Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
PROGRAM SCHEDULE
Please Note: All talks are scheduled in the Bush Memorial Center. Registration, Breaks, and
the Keynote Reception are in the Buchman Pavilion. Dinner (Tues, 6th) and the poster session
are in the Multipurpose Room of the Robison Athletic and Recreation Center. For lunch and
dinner on Wednesday the 7th, a local restaurant guide will be available.
Tuesday, June 6, 2006
12:00 – 1:15 PM
Registration Buchman Pavilion
1:15 – 1:30
Jack Harris
Opening comments
Bush Memorial Center
1:30 – 2:00
M. E. Dueker, and Raymond Sambrotto. Summer microbial populations in
the lower Hudson River estuary and their relationship to dissolved organic nutrients
2:00 – 2:30
O. Roger Anderson. A Method for Estimating Volume-based Carbon
Content of Amoeboid Protists and its Application to the Fractional Analysis of Carbon in
Amoebae in Some Protistan Communities.
2:30 – 3:00
Katharine Haberlandt and George McManus. Potential use of the
oligotrich ciliate, Strombidium stylifer, as a food source for marine aquaculture.
3:00 – 3:30
foraminifera
John J. Lee and Megan Cervasco. Species of Symbiodinium from soritid
3:30 – 4:00
Break
4:00 – 4:30
Dunaliella
Visviki, I., Judge, M.L., and Mahony, J. High Arsenate Resistance in
4:30 – 5:00
its role in biofilms.
David S. Domozych. A different view of the protistan extracellular matrix:
5:00 – 6:00
President’s Address
Buchman Pavilion
D. Wayne Coats
6:00 – 9:00+
Microbial Controls of Chesapeake Bay
Phytoplankton
Dinner and Poster Session
Multipurpose Room, Robison Athletic and Recreation Center
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Wednesday, June 7, 2006
8:30 – 9:00 AM
John C. Clamp. 18s rRNA Gene Sequences Reveal Diversity Within the
Genus Zoothamnium (Ciliophora, Peritrichia)
9:00 – 9:30
C. Chantangsi, M.C. Strüder-Kypke, and D.H. Lynn. Barcoding Protists:
A Trailblazing Study on a Ciliate Genus of Closely Related Species, Tetrahymena
9:30 – 10:00
Joe Salas-Marco, Hua Fan-Minogue, Adam K. Kallmeyer, Lawrence A.
Klobutcher, Philip J. Farabaugh, and David M. Bedwell. Distinct Paths to Stop Codon
Reassignment in the ciliates Tetrahymena thermophila and Euplotes octocarinatus.
10:00 – 10:30
Break
Buchman Pavilion
10:30 – 11:00
Mary Ellen Jacobs, Leroi DeSouza, Haresha Samaranayake, K. W.
Michael Siu and Lawrence A. Klobutcher. The Tetrahymena thermophila phagosome proteome
11:00 – 11:30
Ethan Fremen and Linda A. Hufnagel. Progress on a new web-accessible
archiving system for cilioprotists of southern New England
11:30 – 12:00
history
William Reynolds. Voyages of Discovery: Science learning and living
12:00 – 1:30
Lunch Break (a list of local restaurants is available)
1:30 – 2:00 PM
H. Samaranayake and L. A. Klobutcher. A Tetrahymena thermophila
TtVPS13A antisense ribosome mutant is impaired in phagocytosis and has a temperature sensitive growth defect.
2:00 – 2:30
Guerra, C., Bell, A., Awan, A., Christensen, S.T. and Satir, P. Growth
Control Signaling Pathways in Tetrahymena Cilia II
2:30 – 3:00
Aaron J Bell, Peter Satir, Gary W. Grimes (posthumously). Mirror-imaged
doublets of Oxytricha fallax: implications for the development of left-right asymmetry
3:30 – 4:00
Break
Buchman Pavilion
3:00 – 3:30
Megan Noyes, Spencer J. Greenwood, Denis H. Lynn. Chemotactic
responses of two philasterine scuticociliates, endoparasites of different crustacean hosts
4:00 – 4:30
Andrea Habura, Susan T. Goldstein, Sarah Broderick, and Samuel S.
Bowser. Hidden diversity of allogromiid foraminiferans in low-latitude environments.
4:30 – 5:00
Harris, J.K., Blair, J., Mastroianni, J., Hansen, A., Walker, S.E., Miller,
M., and Bowser, S.S. In situ time-lapse observation of Antarctic foraminifera
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Wednesday, June 7, 2006
5:00 – 6:00PM
Keynote Address, Bush Memorial Center
Laura Landweber
6:00 – 7:00
Keynote Reception
Detangling scrambled genes
Buchman Pavilion
Thursday, June 8, 2006
8:30 – 9:00 AM
Daniela Brito, Irina Tikhonenko, Alexey Khodjakov, and Michael
Koonce. Motile Microtubules in Dictyostelium
9:00 – 9:30
Avelina Espinosa, Adeline Ashmore, Chelsea Fitzsimmons-Diaz and
Melissa Roberto. Identification of a novel bifunctional EhADH2-like enzyme in Entamoeba
invadens
9:30 – 10:00
Rebecca C. MacLean, Leslie Y. Whiteman, and Francine MarcianoCabral. Sequencing of a N. fowleri genomic DNA library and identification of a gene encoding a
putative patatin-like serine hydrolase protein
10:00 – 10:30
Susan T Goldstein, Samuel S. Bowser, Andrea Habura, and Elizabeth A.
Richardson. Fine structure and constructional themes of modern “Allogromiid” foraminifera: an
overview.
10:30 – 11:00
Break
Buchman Pavilion
11:00 – 11:30
A.Teal, J. Ennis, M. Marchewka, J. Hans, S. Madison-Antenucci, and J.
Keithly. A New York State waterpark outbreak of human cryptosporidiosis
11:30 – 12:00
Mary Marchewka, Jennifer Hans, and Susan Madison-Antenucci. Using
real time qRT-PCR to evaluate loss of RNA Editing Associated Protein-1 on editing
12:00 – 12:30
Janet Keithly, Vlasta Ctrnacta, Frantisek Stejskal, Jeffrey Ault, Karolyn
Buttle, Carmen Mannella, and Susan Langreth. Novel Compartmentalization of Core
Metabolism in Cryptosporidium parvum.
12:30 – 1:00
Ulrich, P.N. Marsh, A.G. and S.S. Bowser. Respiration of Antarctic
benthic foraminifera - optical microplate respirometry and effects of oxygen limitation.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
TALK ABSTRACTS
SUMMER MICROBIAL POPULATIONS IN THE LOWER HUDSON RIVER
ESTUARY AND THEIR RELATIONSHIP TO DISSOLVED ORGANIC NUTRIENTS.
1
M. E. Dueker, and 2Raymond Sambrotto
1
Polgar Fellow, Hudson River Foundation, 1,2Lamont Doherty Earth Observatory, Columbia
University, NY
Four sites in the lower Hudson River Estuary (HRE) were sampled repeatedly during the summer
of 2005 (Piermont, NY; lower Manhattan at Pier 26; Newtown Creek; and Gowanus Creek) to
assess the relationships among the levels of nutrients, bacteria, and the upper trophic levels of the
microbial loop represented by ciliates and amoebae. The dissolved organic nitrogen and
phosphorus pools (DON & DOP) dominated dissolved nitrogen and phosphorus at most sites.
Piermont had the lowest phosphate levels and nitrate levels were higher along the main trunk of
the River than in the eastern tributaries. The sewage inputs to Newtown may be the cause of its
unique aspects such as: high bacteria, phosphate and ammonium levels and low ciliate, amoebae,
and DON levels. The lower total dissolved nitrogen there may reflect nitrogen removal during
tertiary treatment and the low-oxygen conditions may account for its lower microbial diversity.
This diversity, along with the inverse relationship between DON and DOP, may be a useful
diagnostic for the relative amount of microbial grazing and overall HRE health. Centric diatom
blooms appeared as small-scale (10s to 100s of meters) patches advected through the lower
HRE. Diurnal sampling at Gowanus showed that run-off was enriched in nitrogenous nutrients,
while phosphorus was mainly associated with the water from the upper Bay. We also assessed
the suitability of various environments for microbial growth with cross-inoculation experiments
that suggested that sites on the main trunk of the River had similar microbial growth conditions.
This contrasted to crosses between the tributaries in which aloricate ciliates and Mesodinium spp.
exhibited large changes.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
A Method for Estimating Volume-based Carbon Content of Amoeboid Protists and its
Application to the Fractional Analysis of Carbon in Amoebae in Some Protistan
Communities.
O. ROGER ANDERSON
Columbia University: Lamont-Doherty Earth Observatory, Palisades, NY
A novel method for estimating the volume of amoebae based on the length of a motile cell has
been developed. A complete analysis of the carbon content of protistan communities has been
limited by lack of a convenient method of estimating amoeba cell volume to be used in
calculating volume-based cell carbon content. A statistically significant, simple linear regression
equation was obtained relating the sphered-up cell diameter (D) and motile cell length (L): D =
0.6 L (r = 0.993, df = 38, p < 0.001). This relationship holds for a wide variety of
gymnamoebae when certain rather simple measurement rules are applied. A topological rationale
for this relationship is presented as a “Length:volume conservation rule.” A physiological
hypothesis for the constancy of the D:L relationship, based on cell morphology and cytoplasmic
pressure flow theory, will be presented. Using this formula, and previously published formulas
for the relation of cell carbon content to cell volume, the carbon content of amoebae in some
terrestrial and aquatic protistan communities was assessed. The fractional carbon content of
amoebae, compared to the carbon content of bacterial prey, is presented for samples taken from
terrestrial moss and from freshwater aquatic environments.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Potential use of the oligotrich ciliate, Strombidium stylifer, as a food source for marine
aquaculture.
Katharine Haberlandt and George McManus
Department of Marine Sciences, University of Connecticut. Groton, CT
This project aims to examine the suitability of the marine oligotrich ciliate, Strombidium stylifer,
as a larval food in marine aquaculture. We have isolated this planktonic, mixotrophic ciliate
from nearshore waters of Brazil, Scotland, and the USA (New England). In culture, we have
evaluated changes in growth rate relative to food concentration, light intensity, and culture age,
grazing rates relative to food concentration, and effects of different microalgal diets on growth.
S. stylifer has a higher maximum growth rate and lower food concentration at which it is at half
of the maximum growth rate, compared to a rotifer species (Brachionus sp.) commonly used as a
larval feed in marine aquaculture. Furthermore, there does not appear to be a declining trend in
growth rate over hundreds of generations in culture or at different light intensities. S. stylifer had
a higher growth rate when fed a mixed diet of algae consisting of a Tetraselmis sp.
(prasinophyte) and Rhodomonas sp. (cryptophyte). Biochemical analysis of S. stylifer is
currently in progress to estimate protein, lipid, fatty acid, carbon, and nitrogen content and assess
its nutritional value as a larval feed. Future work will include experimentation using S. stylifer as
a larval feed for marine larval species yet to be determined.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Species of Symbiodinium from soritid foraminifera
John J. Lee and Megan Cervasco
Department of Biology, City College of City University of New York, and Division of
Invertebrates, The American Museum of Natural History, New York, NY
The Soritinae are a subfamily of large imperforate foraminifera (Phylum Granuloreticulosea)
with discoid annular tests that in some species reach diameters of ~15mm. All members of the
subfamily are hosts for endosymbiotic dinoflagellates belonging to the genus Symbiodinium.
