I. Ecology
| 1-B. Comparison of nestling and fledgling ferruginous hawk (Buteo regalis) diets. |
Scott Wilson, 499
Juvenile foraging efficiency of avian species is limited by experience and underdeveloped flight skills. A consequence of limited foraging efficiency is selection of small prey items. Nesting ferruginous hawks (Buteo regalis) of the grassland natural region of Alberta are highly specialized to Richardson ground squirrels (Urocitellus richardsonii), but microtine, peromyscus and caelifera prey exist in their diet. The objective of this study is to determine how post-fledging and nestling diet differ. Under the constraint of underdeveloped flight and foraging skills, juveniles will consume a larger array of small prey items than nestling ferruginous hawks. Nestling diet composition is determined from video footage of nests collected in 2011-2012 from the Grassland region of Alberta and Saskatchewan. Pellets, stomach, and crop content are used to determine fledgling diet. Prey deliveries to nestlings were primarily Richardson ground squirrel, with small proportions of waterfowl, songbird and other small mammal prey. Insects, feathers and small mammal bones were present in preliminary juvenile pellet analysis. Juvenile diet composition studies are uncommon yet necessary since foraging efficiency and body condition predict first year survival of avian species. Management implications specific to preferred prey should be beneficial to juvenile survival of the declining ferruginous hawk.
2-M. Home Range and Resource Use of Ferruginous Hawks Nesting in Association with Oil and Gas Development
Jesse L. Watson, M.Sc.
Studies have shown historic declines in ferruginous hawks (Buteo regalis) throughout Canada are linked to degradation of native grassland. Recent speculation suggests increased anthropogenic development may also play a role in continuing declines. Understanding the relationship between ferruginous hawk movement patterns at several temporal and spatial scales may lead to important conclusions regarding home ranges and associated mitigation or conservation actions. Our goal is to study hawk movements in an anthropogenic landscape using satellite telemetry. In 2011 and 2012, home ranges were estimated for 15 hawks using minimum convex polygons, kernel density estimations, and the Brownian bridge movement model. Using telemetry data, we will analyze spatial and temporal use patterns, focusing on use of various habitat types (e.g., native grassland and cropland), soil and topography, and areas with high and low industrial impacts (e.g., oil and gas wells and transmission lines). Additionally, we will examine migratory movements and identify similarities or differences between characteristics of breeding and wintering ranges selected by hawks. This research will provide fundamental scientific advancement in the study of ferruginous hawks throughout the Canadian prairies. Conclusions from this study will also help guide the management of the remaining ferruginous hawk habitat throughout all of North America. 3-M. Choose Your Weapon: Cnidae sequestration of the nudibranch Hermissenda crassicornis in the presence of predators
Susan Anthony, M.Sc.
Nudibranchs (sea slugs) evolved from an ancestor with a protective shell. Losing this effective defense would seem disadventageous, but one group of nudibranchs, the aeolids, have a novel defensive strategy whereby they steal stinging organelles (cnidae) from cnidarian prey, such as anemones and corals, and use them for their own defense. Cnidae function in cnidarians as a means for food capture, defense, and adhesion. Therefore, not all cnidae are created equal. Of the 26 different types of cnidae, some are better at piercing hard flesh, others for sticking to a substrate. It has been hypothesized that some cnidae are more effective against one type of predator over another. If it is preferable to an aeolid to have a specific cnidae for defense, will they switch their food to gain this cnida? I conducted my experiment on the aeolid nudibranch Hermissenda crassicornis, a generalist consumer that sequesters cnidae. If different groups of H. crassicornis are given the same cnidarians as food, will they sequester different cnidae based upon which predator they are exposed? Unlike the results of a similar experiment with the aeolid Flabellina verrucosa, H. crassicornis does not preferentially select one cnidarian prey over another when exposed to different predators.
4-M. The impact of human disturbance stimuli on Ferruginous Hawk nesting behaviour
Cameron Nordell, M.Sc.
Human disturbance stimuli, in the form of industry, infrastructure, agriculture and recreation, permeate the Canadian prairies. Organisms which are unable to cope with the considerable human disturbance require special conservation effort. The ferruginous hawk (Buteo regalis) is listed as Threatened federally and Endangered in Alberta. Furthermore, ferruginous hawks have demonstrated reduced nesting success on urbanized landscapes and are thought to respond negatively to human presence at the nest. However, the actual behavioural impact of these disturbance stimuli are largely speculative. Our study examines the fine scale response of ferruginous hawks to human disturbance stimuli by installing 28 video monitoring systems at ferruginous hawk nests across southern Alberta and Saskatchewan in 2012. We will use video recordings to assess precise behavioural responses to disturbance, such as latency to return and changes in prey delivery rates. Additionally, we documented ferruginous hawk defensive behaviours while monitoring 250 nests in 2012. We will present preliminary analyses which are underway, as well as future directions for our research in the 2013 field season.
5-B. The influence of adverse weather events on the behaviour of Ferruginous Hawks (Buteo regalis)
Holly Stemberger, 499
Weather events, such as high winds and heavy rain, can alter the foraging patterns of animals and change parental care activities. The Ferruginous Hawk (Buteo regalis) is an endangered species that breeds in the mixed-grasslands of southern Alberta and Saskatchewan. Many raptors are known to mimic the diurnal activity of small mammals, hunting more prevalently at dawn and dusk when activity levels of prey are higher. However, the threat of bad weather conditions may result in parents foregoing hunting in favour of nest attendance in order to protect nestlings. The subsequent reduction in hunting effort may then negatively impact nestling health. My objective with this study is to assess the influence of storms, characterized by high winds over 50 km/h and precipitation above 15 mm, on the behavior of parent Ferruginous hawks. I will compare daily and hourly weather conditions with two main behavioural categories; nest attendance and prey delivery rates. Comparing these behaviours in response to these weather conditions could provide for crucial information on how these birds respond to extreme weather events.
