Transcriptional Sensitivity of Southern Ocean Plankton Communities to Changes in Temperature and Micronutrient Availability

Photo: Jeff McQuaid

Andrew AllenErin BertrandJohn McCrowHong ZhengAhmed MoustafaJeff McQuaidSIO and JCVI

David HutchinsKai XuNathan WalworthUSC

Deborah BronkRachel SiplerJenna SpackeenVIMS

Mak SaitoDawn MoranAbigail NobleWHOI

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

Jan Nov

Ice

Diatoms

18S rRNA analysis of Spring and Summer water column and sea ice

McMurdo Sound communities

Alveolata

Chlorophyta

Haptophyta

Saito Lab, WHOIAhmed Moustafa, JCVI, AUC Egypt

Cryptophyta

Dawn Moran, Jeff McQuaid, Greg WangerOpisthokonta10%

1%0.1%

Alphaproteobacteria

Gammaproteobacteria

CFB

Jan Nov

Ice 16S rRNA analysis of Spring and

Summer water column McMurdo

Sound communities

Saito Lab, WHOIAhmed Moustafa, JCVI, AUC Egypt

2 µm

Sea Ice diatom and bacteria(Greg Wanger)

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

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 of the Ross Sea, late austral summer 2012/13

MODIS

Ross Island

16 Jan 2013: Collect 1000 L trace metal clean seawater; return via helicopter to McMurdo

Station, Crary Lab

Fe and B12 Dynamics in the Ross Sea

Bertrand et al 2007, 2011

Pseudonitzchia

Phaeocystis +

Chaetoceros

Pseudonitzchia

Phaeocystis 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

Eukaryotic phytoplankton

Cyanobacteria

Heterotrophic bacteria and archaea

MetE

MetH

MetE

MetEMetE

MetH

MetH

MetH

MetH

Photodegradation

B12

Summer

Spring

Short term bioassays: B12 and iron

Bertrand et al in prep

Harvest trace metal clean seawater, return to Crary Lab

Control +B12

+Fe +B12Fe

Incubate 0°C, 24h• RNA samples• Nitrate, bicarbonate

uptake (Bronk Lab)• Chla measure

After 96h, Chla measure

Fill 2.7 L bottles and supplement

Characterize transcriptional response of initial community to micronutrient additions at 0°C• 1 nM FeCl3, 200 pM B12

Photos: Jen Erxleben

96 h24 h

+B12+Fe +B12Fe

*

**

Cont +Fe +B12 +B12Fe

N

O3

- ( m

ol N

L-1

h-1

)

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

H

CO

3- (

mo

l C L

-1 h

-1)

0.00

0.05

0.10

0.15

0.20

t=0

Chl

a ( g

L-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

*

*

0 h

Cont

At 24h: Nitrate uptake rate increased in +Fe treatments No change in chlorophyll a or primary productivity

Sequence community RNA after 24 hours rRNA removal to enrich mRNAtarget bacteria and phytoplankton

Bertrand et al in prep

Chlorophyll a at 96h: community was independently B12 and Fe limited

Metatranscriptome analysis pipeline

Sample collection

200-2000 mL filtered onto

0.2 um cartridge filters

RNA extraction

and cleanup plus rRNA removal

Library prep

NuGen cDNA synthesis and Truseq library preparation

Sequencing

Paired end Illumina Hiseq 2000

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 Annotation

(1)Taxonomy via best blast hit(2) functional domains (PFams/TIGRFams), KEGG, KOG, GO, EC, pathway, transporter and transmembrane domains, organelle assignment

PhyloDB: • 25 million peptide sequences• Does not rely on blast hit for functional annotation• MMETSP transcriptome sequencing included

Targets Bacteria, Archaea, Eukaryotic nuclear and organelle mRNA

Cont ACont B

Cont C Fe AFe B

Fe CB12 A

B12 BB12 C

B12Fe A

B12 Fe B

B12 Fe C

% r

RN

A, %

map

ped

0

20

40

60

80

100

% s

pike

1

0.0

0.2

0.4

0.6

0.8

1.0% mapped% rRNA% spike

Reads ORFSRaw 441,425,648Trimmed 414,698,752Mapped 299,185,770rRNA 21,624,539Assigned 171,783,433 194,173

Eukaryotes 141,024,961 136,144Bacteria 28,424,075 56,031Archaea 214,737 245

Virus 954,416 1096Unknown phylogeny 1,165,244 657

Sequencing and Assembly Summary

82% eukaryotic

16% bacterial

Assigned Reads:

Percent of trimmed reads

Pooled libraries

% of reads assigned to bacteria and archaea

0 10 20 30 40 50 60

Actinobacteria

Bacteroidetes/Chlorobi

Cyanobacteria

Firmicutes

Alphaproteobacteria

Gammaproteobacteria

Betaproteobacteria

Other Bacteria

Archaea Control+Fe+B

12

+B12Fe

B.