Previous research by others (Garcia-Cuetos et al 2005) recognized phylotypes of Symbiodinium
spp which could be categorized into 3 clades and 5 subclades that were found as symbionts in 22
phylotypes of Soritinae. Until the present, we were unable to link specific morphotypical epithets
to the molecularly distinguished phylotypes. Cultured isolates from soritines were found to
differ from each other in a number of ways. A greater number of the isolates in culture are in the
vegetative stage of their life cycle throughout a batch culture cycle. In exception, but in common
with our isolates from Casseopea spp, one soritid isolate has some actively swimming zoospores
through the culture cycle. Zoospore formation in some isolates can be triggered by enriching the
culture with µM levels of nitrate, ammonia or phosphate. Motility in other isolates can be
triggered by diluting the medium. The vegetative cells of our isolates characteristically differ in
size from averages of 7 to 14 µm. Some isolates form a characteristic ring on the surface of
batch cultures. There are two gross plastid architectures in our isolates. Some of our isolates
have petal-like plastids and others reticulate plastids. Some isolates have large assimilation
bodies, others have very small ones. None of our isolates have fuzzy surfaces similar to the kind
found of Symbiodinium pilosum. Their surfaces are all smooth. None of the vegetative cells of
our isolates bears surface plates.
Supported by PSC-CUNY grant 65713-00 34
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
High Arsenate Resistance in Dunaliella
1
Visviki, I., 1Judge, M.L., and 2Mahony, J.
1
Biology Department, College of Mount Saint Vincent/Manhattan College
2
Department of Civil and Environmental Engineering, Manhattan College, NY
Arsenic is a metalloid that can occur in aquatic ecosystems naturally or it can accumulate due to
influx from anthropogenic sources. Arsenic is the number one pollutant of concern on EPA’s
National Priorities List due to its effects on human health. Thus, it is of paramount importance to
identify promising plant species for phytoremediation of polluted ecosystems. Such species must
exhibit high arsenic resistance, arsenic accumulation and detoxification. In this study the
resistance to arsenate of two unicellular chlorophytes of the genus Dunaliella is evaluated using
a combination of individual and population parameters. 96 hour exposure of D. tertiolecta and D.
primolecta to 0, 153, 200 and 307 mg/L of arsenate in Erdschreiber (enriched) medium had no
effect on growth, total chlorophyll, chlorophyll a, chlorophyll b, motility, or cell size
distribution. Similar results were obtained with arsenate exposure in unenriched seawater. High
arsenate resistance makes these marine chlorophytes promising candidates for phytoremediation.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
A different view of the protistan extracellular matrix: its role in biofilms.
David S. Domozych
Department of Biology and Skidmore Microscopy Imaging Center. Skidmore College
Saratoga Springs, NY
The protistan extracellular matrix (ECM) consists of a diverse assortment of macromolecules
that are organized to manifest such structures as scales, thecal plates, shells and cell walls. These
coverings often require complex synthesis/secretory mechanisms, contribute to numerous
cellular functions and may represent the bulk of fixed carbon for a cell. However, the ECM of
many protists also consists of often-overlooked extracellular polymeric substances (EPS) that are
found external to the more recognizable ECM structures. The EPS of many protists is directly
involved in the organism’s ability to enter into, and live in, a complex consortium of microbes
attached to a substrate, or the biofilm. While recent research dealing with biofilm prokaryotes
has revolutionized our understanding of microbial community ecology, we know surprisingly
little about the role of eukaryotes in biofilms and the biochemistry/structure/role of their
EPS/ECM in biofilm dynamics. Recently, my laboratory began an extensive examination of
biofilms found in transient wetlands of the Adirondack region of New York. Amongst the wide
array of biofilm protists are the desmids, advanced members of the green algae. Desmids secrete
an EPS outside their cell walls that consists of fucoxyloglucuronans, other polysaccharides and
some protein components. The macromolecules of the EPS are involved in adhesion to a
substrate, post-adhesion gliding mechanisms and final ensheathment within the biofilm. The EPS
sheath is often produced in large amounts (3 µg/cell) and may serve as an “oasis” for biofilm
heterotrophs. The EPS is synthesized in the Golgi Apparatus (GA) and transported to the cell
surface in large vesicles. These vesicles enter into subplasmamembrane channels of actinmediated cytoplasmic streaming. This peripheral vesicular reservoir of vesicles serves as a
readily-available source of EPS when the cell requires its immediate secretion after contact with
a substrate or during the gliding process.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
PRESIDENT’S ADDRESS
Microbial Controls of Chesapeake Bay Phytoplankton
D. Wayne Coats
Smithsonian Environmental Research Center
Microbial pathogens including viruses, bacteria, protists, and fungi act at all trophic levels within
the global ecosystem to exert top-down control on host populations. For terrestrial
environments, the potentially devastating impact of disease on humans, their domestic animals,
and crops is well established. Marine pathogens, however, are less well understood, with most
knowledge coming from studies of important fishery species. Formulation of the microbial-loop
concept over a quarter century ago brought increased awareness to the significance of microbes
in the flow of energy and matter within planktonic ecosystems, but did not address the issue of
disease. Over the past decade, viral and parasitic infections of marine plankton have received
increased attention and are now seen as major pathways for the decline of bloom-forming algae.
Dinoflagellates that bloom in Chesapeake Bay are commonly infected by parasites, with
epizootic outbreaks inducing sufficient mortality to dissipate blooms. Similarly, diatom viruses
exhibit a seasonal pattern in Chesapeake Bay that reflects the annual bloom cycle of host species.
The ability of microbial pathogens to cause rapid declines in phytoplankton populations has lead
some to consider their possible use as biological controls for harmful algal blooms. Prospects,
questions, and concerns related to that strategy will be discussed.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
18s rRNA Gene Sequences Reveal Diversity Within the Genus Zoothamnium (Ciliophora,
Peritrichia)
John C. Clamp
Department of Biology, North Carolina Central University, Durham, NC
Zoothamnium is a genus of colonial, sessiline peritrichs with free-living and ectosymbiotic
species occurring both in marine and freshwater habitats. It has a contractile stalk that folds into
a “zig zag” conformation rather than a helical one when contracted. The genus is the type of the
family Zoothamniidae, which includes other genera with “zig zag” stalks. Miao, et al. [2004.
Phylogenetic relationships of the Subclass Peritrichia (Oligohymenophorea, Ciliophora) inferred
from small subunit rRNA gene sequences. J. Eukaryot. Microbiol. 51:180-186] demonstrated a
close relationship between the freshwater species Zoothamnium arbuscula and Carchesium
polypinum, a colonial member of the family Vorticellidae with a stalk that contracts into a helical
conformation, using 18S ssu rRNA sequences. In the present study, the 18S rRNA genes of
seven marine species of Zoothamnium (Z. niveum, Z. alternans, Z. pelagicum, and 4 unidentified
species) were sequenced. Based on this evidence, the seven species constitute a poorlysupported, possibly polyphyletic clade allied to the noncontractile, colonial genus Epistylis but
separate from both C. polypinum and Z. arbuscula. However, within this clade, the species
cluster into three, well-supported, small clades of 2-3 species each. Future research is planned
that will utilize gene sequences (including ones coding for proteins) from more species of
Zoothamnium and more thorough description of morphological characteristics, especially the
oral infraciliature, that are still incompletely known for most species in the genus.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Barcoding Protists: A Trailblazing Study on a Ciliate Genus of Closely Related Species,
Tetrahymena
C. CHANTANGSI, M.C. STRÜDER-KYPKE, and D.H. LYNN
Department of Integrative Biology, University of Guelph, Ontario, Canada.
A mitochondrial cytochrome c oxidase subunit 1 (COI) gene has been proposed for use as a
DNA barcode in identifying animal species. To test the applicability of this gene in identifying
protists, the ciliate genus Tetrahymena was selected. Tetrahymena comprises about 42 species
that are highly similar in morphology, and thus very difficult to discriminate. Some of these
species belong to several sibling species complexes, while other species exhibit some
morphological diversity in their polymorphic life cycles. In this study, the COI genes of all
Tetrahymena species, whose cultures are available, and three non-Tetrahymena ciliates,
Colpidium campylum, Colpidium colpoda, and Glaucoma chattoni were amplified and
sequenced. Nucleotide sequence analysis of 1,821 bp of the COI gene among these species
showed an average 10.9% sequence divergence. Furthermore, sequence analysis of the proposed
barcoding region of about 650 bp, which is currently used as diagnostic site for identifying
animal species, showed an average 10.4% sequence divergence. Amplification and sequencing of
the COI gene of intraspecific isolates of the following species from geographically diverse
regions, T. borealis, T. lwoffi, T. patula, T. pyriformis, T. thermophila, and T. tropicalis were
also performed to determine the degree of intraspecific variation. Except for T. tropicalis, which
showed a higher level of intraspecific variation and clustered with other species, the other five
species showed low levels of intraspecific variation, and all intraspecific isolates grouped
together. This study demonstrates the feasiblity of the mitochondrial COI gene as a taxonomic
marker for “barcoding” and identifying ciliated protists.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Distinct Paths to Stop Codon Reassignment in the ciliates Tetrahymena thermophila and
Euplotes octocarinatus.
1
Joe Salas-Marco, 1Hua Fan-Minogue, 1Adam K. Kallmeyer, 2Lawrence A. Klobutcher, 3Philip J.
Farabaugh, and 1David M. Bedwell
1
Dept. of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 2Dept. of
Molecular, Microbial and Structural Biology, University of Connecticut Health Center,
Farmington, CT, and 3Dept. of Biological Sciences, University of Maryland-Baltimore County,
Baltimore, MD
Multiple independent reassignments of universal cod stop codons to sense codons have occurred
among ciliate species. For example, Euplotes has reassigned the standard UGA stop codon to
cysteine, while the UAA and UAG stops encode glutamine in Tetrahymena. Such changes in
“stop codon” meaning are thought to require changes to the eukaryotic release factor 1 protein
(eRF1), whose domain 1 is known to be involved in stop codon recognition during termination of
translation. To determine if changes to ciliate eRF1 domain 1 have indeed occurred, we
constructed hybrid eRF1 genes encoding domain 1 from either Euplotes octocarinatus or
Tetrahymena thermophila, and domains 2 and 3 from Saccharomyces cerevisiae. The constructs
were then examined for their ability to complement a yeast strain lacking eRF1, as well as their
ability to recognize each of the three standard stop codons using an in vivo dual luciferase
reporter system. The Euplotes hybrid protein failed to complement, recognizing the UAA and
UAG stop codons, but not UGA. Surprisingly, the Tetrahymena hybrid protein complemented
the eRF1 deficient yeast and recognized all three stop codons. Thus, domain 1 of eRF1 is not the
sole determinant of stop codon recognition, and Euplotes and Tetrahymena appear to have
developed stop codon reassignment by distinct mechanisms. Possible routes to stop codon
reassignment in Tetrahymena, and the implications of the results for previous models of stop
codon reassignment, will be discussed.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
The Tetrahymena thermophila phagosome proteome
1
Mary Ellen Jacobs, 2Leroi DeSouza, 1Haresha Samaranayake, 2K. W. Michael Siu and
1
Lawrence A. Klobutcher.
