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

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 works2 = Bumpass Hell

3 = Devil’s Kitchen4 = Boiling Springs Lake

Bumpass Hell: pH 1.4-3.2

11-68 oC

Upper Sulfur works: pH 2, 96 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?

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

Low vs. high18S rRNA RFLP diversity

pH 4.6, 37 C

pH 1.8, 68 C

pH 1.7, 30 C

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

Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703

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

Cyanidium-like alga cultivated from Boiling Springs Lake

Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703

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 

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

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

Baldauf, 2003. The deep roots of eukaryotes. Science 300: 1703

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

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

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

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.

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

pH 6

pH 2

pH 6pH 2

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.