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ASLO 2001 Poster (Black)

This document summarizes a study that analyzed the diversity of flagellated protists at four deep-sea hydrothermal vents in the eastern Pacific Ocean using both culture-dependent and culture-independent methods. Eighteen strains from nine species were isolated in culture and belonged to six taxonomic orders. Molecular analysis using PCR and DGGE supported the culture findings and detected additional species. Experiments showed some species have high tolerance to extreme vent conditions like high pressure, sulfide, and metals. Combining culture and molecular methods provided a more comprehensive assessment of flagellate diversity at these remote deep-sea vent environments.

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T = 69░C T = 70░C M 1,000 500 250 base pair s 10,000 1,000 500 250 base pair s 10,000 1,000 500 250 base pair s 10,000 1,000 500 250 base pair s 10,000 T = 68 ░C T = 68.5 ░C T = 69 ░C T = 70 ░C M 1 2 3 4 5 6 7 8 9 10 11 12 M 13 14 15 16 17 18 19 20 21 22 23 Neg Vent Juan De Fuca 21░N 9░N Specific Site LFS CAS BRM BSZ Organism Bodo caudatus Bodo saliens D C D Rhynchomonas nasuta D D C D Isolate LFS2 C D Other euglenozoa Non-euglenozoa C (2) C (3) C (1) C (3) C = cultured D = DGGE1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 ViableCells/1E5Cells CuFeMnZn 0 1 3 7 0 1 3 7 0 1 3 7 0 1 3 7 Caecitellus parvulus strain NBH4 Cafeteria sp. strain EPM1 Cafeteria sp. strain VENT1 Rhynchomonas nasuta strain CBR1 0 M 1E-5 M 1E-4 M 1E-3 M 1E-2 M Time (Days) Culturing Molecular Analysis Microscopical Identification Physiological Experiments DNASequencing Database Comparison & Identification Primer & Probe Design DGGE PCRAmplification of Total Community DNA ╒ s PCRAmplification of Clade-Specific DGGE Fragments DGGE DNASequencing Southern Blotting Probe Hybridization 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. strainTPC1 Ochromonas danica Mallomonas papillosa Synura spinosa Paraphysomonas vestita Paraphysomonas foraminifera Paraphysomonassp. strainTPC2 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 Blastocystissp. Cafeteriasp. strain EPM1 Cafeteria roenbergensis Cafeteriasp. strainVENT1 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 Diplonemasp. Massisteriasp. strain DFS1 ANCYROMONADS APUSOMONADS FUNGI DRIP's CHOANOFLAGELLATES CERCOMONADS STRAMENOPILES KINETO- PLASTIDS DIPLONEMIDS EUGLENIDS E U G L E N O Z O A 99/98 100/100 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 Cu2+ , Fe2+ , Mn 2+ , Zn2+ 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-, S2- CuFeS2 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 J ack Cook,WHOI Graphics 62íC 67íC 72íC 68íC 62íC 67íC 72íC 68íC Euglena gracilis Bodo caudatus Caecitellus parvulus strain EWM1 Monosiga sp. strain BSZ6 Cafeteria sp. strain EWM2 Massisteria marina strain LFS1 Mallomonas papillosa Bodo caudatus Bodo saliens M ixed PC R Products BioventR iftia and M ussels (9░N ) BioventSurpulid Zone (9░N ) Lobo Flange Substrate (JD F) C lam Acres Spire (21░N ) M ixed PC R Products R hynchom onas nasuta Isolate LFS2 Euglena gracilis Environmental Samples BRM band 1= Bodo saliens BRM band 2= Rhynchomonas nasuta BSZ band 1= Rhynchomonas nasuta LFS band 1= Isolate LFS2 CAS band 1= Bodo saliens CAS band 2= Rhynchomonas nasuta Results of sequencing DGGE bands: Favella* Balanion* Thalassiosira* Rhizosolenia* Umbilicosphaera* Thoracosphaera* Synechococcus* Prochlorococcus* Caecitellusá Cafeteriaá Rhynchomonasá Toxicity Threshold of Free Copper (M) 10 101010101010101010 10 1010 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 * as determined by 50% decrease in growth rate á as determined by 50% decrease in survival rate ASSESSMENT OF FLAGELLATE DIVERSITY AT DEEP-SEA HYDROTHERMAL VENTS USING THE COMBINED APPROACH OF CULTURE-DEPENDENT AND CULTURE-INDEPENDENT METHODS Michael S. Atkins1, Andreas P. Teske1, Craig D. Taylor1, Carl O. Wirsen1, and O. Roger Anderson2 1Woods Hole Oceanographic Institution, Woods Hole, MA USA 2Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY USA Funding for this research was provided in part by: The National Science Foundation The Ocean Ventures Fund/Woods Hole Oceanographic Institution The Rhinehart Coastal Research Center The PADI Foundation Publications from this work: Atkins, M.S. and A.P. Teske. Detection and distribution patterns of kinetoplastid flagellates at deep-sea hydrothermal vents as determined by cul turing and denaturing gradient gel electrophoresis. Applied and Environmental Microbiology, in preparation. Atkins, M.S., M.A. Hanna, E.A. Kupetsky, M.A. Saito, C.D. Taylor and C.O. Wirsen. Tolerance of flagellated protozoa to extreme environmental conditions potentially encountered at deep-sea hydrothermal vents: I. High sulfide; II. High concentrations of Cu, Fe, Mn, and Zn. Marine Ecol ogy Progress Series, submitted. Atkins, M.S., A.G. McArthur and A.P. Teske. 