1
Department of Molecular, Microbial and Structural Biology, University of Connecticut Health
Center, Farmington, CT, 2Department of Chemistry and Centre for Research in Mass
Spectrometry, York University, Toronto, ON, Canada
Phagocytosis is used by single-cell organisms for nutrient uptake, and by vertebrate immune
cells to ingest pathogens, to clear apoptotic cells, and for tissue remodeling during development.
Although a number of the hundreds of proteins predicted to be involved in this complex process
have been characterized, the identity and function of many others remain to be elucidated. To
enhance the utility of the ciliate Tetrahymena thermophila as a model organism for studying
phagocytosis, we have analyzed its phagosome proteome. Cells were fed latex beads, lysed, and
the bead-filled phagosomes were purified by sucrose gradient centrifugation. Three different
two-stage fractionation procedures followed by tandem mass spectrometry (MS/MS) were then
carried out on proteins and/or tryptic peptides obtained from the isolated phagosomes. The
analysis led to the identification of 96 proteins or predicted proteins in the Tetrahymena genome
database, and more than 84 of these were found to share significant sequence similarity to
proteins of known function. Twenty-five of the proteins were previously identified in the
phagosome proteomes of other organisms, indicating that phagocytosis in Tetrahymena shares
similarities with the process in these other organisms. In addition, 12 novel proteins were
uncovered in our analysis, as well as two proteins implicated in human genetic diseases. We
have also begun using Tetrahymena's genetic tools to create GFP-tagged versions of the
identified proteins to obtain insight into their specific roles in phagocytosis, and preliminary data
on four proteins will be presented.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Progress on a new web-accessible archiving system for cilioprotists of southern New
England
Ethan Fremen and Linda A. Hufnagel
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI
The Rhode Island Coastal Micrograzers web site (http://athena.cs.uri:8080/ricms) was
established to provide access to the Rhode Island Cilioprotist Micrograzer Survey (RICMS), a
database containing information about the distribution, diversity, and systematics of cilioprotists
of coastal regions of Rhode Island and nearby southern New England. The original plan was to
archive digital videoclips collected over a period of years in a web-accessible database.
However, as we proceeded, additional goals emerged. These remain to: 1) include data for
species identification, bar-coding, and geographical localization; 2) streamline processing and
entry of data; and 3) provide flexibility, accessibility and searchability, for the end-user. To meet
these goals, we are now developing an entirely new Rhode Island Coastal Micrograzers web site,
incorporating intuitive methods to enter geographic, video, photographic, bar-coding, provisional
classifications and other types of data. Entered data will be reviewed by the web site
administrator/curator, who will associate approved data with one or more groups in the most
recent phylogenetic schema for the cilioprotists. However, older phylogenetic schemes will also
be incorporated, to aid understanding. The database will be searchable by a variety of criteria,
using an extensible metadata system and integrated live search functions that allow all relevant
data fields to be searched. Search results will be filtered according to the administrator’s
permissions, so in-progress works will only be visible to those working on them. A history of
edits will be preserved, and no html coding or database experience will be required, by the
administrator, data providers or end-users.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Voyages of Discovery: Science Learning and Living History.
William Reynolds
New Netherland Museum, Albany, NY
Abstract to come??
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
A Tetrahymena thermophila TtVPS13A antisense ribosome mutant is impaired in
phagocytosis and has a temperature -sensitive growth defect.
H. Samaranayake and L. A. Klobutcher
Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health
Center, Farmington, CT
Vacuole sorting protein 13 (VPS13) genes are found in a broad range of organisms, but its
function(s) is incompletely understood. In yeast, VPS13 appears to be involved in trafficking of
membrane proteins from the Golgi to the endocytic pathway and the vacuole. In humans, two
related VPS13 genes have been implicated in the genetic disorders Chorea-acanthocytosis and
Cohen syndrome, which result in defects of the central nervous, cardiovascular, and skeletal
muscle systems. We recently identified one of at least 7 predicted VPS13 proteins encoded in the
Tetrahymena thermophila genome, TtVPS13A, as a component of the phagosome proteome,
suggesting it might play a role in phagosome-related trafficking. As a first step in investigating
the function of TtVPS13A, we have used RT-PCR to characterize the VPS13A mRNA. The
results identify a number of errors in the VPS13A gene prediction, and indicate that the
TtVPS13A gene encodes a protein of 3467 amino acids with significant N-terminal and Cterminal similarity to VPS13 proteins in other organisms. There also appears to be little or no
alternative splicing of the TtVPS13A transcript. The antisense ribosome approach was then used
to generate TtVPS13A knockdown mutants, and preliminary analyses indicate that the mutant
strains have a temperature-sensitive growth defect in SPP medium, as well as impaired
phagocytosis as measured by a latex bead uptake assay. To obtain further insight into the role of
VPS13A, and its possible role in phagocytosis, we are now generating strains carrying either
knockout mutations or encoding a GFP-tagged version of the protein.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Growth Control Signaling Pathways in Tetrahymena Cilia II
Guerra, C., Bell, A., Awan, A., *Christensen, S.T. and Satir, P.
Dept. of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY,
*Institute of Molecular Biology and Physiology, Dept. of Biochemistry, Univ. Copenhagen,
Denmark
We have previously reported on the existence of phospho-tyrosine signaling cascade members
discovered in the Tetrahymena genome. One such proposed receptor tyrosine kinase protein
(TtPTK1, Christenesen et al, 2003), has been isolated to Tetrahymena cilia, indicating that the
signaling cascade originates in the cilium. Here we test the ciliary localization, via RNA upregulation during ciliogenesis, of a series of specific proteins required for the establishment and
function of a phospho-tyrosine cascade signaling pathway from the cilium to the nucleus. RNA
up-regulation of greater than two fold is seen for most of the proteins studied in pathways
involving PI3kinase or RAD51. The molecular evidence of such pathways links the sensing
abilities of Tetrahymena cilia to gene expression control in the nucleus for cell growth, cell
division and cell survival. The presence of phospho-tyrosine signaling pathways in Tetrahymena
suggests that they evolve early in eukaryotic cell evolution and are retained for similar functions
in metazoan cells.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
MIRROR-IMAGED DOUBLETS OF OXYTRICHA FALLAX: IMPLICATIONS FOR
THE DEVELOPMENT OF LEFT-RIGHT ASYMMETRY
Aaron J Bell, Peter Satir, Gary W. Grimes (posthumously)
Albert Einstein College of Medicine, Bronx, NY
Ciliated protozoa possess cellular axes that are reflected in the arrangement of their ciliature.
Cells divide by transverse fission, with each daughter cell possessing identical ciliary patterns;
this ensures perpetuation of the cellular phenotype. Experimentally manipulated cells can be
induced to form atypical phenotypes, some of which are capable of intraclonal propagation. One
such phenotypic variant in the hypotrich ciliates, which includes Oxytricha fallax, is the mirrorimaged doublet. These cells possess two distinct (although deficient) sets of ciliature, juxtaposed
on the surfaces, arranged in mirror-image symmetry. The two halves share a common anteriorposterior axis, but the asymmetry of the two halves is different. We have examined whether
individual ciliary components in mirror-image doublets of Oxytricha fallax are themselves
mirror-imaged. Ultrastructural analysis indicates that despite the global mirror-imaging of the
ciliature, asymmetry of the individual ciliary components is identical in both halves of the
doublets. Mirror-image doublets do not appear to possess mirror-imaged forms of each protein
component used in assembly of the oral apparatuses. The overall symmetry of the mouth thus
appears to be separable from the more localized assembly of individual basal bodies. “True”
mirror-imagery of the individual ciliary components would probably require enantiomorph forms
of the proteins that comprise the oral apparatus, particularly the basal bodies, which is never
observed. These observations permit us to suggest a mechanistic hypothesis with implications
for the development of left-right asymmetry not only in ciliates but perhaps development of leftright asymmetry in general.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Chemotactic responses of two philasterine scuticociliates, endoparasites of different
crustacean hosts
a
Megan Noyes, bSpencer J. Greenwood, aDenis H. Lynn
a
Department of Integrative Biology, University of Guelph, Guelph, ON, and bLobster Science
Centre, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE,
Canada
As aquaculture has expanded over the past decades, there are increasing reports that
scuticociliates are pathogenic to both aquaculture-raised finfish and intensively harvested and
stored invertebrates. The philasterine scuticociliates, Mesanophrys chesapeakensis (MC) and
Anophryoides haemophila (AH) are endoparasites of the blue crab, Callinectes sapidus, and the
American lobster, Homarus americanus, respectively. Preliminary experimental cross-infections
suggest that these parasites may be host specific. However, little is known about the host
specificity or mode of infection of these parasites. To determine if differences in chemotactic
behavior of these parasites could be used to explain host specificity, the chemotactic preferences
of both scuticociliates were determined using a round capillary assay with the following
compounds at varying concentrations (i.e. 10-3 M, 10-4 M, 10-5 M, and 10-6 M): L-leucine, Lhistidine, agmatine SO4, betaine hydrochloride, L-methionine, choline chloride, and the
dipeptide, proline-phenylalanine. These ciliates differ in that MC is attracted to agmatine SO4
while AH is repulsed, and conversely MC is repulsed by proline-phenylalanine while AH is
attracted. There are also differences in sensitivity to these compounds as MC shows a higher
sensitivity to agmatine SO4 while AH shows a higher sensitivity to L-methionine and choline
chloride. Experiments are in progress to test the response of each species to whole blood
products of each crustacean host.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
HIDDEN DIVERSITY OF ALLOGROMIID FORAMINIFERANS IN LOW-LATITUDE
ENVIRONMENTS
ANDREA HABURA1,3, SUSAN T. GOLDSTEIN2, SARAH BRODERICK1, AND SAMUEL S.
BOWSER1,3
1
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health,
Albany, NY, 2Department of Geology, University of Georgia, Athens, GA,, 3Department of
Biomedical Sciences, University at Albany, NY.
Foraminiferal species distribution and richness is usually judged by examining tests (Sen Gupta,
1999). It has long been known that some groups of foraminiferans, particularly the allogromiid
taxa, are not easily identified in this manner (Gooday, Bowser and Bernhard 1996, Pawlowski et
al. 2002). Previously, an environmental DNA study was used to explore the comprehensiveness
of morphological sampling methods. This analysis showed that in a deep-sea-like high-latitude
environment (Explorers Cove, Antarctica), at least 75% of the foraminiferal species present were
not identified by traditional techniques, and most of these "hidden" species were allogromiids
(Habura et al. 2004).
In order to investigate whether this domination of the foraminiferal assemblage by allogromiids
is a feature of high-latitude environments alone, we used a series of targeted DNA-based surveys
and allogromiid-focused morphological searches at several locations along the US eastern
seaboard. Both methods of survey revealed the presence of large numbers of new allogromiid
taxa. Morphological screens and specific PCR primers targeted against particular foraminiferal
clades also documented substantial differences in the allogromiid assemblage between the
locations. The fact that these low-latitude sites exhibit considerable cryptic allogromiid diversity
has significant implications for nutrient cycling in the benthos and reconstruction of
paleoenvironments.
