1. 0.1
Salpingoeca infusonum
Monosiga brevicollis
Monosigasp. strain BSZ6
Acanthocoepsis unguiculata
Diaphanoeca grandis
Dermocystidium salmonis
Rosette agent of Chinook salmon
Rhinosporidium seeberi
Anurofeca richardsi
Ichthyophonus hoferi
Psorospermium haeckelii
Chytridium confervae
Neocallimastix frontalis
Spizellomyces acuminatus
Bullera crocea
Saccharomyces castellii
Geosmithia putterillii
Neurospora crassa
Ancyromonas sigmoidesATCC50267
Apusomonas proboscidea
Cercomonassp. ATCC50316
Thaumathomonassp.
Heteromita globosa
Massisteria marinaATCC50266
Massisteriasp. strain GBB2
Massisteriasp. strain LFS1
Massisteriasp. strain CAS1
Massisteriasp. strain TPC1
Ochromonas danica
Mallomonas papillosa
Synura spinosa
Paraphysomonas vestita
Paraphysomonas foraminifera
Paraphysomonassp. strain TPC2
Aureococcus anophagefferens
Fucus distichus
Ectocarpus siliculosus
Bolidomonas pacifica
Thalassiosira eccentrica
Bacillaria paxillifer
Hypochytrium catenoides
Phytophthora megasperma
Caecitellus parvulusstrain NBH4
Caecitellus parvulusstrain EWM1
Adriamonas peritocrescens
Siluania monomastiga
Blastocystis hominis
Blastocystis
Cafeteria
sp.
Cafeteria roenbergensis
Cafeteria
sp. strain EPM1
sp. strain VENT1
Cafeteriasp. strain EWM2
Labyrinthuloides minuta
Labyrinthuloides haliotidis
Ulkenia profunda
Rhynchobodosp. ATCC 50
Leishmania tarentolae
Endotrypanum monterogeii
Herpetomonas muscarum
Phytomonassp.
Crithidia oncopelti
Blastocrithidia culicis
Bodo caudatus
Trypanoplasma borreli
Cryptobia catostomi
Dimastigella trypaniformis
Rynchomonas nasutaBSZ1
Rynchomonas nasutaCBR1
Bodo saliensATCC 50358
Dutch environmental isolate
Trypanosoma brucei
Kinetoplastid isolate LFS2
Petalomonas
cantuscygniKhawkinea quartana
Euglena gracilis
Lepocinclis ovata
Diplonema papillatum
Massisteria
Diplonema sp.
sp. strain DFS1
ANCYROMONADS
APUSOMONADS
FUNGI
DRIP's
CHOANOFLAGELLATES
CERCOMONADS
STRAMENOPILES
KINETO-
PLASTIDS
DIPLONEMIDS
EUGLENIDS
E
U
G
L
E
N
O
Z
O
99/98
A
100/100
22/53
97/99
86/73
96/94
99/95
53/64
87/89
93/93
100/100
79/45
17/56
91/55
41/18
96/51
67/70
100/100
98/84
100/96
89/91
91/95
98/98
98/100
21/
64
99/99
83/87
70/62
100/100
100/100
100/100
94/95
100/100
56/
68
100/100
85/91
51/67
100/100
88/97
92/70
100/100
100/100
67/98
87/78
98/100
57/80
70/98
94/100
44/
77
53/<50
100/100
parasitic/
pathogenic
free-
living
89/40
78/45
65/23
59/31
59/29
65/<5
ME/MP
MeanGrowthRate(perday)
Hydrostatic Pressure (Atmospheres)
vent strain EWM 1 (2500 m)
n = 3
1 atm ≈ 10 m depth
shallow-water strain NBH 4 (surface)
Caecitellus parvulus
MeanGrowthRate(perday)
Hydrostatic Pressure (Atmospheres)
vent strain BSZ 1 (2500 m)
shallow-water strain CBR 1 (surface)
n = 3
1 atm ≈ 10 m depth
Rhynchomonas nasuta
MeanGrowthRate(perday)
Hydrostatic Pressure (Atmospheres)
n = 3
1 atm ≈ 10 m depth
vent strain BSZ 6 (2500 m)
Monosiga sp.