6-M. If Memory Serves: Evaluating animal cognition and optimal foraging theory as mechanisms for home range development in elk (Cervus elaphus)
Dana Seidel, M.Sc.
Despite being documented across many taxa, home ranges and the mechanisms behind their development are not fully understood. Recent theories suggest that foraging behavior, resource selection, and memory are responsible factors in the emergence of stable home ranges, but these theories have seldom been tested. To explore the influence of foraging behavior on home range, we investigated how a large browsing herbivore uses and returns to particular areas of its home range throughout the growing season. Using GPS data from 20 elk (Cervus elaphus) and a spatial temporal clustering algorithm, areas of foraging were identified within natural home ranges in the montane grasslands of Southwestern Alberta. Vegetation surveys and pellet counts were used to evaluate and compare selected foraging patches to other areas of the home range visited but not selected for foraging. This research documents patch selection throughout the growing season, highlighting fidelity and a cyclic return pattern through areas of the summer range. Investigation into the mechanisms of stable home range development may help us to anticipate and understand range change and how home ranges are influenced by conspecifics, competitors, invaders, and land use change.
7-M Toxicological effects of tailings pond water on waterfowl using captive mallards (Anas platyrhynchos domestica) as an experimental model
Elizabeth Beck, M.Sc.
In Alberta’s oil sands large quantities of waste water are produced during bitumen extraction and by-products are stored in tailings ponds which are attractive to waterfowl. Experts contend ponds pose little threat to birds provided they do not encounter bitumen, and do not remain on the water for long periods. Very little data exists to test this assumption and many toxicological studies have used very high concentrations of toxic material and exposes birds by invasive methods such as intubation, methods not necessarily representative of natural processes. In order to simulate repeated short-term exposures to tailings water that birds are thought to experience in the oil sands, Pekin ducks (Anas platyrhynchos domestica) were exposed to repeat multi-hour baths in either control or recycled tailings pond water over two years. Each trial consisted of three exposures over three weeks; weights, body measurements, and blood samples were collected. Toxicity will be evaluated through clinical biochemistry, endocrinology, hematology, and heavy metal analyses. If recycled tailings pond water is found to be non-toxic then deterrent practices in the oil sands should reflect this, for example by creating bitumen free safe zones in the centre of ponds, and intensifying perimeter deterrent efforts, where bitumen accumulates most.
8-M. Avian deterrent methods for application in the oil sands region of Alberta
Ffion Cassidy, M.Sc.
The Alberta oil sands are among the largest sources of bitumen in the world, and the exploitation of this resource is a massive and rapidly expanding industry. Because this area is located directly under a major flyway for migratory waterfowl, birds are at risk of landing on tailings ponds and coming into contact with bitumen. I will assess the efficacy of ecologically relevant visual and auditory avian deterrents, and investigate new ideas to complement the devices and practices already in use. By applying knowledge of sensory and behavioral ecology it may be possible to capitalise on stimuli to which birds have evolved or learned to respond. This may also help to minimise habituation, which is a major limiting factor in the effectiveness of current practices. Initial trials were carried out during the fall of 2012 in the Edmonton area, and will be used to guide further work in 2013. Results will be examined across guilds of affected birds so that deterrent response may be tailored to certain species and conditions. The aim of my research is to make recommendations for future deterrent type, placement, and activity to minimise bird contact with the bitumen found in oil sands tailings ponds.9-B. Attraction of Pseudaletia unipuncta to acetic acid and 3-methyl-1-butanol
Amanda St. Onge, 499
Lepidopteran pests are monitored with semiochemical traps; these are usually sex pheromones but can also be host volatiles. Recently, field studies have shown a food-based lure (acetic acid and 3-methyl-1-butanol) is attractive to the true armyworm, Pseudaletia unipuncta (Lepidoptera: Noctuidae). The objective of this study was to assess the nature of this attraction and how it differs with sex and feeding status. Male and female, starved and fed moths were flown in the wind tunnel to a baited trap and an unbaited control. We have had some success in capturing moths in baited traps.
II. Molecular Biology and Genetics
| 10-M. Identification and characterization of mel-43, a gene required for early embryonic development in C. elegans |
Maryam Ataeian, M.Sc.
Formation of the spindle is required for segregation of chromatin during meiotic and mitotic divisions. The female meiotic spindle forms around chromatin without the contribution of centrosomes, even though the centriole pair is introduced into the oocyte with the sperm upon fertilization. Since meiosis and mitosis divisions occur in the same cytoplasm, regulation of the microtubule cytoskleleton is important to ensure that these two types of spindles do not form at the same time in the newly fertilized embryo. mel-43(sb41) is a dominant, temperature-sensitive, maternal-effect mutation (Mitenko, 1997) that causes the failure in segregation of chromatids in meiosis II and display a significant delay in transition from metaphase II to anaphase II. mel-43 is predicted to encode a novel, potentially nematode-specific protein. mel-43 has two very similar paralogues. RNAi directed against all paralogues results in the failure to extrude the first polar body and no meiosis II spindle assembly before entering mitosis. MEL-43 has high cytoplasmic signals in meiosis, which degrades prior to mitosis through a cullin-based E3 ligase pathway. Immunostaining of mel-43(sb41) embryos revealed that failure in anaphase II correlates with the persistence of REC-8 cohesin on sister chromatids.