2000b. Ancyromonadida: a new phylogenetic lineage among the protozoa closely related to the com mon ancestor of Metazoans, Fungi, and Choanoflagellates (Opisthokonta). Journal of Molecular Evolution 51:278-285. Atkins, M.S., A.P. Teske and O.R. Anderson. 2000a. A survey of flagellate diversity at four deep-sea hydrothermal vents in the Eastern Pacific Ocean using structural and molecular approaches. Journal of Eukaryotic Microbiology 47(4):400-411. Atkins, M.S., O.R. Anderson and C.O. Wirsen. 1998. Effect of hydrostatic pressure on the growth rates and encystment of flagellated protozoa isolated from a deep-sea hydrothermal vent and a deep shelf region. Marine Ecology Progress Series 171: 85-95. Abstract: Eighteen strains of flagellated protists representing 9 species were isolated and cultured from four deep-sea hydrother mal vents in the Eastern Pacific Ocean: Juan de Fuca Ridge, Guaymas Basin, and both 21 N and 9 N on the East Pacific Rise (EPR). The hydrothermal vent flagellates belonged to six different taxonomic orders: the Ancyro monadida, Bicosoecida, Cercomonadida, Choanoflagellida, Chrysomonadida, and Ki netoplastida. Many of the vent isolates were ubiquitous members of marine, freshwater, and terrestrial ecosystems worldwide, sug gesting a global distribution of these flagel late species. This discovery advanced the hy pothesis that ubiquity in distribution patterns among heterotrophic flagellates implies high tolerance and/or adaptability to a wide range of environmental 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 conditions. Deep-sea vent samples were both cultured to select for kinetoplastid flagellates and ana lyzed without culturing by denaturing gradient gel electrophoresis (DGGE) using PCR pri mers specific to the kinetoplastid clade. By comparing these two different methods of analysis, my goal was to decrease the biases and/or errors inherent in either method alone and to improve our ability to assess flagellate diversity and distribution in samples from re mote vent environments. PCR and DGGE were used to specifically isolate and amplify target DNA's from all cultured kinetoplastid species in matching vent samples, thus cor roborating the findings of culturing. Molecu lar methods had the additional ability to de tect species presence where culturing did not, thereby providing a better indication of the distribution of these species. Species Strain Collection Location Vent Ancyromonadida, Cavalier-Smith, 1998 Ancyromonas sigmoides, Kent, 1880 50267 American Type Culture Collection Ancyromonas sigmoides, Kent, 1880 BRM2 Biovent Riftia and Mussels Bed 9N Bicosoecida, GrassÄ and Deflandre, 1952 Cafeteria sp. VENT1 9N vent water - H2S reactors 9N Cafeteria sp. EWM2 East Wall Mussels Bed 9N Cafeteria sp. EPM1 Eel Pond Marsh - H2S reactors surface Caecitellus parvulus, Patterson et al.,1993 BSZ7 Biovent Serpulid Zone 9N Caecitellus parvulus, Patterson et al.,1993 EWM1 East Wall Mussels Bed 9N Caecitellus parvulus, Patterson et al.,1993 NBH4 New Bedford Harbor, MA surface Cercomonadida, Vickerman, 1983 Massisteria marina, Larsen and Patterson, 1990 50266 American Type Culture Collection Massisteria marina, Larsen and Patterson, 1990 BSZ3 Biovent Serpulid Zone 9N Massisteria marina, Larsen and Patterson, 1990 GBB2 Guaymas Basin Beggiotoa Mat GBB Massisteria marina, Larsen and Patterson, 1990 DFS1 Dante Flange Substrates JDF Massisteria marina, Larsen and Patterson, 1990 LFS1 Lobo Flange Substrates JDF Massisteria marina, Larsen and Patterson, 1990 CAS1 Clam Acres Spire 21N Massisteria marina, Larsen and Patterson, 1990 TPC1 Twin Peaks Chimney 21N Choanoflagellida, Kent, 1880 Monosiga sp. BSZ6 Biovent Serpulid Zone 9N Chrysomonadida, Engler, 1898 Paraphysomonassp. TPC2 Twin Peaks Chimney 21N Kinetoplastida, Honigberg, 1963 