Error bars: 1SD, triplicates

% of reads assigned to eukaryotes

0 10 20 30 40 50 60

Pennate Diatom

Centric Diatom

Pelagophyte

Other Stramenopiles

Ciliophora

Dinophyta

Chlorophyta

Streptophyta

Cryptophyta

Haptophyta

Fungi

Metazoa

Other Eukayotes Control+Fe+B12

+B12Fe

A.

Community contributions to mRNA pool

Transcriptome differences reflect changes in gene expression levels within the community, NOT community composition changes

Eukaryotes Bacteria and Archaea

% of diatom reads assigned

0 10 20 30 40 50 60

Fragilariopsis

Pseudo-nitzchia

other pennate

Chaetoceros

Thalassiosira

Coscinodiscophyceae

other centricControl+Fe+B

12

+B12Fe

C.

Error bars: 1SD, triplicates

% of reads assigned to eukaryotes

0 10 20 30 40 50 60

Pennate Diatom

Centric Diatom

Pelagophyte

Other Stramenopiles

Ciliophora

Dinophyta

Chlorophyta

Streptophyta

Cryptophyta

Haptophyta

Fungi

Metazoa

Other Eukayotes Control+Fe+B12

+B12Fe

A.

Community contributions to mRNA pool

Fragilariopsis- dominated diatom community, no significant differences: changes reflect transcriptome responses to micronutrients, not community composition changes

Eukaryotes Diatoms

Transcriptional changes induced by micronutrient additions

Percentage of differentially expressed

ORFs (FDR< 0.05)

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Pennate diatoms

Centric diatoms

Dinophyta

Alphaproteobacteria

Gammaproteobacteria

Bacteroidetes/Chlorobi

Cont vs FeB12 vs B12 FeCont vs B12Fe vs B12Fe

Percentage of reads mapped to diff.

expressed ORFs (FDR< 0.05)

0.0 0.5 1.0 1.5 2.0 2.5

Pennate diatoms

Centric diatoms

Dinophyta

Alphaproteobacteria

Gammaproteobacteria

Bacteroidetes/Chlorobi

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

Diatom responses to changes in B12 availability

MetE and CBA1(Bertrand et al 2012, 2013)

High B12 Low B12

Figure: Amy Caracappa-Qubeck, WHOI

MetE

fmol

Met

E p

er

g to

tal p

rote

in0

1

2

3

4

CBA1

+ B12

fmol

CB

A1

per g

tota

l pro

tein

0

2

4

6

8

10

- B12

MetE and CBA1 protein in P. tricornutum cultures

Hypothesized B12- starvation biomarkers from culture work:

(Bertrand et al L and O, 2013)

MetE

log

2(t

rea

tme

nt/co

ntr

ol)

-10

-5

0

5

10

Fe/controlB12/controlB12Fe/control

CBA1

log

2(t

rea

tme

nt/co

ntr

ol)

-6

-4

-2

0

2

4

6 Fe/controlB12/controlB12Fe/Fe

Showing three log 2 fold change values for each ORF annotated as diatom MetE, CBA1 with >50 total reads mapped

MetE and CBA1: repressed by B12 and not driven by iron- confirmed as biomarkers for B12 starvation

Diatom responses to changes in B12 availability

High B12 Low B12

Figure: Amy Caracappa-Qubeck, WHOI

Flavodoxin, clade 2

log2

(tre

atm

ent/c

ontr

ol)

-4

-2

0

2

4 Fe/controlB12/control

ISIP2A

log

2(t

rea

tme

nt/c

on

tro

l)

-4

-2

0

2

4 Fe/controlB12/control

12% of flavodoxin c2 ORFs , 16% of ISIP2A ORFS with > 500 reads assigned:down-regulated (FDR <0.05) by Fe addition

Behavior of known Fe- responsive transcripts

% of reads assigned to bacteria and archaea

0 10 20 30 40 50 60

Actinobacteria

Bacteroidetes/Chlorobi

Cyanobacteria

Firmicutes

Alphaproteobacteria

Gammaproteobacteria

Betaproteobacteria

Other Bacteria

Archaea Control+Fe+B

12

+B12Fe

B.

