
0
10
CBA1
8
6
4
Figure: Amy Caracappa-Qubeck, WHOI
2
0
+ B12
- B12
MetE and CBA1 protein in
P. tricornutum cultures
(Bertrand et al L and O, 2013)
MetE and CBA1
(Bertrand et al 2012, 2013)
Diatom responses to changes in B12 availability
log2(treatment/control)
10
5
Fe/control
B12/control
B12Fe/control
MetE
High B12
Low B12
0
-5
-10
Figure: Amy Caracappa-Qubeck, WHOI
log2(treatment/control)
6
4
2
0
-2
-4
Fe/control
B12/control
B12Fe/Fe
CBA1
MetE and CBA1:
repressed by B12 and not
driven by iron- confirmed
as biomarkers for B12
starvation
-6
Showing three log 2 fold change values for each ORF annotated as diatom MetE,
CBA1 with >50 total reads mapped
Behavior of known Fe- responsive transcripts
log2(treatment/control)
4
Fe/control
B12/control
Flavodoxin, clade 2
2
0
-2
-4
log2(treatment/control)
4
Fe/control
B12/control
ISIP2A
2
0
-2
-4
12% of flavodoxin c2 ORFs , 16% of ISIP2A ORFS with > 500 reads assigned:
down-regulated (FDR <0.05) by Fe addition
Dominated by phyla we expect for the season
Bacteria and Archaea
(Ghiglione and Murray 2012; Williams et al 2012; Williams et al 2013;
Grzymski et al 2012; Brown et al 2012)
Control
+Fe
+B12
Archaea
Other Bacteria
B.
Bacteroidetes: 53% of reads best hit
to 4 genomes
+B12Fe
Polaribacter
Betaproteobacteria
Aequorivita
Gammaproteobacteria
Ulvibacter sp. SCB49
Alphaproteobacteria
Other CFB
Firmicutes
Cyanobacteria
Bacteroidetes/Chlorobi
Actinobacteria
Gammaproteobacteria: 52% of reads best hit
0
10
20
30
40
50
% of reads assigned to bacteria and archaea
Alphaproteobacteria: 42% of reads best hit to
10 genomes
60
to 7 genomes
SAR 92
other Alteromonadales
Neptinibacter
other Oceanospirillales
OMG
Methylophaga
other gamma
SAR11
Rhodobactereaceae
uncultured marine alphas
other alpahs
Clear documentation of B12- starvation
and Fe limitation in diatoms…
B12 sources, Fe demand in bacteria?
Uroporphyrinogen-III
cysG
Precorrin 2
Heme and Chla synthesis
cbiX
(Cobalt) Precorrin 2
Transcripts from each enzyme in B12
biosynthesis pathway detected
cobI/cbiL
(Cobalt) Precorrin 3
ORFs detected (≥1)
with best blast hits to:
cobGJ/cbiHG
Gammaproteobacteria
Alphaproteobacteria
(Cobalt) Precorrin 4
Bacteroidetes
cobM/cbiF
(Cobalt) Precorrin 5
ORFs B12-repressed
(FDR≤0.1)
cobF/cbiD
Cob(II)yrinic acid a,c diamide
(Cobalt) Precorrin 6x
cobK/cbiJ
reductase
Cob(I)yrinic acid a,c diamide
(Cobalt) Precorrin 6y
cobO/btuR
cobL/cbiET
Adenosylcobyrinic acid a,c diamide
(Cobalt) Precorrin 8x
Transport and uptake
btuB
btuF
cobQ/cbiP
cobH/cbiC
cobC/D
Aminopropanol-2P
Hydrogenobyrinic acid/Cobyrinic acid
Adenosylcobyrinic acid
cobD/cbiB
Cobinamide
cobB/cbiA
Hydrogenobyrinic acid
a,c diamide
cobNST
DMB + NHN
cobT
α-ribazole 5P
cobC
α-ribazole
Adenosylcobinamide
btuR
cobU/cobP
Adenosyl-GDP-cobinamide
Cobyrinic acid
cobS
btuR
Pathway modified from Rovionov et al 2003
Cobalamin
Adenosylcobalamin
Control +Fe
+B12
+B12Fe
Up
Down
Iron- induced
Differentially expressed
Gammaproteobacterial
transcripts
differentially expressed: edgeR pairwise
comparisons between any treatments FDR< 0.05
MeV hierarchical clustering: genes
Key Iron Induced transcripts
