Transcript Heterotrophic flagellates - Institut de Ciències del Mar

Looking for the dominant marine bacterivores
Ramon Massana
Institut de Ciències del Mar (CSIC), Barcelona
MEDOCEAN meeting, 28 November 2013
Looking for the dominant marine bacterivores
Smetacek 2002. Nature
Heterotrophic flagellates - Main bacterial grazers
Unicellular eukaryotes, colorless, and with flagella
Ubiquitous in the plankton at abundances around 1000 cells ml -1
Most are 1-3 µm in size
Exhibit phylogenetic and functional diversity
Beyond the simplified microbial loop …
Heterotrophic flagellates as algivorous and osmotrophs
Mixotrophic flagellates as bacterivorous
Viruses as a factor for bacterial mortalityå
Azam et al. 1983. Mar. Ecol. Progr. Ser
Heterotrophic flagellates - Microscopy
DAPI-stained epifluorescence microscopy
The only quantitative approach
Very poor identification power
5 µm
Heterotrophic flagellates – Culturing bias
The most common in pelagic marine waters (by culturing, enrichments and direct observations)
Stramenopiles
Bicosoecida
Chrysopytes
Pedinellids
Caecitellus parvulus
Cafeteria roenbergensis
Boroka karpovii
Paraphysomonas imperforata
Pteridomonas danica
Kinetoplastids
Neobodo designis
Rynchomonas nasuta
Inserta sedis
Ancyromonas sigmoides
Amastigomonas debruynei
Paraphysomonas imperforata
Dominates (up to 98% of cells) in flagellate
enrichments in the US East Coast
Represents <1% in natural samples
Lim et al. 1999. Limnol. Oceanogr.
Heterotrophic flagellates – Environmental molecular surveys
Analysis of 18S rDNA sequences retrieved from the environment
• 40 studies on marine microeukaryotes since 2001
• 8719 environmental sequences
Alveolata
Stramenopiles
Rhizaria
Archaeplastida
CCTH
Excavata
Opisthokonta
Amoebozoa
Incertae sedis
Massana 2011. Annu Rev. Microbiol.
MALV-1
Dinophyceae
MALV-2
MALV-1
Ciliophora
MALV-2
MALV-1
Dinophyceae
Ciliophora
MALV-3
Dinophyceae
Ciliophora
MALV-2
MALV-1
MALV-3
MALV-5
MALV-1
MALV-3
Dinophyceae
Ciliophora
MALV-2
MALV-5
Other alveolates
Ciliophora
MALV-5
MALV-1
MALV-3
Dinophyceae
MALV-2
Other
alveolates
Bacillariophyta
MALV-3
Other
alveolates
Ciliophora
MALV-5
MALV-1
Dinophyceae
Bacillariophyta
MAST-4
(6-11)
MALV-5
Bacillariophyta
MALV-3
Other
alveolates
Ciliophora
MALV-1
MAST-4
MAST-3(6-11)
(12)
Other
alveolates
MAST-4
(6-11)
MALV-5
Bacillariophyta
MALV-3
Ciliophora
MAST-3
(12)
Chrysophyceae
Bacillariophyta
MAST-3
(12)
Other
alveolates
MAST-4
(6-11)
MALV-5
MALV-3
Chrysophyceae
MAST-1
MAST-4
(6-11)
Chrysophyceae
Bacillariophyta
MAST-3
(12)
Other
alveolates
MALV-5
MAST-1
Dictyophyceae
MAST-3
(12)
MAST-1
MAST-4
(6-11)
Chrysophyceae
Bacillariophyta
Other
alveolates
Dictyophyceae
Bicosoecea
Chrysophyceae
Dictyophyceae
MAST-3
(12)
MAST-1
MAST-4
(6-11)
Bacillariophyta
Bicosoecea
Bolidophyceae
MAST-1
Chrysophyceae
Bicosoecea
Dictyophyceae
MAST-3
