0.1 Salpingoeca infusonum Monosiga brevicollis Monosiga sp. 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 sigmoides ATCC50267 Apusomonas proboscidea Cercomonas sp. ATCC50316 Thaumathomonas sp. Heteromita globosa Massisteria marina ATCC50266 Massisteria sp. strain GBB2 Massisteria sp. strain LFS1 Massisteria sp. strain CAS1 Massisteria sp. strain TPC1 Ochromonas danica Mallomonas papillosa Synura spinosa Paraphysomonas vestita Paraphysomonas foraminifera Paraphysomonas sp. strain TPC2 Aureococcus anophagefferens Fucus distichus Ectocarpus siliculosus Bolidomonas pacifica Thalassiosira eccentrica Bacillaria paxillifer Hypochytrium catenoides Phytophthora megasperma Caecitellus parvulus strain NBH4 Caecitellus parvulus strain EWM1 Adriamonas peritocrescens Siluania monomastiga Blastocystis hominis Blastocystis Cafeteria sp. Cafeteria roenbergensis Cafeteria sp. strain EPM1 sp. strain VENT1 Cafeteria sp. strain EWM2 Labyrinthuloides minuta Labyrinthuloides haliotidis Ulkenia profunda Rhynchobodo sp. ATCC 50 Leishmania tarentolae Endotrypanum monterogeii Herpetomonas muscarum Phytomonas sp. Crithidia oncopelti Blastocrithidia culicis Bodo caudatus Trypanoplasma borreli Cryptobia catostomi Dimastigella trypaniformis Rynchomonas nasuta BSZ1 Rynchomonas nasuta CBR1 Bodo saliens ATCC 50358 Dutch environmental isolate Trypanosoma brucei Kinetoplastid isolate LFS2 Petalomonas cantuscygni Khawkinea 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 Mean Growth Rate (per day) Hydrostatic Pressure (Atmospheres) vent strain EWM 1 (2500 m) n = 3 1 atm ≈ 10 m depth shallow-water strain NBH 4 (surface) Caecitellus parvulus Mean Growth Rate (per day) Hydrostatic Pressure (Atmospheres) vent strain BSZ 1 (2500 m) shallow-water strain CBR 1 (surface) n = 3 1 atm ≈ 10 m depth Rhynchomonas nasuta Mean Growth Rate (per day) Hydrostatic Pressure (Atmospheres) n = 3 1 atm ≈ 10 m depth vent strain BSZ 6 (2500 m) Monosiga sp. Cu 2+ , 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 H 2 S, HS - , S CuFeS 2 2- ZnS CuS 2 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 Fluid 350°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. Atkins 1 , Andreas P. Teske 1 , Craig D. Taylor 1 , Carl O.W irsen 1 , O.Roger Anderson 2 1 W oods HoleOceanographic Institution, W oods HoleMA, USA • 2 Lamont-Doherty EatrhObsev r ator y, Palisades NY ,USA Flagellate Growth and Survival Under Conditions Potentially Encountered at Deep Sea Hydrothermal Vents A STR O B IO LO G Y A STR O B IO LO G Y Thiw s orkisuppotredbyNASA ’ sAsto rbiologIynstitute CooperativAegreementwith theMarine Biological Laboratoya r W t oodsHoleandtheW oodsHoleOceanographIin c 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 tropwh itsh 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.as in t 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. Viable Cell/ 1E5 Cells Time (Days) Total Metals Cu Fe Mn Zn A B C D E