Connell Lab - Publication Abstracts

Connell Lab: Publication Abstract

Slemmons C, Connell LB, Beazley W, Redman RS and RJ Rodriguez. (2010) Automated ribosomal intergenic spacer analysis (ARISA) library of Antarctic soil fungi. International Society for Microbial Ecology 2010. Seattle, WA.

A major stumbling block in determining the change in total fungal diversity to assess their potential influence on ecosystem function is determination of which species are present. Several molecular methods have been used in these studies. Of these, automated ribosomal intergenic spacer analysis (ARISA) has been shown to give the greatest number of bands (e.g. ITS diversity) and can give a good “snapshot” of fungal diversity. This method is based on the heterogeneity of length of the entire ITS region, and utilizes PCR amplification of the spacer regions coupled with automated flurometric analysis of the fragments. We adapted this assay to use as a rapid method of species determination from cultured fungal colonies, thus providing both abundance and diversity information faster and at a lower cost than standard sequence analysis.

Duy J, Connell LB, Eck W, Collins SD and R Smith. (2010) Preparation of a surfactant-stabilized gold nanoparticle-peptide nucleic acid conjugates. J Nanopart Res.

A simple, two-step method of producing stable and functional peptide nucleic acid (PNA)-conjugated gold nanoparticles using a surfactant stabilization step is presented. PNA are DNA analogs with superior chemical stability and target discrimination, but their use in metallic nanoparticle systems
has been limited by the difficulty of producing stable colloids of nanoparticle–PNA conjugates. In this work, the nonionic surfactant Tween 20 (polyoxyethylene (20) sorbitan monolaurate) was used to sterically shield gold surfaces prior to the addition of thiolated PNA, producing conjugates which remain dispersed in solution and retain the ability to hybridize to complementary nucleic acid sequences. The conjugates were characterized using transmission electron microscopy, dynamic light scattering, and UV–visible absorbance spectroscopy. PNA attachment to gold nanoparticles was confirmed with an enzyme-linked immunoassay, while the ability of nanoparticle-bound PNA to hybridize to its complement was demonstrated using labeled DNA.

Connell LB, Redman R, Rodriguez R, Barrett A, Iszard M, Fonseca A.(2009) Dioszegia antarctica sp. nov. and Dioszegia cryoxerica sp. nov., novel psychrophilic basidiomycetous yeasts from polar desert soils in Antarctica.Int J Syst Evol Microbiol

During a survey of the culturable soil fungal population in samples collected in Taylor Valley, South Victoria Land, Antarctica, thirteen basidiomycetous yeast strains with orange-coloured colonies were isolated. Phylogenetic analyses of ITS and partial LSU rRNA gene sequences showed that the strains belong to the Dioszegia clade of the Tremellales (Tremellomycetes, Agaricomycotina), but did not correspond to any of the hitherto recognised species. Two novel species, Dioszegia antarctica (CBS 10920T = PYCC 5970T) and D. cryoxerica (CBS 10919T = PYCC 5967T) are described to accommodate ten and three of those strains, respectively. Analysis of ITS sequences demonstrated intra-strain sequence heterogeneity in D. cryoxerica. The latter species is also notable for producing true mycelium with clamp connections and haustoria. However, no sexual structures were observed. The two novel species can be considered obligate psychrophiles since they failed to grow above 20 degrees C and grew best between 10 and 15 degrees C.[VIEW PDF]

Scott Hamilton and Laurie Connell (2009) Improved methodology for tracking and genetically identifying the softshell clam, Mya arenaria. Journal of Shellfish Research, Vol. 28: 4, 747-750

Attempts to characterize the population dynamics of the softshell clam Mya arenaria are complicated by a lack of nonlethal genotyping techniques and reliable tagging methods. A straightforward and nonlethal technique for clam genotyping is presented here, as well as a new method for the long-term tagging of clams. Hemolymph extracted from M. arenaria was used directly in a polymerase chain reaction to amplify successfully a DNA fragment suitable for sequencing. Tested M. arenaria showed 100% (n 1/4 10) survival after a period of 4 wk. In a separate experiment, passive integrated transponder (PIT) tags were inserted between the mantle and shells of 72 clams that were monitored for tag retention and mortality. Among all PIT-tagged clams, there was 100% survival and 92% tag retention. These methods provide a mechanism by which softshell clams can be genotyped and individually monitored during field experiments. [VIEW PDF]

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Connell, LB, Barrett A, Templeton A and Staudigel H. (2009) Fungal diversity associated with an active deep sea volcano: Vailulu'u Seamount, Samoa', Geomicrobiology Journal, 26: 8, 597-605

Active undersea volcanoes generate complex hydrothermal environments that provide microbial habitats rich in reduced metals. These habitats harbor a substantial microbial communities functionally capable of Fe (II) and Mn (II) oxidation. The role of eukaryotes in these settings remains largely unknown. We explored the presence of fungi in actively growing Fe-oxide mats and basalt rock surfaces from the active volcano, Vailulu’u seamount (Samoan chain). Here we document the presence of a diverse fungal community including eight yeasts and yeast-like fungal species isolated from cold hydrothermal environments and basalt rock surfaces. Many of the isolates produce siderophores, a class of molecules used to acquire and utilize Fe (III), and one isolate, Rhodotorula graminis oxidizes Mn(II). These results suggest that fungi may also play a functional role in seafloor alteration and biomineralization processes. [VIEW PDF]

Duy J, Connell LB, and Smith RL. (2008) Development of a direct detection method for potato wart fungus, Synchytrium endobioticum. In Northeast Potato Technology Forum 2008. Fredericton, NB, Canada: Northeast Potato Technology Forum.73.