11-B. The response of microbial community in multiyear sea ice to enviromental temperature and salinity stress
Annabella Yee, 499
Microorganisms in sea ice play a significant role in the biogeochemical cycles of the Arctic Ocean and provide year-round support to Arctic Ocean productivity, directly influencing its carbon and energy fluxes. They serve as an important base for the entire polar food web. Historically, sea ice in the Arctic has been predominantly multiyear ice (MYI), which is ice that survived at least one melt period. MYI has a distinct top to bottom gradient in dissolved matter concentrations. Similarly, a temperature gradient is generated since the upper portions of sea ice sheets are exposed to atmospheric temperatures that can get as low as -40˚C, whereas the bottom of the ice sheet is exposed to -1.86˚C. I propose to test the response of MYI microbial community to environmental stress generated by the inversion of MYI ice, thus disrupting the established top-bottom salinity and temperature gradients. I hypothesize that microbial communities are adapted to the specific physical and chemical conditions found in the relatively stable environments at different depths in MYI. Currently, I am characterizing physical and chemical attributes of the ice, determining the microbial composition of different ice layers, and assessing how they correlate in ice cores that were incubated in situ.
12-P. Characterizing Laulimalide using C. elegans embryo
Megha Bajaj, Ph.D.
Marine organisms have served as an important source for chemicals that target eukaryotic microtubules and hence they have been used to isolate new anti-cancer molecules. In one such screen done using the marine sponge, Cacospongia mycofijiensis, a crude lipophilic extract yielded the compound laulimalide (Mooberry S.L., et al 1999, Pryor D.E., et al 2002). laulimalide is known to disrupt the normal microtubule assembly and cause bundling of microtubules. It is known to inhibit cell proliferation and initiate mitotic arrest. Studies done using mammalian cell lines suggests that laulimalide leads to failure in proper chromosome segregation and cytokinesis. In some cases it has been observed that, similar to paclitaxel, it induces microtubule stabilization. Studies done so far using mammalian cell lines show that laulimalide causes cytotoxicity however, its precise effect on cytoskeletal structures and mode of action is unclear. Mammalian cells can take up to 20 hours to divide; therefore acute effects of the drug are often not readily apparent. In the present study we have used the first mitotic division in the Caenorhabditis elegans embryo to characterize laulimalide. The early cell division events in C. elegans have been studied in detail. The C. elegans embryo serves as an excellent system for the preliminary characterization of microtubule-targeted drugs because morphological changes to the cytoskeleton occur rapidly and are highly stereotypical. In C. elegans embryo the use of small molecule inhibitors is limited by eggshell impermeability. We have used an RNAi-based approach to create a permeable eggshell (Carvalho A., et al 2011). We have observed dose-dependent phenotypes for laulimalide, whereby different concentrations lead to stabilization or destabilization of microtubules. We have also carried out experiments to look for synergistic effects of laulimalide and paclitaxel on microtubules. In the present study, results will be presented to characterize the similarities and differences between paclitaxel and laulimalide with respect to their effects on the microtubule cytoskeleton in the one-cell C. elegans embryo.
13-B. Analyzing heme and pathways in Drosophila melanogaster
Pendleton Cox, 499
How heme biosynthesis is regulated is largely unclear. To study heme biosynthetic pathways, genes in the production pathway will be knocked down via RNAi with different Gal4 drivers in different tissues. As well, heme will be measured with a stain in wild type and mutant flies to further understand the level of heme required at the third instar larval stage. Finally, RNAi knockouts of genes used for iron synthesis will be done to view the effects on the fly and its relation to heme.
14-P. Studying Phage-Antibiotic Synergy in the cystic fibrosis pathogen Burkholderia cenocepacia
Fatima Kamal, Ph.D.
The Burkholderia cepacia complex (BCC) is a group of at least 17 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. BCC organisms have high innate antimicrobial resistance and a proposed alternative treatment is phage therapy. Recently, some phages have been observed to form larger plaques in the presence of sub-lethal concentrations of certain antibiotics; this effect has been coined Phage-Antibiotic Synergy (PAS). These reports suggest that some antibiotics have the ability to stimulate bacteria to produce increased concentrations of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkolderia cenocepacia strains- C6433 and K56-2. BCC phages KS5, KS12 and KS14 were tested for PAS. Of the antibiotics tested, the most pronounced effect was observed for meropenem, ciprofloxacin, and tetracycline. Electron micrographs of the B. cenocepacia host cells permitted visualization of the antibiotic effect. Cells appear as chains in the presence of meropenem, are elongated in the presence of ciprofloxacin and appear in groups in the presence of tetracycline. Plaque sizes and the corresponding phage titers both show increases with higher antibiotic concentrations. K56-2-infected Galleria mellonella larvae treated with phage KS12 and sub-lethal levels of meropenem demonstrate increased survival over controls without meropenem. These results suggest that BCC phage therapy can be combined with low doses of antibiotics to stimulate increased phage production and/or activity and thus improve the efficacy of phage therapy.
15-P. CECR2: a protein involved in tissue-specific chromatin remodelling complexes
Farshad Niri, Ph.D.
ATP-dependent chromatin remodelling complexes play a very important role in
many cellular processes such as transcription, DNA replication and DNA repair. The
complexes bind to chromatin and use the energy of ATP to rearrange chromatin to make DNA accessible/inaccessible to the regulatory factors. CECR2 is a subunit of CERF
chromatin remodelling complex, and binds to the ATPase subunits SNF2H or SNF2L.
