Sandra A. Nierzwicki-Bauer

Bacterial Community Structure of Acid-Impacted Lakes: What Controls Diversity?†

ABSTRACT Although it is recognized that acidification of freshwater systems results in decreased overall species richness of plants and animals, little is known about the response of aquatic microbial communities to acidification. In this study we examined bacterioplankton community diversity and structure in 18 lakes located in the Adirondack Park (in the state of New York in the United States) that were affected to various degrees by acidic deposition and assessed correlations with 31 physical and chemical parameters. The pH of these lakes ranged from 4.9 to 7.8. These studies were conducted as a component of the Adirondack Effects Assessment Program supported by the U.S. Environmental Protection Agency. Thirty-one independent 16S rRNA gene libraries consisting of 2,135 clones were constructed from epilimnion and hypolimnion water samples. Bacterioplankton community composition was determined by sequencing and amplified ribosomal DNA restriction analysis of the clone libraries. Nineteen bacterial classes representing 95 subclasses were observed, but clone libraries were dominated by representatives of the Actinobacteria and Betaproteobacteria classes. Although the diversity and richness of bacterioplankton communities were positively correlated with pH, the overall community composition assessed by principal component analysis was not. The strongest correlations were observed between bacterioplankton communities and lake depth, hydraulic retention time, dissolved inorganic carbon, and nonlabile monomeric aluminum concentrations. While there was not an overall correlation between bacterioplankton community structure and pH, several bacterial classes, including the Alphaproteobacteria, were directly correlated with acidity. These results indicate that unlike more identifiable correlations between acidity and species richness for higher trophic levels, controls on bacterioplankton community structure are likely more complex, involving both direct and indirect processes.

Differences in mRNA levels in Anabaena living freely or in symbiotic association with Azolla.

Azolla is a small water fern in whose leaf cavities the filamentous nitrogen‐fixing cyanobacterium Anabaena azollae is symbiotically associated. Using cloned genes from Anabaena 7120 for glutamine synthetase (GS), ribulose‐1,5‐bisphosphate (RuBP) carboxylase, nitrogenase and the 32‐kd protein of photosystem II, mRNA levels of the corresponding genes in the Anabaena endosymbiont were studied by Northern hybridization. In RNA isolated from the endosymbiont there is a 10‐fold reduction of GS transcript levels, a greater than 5‐fold increase in 32‐kd transcript levels and a greater than 5‐fold decrease in RuBP carboxylase transcript levels, compared with levels in the free‐living Anabaena azollae. In the endosymbiont and in heterocysts of the free‐living Anabaena azollae the nif H, nif D, and nif K genes are transcribed from a single nif HDK operon.

Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.

Specific amplification of the 18S rRNA gene as a method to detect zebra mussel (Dreissena polymorpha) larvae in plankton samples

An important issue in the management of zebra mussel (Dreissena polymorpha) populations is early, rapid, and accurate detection of the planktonic larvae (veliger) of the zebra mussel. The goal of this study was to explore the feasibility of developing a molecular approach for the detection of zebra mussel larvae in diverse environments. In this study a Dreissena polymorpha-specific 18S ribosomal RNA gene targeted oligonucleotide primer (ZEB-715a) and Polymerase Chain Reaction (PCR) assay was developed and compared with cross-polarized microscopy as a means to detect zebra mussel veligers in plankton samples. The design of the zebra mussel-specific primer was facilitated by sequencing nearly the complete 18S rRNA gene from the zebra mussel and three other closely related freshwater Veneroids including the quagga mussel (D. bugensis), the dark false mussel (Mytilopsis leucophaeata), and the Asian freshwater clam (Corbicula fluminea). The specificity of the primer for the zebra mussel was empirically tested by using the primer as a direct probe in a blot hybridization format. A single veliger in a plankton sample could be detected by PCR using this approach. Veliger detection sensitivity using the PCR approach was estimated to be over 300 times more sensitive than cross-polarized light microscopy based techniques. Cross-polarized light microscopy and the PCR technique were used to identify the presence of zebra mussel larvae in plankton samples that were collected from a variety of natural and industrial water sources. Detection results (presence or absence) were generally consistent between the two methods. Although additional studies will be required before routine application of molecular based veliger detection technology is available, a long-term goal of this work is the application of molecular technology to the development of a field device for the routine detection and quantification of zebra mussel veligers.

Chronic and episodic acidification of Adirondack streams from acid rain in 2003-2005.