The chytridiomycete Synchytrium endobioticum is a soil-borne pathogen that infects susceptible potato cultivars and infests the soil by releasing thick-walled sporangia which are easily transported and are viable for at least 15 years. Potato plants that are infected develop wart tissue on tubers and root systems, rendering crops inedible. The pest is endemic in the Netherlands and parts of Canada; therefore it is of prime importance to detect the presence and restrict the entry of the organism into uninfected regions since its spread could have a substantial economic impact on the agricultural industry. Infected plots are checked periodically over 20 years to determine the number of disease-causing sporangia in the soil; current testing methods rely on microscopic identification by skilled trained personnel and take about 1 day to complete. For this reason the need exists to develop a quick, easy, and inexpensive method of detecting the presence of S. endobioticum. Our proposed detection mechanism involves functionalizing gold surfaces with peptide nucleic acid (PNA) probes complementary to S. endobioticum pathotypes and then sensing binding (hybridization) of target sequences through surface plasmon resonance (SPR). PNAs, which are DNA analogs with a backbone composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds, were selected for the probe layer because of their suitability for field use: PNAs are resistant to cleaving by nucleases and proteases and hybridize under a broad variety of conditions. SPR is a good choice for the detection of target hybridization because this technique can accurately measure thickness changes on adsorbed species on surfaces such as gold, with a detection limit down to the femtomolar range. Preliminary experiments using atomic force microscopy (AFM) imaging and ellipsometry show that cysteine-terminated PNA sequences attach to flat gold surfaces through gold-thiol bonds. The PNAs used are biotin-terminated and surface attachment has been verified through biotin quantitation, and hybridization has been confirmed through binding of complementary labeled DNA. Experiments using a field-ready SPR instrument have confirmed both PNA probe binding to the (gold) sensor surface and complementary DNA hybridization at several concentrations. [VIEW PDF]

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Connell LB, Barrett A, Redman R, and Rodriguez R. (2008) The utility of 5-bromo-2-deoxyuridin (BrdU) for identification of active micro-fungi from Antarctic soil. Polar and Alpine Microbiology Conference 2008. Banff, BC, Canada

The thymidine analog 5-bromo-2-deoxyuridin (BrdU) has been used primarily for the study of DNA replication in tissues and more recently for determination of microbial populations that are active under specific environmental conditions. BrdU is incorporated into the newly synthesized DNA during the S phase. DNA extracted from cells that have incorporated BrdU can be isolated using an anti-BrdU antibody for use in the generation clone libraries. Therefore, the use of BrdU directly in environmen¬tal samples could be a powerful tool in establishing which microbes are most actively dividing under specific conditions. However, it has been shown that some organisms, most notably laboratory strains of the bakers yeast Saccharomyces cerevisiae, do not incorporate BrdU. This study determined the BrdU incorporation of 20 microfungal species isolated from Antarctic mineral soils. Cultured cells were grown with BrdU and extracted DNA was used in subsequent PCR reactions using internal transcribes spacer (ITS) ribosomal gene primers ITS4 and ITS5. Additionally, Antarctic soils samples were inoculated with BrdU and extracted DNA was cloned for comparison to both cell culture and pre-BrdU inoculation data. The use of BrdU for determination of micro-fungal activity from Antarctic soils should provide a powerful new tool.

Connell LB, Redman R, Craig SD, Scorzetti G, Iszard M, and Rodriguez R, (2008) Diversity of soil yeasts isolated from South Victoria Land, Antarctica. Microbial Ecology. 56:448-459

Unicellular fungi, commonly referred to as yeasts, were found to be components of the culturable soil fungal population in Taylor Valley, Mt. Discovery, Wright Valley, and two mountain peaks of South Victoria Land, Antarctica. Samples were taken from sites spanning a diversity of soil habitats that were not directly associated with vertebrate activity. A large proportion of yeasts isolated in this study were basidiomycetous species (89%), of which 43% may represent undescribed species, demonstrating that culturable yeasts remain incompletely described in these polar desert soils. Cryptococcus species represented the most often isolated genus (33%) followed by Leucosporidium (22%). Principle component analysis and multiple linear regression using stepwise selection was used to model the relation between abiotic variables (principle component 1 and principle component 2 scores) and yeast biodiversity (the number of species present at a given site). These analyses identified soil pH and electrical conductivity as significant predictors of yeast biodiversity. Species-specific PCR primers were designed to rapidly discriminate among the Dioszegia and Leucosporidium species collected in this study. [VIEW PDF]

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Staudigel H, Furnes H, McLoughlin N, Banerjee NR, Connell LB, and Templeton A. (2008) 3.5 billion years of glass bioalteration: volcanic rocks as a basis for microbial life? Earth Science Reviews 89:156-176