Rhynchomonas nasuta, Klebs, 1892 CBR1 Chesapeake Bay, MD surface Rhynchomonas nasuta, Klebs, 1892 BSZ1 Biovent Serpulid Zone 9N Rhynchomonas nasuta, Klebs, 1892 BSZ2 Biovent Serpulid Zone 9N Rhynchomonas nasuta, Klebs, 1892 BSZ8 Biovent Serpulid Zone 9N Bodo saliens, Larsen and Patterson, 1990 50358 American Type Culture Collection Bodo saliens, Larsen and Patterson, 1990 BRM1 Biovent Riftia and Mussels Bed 9N Unidentified LFS2 Lobo Flange Substrates JDF Caecitellus parvulus strain EWM1 Cafeteria sp. strain VENT1 Rhynchomonas nasuta strain BSZ1 Caecitellus parvulus strain NBH4 Cafeteria sp. strain EPM1 Rhynchomonas nasuta strain CBR1 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 0 1 3 6 24 0 1 3 6 24 0 1 3 6 24 0 1 3 6 24 0 1 3 6 24 168 0 1 3 6 24 168 Time (hours) Time (hours) ViableCells/1E5Cells 0.0 mM 1.0 mM 10.0 mM 0.1 mM 2.0 mM 20.0 mM 0.5 mM 5.0 mM 30.0 mM 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1 50 100 150 200 250 300 1 50 100 150 200 250 300 1 50 100 150 200 250 300 1.5 1.0 0.5 0.0 0.5 0.4 0.3 0.2 0.1 0.0 Vent Shallow Caecitellus parvulus Rhynchomonas nasuta Monosiga sp. MeanGrowthRatePerDay MeanGrowthRatePerDay MeanGrowthRatePerDay Hydrostatic Pressure (Atmospheres) Hydrostatic Pressure (Atmospheres) Hydrostatic Pressure (Atmospheres) Figure 1. Vent map with flagellate species collected Figure 2. Flow diagram of culture-dependent and culture-independent methods used on vent samples in this research. Figure 3. (A) Cafeteria sp. strain VENT 1 showing mastigonemes on anterior flagellum; (B) Cafeteria sp. strain EPM 1; (C) light micrograph of Caecitellus parvulus trophs with characteristic gliding morphology; (D) Caecitellus parvu lus strain NBH 4 (arrow, acronematic flagellar tip); (E) light micrograph of Rhynchomonas nasuta strain BSZ 1 trophonts with characteristic proboscis; (F) Rhynchomonas nasuta strain BSZ 1; (G) light micrograph of Rhynchomo nas nasuta strain CBR 1 trophonts with characteristic proboscis; (H) Rhyncho monas nasuta strain CBR 1 showing long posterior and short anterior flagella and proboscis emerging from groove at the base of the snout; (I) unidentified kinetoplastid flagellate LFS 2 showing two heterokont flagella; (J, K) thin-sec tion 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 flagel lum; (M) apical flagellum of Monosiga sp. strain BSZ 6; (N) mastigoneme-cov ered anterior flagellum of Paraphysomonas sp. strain TPC 2; (O, P) Ancyromo nas 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 m. Table 1. Pure culture isolates obtained from the American Type Culture Collection (ATCC), shallow, coastal waters (Chesapeake Bay, MD (CBR), Eel Pond, MA (EPM) and New Bedford Harbor, MA (NBH)) and four deep-sea hydrothermal vents in the Eastern Pacific Ocean (Juan De Fuca (JDF), Guaymas Basin (GBB), 21 N and 9 N). Shown are taxonomic classification, species and strain names, specific collection locations and vent sites (see Figure 1). All cultures were grown at atmospheric pressure. Figure 4. Distance tree of hydrothermal vent flagellates based on analysis of near-complete small subunit ribosomal DNA sequences using euglenozoan flagellates as the outgroup. The evo lutionary distance between two organisms is obtained by the summation of the length of the con necting branches along the horizontal axis, using the scale at the bottom. Numbers at nodes show percent bootstrap support with distance (minimum evolution) followed by maximum parsi mony (1,000 replicates each). Organisms sequenced in this study are in larger, bold font. Figure 5. Mean growth rates of vent and shallow-water flagellates with increasing hydrostatic pressure. Error bars are 1 SD. Figure 6. Light microscopic images of Caecitellus parvulus strain EWM 1 (A-C) and Rhynchomonas nasuta strain BSZ 1 (D), and transmission electron microscopic images 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 show ing typical proboscis and trailing flagellum. (E) A carbon-platinum, shadowed flagellum (F) with trail ing 30 nm thick filaments (arrow) and characteristic swollen tip (T). (F) Negatively stained motile cell showing the proboscis (P) and curved flagellum (F) with a densely-stained, rod-shaped bacterium near the tip. (G) Carbon-platinum, 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 en larged view of the edge of a cyst showing the smooth surface with a thin negatively stained outer