The 41st Annual BIO481Y5

Symposium

March 29, 2019 &

April 5, 2019 UTM Room

8:55am to 3pm

Reception April 5, 2019 UTM Room

3pm

BIO481 – March 29, 2019 – Abstracts and Presentations Session I – 9:00 am to 12:00 pm – Moderator: Rob Ness 9:00 am Amy Dao (Bailey McMeans) 9:15 am Shanza Ayub (Rob Ness) 9:30 am Jenna Black (Steven Short) 9:45 am Eric Yung (Shannon McCauley/Bailey McMeans) 10:00 am Eniolaye Balogun (Robert Ness)

Break 10:30 am Samuel Fung (Mary Cheng) 10:45 am Stuti Joshi (Angela Lange) 11:00 am Tommy Lin Weng (Robert Ness) 11:15 am Safa Ahmad (Mary Cheng) 11:30 am Trevor Schmahl (Rob Ness/James Santangelo) 11:45 am Kayvan Aflaki (Angela Lange)

Session II – 1:00 pm to 3:00pm – Moderator: Mike Phillips 1:00 pm Kerman Sekhon (Jayson Parker) 1:15 pm Ibadat Bajwa (Mike Phillips) 1:30 pm Jona Gjevori (Helene Wagner) 1:45 pm Andrew Crozier (Sanja Hinic-Frlog) 2:00 pm Ravneet Jaura (Ho Sung Rhee) 2:15 pm Madina Nouren (Glenn Morris) 2:30 pm Ahmed Hanif (Jodie Jenkinson) 2:45 pm Anmol Chahal (Mike Phillips)

BIO481 – April 5, 2019 – Abstracts and Presentations Session I – 9:00 am to 10:05 am – Moderator: Robert Gerlai 9:00 am Agatha Tymczak (Voula Kanelis) 9:15 am Mario Filice (Tim Westwood) 9:30 am Khadija Lakdawala (Joshua Milstein/Emilius Visser) 9:45 am Ishti Paul, Zelaikha Najmi and Amira Abozaid (Robert Gerlai) Session II – 10:15 am to 12:00 pm – Moderator: Bryan Stewart 10:15 am Eun-Kyung Jung (Jumi Shin) 10:30 am Arif Sarowar (Bryan Stewart) 10:45 am Ifeoluwa Egbeniran (Tim Westwood) 11:00 am Anuj Kaushal (Bryan Stewart) 11:15 am Zaleena Akheralie (Ted Erclik) 11:30 am Denia Hurst (Tim Westwood) 11:45 am Gill Onate (Ted Erclik)

Session III – 1:00 pm to 3:00pm – Moderator: Baohua Liu 1:00 pm Fagun Kaushik (Joel Levine) 1:15 pm Celine Bailleul and Samuel Nguyen (Robert Gerlai) 1:35 pm Omar Alhalabi (Baohua Liu) 1:50 pm Ramsha Awan (Baohua Liu) 2:05 pm Kyshona Edwards (Baohua Liu) 2:20 pm Sigi Maho (Robert Reisz/Diane Scott)

BIO481 – March 29, 2019 – Abstracts and Presentations

Session I – 9:00 am to 12:00 pm – Moderator: Rob Ness

9:00 am Amy Dao (Bailey McMeans) Seasonal Plasticity in the Gut of Salvelinus namaycush and Micropterus dolomieu The digestive tract is one of the body’s more expensive systems to maintain, due to its high energy requirements. In order to compensate, many organisms have shown plasticity in their digestive tract in response to changing environments. In particular, the literature has suggested that many fish will downregulate their digestive tract during fasting, which results in lower intestinal length, intestine wet mass and an overall lower maintenance cost. In natural lakes, fish are subjected to varying levels of resources and environmental conditions during each season. Very few studies have observed fish’s activity levels and foraging behaviour during the winter. During the winter there is a decrease in temperature, sunlight and productivity, which limits food availability. Therefore, we predicted a decrease in the size of the digestive tract during the winter. If the variation in food abundance is predictable throughout the seasons, then the fish should show plasticity in their gut size to converse the energy that’s associated with maintaining a large digestive tract. In this study we will be observing a cold water species, Salvelinus namaycush and a warm water species, Micropterus dolomieu over four seasons between the years of 2017-2018. Samples were dissected, and their intestine length was measured. Based on the findings of this study, it will help furthermore our understanding of fish behaviour in the winter and how they respond to their changing environment.