Alteration textures in volcanic glass from the seafloor fall into two classes, one suggestive of abiotic/diffusive hydration and chemical exchange, and another likely to be caused by microbial, cavity-forming, congruent dissolution. Glass bioalteration is common in submarine lavas throughout the world's ocean, dominant in the upper 300 m of the oceanic crust, and found in all well-preserved ophiolites and greenstone belts dating back to 3.5 Ga. It may yield a significant fraction of the global biomass and geochemical fluxes and is relevant to the development of the earliest life on Earth. We present a critical review concerning these glass bioalteration textures and present new data on their microchemical environment. We explore arguments for their biogenicity and further develop the prevalent model for their formation by relating corrosion morphology to the mechanism of microbial dissolution. Biological alteration produces conspicuous micron-scale granular and tubular textures. Granular glass alteration is well explained by colonizing microbes that selectively dissolve the glass in their contact area, forming a sponge-like interconnected network of micronsized cavities along glass surfaces. Tubular alteration meanwhile, is more likely to be caused by filamentous cell extensions in a process similar to fungal tunneling of soil feldspars and marine carbonates. While we see clear functional similarities to fungal dissolution behavior, we do not know whether fungal or prokaryotic organisms are involved. However, this functional constraint may eventually help to identify potential microbes responsible for these features, potentially including eukaryotic or prokaryotic organisms. Yet, we caution that these organisms may be difficult to identify and to study, because they are likely to be sparsely distributed, slow growing, and difficult to cultivate.

Staudigel H, Furnes H, McLoughlin N, Banerjee NR, Connell LB, and Templeton A. (2008) Glass Bioalteration: Inferring microbial function from trace fossil morphology. In International Geological Congress 33 Geomicrobiology: Low-temperature alteration, mineralization, and microbial interactions. Oslo Norway.

The interaction of microbes with volcanic glass may have a significant impact on a range of first-order earth science issues, including the geochemical fluxes between seawater and the oceanic crust, a substantive contribution to global biomass and the earliest life on earth. Glass bioalteration has been recognized on the basis of their distinct textures, and supported by geochemical fingerprinting and characteristic DNA accumulations associated with biotextures in altered glass. Textural arguments rely on the observation of two distinct types of textures associated with volcanic glass alteration: (1) annular bands that propagate from the exterior into the glass towards its fresh core indicating abiotic/diffusive hydration and chemical exchange, and (2) micron-sized tunnels or agglomeration of spherical cavities in glass surfaces that are likely to be caused by microbial, cavity-forming, congruent dissolution. It has been proposed that these textures are formed by colonizing prokaryotic microbes that locally dissolve glass by changing the pH in their contact area. These bioalteration textures are common in submarine lavas throughout the world’s ocean, and the dominant glass alteration mode in the upper 300 m of the oceanic crust. They can be found in nearly all well-preserved ophiolites and greenstone belts dating back to 3.5 Ga. Chemical erosion by colonizing microbes, however, is an unlikely mechanism leading to tubular features that are 100µm deep and less than 1 µm wide tunnels. A microbe deep inside this tunnel has to acquiring the organic carbon and energy necessary to promote dissolution and it has to remove the byproducts of its activity. Furthermore, individual microbes are unlikely to have the sense of “directionality” needed to make straight tubes, or regularly spiraling tubes. To address these issues, we propose that the distinct morphology of tubular bioalteration implies a different mechanism of microbial dissolution. Granular glass alteration is well explained by colonizing microbes that selectively dissolve the glass in their contact area, forming a sponge-like interconnected network of micron sized cavities along glass surfaces exposed to water. Tubular alteration, however, is more likely to be caused by processes similar to fungal tunneling as observed in soil feldspars and marine carbonates. Fungal hyphae clearly have the dimensions and can take on shapes that could generate the diversity of tunneling morphologies observed. Fungal hyphae have been shown to excrete oxalic acid at their tips and to recover nutrients that are brought back into the fungal host cell. Hyphae like organelle are not unique to fungi, however, whereby some bacterial groups (e.g. Acetomycetes) may display a similar function. While any of these processes remain poorly studied, we suggest that tubular textures are caused by such cell extensions offering us some important new clues in the search for identifying the microbes involved in this process.

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Duy J and Connell LB. (2007) Development of fast and accurate detection of Alexandrium species using peptide nucleic acid probes and surface plasmon resonance. In Fourth Symposium on Harmful Algae in the U.S. Woods Hole, MA, USA. 103.

Rapid detection of some members of the toxigenic alga Alexandrium is of paramount importance in cool temperate waters since these organisms produce a suite of toxins responsible for paralytic shellfish poisoning (PSP). Since Alexandrium blooms may not produce cell populations large enough to be visible and these may be below the water surface, early detection can lead to beach closures and fishing restrictions which can prevent human ingestion of contaminated shellfish. Current identification for filed groups still is based on microscopy, which is time consuming and requires skilled personnel. Our proposed detection mechanism involves functionalizing gold surfaces with peptide nucleic acid (PNA) probes and then sensing binding (hybridization) of target sequences through surface plasmon resonance (SPR). PNAs, which are DNA analogs with a backbone composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds, were selected for the probe layer because of their suitability for field use: PNAs are resistant to cleaving by nucleases and hybridize under a broad variety of conditions. SPR is a good choice for the detection of target hybridization because this technique can accurately measure thickness changes on adsorbed species on surfaces such as gold. Preliminary experiments using atomic force microscopy (AFM) imaging and ellipsometry show that cysteine-terminated PNA sequences attach to flat gold surfaces through gold-thiol bonds. The PNAs used are biotin-terminated and surface attachment can be verified through biotin quantitation, while hybridization efficacy can be confirmed through binding of complementary labeled DNA.