• Multiple Bacterioferritins
• Recombinases
• Sigma factors and kinases
Regulated Fe uptake and
storage responses: signs of
Fe-limitation
Key Iron repressed transcripts
• Multiple TonB-dependent iron
complex, Fe3+ dicitrate
receptor domains
Iron- repressed
Control +Fe
+B12
+B12Fe
Up
Down
Differentially expressed
Gammaproteobacterial
transcripts
differentially expressed: edgeR pairwise
comparisons between any treatments FDR< 0.05
MeV hierarchical clustering: genes
B12- /B12Fe
induced
B12- repressed
Key B12 / B12Fe Induced:
• RNA polymerase
• multiple pilA
• Chaperonins
• outer membrane proteins
Key B12 repressed:
• B12- transporters and binding
domains
• B12- biosynthesis proteins
• B12-dependant RNR
Regulated B12 uptake,
production, and use
Bacterial group possibly responsible for B12- production:
signs of Fe- limitation
Gammaproteobacteria
Key Iron repressed:
Multiple TonB-dependent
iron complex, Fe3+ dicitrate
receptor domains
Ross Sea Springtime
Bacterial growth independently Fe-limited (Bertrand et al 2011)
MetE MetH
MetE
MetE
Molecular evidence for
multiple layers of
micronutrient interactions
and starvation
Key B12 repressed:
• B12- transporters and
binding domains
• B12- biosynthesis proteins
• B12-dependant RNR
Cyanobacteria
MetE
B12
MetH
MetH
Eukaryotic
MetHphytoplankton
MetH
Heterotrophic bacteria
and archaea
Photodegradation
McMurdo Sound of the Ross Sea, late austral summer 2012/13
Ross Island
MODIS
16 Jan 2013: Collect 1000 L trace metal clean
seawater; return via helicopter to McMurdo
Station, Crary Lab
Three types of manipulative experiments
• Short term batch- manipulate Fe, B12
• Semicontinuous dilution- manipulate temperature, Fe
• Continuous flow- “ecostats”- manipulate temperature, Fe, CO2
McMurdo Sound Semicontinuous Experiment
20
0 C cont
0 C +Fe
4 C cont
4 C +Fe
Chl a (g/L)
15
Iron: +/- I nM Fe
Temperature: 4°C or 0°C
Triplicates, outdoor incubation
at 15% of ambient light
t=0 same as short term Fe and
B12 addition experiment
10
5
0
2
4
Micronutrient nutritional status and interaction changes
8
Dilute at arrows
(1:6 to 1:2)
Sequence transcriptomes
Short term B12 and
Fe experiment (t = 0)
Transcriptomes to assess:
•
Identity and physiology of major groups responding favorably to temperature increase
•
6
10
12
14
16
18
Days
Temperature and Iron:
strong drivers of
phytoplankton growth
McMurdo Sound Semicontinuous Experiment:
Community mRNA contributions
% assigned reads
80
t=0
0C, low Fe
0C, high Fe
4C, low Fe
4C, high Fe
60
40
20
0
s
se
u
r
Vi
h
Arc
a
ae
ia
te r
c
Ba
o
y
r
a
k
Eu
n
s
e
t
a
cle
t
r
u
C
r
hlo
o
s
a
l
p
h
ti oc
M
a
o
ri
d
n
High temperature: Eukaryotes make a larger contribution
to mRNA pool at the expense of Bacteria
t = 0: all 12 libraries from short term experiment
Diatom gene expression shifts
% ORFs Significantly
Different (FDR < 0.05)
50
4C -Fe vs 0C -Fe
4C +Fe vs 0C +Fe
+Fe 0C vs -Fe 0C
+Fe 4C vs -Fe 4C
40
30
20
10
0
tz
Pse
ros
chia
z
/Nit
a
i
h
c
- ni
udo
Ch
ce
aeto
ario
agil
psis
Fr
EdgeR pairwise comparisons; triplicate treatments
• Temperature has a larger influence than Fe on Pseudo-nitzchia and Chaetoceros