9:15 am Shanza Ayub (Rob Ness) The Genomic Landscape of Polymorphic Transposable Elements in the Chlamydomonas reinhardtii Genome

Transposable elements (TEs) are a class of repetitive DNA elements that have the ability to move around in the genome. When TEs are copied or excised from the genome, they introduce new mutations, and have been linked to disease, adaptations and changes in overall genome size and structure. Most of our knowledge of TEs has come from studies comparing the genome of related species, and how TE insertions impact the overall fitness of an individual. However, little is known about the selective pressures acting on TEs themselves. When TEs arise, they are polymorphic until they are either purged or fixed in the population. The frequencies of polymorphic TEs can provide insights into the evolutionary force that influence the fixation of TEs. In our study, we used bioinformatics tools and novel TE calling algorithms to call TE variants in 27 Chlamydomonas reinhardtii genomes. We then classified those calls based on their location and genetic annotation in the genome. We found that each strain had an average of 450 insertions, and most of them were rare in the population. TEs also tended to be rarer in genic regions, while the ones found in intergenic regions were shared by >5 strains in the population. These results demonstrate differences of TE dynamics in different strains, and provide insights into how TEs arise in a population, and how they might be removed or fixed in the population.

9:30 am Jenna Black (Steven Short) The Dynamics of Viruses and Virophages that Infect C. parva, a Freshwater Algae

The freshwater haptophyte Crysochromulina parva was first isolated from water samples of Lake Ontario. This primary producer is infected by the virus CpV, a giant virus of which there are two versions: CpV-BQ1 and BQ2. Genome sequences also point to the existence of three versions of a virophage known as CpVV Larry, Curly and Moe which likely infect CpV during infection of C. parva. This research aims to confirm the existence and compare the relative abundance of these five viral particles. First, viral concentrates of lysates were hot/cold treated prior to testing. PCR primers were designed to confirm the presence of the viruses and virophages by amplifying marker genes. QPCR primers and probes were also designed to examine the spatial and temporal variation in relative abundance of the viruses and virophages. All viral agents were confirmed to be present in lysates. CpV-BQ1 has a wider range than BQ2 both spatially and temporally, potentially indicating that BQ1 has a second host. The abundance of CpVV Larry and Moe are more closely correlated to CpV-BQ2, while Curly has no significant relation to either virus. A C. parva qPCR assay is in the validation stages and will be used to determine the abundance of the host. After validation, the relationships of this important primary producer will be further elucidated.

9:45 am Eric Yung (Shannon McCauley/Bailey McMeans) Investigating Implications of Sublethal Road Salt (NaCl) Concentrations on Freshwater Trichopteran Sugar Maple Leaf Processing Rates Road salt application during winter season is typically used in ice removal. However, much of this salt run offs into nearby waterways due to impermeable road surfaces. The Trichoptera (caddisflies) from a group of taxa called “EPT” serve as bioindicators of stream health. Much is known about salt tolerance ranges of EPT but knowledge gaps remain regarding their sublethal effects. Specifically, leaf processing rates were investigated in these organisms. Leaf loss was measured by allocating caddisflies to varying concentrations of road salt. Observations were additionally made to gauge survival status, overall leaf consumption, and activity levels. A Kruskal-Wallis test measuring extent of removed leaf area over days survived (cm2/days alive) presented insignificant results. Observations however suggest that when caddisflies are subjected to higher salt concentrations, there may be greater allocation of metabolic resources towards survival at the cost of their ability to carry out basic functions such as leaf scavenging. Understanding whether or not these observations still apply under winter temperatures (i.e. 4˚C) and how road salt can indirectly influence other life stages such as reproduction (e.g. clutch size, number of offspring) will be critical in advancing our knowledge in the sublethal implications of road salt.