Connell LB, Hamilton SA, and Bratcher A. (2007) Population structure of paralytic shellfish poisoning (PSP) resistant softshell clam, Mya arenaria, in Eastern Maine, USA In Fourth Symposium on Harmful Algae in the U.S. Woods Hole, MA, USA. 98.

Atlantic Canadian populations of the softshell clam, Mya arenaria, with repeated exposure have been shown to be resistant to paralytic shellfish poisoning blooms (PSP) caused by the toxigenic algae Alexandrium spp. (MacQuarrie and Bricelj 2000; MacQuarrie 2002; Bricelj et al. 2005; Connell et al. 2006). This resistance is correlated with a mutation in the sodium channel gene pore region, the binding site for saxitoxin, a principle component of paralytic shellfish toxins (PSTs) (Bricelj et al. 2005). This mutation occurs at one base in Domain II resulting in a single amino acid change (Bricelj et al. 2005). A survey of geographically distant M. arenaria populations along the eastern seaboard and northwest coast of North America revealed that the resistant genotype occurs in many populations and that the mutation may have occurred multiple times in separate populations. Selective pressures exerted on clam populations by PSTs have not yet been specifically measured, but a survey of Alexandrium spp. bloom history coupled with the extent of PST exposure to various Mya arenaria populations may provide clues to the rate of expansion of a STX resistant phenotype. This study surveyed Mya arenaria populations from eight locations in Eastern Maine, half of which have a history of repeated PSP blooms. The Domain II sodium channel pore region was sequenced and the populations were compared for percent of homozygous (resistant or sensitive) and heterozygous individuals. These data were then compared with PSP history and dominant ocean currents that can strongly effect larval recruitment. Results from this survey effect management decisions for local shellfish committees on the source of spat to be set in each bay as well as The Maine Department of Marine Resources biotoxin monitoring program.

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Hamilton SA and Connell LB. (2007) New techniques for non-lethal DNA extraction from, and passive integrated transponder (PIT) tagging of, the softshell clam Mya arenaria. In Fourth Symposium on Harmful Algae in the U.S. Woods Hole, MA, USA. 119.

Attempts to characterize the population dynamics of the soft-shell clam Mya arenaria in relation to paralytic shellfish toxins are complicated by a lack of non-lethal genotyping techniques and reliable tagging methods. An easier and non-lethal technique for clam genotyping is presented here. In addition, I propose a new method for clam tagging, which will increase the consistency of identification when retrieving tagged clams. Both of these techniques will be useful in current and future studies on the effects of red tide toxins on the population genetics of soft-shell clams along the coast of Maine. A non-lethal method of genotyping, using small amounts of hemolymph, was tested for the first time with Mya arenaria. A small syringe was used to extract 200?l of hemolymph from the clams’ anterior abductor muscle, which was then applied directly in a polymerase chain reaction (PCR) to successfully amplify a 172 bp DNA fragment for sequencing. Afterwards, all tested clams survived. Using this method, clams can easily be genotyped before placement back in natural conditions for observation in relation to red tide toxins. PIT tags are a useful way to reliably track individual animals in the field. By inserting PIT tags into softshell clams between the mantle and shell, the loss of clam identification could be consistently avoided. This project was designed to determine a method in which PIT tags can be non-lethally inserted, and remain in the clam without rejection. Three groups of clams were acclimatized in a natural sea water flow-through tank. One group acted as a control without tags; while the experimental group had tags inserted. A third group received the same treatment as the tagged group without tag insertion. The clams were then monitored for a number of weeks for death and tag rejection.

Wark M, Ellis L, Fick J, Neivandt D, Connell LB, and Vetelino JF. (2007) A Lateral Field Excited Acoustic Wave Sensor for the Detection of Saxitoxin in Water. In IEEE Ultrasonics Symposium. New York, New York.

In the United States, approximately 20% of all foodborne disease outbreaks result from the consumption of seafood products. Specifically, the disease Paralytic Shellfish Poisoning (PSP) is caused by consuming molluscan shellfish contaminated with a suite of neurotoxins the most potent of which is saxitoxin (STX). The current method for detecting STX is the mouse bioassay in which a mouse is exposed to a shellfish sample and the time required for the mouse to perish is noted. The length of time required for the mouse to die is used to estimate the level of STX in the original sample. Since this technique is a time consuming and costly laboratory-based procedure, a rapid in situ sensor is needed to detect STX levels in shellfish and in sea water so timely closures of shellfish grounds can be made to protect public health. In this work, a novel Lateral Field Excited (LFE) acoustic wave sensor, which has been successfully used for chemical and biological sensing, is employed to detect STX in water, proving itself as a feasible alternative to the mouse bioassay in STX detection. [VIEW PDF]