Mutations in mouse Cecr2 cause a lethal neural tube defect in ~60% of mutant embryos. In nonpenetrant Cecr2 mutants that close their neural tube, both males and females show subfertility, having smaller litters than wild-types. I hypothesize that CECR2 is part of different complexes in neural tissues and gonads and these complexes function differently. In order to study the role of CECR2 in neurulation and reproduction, I have developed a CECR2-specific antibody and successfully immunoprecipitated CECR2- containing complexes. This confirmed a size difference between CECR2-containing complexes in mouse ES cells and testis, suggesting additional tissue-specific components in the two complexes. I am identifying additional components with mass spectrometry. Characterization of CERF complexes in different tissues may indicate differences in function for Cecr2 during neurulation and reproduction.
III. Microbiology and Biotechnology
| 16-P. Influence of vertically inherited symbionts and environmental bacteria on microbial communities in glass sponges. |
Anna Bramucci, Ph.D
Freshwater sponges (Porifera, Spongillidae) are basal animals that lack any kind of internal organs. They exhibit a sturdy endoskeleton composed of fused silica spicules. Freshwater sponges feed by sucking up water through small pores into their inner cavity, filtering it for bacteria and other microorganisms and ejecting it again through a tube-like structure called an osculum [1]. Besides serving as a source of food, microbial communities can comprise up to 40% of the total sponge tissue volume [2]. Under unfavourable conditions, many sponges form internal gemmules consisting of dormant, unspecialized cells surrounded by a protective layer of spicules. When conditions improve, the gemmules hatch and new sponges can grow from them [3].mIn this work, we hatched gemmules coming from a single freshwater sponge of the species Ephydatia muelleri and grew them in different media: Water from a similar freshwater lake environment, from the North Saskatchewan River, snow melt water and double distilled water as a control. Using DGGE (Denaturing Gradient Gel Electrophoresis), we investigated the composition of bacterial communities extracted from sponges after 3 weeks of growth. After 3 weeks, we observed notable variation in the growth of sponges kept in different media. Sponges grown in sterile water without bacterial food sources were less developed than those kept in water from environmental sources. DGGE banding patterns showed several PCR products that were common in all sponges. These probably derived from either sponge mitochondria, chloroplasts of algal symbionts or bacterial symbionts. Furthermore, we observed patterns specific to sponges grown in different water sources and even specific to single sponges. These likely represent bacteria that were taken up from the environment. [1] Weissenfels, N.(1992). The filtration apparatus for food collection in freshwater sponges (Porifera, Spongillidae). Zoomorphology 112(1):51-55 [2] Taylor MW, Radax R, Steger D, Wagner M.(2007). Sponge-Associated Microorganisms: Evolution, Ecology, and Biotechnological Potential. Microbiology and Molecular Biology Reviews 71(2): 295-347. [3] Simpson TL, Fell PE.(1974) Dormancy among the Porifera: Gemmule Formation and Germination in Fresh-Water and Marine Sponges. Transactions of the American Microscopical Society 93(4):544-577.
17-M. Autoregulation of RNA helicase levels by proteolysis in response to abiotic stress.
Oxana Tarassova, M.Sc.
RNA helicases are involved in rearrangement of the RNA secondary structures and have been shown to participate in almost every aspect of RNA metabolism. CrhR is DEAD-box RNA helicase in cyanobacterium Synechocystis sp. PCC 6803, whose expression is regulated by the redox status of the electron transport chain (Kujat et.al., 2000) and therefore can be affected by various abiotic stresses. It was observed that CrhR expression is at the basal level while growing cells at 30oC and it gets induced when the temperature is decreased to 20oC. Thus, it was considered for a long time that CrhR expression is inducible by the temperature shift from warm to cold. However, my recent findings show that we observe reduced levels of this RNA helicase at 30oC due to the presence of active degradation machinery, which can deliberately degrade CrhR at higher temperatures. Trying to unravel this degradation process we noticed that transcription (Georg, unpublished data) and protein levels for a number of genes including FtsH protease are different in CrhR mutant compared to the wild type, suggesting its possible role in the regulation of CrhR protein levels. Other experiments are still in progress in order to understand regulation of CrhR cellular levels.
18-M. Microbial Community Analysis of Wastewater Tertiary Effluent Using 16S rRNA PCR-DGGE
Albert Remus Rosana, M.Sc.
The stability of microbial communities from effluents of five different sections of the tertiary wastewater treatment plant (biological nutrient removal system) was investigated using denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplicons of the 16S rRNA gene. The EPCOR’s continuous bioreactor system with specialized microbial zones (pre-anoxic, anaerobic, anoxic, aerobic, pre-UV) showing variable oxygen tension collectively reduced the levels of phosphorus and ammonia in the effluent. Results suggest a high microbial diversity with shared banding patterns in its assemblage with “generalist” microorganisms observed across all stages of the tertiary treatment plant. Furthermore, oxygen tension is found to be the key driving force that affects the dominance of certain microbial groups as well as “specialist” microorganisms. These results implicate that a stable microbial community structure in a tertiary treatment phase contributes to an efficient nutrient removal in a wastewater treatment plant.
19-M. Role of the N-linked protein glycosylation pathway in the survival of campylobacter species under osmotic stress
Ritika Dwivedi, M.Sc.