Supported by NSF grants OPP0003639, ANT0440769, and DEB0445181
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
In situ time-lapse observation of Antarctic foraminifera
Harris, J.K.1, Blair, J.2, Mastroianni, J.2, Hansen, A.2, Walker, S.E.3, Miller, M.4, and Bowser,
S.S.5
1
The Sage Colleges, Troy, NY, 2Magee Scientific Company, Berkeley, CA, 3Department of
Geology, University of Georgia, Athens, GA, 4Department of Earth and Environmental Sciences,
Vanderbilt University, Nashville, TN 5Wadsworth Center, NY State Dept. Health, Albany, NY
ROMEO6 -- the world’s southernmost cabled-underwater observatory -- was used to image the
protist Cibicides refulgens in situ at Explorers Cove, Antarctica. This trophically-complex
foraminiferan lives epizoically on the Antarctic scallop Adamussium colbecki. Long-term data on
Cibicides life habits (e.g., mobility, reproduction, and encounters with megafauna) were gathered
using ROMEO, as were the physical parameters in this natural setting. Time-lapse movies
directly demonstrate the biofouling ecology of this abundant protist and document its resistance
to disturbance by megafauna. Enhanced understanding of the Explorers Cove ecosystem, enabled
by year-round use of remote instrumentation like ROMEO, will provide a modern analogue for
the paleoenvironmental interpretation of Holocene fossiliferous sediments in the Taylor Valley,
especially the paleoecology of the foraminiferan assemblage and associated macrobenthos.
Supported by NSF grants OPP0216043 and ANT0440769
6
Remotely Operable Micro-Environmental Observatory:
www.bowserlab.org/Antarctica/romeo.html.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
KEYNOTE ADDRESS
Laura Landweber:
Detangling scrambled genes
Ciliated protozoa house two types of genomes in one cell: a somatic macronucleus that encodes
the proteome and a germline micronucleus used in conjugation. The somatic genomes of some
ciliates are composed of short "nano-chromosomes", each encoding only one or a few genes. In
Oxytricha trifallax the macronuclear genome is ~50 Mb, divided among ~24,500 types of nanochromosomes, ranging from 0.25 kb to ~40 kb long, each present in approximately 1000 copies.
Individual nanochromosome contains short coding and regulatory sequences, few if any introns,
and 20bp telomeres at both ends. The observation that most macronuclear chromosomes bear 1-3
genes suggests the presence of >26,000 genes, or a relatively high genome complexity.
Furthermore, comparisons between the germline and somatic versions of DNA genes suggest
that ~20-30% of all Oxytricha genes are scrambled; i.e. both fragmented and permuted in the
germline. In one case, the segments for two independent transcripts are intertwined on two
separate germline loci. I will describe our current understanding of scrambled genes, based on
experiments in our lab that surveyed the origin, evolution, and developmental processing of
scrambled genes. I will also review progress on the Oxytricha genome project.
LAURA LANDWEBER is an Associate Professor in the Department of Ecology &
Evolutionary Biology and Molecular Biology at Princeton University. She received her A.B.
from Princeton in 1989 and her Ph.D. from Harvard in 1993. Before starting her faculty position
in 1994, she was a Junior Fellow of the Harvard Society of Fellows. She has served on several
panels, working groups, and advisory committees for the NSF, NIH, NHGRI, and NASA and has
co-chaired the NHGRI Comparative Genome Evolution Working Group since 2003. She is an
author of 100 publications in molecular and evolutionary biology and editor of 3 books, ranging
from Genetics and the Extinction of Species to DNA Based Computers. She is currently CoEditor-in-Chief of Biology Direct (biology-direct.com), a new journal experimenting with open
peer review. She is also an associate editor for Journal of Molecular Evolution, on the advisory
board for Genome Biology, and a member of Faculty of 1000. She has organized more than ten
workshops and conferences, including Nucleic Acid Selection (1999), FASEB Ciliate Molecular
Biology (2001), and a symposium on Evolution of Dynamic Gene Rearrangements at the 1999
SMBE meeting in Australia. A recipient of Burroughs-Wellcome Fund (1994) and Sigma Xi
(1999) young investigator awards, she was recently elected a Fellow of AAAS for probing the
diversity of genetic systems in microbial eukaryotes, including scrambled genes, RNA editing,
variant genetic codes, and comparative genomics in protests
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Motile Microtubules in Dictyostelium
1,2
Daniela Brito, 1Irina Tikhonenko, 1,2Alexey Khodjakov, and 1,2Michael Koonce
1
Wadsworth Center, 2School of Public Health, University at Albany, Albany, NY
The interphase microtubule cytoskeleton in Dictyostelium is firmly anchored at one end to the
centrosome and extends to form a radial cytoplasmic array that is common in most eukaryotic
cells. We have examined microtubule motions in Dictyostelium using a combination of GFPtubulin, dominant negative dynein mutants, and a laser microbeam. Individual microtubules in
Dictyostelium undergo striking, rapid, lateral bending and sweeping motions through the
cytoplasm. Although part of this motion is driven by a robust amount of organelle movement,
there are clear force-producing activities at the cell cortex. Laser ablation of the centrosome
results in full-scale depolymerization of the microtubule array, from the newly-exposed minus
ends. In dynein mutant cells, the centrosome is motile, and moves through the cytoplasm at rates
up to 2.5 m/second. Irradiation of the trailing, comet-like tail of microtubules suggests that a
pushing force contributes significantly to this movement. Our data support the existence of
cortical forces acting on the interphase microtubules: a dynein dependent pulling force and a
kinesin-like pushing action. We feel that these activities are part of a common mechanism to
position microtubule arrays in eukaryotic cells. In Dictyostelium, a combination of limited
numbers of microtubules and highly motile cells accentuates this motility, making this organism
an opportune model to characterize the forces and their regulation. Taking advantage of the
ability to grow significant quantities of genetically modified Dictyostelium, we have begun to
biochemically characterize the regulation of the dynein motor in this motility. (work supported
by the National Science Foundation, MCB-0542713)
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Identification of a novel bifunctional EhADH2-like enzyme in Entamoeba invadens
Avelina Espinosa, Adeline Ashmore, Chelsea Fitzsimmons-Diaz and Melissa Roberto
Laboratory of Molecular Microbiology, Department of Biology, Roger Williams University,
Bristol, RI
The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy
from the fermentation of glucose to ethanol. Entamoeba histolytica alcohol dehydrogenase 2
(EhADH2), a bifunctional enzyme with both aldehyde dehydrogenase (ALDH) and alcohol
dehydrogenase (ADH) activities, constitutes a key enzyme in this pathway. We have previously
shown that EhADH2 expression is required for the growth and survival of E. histolytica
trophozoites. EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and Cterminal ADH domains. Molecular and biochemical studies suggest these fermentation enzymes
were transferred horizontally from bacteria. ADHE enzymes have been reported in firmicutes,
proteobacteria, cyanobacteria, and a few eukaryotes (E. histolytica, Giardia intestinalis,
Mastigamoeba balamuthi, chlorophyte algae, and Piromyces sp. 2). A likely different
evolutionary origin from vertebrate enzymes designates them as appealing antimicrobial targets.
We have recently identified a new member of this family in Entamoeba invadens, a reptilian
pathogen. Molecular and biochemical studies are currently in progress.
Funded by NIH-NCRR Grant # 2 P20 RR16457-04 “Anti-Amoebic Drugs with less toxicity in
Humans”
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Sequencing of a N. fowleri genomic DNA library and identification of a gene encoding a
putative patatin-like serine hydrolase protein
1
Rebecca C. MacLean, 2Leslie Y. Whiteman, 1Francine Marciano-Cabral
1
Department of Microbiology and Immunology, Virginia Commonwealth University School of
Medicine, Richmond, VA, 2Department of Natural Sciences, Virginia Union University,
Richmond, VA
Naegleria fowleri, a free-living amoeboflagellate, is the causative agent of primary amebic
meningoencephalitis. The genome of this organism has not been sequenced, therefore, we have
constructed a genomic DNA library to search for putative virulence factors. We have performed
partial sequencing of 155 plasmids and using protein prediction software, we have identified
putative genes for protein regulation, cell signaling, gene regulation, protein synthesis,
respiration, energy production, DNA repair, cell motility and chromosome segregation, as well
as genes with unknown functions. We have also identified a putative virulence factor, a patatinlike protein. Patatin has been shown to exhibit phospholipase A2 activity in other organisms and
has been shown to be involved in invasion into human tissue in certain pathogens. The N.
fowleri patatin-like protein contains conserved domains including a patatin domain as well as a
predicted esterase of the alpha-beta hydrolase superfamily, which is similar to other patatin-like
proteins. Northern analysis using a cDNA probe demonstrated hybridization with N. fowleri
RNA at 3kb. There was no hybridization of the patatin probe to N. gruberi, N. austrailiensis, A.
culbertsoni, A. astronyxis, or Balamuthia mandrillaris. There was a slightly smaller product
observed in N. lovaniensis. RT-PCR analysis using two different primer sets was positive for
pathogenic N. fowleri and negative for nonpathogenic N. gruberi and N. lovaniensis. Further
studies are needed to determine whether the patatin-like protein in N. fowleri serves as a
virulence factor and plays a role in invasion in human tissue.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
FINE STRUCTURE AND CONSTRUCTIONAL THEMES OF MODERN
“ALLOGROMIID” FORAMINIFERA: AN OVERVIEW
1
SUSAN T. GOLDSTEIN, 2SAMUEL S. BOWSER, 2ANDREA HABURA, AND
3
ELIZABETH A. RICHARDSON
1
Department of Geology, University of Georgia, Athens, GA, 2Wadsworth Center, New York
State Department of Health, Albany, NY, 3Department of Plant Biology, University of Georgia,
Athens, GA
The Foraminifera are well known for the long fossil record of their mineralized shells or tests.
Most “allogromiids” (members of the basal clades), however, have tests that consist of just a
single organic or agglutinated, non-mineralized chamber that range in shape from domed or
spherical to tubular or stellate. Although test morphology underpins foraminiferal classification,
molecular phylogenetic studies demonstrate that these simple allogromiid traits are inadequate
for classification, and that the molecular clades bear little resemblance to the taxa defined by
traditional criteria. This simplistic allogromiid morphology, however, belies the intricate
patterns of test construction found at the ultrastructural level. Here we apply ultrastructural
methods to delineate several fundamental themes in allogromiid test construction: herringbone
structure in an organic test (e.g., Allogromia laticollaris), a herringbone organic inner test or
“theca” with an organic (e.g., Iridia lucida) or agglutinated outer covering (e.g., Astrammina
spp.), a granulofibrilar inner theca with an agglutinated outer covering (e.g., Notodendrodes
spp.), a flexible agglutinated test with a featureless to fibrous inner organic lining (e.g.,
Cribrothalammina alba), an “agglutinated” covering that lacks an organic lining or bioadhesive
(e.g., Crithionina), and a “hyperamminid” construction with an agglutinated layer and inner
organic lining of crescent-shaped fibers. Our survey of constructional themes is currently far
from complete, and we know even less about the composition of the various test components.
Nonetheless, results obtained thus far tend to support certain molecular clades. Supported in part
by NSF DEB0445181 and ANT0440769.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
A New York State Waterpark Outbreak of Human Cryptosporidiosis
A.Teal, J. Ennis, M. Marchewka, J. Hans, S. Madison-Antenucci, J. Keithly
Division of Infectious Diseases, Parasitology Laboratory, Wadsworth Center New York State
Department of Health, Albany, NY.
Cryptosporidiosis is one of the most common enteric infections in humans worldwide, i.e. about
6% of all diarrheal diseases are caused by this parasite. Cryptosporidium hominis and C. parvum
are unicellular, obligatory intracellular parasites that belong to the Apicomplexa. These
organisms infect the gastrointestinal (GI) epithelium of humans and/or domestic mammals
(cattle), to produce a diarrhea that is self-limited in immunocompetent persons, but potentially
life-threatening in immunocompromised persons, especially those with acquired
immunodeficiency syndrome (AIDS), the very young, or the very elderly.