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Adams B, Bardgett R, Aislabie J, Ayres E, Bamforth S, Bargagli R, Cary C, Cavacini P, Connell LB, Convey P, Fell JW, Frati F, Hogg I, Newsham K, O'Donnell T, Russell N, Seppeldt R, Stevens M, Wall D (2006) Diversity and Distribution of Victoria Land Biota. Soil Biology & Biochemistry 38: 3003-3018

Understanding the relationship between soil biodiversity and ecosystem functioning is critical to predicting and monitoring the effects of ecosystem changes on important soil processes. However, most of Earth's soils are too biologically diverse to identify each species present and determine their functional role in food webs. The soil ecosystems of Victoria Land (VL) Antarctica are functionally and biotically simple, and serve as in situ models for determining the relationship between biodiversity and ecosystem processes. For a few VL taxa (microarthropods, nematodes, algae, mosses and lichens), species diversity has been intensively assessed in highly localized habitats, but little is known of how community assemblages vary across broader spatial scales, or across latitudinal and environmental gradients. The composition of tardigrade, rotifer, protist, fungal and prokaryote communities is emerging. The latter groups are the least studied, but potentially the most diverse. Endemism is highest for microarthropods and nematodes, less so for tardigrades and rotifers, and apparently low for mosses, lichens, protists, fungi and prokaryotes. Much of what is known about VL diversity and distribution occurs in an evolutionary and ecological vacuum; links between taxa and functional role in ecosystems are poorly known and future studies must utilize phylogenetic information to infer patterns of community assembly, speciation, extinction, population processes and biogeography. However, a comprehensive compilation of all the species that participate in soil ecosystem processes, and their distribution across regional and landscape scales is immediately achievable in VL with the resources, tools, and expertise currently available. We suggest that the soil ecosystems of VL should play a major role in exploring the relationship between biodiversity and ecosystem functioning, and in monitoring the effects of environmental change on soil processes in real time and space.

Connell LB, MacQuarrie SP, Twarog BM, Iszard M, Bricelj VM (2006) Population differences in nerve resistance to paralytic shellfish toxins in softshell clam, Mya arenaria, associated with sodium channel mutations. Marine Biology 150:1227-1236

The softshell clam, Mya arenaria, is a commercially important bivalve with wide latitudinal distribution in North America. Populations of clams with a history of repeated exposure to toxic Alexandrium spp. have developed a natural resistance to the paralytic shellfish toxins (PSTs) produced by these algae. An association between PST resistance in individual clams and a single mutation in the saxitoxin (STX) binding region of the alpha-subunit of the voltage-gated sodium (Na+) channel gene was previously identified. Here we establish that more than one mutation associated with nerve resistance to STX occurred at this locus. Both cDNA from mRNA and genomic DNA sequences from individual clams are identical demonstrating that both alleles are expressed simultaneously. In addition, one resistant allele per individual is sufficient to confer neural resistance to STX even though heterozygous individuals show an intermediate level of resistance to STX in vitro nerve trunk assays.

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Connell LB, Ray J, Litaker WW, Tester P (2006) Enhanced detection levels in a semi-automated sandwich hybridization assay using a peptide nucleic acid (PNA) signal probe. African Journal of Marine Biology 28: 237-239

A peptide nucleic acid (PNA) signal probe was tested as a replacement for a typical DNA oligonucleotide-based signal probe in a semi-automated sandwich hybridisation assay designed to detect the harmful phytoplankton species Alexandrium tamarense. The PNA probe yielded consistently higher fluorescent signal intensities than the DNA-based signal probes without any loss of specificity. A combination of species-specific DNA-based capture probes and more generic PNA signal probes are suggested as an optimal configuration for semi-automated sandwich hybridisation assays, designed to detect harmful algal species. The DNA-based probe provides a cost-effective means of capturing RNA from different species, whereas the more expensive PNA signal probe provides enhanced signal intensity compared with traditional DNA probes. Configuring the PNA signal probe to bind multiple species can facilitate the development of new assays and reduce the per-assay cost of using PNA probes.[VIEW PDF]

Connell LB, Redman R, Craig SD, Rodriguez R (2006) Distribution and abundance of fungi in the soils of Taylor Valley, Antarctica. Soil Biology & Biochemistry 38: 3083–3094

The occurrence and distribution of culturable fungi in Taylor Valley, Antarctica was assessed in terms of soil habitat. Soil transects throughout the valley revealed differential habitat utilization between filamentous and non-filamentous (yeast and yeast-like) fungi. In addition, there were significant differences in species distribution patterns with respect to soil pH, moisture, distance from marine coastline, carbon, chlorophyll a, salinity, elevation and solar inputs. Filamentous fungal abundance is most closely associated with habitats having higher pH, and soil moistures. These close associations were not found with yeast and yeast-like fungi demonstrating that yeast and yeast-like fungi utilize a broader range of habitat. An intensive survey of the Victoria Land is necessary to gain a better understanding of their role in soil functioning and nutrient cycling processes.