layer (ar row). Scale bars in (A), (B), and (D) 5 m; (C) 2 m; (E) and (I) 0.3 m; and (F-H) 2 m. Figure 7. Ultrathin sections of choanoflagellates cultured at ambient atmospheric pressure (A) and at 300 atm (B-E). (A) Normal cell with prominent nucleus (N), mitochondria with flattened cristae and lightly granu lar matrix (M), osmiophilic, reserve bodies that appear 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 evidence of encyst ment (note light deposit of granular material on the cell surface, arrow) with irregularly shaped nucleus (N), enlarged digestive vacuoles with loosely ar ranged 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 irregular in shape. Digestive vacuoles (V), when present, are in late stages with only membranous 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 charac teristic of kinetoplastid cysts as in Figure 6 (I). Scale bars in (A) and (E) 0.5 m, others 1 m. Figure 8. Survival in sulfide toxicity experiments. Deep-sea vent strains are in the left column; shallow-water strains are in the right column. All sulfide concentrations shown in the figure legend were tested on each organism; overlaying of lines occurred at lower concentrations of sulfide for Caecitel lus and Rhynchomonas up to 24 hr and for all concentrations of sulfide up to 24 hr for Cafeteria. 95% confidence interval. Figure 9. 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. Figure 10. Copper toxicity data for a variety of marine organisms. It is important to note that data on species marked with an aster isk were taken from studies that measured toxicity by decreases in growth rate, while our study measured toxicity by decreases in survival in the absence of growth. Figure 11. Agarose gel results of a temperature gradient PCR with pri mers Kin F/R, to determine an ap proximate annealing temperature that would specifically amplify eu glenozoan flagellates while exclud ing non-euglenozoa. Euglenozoans: E. gracilis and B. caudatus; Non-eu glenozoans: cercomonads, M. mari na; choanoflagellates, Monosiga sp.; stramenopiles, C. parvulus, Cafeteria sp., and M. papillosa. Fragment sizes range from 329-432 base pairs. Vertical lines indicate approximate annealing tempera tures along the PCR block gradient; the heavy line (68 C) corresponds to the approximate temperature at which specificity occurs. Figure 12. Agarose gel results of PCR products amplified while optimiz ing annealing tempera ture for euglenozoan specificity using pri mers KinF/R. Taking the results from tempera ture gradient PCR (Fig ure 4.2), the optimal annealing temperature for the desired specifici ty was determined to be 68.5 C. The differ ence of 0.5 C between specific and non-specif ic results is within the accuracy of the instru ment used. M = 1 kb ladder marker (Prome ga Corp.); Lanes: eu glenozoa: 1 = Euglena gracilis; 2 = Bodo cau datus; 3 = Rhynchomo nas nasuta strain CBR1; 4 = Rhynchomo nas nasuta strain BSZ1; 5 = kinetoplastid isolate LFS2; non-euglenozoa: 6 = Ancyromonas sigmoides; 7-12 = Massisteria marina strains GBB2, DFS1, LFS1, CAS1 and TPC1; 13 = Monosiga sp. strain BSZ6; 14 = Cafeteria sp. strain EWM2; 15 = Cafeteria sp. strain VENT1; 16 = Caecitellus parvulus strain EWM1; 17 = Caecitellus parvulus strain NBH4; 18 = Mallomonas papillosa; 19 = Cafeteria sp. strain EPM1; 20 = Cafeteria sp. strain EWM2; 21 = Jakoba libera; 22 = unidentified vent isolate GBB1; 23 = Paraphysomonas sp. strain TPC2; Neg = negative control. Figure 13. Results of denaturing gradient gel electrophoresis (DGGE) of PCR-amplified products. Mixed product lanes were run to show that discrete band resolution occurs with complex mixed samples. Table 2. A comparison between cul turing (C) and DGGE (D) methods of determining the presence of kineto plastid flagellates at different vent sites. Also shown are other eugleno zoan and non-euglenozoan flagel lates detected at these sites using the methods indicated. Numbers in parenthesis indicate the number of flagellates (> 1) detected by that method. LFS = Lobo Flange Sub strates; CAS = Clam Acres Spire; BRM = Biovent Riftia and Mussels; BSZ = Biovent Serpulid Zone. Figure 14. A diagram summarizing the results of this thesis, which support the hypothesis that ubiquity in occurrence pat terns among heterotrophic flagellates implies high tolerance and/or adaptability to a wide range of environmental conditions.