Dominated by phyla we expect for the season(Ghiglione and Murray 2012; Williams et al 2012; Williams et al 2013; Grzymski et al 2012; Brown et al 2012)

Bacteria and Archaea

PolaribacterAequorivitaUlvibacter sp. SCB49Other CFB

Bacteroidetes: 53% of reads best hit to 4 genomes

SAR 92other AlteromonadalesNeptinibacterother OceanospirillalesOMGMethylophagaother gamma

Gammaproteobacteria: 52% of reads best hit to 7 genomes

SAR11

Rhodobactereaceae

uncultured marine alphas

other alpahs

Alphaproteobacteria: 42% of reads best hit to 10 genomes

Clear documentation of B12- starvation and Fe limitation in diatoms… B12 sources, Fe demand in bacteria?

Uroporphyrinogen-III

cysGHeme and Chla synthesis

Precorrin 2 cbiX(Cobalt) Precorrin 2

cobI/cbiL(Cobalt) Precorrin 3

cobGJ/cbiHG

cobM/cbiF

(Cobalt) Precorrin 4

(Cobalt) Precorrin 5

cobF/cbiD

(Cobalt) Precorrin 6x

cobK/cbiJ

(Cobalt) Precorrin 6y

cobL/cbiET

(Cobalt) Precorrin 8x

cobH/cbiCHydrogenobyrinic acid/Cobyrinic acid

cobB/cbiA

Hydrogenobyrinic acida,c diamide

GammaproteobacteriaAlphaproteobacteria

Bacteroidetes

ORFs detected (≥1) with best blast hits to:

ORFs B12-repressed(FDR≤0.1)

reductase

Cob(I)yrinic acid a,c diamide

cobO/btuRAdenosylcobyrinic acid a,c diamide

cobQ/cbiPAdenosylcobyrinic acid

cobD/cbiB

Adenosylcobinamide

cobU/cobPAdenosyl-GDP-cobinamide

Aminopropanol-2PcobC/D

cobS

DMB + NHN

cobT

α-ribazole

α-ribazole 5P

cobC

Adenosylcobalamin

Transport and uptakebtuBbtuF

btuR

btuR

Cobyrinic acid

Cobinamide

Cobalamin

Cob(II)yrinic acid a,c diamide

cobNST

Transcripts from each enzyme in B12 biosynthesis pathway detected

Pathway modified from Rovionov et al 2003

Down

UpDifferentially expressed Gammaproteobacterial

transcripts

Control +Fe +B12 +B12Fe

Iron- induced

Iron- repressed

Key Iron Induced transcripts• Multiple Bacterioferritins• Recombinases• Sigma factors and kinases

Key Iron repressed transcripts• Multiple TonB-dependent iron

complex, Fe3+ dicitrate receptor domains

Regulated Fe uptake and storage responses: signs of Fe-limitation

differentially expressed: edgeR pairwise comparisons between any treatments FDR< 0.05

MeV hierarchical clustering: genes

Control +Fe +B12 +B12Fe

B12- repressed

B12- /B12Fe induced

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

Differentially expressed Gammaproteobacterial

transcriptsdifferentially expressed: edgeR pairwise

comparisons between any treatments FDR< 0.05MeV hierarchical clustering: genes

Down

Up

Bacterial group possibly responsible for B12- production: signs of Fe- limitation

Ross Sea Springtime Bacterial growth independently Fe-limited (Bertrand et al 2011)

Eukaryotic phytoplankton

Cyanobacteria

Heterotrophic bacteria and archaea

MetE

MetH

MetE

MetEMetE

MetH

MetH

MetH

MetH

Photodegradation

B12

Key Iron repressed:Multiple TonB-dependent iron complex, Fe3+ dicitrate receptor domains

Molecular evidence for multiple layers of

micronutrient interactions and starvation

Key B12 repressed:• B12- transporters and

binding domains• B12- biosynthesis proteins • B12-dependant RNR

Gammaproteobacteria

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 of the Ross Sea, late austral summer 2012/13

