Mark R. Dennett

Defining DNA-Based Operational Taxonomic Units for Microbial-Eukaryote Ecology

ABSTRACT DNA sequence information has increasingly been used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, studies of the autecology of ecologically relevant species, and identification and enumeration of species of interest for human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is in part a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes and in part a consequence of the problematic nature of and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and there is a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximately species-level distinctions. This approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small-subunit (18S) rRNA gene sequences and partial gene sequences obtained from the GenBank database for morphologically described protistan species. The program was tested using environmental 18S rRNA data sets for two oceanic ecosystems. A total of 388 operational taxonomic units were observed for 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific oceans.

Susceptibility of some marine phytoplankton species to cell breakage during filtration and post-filtration rinsing

Abstract Several fragile phytoplankton species among a diverse group of 13 species were found to be very susceptible to cell breakage when exposed to the air under vacuum between the filtration and rinsing steps used to terminate 14C fixation experiments and remove residual [ 14 C]HCO 3 − . Up to 60% of fixed carbon after 15-min incubation was found in the rinse. Losses were even greater when cultures were pulsed with NH 4 + at the start of the incubation, probably because rapid NH 4 + uptake leads to the accumulation of large pool of soluble and low molecular weight compounds. Most likely, the cells, when exposed to the air, are subject to extreme osmotic shock and rupture. Unaccountable losses of 14 C occurred with polycarbonate filiters relative to glass-fiber filters. In addition, vacuum pressure differentials >25–100 mmHg across polycarbonate filters also caused cell breakage that led to the accumulation of 14 C in the filtrate. Avoiding air exposure of the filter between the filtration and rinsing steps or eliminating the rinsing step entirely and acid-soaking or fuming the filters led to virtually complete recovery of fixed carbon. Our results based on 15-min incubations may not be directly comparable with longer-term incubations, but they do serve highlight our concerns about filtration procedures in general.

Upward transport of oceanic nitrate by migrating diatom mats

The oligotrophic gyres of the open sea are home to a flora that includes the largest known phytoplankton. These rare species migrate as solitary cells or aggregations (mats) between deep nutrient pools (below 80–100 m) and the surface. This migration contributes to new production because of the concomitant upward transport of nitrate. But just how significant this contribution is remains uncertain because of the difficulty of making quantitative measurements of these rare cells. Here we report remote video observations of a previously undersampled class of diatom (Rhizosolenia) mats throughout the upper 150 m of the central North Pacific Ocean. These mats are virtually invisible to divers, and their presence increases the calculated phytoplankton-mediated nitrate transport into the surface ocean by up to a factor of eight. Cruise averages indicate that Rhizosolenia mats transport 18–97 µmol N m−2 d−1; however, this value reached 171 μmol N m−2 d−1 at individual stations, a value equivalent to 59% of the export production. Although considerable temporal and spatial variability occurs, this means of upward nutrient transport appears to be an important source of new nitrogen to the surface ocean, and may contribute to other regional elemental cycles as well.

Experimental demonstration of the roles of bacteria and bacterivorous protozoa in plankton nutrient cycles

We have used a model food chain composed of a natural bacterial assemblage, a pennate diatom and a bacterivorous microflagellate to investigate the factors controlling the relative importance of bacteria and protozoa as sources for regenerated nitrogen in plankton communities. In bacterized diatom cultures in which diatom growth was nitrogen-limited, the carbon:nitrogen (C:N) ratio of the bacterial substrate greatly affected which population was responsible for the uptake of nitrogen. When nitrogen was added as NH4+ and the cultures were supplemented with glucose, the bacteria competed successfully with the algae for NH4+ and prevented the growth of algae by rapidly assimilating all NH4+ in the cultures. Bacterivorous protozoa inoculated into these cultures grazed the bacterial population and remineralized NH4+, thus relieving the nitrogen limitation of algal growth and allowing an increase in algal biomass. In contrast, bacteria in cultures supplemented with the amino acid glycine (C:N = 2) were major remineralizers of nitrogen, and the influence of protozoan grazing was minimal. We conclude that the relative importance of bacteria and protozoa as nutrient regenerators in the detrital food loop is dependent largely on the overall carbon:nutrient ratio of the bacterial substrate. The role of bacterivorous protozoa as remineralizers of a growth-limiting nutrient is maximal in situations where the carbon:nutrient ratio of the bacterial substrate is high.

Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis

ABSTRACT The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.

Isotopic fractionation of oxygen by respiring marine organisms

We measured the respiratory isotope effect ϵresp for seven representative unicellular marine organisms. The bacterium Pseudomonas halodurans, the diatom Phaeodactylum tricornutum, the phytoflagellates Cryptomonas baltica and Dunaliella tertiolecta, the heterotrophic flagellates Paraphysomonas imperforata and Bodo sp., and the ciliate Uronema sp. exhibit ϵresp values in the range 14-26‰. We also measured ϵresp for three metazoans. The ϵresp for the copepod Acartia tonsa ranged from 17 to 25‰, while two larger organisms, the mollusk Mercenaria mercenaria and the salmon Salmo salmar, respire with a smaller ϵresp of 5-10‰. The average respiratory isotope effect of the dominant marine respirers (the bacteria, microalgae and zooplankton) is about 20 ± 3‰. An ϵresp of this magnitude supports the hypothesis that the photosynthesis-respiration cycle is responsible for the 23.5‰ enrichment in the δ18O ratio of atmospheric O2 relative to seawater (the Dole effect). The large value and high variability in the average ϵresp limits the usefulness of a proposed method using the δ18O of naturally fractionated dissolved O2 in seawater as a tracer of primary production in the oligotrophic ocean.

The effect of pH in intensive microalgal cultures. I. Biomass regulation

Two freshwater and two marine algal species were grown in intensive continuous cultures at a fixed dilution rate of 0.5 day −1, but at varying pH levels in the range 7.6 to 10.6. Both freshwater species, Scenedesmus obliquas (Turp.) Kutz. and Chlorella vulgaris Beij., grew up to pH 10.6 although C. vulgaris was more adversely affected by alkaline pH than was Scenedesmus obliquas. Of the marine species, Phaeodactylum tricornutum (TFX-1) Bohlin was hardly affected by varying pH up to its maximum tolerable level of 10.3, whereas growth of Dunaliella tertiolecta (Dun) Butcher was adversely affected by increasing pH and ceased when the pH exceeded 9.3. These results are consistent with the general observations that many marine species cannot tolerate alkaline pH values much above 9.5. Moreover, the unique ability of Phaeodactylum tricornutum to grow at pH > 10 probably is a major factor contributing to its well documented success in large-scale outdoor cultures that are poorly buffered. It is difficult to separate metabolic from purely chemical factors that influence the pH tolerance limits of the individual species. The lower pH limits were, however, distinctly controlled by the production of alkalinity concomitant with NO−3 uptake, whereas the upper pH limits in the case of Scendesmus obliquus and Phaeodactylum tricornutum seemed to be regulated primarily by metabolic control. In no case was the availability of inorganic carbon an influencing factor in setting the maximum attained pH.

Surfactant effects on air-sea gas exchange under turbulent conditions

Abstract In a series of laboratory gas exchange studies we found that under turbulent conditions additions of two synthetic surfactants (polyethylene oxide and oleyl alcohol) to distilled water and seawater led to reductions in oxygen evasion at the air-liquid interface. The oxygen exchange coefficient relative to that of a distilled water control asymptotically reached a lower limit of ≈50% as surfactant concentration was increased. For natural seawater samples, an asymptotic reduction in relative gas exchange rate was demonstrated for increasing amounts of surface-active material as determined from surface pressure-area isotherms. Possibly surfactants act to reduce gas exchange by creating surface pressure forces that oppose and reduce turbulent eddy velocities and, correspondingly, reduce surface renewal. However, the greatest reductions in the oxygen exchange coefficient occurred at initial surface pressure ( π i ) less than 0.5 mN m −1 . This result may have been due to the presence of soluble surfactants, which are known to be very effective in reducing gas exchange but which do not display concentration-dependent surface pressures. Based on results from an opportunistic sampling survey of marine waters and a cruise to the Sargasso Sea, a gradient in gas exchange reductions from 5–15% in oceanic waters to 50% in nearshore waters was found. However, reductions of 50% occured for surface film material obtained from the Sargasso Sea. Our results provide some measure of the potential for reduction of gas exchange at high turbulence in marine waters in the presence of natural surfactants.