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ASLO 2001 Poster (Black)

  • 1.
    T = 69░C T= 70░C M 1,000 500 250 base pair s 10,000 1,000 500 250 base pair s 10,000 1,000 500 250 base pair s 10,000 1,000 500 250 base pair s 10,000 T = 68 ░C T = 68.5 ░C T = 69 ░C T = 70 ░C M 1 2 3 4 5 6 7 8 9 10 11 12 M 13 14 15 16 17 18 19 20 21 22 23 Neg Vent Juan De Fuca 21░N 9░N Specific Site LFS CAS BRM BSZ Organism Bodo caudatus Bodo saliens D C D Rhynchomonas nasuta D D C D Isolate LFS2 C D Other euglenozoa Non-euglenozoa C (2) C (3) C (1) C (3) C = cultured D = DGGE1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 ViableCells/1E5Cells CuFeMnZn 0 1 3 7 0 1 3 7 0 1 3 7 0 1 3 7 Caecitellus parvulus strain NBH4 Cafeteria sp. strain EPM1 Cafeteria sp. strain VENT1 Rhynchomonas nasuta strain CBR1 0 M 1E-5 M 1E-4 M 1E-3 M 1E-2 M Time (Days) Culturing Molecular Analysis Microscopical Identification Physiological Experiments DNASequencing Database Comparison & Identification Primer & Probe Design DGGE PCRAmplification of Total Community DNA ╒ s PCRAmplification of Clade-Specific DGGE Fragments DGGE DNASequencing Southern Blotting Probe Hybridization 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. strainTPC1 Ochromonas danica Mallomonas papillosa Synura spinosa Paraphysomonas vestita Paraphysomonas foraminifera Paraphysomonassp. strainTPC2 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 Blastocystissp. Cafeteriasp. strain EPM1 Cafeteria roenbergensis Cafeteriasp. strainVENT1 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 Diplonemasp. Massisteriasp. strain DFS1 ANCYROMONADS APUSOMONADS FUNGI DRIP's CHOANOFLAGELLATES CERCOMONADS STRAMENOPILES KINETO- PLASTIDS DIPLONEMIDS EUGLENIDS E U G L E N O Z O A 99/98 100/100 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 Cu2+ , Fe2+ , Mn 2+ , Zn2+ 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-, S2- CuFeS2 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 J ack Cook,WHOI Graphics 62íC 67íC 72íC 68íC 62íC 67íC 72íC 68íC Euglena gracilis Bodo caudatus Caecitellus parvulus strain EWM1 Monosiga sp. strain BSZ6 Cafeteria sp. strain EWM2 Massisteria marina strain LFS1 Mallomonas papillosa Bodo caudatus Bodo saliens M ixed PC R Products BioventR iftia and M ussels (9░N ) BioventSurpulid Zone (9░N ) Lobo Flange Substrate (JD F) C lam Acres Spire (21░N ) M ixed PC R Products R hynchom onas nasuta Isolate LFS2 Euglena gracilis Environmental Samples BRM band 1= Bodo saliens BRM band 2= Rhynchomonas nasuta BSZ band 1= Rhynchomonas nasuta LFS band 1= Isolate LFS2 CAS band 1= Bodo saliens CAS band 2= Rhynchomonas nasuta Results of sequencing DGGE bands: Favella* Balanion* Thalassiosira* Rhizosolenia* Umbilicosphaera* Thoracosphaera* Synechococcus* Prochlorococcus* Caecitellusá Cafeteriaá Rhynchomonasá Toxicity Threshold of Free Copper (M) 10 101010101010101010 10 1010 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 * as determined by 50% decrease in growth rate á as determined by 50% decrease in survival rate ASSESSMENT OF FLAGELLATE DIVERSITY AT DEEP-SEA HYDROTHERMAL VENTS USING THE COMBINED APPROACH OF CULTURE-DEPENDENT AND CULTURE-INDEPENDENT METHODS Michael S. Atkins1, Andreas P. Teske1, Craig D. Taylor1, Carl O. Wirsen1, and O. Roger Anderson2 1Woods Hole Oceanographic Institution, Woods Hole, MA USA 2Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY USA Funding for this research was provided in part by: The National Science Foundation The Ocean Ventures Fund/Woods Hole Oceanographic Institution The Rhinehart Coastal Research Center The PADI Foundation Publications from this work: Atkins, M.S. and A.P. Teske. Detection and distribution patterns of kinetoplastid flagellates at deep-sea hydrothermal vents as determined by cul turing and denaturing gradient gel electrophoresis. Applied and Environmental Microbiology, in preparation. Atkins, M.S., M.A. Hanna, E.A. Kupetsky, M.A. Saito, C.D. Taylor and C.O. Wirsen. Tolerance of flagellated protozoa to extreme environmental conditions potentially encountered at deep-sea hydrothermal vents: I. High sulfide; II. High concentrations of Cu, Fe, Mn, and Zn. Marine Ecol ogy Progress Series, submitted. Atkins, M.S., A.G. McArthur and A.P. Teske. 