Fell JW, Scorzetti G, Connell LB, Craig SD (2006) Biodiversity of micro-eukaryotes in Antarctic Dry Valley soil with <5% soil moisture. Soil Biology & Biochemistry 38: 3107–3119

Soils in the Dry Valleys of Antarctica are considered to be among the world's most extreme environments. These soils are old, cold and dry with low contents of organic carbon and nitrogen. Habitats adjacent to water (lakes and ice melts) have significant biological activity as demonstrated by the presence of algal mats, lichens and small invertebrates, particularly nematodes, tardigrades and rotifers. In contrast, there are extensive areas in the Dry Valleys that are extremely dry with less than 5% moisture content. These soils are often salty and appear to be barren of life as they have a coarse texture due to their lack of plant organic material. In contrast, molecular techniques (DNA extraction from soils, cloning and rDNA sequence analysis) demonstrated the presence of a complex micro-eukaryotic food web whose structure and composition varied with moisture content and location. Micro-eukaryotic communities in soils with 0.2–1.3% moisture were represented by species of the yeast genus Trichosporon and an unidentified clade of micro-eukaryotes, whereas levels from 3.1% to 4.9% contained complex food webs including primary producers (chlorophytes and stramenopiles), symbionts (lichen associated fungi), saprophytes (fungi), predators (alveolates and cercozoans) and fungal nematode parasite/pathogens. The soils had a diversity of species (80 species from 15 sites) with a restricted number (3–21 species) at each site. The sensitive and measurable community structure of the low moisture Dry Valley soils provides an unparalleled opportunity to examine local and global environmental effects on micro-eukaryotic community dynamics with multiple trophic levels.

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Bricelj, V. M., L. B. Connell, et al. (2005). Sodium channel mutation leading to saxitoxin resistance in clams increases risk of PSP. Nature. 434: 763-767.

Bivalve molluscs, the primary vectors of paralytic shellfish poisoning (PSP) in humans, show marked inter-species variation in their capacity to accumulate PSP toxins (PSTs) which has a neural basis. PSTs cause human fatalities by blocking sodium conductance in nerve fibers. Here we identify a molecular basis for inter-population variation in PSP resistance within a species, consistent with genetic adaptation to PSTs. Softshell clams (Mya arenaria) from areas exposed to 'red tides' are more resistant to PSTs, as demonstrated by whole-nerve assays, and accumulate toxins at greater rates than sensitive clams from unexposed areas. PSTs lead to selective mortality of sensitive clams. Resistance is caused by natural mutation of a single amino acid residue, which causes a 1,000-fold decrease in affinity at the saxitoxin-binding site in the sodium channel pore of resistant, but not sensitive, clams. Thus PSTs might act as potent natural selection agents, leading to greater toxin resistance in clam populations and increased risk of PSP in humans. Furthermore, global expansion of PSP to previously unaffected coastal areas might result in long-term changes to communities and ecosystems.

Connell, L. B., R. Redman, S. D. Craig and R. Rodriguez (2005). Abundance and distribution of yeast and yeast-like fungi in Taylor Valley Antarctica. Synthesis of Soil Biodiversity and Ecosystem Functioning in Victoria Land, Antarctica, Jekyll Island, GA USA.

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Costas, B., L. B. Connell, et al. (2005). Understanding micro-zooplankton community structure both temporally and spatially in Long Island Sound using species-specific molecular probes. ASLO.

Microzooplankton, specifically oligotrich and choretrich ciliates are an integral part of the ecosystem dynamics of Long Island Sound, playing key roles in the food web as prey, grazers, and nutrient recyclers. Since ciliate communities are diverse and dynamic, with rapid changes in abundance and species composition over short temporal and spatial scales, present techniques limit our understanding of their structure, dynamics, and how they correlate with the physical/chemical environment. Using species specific molecular probes, we have been developing a method for quick identification and quantification of ciliates in a large number of samples. To date, we have focused on the oligotrich Laboea strobila and the tintinnid Favella ehrenbergii. At each sample site 60 liters of water are processed, first concentrating the 20-200 micrometer size fraction, and then using the species specific molecular probes. Our methodology allows us to quickly process a large number and to identify presence of a particular species even when it may be relatively rare in the water column. Methods for concentrating and probing ciliate DNA from environmental samples and preliminary results will be discussed.

Bill, B. D., K. Baugh, et al. (2003). ECOHAB-PNW: Pseudo-nitzscia species composition, abundance and specific toxicity during early and late summer cruises. Second Symposium on Harmful Marine Algae in the U.S., Wood's Hole MA USA.

The ECOHAB-Pacific Northwest project was designed to determine the composition, relative abundance, and toxicity of Pseudo-nitzschia species off the coast of Washington State. This region contains a seasonal oceanographic feature called the Juan de Fuca eddy, which may serve as an initiation site for toxic Pseudo-nitzschia blooms that toxify coastal Washington razor clam populations. Formalin preserved seawater and net tow samples were collected from both the eddy and the coast during cruises in early (2-23 June) and late (30 August-19 September) summer, 2003. Analysis of these samples using a variety of techniques to identify Pseudo-nitzschia species assemblages and to assess species abundance indicated that several Pseudo-nitzschia species were present in both the eddy and the coastal regions. Validation of molecular-based techniques performed onboard ship (whole cell and sandwich hybridization assays), was achieved using both light microscopy and scanning electron microscopy in the laboratory. Specific levels of cellular domoic acid were determined in growth studies using clonal cultures in which toxin levels were assessed using a receptor-binding assay.