The N-linked protein glycosylation pathway (Pgl) of Campylobacter jejuni is necessary for the biosynthesis of a unique heptasaccharide, GalNAc-GalNAc-(Glc)-GalNAc-GalNAc-GalNAc-diNAcBac (where diNAcBac is 2,4-di-acetamido-2,4,6-trideoxy-D-Glc) in the cytoplasm followed by the transfer onto the sequon D/E-X-N-X-S/T by the oligosaccharyltransferase, PglB. In addition, PglB exhibits hydrolase activity and releases the heptasaccharide in its free oligosaccharide (fOS) form into the periplasmic space. Recently, it was discovered that fOS levels are dependent on the osmotic conditions that C. jejuni encounters in its environment [1]. A related Campylobacter species, C. lari , is a halotolerant organism colonizing hosts found in high salinity environments (eg. marine animals and seagulls) and is able to tolerate 1.5% NaCl levels under laboratory conditions [2]. We are currently investigating how fOS contributes to the halotolerant phenotype of C. lari. Interestingly, MS and H1-NMR analyses indicate that C. lari fOS are hexasaccharides with a phosphate at the reducing end. In contrast, hexasaccharides are added directly to asparagine residues without further modification. As expected, MS analysis revealed that a C. lari pglB mutant does not produce fOS or N-linked glycans. In order to confirm that C. lari PglB in not able to cleave lipid-linked oligosaccharides in two positions, the C. lari PglB enzyme was expressed in a C. jejuni pglB mutant. In this experiment, only unphosphorylated fOS consistent with the C. jejuni heptasaccharide structure was detected, indicating that phosphorylation of fOS in C. lari is PglB independent. We are currently investigating into the mechanism that leads to phosphorylated fOS in C. lari. It is speculated that the anionic fOS modification in this species increases the periplasmic retention of the hexasaccharide. Future studies will focus on understanding the role of the fOS phosphate modification in the osmotolerance of C. lari. References: [1] Nothaft, H et al. (2009) Proc Natl Acad Sci U S A 106:15019-15024., [2] Miller, WG et al. (2008) Foodborne Pathog Dis, 5:371-386.
20-B. A general O-linked glycosylation system of Burkholderia cenocepacia
Karen Lithgow, 499
The Burkholderia cepacia complex (Bcc) is a group of gram-negative beta-proteobacteria with important roles in soils and the propensity to cause opportunistic infections in the lungs of immuno-compromised patients1. O-linked glycosylation is the enzymatic addition of glycan substrates onto hydroxylated amino acids (AAs) of acceptor proteins, purportedly contributing to immune evasion, stabilization from proteolysis, and adhesion2 in bacteria. During the en bloc transfer mechanism, sugars are sequentially built upon an inner membrane lipid carrier, which is translocated to the periplasm where an oligosaccharyltransferase (OTase) transfers the sugar to a target protein3. Although protein glycosylation is known to contribute to virulence in other beta-proteobacteria, it remains unexplored among Bcc bacteria. In this study we characterize the B. cenocepacia OTase (Bcal0960), demonstrating the presence of a general O-linked glycosylation system which functions by means of Bcal0960 in an en bloc transfer mechanism. Phenotypic work on an insertional mutant, bcal0960::Tet, showed important roles for this gene in virulence and motility. An enrichment for glycoproteins modified by Bcal0960 revealed fourteen glycoproteins in the WT that were absent from bcal0960::Tet; notably, the glycan-modified AA residue was identified from six of the fourteen glycoproteins. Further works aims to characterize Bcal0960 and the general O-linked glycosylation system. 1. Mahenthiralingam, E., et al. (2005) The multifarious…Burkholderia cepacia complex. Nat. Rev. Microb. 3:144-156. 2. Nothaft, H., et al. (2010). Protein glycosylation in bacteria: sweeter than ever. Nat. Rev. Microb. 8(11):765-778. 3. Faridmoayer, A., et al. (2007). Functional characterization of bacterial… J. Bact 189:8088-8098.
21-M. Genetic and Functional Analysis of the Type VI Secretion System in Acinetobacter
Brent Weber, M.Sc.
The genus Acinetobacter is comprised of a diverse group of organisms, several of which are pathogens. Our genetic analysis of various species of Acinetobacter indicated that many of these organisms possess the genes required to assemble a functional type VI secretion system (T6SS), a widespread virulence system among Gram negative bacteria. A. baumannii, the most studied species within this genus, is emerging as a major nosocomial pathogen and presents a significant threat for healthcare systems worldwide. The propensity of A. baumannii to develop multidrug resistance has severely reduced treatment options. Despite the widespread dissemination of A. baumannii, little is known about its pathogenic mechanisms. We determined that the T6SS is conserved among A. baumannii strains, which express and secrete the hallmark Hcp protein in a manner dependent on other structural components. Unlike other bacteria, A. baumannii ATCC 17978 does not appear to use its T6SS to kill Escherichi!
a coli. Deletion of the structural component TssM does not affect virulence in two eukaryotic infection models nor biofilm formation. Although none of the typical roles seem applicable to A. baumannii, the conservation of the T6SS indicates that this system may fulfil an important role in the various lifestyles of the Acinetobacter spp.
22-M. Analysis of Acinetobacter baumanni o-glycan variability
Rachel Kinsella, M.Sc.
Acinetobacter baumannii (Ab), a Gram-negative, opportunistic, nosocomial pathogen is progressively more difficult to treat, due to increasing antibiotic resistance. Recently our lab showed Ab ATCC 17978 has an active O-linked protein glycosylation system, modifying proteins with a pentasaccharide containing acetylated glucuronic acid. O-glycosylation, the en bloc transfer of a lipid-linked glycan to Serine or Threonine by an O-oligosaccharyltransferase (O-OTase), is a virulence trait in some bacteria including Ab ATCC 17978.