Victims or vectors: a survey of marine vertebrate zoonoses from coastal waters of the Northwest Atlantic

Surveillance of zoonotic pathogens in marine birds and mammals in the Northwest Atlantic revealed a diversity of zoonotic agents. We found amplicons to sequences from Brucella spp., Leptospira spp., Giardia spp. and Cryptosporidium spp. in both marine mammals and birds. Avian influenza was detected in a harp seal and a herring gull. Routine aerobic and anaerobic culture showed a broad range of bacteria resistant to multiple antibiotics. Of 1460 isolates, 797 were tested for resistance, and 468 were resistant to one or more anti-microbials. 73% (341/468) were resistant to 1-4 drugs and 27% (128/468) resistant to 5-13 drugs. The high prevalence of resistance suggests that many of these isolates could have been acquired from medical and agricultural sources and inter-microbial gene transfer. Combining birds and mammals, 45% (63/141) of stranded and 8% (2/26) of by-caught animals in this study exhibited histopathological and/or gross pathological findings associated with the presence of these pathogens. Our findings indicate that marine mammals and birds in the Northwest Atlantic are reservoirs for potentially zoonotic pathogens, which they may transmit to beachgoers, fishermen and wildlife health personnel. Conversely, zoonotic pathogens found in marine vertebrates may have been acquired via contamination of coastal waters by sewage, run-off and agricultural and medical waste. In either case these animals are not limited by political boundaries and are therefore important indicators of regional and global ocean health.

The temporal dynamics of the flagellated and colonial stages of Phaeocystis antarctica in the Ross Sea

Abstract Phaeocystis antarctica in the Ross Sea forms colonies, and blooms of colonial P. antarctica often occur over large areas in the southern Ross Sea. Sites where colonies occur often have significant vertical fluxes of carbon in the form of aggregated and flocculent material. P. antarctica also is a key component of the sulfur cycle; therefore, the species is critically important in many of the biogeochemical cycles in the Ross Sea. Despite this fundamental role, the life history and temporal dynamics of this species are poorly known. This study investigated the contribution of solitary, flagellated forms and colonial cells of P. antarctica to phytoplankton abundance and autotrophic carbon, and the factors that might control the relative importance of these two morphological forms. Solitary P. antarctica cells numerically dominated the phytoplankton assemblage early in austral spring, although colony formation occurred almost immediately upon the onset of net population growth. The percentage of solitary cells relative to total cells (colonial+solitary) was high in early austral spring but decreased to a minimum during late spring; specifically, nearly 98% of the P. antarctica cells were in colonies in late spring, coinciding with the seasonal chlorophyll maximum. Significant phytoplankton mortality rates were positively correlated with high ratios of solitary: total P. antarctica cells. Highest mortality rates were observed during austral spring when solitary cells dominated the P. antarctica population. The abundance of solitary P. antarctica cells began to increase again during late summer, but colonial cell numbers were always greater than those of solitary cells during this period. This increase in the contribution of solitary forms during summer may have been a consequence of more severe micronutrient limitation for the colonies (relative to solitary cells), life history processes of P. antarctica , reduced microzooplankton grazing at that time, or a combination of these and other factors. We conclude that the relative abundance of solitary and colonial forms of this prymnesiophyte alga may be a consequence of seasonal changes in these factors. The outcome of these interactions affects the contribution of this alga to the vertical flux of carbon and the degree to which P. antarctica participates in the microbial food web of the Ross Sea.