In 2005, the Parasitology Laboratory of New York State began screening patient specimens
associated with visitors at a Spraypark in Upstate New York from June - August. Approximately
3039 people presented with gastrointestinal illness that included diarrhea, fever, nausea, and in
some cases vomiting. This outbreak affected persons within 37 NYS counties. Unlike the 1993
Milwaukee waterborne outbreak in which 104 of 403,000 patients died, all of the 41 patients
hospitalized in the NYS outbreak recovered from their illness. Routine diagnostic analyses using
an Enzyme-Linked Immunosorbent Assay (ELISA) determined that 850 of 884 formalin-fixed
samples were positive for Cryptosporidium (96%). In addition to routine testing, the laboratory
tested 147 fresh samples transported in Cary-Blair medium by PCR. Of these, 134 were human
stool samples and 8 were environmental samples. All 142 of these samples were determined by
Real Time PCR to contain Cryptosporidium DNA.
To determine which of the human species of Cryptosporidium, C. hominis or C. parvum, was
responsible for the waterpark outbreak, the Cary-Blair specimens were typed by Restriction
Fragment Length Polymorphism (RFLP)-PCR. Primers which amplify the Cryptosporidium
Oocyst Wall Protein (COWP) gene were used, and the resulting amplicons were then digested
with the restriction enzyme Rsa1. Of these, 129 of134 patient samples were RFLP-PCR positive
for C. hominis (96%), the parasite primarily found in humans. All 6 of the environmental water
samples (100%) also contained C. hominis, and the two samples from geese were negative for
any species of Cryptosporidium. A subset of samples determined epidemiologically not to be
part of the outbreak, were also genotyped using small subunit rDNA, which discriminates
between diverse species of animals. When these amplicons were digested with Ssp1 and Vsp1,
four were genotyped to C. felis (cats), and one to C. parvum. These results suggest that the
species responsible for the NYS waterborne cryptosporidiosis outbreak was Cryptosporidium
hominis, the genotype found primarily in humans.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Using real time qRT-PCR to evaluate loss of RNA Editing Associated Protein-1 on editing
Mary Marchewka, Jennifer Hans, and Susan Madison-Antenucci
NYS DOH, Wadsworth Center
Trypanosoma brucei is a blood-borne pathogenic parasite transmitted by tse-tse flies. The
parasite causes African trypanosomiasis, or sleeping sickness, an infection that is lethal if left
untreated and for which there is no prophylactic chemotherapy. One unique aspect of
trypanosome biology is RNA editing carried out in the mitochondrion of the parasite. While
RNA editing is not restricted to kinetoplastids, uridine insertion and deletion editing is exclusive
to these organisms. RNA editing is carried out by a large ribonucleoprotein complex. One of the
proteins involved in RNA editing is REAP-1, an RNA binding protein that specifically
recognizes RNAs requiring editing. Based upon this activity we think that REAP-1 acts as a
recruitment factor to deliver RNAs to the complex. In order to investigate the role of REAP-1 in
editing, we generated double-knockout cell lines and measured RNA levels using reverse
transcriptase real time PCR. RNA levels for pre-edited, edited, and non-edited RNAs were
determined relative to two endogenous normalizer genes (tubulin, 18s rRNA). Using a software
program LinRegPCR to generate PCR efficiency values, the data were analyzed using the Pfaffl
mathematical equation. These results are then expressed as the relative ratio of target RNA to
normalizer RNA. The results showed, as expected, that REAP-1 RNA is not present. They also
showed levels went up for all mitochondrial RNAs tested in the null mutants and indicate the
loss of REAP-1 affects mitochondrial RNA processing.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Novel Compartmentalization of Core Metabolism in Cryptosporidium parvum.
Janet Keithly1, Vlasta Ctrnacta2, Frantisek Stejskal2, Jeffrey Ault1, Karolyn Buttle1,3, Carmen
Mannella1,3, Susan Langreth4
Wadsworth Center New York State Department of Health1 and Resource for the Visualization of
Biological Complexity3, Albany, NY, Department of Tropical Medicine, Charles University in
Prague, Czech Republic2; Uniformed Services University of the Health Sciences, Bethesda, MD.
Sporozoites of the apicomplexan Cryptosporidium parvum possess a small, double membranebounded organelle sandwiched between the nucleus and crystalloid body. This organelle has
been identified as a relic mitochondrion (mitosome). Transmission electron microscopy (TEM)
and tomographic reconstruction reveal the complex arrangement of membranes in the vicinity of
this organelle, as well as its internal organization. The infoldings of the inner membrane lack the
tubular “crista junctions” found in typical metazoan, fungal and protist mitochondria. The
absence of this highly conserved structural feature is congruent with the loss, through reductive
evolution, of the normal oxidative phosphorylation machinery in the C. parvum mitosome.
Extended glycolysis in C. parvum is the primary source of ATP, yet virtually nothing is
known about its compartmentalization. In many microaerophilic protists, oxygen-sensitive
pyruvate:ferredoxin oxidoreductase (PFO), a key enzyme in core metabolism, is
compartmentalized either within the cytosol (Giardia, Entamoeba) or within a double
membrane-bounded hydrogenosome (Trichomonas, Nyctotherus). In C. parvum, PFO is the Nterminal domain of a unique fusion with NAD(P)H-cytochrome P450 reductase forming
pyruvate:NADP+ ferredoxin oxidoreductase (CpPNO), an enzyme rarely found in eukaryotes.
CpPNO shares significant sequence homology and domain organization with Euglena gracilis
PNO (EgPNO), but not a typical N-terminal mitochondrial targeting sequence. That CpPNO is a
cytosolic protein was confirmed by both confocal immunofluorescence and immunogold
labeling, using polyclonal anti-CpPFO, the cytosolic marker anti-CpPPTase, and the
mitochondrial chaperone anti-CpCPN60. These new data are congruent with the suggestion that
the mitosome of C. parvum serves primarily as an organelle for the import and maturation of FeS clusters, and that it plays no major role in core energy metabolism.
On the other hand, and in contrast to other apicomplexans, C. parvum sporozoites possess
an additional posterior organelle, the crystalloid body (CB), whose function is completely
unknown. Somewhat unexpectedly, both confocal images and immunogold labeling showed that
CpPNO localized within this organellar subcompartment, as well as within the cytosol.
Furthermore, although neither TEM nor tomographic reconstruction could resolve whether the
CB is membrane-bounded, the uptake and localization of DiOC6, Mitotracker Green FM,
Rhodamine B and Rhodamine 123 into both the CB and mitosome indicate an organellar
membrane potential. These data, together with the close juxtaposition of the CB and mitosome,
as well as their distinctive internal structure, suggest there may be some interesting evolutionary
implications for why the CB remains a unique subcompartment within C. parvum. Because
CpPNO is compartmentalized in a novel way, it opens the possibility that this apicomplexan may
display yet another unique type of core energy metabolism in microaerophilic protists that might
lead to new strategies for drug development against human cryptosporidiosis.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Respiration of Antarctic benthic foraminifera - optical microplate respirometry and effects
of oxygen limitation.
1
Ulrich, P.N. 1Marsh, A.G. and 2S.S. Bowser
1
College of Marine Studies, University of Delaware, Lewes, DE
2
Wadsworth Center, NY State Dept. Health, Albany, NY
Larger agglutinated foraminiferan protists comprise a significant proportion of benthic biomass
in Explorers Cove, McMurdo Sound, Antarctica. Despite high abundance and potential relevance
to oceanic carbon cycling, few replicated estimates of respiration of benthic foraminifera are yet
available. In this study, we measured respiration rates of individual Astraminna rara, a large (1-3
mm diameter) species that builds a single-chambered agglutinated test, with microplate
respirometers built with glass-bottom well plates containing ruthenium optodes. Whole
specimens consumed 573 ± 336 pmol O2 h-1 (n = 10). To quantify the contribution of
pseudopodia to total respiration, we measured respiration rates of both tests vested with
pseudopodia and cell body with a subset of A. rara which we had carefully dissected.
Surprisingly, tests with adherent pseudopodia contributed significantly to total respiration, and
the total respiration of paired tests and shucked A. rara (1146 ± 458 pmol O2 h-1) was
significantly higher (p = .003, n = 10) than that of intact specimens. These data indicate that A.
rara respiration may normally be limited by oxygen diffusion across the test and throughout the
large cell body. In this scenario, limited maintenance metabolism in the cell body could conserve
energy without compromising the foraging performance of metabolically active pseudopodia.
Supported by NSF grants ANT023828 and ANT0440769.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
POSTER ABSTRACTS (Random Order)
VOYAGE OF DISCOVERY: AN INITIAL FIELD SURVEY OF FORAMINIFERA IN
THE THREE ESTUARIES OF NEW NETHERLAND
1
William T. Reynolds, 1Alison S. Laufer, 2Andrea Habura, and 2Samuel S. Bowser
1
New Netherland Museum, Albany, NY
2
Wadsworth Center, NY State Department of Health, Albany, NY
Foraminifera, widely distributed and studied in the oceans of the world, are not well studied in
low salinity environments. In June, 2006 the square rigged sailing ship Half Moon will complete
an historical and educational voyage, traveling from Athens, NY, downstream into New York
Harbor, through the East River and western Long Island Sound, upstream in the Connecticut
River, thence to the Delaware River, concluding in Wilmington, DE. The crew of the ship,
including students from Union College (Schenectady, NY), will sample the top cm of sediment
along the high, middle, and low saline sections of the Hudson, Connecticut, and Delaware
estuaries. Each collection site will be a low-energy shoreline, exposed at low tide, comprised of
sediments rich in organic material and detritus, with typical associated plants of the low marsh
(Spartina alterniflora) and adjacent flourishing vegetation. Environmental DNA will be
extracted from these samples, and probed using PCR primers specific for various basal
foraminiferal clades. The results will provide a baseline for future research on foraminiferal
species composition, population densities, and distribution in estuarine and riverine
environments of eastern North America. This project also represents a novel collaboration
between research scientists, undergraduate students, and historians.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Evaluating Support for Eukaryotic Supergroups and the Search for Sister Groups of
Foraminifera
1
Laura Wegener Parfrey, 2Erika Barbero, 3David J Patterson and 1,2Laura A Katz
1
Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, MA,
2
Department of Biological Sciences, Smith College, 3Bay Paul Center for Genomics, Marine
Biological Laboratory, MA
Perspectives on eukaryotic diversity have recently shifted towards a supergroup system. We
analyze two facets of supergroup robustness and find variable levels of support from molecular
genealogies and considerable taxonomic instability (Parfrey LW, Barbero E, Dunthorn MS,
Lasser E, Bhattacharya D, Patterson DJ, Katz LA, in prep). Destabilized factors include issues of
eukaryotic complexity, limited data, nomenclatural ambiguity, and sparse taxonomic sampling.
We argue that low taxonomic sampling of diverse free living microbial lineages is the most
critical factor, as the resulting molecular genealogies substantially underrepresent diversity.