2000b. Ancyromonadida: a new phylogenetic lineage among the protozoa closely related to the com mon ancestor of Metazoans, Fungi, and Choanoflagellates (Opisthokonta). Journal of Molecular Evolution 51:278-285. Atkins, M.S., A.P. Teske and O.R. Anderson. 2000a. A survey of flagellate diversity at four deep-sea hydrothermal vents in the Eastern Pacific Ocean using structural and molecular approaches. Journal of Eukaryotic Microbiology 47(4):400-411. Atkins, M.S., O.R. Anderson and C.O. Wirsen. 1998. Effect of hydrostatic pressure on the growth rates and encystment of flagellated protozoa isolated from a deep-sea hydrothermal vent and a deep shelf region. Marine Ecology Progress Series 171: 85-95. Abstract: Eighteen strains of flagellated protists representing 9 species were isolated and cultured from four deep-sea hydrother mal vents in the Eastern Pacific Ocean: Juan de Fuca Ridge, Guaymas Basin, and both 21 N and 9 N on the East Pacific Rise (EPR). The hydrothermal vent flagellates belonged to six different taxonomic orders: the Ancyro monadida, Bicosoecida, Cercomonadida, Choanoflagellida, Chrysomonadida, and Ki netoplastida. Many of the vent isolates were ubiquitous members of marine, freshwater, and terrestrial ecosystems worldwide, sug gesting a global distribution of these flagel late species. This discovery advanced the hy pothesis that ubiquity in distribution patterns among heterotrophic flagellates implies high tolerance and/or adaptability to a wide range of environmental 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 conditions. Deep-sea vent samples were both cultured to select for kinetoplastid flagellates and ana lyzed without culturing by denaturing gradient gel electrophoresis (DGGE) using PCR pri mers specific to the kinetoplastid clade. By comparing these two different methods of analysis, my goal was to decrease the biases and/or errors inherent in either method alone and to improve our ability to assess flagellate diversity and distribution in samples from re mote vent environments. PCR and DGGE were used to specifically isolate and amplify target DNA's from all cultured kinetoplastid species in matching vent samples, thus cor roborating the findings of culturing. Molecu lar methods had the additional ability to de tect species presence where culturing did not, thereby providing a better indication of the distribution of these species. Species Strain Collection Location Vent Ancyromonadida, Cavalier-Smith, 1998 Ancyromonas sigmoides, Kent, 1880 50267 American Type Culture Collection Ancyromonas sigmoides, Kent, 1880 BRM2 Biovent Riftia and Mussels Bed 9N Bicosoecida, GrassÄ and Deflandre, 1952 Cafeteria sp. VENT1 9N vent water - H2S reactors 9N Cafeteria sp. EWM2 East Wall Mussels Bed 9N Cafeteria sp. EPM1 Eel Pond Marsh - H2S reactors surface Caecitellus parvulus, Patterson et al.,1993 BSZ7 Biovent Serpulid Zone 9N Caecitellus parvulus, Patterson et al.,1993 EWM1 East Wall Mussels Bed 9N Caecitellus parvulus, Patterson et al.,1993 NBH4 New Bedford Harbor, MA surface Cercomonadida, Vickerman, 1983 Massisteria marina, Larsen and Patterson, 1990 50266 American Type Culture Collection Massisteria marina, Larsen and Patterson, 1990 BSZ3 Biovent Serpulid Zone 9N Massisteria marina, Larsen and Patterson, 1990 GBB2 Guaymas Basin Beggiotoa Mat GBB Massisteria marina, Larsen and Patterson, 1990 DFS1 Dante Flange Substrates JDF Massisteria marina, Larsen and Patterson, 1990 LFS1 Lobo Flange Substrates JDF Massisteria marina, Larsen and Patterson, 1990 CAS1 Clam Acres Spire 21N Massisteria marina, Larsen and Patterson, 1990 TPC1 Twin Peaks Chimney 21N Choanoflagellida, Kent, 1880 Monosiga sp. BSZ6 Biovent Serpulid Zone 9N Chrysomonadida, Engler, 1898 Paraphysomonassp. TPC2 Twin Peaks Chimney 21N Kinetoplastida, Honigberg, 1963 Rhynchomonas nasuta, Klebs, 1892 CBR1 Chesapeake Bay, MD surface Rhynchomonas nasuta, Klebs, 1892 BSZ1 Biovent Serpulid Zone 9N Rhynchomonas nasuta, Klebs, 1892 BSZ2 Biovent Serpulid Zone 9N Rhynchomonas nasuta, Klebs, 1892 BSZ8 Biovent Serpulid Zone 9N Bodo saliens, Larsen and Patterson, 1990 50358 American Type Culture Collection Bodo saliens, Larsen and Patterson, 1990 