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Connell, L. B. (2002). "Rapid identification of marine algae (Raphidophyceae) using three-primer PCR amplification of nuclear internal transcribed spacer (ITS) regions." Phycologia 41(1): 15-21.

The internal transcribed spacer region of the nuclear rRNA gene was used to discriminate among the marine raphidophyte species Chattonella antiqua, Chatonella subsalsa, Fibrocapsa japonica, Heterosigma akashiwo, and Olislhodiscus IUleus. These microalgae can be difficult to identify from natural water samples, owing to their fragile nature and pleomorphic morphology. Several of these species have been associated with massive fin-fish kills throughout the world, and positive identification is therefore of great economic importance. Species-specific diagnostic polymerase chain reaction (PCR) fragment sizes resulting from three-primer amplification reactions can reproducibly detect as few as five cells per fixed sample and as few as one cell from fresh or frozen samples. Recently, an active area of ecological research has been retrospective studies from archival material to determine population dynamics and species diversity. The PCR primers were successfully used with archived material containing either formalin or Lugol's iodine, as well as with fresh cultures. Because of the existence of other techniques more conducive to monitoring for aquaculture, this assay was developed primarily as a research tool for identification of Raphidophyceae from both cultured and archived field samples where cell integrity has been compromised. It has proved to be a valuable technique for detection and discrimination among marine Raphidophyceae, especially when cells cannot be identified morphologically and fixation methods have destroyed cellular RNA content. [VIEW PDF]

Connell, L. B., W. Litaker, et al. (2002). Comparison of PNA vs DNA probes in a sandwich hybridization assay format. Tenth International Conference on Harmful Algal Blooms, St Pete Beach, FL USA.

Identification of harmful algal bloom (HAB) organisms directly from environmental samples is currently a growing field of research. Water quality managers, researchers and aquaculture require accurate detection of possible blooms in a timely manner to reduce losses. A number of new molecular techniques are being developed for the detection of HAB species, most are based on RNA targeted DNA probes. One of the promising new molecular tools are the DNA analogs peptide nucleic acids (PNA). This feasibility study compared traditional DNA based probes for detection of Alexandrium to analogous PNA probes for use in a sandwich hybridization assay (SHA). Both SHA and direct plate assays were used to compare binding and sensitivity of PNA vs. DNA signal and capture probes. Conditions under which PNA probes were stable and effective (such as strong chaotropic agents) were also explored. Sensitivity of labeled probes (bi- and tri- labeled DNA vs. bi—labeled PNA) assays revealed that the PNA signal probes were superior to the DNA signal probe. However, generally the results of these experiments were mixed, demonstrating that the decision to choose DNA over PNA probes must be made on a case by case basis.

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Connell, L. B., H. Stender, et al. (2002). "Rapid detection and identification of Brettanomyces from winery air samples based on peptide nucleic acid analysis." American Journal of Enology & Viticulture 53(4): 322-324

A new filter-based chemiluminescent in situ hybridization method using peptide nucleic acid probes for rapid and simultaneous detection, identification, and enumeration of Brettanomyces from air samples was developed. The method also allows for detection of the yeast prior to visual appearance of large colonies. Using this technique, air samples from various locations within a winery (crush, tank, barrel, and bottling rooms) were analyzed. Brettanomyces was identified in samples taken from all four rooms, confirming that this spoilage yeast can be spread through a winery by air currents.[VIEW PDF]

Connell, L. B., V. L. Trainer, et al. (2002). A molecular basis for variation in resistance to paralytic shellfish toxins in bivalve molluscs. Tenth International Conference on Harmful Algal Blooms, St Pete Beach FL.

Paralytic shellfish toxins (PSTs) impair normal nerve function by specifically binding to the voltage-sensitive sodium (Na+) channel, thereby blocking the channel opening and causing paralysis. A paradox in the marine environment is that PSTs are produced by the algae that bivalves eat, yet can be toxic to those same organisms. Some bivalves, however, clearly have adapted to tolerate toxins in their food. Prior research demonstrated significant intraspecific variation in burrowing, feeding, toxin uptake and nerve responses to PSTs in the softshell clam, Mya arenaria, and suggested a genetic basis for this variation. Our current study investigates the molecular mechanism of resistance to PSTs within and among bivalve species. We have completed the sequence for the functional domains (DI-DIV) of the Na+ channel gene in M. arenaria using nested PCR and degenerate primers derived from published Na+ channel amino acid sequences. The predicted amino acid alignment of the M. arenaria gene with known Na+ channels reveals a high degree of homology within the functional domains (for example up to 100% for DI in squid). Because the SS1-SS2 amino acid sequence in each functional domain contains residues essential for saxitoxin (STX) binding, we have sequenced these regions in individual clams (intraspecific characterization) that have been characterized by nerve response as either resistant or sensitive to STX. A predicted amino acid residue in the DII pore region (SS1-SS2) that is unique in M. arenaria compared to all other known Na+ channel sequences is a candidate for conferring differential response to STX in this clam. In future, comparison of "resistant" subtypes across species will allow us to develop specific molecular probes for this genotype. Identification of inter- and intraspecific genetic differences will contribute to our fundamental understanding of toxin resistance mechanisms and perhaps open future avenues for selective breeding.