Limited information is available on streams in the Adirondack region of New York, although streams are more prone to acidification than the more studied Adirondack lakes. A stream assessment was therefore undertaken in the Oswegatchie and Black River drainages; an area of 4585 km(2) in the western part of the Adirondack region. Acidification was evaluated with the newly developed base-cation surplus (BCS) and the conventional acid-neutralizing capacity by Gran titration (ANC(G)). During the survey when stream water was most acidic (March 2004), 105 of 188 streams (56%) were acidified based on the criterion of BCS < 0 microeq L(-1), whereas 29% were acidified based on an ANC(G) value < 0 microeq L(-1). During the survey when stream water was least acidic (August 2003), 15 of 129 streams (12%) were acidified based on the criterion of BCS < 0 microeq L(-1), whereas 5% were acidified based on ANC(G) value < 0 microeq L(-1). The contribution of acidic deposition to stream acidification was greater than that of strongly acidic organic acids in each of the surveys by factors ranging from approximately 2 to 5, but was greatest during spring snowmelt and least during elevated base flow in August. During snowmelt, the percentage attributable to acidic deposition was 81%, whereas during the October 2003 survey, when dissolved organic carbon (DOC) concentrations were highest, this percentage was 66%. The total length of stream reaches estimated to be prone to acidification was 718 km out of a total of 1237 km of stream reaches that were assessed.

Acidification in the Adirondacks: Defining the Biota in Trophic Levels of 30 Chemically Diverse Acid-Impacted Lakes

The Adirondack Mountains in New York State have a varied surficial geology and chemically diverse surface waters that are among the most impacted by acid deposition in the U.S. No single Adirondack investigation has been comprehensive in defining the effects of acidification on species diversity, from bacteria through fish, essential for understanding the full impact of acidification on biota. Baseline midsummer chemistry and community composition are presented for a group of chemically diverse Adirondack lakes. Species richness of all trophic levels except bacteria is significantly correlated with lake acid-base chemistry. The loss of taxa observed per unit pH was similar: bacterial genera (2.50), bacterial classes (1.43), phytoplankton (3.97), rotifers (3.56), crustaceans (1.75), macrophytes (3.96), and fish (3.72). Specific pH criteria were applied to the communities to define and identify acid-tolerant (pH<5.0), acid-resistant (pH 5.0-5.6), and acid-sensitive (pH>5.6) species which could serve as indicators. Acid-tolerant and acid-sensitive categories are at end-points along the pH scale, significantly different at P<0.05; the acid-resistant category is the range of pH between these end-points, where community changes continually occur as the ecosystem moves in one direction or another. The biota acid tolerance classification (batc) system described herein provides a clear distinction between the taxonomic groups identified in these subcategories and can be used to evaluate the impact of acid deposition on different trophic levels of biological communities.

Metal and proton toxicity to lake zooplankton: A chemical speciation based modelling approach

The WHAM-FTOX model quantifies the combined toxic effects of protons and metal cations towards aquatic organisms through the toxicity function (FTOX), a linear combination of the products of organism-bound cation and a toxic potency coefficient for each cation. We describe the application of the model to predict an observable ecological field variable, species richness of pelagic lake crustacean zooplankton, studied with respect to either acidification or the impacts of metals from smelters. The fitted results give toxic potencies increasing in the order H(+) < Al < Cu < Zn < Ni. In general, observed species richness is lower than predicted, but in some instances agreement is close, and is rarely higher than predictions. The model predicts recovery in agreement with observations for three regions, namely Sudbury (Canada), Bohemian Forest (Czech Republic) and a subset of lakes across Norway, but fails to predict observed recovery from acidification in Adirondack lakes (USA).

Extraction and purification of PCR amplifiable DNA from lacustrine subsurface sediments

Abstract An extraction procedure which recovers high quality DNA from microbial communities in lacustrine type sediments has been developed. This method employs direct lysis of cells in an agarose-sediment mixture, electroelution of community DNA, followed by ammonium acetate precipitation for further purification. The extracted community DNA was found to be suitable for PCR amplification with 16S rRNA gene-specific primers when T4 gene 32 protein was present in amplification reactions.

Introduction Pathways, Differential Survival of Adult and Larval Zebra Mussels (Dreissena polymorpha), and Possible Management Strategies, in an Adirondack Lake, Lake George, NY

Abstract The introduction pathway and source of zebra mussel larvae into Lake George, NY were determined and a general bioassay was developed to assess the zebra mussel colonization risk of a water body. The presence of zebra mussel larvae (veligers), recruitment, and adults were monitored in Lake George from 1995–2003. All observations of zebra mussel veligers were at marinas, boat ramps, or areas heavily used by fishing boats. Models and observation suggest that human activity is the primary mechanism by which zebra mussels are transported overland. Although one small colony of zebra mussels was discovered during this period, no evidence was found of recruitment or permanent colonization by zebra mussels in Lake George. A series of bioassays to assess both larval and adult growth and survival was developed and indicated that Lake George water limited the survival of zebra mussel larvae but not of adults. These bioassays confirmed model predictions that zebra mussel recruitment is limited by the moderate water alkalinity in Lake George, but that adults were able to survive and grow. The unique water chemistry that limits zebra mussel colonization, and the close proximity of Lake George to other mussel-populated waters, make Lake George an ideal natural laboratory to study the introduction process of zebra mussel adults and/or larvae into a landlocked lake. Although zebra mussels have colonized the major waterways of Eastern North America, the establishment of zebra mussel populations in landlocked lakes is occurring much more slowly. Public outreach and education efforts that appear to aid in limiting the introduction of zebra mussels are also discussed.