Extreme Eukaryotes in the Hydrothermal Environments of Lassen Volcanic National
Park
Gordon Wolfe
Dept. Biological SciencesCalifornia 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
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
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?
18S rDNA eubacterial 16S rDNA
USW 123pH 3.3, 25
C
SW114pH 1.9, 69
C
USW 123pH 3.3, 25
C
SW114pH 1.9, 69
C
CTAB UC FD
0
1
2
3
4
5
6
0 20 40 60 80 100p
H
Bumpass Hell
a
0
1
2
3
4
5
6
0 20 40 60 80 100
T (oC)
pH
Devil's Kitchen c
0
1
2
3
4
5
6
0 20 40 60 80 100
pH
Sulfur Works / Upper Sulfur Works
b
0%
10%
20%
30%
40%
50%
60%
70%
80%
BH100
BH105
BH105
BH106
BH107
BH107
BH118
BH118
DK102
DK105
DK107
DK110
DK110
DK112
DK112
DK116
DK116
USW123
USW123
USW124
USW124
USW125
USW126
USW127
site
% u
niq
ue
RF
LP
clo
nes
0%
10%
20%
30%
40%
50%
60%
70%
80%
0 2 4 6 8
site pH
% c
lone
s w
ith
uniq
ue R
FL
Ps
0%
10%
20%
30%
40%
50%
60%
70%
80%
0 20 40 60 80
site T (oC)%
clo
nes
wit
h un
ique
RF
LP
s
Viridiplantae Closest BLAST identities % sim. Site pH T (oC)
Chlorophyta
Chlorophyceae Chlorophyceae sp. 97 USW124BH105
5.33.2
4414
Chlamydomonadales
Chlamydomonadales sp. 97-99 BH107BSL101
1.82.5
6837
Chlamydomonadaceae
Chlamydomonadaceae sp. 97 USW126 5.8 45
Chlamydomonas sp. 9796
SW117USW126
2.25.8
3445
Chlamydomonas acidophila / clone RT1n1
9896-99
DK102BH118
mat2.2
19
Dunaliella sp. 98 BH100 1.7 30
Haematococcaceae
Chlorogonium sp. 98 BSL008 2.4 28
Sphaeropleales Scenedesmaceae sp. / clone 18S-AK-W-5
97 USW124 5.3 44
Scenedesmus sp. USW124 5.3 44
Viridiplantae Closest BLAST identities % sim. Site pH T (o
C)
Chlorophyta
Trebouxiophyceae Chlorellales
Chlorellales sp. 95 USW126 5.8 45
Chlorellaceae Chlorella sp. 99 DK107 mat
Chlorella sp. / Nannochloris sp.
99989998
BH100USW124DK116DK112
1.75.34.03.0
30442245
Streptophyta 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
Rhodophyta Closest BLAST identities
% sim.
Site pH
T (oC)
Bangiophyceae Cyanidiaceae
Cyanidioschyzon merolae 99 BSL006BSL009
2.3
53
Stramenopiles Closest BLAST identities % sim.Sit
e pH T (oC)
Bacillariophyta Bacillariophyceae
Bacillariophycidae Naviculales
Pinnularia sp. / clones RT7iin2 or RT7in48
9596969795979795939593
USW123
BH105BH106BH107DK112DK116USW12
5USW12
4USW12
6USW12
7BSL00
8DK110
3.33.23.21.83.04.03.35.35.84.72.44.5
25
14
15
68
45
22
44
44
45
36
28
60
Pinnularia sp. BH105USW12
7
3.24.7
14
36
Coscinodiscophyceae
Aulacoseira sp. 989896
BH105BH106BH107
3.23.21.8
14
15
68
StramenopilesClosest BLAST identities % sim. Site pH
T (oC)
Chrysophyceae Hibberdiales sp. 89 BH107 1.8 68
Chrysosphaerales 96 BH105 3.2 14
Spumella-like flagellate / clone LG22-12
96 BH105BH107
3.21.8
1468
Ochromonadales Ochromonadaceae
clone RT1n9 similar to Poterioochromonas, Chrysocapsa
96 BSL014
2.7 25
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
Scuticociliatia similar to Philasterides, Miamiensis
90 BH100 1.7
30
Spirotrichea Spirotrichea sp. 97 BH107 1.8
68
Metopus sp. / clone A1_E041
98 USW127
4.7
36
Opisthokonts Closest BLAST identities % sim. Site pH T (oC)
Fungi Fungi sp. 96 / 90 DK110 4.5 60
Chytridiomycota Chytridiomycota sp. 91 BH107 1.8 68
Rhizophydium sp. 99 USW127 4.7 36
Ascomycota 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
Opisthokonts Closest BLAST identities
% sim. Site pH T (oC)
Metazoa
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 DK112DK116
3.04.0
4522
Collembola
Symphypleona sp. 969596
DK110DK107BSL013
4.5mat4.0
6045
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
Cercozoa Vampyrellidae sp. 93 BH107 1.8 68
clone 18S-AK-B-47 / Cercozoa sp.
clone RT5iin44 / Cercozoa sp. BH100 1.7 30
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)
Viridiplantae
Chlorophyta
Chlamydomonadales Chlamydomonadales sp. 97-99 1(4)
Stramenopiles
Bacillariophyta Bacillariophyceae Bacillariophycidae Naviculales
Pinnularia sp. / clones RT7iin2 or RT7in48
97 2(D2), 3G
Coscinodiscophyceae
Aulacoseira sp. 98 2(A9), 3(D)
Chrysophyceae Hibberdiales sp. 89 3(E)
Spumella-like flagellate / clone LG22-12
96 2(A4), 3(E)
Alveolata
Ciliophora
Spirotrichea Spirotrichea sp. 97 3(I)
Opisthokonts
Fungi
Chytridiomycota Chytridiomycota sp. 91 2(E)
Basidiomycota clone dpeuk9 / Basidiomycota sp. 90 2(I)
Cercozoa Vampyrellidae sp. 93 2(C)
clone 18S-AK-B-47 / Cercozoa sp.
BH107:
pH 1.8 68 oC
Problems and biases
1. Incomplete survey2. Biases in extraction, amplification and cloning3. Are the genes from living organisms?
Patty Siering and Mark Wilson Humboldt State UniversityRachel Whitaker, Scott Dawson UC Berkeley
CSUC graduate students: Patricia BrownCSUC undergraduate students: Mary Ellen SandersCSUC classes: Microbial Ecology, Microbiology
Lassen Volcanic National Park
Acknowledgements
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
Some unusually large genomes (300 – 1200 kb)Unusual genes (transcription factors, K ion channels, signal transduction factors)
Suttle, 2005. Viruses in the sea. Nature 437: 356
Paramecium bursaria chlorella virus (PBCV-1)
large, icosahedral, plaque-forming, dsDNA viruses
Chlorella host is a hereditary endosymbiont of the ciliate Paramecium bursaria.
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.
http://plantpath.unl.edu/facilities/virology/intro.html
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 self-splicing intron in a transcription factor-like gene and a splicesomal processed type of intron in its DNA polymerase
Eukaryotes: dominant photoautotrophs below pH 4
Eukaryote vs. prokaryotes as hosts for viruses in acidic thermal sites
One of the smallest known eukaryotic genomes (Cyanidioschyzon merolae: 16.5 Mb)
Cyanidium sp. from BSL
Cytosolic pH of acidophilic eukaryotes is ~neutral
Messerli et al., 2005. Life at acidic pH imposes an increased energetic cost for a eukaryotic acidophile. J.
Exp. Biol. 208: 2569
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.