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Ster, C.M., L.B. Connell et al (2002) Morphological, toxicological, and genetic differences among Pseudo-nitzschia (Bacillariophyceae) species in inland embayments and outer coastal waters of Wahington State, USA. Journal of Phycology 38: 55-65

Plankton samples from three inland embayments and several outer coastal sites of Washington State were collected from 1997 through 1999 and were examined for the presence of diatoms of the genus Pseudo-nitzschia and levels of the toxin, domoic acid (DA). Seven species were observed, including Pseudonitzschia pungens(Grunow ex Cleve) Hasle, P. multiseries(Hasle) Hasle, P. australis Frenguelli, P. fraudulenta (Cleve) Hasle, P. cf. heimii Manguim, P. pseudodelicatissima(Hasle) Hasle, and P. delicatissima(Cleve) Heiden. The coastal Pseudo-nitzschia species assemblages differed significantly from those observed within embayments. The dominant species observed at coastal sites were P. pseudodelicatissima and P. cf. heimii. Pseudo-nitzschia assemblages found in embayments included one or more of the following species: P. pungens, P. multiseries, P. australis, P. pseudodelicatissima, and P. fraudulenta. The nuclear large subunit rRNA gene was sequenced for six of the seven species identified. This sequence revealed that P. multiseries, P. pungens, P. australis, and P. heimii were genetically similar to those found in California, whereas P. delicatissima and P. pseudodelicatissima were distinct from the California isolates. Although the concentrations of DA in razor clams along Washington State coasts have exceeded regulatory limits several times since 1991, levels of DA in shellfish from Washington State embayments have not yet exceeded regulatory limits. The widespread presence of toxin-producing Pseudonitzschia species suggests, however, that toxic blooms are likely to occur within embayments in the future. In conjunction with the monitoring of environmental conditions conducive to toxic bloom formation, the development of species-specific probes for rapid and accurate detection of potentially toxic Pseudo-nitzschia species in this region would enable the forecasting of a toxic event before DA accumulates in shellfish, thereby reducing the impacts to coastal communities.[VIEW PDF]

Tyrrell, J., L. B. Connell, et al. (2002). "Monitoring for Heterosigma akashiwo using a sandwich hybridization assay." Harmful Algae 1(2): 205-214.

Field testing of a ribosomal RNA (rRNA)-targeted sandwich hybridization assay (SHA) for Heterosigma akashiwo (Raphidophyceae) in Puget Sound,WA, USA, has showed that the lower limit of detection is well below the level at which cells pose a danger to fish. Moreover, the assay has proven to be both rapid and easy-to-use. Isolates of H. akashiwo from Australia, Japan, New Zealand, South Korea, Spain and USA were correctly identified using the SHA, indicating that this diagnostic tool could be deployed globally. Samples containing H. akashiwo can be preserved for subsequent SHA analysis using several methods: fixation with acidic Lugol’s iodine followed by room temperature storage, collection onto Durapore filters followed by storage at -70 C or, alternatively, the filters are mixed with a lysis solution buffer and the sample lysate stored at -70 C. Additionally, we sought to determine whether the SHA could successfully detect H. akashiwo in the presence of clay that might some day be used to mitigate the impacts of natural H. akashiwo blooms. Results from preliminary laboratory trials indicate that clay at the maximum proposed dosage rate does not interfere with the assay. Thus, it may be possible to use the SHA as a simple means of following the fate of H. akashiwo cells during larger-scale clay mitigation trials.[VIEW PDF]

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Kooistra, W. H. C. F., M. K. de Boer, et al. (2001). "Variation along ITS markers across strains of Fibrocapsa japonica (Raphidophyceae) suggests hybridization events and recent range expansion." Journal of Sea Research 46: 213-222.

The flagellate micro-alga Fibrocapsa japonica can form harmful algal blooms along all temperate coastal regions of the world. The species was first observed in coastal waters of Japan and the western US in the 1970s; it has been reported regularly worldwide since. To unravel whether this apparent range expansion can be tracked, we assessed genetic variation among nuclear ribosomal DNA ITS sequences, obtained from sixteen global strains collected over the course of three decades. Ten sequence positions showed polymorphism across the strains. Nine out of these revealed ambiguities in several or most sequences sampled. The oldest strain collected (LB-2161) was the only one without such intra-individual polymorphism. In the others, the proportion of ambiguities at variable sites increased with more recent collection date. The pattern does not result from loss of variation due to sexual reproduction and random drift in culture because sister cultures CS-332 and NIES-136 showed virtually the same ITS-pattern after seven years of separation. Neither are the patterns explained by recent range expansion of a single genotype, because in that case one would expect lowest genetic diversity in the recently invaded North Sea; instead, polymorphism is highest there. Recent ballast-water-mediated mixing of formerly isolated populations and subsequent ongoing sexual reproduction among them can explain the increase in ambiguities. The species’ capacity to form harmful blooms may well have been enhanced through increased genetic diversity of regional populations. [VIEW PDF]