Cu2+
, Fe 2+
, Mn 2+
, Zn 2+
pH ~4.5
Mussel/worm beds
provide a good
habitat for flagellates
(Atkins et al. 2000)
pH 6-8.2, 2-30 °C
Metal sulfide
precipitates
Vent field
H2S, HS - , S
CuFeS2
2-
ZnS
CuS2
FeS
Hypersaline ponds
Freshwater lakes,
ponds, streams
Terrestrial environments
(Ekelund & Patterson 1997)
Sinking particulate
matter with flagellates
(Silver & Alldredge 1981)
Cyst or cell
entrainment
in plume waters
reseeds water
column
Hydrothermal Fluid350°C
Sulfides: Metals:
(Lee & Patterson 1998)
Heterotrophic flagellates are
integral components of
microbial food webs
(Fenchel 1982)
(Caron et al. 1982)
(Azam et al 1983)
(Patterson & Simpson 1996)
Water Column
(Caron et al. 1993)
(Patterson et al. 1993)
Deep-Sea Benthos
(Small & Gross 1985)
(Turley et al, 1988)
(Atkins et al. 1998)
Flagellate community
density decreases
with depth in both
soil and sediments
Illustration by Jack Cook, WHOI Graphics
Michael S. Atkins1
, Andreas P. Teske1
, Craig D. Taylor1
, Carl O.W irsen1
, O.Roger Anderson2
1
W oodsHoleOceanographic Institution, W oodsHoleMA, USA •2
Lamont-Doherty EatrhObsevr ator y, Palisades NY ,USA
Flagellate Growth and Survival Under Conditions
Potentially Encountered at Deep Sea Hydrothermal Vents
A S T R O B IO L O G YA S T R O B IO L O G Y
ThiwsorkissuppotredbyNASA ’sAstorbiologIynstitute CooperativAegreementwiththeMarine
Biological LaboratoyarWt oodsHoleandtheW oodsHoleOceanographIincstitution.
ABSTRACT
Eighteen strains of flagellated protists, representing 9 species from 6 taxonomic orders, were isolated
and cultured from four deep-sea hydrothermal vents. Many of the vent isolates are ubiquitous mem-
bers of marine, freshwater, and terrestrial ecosystems worldwide, suggesting a global distribution of
these flagellate species. This discovery advanced the hypothesis that ubiquity in distribution patterns
among heterotrophic flagellates implies high tolerance and/or adaptability to a wide range of environ-
mental conditions. Experiments under vent conditions of high pressure and high concentrations of
metals and sulfide showed that some of these species are very tolerant to extreme environmental con-
ditions.
Three isolates of deep-sea flagellates were grown in culture at 1-300 atm to measure their growth re-
sponse to increasing hydrostatic pressure. The growth rates of two vent flagellates, Caecitellus parvu-
lus and Rhynchomonas nasuta, were compared to the growth rates of shallow-water strains of the
same species. Deep-sea isolates of C. parvulus and R. nasuta had a higher rate of growth at higher
pressures than did their shallow-water counterparts. Vent strains of C. parvulus and R. nasuta were
capable of growth at pressures corresponding to their respective depths of collection, indicating that
these species could be metabolically active at these depths. However, C. parvulus and R. nasuta en-
cysted at pressures greater than their depth of collection. The choanoflagellate isolate was observed
to encyst at pressures greater than 50 atm.