Hence, statements of monophyly may be premature. In conjunction the EuTree consortium, we
are testing organismal relationships within the putative supergroup Rhizaria‚ by expanded
sampling of understudied lineages and a multigene approach. Rhizaria‚ encompasses
Foraminifera, members of the polyphyletic Radiolaria, and a heterogeneous collection of
amoebae and flagellates. Support for Rhizaria‚ comes entirely from molecular studies, although
the resulting tree topologies vary tremendously with taxon sampling and the method of tree
construction. Our work aims to elucidate the sister groups of the Foraminifera, as molecular
studies have pointed to at least four groups as the sister taxon. This incongruity is largely due to
highly divergent Foraminifera genes (Habura A, et. al. 2005 Mol. Biol. and Evo. 22: 2000-2009).
Expanding sampling of taxa and genes may clarify these relationships.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
EFFECTS OF INHIBITORS OF MYOSIN LIGHT CHAIN KINASE AND PI 3-KINASE
ON TETRAHYMENA PHAGOCYTOSIS
1
ROBERT V. ZACKROFF, 2LEAH OCTAVIO, 2ASHKAN BIGDELI, AND 2LINDA A.
HUFNAGEL
1
Division of Arts and Sciences, Massachusetts College of Pharmacy and Health Sciences,
Boston, MA, 2Department of Cell and Molecular Biology, University of Rhode Island, Kingston,
RI
Wortmannin is a PI 3-kinase and myosin light chain kinase (MLCK) inhibitor. While many
drugs inhibit ciliate phagocytosis, wortmannin is the only drug (other than those that bind actin)
reported to block nascent phagosome closure in Tetrahymena, as evidenced by the formation of
oversized or elongated nascent phagosomes. However, while the actin inhibitors latrunculin B
and cytochalasin D abolish actin antibody staining of the post-oral fiber, wortmannin has no
effect. In order to determine which of wortmannin’s enzyme inhibitory activities might be
responsible for inhibition of nascent phagosome closure, we compared its effects on
phagocytosis with those of LY 294002 (a structurally unrelated inhibitor specific for PI 3-kinase,
but not MLCK) and ML-7 (an inhibitor specific for MLCK). Wortmannin reduced the rate of
phagosome formation by about 50% at 0.1 M, and induced oversized nascent phagosomes in
most cells in the 0.2-2 M concentration range. LY 294002 reduced the rate of phagosome
formation between 4-40 M, but did not induce oversized nascent phagosomes. ML-7, up to 10
M, had no detectable effect on phagocytosis. Lack of effect of ML-7 was not due to generalized
resistance, since 10 M ML-7 caused a marked division delay in log phase cells. Thus, two
different PI 3-kinase inhibitors affected Tetrahymena phagocytosis, while a specific MLCK
inhibitor did not. These results suggest that PI 3-kinase, but not MLCK, is required for
Tetrahymena phagocytosis, but that wortmannin’s ability to inhibit phagosome closure is not due
an effect on either of these enzymes.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Structural and Functional Analysis of the -Hemoglobin Encoding Gene in Paramecium
tetraurelia
Chrissa Wright
State University of New York at Plattsburgh, NY
Mentor: Dr. Nancy L. Elwess
Prior to the start of this investigation, the presence of -Hemoglobin genes in unicellular
eukaryotic organisms was thought to be either an evolutionary artifact or serve a functional
purpose in cell survival. The initial presence of the gene in isolated Paramecium tetraurelia
mRNA indicated a probable functional purpose of the gene and subsequent protein expression.
Genetic expression of the gene in P. tetraurelia was examined under oxidative and hypoxic
conditions. Methods of RNA isolation, cDNA production, gene isolation and amplification and
systematic variance of Polymerase Chain Reaction revealed a significantly higher rate of genetic
expression in cell cultures grown under hypoxic conditions. Real Time PCR was then used to
quantify levels of expression under the different oxidative conditions. Previously published
literature specifies a single 29 bp intron structure particular to the -Hemoglobin gene in P.
tetraurelia. cDNA and genomic DNA as sequenced in our lab, experimental results aligned
exactly with the published structure. Future experiments to fully characterize the gene will
include a western blot analysis, determination of -Hemoglobin protein location in P. tetraurelia
cells with the use of a fluorometer, and RNA interference to produce a “knock out” culture. This
in-depth analysis of the -Hemoglobin encoding gene in Paramecium tetraurelia will provide
additional insight into the comparative evolution of the hemoglobin gene through generations of
plants, prokaryotes, unicellular and complex eukaryotes. It will also serve to expand on the
ability of this unique unicellular eukaryotic organism to act as a model system in research
settings and academia.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Prediction of 3D models and active site structures of Icd1p and Icd2p, two NADP+dependent isocitrate dehydrogenases from Tetrahymena pyriformis, based on homology
modeling
Anna K. Kaczyska and John B. Ferguson
Biology Program, Bard College, Annandale-on-Hudson, NY
Three-dimensional models of Icd1p and Icd2p, two NADP+-dependent isocitrate
dehydrogenases from Tetrahymena pyriformis, were constructed by homology modeling.
Models were built using the comparative modeling program Modeller, using as templates the
only known 3D structures of eukaryotic NADP+-dependent isocitrate dehydrogenases, 1LWD
(that of an enzyme from the pig heart mitochondria), and 1T09 and 1T0L (both for the same
human cytosolic enzyme). Models were independently evaluated for Ramachandran plot quality
by PROCHECK, for interaction energies by PROSA2003, and for packing quality by WHATIF,
and the best model for each protein was chosen. The active sites of the best models were
compared to those of their templates by identifying amino acid residues implicated by the
literature to be involved in substrate binding and catalysis. The Tetrahymena models had
identical amino acid residues at all positions except one: Icd2p had an amino acid substitution for
a residue that had been suggested to be involved in the transition from a putatively inactive form
(1T09) to the active form (1T0L) of the human cytosolic isocitrate dehydrogenase brought about
by the addition of substrates to the enzyme. This result implies that the mechanism of activation
that was proposed for the human cytosolic isocitrate dehydrogenase may not apply to Icd2p.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Successful in vitro treatment of Acanthamoeba castellanii with pentamidine and 2,6dichlorobenzonitrile
1
Nripesh Dhungel, 2Ricky Dudley, 1John B. Ferguson, and 2Naveed A. Khan
1
Biology Program, Bard College, Annandale-on-Hudson, NY.
2
School of Biological & Chemical Sciences, Birkbeck College, University of London, London,
England
The protozoan Acanthamoeba castellanii is an opportunistic pathogen that causes disease in
immunocompromised patients. The pathogenicity of Acanthamoeba partly depends on its ability
to differentiate from a dormant cyst into an active and pathogenic trophozoite. Treatment
options for Acanthamoeba are usually futile due to its ability to encyst via a cellulose synthesis
pathway in response to treatment. By using a dual drug treatment involving pentamidine
isethionate, a common therapeutic option for amoebae, and 2,6-dichlorobenzonitrile, a cellulose
biosynthesis inhibitor, this study was able to demonstrate strong amoebicidal effects in vitro.
Whereas separate treatments of 2,6-dichlorobenzonitrile and pentamidine are shown to be
amoebastatic, together in a dual treatment they are dramatically amoebicidal. Dual treatments
have been shown to reduce the number of amoebae by as much as 95% and to reduce the number
of cysts by 99%. These results indicate a possible role for pentamidine and 2,6dichlorobenzonitrile as dual treatments in therapeutic intervention of Acanthamoeba related
infections.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Inhibition of Exocytosis by Paromomycin in Tetrahymena
Maj-Britt Nielsen and Birgit H. Satir
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx,
NY
The ciliate Tetrahymena thermophilia is a favorite model system for studies of exocytosis. An
assay used for characterizing exocytosis is causpule formation in which cells are stimulated with
Alcian blue (0.2%) after a 5 hours starvation period in phosphate buffer. This stimulation leads
to synchronous release of the content of all docked mucocysts, forming a blue capsule
surrounding the living cell. This assay gives information on release per cell in addition to the %
cells releasing in a population. We have earlier hypothesized the involvement of a
phosphoglucomutase (PGM) superfamily member, parafusin (PFUS), in exocytosis
(Subramanian and Satir, Proc. Natl. Acad. Sci. 89, 1129, 1992). A Tetrahymena mutant was
made in which 80% of the PGM1 gene, (deltaPGM1), was replaced by an insert containing a
neo2 cassette conveying paromomycin resistance. The mutant selected with this drug showed no
effect on the ability to exocytose when stimulated and observed with immunofluorescence
(Childcoat and Turkewitz. J. Cell Biol. 139, 1192, 1997). We have examined the effect of
paromomycin on exocytosis in several strains of Tetrahymena thermophila: SB210 (wt),
Cu428.2 (wt) and deltaPGM1. The ability to exocytose was abolished in all three strains when
paromomycin was present during starvation. All strains starved without the drug showed
exocytosis. In wt cells starved without the drug exocytose was abolished when stimulated with
Alcian blue containing paromomycin. Wt cells starved with paromomycin and washed before
stimulation showed no capsule formation, a recovery time is needed. These results suggest that
paromomycin when present is a potent inhibitor of exocytosis.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Hans Ris: Contributions to Protistology
Michael F. Dolan
Department of Geosciences, University of Massachusetts, Amherst, MA
Professor Hans Ris (1914-2004) of the University of Wisconsin, Madison, one of the foremost
electron microscopists of the 20th century, is well known by cell biologists for his studies of
chromatin, the cytoskeleton and the nuclear pore complex. Although he would not have
considered himself a protistologist, he studied a wide variety of organisms, many of which were
protists. Working with the Ris family I have arranged for the disposition of his research materials
for use by other scientists. His collection of transmission electron micrographs, negatives,
stereomicrographic slides, and TEM blocks have been deposited at the American Museum of
Natural History’s Special Collections Library. The protist files in his collection include Amoeba,
Amphidinium, Aulacantha (with Syndinium), Blastocladiella, Chara, Chlamydomonas,
Collozoum DNAse, Collozoum Hawaii Collozoum Villefranche, Collozoum spindle (serial),
Collozoum spindle (cross), Cryptomonas, Cyanophora, dinoflagellates, Euglena, Euplotes,
Kappa particles, Micromonas, Oodinium, Paramecium, Physarum, Porphyridium, Spirogyra,
Syndinium centriole, Syndinium development, Syndinium chromosome attachment,
Trichonympha, and zooxanthellae. Most of this material has not been published, and should be of
use to researchers studying the ultrastructure of these organisms.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Monophyly and Paraphyly Within the class Colpodea (phylum Ciliophora)
1
Dunthorn, M.S., 2W. Foissner, and 1,3L.A. Katz
1
University of Massachusetts, Amherst, MA, 2University of Salzburg, Austria, 3Smith College,
MA.
The class Colpodea is one of eleven classes of ciliates (phylum Ciliophora), and is a common
member of soil microbial communities. While relationships among Colpodea species and higher
taxa have been monographed using morphology, molecular analyses to test these relationships
were based on sparse taxon sampling. Although morphological evidence is strong for the
monophyly of the class, previous molecular evidence shows only weak support or none at all, but
did point to unsuspected relationships that challenge the morphological-based ordinal groups.
