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Epifluorescence microscopy image of DAPI stained cells (left) under UV excitation and blue emission, and Group 1 marine Archaea hybridized with FITC-labelled polynucleotide probes (right) under blue excitation and green emission. Samples are from the Hawai‘i Ocean Time-series station ALOHA, 1000 m depth, Aug. 1998. Both images show the same microscopic field. Counts were made comparing the DAPI image to the FITC image and calculating percentages of probe-positive cells with respect to the DAPI count of the same microscopic field (double staining).

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Sums of abundances of pelagic microbes relative to DAPI count. Bacteria, Group 1 and Group 2 Archaea, and Negative (autofluorescence) counts were obtained by fluorescence in situ hybridization with polynucleotide probes of whole, fixed cells immobilized on polycarbonate filters. Samples were collected at six depths from the Hawai‘i Ocean Time-series station ALOHA, in the North Pacific Ocean.

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Influence of preservation method on counts of marine Group 1 Archaea in fluorescent in situ hybridization.

Samples were formalin fixed then stored either as whole sea water (“liquid”) or after filtration onto 0.2 m polycarbonate filters.

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DiscussionDirect counts of Archaea with parallel enumeration of Bacteria in pelagic open ocean environments have remained elusive for the past decade. Existing methods have serious limitations in their detection thresholds in oligotrophic waters. Polyribonucleotides targeting rRNA have allowed us along with Taylor et al. (1999) to now present single cell time-series data sets identifying and enumerating pelagic Archaea in the open ocean. Figure 1 demonstrates the high fluorescence yield on this 1000 m depth sample for the G1 Archaea probe. Since advanced molecular biology techniques and epifluorescence enumeration and have an inevitable operator-related subjective component, we tested for differences in these factors using the same samples. The results, largely gathered while methodology development and optimization were still underway, indicate that the method is fairly robust to this challenge (Figures 5, 6, and 7).

While Bacteria dominate the upper 250 m of the ocean, G1 Archaea consistently seem to match bacterial numbers at deeper layers. With a few exceptions over the year-long study, G1 Archaea probe positive cells represented about 1/4 of the total DAPI numbers at 250 m and 1000 m, roughly equal to the Bacteria probe positive proportion. G2 Archaea were much lower in relative abundance and more often found at near surface depths. Figures 2a and 2b, representing distributions of Bacteria and G1 Archaea, correspond in that a rise in the layers where G1 Archaea are abundant is reflected by a corresponding decrease in Bacteria probe positive cell abundances (e.g., Jan.–Feb. 1998 in Figures 2a and 2b). The sum of G1 and G2 Archaea, Bacteria and Negative (blank) counts are fairly constant at about 80%, and the maxima of the different archaeal groups occurred at different depths. These data, in combination dual labeling experiments using probes labeled with different fluorochromes , indicated that the probes were indeed specific for their targeted groups. The observation that less than 100% of the DAPI positive cells are accounted for is not unexpected. Other means of detecting microbial metabolic activity correlated well with universal oligonucleotide probe assays and never accounted for the full DAPI count (Karner and Fuhrman, 1997). Cells that were undetectable by either probe but visible with DAPI staining could be too poor in rRNA (low metabolic activity) or alternately, dead cells or “ghosts” (Zweifel and Hagström, 1995). The more variable total sum of probe detectable cells (Figure 4) at greater depths may be related to episodic sedimentation leading to periodically increasing metabolic activities and thus increasing detectability. Further, the preliminary deep cast data (Figure 3) indicate that the relative numbers of Archaea stay relatively constant from the 250 m depth layer all the way to the greatest depths sampled (4750 m). Note that our data are relative numbers compared to the DAPI count. Since total microbial abundances decrease exponentially with depth, so does the total number of Archaea. Compared to the numbers of probe detectable Bacteria though, pelagic deep sea Archaea are an equal part of the microbial deep sea environment.

References

Baumann, J.G. J, and Bentvelzen, P. 1988. Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization. Cytometry 9: 517–524

DeLong, E.F. 1998. Archaeal means and extremes. Science 280: 542–543

DeLong, E.F., G.S. Wickham, and N.R. Pace. 1989. Phylogenetic stains: Ribosomal RNA-based probes for the identification of single cells. Science 243: 1360–1363.