(12)
MAST-4
(6-11)
Bolidophyceae
Pelagophyceae
Dictyophyceae
Bolidophyceae
MAST-1
Bicosoecea
Chrysophyceae
MAST-3
(12)
Pelagophyceae
Pirsonia
Bicosoecea
Dictyophyceae
Pelagophyceae
Bolidophyceae
MAST-1
Chrysophyceae
Pirsonia
Labyrinthulidae
Bolidophyceae
Bicosoecea
Pirsonia
Pelagophyceae
Dictyophyceae
MAST-1
Labyrinthulidae
Other stramenopiles
Pelagophyceae
Bolidophyceae
Pirsonia
Labyrinthulidae
Bicosoecea
Dictyophyceae
Other
stramenopiles
Radiolaria/Polycystinea
Pirsonia
Pelagophyceae
Labyrinthulidae
Other
stramenopiles
Bolidophyceae
Bicosoecea
Radiolaria/Polycystinea
Radiolaria/Acantharea
Labyrinthulidae
Pirsonia
Radiolaria/Polycystinea
Other
stramenopiles
Pelagophyceae
Bolidophyceae
Radiolaria/Acantharea
Radiolaria/RAD
B-sticolonche
o
Other
stramenopiles
Labyrinthulidae
Radiolaria/Polycystinea
Radiolaria/Acantharea
Pelagophyceae
Radiolaria/RAD
B-sticolonche
o Pirsonia
Cercozoa/Monadof
ilsa
Radiolaria/Polycystinea
Other
stramenopiles
Radiolaria/Acantharea
Radiolaria/RAD
B-sticolonche
o Pirsonia
Labyrinthulidae
Cercozoa/Monadof
ilsaA
Radiolaria/RAD
Radiolaria/Acantharea
Radiolaria/Polycystinea
Radiolaria/RAD
B-sticolonche
o
Cer
cozoa/Monadof
ilsaA
Other
stramenopiles
Labyrinthulidae
Radiolaria/RAD
Cercozoa/Chloroarachniophyta
Radiolaria/Acantharea
Radiolaria/RAD
B-sticolonche
o
Cer
cozoa/Monadof
ilsaA
Radiolaria/RAD
Radiolaria/Polycystinea
Other Other
stramenopiles
Cercozoa/Chloroarachniophyta
rhizaria
Radiolaria/RAD
B-sticolonche
o
Cer
cozoa/Monadof
ilsaA
Radiolaria/RAD
Cercozoa/Chloroarachniophyta
Radiolaria/Acantharea
Radiolaria/Polycystinea
Other
rhizaria
Prasinophyceae
Radiolaria/RAD
Cer
cozoa/Monadof
ilsaA
Cercozoa/Chloroarachniophyta
Other
rhizaria
Radiolaria/RAD
B-sticolonche
o
Radiolaria/Acantharea
Prasinophyceae
Trebouxiophyceae
Radiolaria/RAD
Cercozoa/Chloroarachniophyta
Other
rhizaria
Prasinophyceae
Cer
cozoa/Monadof
ilsaA
Radiolaria/RAD
B-sticolonche
o
Trebouxiophyceae
Other
archaeplastida
Cercozoa/Chloroarachniophyta
Other rhizaria
Prasinophyceae
Trebouxiophyceae
Radiolaria/RAD
CerOther
cozoa/Monadof
ilsaA
archaeplastida
Prymnesiophyceae
Other
rhizaria
Prasinophyceae
Trebouxiophyceae
Other
archaeplastida
Cercozoa/Chloroarachniophyta
Radiolaria/RAD
A
Prymnesiophyceae
Cryptophyceae
Prasinophyceae
Trebouxiophyceae
Other
archaeplastida
Prymnesiophyceae
Other
rhizaria
Cercozoa/Chloroarachniophyta
Cryptophyceae
Telonemia
Trebouxiophyceae
Prymnesiophyceae
Other
archaeplastida
Prasinophyceae
Other
rhizaria
Cryptophyceae
Telonemia
Picobiliphyta
Other
archaeplastida
Prymnesiophyceae
Cryptophyceae
Trebouxiophyceae
Prasinophyceae
Telonemia
Picobiliphyta
Other CCTH
Prymnesiophyceae
Cryptophyceae
Other
archaeplastida
Trebouxiophyceae
Telonemia
Picobiliphyta
Other
CCTH
Diplonemea
Cryptophyceae
Prymnesiophyceae
Other
archaeplastida
Telonemia
Picobiliphyta
Other
CCTH