10:00 am Eniolaye Balogun (Robert Ness) Is recombination mutagenic: evidence from whole genome sequencing of Chlamydomonas reinhardtii tetrads

It is commonly hypothesized that recombination may be mutagenic, but the evidence behind this theory has been tentative. Various papers since the 1960s have shown evidence of a higher mutation rate in meiosis as opposed to mitosis. In yeast experiments, a higher frequency of mutations surrounding crossover sites was observed, which supported the theory that the breakdown of the DSB DNA repair system was responsible for the disparity in meiotic and mitotic mutation rates. In our study, we will test for a positive correlation between recombination and de novo mutations in the eukaryotic unicellular algae Chlamydomonas reinhardtii. We use whole-genome sequence mutation accumulation from 108 individuals representing 27 tetrads derived from 3 parental crosses. We discovered, on average, approximately 100 novel mutations per parental cross establishing a higher meiotic mutation rate than the previously documented mitotic mutation rate of 11.5 x 10-10 /site/generation. We also find a significant association between the locations of mutations and crossover break points. Our results support the idea that recombination is mutagenic and may be a driver of patterns of sequence variation that drive evolution. In addition we tested whether the de novo mutations we identified tended to be more common surrounding the position of recombination breakpoints. Our results will provide valuable insights into the influence that recombination and meiosis have on the generation of genetic variation.

Break

10:30 am Samuel Fung (Mary Cheng) Abstract attached to the BIO481 Program

10:45 am Stuti Joshi (Angela Lange) Investigating the Role of Myoinhibiting Peptides in Feeding of the kissing bug, Rhodnius prolixus

The progression of the Chagas disease vector, Rhodnius prolixus, through the various developmental stages and reproductive maturity, is governed by the consumption of large blood meals. In part, salivary secretions provide the ability to consume large volumes of blood during feeding by ensuring continuous blood flow. Previous studies on R. prolixus have shown myoinhibiting peptides (MIPs), a family of inhibitory neuropeptides, in neurons in the central nervous system (CNS), and in processes on the salivary glands/ducts, and also on the female oviducts and the male accessory glands. The present study was conducted to study the role of MIPs in feeding and reproduction. Contraction assays on the dorsal vessel (heart) showed that MIPs lead to a dose-dependent decrease in the frequency of the heartbeat and that MIPs could modulate contractions produced by stimulatory peptides. Using quantitative PCR, MIP receptor transcript levels were observed to increase in the CNS after feeding, indicating a possible role of MIPs in satiation. Immunohistochemistry on the CNS was also performed to study MIP expression during the fifth instar and adult stage, and revealed differences in the neurons containing MIP-like staining that may be attributed to reproductive maturation. This data suggests that MIPs play a role in regulating feeding and reproduction.

11:00 am Tommy Lin Weng (Robert Ness) Effect of cold stress on neutral lipid accumulation of Chlamydomonas reinhardtii

Microalgae have recently garnered a lot of attention because of their ability to produce lipids that are easily converted to biofuels. Triacylglyceride (TAG) is a neutral lipid that can be converted into fatty acid methyl esters (FAME) through a transesterification process to produce biodiesel. TAG is produced and accumulated in lipid bodies in algae under many conditions. Previous studies have found multiple ways to induce neutral lipid accumulation in Chlamydomonas reinhardtii and other algae species, such as exposing the algae to a N-deficient media, high salinity, increasing temperatures, photoperiod changes, etc. However, the effect of cold stress on C. reinhardtii’s have not been investigated yet. In our study, C. reinhardtii cultures of five strains were exposed to stressful temperatures of 4°C with and without light. The amount of neutral lipid of C. reinhardtii cold cultures were compared to cultures kept at 25°C with and without light. The amount of neutral lipid per cell was determined by staining the algae’s neutral lipids with Nile Red and measuring fluorescence of individual cells with a flow cytometer. We explore fluorescence patterns to test for effects of cold stress and whether individual strains responses to cold are related to the latitude from where the strains were sampled.