DeLong, E., K.Y. Wu, B.B. Prézlin, and R.V.M. Jovine. 1994. High abundance of Archaea in Antarctic marine picoplankton. Nature 371: 695–697.

Fuhrman, J.A. and A.A. Davis. 1997. Widespread Archaea and novel Bacteria from the deep sea as shown by 16S rRNA gene sequences. Mar. Ecol Prog. Ser. 150: 275–285.

Glöckner, O.F., R. Amman, A. Alfreider, J. Pernthaler, R. Psenner, K. Trebesius, and K-H. Schleifer. 1996. An in situ hybridization protocol for detection and identification of planktonic bacteria. System. Appl. Microbiol. 19: 403–406.

Karl, D.M. and R. Lukas. 1996. The Hawaii Ocean Time-series (HOT) program: Background, rationale and field implementation. Deep-Sea Res. 43: 129–156.

Karner, M. and J. Fuhrman. 1997. Determination of active marine bacterioplankton: a comparison of universal 16S rRNA probes, autoradiography, and nucleoid staining. Appl. Environ. Microbiol. 63:1208–1213.

Moyer, C.L., J.M. Tiedje, F.C. Dobbs and D.M. Karl. 1998. Diversity of deep-sea hydrothermal vent Archaea from Loihi Seamount, Hawaii. Deep-Sea Res. II 45: 303-317.

Porter, K., and Y.S. Feig. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943–948.

Stein, J.L., and M.I. Simon. 1996. Archaeal ubiquity. Proc. Natl. Acad. Sci. USA 93: 6228–6230.

Taylor, L.T., C.M. Preston, and E.F. DeLong. 1999. Visualization and enumeration of marine picoplankton using RNA targeted polyribonucleotide probes. Presentation at the 1999 Aquatic Sciences Meeting of the American Society of Limnology and Oceanography, Santa Fe, New Mexico.

Woese, C.R., O. Kandler, and M.L. Wheelis. 1990. Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci., USA 87: 4576–4579.

Zweifel, U.L., and Å. Hagström. 1995. Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts). Appl. Environ. Microbiol. 61: 2180-2185.

AcknowledgmentsWe thank the crew of the R/V Moana Wave and L. Tupas for help with sampling. L. Fujieki prepared the interpolation and contour plots on Matlab 4.2c (UNIX, interpolation algorithm: E. Firing and R. Lukas). This work was supported by grants from NSF OCE # 9529804 and the Monterey Bay Aquarium Research Institute to E.F.D., and NSF OCE # 9315392 to D.M.K.

BaBacterial and Archaeal Districterial and Archaeal Distribubutionstionsat the Hawai‘i Ocean Time-Series Station ALOHAin the North Pacific Ocean

M.B. M.B. KarneKarnerrUniversity of Hawai‘i, Honolulu, HI 96822, USAkarner@soest.hawaii.edu

L.T. TaylorL.T. TaylorMonterey Bay Aquarium Research Institute, Moss Landing, CA 95039, USAtaylor@mbari.org

E.F. DeLongE.F. DeLongMonterey Bay Aquarium Research Institute, Moss Landing, CA 95039, USAdelong@wave.mbari.org

D.M. D.M. KarlKarlUniversity of Hawai‘i, Honolulu, HI 96822, USAdkarl@soest.hawaii.edu

AbstractABUNDANCE OF MAJOR MICROBIAL GROUPS was assessed in the North Pacific subtropical gyre using fluorescently labeled polyribonucleotide probes (>100 bp) for ribosomal RNA targeted in situ hybridization. Monthly cruises to the Hawai‘i Ocean Time-series (HOT) benchmark station ALOHA revealed relative abundances of Marine Group 1 (G1) Archaea, Marine Group 2 (G2) Archaea and Bacteria, as compared to total cell counts using DAPI staining. G1 Archaea relative abundance was found to increase in the 250–1000 m depth range but was low or nil at shallow depths. G1 Archaea relative abundance was up to one third of total cells at 1000 m, sometimes equaling bacterial numbers at this depth. G2 Archaea generally peaked in the surface layer but their relative abundance stayed below 10%. Bacteria were dominant at shallow depths, with abundance between 80 and 90% of the DAPI count. Archaeal and bacterial numbers when summed were sufficient to account for the independent DAPI count. Relative archaeal abundances showed some marked differences between the monthly cruises, namely G1 Archaea seemed to appear at higher relative abundances and at shallower depths during Winter 1997/1998.