Here we present a more detailed molecular analysis using small subunit ribosomal DNA
sequences combined with more extensive taxon sampling, including previously unsampled
orders. Our initial results show to the monophyly of the class is still not well supported, and that
the Colpodida and the Cyrtolophosidida are paraphyletic. Furthermore, there is evidence for the
paraphyly of the genus Colpoda. A discussion of morphological evolution as implied by the
molecular tree is offered to help explain the discrepancy between previous classifications and the
phylogeny.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Identification of Copine A Target Proteins in Dictyostelium
Marina Bayeva, Lauren C. Naliboff, and Cynthia K. Damer
Vassar College, Poughkeepsie, NY
Copines make up a novel family of highly conserved, Ca2+-dependent, phospholipid-binding
proteins found in a wide range of eukaryotic organisms. At the N-terminus, copines contain two
C2 domains capable of binding to cellular membranes in response to elevated calcium levels.
This is followed by a C-terminal protein-binding A domain. Although the biological role of
copines is not yet known, recent studies suggest that copines function as chaperone proteins,
bringing target proteins associated with their A domains to particular membranes in response to
stimuli. We are studying copine protein function in the model organism, Dictyostelium
discoideum, and have identified six copine genes in the Dictyostelium genome, cpnA-cpnF.
Identification of the proteins that bind to copines is critical to the understanding of the role
copines play in cellular function. We are currently using affinity chromatography and MALDITOF-mass spectrometry to isolate and identify proteins that bind to Dictyostelium Copine A. In
addition, we have created a cpnA-knockout cell line. The cpnA- cells exhibit a cytokinesis
defect when grown in suspension, exhibit a reduced rate of exocytosis, and normal rates of
endocytosis and phagocytosis.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
An Examination of Copine Expression During Development of Dictyostelium discoideum
Pamela S. Kim, Dr. Eric S. Eberhardt and Dr. Cynthia K. Damer
Departments of Biology and Chemistry, Vassar College, Poughkeepsie, NY
Copines are a class of calcium-dependent membrane binding proteins found in a variety of
eukaryotic organisms. The characteristic C2 domains found at the N-terminus region of copines
suggests involvement in cell signalling pathways or membrane trafficking. Although not much is
known about the function of copines, the slime mold Dictyostelium discoideum has served as an
ideal system for study. In this model organism, six copine genes (cpnA-cpnF) have been
identified, each of which are diverse in sequence and hold the possibility of carrying out distinct
functions. During Dictyostelium development, the cells signal each other by release of cAMP
and aggregate into hemispherical mounds and fruiting bodies. To investigate whether the copine
genes may be important regulators of development, we used real-time RT-PCR to determine the
expression levels of each of the copine genes during development. Dictyostelium cells were
developed on filters and total RNA was isolated every two hours during their 24-hour
development program. All six copine genes are expressed in vegetative cells and are expressed
at varying levels throughout development. All of the copine genes, except cpnF show upregulation at one or two major developmental transitions. cpnC expression is up-regulated very
early in development prior to aggregation, cpnA and cpnB are up-regulated immediately prior to
mound formation, and cpnA, cpnC, cpnD, and cpnE are up-regulated immediately prior to
culmination.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
The coat of Bacillus subtilis spores is required for “eat resistance” during phagocytic
predation by the ciliate Tetrahymena thermophila
Lawrence A. Klobutcher, Katerina Ragkousi and Peter Setlow
Department of Molecular, Microbial and Structural Biology, University of Connecticut Health
Center, Farmington, CT
Spores of Bacillus bacteria are highly resistant to many environmental stresses (e.g., heat,
radiation, desiccation), owing in part to the presence of multiple “extracellular” layers. A number
of these layers have been implicated in various types of spore resistance, but the role of the
outermost layer, the spore coat, is not completely understood. In this study we examined the role
of the spore coat in resistance to predation by the ciliated protozoan Tetrahymena thermophila,
which uses phagocytosis to ingest and degrade other microorganisms. Wild-type dormant spores
of Bacillus subtilis were efficiently ingested by Tetrahymena, but were not digested and failed to
support Tetrahymena growth. In contrast, spores bearing mutations in a number of genes that are
required for normal coat formation supported Tetrahymena growth, and were killed and digested,
leaving only an outer shell termed a rind. The undigestable rinds were similar in appearance to
structures generated by treatment of coat-defective spores with lysozyme. Moreover, the
lysozyme sensitivity of coat-defective spores correlated with their ability to support Tetrahymena
growth. Spore killing by T. thermophila was via lytic enzymes within the protozoal phagosome,
not by initial spore germination followed by killing. These findings indicate the coat plays a
major role in protecting spores from predation, and demonstrate the usefulness of the
Tetrahymena system for assessing the integrity of the spore coat.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Reassessing the phylogeny of euplotid ciliates using ribosomal sequences
Yara K. Haddad and John A. Kloetzel
Department of Biological Sciences, University of Maryland Baltimore County
Baltimore, MD
Within the ciliate family Euplotidae, the genus Euplotes is a cosmopolitan and widely studied
taxon, with over 60 named species. In 1995 Borror and Hill split members of this traditional
genus into four subgroups, based on morphological, developmental and ecological criteria. Each
subgroup was raised to the level of genus, with new generic names provided (Moneuplotes,
Euplotopsis, and Euplotoides, to accompany the retained genus Euplotes). Using polymerase
chain reaction (PCR) with universal eukaryotic ribosomal primers (and others designed from
known Euplotes sequences), we have determined the sequence of the ribosomal small subunit
gene (SSU-rDNA) from Euplotes (“Euplotopsis”) muscorum, an uncommon small euplotid none
of whose genes are available on public databases. We have used this sequence, in combination
with online genomics software (WorkBench; http://workbench.sdsc.edu) and SSU-rDNA
sequences for many Euplotes species available on GenBank, to construct a neighbor-joining
phylogenetic tree for these species. We find significant differences from the taxonomic
groupings suggested by Borror and Hill. In agreement with Petroni et al. (2002), E. raikovi in
our phylogeny seems to branch well away from all other species analyzed, rather than being
grouped within Euplotopsis (with muscicola and muscorum). The closest relatives of these latter
two species (based on our molecular phylogeny, including our new SSU-rDNA sequence for
muscorum) include charon and rariseta, which in the Borror and Hill scheme are placed within a
different genus. At the present time, the “clades” assigned by Petroni et al. (2002) appear to be
the most reasonable groupings on molecular grounds, and are in general concordance with our
preliminary analysis.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Geographic Expansion of Human Babesiosis in New York State
Dimitra Mitsani1, Federico Viganego1, Ellis Tobin2, David Liebers3, abd Allen Teal4
1
Albany Medical College, Albany, NY; 2Albany Medical College and Upstate Infectious
Diseases Associates, Albany, NY; 3Ellis Hospital, Division of Infectious Diseases, Schenectady,
NY; 4Parasitology Laboratory, Division of Infectious Diseases, Wadsworth Center, New York
State Department of Health, Albany, NY.
Babesiosis is a zoonotic disease transmitted by Ixodid ticks that is caused when erythrocytes
become infected by the apicomplexan Babesia microti. Haemolysis is commonly observed.
Although infection is often subclinical, it can present as a febrile illness that is occasionally fatal
in immunocompromised hosts. Babesia microti is the most commonly identified species of
Babesia in the United States. The geographic distribution has, until recently, remained limited to
coastal areas of the northeast and parts of the upper Midwest where tick vectors cluster. Human
babesiosis has been a reportable disease in NYS since 1986. The majority of cases in upstate NY
were associated with travel to known endemic locations, or through blood transfusion. Locallyacquired cases of human babesiosis in upstate NY are sporadic and have been previously
described as far north as Columbia County. Here we report two cases of babesiosis occurring in
Rensselaer and Schenectady Counties. These cases provide new evidence of northern expansion
of endemic human babesiosis in NYS.
Cases. Patient 1 was a 56 year old male, Rensselaer County resident, who presented with
nonspecific febrile illness associated with chest pain and haemolysis. His course was
complicated by myocardial infarction. Pateint 2 was a 70 year old Ukranian male, who resided in
Schenectady County for the preceding two months, and presented with fever and haemolysis.
Neither were immunocompromised, had recent travel to endemic areas, nor had received blood
transfusions. Intraerythrocytic parasites were seen on blood smears. Babesia microti was
detected by PCR and confirmed by DNA sequencing.
Discussion. Recent data suggest continual expansion of human babesiosis to previously nonendemic areas. This appears to be related to the increase in the density of white-tailed deer that
are thought to be the most important host for Ixodid ticks.
Conclusion. Although further epidemiologic studies are needed, our cases provide evidence of
an increase in the northeastern range of endemic human babesiosis in NYS. Physicians and
Public Health officials should be alerted to the geographic expansion of babesiosis so that rapid
detection, treatment, and preventive measures are carried out.
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
COLD-ADAPTATION OF FORAMINIFERAL MICROTUBULES: AN
ENVIRONMENTAL APPROACH
ANDREA HABURA,1,2 SARAH BRODERICK1, AND SAMUEL S. BOWSER1,2
1
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health,
Albany, NY, 2Department of Biomedical Sciences, University at Albany, Albany, NY
Foraminiferans are known to be highly divergent in several normally conserved genes. Although
the best-understood example of this phenomenon is the ribosomal small subunit (Pawlowski
2000, Habura et al. 2004), other genes also appear to have been altered from the eukaryotic
consensus. For example, foraminiferal -tubulins are highly modified compared to those from
other organisms, in ways which have implications for foraminiferal microtubule assembly
(Habura et al. 2005).
Because these genes are so unusual, specific primers can be used to identify foraminiferal
-tubulins directly from environmental DNA samples. This approach allows rapid testing of
hypotheses about environmental influences on tubulin assembly, such as low temperatures, and
also helps to reduce possible effects from sample bias. Our results suggest that microtubule coldstability in foraminiferans is probably extrinsic to the tubulins. We also use this data, in
combination with data from other organisms, to draw more global conclusions about the
generation of cold-stable microtubules in eukaryotes.
Supported by NSF grants OPP0003639, ANT0440769, and DEB0445181
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
REGISTERED PARTICIPANTS
Anderson, O. Roger
Columbia University
Bio, Lamont-Doherty Earth Observ.
Palisades, NY 10964
845-364-8452
ora@LDEO.columbia.edu
Clamp, John C.
NC Central Univ
Dept Biology
Durham, NC 27707
919-530-6395
jclamp@nccu.edu
Bell, Aaron
Albert Einstein College of Medicine
1300 Morris Park Avenue
Forch 610
Bronx, NY 10461
718-430-4062
abell@aecom.yu.edu
Coats, D. Wayne
Smithsonian Environ Res Ctr
647 Contees Wharf Rd PO Box 28
Edgewater, MD 21307
443-482-2271
coiatsw@si.edu
Bigteli, Ashkan
University Rhode Island
431 Conshohaken State Road
Gladwyne
PA 19035
610-331-8883
ashbig@mail.uri.edu
Bleyman, Lea K Dr
Baruch College, CUNY
Department of Natural Sciences
Box A-0506
One Bernard Baruch Way
New York, NY 10010-5585
646-660-6236
Leableyman@baruch.cuny.edu
Cabral, Guy
VCU School of Med
PO Box 980678
Richmond, VA 23298
804-828-2306
gacabral@hsc.vcu.edu
Chantangsi, Chitchai
University of Guelph
Department of Integrative Biology
Guelph, Ontario, Canada N1G 2W1
519-824-4120
cchantan@uoguelph.ca
Dhungel, Nripesh
Bard College
PO Box 5000
Annondale-on-Hudson, NY 12504-5000
845-752-5237
nd332@bard.edu
Dolan, Michael F
UMASS Amherst
Department of Geosciences
Amherst, MA 01003
413-545-3244
mdolan@geo.umass.edu
Dolan, Sona
Holyoke Community College
Division of Science and Mathematics
Holyoke, MA 01040
413-545-3244
mdolan@geo.umass.edu
Domozych, David
Skidmore College
DanaSciCtr
815 Broadway
Saratoga, NY 12866
518-580-5075
ddomoz@skidmore.edu
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Dueker, M.E.