11:15 am Safa Ahmad (Mary Cheng) Abstract attached to the BIO481 Program

11:30 am Trevor Schmahl (Rob Ness/James Santangelo) Estimating Effective Population Size – Using Population Genetic Parameters Theta & Rho One of the most important measures of a population in evolutionary genetics is the effective population size (𝑁𝑁𝑒𝑒). A concept introduced by Sewall Wright, 𝑁𝑁𝑒𝑒 informs you of the degree to which a populations genetic composition is being influenced by genetic drift, as well the extent to which inbreeding is occurring in a population. The lower the value of 𝑁𝑁𝑒𝑒, the greater the degree to which drift is said to be operating, or inbreeding occurring. There are various ways to estimate the value of 𝑁𝑁𝑒𝑒. Two of the most common parameters, and the ones we investigate in this study, are theta (𝜃𝜃 = 4𝑁𝑁𝑒𝑒𝜇𝜇) the population scaled mutation rate, and rho (𝜌𝜌 = 4𝑁𝑁𝑒𝑒𝑟𝑟) the population scaled mutation rate. Once theta and rho are obtained from sequence data, a simple relation makes it convenient to estimate 𝑁𝑁𝑒𝑒 as 𝜃𝜃/4𝜇𝜇, or 𝜌𝜌/4𝑟𝑟. One is tempted to assume these values of 𝑁𝑁𝑒𝑒 should be equivalent. If a population is at equilibrium, there is a basis for this assumption, however in the real world, where populations are rarely (if ever) in equilibrium, this assumption may not be very reliable. The existence of this real potential for rho and theta to be significantly out of agreeance with each other in their estimate of 𝑁𝑁𝑒𝑒 when a population is in disequilibrium, is the primary focus of this paper. To investigate this, we used an evolutionary simulation package “SLiM” (Selection on Linked Mutations) which is a general purpose forward genetic simulation framework. A simple neutral model of a Wright-Fisher population which experiences a bottleneck after which returns to its original population size was simulated. Once equilibrium is reached, the bottleneck disturbs the equilibrium state. Once the population recovers to its original size, theta and rho could follow “different paths” to recovery, before the population reaches equilibrium again. The simulation was run at varying population sizes, with varying bottleneck strengths. Output from each simulation, before, during, and after the bottleneck was used to generate the allele Site Frequency Spectrum (SFS) from which theta was calculated, and 𝑁𝑁𝑒𝑒 estimated. Rho was estimated from output using LDhat software. Estimates of 𝑁𝑁𝑒𝑒 from both parameters were then compared to determine whether or not theta and rho agree in their estimates when a population is no longer in equilibrium.

11:45 am Kayvan Aflaki (Angela Lange) The Role of Octopamine and Tyramine in the Egg-Laying Behaviour of Adult Female Reproductive System of Rhodnius prolixus

Octopamine and tyramine are biogenic amines involved in diverse physiological processes. In insects, octopamine and tyramine act analogously to epinephrine and norepinephrine in vertebrates, binding to G protein-coupled receptors (GPCRs) leading to changes in second messenger levels and thereby modifying the function in target tissues and insect behavior. In a previous study, the role of octopamine and tyramine in regulating contractions of reproductive tissues was examined in the female Rhodnius prolixus. Both octopamine and tyramine were found to be inhibitory on contractions of the oviducts and bursa. In other insects, this inhibition of contractions of the reproductive tissue was found to decrease the passage of eggs during egg-laying leading to sterile insects. In this project, the effects of both amines will be examined on the ability of mated adult female R. prolixus to egg-lay.

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Session II – 1:00 pm to 3:00pm – Moderator: Mike Phillips

1:00 pm Kerman Sekhon (Jayson Parker) The Association Between Activity During Sleep, Breakfast Composition and Workout Condition

There is a lack of research considering the association between activity during sleep, breakfast composition and physical activity. Previously, the association between high blood sugar and sleep disorders involving larger and more rapid movements, such as periodic limb movements (PLMs) during sleep has been considered. However, research regarding differential activity during sleep in younger populations (sleep quality) and breakfast composition (sugar-based, baseline, no-breakfast) is lacking. This is an exploratory, case-study, considering one participant. Data was collected during 12 different 36-hour periods using the Hexoskin, a wearable shirt that collects biometric data. Six different conditions were measured using all possible combinations of the following variables: ‘Breakfast’, ‘No Breakfast’ and ‘Sugar-Breakfast’ with ‘Workout’ and ‘No Workout’. Data analysis consisted of the 2nd sleep cycle (1:40 minutes into sleep and 1:40 minutes long) being analyzed in comparison with activity during sleep from the night before. It was seen that ‘Sugar Breakfast’ with ‘Workout’ had an association as compared to ‘No Workout’. In the future, machine learning should be used to predict lifestyle outcomes.