IntroductionPlanktonic micro-organisms, both autotrophic and heterotrophic, play a key role in the world’s oceans. At the same time, qualitative understanding of community structures has remained very limited due to lack of culturability of many marine micro-organisms. The discovery of a major hitherto unknown domain of organisms, Archaea, has prompted new questions (Woese et al., 1990). Archaeal rRNA genes have been recovered from a variety of environments (DeLong et al., 1994, Stein and Simon, 1996, Fuhrman and Davis, 1997, Moyer et al., 1998). Archaea could constitute both numerically and ecologically a major part of picoplankton, previously thought to be Bacteria (using non-specific staining and direct counts). Tools such as fluorescent in situ hybridization give access to direct cell counts of specific classes of organisms, determined by 16S rRNA sequences targeted. The most widely used method to date employs fluorescently labeled oligonucleotides targeting microbial 16S rRNA (DeLong et al., 1989). In open ocean environments, however, fluorescently-labeled rRNA-targeted oligonucleotide probes are often below threshold detection limits. Here we report results from a year long study of the occurrence of planktonic Archaea at the Hawai‘i Ocean Time-series (HOT) station ALOHA in the North Pacific (Karl and Lukas, 1996) using rRNA targeted polyribonucleotide probes and filter-based in situ hybridization assays (Taylor et al., 1999, DeLong et al., in prep.). This approach is a modification of a previously reported probe preparation method (Baumann and Bentvelzen, 1988) and hybridization technique (Glöckner et al., 1996). The modified procedure used in this study (DeLong et al., in prep.) allowed for routine and specific detection and enumeration of two different groups of planktonic Archaea to depths of 1000 m at station ALOHA.

Materials and MethodsSampling and sample storage

Water samples were taken with a rosette sampler at approximately monthly intervals at station ALOHA (22º45'N 158º00'W). Sample depths were 5, 25, 45, 100, 250 or 300 and 1000 m. One deep profile was taken in July 1998 with additional depths of 2000 m, 3000 m and 4750 m. Samples were drawn from sampling bottles, immediately fixed with 0.2 m filtered formalin to a final concentration of 2%, filtered onto 0.2 m polycarbonate filters and the filters stored at -20ºC until analyzed. Prior to freezing, the filters were treated with a 50/50 (v/v) solution of 2% NaCl and ethanol for 2 min. Selected samples were also stored as frozen whole fixed sea water prior to filtration to compare effects of either preservation method.

