Extreme Eukaryotes in the Hydrothermal
Environments of Lassen Volcanic National Park
Gordon Wolfe
Dept. Biological Sciences
California State University Chico
Outline
1. My life as a virologist
2. Protist molecular diversity in LVNP hydrothermal sites
3. Possible eukaryotic viral hosts
eukaryotes vs. prokaryotes as viral hosts
Oregon State, 1989: marine
coccolithophorid alga
Emiliania huxleyi
~90 nm hexagonal
particles in algal lysate 
Brussaard et al. 1996. Virus-like
particles in a summer bloom of
Emiliania huxleyi in the North Sea.
Aquatic Microbial Ecology 10: 105
Castberg et al. (2002). Isolation and characterization of a virus that infects Emiliania
huxleyi (Haptophyta). J. Phycology 38: 767
Wilson et al. (2005). Complete genome sequence and lytic phase transcription
profile of a Coccolithovirus. Science 309: 1090
Suttle, 2005. Viruses in the sea. Nature 437: 356
Humans of the world
Protist Molecular Diversity
Non-thermal sites
Neutral
Lakes, rivers and oceans, groundwater
Acidic
Acid mine drainage
Thermal sites
Neutral
Non-sulfidic hydrothermal
Acidic
Sulfidic hydrothermal
Rio Tinto, Spain
pH 2; heavy metals
• chlorophytes, diatoms
• ciliates, amoebae,
cercomonads
• fungi
1 = Sulfur works
2 = Bumpass Hell
3 = Devil’s Kitchen
4 = Boiling Springs Lake
Upper Sulfur works:
pH 2, 96 oC
Bumpass Hell:
pH 1.4-3.2
11-68 oC
Questions
• What eukaryotic rDNA sequences exist in
LVNP hydrothermal environments?
• Is genetic diversity dominated by
autotrophs or heterotrophs?
• What factors determine the composition
and diversity of protists in LVNP?
eubacterial 16S rDNA
18S rDNA
CTAB UC FD
USW 123
pH 3.3, 25 C
SW114
pH 1.9, 69 C
USW 123
pH 3.3, 25 C
SW114
pH 1.9, 69 C
a
6
5
Bumpass Hell
pH
4
3
2
1
0
0
20
40
60
80
100
6
b
Sulfur Works / Upper Sulfur Works
5
pH
4
3
2
1
0
0
20
40
60
80
100
6
c
Devil's Kitchen
5
pH
4
3
2
1
0
0
20
40
60
T (oC)
80
100
Low vs. high
18S rRNA RFLP
diversity
pH 4.6, 37 C
pH 1.8, 68 C
pH 1.7, 30 C
BH
BH100
BH105
BH105
BH106
BH107
BH107
BH118
11
8
D
K
D 102
K
D 105
K
D 107
K1
D 10
K
D 110
K
D 112
K1
D 12
K
D 116
K1
16
U
SW
U 1
S 23
U W1
S 2
U W1 3
S 2
U W1 4
SW 24
U 1
S 25
U W1
SW 26
12
7
% unique RFLP clones
80%
70%
60%
50%
40%
30%
20%
10%
0%
site
80%
70%
70%
% clones with unique RFLPs
% clones with unique RFLPs
80%
60%
50%
40%
30%
20%
10%
60%
50%
40%
30%
20%
10%
0%
0%
0
2
4
site pH
6
8
0
20
40
site T (oC)
60
80
Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703
Viridiplantae
Closest BLAST identities
% sim.
Site
pH
T
(oC)
Chlorophyceae sp.
97
USW124
BH105
5.3
3.2
44
14
Chlamydomonadales sp.
97-99
BH107
BSL101
1.8
2.5
68
37
Chlamydomonadaceae sp.
97
USW126
5.8
45
Chlamydomonas sp.
97
96
SW117
USW126
2.2
5.8
34
45
Chlamydomonas acidophila / clone
RT1n1
98
96-99
DK102
BH118
mat
2.2
19
Dunaliella sp.
98
BH100
1.7
30
Chlorogonium sp.
98
BSL008
2.4
28
Scenedesmaceae sp. / clone 18S-AKW-5
97
USW124
5.3
44
USW124
5.3
44
Chlorophyta
Chlorophyceae
Chlamydomonadales
Chlamydomonadaceae
Haematococcaceae
Sphaeropleales
Scenedesmus sp.
Viridiplantae
Closest BLAST identities
% sim.
Site
pH
T
(
o
C
)
Chlorophyta
Trebouxiophyceae
Chlorellales
Chlorellales sp.
95
USW126
5.8
Chlorella sp.
99
DK107
mat
Chlorella sp. / Nannochloris sp.
99
98
99
98
BH100
USW124
DK116
DK112
1.7
5.3
4.0
3.0
30
44
22
45
Ericales sp.
98
BH118
2.2
19
Bryopsida sp.
99
BSL014
2.7
25
Klebsormidiophyceae
Klebsormidium sp.