In silico analysis revealed the ubiquity of a putative O-OTase in Ab strains. We identified a cluster of genes including glycosyltransferases, a flippase, and sugar modifying enzymes predicted to synthesize the lipid-linked glycan. Organization of the sugar synthesis locus and position adjacent to capsule export genes is conserved, varying only in sugar synthesis enzymes. Several Ab sugar synthesis loci contain enzymes for synthesis of a negatively charged sugar including glucuronic acid or legionaminic acid. We used in silico analysis and Mass Spectrometry analysis to evaluate glycan variability in Ab strains including ATCC 17978, ATCC 19606, SDF and clinical isolates. Further studies will illuminate the extent of glycan diversity and whether glycan diversity can be utilized in diagnostics or glycoconjugate vaccine design for identification and treatment of Ab.
23-M. Identification of a specific RNA regulon of CrhR RNA helicase
Denise Whitford, M.Sc.
RNA helicases play an important role in the cell, unwinding and annealing RNA secondary structures to either protect the RNA or make it available to other proteins. RNA helicases are involved in processes that include ribosome biogenesis, translation and RNA degradation. The RNA molecules that associate with the helicase during these process can be either specific, as in the case of ribosome biogenesis, or more general, such as during translation. In the cyanobacterium Synechocystis sp. PCC 6803, the physiological role of its RNA helicase, CrhR, is unknown. Here we describe a methodology to identify RNA molecules that interact with CrhR, with the goal of identifying a role for this helicase. We propose to extract associated RNA molecules from purified CrhR protein, which will be sequenced on the Iontorrent platform. These results will be confirmed though EMSA RNA helicases play an important role in the cell, unwinding and annealing RNA secondary structures to either protect the RNA or make it available to other proteins. RNA helicases have been shown to be involved in processes that include ribosome biogenesis, translation and RNA degradation. The RNA molecules that associate with the helicase during these process can be either specific, as in the case of ribosome biogenesis, or more general, such as during translation. In the cyanobacteria Synechocystis sp. PCC 6803, the physiological role of its RNA helicase, CrhR, is unknown. Here we describe a methodology to identify RNA molecules that interact with CrhR, with the goal of identifying a role for this helicase. We propose to extract associated RNA molecules from purified CrhR protein, which will be sequenced on the Iontorrent platform. These results will be confirmed though EMSA assays using CrhR protein. Early data supports prior observations that CrhR protein interacts with crhR mRNA. using CrhR protein. Early data supports prior observations that CrhR protein interacts with crhR mRNA, perhaps as part of a feedback loop.
24-M. Development of a high-throughput assay for the identification of bacteriophage receptor binding proteins
Jessica Sacher, M.Sc.
Bacteriophages, or bacterial viruses, specifically target their bacterial hosts through receptor binding proteins (RBPs). The Campylobacter jejuni phage NCTC 12673 RBP has been used previously to demonstrate that phage RBPs can be effectively employed as bacterial diagnostics. However, in spite of the rapid progress in DNA sequencing technologies, phage genome sequencing remains a challenge. Furthermore, phage RBPs are difficult to identify through homology due to their wide ranges of host receptors. Thus, an alternative means of identifying phage RBPs is necessary in order to further exploit RBP-based technologies. We are developing a high-throughput method to screen for putative RBP genes in a phage genome. We expressed a C. jejuni phage RBP in Escherichia coli, immobilized cell lysates on a synthetic membrane, probed with intact C. jejuni cells and examined growth of bound colonies. Our preliminary results indicate that this method can be successfully employed to detect E. coli colonies expressing recombinant RBPs. We are now extending this methodology to examine total expression libraries of NCTC 12673 phage genomic DNA. Further progress in this direction would result in a rapid and inexpensive means of RBP identification, providing access to an untapped natural source of bacterial diagnostic tools.
VI. Physiology and Cell Biology
| 25-M. Examination of the biochemical signaling cascades induced by channel catfish leukocyte immune-type receptors |
Myron Zwozdesky, M.Sc.
Channel catfish (Ictalurus punctatus) is a Teleost family member, whose immune system contains a variety of immunoregulatory receptor types, including the leukocyte immune-type receptors (LITRs). As previously demonstrated by our lab, these receptors are capable of regulating immune cell effector responses via different intracellular signaling pathways. For example, LITR 2.6b, a stimulatory receptor, can induce degranulation and phagocytosis while LITR 1.1b, an inhibitory receptor, abrogates natural killer cell-mediated killing responses. In this study, LITRs 2.6b and 1.1b were transfected and stably expressed in rat basophilic leukaemia cells. This system was employed to elucidate the recruitment capabilities of the different receptors by examining their interactions with various intracellular signaling adaptor proteins to provide a mechanistic basis for their differential evocation of effector responses. Recently, LITR 1.1b was shown to induce phagocytosis in this model system at levels higher than 2.6b, even at low temperatures. Co-immunoprecipitation and Western blotting experiments demonstrate that such observed differences in effector function between receptor constructs may be traced, in part, to differential recruitment of phosphoinositide 3-kinase p85 and potentially Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1), SHP-2 and/or C-Src tyrosine kinase (CSK). Evaluation of candidate adaptor protein signaling cascades is necessary to provide an understanding of the myriad capabilities of channel catfish LITRs.
26-P. Integration of PI3K signal transduction in the control of GnRH actions using the goldfish (Carassius auratus) pituitary cell model system
Joshua G. Pemberton, Ph.D.