Fluorescent in situ hybridization and cell counts

We used two archaeal probes, for marine Group 1 (G1) and marine Group 2 (G2) Archaea that represent marine planktonic forms within Crenarchaeota and Euryarchaeota, respectively (DeLong, 1998). Samples were filter hybridized with polyribonucleotide probes targeting G1 Archaea, G2 Archaea, Bacteria or a non-specific probe (“Negative”). The Negative probe was prepared identically to the Group 1 probe, except that it was complementary to the opposite (non-rRNA) strand. A hybridization buffer of 10% (w/v) dextran sulfate, 0.01% PolyA and 0.1% SDS in 5X SET (1X SET is 150 mM NaCl, 1mM EDTA, 20 mM Tris, pH 8.0) was used with either 50 % formamide (Bacteria, Negative) or 70% formamide (G1 and G2 Archaea). Hybridization conditions were established empirically for highest possible stringency while conserving optimal signal strength. Hybridization temperatures were thus 65ºC for the archaeal probes and 55ºC for Bacteria and Negative, all overnight. This was followed by a 2 h post-hybridization wash in 0.2X SET and 50% formamide at 50ºC and 45ºC for archaeal and Bacteria/Negative probes, respectively. DAPI staining (Porter and Feig, 1980) was performed just prior to slide mounting of the filters. This double staining provided a direct comparison of DAPI cell counts with probe counts. Polyribonucleotide probe preparation was performed essentially as previously described by Baumann and Bentvelzen (1988), with minor modifications. Briefly, polyribonucleotide probes complementary to 16S and 23S rRNA were synthesized using T7 RNA polymerase, and fluorescein labeled UTP and CTP. Cloned rRNA operons from G1 Archaea, or cloned 23S rRNA fragments from G2 Archaea, were used as DNA templates for in vitro RNA synthesis. For the Bacteria probe, rRNA operons were amplified directly from bacterioplankton DNA samples, and the mixed bacterial rRNA operon DNA was used as template for bacterial in vitro RNA synthesis. The multiply labeled polyribonucleotide probes were hydrolyzed to an average size of approximately 100 bp. The probe was added to the hybridization mixture at a final concentration of 2 ng/l. Counting was done on a Zeiss microscope with 100 W UV illumination and a 100x objective. DAPI stained cells were counted under UV excitation and blue emission, FITC signal from probe bound to cells was counted under blue excitation and green emission filter sets suitable for FITC. Ten fields were counted for each DAPI and FITC image, respectively, amounting in most cases to about 200 cells counted per sample. The Negative count, essentially accounting for non specific dye binding and autofluorescence, was subtracted from the counts obtained for probe positive cells. We also assessed operator influence on counts and performed a cross laboratory comparison of hybridization and enumeration.

ResultsThe actual microscopic image of G1 probe positive archaeal cells at 1000 m depth is shown in Figure 1. Contour plots of relative abundances (expressed as percentages of the DAPI total cell count) are shown in Figures 2a, b and c for Bacteria, G1 Archaea and G2 Archaea, respectively. Cells hybridizing with the Bacteria-targeted polynucleotide probe dominated the population in the upper 250 m of the water column (Figure 2a) and generally comprised about 80 % of the DAPI count above 250 m depth, and at around 50% at 1000 m depth. G1 Archaea (Figure 2b) matched Bacteria in relative abundance below the 250 m depth layer and represented a significant fraction of the microbial community at 250 and 1000 m depths over the entire sampling year. G1 Archaea generally comprised 20–30% of the DAPI count at these depths. G2 Archaea (Figure 2c) were sporadically present in the near surface layer but generally remained at a few percent of the total count. One preliminary deep profile from surface to sea floor depth (4750 m) indicates that the relative abundance of G1 Archaea at depth remains at levels similar to those found at 250 m and 1000 m (Figure 3).

Summation of the relative abundances of Bacteria, G1 and G1 Archaea (Figure 4) and Negative counts (non-specific binding or autofluorescence) generally constituted 80% of the DAPI count above 250 m (e.g., 82 6% at 45 m, mean 1 SD of the 12 sampling dates). For the deeper samples, this number was more variable but still high, amounting to a mean and SD of 70 13% at 1000 m depth.

Differences in preservation method (frozen fixed cells on filters vs. frozen whole fixed sea water, Figure 5), differences in hardware and laboratory of analysis with same operator (Figure 6), and different operators for the entire hybridization and counting process (Figure 7) yielded comparable results for both Archaea and Bacteria.

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Contour plots of relative abundances of (a) Bacteria, (b) Group 1 marine Archaea, and (c) Group 2 marine Archaea. Samples were taken at the Hawai’i Ocean Time-series station ALOHA in the North Pacific Ocean at six depths (5 to 1000 m) for a year at approximately monthly intervals. Plots show relative abundances of the above microbial groups as assessed by fluorescent polyribunucleotide in situ hybridization, in percent of the corresponding DAPI count. Note logarithmic depth scale.

Deep water profile of G1 Archaea at HOT station ALOHA in July 1998. Values are raw counts (Negative not subtracted). Note logarithmic depth scale.

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