LDEO
Columbia Univ
248 9th St #3
Brooklyn, NY 11215
347-645-2916
med21D9@columbia.edu
Guerra, Charles
Albert Einstein College of Medicine
1300 Morris Park Avenue
Forch 610
Bronx, NY 10461
718-430-4062
guerra@aecom.yu.edu
Ennis, Jill
NYS DOH Wadsworth Center
DAI 120 New Scotland Ave
Albany, NY 12208
518-474-4177
jxe02@health.state.ny.us
Haberlandt, Katharine
University of Connecticut
Dept Marine Sciences
1084 Shennecossett Road
Grptn, CT 06340
860-961-9863
katharinehaberlandt@yahoo.com
Ferguson, John B
Bard College
PO Box 5000
Annondale-on-Hudson, NY 12504-5000
845-758-7300
ferguson@bard.edu
Fremen, Ethan
University Rhode Island
Dept Cell and Molecular Biology
Kingston, RI 02881
818-357-6747
i@mindlace.net
Gentekaki, Eleni
University of Guelph
Department of Integrative Biology
Guelph, Ontario, Canada N1G 2W1
519-824-4120
egenteka@uoguelph.ca
Goldstein, Susan T
University of Georgia
Department of Geology
210 Field Street
Athens, GA 30602-2501
706-542-2652
sgoldst@gly.uga.edu
Hans, Jennifer
NYS-DOH, Wadsworth Center
DAI 120 New Scotland Ave
Albany, NY 12208
518-474-4177
jhans@wadsworth.org
Harris, Jack
The Sage Colleges
Department of Biology
45 Ferry Street
Troy, NY 12180
518-244-2291
harrij@sage.edu
Hufnagel, Linda A
University of Rhode Island
Dept Cell and Molecular Biology
Kingston, RI 02881
401-742-5358
lhufnagel@uri.edu
Jacobs, Mary Ellen
Univ.of Connecticut Health Center
263 Farmington Ave MC-3305
Farmington, CT 06030-3305
860-679-2224
jacobs@neuron.uchc.edu
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Kaczynska, Anna K.
Bard College
PO Box 5000
Annondale-on-Hudson, NY 12504-5000
917-834-2291
ak471@bard.edu
Lynn, Denis H Dr
Univeristy of Guelph
Department of Integrative Biology
Guelph, ON, Canada N1G 2W1
519-824-4120
519-767-1656
Keithly, Janet, Dr
NYS DOH, Wadsworth Center
DAI 120 New Scotland Ave
Albany, NY 12208
518-474-4177
keithly@wadsworth.org
MacLean, Rebecca
VCU School of Med
PO Box 980678
Richmond, VA 23298
804-828-9742
macleanrc@vcu.edu
Klobutcher, Lawrence
Univ. of Connecticut Health Center
Dept Mol., Micro., and Structural Biology
263 Farmington Ave MC-3305
Farmington, CT 06030-3305
860-679-2816
klobutcher@nso2.uchc.edu
Madison-Antenucci, Susan Dr
NYS DOH, Wadsworth Center
DAI 120 New Scotland Ave
Albany, NY 12208
518-474-4177
susanma@wadsworth.org
Koonce, Michael
Wadsworth Center
Empire State Plaza, PO Box 509
Albany, NY 12201-0509
518-486-1490
koonce@wadsworth.org
Leach, Steven C
Novozymes Biologicals
408 Scott Lane
Wallingford, PA 19086
215-327-4306
scle@novozymes.com
Lee, John J
City College CUNY
Convent Ave @138 St
New York, NY 10031
212-650-9545
jlee@sci.ccny.cuny.edu
Marchewka, Mary
NYS DOH, Wadsworth Center
DAI 120 New Scotland Ave
Albany, NY 12208
518-474-4177
marchewk@wadsworth.org
Marciano-Cabral, Francine
VCU School of Med
PO Box 980678
Richmond, VA 23298
804-828-9742
fmcabral@hsc.vcu.edu
Nielsen, Maj-Britt
Albert Einstein College of Med
1300 Morris Park Ave
Bronx, NY 10461
718-430-4064
mnielsen@aecom.yu.edu
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Noyes, Megan
University of Guelph
Department of Integrative Biology
Guelph, Ontario, Canada N1G 2W1
519-824-4120
noyesm@uoguelph.ca
Octavio, Leah
University Rhode Island
Dept Cell and Molecular Biology
Kingston, RI 02881
401-339-3279
loct4817@postoffice.uri.edu
Samaranayake, Haresha
Univ. of Connecticut Health Center
263 Farmington Ave MC-3305
Farmington, CT 06030-3305
860-679-2224
hsamaranayake@student.uchc.edu
Satir, Birgit
Albert Einstein College of Medicine
Dept Anatomy and Structural Biology
1300 Morris Park Ave
Bronx, NY 10461
718-430-4063
bsatir@aecom.yu.edu
Satir, Peter
Albert Einstein College of Medicine
Dept Anatomy and Structural Biology
1300 Morris Park Ave
Bronx, NY 10461
718-430-4061
satir@aecom.yu.edu
Teal, Allen
NYS DOH, Wadsworth Center
120 New Scotland Ave
Albany, NY 12208
518-474-4177
teal@wadsworth.org
Visviki, Ioanna
College of Mount Saint Vincent
Manhattan College
31-29 36th Street Apt 2
Astoria, NY 11106
718-405-3482
Ioanna.visviki@mountsaintvincent.edu
Wright, Chrissa
SUNY Plattsburgh
8 Willard Drive
Berhards Bay, NY 13028
315-675-8841
wrig2032@mail.plattsburgh.edu
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
Planning Committee
Samuel S. Bowser – Wadsworth Center
Michael Dolan - University of Massachusetts, Amherst
Andrea Habura – Wadsworth Center
Jack Harris - Russell Sage College
Jan Keithly – Wadsworth Center
Michael Koonce – Wadsworth Center
Jeff Travis – University at Albany
Also, special thanks goes to:
Janice Bonaccorso
Lab Coordinator – Russell Sage
Libby Mahoney
Admin. Assistant. Russell Sage Dean's Office
Cindy Graham – Wadsworth Center
Photography and Illustrations – Wadsworth Center
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
About Russell Sage College
Women of Influence - Mission and Purpose
The mission of Russell Sage College is to prepare
women to participate fully and actively in the life and
work of society. The College seeks to provide women
of all ages and backgrounds with a strong vision of
their own power and potential.
Within the context of societal changes, the Russell
Sage College mission remains consistent in principle
yet diverse in expression. The College's commitment
to integrating liberal and career-focused study reflects
the fundamental belief that each complements the
other. Through both its academic program and
supportive environment, Russell Sage College creates
an intellectually challenging and caring community to
facilitate the development of knowledge, skills, and
attitudes essential in an increasingly interdependent
world.
History of Russell Sage College
Founded by Margaret Olivia Slocum Sage and named
for her husband, Russell Sage College is the outgrowth of the Emma Willard School, the oldest
preparatory school for women in the United States. An 1847 graduate of Emma Willard, Mrs.
Sage began a teaching career before marriage. In later life she became the custodian of a large
fortune bequeathed by her financier husband and became America's foremost woman
philanthropist. When she was almost 88 years old, Mrs. Sage decided that the three original
Willard School buildings in downtown Troy, N.Y. should e the nucleus of something "entirely
different" from the preparatory school.
With Eliza Kellas, principal of Emma Willard, Mrs. Sage approved a blueprint for a curriculum
that offered women students a new concept in American education: a combination of traditional
liberal arts study with preparation for specific careers. Russell Sage College opened its doors in
September 1916 as a “School of Practical Arts”, and students arrived from as far away as Oregon
and Hawaii to enroll in its program.
By the time of its first four-year graduation in 1920, its academic program was solidly
established, and the extracurricular life on campus was lively, complete with student
government, student publications, clubs, and social events. On December 29, 1927, the New
York Board of Regents granted a new and separate charter for Russell Sage College, reaffirmed
the status of Emma Willard as a secondary school, and approved the legal transfer of property to
the college.
Throughout its history, Russell Sage College has remained true to its founding mission.
However, during World War II an Emergency Men’s Division was created which later became a
coeducational Albany Division established in 1949 to offer associate, bachelor’s and master’s
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
degrees to government workers and returning veterans. Today, Russell Sage joins with the
undergraduate Sage College of Albany and Sage Graduate School to make up The Sage
Colleges.
A Russell Sage Education
Russell Sage College is a distinctive academic community: a small, professionally oriented
college for women, where individuals count and are consistently challenged to develop their
strengths, self-confidence, and autonomy. Academic standards are high at Russell Sage, as
students learn to think critically, make sound value judgments, and develop a love of learning
that will be theirs for life. Study in both the professional and liberal arts provides excellent
preparation for successful careers and contributive lives.
Sage students benefit from recent research into areas of special significance to women. Female
students tend to do particularly well in an environment valuing collaborative teaching and
learning. Small classes and a college philosophy that emphasizes active learning and discussion
foster such an environment here.
Interdisciplinary core courses required as part of the comprehensive general education program
assist students in gaining an understanding of
the status, conditions, and contributions of
women around the world. The courses enable
students to see themselves as potential agents
for change, whether locally or globally, in the
world beyond Sage.
Beyond the curricular focus on women,
numerous programs and initiatives are provided
each year to help students develop skills and
self-confidence. Such efforts are sponsored by
the Helen M. Upton Center for Women's
Studies, the Allies Center for the Study of
Social Responsibility, the Opportunities for
Women in Leadership Program (OWL), and the
Returning Women's Program, among others.
Highly credentialed, talented, and caring faculty
members are teachers first and foremost, but
they are also authors, researchers, and leaders in
their fields of study and practice. They
encourage, challenge, and support students and
provide programs of study that systematically
connect the classroom to the world beyond the
campus.
College programs have reflected the RSC motto, "To Be, To Know, To Do," since the college's
inception in 1916. Knowledge becomes valuable as it becomes action; RSC stresses the
Thirteenth East Coast Meeting on Protistology, Russell Sage College, Troy, NY
importance of practical experiences as a crucial component of the learning process. Russell Sage
offers internships at more than 900 locations in New York and New England, with internship
opportunities also available overseas. Sage students learn and work in hospitals, social service
agencies, laboratories, courtrooms, museums, and art centers, as well as for newspapers,
department stores, the New York State Legislature, state agencies, television and radio stations,
banks, law enforcement agencies, schools, and halfway houses. Many students also collaborate
with professors on original research. Some students present the results of their research at
national meetings or publish in refereed journals.
Another hallmark of Russell Sage is the strong connection between undergraduate and graduate
study. Several programs, such as physical therapy, occupational therapy and nursing, offer
accelerated bachelor’s-to-master’s or bachelor’s to doctorate options. Any student at Russell
Sage with an academic record that qualifies her for graduate entry may develop an individualized
plan to combine undergraduate and graduate degrees. Russell Sage also offers joint degrees with
other institutions, including a 3-2 program in mathematics and engineering with Rensselaer
Polytechnic Institute and a 3-3 program with Albany Law School.