BRM1 Biovent Riftia and Mussels Bed 9N Unidentified LFS2 Lobo Flange Substrates JDF Caecitellus parvulus strain EWM1 Cafeteria sp. strain VENT1 Rhynchomonas nasuta strain BSZ1 Caecitellus parvulus strain NBH4 Cafeteria sp. strain EPM1 Rhynchomonas nasuta strain CBR1 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 1E6 1E5 1E4 1E3 1E2 1E1 1E0 0 1 3 6 24 0 1 3 6 24 0 1 3 6 24 0 1 3 6 24 0 1 3 6 24 168 0 1 3 6 24 168 Time (hours) Time (hours) ViableCells/1E5Cells 0.0 mM 1.0 mM 10.0 mM 0.1 mM 2.0 mM 20.0 mM 0.5 mM 5.0 mM 30.0 mM 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1 50 100 150 200 250 300 1 50 100 150 200 250 300 1 50 100 150 200 250 300 1.5 1.0 0.5 0.0 0.5 0.4 0.3 0.2 0.1 0.0 Vent Shallow Caecitellus parvulus Rhynchomonas nasuta Monosiga sp. MeanGrowthRatePerDay MeanGrowthRatePerDay MeanGrowthRatePerDay Hydrostatic Pressure (Atmospheres) Hydrostatic Pressure (Atmospheres) Hydrostatic Pressure (Atmospheres) Figure 1. Vent map with flagellate species collected Figure 2. Flow diagram of culture-dependent and culture-independent methods used on vent samples in this research. Figure 3. (A) Cafeteria sp. strain VENT 1 showing mastigonemes on anterior flagellum; (B) Cafeteria sp. strain EPM 1; (C) light micrograph of Caecitellus parvulus trophs with characteristic gliding morphology; (D) Caecitellus parvu lus strain NBH 4 (arrow, acronematic flagellar tip); (E) light micrograph of Rhynchomonas nasuta strain BSZ 1 trophonts with characteristic proboscis; (F) Rhynchomonas nasuta strain BSZ 1; (G) light micrograph of Rhynchomo nas nasuta strain CBR 1 trophonts with characteristic proboscis; (H) Rhyncho monas nasuta strain CBR 1 showing long posterior and short anterior flagella and proboscis emerging from groove at the base of the snout; (I) unidentified kinetoplastid flagellate LFS 2 showing two heterokont flagella; (J, K) thin-sec tion 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 flagel lum; (M) apical flagellum of Monosiga sp. strain BSZ 6; (N) mastigoneme-cov ered anterior flagellum of Paraphysomonas sp. strain TPC 2; (O, P) Ancyromo nas 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 m. Table 1. Pure culture isolates obtained from the American Type Culture Collection (ATCC), shallow, coastal waters (Chesapeake Bay, MD (CBR), Eel Pond, MA (EPM) and New Bedford Harbor, MA (NBH)) and four deep-sea hydrothermal vents in the Eastern Pacific Ocean (Juan De Fuca (JDF), Guaymas Basin (GBB), 21 N and 9 N). Shown are taxonomic classification, species and strain names, specific collection locations and vent sites (see Figure 1). All cultures were grown at atmospheric pressure. Figure 4. Distance tree of hydrothermal vent flagellates based on analysis of near-complete small subunit ribosomal DNA sequences using euglenozoan flagellates as the outgroup. The evo lutionary distance between two organisms is obtained by the summation of the length of the con necting branches along the horizontal axis, using the scale at the bottom. Numbers at nodes show percent bootstrap support with distance (minimum evolution) followed by maximum parsi mony (1,000 replicates each). Organisms sequenced in this study are in larger, bold font. Figure 5. Mean growth rates of vent and shallow-water flagellates with increasing hydrostatic pressure. Error bars are 1 SD. Figure 6. Light microscopic images of Caecitellus parvulus strain EWM 1 (A-C) and Rhynchomonas nasuta strain BSZ 1 (D), and transmission electron microscopic images 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 show ing typical proboscis and trailing flagellum. (E) A carbon-platinum, shadowed flagellum (F) with trail ing 30 nm thick filaments (arrow) and characteristic swollen tip (T). (F) Negatively stained motile cell showing the proboscis (P) and curved flagellum (F) with a densely-stained, rod-shaped bacterium near the tip. (G) Carbon-platinum, 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 en larged view of the edge of a cyst showing the smooth surface with a thin negatively stained outer layer (ar row). Scale bars in (A), (B), and (D) 5 m; (C) 2 m; (E) and (I) 0.3 m; and (F-H) 2 m. Figure 7. Ultrathin sections of choanoflagellates cultured at ambient atmospheric