MODIS

Ross Island

16 Jan 2013: Collect 1000 L trace metal clean seawater; return via helicopter to McMurdo

Station, Crary Lab

McMurdo Sound Semicontinuous Experiment

Iron: +/- I nM FeTemperature: 4°C or 0°CTriplicates, outdoor incubationat 15% of ambient light

t=0 same as short term Fe and B12 addition experiment

Dilute at arrows (1:6 to 1:2)

Short term B12 and Fe experiment (t = 0)

Sequence transcriptomes

Days

2 4 6 8 10 12 14 16 18

Ch

l a (

g/L

)

0

5

10

15

20

0 C cont0 C +Fe4 C cont4 C +Fe

Temperature and Iron: strong drivers of

phytoplankton growth

Transcriptomes to assess:• Identity and physiology of major groups responding favorably to temperature increase

• Micronutrient nutritional status and interaction changes

Viruses

Archaea

Bacteria

Eukaryotes nuclear

Chloroplast

Mitochondria

% a

ssig

ned

read

s

0

20

40

60

80

t = 00C, low Fe0C, high Fe4C, low Fe4C, high Fe

McMurdo Sound Semicontinuous Experiment: Community mRNA contributions

High temperature: Eukaryotes make a larger contribution to mRNA pool at the expense of Bacteria

t = 0: all 12 libraries from short term experiment

Pseudo-nitzchia/Nitzchia

Chaetoceros

Fragilariopsis

% O

RF

s S

igni

fican

tly

Diff

eren

t (F

DR

< 0

.05)

0

10

20

30

40

50

4C -Fe vs 0C -Fe4C +Fe vs 0C +Fe+Fe 0C vs -Fe 0C+Fe 4C vs -Fe 4C

Diatom gene expression shifts

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

Down

Up

High Fe- induced regardless of temperature

High Fe- Repressed

Fragilariopsis responses to variability in iron

CBA1

Plastocyanin*

Bacteriorhodopsin

Cation transporter

ISIP2A

Lhcf 1, 4, 8

Lhcf 10, 11

Lhcf 5, 12, histones

SAH-ase, Betaine transporter, Lhc 12, ATP-ases

0 -Fe 0 +Fe 4 -Fe 4 +Fe Group- normalized Z- scores for top 100 most abundant Fragilariopsis transcripts (nuclear)differentially expressed in high vs low Fe (EdgeR FDR < 0.05)

Flavodoxin*

High Fe- induced only at high temperature

Diatom B12- starvation indicators

High Fe and temperature appear to enhance B12 limitation in diatoms

Sum

Gro

up

Nor

mal

ized

E

xpre

ssio

n V

alue

0

2000

4000

6000

8000

10000

0C -Fe0C +Fe4C -Fe4C +Fe

CBA1Centric

CBA1Pennate

MetECentric

MetEPennate

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.

0 2 4 6 8 10 120

1020304050607080

10 nM B1 + 0 B12

0 B1 + 100 pM B12

10 nM B1 + 100 pM B12

Days

Fluo

resc

ence

Preliminary data, post antibiotic treatment, not axenic

One Pseudo- nitzchia isolate is a confirmed B12 auxotroph

Mixed community, Arrow: MCM Pseudo-nitzchiaImage Credit: Jeff McQuaid

Pseudo-nitzchia dominate at high Fe high temp despite documented B12 limitation• Bacterial partners?• Strain variability?

cells mL-1

0 2000 4000 6000 8000 10000

Chaetoceros sp.

Pseudo-nitzschia sp.

Nitzschia sp.

Fragilariopsis sp.

Undetermined

t = 00C, low Fe0C, high Fe4C, low Fe4C, high Fe

96 h24 h

+B12+Fe +B12Fe

*

**

+Fe +B12 +B12Fet=0

Chl

a ( g

L-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.60 h

ContCont

Days

2 4 6 8 10 12 14 16 18

Ch

l a (

g/L

)

0

5

10

15

20

0 C cont0 C +Fe4 C cont4 C +Fe

Summary- McMurdo Sound Incubation Metatranscriptomics

Short term B12 and Fe experiment• 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

Semicontinuous Fe and Temperature experiment

• 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

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 viruseso Viruses and domoic acid (DA) producing phytoplankton (HABs)