1:15 pm Ibadat Bajwa (Mike Phillips) Untargeted metabolomics analysis of isotopically labeled Arabidopsis thaliana using chemical and electron ionization mass spectrometry Carbon is an essential element used by plants. At the metabolome level, carbon is a vital element of all plant primary metabolism. Here we explore an untargeted chemical ionization gas chromatography-mass spectrometry (CI-GCMS) method to monitor variation in metabolism in Arabidopsis thaliana rosettes subjected to whole plant 13CO2 labeling. We have enlisted a standard procedure for preparing metabolic extracts of Arabidopsis thaliana leaf tissue for GCMS analysis using MeOX/TMS derivatization. However, we have optimized the data acquisition phase to also capture information on the kinetic rates of central metabolic pathways, as judged by the appearance of 13C label in downstream metabolite pools. To do this, we employed ammonia CI-GCMS in positive mode as an analytical tool to

simplify the calculation of label incorporation. This unbiased approach allows for the simultaneous relative quantification of hundreds of metabolites as well as turnover rates of about a dozen metabolites showing label incorporation in short term labeling experiments (≤ 2h). This quantified data was compared to the total 13C pool of the plant acquired via isotope-ratio mass spectrometry, to calculate the ultimate allocation of carbon in a given metabolite and hence the metabolic cycle.

1:30 pm Jona Gjevori (Helene Wagner) Potential evidence supporting Halictus confusus as an obligately social species found at its northernmost range in Alberta

Halictus confusus is a sweat bee commonly found in North America and classified as eusocial; however, detailed social organization information is lacking at its northernmost ranges. Sweat bees show varying degrees of sociality; some species are obligates while others are able to use both social and solitary strategies as a plastic response to environmental variation. Understanding sociality can reveal how sweat bees respond to climate change. We investigated the sociality of Halictus confusus in Alberta at a Southern and Northern habitat using physiological measurements and dissections from pan trap samples in 2015. The South site is typical of temperate conditions, while the North site represents a harsher climate. If this species is socially polymorphic, we would expect to see a lack of a worker brood at the North site. If obligately social, these colonies are expected to show a stronger sociality compared to the South. Both sites showed a bimodal bee capture distribution over time. 2-way ANOVAs revealed Northern queens were larger on average compared to the South (n=12). The same was true for the respective worker broods (n=11) with statistically significant interactive effects of caste and region. Ovarian development of Northern queens was comparable to that of her workers, potentially due to disease, and did not support a reproductive skew towards queens. Taken together, these results could suggest some evidence for Halictus confusus as obligately eusocial; however, further analysis of additional Northern sites are required to confirm.

1:45 pm Andrew Crozier (Sanja Hinic-Frlog) Problem-Solving in Black-Capped Chickadees, Poecile atricapillus, across an Urbanization Gradient along the Credit River of Mississauga, Ontario

Birds exhibit various morphological, physiological, and behavioural responses to urbanization. To date, most work related to urbanization and avian problem-solving have focused on laboratory trials of captive individuals. We instead conducted an observational field study where we assessed problem-solving of black-capped chickadees, Poecile atricapillus, across an urbanization gradient. If urbanization affects problem-solving in chickadees, we expect individuals in urban areas to solve more problems or solve problems more quickly than individuals in non-urban areas. We conducted field observations in Terra Cotta, Brampton, and Mississauga, Ontario while exposing chickadees to a novel foraging innovation task. This innovation task consisted of an inverted test-tube blocked by a cotton ball, filled with seeds, and clamped to a bird feeder pole. We observed individuals at each location for a 30-minute period during which we measured problem-solving latency and number of problem-solving attempts. We documented the only single successful problem-solving event at one of the urban sites. Furthermore, our preliminary data indicate that chickadee populations at urban sites show a trend of more problem-solving attempts than the chickadees at the non-urban sites.