pressure (A) and at 300 atm (B-E). (A) Normal cell with prominent nucleus (N), mitochondria with flattened cristae and lightly granu lar matrix (M), osmiophilic, reserve bodies that appear 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 evidence of encyst ment (note light deposit of granular material on the cell surface, arrow) with irregularly shaped nucleus (N), enlarged digestive vacuoles with loosely ar ranged 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 irregular in shape. Digestive vacuoles (V), when present, are in late stages with only membranous 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 charac teristic of kinetoplastid cysts as in Figure 6 (I). Scale bars in (A) and (E) 0.5 m, others 1 m. Figure 8. Survival in sulfide toxicity experiments. Deep-sea vent strains are in the left column; shallow-water strains are in the right column. All sulfide concentrations shown in the figure legend were tested on each organism; overlaying of lines occurred at lower concentrations of sulfide for Caecitel lus and Rhynchomonas up to 24 hr and for all concentrations of sulfide up to 24 hr for Cafeteria. 95% confidence interval. Figure 9. 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. Figure 10. Copper toxicity data for a variety of marine organisms. It is important to note that data on species marked with an aster isk were taken from studies that measured toxicity by decreases in growth rate, while our study measured toxicity by decreases in survival in the absence of growth. Figure 11. Agarose gel results of a temperature gradient PCR with pri mers Kin F/R, to determine an ap proximate annealing temperature that would specifically amplify eu glenozoan flagellates while exclud ing non-euglenozoa. Euglenozoans: E. gracilis and B. caudatus; Non-eu glenozoans: cercomonads, M. mari na; choanoflagellates, Monosiga sp.; stramenopiles, C. parvulus, Cafeteria sp., and M. papillosa. Fragment sizes range from 329-432 base pairs. Vertical lines indicate approximate annealing tempera tures along the PCR block gradient; the heavy line (68 C) corresponds to the approximate temperature at which specificity occurs. Figure 12. Agarose gel results of PCR products amplified while optimiz ing annealing tempera ture for euglenozoan specificity using pri mers KinF/R. Taking the results from tempera ture gradient PCR (Fig ure 4.2), the optimal annealing temperature for the desired specifici ty was determined to be 68.5 C. The differ ence of 0.5 C between specific and non-specif ic results is within the accuracy of the instru ment used. M = 1 kb ladder marker (Prome ga Corp.); Lanes: eu glenozoa: 1 = Euglena gracilis; 2 = Bodo cau datus; 3 = Rhynchomo nas nasuta strain CBR1; 4 = Rhynchomo nas nasuta strain BSZ1; 5 = kinetoplastid isolate LFS2; non-euglenozoa: 6 = Ancyromonas sigmoides; 7-12 = Massisteria marina strains GBB2, DFS1, LFS1, CAS1 and TPC1; 13 = Monosiga sp. strain BSZ6; 14 = Cafeteria sp. strain EWM2; 15 = Cafeteria sp. strain VENT1; 16 = Caecitellus parvulus strain EWM1; 17 = Caecitellus parvulus strain NBH4; 18 = Mallomonas papillosa; 19 = Cafeteria sp. strain EPM1; 20 = Cafeteria sp. strain EWM2; 21 = Jakoba libera; 22 = unidentified vent isolate GBB1; 23 = Paraphysomonas sp. strain TPC2; Neg = negative control. Figure 13. Results of denaturing gradient gel electrophoresis (DGGE) of PCR-amplified products. Mixed product lanes were run to show that discrete band resolution occurs with complex mixed samples. Table 2. A comparison between cul turing (C) and DGGE (D) methods of determining the presence of kineto plastid flagellates at different vent sites. Also shown are other eugleno zoan and non-euglenozoan flagel lates detected at these sites using the methods indicated. Numbers in parenthesis indicate the number of flagellates (> 1) detected by that method. LFS = Lobo Flange Sub strates; CAS = Clam Acres Spire; BRM = Biovent Riftia and Mussels; BSZ = Biovent Serpulid Zone. Figure 14. A diagram summarizing the results of this thesis, which support the hypothesis that ubiquity in occurrence pat terns among heterotrophic flagellates implies high tolerance and/or adaptability to a wide range of environmental conditions.