Diplonemea
Kinetoplastea
Prymnesiophyceae
Telonemia
Cryptophyceae
Picobiliphyta
Other
CCTH
Diplonemea
Kinetoplastea
Other
excavata
g
Cryptophyceae
Picobiliphyta
Telonemia
Other
CCTH
Diplonemea
Kinetoplastea
Other
excavata
gatea
Choanof
laell
Other
CCTH
Telonemia
Picobiliphyta
Diplonemea
Kinetoplastea
Other
excavata
gatea
Choanof
l
a
ell
OtherKinetoplastea
opisthokonta
Diplonemea
Picobiliphyta
Other
CCTH
Other
excavata
gatea
Choanof
l
a
ell
Other opisthokonta
Amoebozoa
Kinetoplastea
Other
CCTH
Diplonemea
Other
excavata
gatea
Choanof
l
a
ell
Other
opisthokonta
Amoebozoa
Inserta
Other
excavata
Diplonemea
Kinetoplastea
Choanof
laellgsedis
atea
Other
opisthokonta
Amoebozoa
Inserta
sedis
Choanof
laexcavata
ellgsedis
atea
Kinetoplastea
Other
Other
opisthokonta
Amoebozoa
Inserta
0
0
0
0
500
1000
1500
500
1000
1500
Number
500of sequences
1000in molecular
1500surveys
Number
of
sequences
in
molecular
500
1000
1500surveys
2000
2000
2000
2000
Groups with cultured heterotrophic
flagellates
Alveolata
Stramenopiles
Rhizaria
Archaeplastida
CCTH
Excavata
Opisthokonta
Amoebozoa
Incertae sedis
Massana 2011. Annu Rev. Microbiol.
MALV-1
Dinophyceae
MALV-2
MALV-1
Ciliophora
MALV-2
MALV-1
Dinophyceae
Ciliophora
MALV-3
Dinophyceae
Ciliophora
MALV-2
MALV-1
MALV-3
MALV-5
MALV-1
MALV-3
Dinophyceae
Ciliophora
MALV-2
MALV-5
Other alveolates
Ciliophora
MALV-5
MALV-1
MALV-3
Dinophyceae
MALV-2
Other
alveolates
Bacillariophyta
MALV-3
Other
alveolates
Ciliophora
MALV-5
MALV-1
Dinophyceae
Bacillariophyta
MAST-4
(6-11)
MALV-5
Bacillariophyta
MALV-3
Other
alveolates
Ciliophora
MALV-1
MAST-4
MAST-3(6-11)
(12)
Other
alveolates
MAST-4
(6-11)
MALV-5
Bacillariophyta
MALV-3
Ciliophora
MAST-3
(12)
Chrysophyceae
Bacillariophyta
MAST-3
(12)
Other
alveolates
MAST-4
(6-11)
MALV-5
MALV-3
Chrysophyceae
MAST-1
MAST-4
(6-11)
Chrysophyceae
Bacillariophyta
MAST-3
(12)
Other
alveolates
MALV-5
MAST-1
Dictyophyceae
MAST-3
(12)
MAST-1
MAST-4
(6-11)
Chrysophyceae
Bacillariophyta
Other
alveolates
Dictyophyceae
Bicosoecea
Chrysophyceae
Dictyophyceae
MAST-3
(12)
MAST-1
MAST-4
(6-11)
Bacillariophyta
Bicosoecea
Bolidophyceae
MAST-1
Chrysophyceae
Bicosoecea
Dictyophyceae
MAST-3
(12)
MAST-4
(6-11)
Bolidophyceae
Pelagophyceae
Dictyophyceae
Bolidophyceae
MAST-1
Bicosoecea
Chrysophyceae
MAST-3
(12)
Pelagophyceae
Pirsonia
Bicosoecea
Dictyophyceae
Pelagophyceae
Bolidophyceae
MAST-1
Chrysophyceae
Pirsonia
Labyrinthulidae
Bolidophyceae
Bicosoecea
Pirsonia
Pelagophyceae
Dictyophyceae
MAST-1
Labyrinthulidae
Other stramenopiles
Pelagophyceae
Bolidophyceae
Pirsonia
Labyrinthulidae
Bicosoecea
Dictyophyceae
Other
stramenopiles
Radiolaria/Polycystinea
Pirsonia
Pelagophyceae
Labyrinthulidae
Other
stramenopiles
Bolidophyceae
Bicosoecea
Radiolaria/Polycystinea
Radiolaria/Acantharea
Labyrinthulidae
Pirsonia
Radiolaria/Polycystinea
Other
stramenopiles