The survival rates of three species of deep-sea hydrothermal vent flagellates were measured after ex-
posure to chemical conditions potentially encountered in vent environments. The survival rates, meas-
ured as viability through time of Caecitellus parvulus, Cafeteria sp. and Rhynchomonas nasuta were
determined and compared to shallow-water strains of the same species after exposure to increasing
concentrations of sulfide or the metals Cu, Fe, Mn and Zn. Responses were variable but in all cases
these flagellates showed very high tolerance to extreme conditions. Cafeteria spp. were remarkable in
that both strains showed 100% viability after a 24 h exposure to 30 mM sulfide under anoxic condi-
tions. By contrast, the highest naturally-occurring sulfide concentrations ever measured are only 18-
20 mM. There was little effect from metals at concentrations up to 10-3 M total metal, but a sharp de-
crease in viability occurred between 10-3 M and 10-2 M total metal, due either to a rapid increase in
the availability of free metal ions or colloid formation or both. This study is consistent with other previ-
ously reported studies that indicate these flagellate species are present and capable of being active
members of the microbial food webs at deep-sea vents.
Figure 1. Map showing locations and geo-
logical features of four deep-sea hydrother-
mal vents sampled for the present study
(adapted from Heezen and Tharp 1977).
Flagellate species and strain names are list-
ed by collection location.
Figure 2. Light, TEM and SEM micrographs of vent iso-
lates. (A) Cafeteria sp. strain VENT 1 showing mastigo-
nemes on anterior flagellum; (B) Cafeteria sp. strain EPM 1;
(C) light micrograph of Caecitellus parvulus tropwhitsh
characteristic gliding morphology; (D) Caecitellus parvulus
strain NBH 4 (arrow, acrone-matic flagellar tip); (E) light mi-
crograph of Rhynchomonas nasuta strain BSZ 1 trophonts
with characteristic proboscis; (F) Rhynchomonas untaas
strain BSZ 1; (G) light micrograph of Rhynchomonas nasuta
strain CBR 1 trophonts with characteristic proboscis; (H)
Rhyn-chomonas nasuta strain CBR 1 showing long posteri-
or and short anterior flagella and pro-boscis emerging from
groove at the base of the snout; (I) unidentified kinetoplastid
flagel-late LFS 2 showing two heterokont flagella; (J, K) thin-
section TEM images of Monosiga sp. strain BSZ 6 showing
corona of microvilli; (L) light micrograph of Monosiga sp.
strain BSZ 6 showing collar and apical flagellum; (M) apical
flagellum of Monosiga sp. strain BSZ 6; (N) mastigoneme-
covered anterior flagellum of Paraphysomonas spr.asint
TPC 2; (O, P) Ancyromonas sigmoides strains ATCC 50267
and BRM 2, respectively; (Q) detail of papillate projections
from the latero-ventral groove of Ancyromonas sigmoides
strain BRM 2. All markers = 1.0 µm.
Figure 3. Distance tree of hydrothermal vent flagellates based on analy-
sis of near-complete small subunit ribosomal DNA sequences using eu-
glenozoan flagellates as the outgroup. The evolutionary distance between
two organisms is obtained by the summation of the length of the connect-
ing branches along the horizontal axis, using the scale at the bottom.
Numbers at nodes show percent bootstrap support with distance (mini-
mum evolution) followed by maximum parsimony (1,000 replicates each).
Organisms sequenced in this study are in larger, bold font.
Figure 4. Light microscopic images of Caecitellus parvulus strain EWM 1 (A-C) and
Rhynchomonas nasuta strain BSZ 1 (D), and transmission electron microscopic im-
ages of R. nasuta motile cells (E-G) and cysts (H, I) in whole particle preparations. (A)
C. parvulus trophic cells cultured at atmospheric pressure showing normal apical and
trailing flagella. (B) A cell after two days at 300 atm showing early stages of cyst wall
formation (arrow) and resorption of flagella. Note increase in cell size. (C) A fully en-
cysted cell after 5 days at 300 atm. (D) R. nasuta trophic cells cultured at atmospheric
pressure showing typical proboscis and trailing flagellum. (E) A carbon-platinum, shad-
owed flagellum (F) with trailing 30 nm thick filaments (arrow) and characteristic swollen
tip (T). (F) Negatively stained motile cell showing the proboscis (P) and curved flagel-
lum (F) with a densely-stained, rod-shaped bacterium near the tip. (G) Carbon-plati-
num, shadowed motile cell with curved flagellum (F). (H) Carbon-platinum, shadowed
cyst (C), with a smooth surface, casting a typical shadow (S) for a spheroidal body. (I)
An enlarged view of the edge of a cyst showing the smooth surface with a thin nega-
tively stained outer layer (arrow). Scale bars in (A), (B), and (D) µ5m; (C) 2µm; (E)
and (I) 0.3 µm; and (F-H) 2 µm.