In vertebrates, multiple gonadotropin-releasing hormone (GnRH) forms exist in the brain and peripheral tissues but how GnRH isoform-specific actions are manifested is not well studied. Among its many functions, GnRH stimulates luteinizing hormone (LH) and growth hormone release (GH). Goldfish pituitary cells are exposed to two GnRHs, GnRH-2 and GnRH-3, and cell-type- and GnRH-isoform-specific signalling exists in this system. We recently showed that phosphoinositide 3-kinase (PI3K) mediates both GnRH-2 and GnRH-3 stimulation of goldfish LH and GH release, a novel finding in any pituitary cell system. How PI3K interacts with known mediators of GnRH action, including protein kinase C (PKC), is unknown. Using goldfish pituitary cells, we examined whether and how PI3K interacts with PKC and two common PI3K targets, phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (Akt), in GnRH-2 and -3 control of LH and GH release. Overall, our results demonstrate important cell-type- and GnRH-isoform-selective differences in the involvement of PI3K signalling to control pituitary cell functions. These findings also have wider implications for understanding GnRH actions in extra-pituitary targets, including immune and cancer cells. (Supported by NSERC, Alberta Innovates Health Solutions, and the Killam Trusts)
27-M. Expression of goldfish (Carassius auratus L.) acute phase proteins following Trypanosoma carassii infection
Nikolina Kovacevic, M.Sc.
The acute phase response is a rapid physiological reaction to infection, injury or trauma. Acute phase proteins are synthesized in the liver and play a crucial role in immune processes and pathogen elimination. Transferrin, ceruloplasmin and warm temperature acclimation protein 65 (WAP65) are acute phase proteins involved in iron homeostasis, in addition to immune function. Trypanosoma carassii is a protozoan, extracellular parasite infecting economically important fish. In aquaculture settings, increased prevalence can result in significant disease and mortality. In the current study, goldfish (Carassius auratus L.) will be infected with low (6.25X106) or high (1X107) doses of T. carassii to ascertain the acute phase response. Goldfish transferrin, ceruloplasmin and WAP65 will be cloned and sequenced. mRNA levels of will be assessed in blood and body tissues during acute phase of infection (days 3, 7, 14 and 21).
28-B. Influence of insect hair patterning on drop shedding
Delyle Polet, 499
Tapered hair-like structures on insect cuticles can shed water droplets unidirectionally. However, no studies have examined the effect of hair patterning and orientation on two-dimensional drop movement across a wing surface. Two-dimensional control of drop movement may be important for efficiently shedding droplets from the wing. March flies (Penthetria heteroptera) display a dense array of hairs on their wings with locally varied orientation. Drops of water (5-10µL) were placed on the wing and filmed using high-speed video. Their trajectories were mapped and compared to local microstructure. Preliminary results suggest that drops placed at different positions on the wing follow different trajectories, and that these trajectories seem to be associated with local hair orientation and the presence of larger structures such as veins.
VI. Plant Biology
| 29-B. Functional characterization of white spruce CHITINASES |
Jeremy Morris, 499
Woody perennials, such as the forest tree white spruce, are challenged on a seasonal basis by exposure to growth-limiting conditions and, consequently, must temporarily halt growth and most physiological processes. The transition from active growth to the over-wintering state is mediated by changes which occur at the cellular and biochemical level. Previously, an integrated transcriptomic and proteomic study showed that members of the CHITINASE family are differentially expressed in white spruce during this transition. Plant CHITINASES are well known to act in defense against pathogens, however, they have also been associated with signaling, cell wall remodeling, antifreeze and storage functions. The long term objective of this work is to functionally characterize the roles that individual white spruce CHITINASES play during the transition to an overwintering state. I will present fine-scale expression profiles for CHITINASES during this transition, as well as preliminary results concerning heterologous CHITINASE overexpression and sub-cellular localization studies.
30-M. Functional Characterization of Putative Regulators of White Spruce Bud Formation and Dormancy
Amanda Gregoris, M.Sc.
White spruce initiate dormancy by terminating cell proliferation at meristems, which is accompanied by development of a terminal bud. Bud formation involves many developmental processes that occur at the shoot apical meristem, and shares similarities with the transition from vegetative to flowering growth. MADS-box genes encode transcription factors, which play crucial roles in plant development, including regulating the transition from vegetative to reproductive growth. Using phylogenetic and microarray analysis we have identified candidate white spruce MADS-box genes involved in dormancy regulation. In order to determine if these genes have the ability to influence bud formation, we will (1) carry out qRT-PCR to quantify gene expression at different stages of the annual growth cycle, and in response to photoperiod and temperature changes, (2) perform complementation assays using Arabidopsis mutants for these genes to determine if Picea glauca promoters and coding sequences can restore a wildtype phenotype, and (3) conduct in situ hybridization to determine the fine scale spatial and temporal expression patterns of these genes. Through these experiments we hope to uncover genes underlying the molecular basis of bud formation and dormancy regulation in white spruce.
VII. Systematics and Evolution
| 31-B. Pleistocene mustelids of Cathedral Cave, Nevada and their significance in the Great Basin |
Shannon Kraichy, 498
In the arid Great Basin, caves offer glimpses into the changing climate and faunal assemblages of the Pleistocene. Cathedral Cave sits at an elevation of 1950 m, has a maximum age of between 146 and 151 ka, and has produced some surprising fossil remains (e.g., Osteichthyes). This project attempts to identify the mustelids (weasels and their kin) from the 2003 excavation in Room 2, with plans to continue to describe all carnivoran fossils. Many Pleistocene species are larger than their living members, and this has created problem in the literature. Does size difference alone warrant the naming of a separate species in the fossil record? In particular, the extinct noble marten (Martes nobilis) has been considered a larger form of the extant pine marten (Martes americana), and this study may provide insight into the issue. Identifications are being made through comparison to modern material and published descriptions, while minimizing geographic assumptions.
32-P. Polar Bear Genetics
Rene Malenfant, Ph.D.