Pelagophyceae
Bolidophyceae
Radiolaria/Acantharea
Radiolaria/RAD
B-sticolonche
o
Other
stramenopiles
Labyrinthulidae
Radiolaria/Polycystinea
Radiolaria/Acantharea
Pelagophyceae
Radiolaria/RAD
B-sticolonche
o Pirsonia
Cercozoa/Monadof
ilsa
Radiolaria/Polycystinea
Other
stramenopiles
Radiolaria/Acantharea
Radiolaria/RAD
B-sticolonche
o Pirsonia
Labyrinthulidae
Cercozoa/Monadof
ilsaA
Radiolaria/RAD
Radiolaria/Acantharea
Radiolaria/Polycystinea
Radiolaria/RAD
B-sticolonche
o
Cer
cozoa/Monadof
ilsaA
Other
stramenopiles
Labyrinthulidae
Radiolaria/RAD
Cercozoa/Chloroarachniophyta
Radiolaria/Acantharea
Radiolaria/RAD
B-sticolonche
o
Cer
cozoa/Monadof
ilsaA
Radiolaria/RAD
Radiolaria/Polycystinea
Other Other
stramenopiles
Cercozoa/Chloroarachniophyta
rhizaria
Radiolaria/RAD
B-sticolonche
o
Cer
cozoa/Monadof
ilsaA
Radiolaria/RAD
Cercozoa/Chloroarachniophyta
Radiolaria/Acantharea
Radiolaria/Polycystinea
Other
rhizaria
Prasinophyceae
Radiolaria/RAD
Cer
cozoa/Monadof
ilsaA
Cercozoa/Chloroarachniophyta
Other
rhizaria
Radiolaria/RAD
B-sticolonche
o
Radiolaria/Acantharea
Prasinophyceae
Trebouxiophyceae
Radiolaria/RAD
Cercozoa/Chloroarachniophyta
Other
rhizaria
Prasinophyceae
Cer
cozoa/Monadof
ilsaA
Radiolaria/RAD
B-sticolonche
o
Trebouxiophyceae
Other
archaeplastida
Cercozoa/Chloroarachniophyta
Other rhizaria
Prasinophyceae
Trebouxiophyceae
Radiolaria/RAD
CerOther
cozoa/Monadof
ilsaA
archaeplastida
Prymnesiophyceae
Other
rhizaria
Prasinophyceae
Trebouxiophyceae
Other
archaeplastida
Cercozoa/Chloroarachniophyta
Radiolaria/RAD
A
Prymnesiophyceae
Cryptophyceae
Prasinophyceae
Trebouxiophyceae
Other
archaeplastida
Prymnesiophyceae
Other
rhizaria
Cercozoa/Chloroarachniophyta
Cryptophyceae
Telonemia
Trebouxiophyceae
Prymnesiophyceae
Other
archaeplastida
Prasinophyceae
Other
rhizaria
Cryptophyceae
Telonemia
Picobiliphyta
Other
archaeplastida
Prymnesiophyceae
Cryptophyceae
Trebouxiophyceae
Prasinophyceae
Telonemia
Picobiliphyta
Other CCTH
Prymnesiophyceae
Cryptophyceae
Other
archaeplastida
Trebouxiophyceae
Telonemia
Picobiliphyta
Other
CCTH
Diplonemea
Cryptophyceae
Prymnesiophyceae
Other
archaeplastida
Telonemia
Picobiliphyta
Other
CCTH
Diplonemea
Kinetoplastea
Prymnesiophyceae
Telonemia
Cryptophyceae
Picobiliphyta
Other
CCTH
Diplonemea
Kinetoplastea
Other
excavata
g
Cryptophyceae
Picobiliphyta
Telonemia
Other
CCTH
Diplonemea
Kinetoplastea
Other
excavata
gatea
Choanof
laell
Other
CCTH
Telonemia
Picobiliphyta
Diplonemea
Kinetoplastea
Other
excavata
gatea
Choanof
l
a
ell
OtherKinetoplastea
opisthokonta
Diplonemea
Picobiliphyta
Other
CCTH
Other
excavata
gatea
Choanof
l
a
ell
Other opisthokonta
Amoebozoa
Kinetoplastea
Other
CCTH
Diplonemea
Other
excavata
gatea
Choanof
l
a
ell
Other
opisthokonta
Amoebozoa
Inserta
Other
excavata
Diplonemea
Kinetoplastea
Choanof
laellgsedis
atea
Other
opisthokonta
Amoebozoa