gene expression
• Iron is a more important factor for Fragilariopsis gene expression than the
other diatoms
• Suggests different sensitivities/ capabilities for handling change
Fragilariopsis responses to variability in iron
0 -Fe 0 +Fe 4 -Fe 4 +Fe
Up
Group- normalized Z- scores for
top 100 most abundant
Fragilariopsis transcripts (nuclear)
differentially expressed in high vs
low Fe (EdgeR FDR < 0.05)
Down
SAH-ase, Betaine transporter,
Lhc 12, ATP-ases
CBA1
High Fe- induced
regardless of
temperature
Plastocyanin*
Lhcf 5, 12, histones
Flavodoxin*
Lhcf 10, 11
High Fe- induced
only at high
temperature
Lhcf 1, 4, 8
Bacteriorhodopsin
Cation transporter
ISIP2A
High Fe- Repressed
Diatom B12- starvation indicators
Sum Group Normalized
Expression Value
10000
8000
0C -Fe
0C +Fe
4C -Fe
4C +Fe
6000
4000
2000
0
CBA1
Centric
CBA1
Pennate
MetE
Centric
MetE
Pennate
High Fe and temperature appear to enhance B12 limitation in diatoms
Values estimate expression of each ORFs relative to expression of all ORFs from centric
or pennate diatoms
EdgeR TMM values, mean of triplicates +/- 1 SD shown.
Fluorescence
One Pseudo- nitzchia isolate is a confirmed B12 auxotroph
90
80
70
60
50
40
30
20
10
0
10 nM B1 + 0 B12
0 B1 + 100 pM B12
10 nM B1 + 100 pM B12
Mixed community, Arrow:
MCM Pseudo-nitzchia
Image Credit: Jeff McQuaid
0
2
Preliminary data, post antibiotic
treatment, not axenic
4
6
Days
8
10
12
t=0
0C, low Fe
0C, high Fe
4C, low Fe
4C, high Fe
Undetermined
Fragilariopsis sp.
Pseudo-nitzchia dominate at high Fe high
temp despite documented B12 limitation
• Bacterial partners?
• Strain variability?
Nitzschia sp.
Pseudo-nitzschia sp.
Chaetoceros sp.
0
2000
4000
6000
cells mL
-1
8000
10000
Summary- McMurdo Sound Incubation Metatranscriptomics
Short term B12 and Fe experiment
1.6
0h
24 h
-1
Chl a (g L )
1.4
96 h
*
•
B12 and iron limitation of phytoplankton
growth
•
Confirmation of diatom molecular markers
for B12 limitation
•
Gammaproteobacterial B12 source?
•
Interactive micronutrient dynamics: Fe
limitation of B12 production and use
*
*
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Cont +Fe +B12 +B12Fe
t=0
Cont +Fe +B12 +B12Fe
Semicontinuous Fe and Temperature experiment
20
Strong temperature impact on
phytoplankton growth, community
composition, and gene expression
Pseudo-nitzchia
•
Imbalance in bacterial and phytoplankton
response
• Implications for future impact of
temperature on micronutrient
dynamics
0 C cont
0 C +Fe
4 C cont
4 C +Fe
15
Chl a (g/L)
•
10
5
0
2
4
6
8
10
Days
12
14
16
18
Towards understanding diversity and transcriptional baselines and
sensitivities of Antarctic marine microbial communities - three
approaches
• Cultures
• Manipulative experiments
• Surveys
• Powerful synergy developing
o Future work: further exploration of temperature and
micronutrient interactions in co-cultures and manipulative
experiments
o Better and temporally extended microbial surveys: LR-AUV
technology with automated microbial sampling platforms
o Influence of temperature on sea ice algae life cycles and viruses
o Viruses and domoic acid (DA) producing phytoplankton (HABs)