33-B. A turtle-like dinosaur jaw, and its significance for the diet of “egg thieves”
Greg Funston, 499
The oviraptorosaurs were an enigmatic group of toothless dinosaurs from the Cretaceous period of Asia and North America. First known from a specimen sitting on a nest of eggs, they were named the egg thieves. Recent work has shown these eggs to belong to Oviraptor, revealing that it was protecting the eggs, not stealing them. As such, the diet of oviraptorosaurs is currently unknown. One group, the caenagnathids, is especially mysterious. This study compares a well-preserved Caenagnathus mandible with those of turtles, which are similar, to better understand the diet of the group. Three-dimensional computer models of both jaws were created, and simulations were run to determine their stress-handling capabilities. Comparative osteology reveals that Caenagnathus may have been adapted for a quick snapping bite, with an effective shearing ‘beak’. Therefore, Caenagnathus probably would have been herbivorous, but likely retained a degree of omnivory in the diet.
34-P. Growth and Development of varices – How do snails control shell decorations?
Nicole Webster, Ph.D.
Gastropods (snails) produce some of the most beautiful skeletons of any animal, often withcomplex ornamentation in the form of spines and spiral or axial ribs. Strong ornamentation is thought to reduce predation vulnerability, as well as being implicated in other functions, such as stabilizing the shell, feeding, and sensory functions. How snails control the growth and spacing of shell ornamentation is a significant unsolved puzzle of gastropod biology. Developmental mechanisms controlling shell ornamentation have never been studied experimentally. Here I studied the growth of varices, periodic blade-like thickenings of the shell lip, in two predatory marine snails, Ceratostoma foliatum and Nucella lamellosa (Caenogastropoda: Muricidae:Ocenebrinae). Ceratostoma foliatum has regular, synchronized varices with 120° spacing, and N. lamellosa has irregularly spaced varices with 10-50° between them. As a shell grows, previous varices on the body whorl are believed to stimulate the formation of future varices at the proper location in species with constant varix spacing, but this has not been tested. Body whorl varices and/or the apertural varix were removed to determine how their absence affected future varix growth in both species. Nucella lamellosa varices appear to weakly affected by previous varix locations; however in C. foliatum the location of previous varices appears to stimulate the growth of new varices. These results support our prediction: irregular varices may not have specific controls for placement, but regular varices at least partially require information from previous varices to maintain proper spacing.
35-M. Genetic structure and diversity of glass sponge reefs in the Pacific Northwest
Rachel Brown, M.Sc.
Glass sponge reefs provide important habitat to many organisms off the coast of BC, but little is known about their reproduction or genetic fitness. My aim is to determine the distribution and relationship of the reef-building glass sponge Aphrocallistes vastus within and between reefs. Glass sponge larvae are nearly impossible to obtain for reef-building species due to the depth of the reefs, scarcity of larvae and unknown breeding season. Because of this, I will examine their reproductive strategies, the genetic diversity of populations, and their gene flow using a combination of a Genotyping by Sequencing approach on the Ion Torrent PGM and histological observations of embryonic development. This will provide insight into management requirements and lead to predictive models of genetic diversity in other glass sponge species.
36-M. Arctic Grayling Diversity in a Disturbed Landscape
Jessica Reilly, M.Sc.
Arctic grayling (Thymallus arcticus) are cold-water, salt-water intolerant salmonids with a holarctic distribution including most of northern Canada. In Alberta, the abundance of Arctic grayling has been steadily declining since the 1950s and the species is now provincially classified as a Species of Special Concern. Although the suspected factors behind the Arctic grayling decline (e.g., overharvest, reduced habitat quality) have been documented, no genetic studies of Alberta Arctic grayling have been completed. To investigate population structure and genetic diversity, tissue has been collected from approximately 750 Arctic grayling from 24 rivers in the Hay River, Peace River and Athabasca River watersheds. We will develop and use microsatellite markers to investigate broad and fine scale population structure. It is expected that populations will be distinct between watersheds, and that structure within subwatersheds will represent a spatially nested hierarchy of sub-populations linked by infrequent gene flow. Subpopulations are likely because of varying degrees of isolation resulting from barriers, distance between subpopulations, and spawning site fidelity. We also expect genetic diversity in Arctic grayling populations to decrease in a south-eastern direction because these populations are the furthest from putative, northern refugia. In the future, we plan to use these data to investigate correlations between contemporary landscape features and genetic diversity of Arctic grayling subpopulations.
37-B. Differential Morphology of the Caudate Appendicular Skeleton Across Taxa
Aurora Jansen, 499
Members of the amphibian order Caudata inhabit numerous types of environments; terrestrial, aquatic, even arboreal. With the exception of Sirenidae and Amphiumidae, a well-developed appendicular skeleton helps caudates move throughout these habitats. The morphology of this appendicular skeleton has evolved in response to the specific lifestyle of each caudate. Or has it? In many land-dwelling caudate clades, the forelimbs and pectoral girdle play a major role in audition by transmitting seismic vibrations to the inner ear. The anterior appendicular skeleton’s function as a sound-transmission board may elicit a developmental constraint on possible morphologies of its elements. These elements must maintain a specific morphology to remain function in their roles of both locomotion and audition. In this study, I am qualitatively and quantitatively comparing the ossification patterns of the appendicular skeletons of seven caudate families. I predicted that due to the constraint in terrestrial caudates, the anterior appendicular skeletons of ecologically- and phylogenetically-distinct taxa would show little variation in terrestrial taxa and high variation in fully-aquatic taxa. In contrast, the posterior appendicular skeleton would show high variation across all taxa since its only use is locomotion. Posterior appendicular skeleton morphology would follow a phylogenetic trend, with modifications made by environmental pressures.