Inserta
sedis
Choanof
laexcavata
ellgsedis
atea
Kinetoplastea
Other
Other
opisthokonta
Amoebozoa
Inserta
0
0
0
0
500
1000
1500
500
1000
1500
Number
500of sequences
1000in molecular
1500surveys
Number
of
sequences
in
molecular
500
1000
1500surveys
2000
2000
2000
2000
Phototrophic stramenopiles
Pirsonids
Hyphochytrids
MAST-1
Developayella
Marine Stramenopiles - MAST
(novel, uncultured groups)
Oomycetes
MAST-2
MAST-3
Blastocystis
MAST-12
Bicosoecids
Placididea
MAST-7,-8
MAST-4
MAST-11
MAST-6,-9,-10
Labyrinthulids
Thraustochytrids
Massana et al. 2002. Appl. Environ. Microbiol.
Massana et al. 2004. Appl. Environ. Microbiol.
MAST-4 as a model heterotrophic flagellate
Phototrophic stramenopiles
Pirsonids
Hyphochytrids
MAST-1
A bacterivorous heterotrophic picoeukaryote
Developayella
Oomycetes
MAST-2
MAST-3
Blastocystis
MAST-12
Bicosoecids
Placididea
DAPI
10 µm
FISH
Massana et al. 2006. Environ. Microbiol.
MAST-7,-8
MAST-4
MAST-11
MAST-6,-9,-10
Labyrinthulids
Thraustochytrids
Grazing rate:
1–3 bacteria h-1
DAPI
FISH
Ingested FLB
Bacterial mortality:
1 to 10% of production
Massana et al. 2009. ISME J.
MAST-4 as a model heterotrophic flagellate
Phototrophic stramenopiles
Pirsonids
Hyphochytrids
MAST-1
Widely distributed and abundant
Developayella
Oomycetes
MAST-2
FISH
MAST-3
Blastocystis
MAST-12
Bicosoecids
Placididea
MAST-7,-8
MAST-4
MAST-11
MAST-6,-9,-10
Labyrinthulids
Thraustochytrids
q-PCR
Rodríguez-Martínez et al. 2009. Environ. Microbiol.
MAST-4 as a model heterotrophic flagellate
Limited genetic diversity
GenBank sequences
MAST-4 as a model heterotrophic flagellate
Global distribution and environmental selection
18S rDNA 287 bp
5.8S rDNA
ITS1
28S rDNA
ITS2
184-256 bp
~550 bp – 123 ARISA fingerprints
Rodríguez-Martínez et al. submitted
MAST-4 as a model heterotrophic flagellate
Global distribution and environmental selection
Rodríguez-Martínez et al. submitted
Summary
The diversity of heterotrophic flagellates is poorly investigated
The culturing bias a main drawnback in the ecology of heterotrophic flagellates
The novel MAST lineages are important marine heterotrophic flagellates
Single Amplified Genomics provide new opportunities for ecological and evolutionary studies
MAST-4 is a nice model of heterotrophic flagellates
Widespread in euphotic waters
Accounts for ~10% of cells
Active bacterial grazer
Limited diversity (only 5 clades)
Acknowledgments
Institut de Ciències del Mar
Raquel Rodríguez-Martínez
Ramiro Logares
Javier del Campo
Massimo Pernice
Caterina Rodríguez-Giner
Irene Forn
Vanessa Balagué
Station Biologique de Roscoff, France
Fabrice Not
Colomban de Vargas
Instituto Tecnológico de Chascomús, Argentina
Fernando Unrein
University of Washington, USA
Gabrielle Rocap
Bigelow Laboratory for Ocean Sciences, USA
Mike Sieracki
Ramunas Stepanauskas
you for your attention !