Figure 8. Ultrathin sections of choanoflagellates cultured at ambient atmospheric pres-
sure (A) and at 300 atm (B-E). (A) Normal cell with prominent nucleus (N), mitochondria
with flattened cristae and lightly granular matrix (M), osmiophilic, reserve bodies that ap-
pear to be lipid (L), and digestive vacuoles (V) containing early stages of digested food.
(B) Pressure-treated cell with almost normal appearance compared to (A) showing,
however, a somewhat more irregularly-shaped nucleus (N), some reserve bodies (L),
and digestive vacuoles (V) mainly in late stages. (C) A cell showing more advanced evi-
dence of encystment (note light deposit of granular material on the cell surface, arrow)
with irregularly shaped nucleus (N), enlarged digestive vacuoles with loosely arranged
membranous components and few dense reserve bodies. (D) A series of cells showing
signs of increasing encystment (right to left). The nucleus (N) is smaller and more irreg-
ular in shape. Digestive vacuoles (V), when present, are in late stages with only mem-
branous matter; the surface of the cell is increasingly enclosed by an electron-dense
granular deposit that appears to be an early stage of cyst wall deposition (CW). (E) An
electron-opaque section of a wall, apparently a fully-formed cyst, exhibiting a brittle
quality and smooth outer surface as is also characteristic of kinetoplastid cysts as in Fig-
ure 4 I. Scale bars in (A) and (E) 0.5 µm, others 1 µm.
Figure 5. Mean growth rates of Caecitellus
parvulus strain EWM1 from 2500 m and
strain NBH4 from a shallow-water location
with increasing hydrostatic pressure (Error
bars are– 1 SD). Cultures grown at > 250
atm underwent reversible encystment.
Figure 6. Mean growth rates of Rhynchomo-
nas nasuta strain BSZ1 from 2500 m and
strain CBR1 from a shallow-water location
with increasing hydrostatic pressure (Error
bars are– 1 SD). Cultures grown at > 250
atm underwent reversible encystment.
Figure 7. Mean growth rates of the choano-
flagellate Monosiga sp. strain BSZ6 from
2500 m with increasing hydrostatic pressure
(Error bars are – 1 SD). Cultures grown at >
50 atm underwent reversible encystment.
Figure 9 (left). Survival in sulfide toxicity experiments. Deep-sea vent strains are in the left column; shallow-water strains are in the right col-
umn. All sulfide concentrations shown in the figure legend were tested on each organism; overlaying of lines occurred at lower concentrations
of sulfide for Caecitellus and Rhynchomonas up to 24 hr and for all concentrations of sulfide up to 24 hr for Cafeteria.
Figure 10 (above). Survival in metal toxicity experiments. Metals concentrations represent total metals. All metals concentrations shown in
the figure legend were tested on each organism; overlaying of lines occurred at lower concentrations of all metals. (A) C. parvulus strain
EWM 1; (B) C. parvulus strain NBH4; (C) Cafeteria sp. strain VENT1; (D) Cafeteria sp. strain EPM1; (E) R. nasuta strain CBR1.
Figure 11. A diagram summarizing the results of this work, which support the hypothesis
that ubiquity in occurrence patterns among heterotrophic flagellates implies high tolerance
and/or adaptability to a wide range of environmental conditions.
ViableCell/1E5Cells
Time (Days)
Total MetalsCu Fe Mn ZnA
B
C
D
E