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Salpingoeca infusonumMonosiga brevicollisMonosiga sp. strain BSZ6
Acanthocoepsis unguiculataDiaphanoeca grandis
Dermocystidium salmonisRosette agent of Chinook salmonRhinosporidium seeberiAnurofeca richardsiIchthyophonus hoferi
Psorospermium haeckeliiChytridium confervaeNeocallimastix frontalisSpizellomyces acuminatus
Bullera croceaSaccharomyces castellii
Geosmithia putterilliiNeurospora crassa
Ancyromonas sigmoides ATCC50267Apusomonas proboscideaCercomonas sp. ATCC50316Thaumathomonas sp.Heteromita globosa
Massisteria marina ATCC50266Massisteria sp. strain GBB2
Massisteria sp. strain LFS1Massisteria sp. strain CAS1Massisteria sp. strain TPC1
Ochromonas danicaMallomonas papillosaSynura spinosaParaphysomonas vestitaParaphysomonas foraminiferaParaphysomonas sp. strain TPC2
Aureococcus anophagefferensFucus distichusEctocarpus siliculosusBolidomonas pacificaThalassiosira eccentricaBacillaria paxilliferHypochytrium catenoidesPhytophthora megasperma
Caecitellus parvulus strain NBH4Caecitellus parvulus strain EWM1
Adriamonas peritocrescensSiluania monomastiga
Blastocystis hominisBlastocystis
Cafeteria
sp.
Cafeteria roenbergensis
Cafeteria sp. strain EPM1 sp. strain VENT1
Cafeteria sp. strain EWM2
Labyrinthuloides minuta
Labyrinthuloides haliotidisUlkenia profunda
Rhynchobodo sp. ATCC 50
Leishmania tarentolaeEndotrypanum monterogeii
Herpetomonas muscarumPhytomonas sp.
Crithidia oncopeltiBlastocrithidia culicis
Bodo caudatus
Trypanoplasma borreliCryptobia catostomi
Dimastigella trypaniformisRynchomonas nasuta BSZ1Rynchomonas nasuta CBR1
Bodo saliens ATCC 50358
Dutch environmental isolate
Trypanosoma brucei
Kinetoplastid isolate LFS2
PetalomonascantuscygniKhawkinea quartana
Euglena gracilisLepocinclis ovata
Diplonema papillatum
Massisteria
Diplonema sp.
sp. strain DFS1
ANCYROMONADSAPUSOMONADS
FUNGI
DRIP's
CHOANOFLAGELLATES
CERCOMONADS
STRAMENOPILES
KINETO-PLASTIDS
DIPLONEMIDS
EUGLENIDS
EUGLENOZO
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
100/100100/100
100/100
94/95
100/100
56/68
100/10085/91
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
Me
an
Gro
wth
Ra
te (
pe
r d
ay
)
Hydrostatic Pressure (Atmospheres)
vent strain EWM 1 (2500 m)
n = 31 atm ≈ 10 m depth
shallow-water strain NBH 4 (surface)
Caecitellus parvulus
Me
an
Gro
wth
Ra
te (
pe
r d
ay
)
Hydrostatic Pressure (Atmospheres)
vent strain BSZ 1 (2500 m)
shallow-water strain CBR 1 (surface)
n = 31 atm ≈ 10 m depth
Rhynchomonas nasutaM
ea
n G
row
th R
ate
(p
er
day
)
Hydrostatic Pressure (Atmospheres)
n = 31 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 sulfideprecipitates
Vent field
H2S, HS - , S
CuFeS2
2-
ZnSCuS2
FeS
Hypersaline pondsFreshwater lakes,
ponds, streams
Terrestrial environments(Ekelund & Patterson 1997)
Sinking particulatematter with flagellates
(Silver & Alldredge 1981)
Cyst or cellentrainment
in plume watersreseeds water
column
Hydrothermal Fluid350°C
Sulfides: Metals:
(Lee & Patterson 1998)
Heterotrophic flagellates areintegral 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 communitydensity decreases
with depth in bothsoil and sediments
Illustration by Jack Cook, WHOI Graphics
Michael S. Atkins 1, Andreas P. Teske1, Craig D. Taylor1, Carl O.W irsen1, O.R oger Anderson2
1W oods HoleO ceanographic Institution, W oods HoleM A, USA • 2 Lamont-Doherty EatrhO bsevr 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 YThiws orki ssu ppotredb yN ASA ’sA storbiologIyn stitute CooperativAeg reementw ithth eM arine
Biological Laboratoyar Wt oodsH olea ndt heW oodsH oleO ceanographIinc stitution.
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 tro pwhitsh 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.a sint 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) 2 µm; (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.
Via
ble
Cell
/ 1E
5 C
ell
s
Time (Days)
Total MetalsCu Fe Mn ZnA
B
C
D
E