99
DK112
3.0
45
Zygnemophyceae
Desmidiales
Desmidiaceae
Staurastrum sp.
97
BH106
3.2
15
Chlorellaceae
Streptophyta
45
Rhodophyta
Bangiophyceae
Cyanidiaceae
Closest BLAST identities
% sim.
Site
pH T
(oC)
Cyanidioschyzon merolae
99
BSL006
BSL009
2.3
53
Cyanidium-like alga cultivated from Boiling Springs Lake
Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703
Stramenopiles
Bacillariophyta
Bacillariophyceae
Bacillariophycidae
Naviculales
Closest BLAST identities
Pinnularia sp. / clones RT7iin2 or
RT7in48
% sim.
95
96
96
97
95
97
97
95
93
95
93
Pinnularia sp.
Coscinodiscophyceae
Aulacoseira sp.
98
98
96
Site pH
T (oC)
USW123
BH105
BH106
BH107
DK112
DK116
USW125
USW124
USW126
USW127
BSL008
DK110
3.3
3.2
3.2
1.8
3.0
4.0
3.3
5.3
5.8
4.7
2.4
4.5
25
14
15
68
45
22
44
44
45
36
28
60
BH105
USW127
3.2
4.7
14
36
BH105
BH106
BH107
3.2
3.2
1.8
14
15
68
Stramenopiles
Chrysophyceae
Ochromonadales
Ochromonadaceae
Closest BLAST identities
% sim.
Site
pH
T (oC)
Hibberdiales sp.
89
BH107
1.8
68
Chrysosphaerales
96
BH105
3.2
14
Spumella-like flagellate /
clone LG22-12
96
BH105
BH107
3.2
1.8
14
68
clone RT1n9 similar to
Poterioochromonas,
Chrysocapsa
96
BSL014
2.7
25
Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703
Alveolata
Closest BLAST identities
% sim.
Site
pH T
(oC)
Ciliophora
Colpodea
Colpodea sp.
99
BH100
1.7
30
Oligohymenophorea
Oligohymenophorea sp.
91
BH100
1.7
30
Scuticociliatia similar to
Philasterides, Miamiensis
90
BH100
1.7
30
Spirotrichea sp.
97
BH107
1.8
68
Metopus sp. / clone
A1_E041
98
USW127
4.7
36
Scuticociliatia
Spirotrichea
Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703
Opisthokonts
Closest BLAST identities
% sim.
Site
pH
T
(oC)
Fungi sp.
96 / 90
DK110
4.5
60
Chytridiomycota sp.
91
BH107
1.8
68
Rhizophydium sp.
99
USW127
4.7
36
mitosporic Ascomycota sp. /
clone RT5iin23
95
DK107
mat
Ascomycota sp. / clone RT3n5
99
BSL014
2.7
25
Pezizomycotina sp.
97
USW126
5.8
45
Candida sp.
89
BH106
3.2
15
Basidiomycota
clone dpeuk9 / Basidiomycota
sp.
90
BH107
1.8
68
Choanoflagellida
clone P1.39 / Choanoflagellida
sp
95 / 93
SW105
5.1
15
Fungi
Chytridiomycota
Ascomycota
Opisthokonts
Closest BLAST identities
% sim.
Site
pH
T
(oC)
Nematoda
clone 18S-AK-B-43 /
Tripyloidea sp.
88-95
USW126
5.8
45
Annelida
Oligochaeta sp.
96
USW126
5.8
45
Platyhelminthes
Stenostomum sp.
98
USW123
3.3
25
Arthropoda
Hexapoda
Coleoptera
Polyphaga
Hydrophilidae sp.
98
DK112
DK116
3.0
4.0
45
22
Symphypleona sp.
96
95
96
DK110
DK107
BSL013
4.5 60
mat 45
4.0
Metazoa
Collembola
Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703
Classification
Closest BLAST identities
% sim.
Site
pH
T
(oC)
Euglenozoa
Kinetoplastida
Bodonidae
Bodo sp.
92-94
BH100
1.7
30
Euamoebida
Hartmannella sp.
99
USW124
5.3
44
Echinamoeba thermarum
99
DK105
mat
Protacanthamoeba / Acanthamoeba
sp.
95
DK107
mat
Soil amoebae AND32 /
Acanthamoeba sp.
99
BH100
1.7
30
Vampyrellidae sp.
93
BH107
1.8
68
BH100
1.7
30
Cercozoa
clone 18S-AK-B-47 / Cercozoa sp.
clone RT5iin44 / Cercozoa sp.
Observations
1. Few planktonic forms in streams; most benthic
2. Photosynthetic acidophiles dominate
3. Heterotrophic taxa include alveolates, amoebae,
flagellates and fungi
4. Many sequences highly similar to cultured isolates, Rio
Tinto clone library
5. Some unclassified / deeply branching organisms
6. No clear correspondence between sites, or site
conditions, and genetic diversity
Classification
Closest BLAST identities
% sim.
clone library
set (RFLP ID,
#clones)
BH107:
Viridiplantae
pH 1.8
68 oC
Chlorophyta
Chlamydomonadales
Chlamydomonadales sp.
97-99
1(4)
Pinnularia sp. / clones RT7iin2 or RT7in48
97
2(D2), 3G
Aulacoseira sp.
98
2(A9), 3(D)
Hibberdiales sp.
89
3(E)
Spumella-like flagellate /
clone LG22-12
96
2(A4), 3(E)
Spirotrichea sp.
97
3(I)
Chytridiomycota
Chytridiomycota sp.
91
2(E)
Basidiomycota
clone dpeuk9 / Basidiomycota sp.
90
2(I)
Vampyrellidae sp.
93
2(C)
Stramenopiles
Bacillariophyta
Bacillariophyceae
Bacillariophycidae
Naviculales
Coscinodiscophyceae
Chrysophyceae
Alveolata
Ciliophora
Spirotrichea
Opisthokonts
Fungi
Cercozoa
clone 18S-AK-B-47 / Cercozoa sp.
Problems and biases
1.
2.
3.
Incomplete survey
Biases in extraction, amplification and cloning
Are the genes from living organisms?
Acknowledgements
Patty Siering and Mark Wilson
Rachel Whitaker, Scott Dawson
Humboldt State University
UC Berkeley
CSUC graduate students: Patricia Brown
CSUC undergraduate students: Mary Ellen Sanders
CSUC classes: Microbial Ecology, Microbiology
Lassen Volcanic National Park
Trophic Structure in Acidic Hydrothermal Sites
What are biotic limits to primary production?
Lots of ‘meat on the hoof’: chemosynthesis and photosynthesis
At times, high biomass from chemosynthetic environments
Factors that might affect viral ecology:
abiotic conditions
environmental stability
host diversity
environmental vs. host conditions
Protist virus biology
Viruses now known for chlorophytes, stramenopiles, haptophytes, alveolates
Suttle, 2005. Viruses in the sea. Nature 437: 356
Some unusually large genomes (300 – 1200 kb)
Unusual genes (transcription factors, K ion channels, signal transduction factors)
Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703
Paramecium bursaria chlorella virus (PBCV-1)
large, icosahedral, plaque-forming, dsDNA viruses
Chlorella host is a hereditary endosymbiont of the
ciliate Paramecium bursaria.
http://plantpath.unl.edu/facilities/virology/intro.html
prototype of a group (family Phycodnaviridae, genus Chlorovirus)
NC64A viruses (type = PBCV-1): infect Chlorella-NC64A isolated from
Paramecium bursaria originally collected in the southeastern
United States.
Pbi viruses (type = CVA-1): infect Chlorella-Pbi isolated from
Paramecium bursaria originally collected in Germany.
Hydra viruses (type = HVCV-1): infect Chlorella originally isolated
from Hydra viridis. This alga host was never cultured presumably
because the virus is either lysogenic or integrates into the host
genome.
Infection of Chlorella strain NC64A by PBCV-1.
(A) Viral particle in close proximity to the alga
(B and C) Attachment of PBCV-1 to the algal wall and
digestion of the wall at the point of attachment
(D) Viral DNA beginning to enter the host
(E) An empty viral capsid remaining on the surface of
the host
(F) PBCV-1 attachment and dissolution of a Chlorella
cell wall fragment. Note that (i) viral attachment
always occurs on the external side of the wall (i.e., the
internal side of the wall curls inward) and (ii) DNA is
not released from viral particles attached to wall
fragments. Size markers in panel E and F represent
100 nm and 200 nm, respectively.
http://plantpath.unl.edu/facilities/virology/intro.html
330,744-bp genome:
• multiple DNA methyltransferases and
DNA site-specific endonucleases
• part, if not the entire machinery to
glycosylate glycoproteins
• at least two types of introns : a selfsplicing intron in a transcription factorlike gene and a splicesomal processed
type of intron in its DNA polymerase
Eukaryote vs. prokaryotes as hosts for viruses in
acidic thermal sites
Eukaryotes: dominant photoautotrophs below pH 4
Cyanidium sp. from BSL
One of the smallest known eukaryotic genomes
(Cyanidioschyzon merolae: 16.5 Mb)
Messerli et al., 2005. Life at acidic pH imposes an increased
energetic cost for a eukaryotic acidophile. J. Exp. Biol. 208: 2569
Cytosolic pH of acidophilic eukaryotes is ~neutral
pH 2
pH 6
pH 2
pH 6
Conclusions
1. Hydrothermal systems are highly dynamic, variable over time and
space, and contain multiple habitats
2. Diverse protist communities in LVNP hydrothermal sites: likely viral
hosts
3. Impact of viruses on hydrothermal acidophilic eukaryotes completely
unknown. Work in other aquatic systems suggests likely impact.