2:00 pm Ravneet Jaura (Ho Sung Rhee) Understanding the Role of Intergenic DNA Region of Hb9 Gene during Spinal Motor Neuron Differentiation

During the development of motor neurons, embryonic stem (ES) cells differentiate to spinal motor neurons by the activation of a unique combination of transcription factors. The Hb9 gene is the selective marker for the identity of post-mitotic spinal motor neurons. The previous research with Hb9 knockout mice has shown disruptions in the formation of spinal motor neurons. It has yet to be understood if modifications occurred in the intergenic regions of the genome, how would the chromatin organize and stabilize during differentiation, thereby the effect on gene expression. To study the effect on Hb9 gene expression from a change in chromatin organization, CRISPR-Cas9 was utilized to delete a 2.25 kb of the upstream intergenic region of Hb9 gene in motor neuron differentiation system using mouse ES cells. It was hypothesized with this deletion mutant, there would be downregulation in Hb9 gene expression in spinal motor neurons. Low efficiency of CRISPR was observed as out of 56 colonies, a single colony had the homozygous deletion of 2.25 kb intergenic region. After differentiation of these mutant ES cells to spinal motor neurons, real-time polymerase chain reaction results showed 35% of downregulation in Hb9 gene expression in nascent motor neurons and 39% of downregulation in maturing motor neurons for the mutant ES cells as compared to the wildtype. These results indicate that the 2.25 kb intergenic region plays a functional role in Hb9 gene expression.

2:15 pm Madina Nouren (Glenn Morris) Examining the stridulatory mechanisms Katydid species Conocephalus brevipennis and Orchelimum nigripes

Male conocephaline katydids (Tettigoniidae) generate broadband calling sounds by vibrating two forewing cells (speculae) called the harp and mirror (Morris et al. 2016). The calling songs of two conocephaline species Orchelimum nigripes and Conocephalus brevipennis were analysed and are described here for the first time. As expected both species make their sounds with nonresonant rapid-decay pulses (op. cit.). The much smaller species C. brevipennis, produces more ultrasonics than the larger. Both species employ two forms of time-domain pulse-train structure: one is characteristic of ticks, one is characteristic of buzzes. The cantilever model for katydid sound-production (Morris and Pipher 1967) relates principal carrier frequency to the longitudinal diameter of the mirror wing cell: ignoring the possible effect of the harp in its relation to the main supporting wing vein, the radius. I tested whether adopting a cantilever length shifted in the direction of the harp would affect the relationship between carrier frequency plotted against length and projected through the origin.

2:30 pm Ahmed Hanif (Jodie Jenkinson) Addressing undergraduate student misconceptions on generation time in natural selection with an inquiry-based module.

Scientists and other science educators depict evolution to be the central theory of biology. Conversely, there are still a vast number of misconceptions held by undergraduate students regarding generation time and its effect on natural selection. Even after instruction students harbour misconceptions regarding generation time, proposing that traditional teaching methods may be ineffective in teaching concepts in evolutionary genetics. In this study we designed an interactive inquiry-based module that teaches the principle of generation time and its effect on the rate of evolution. We measured undergraduate student misconceptions and understanding of generation time using multiple choice and true and false tests before and after the administration of the module. The pre and post-test questions were divided into 3 categories; Direct knowledge, Indirect knowledge, and general knowledge relating to natural selection. While our inquiry-based module was very effective in remediating misconceptions regarding direct questions (70.5% of students achieved score of 80% or higher on these questions), we did not find evidence to support its efficacy in correcting misconceptions that were indirectly related to generation time and those relating more generally to natural selection. These results suggest that concepts that were directly addressed through the module were effective in remediating student misconceptions, whereas concepts that were indirect and unrelated to the module were not. This study indicates a strong potential of using an inquiry-based approach in teaching direct concepts in evolutionary genetics, further studies must investigate the effectiveness of inquiry-based learning on other biological concepts in order to support traditional teaching methods.

2:45 pm Anmol Chahal (Mike Phillips) Engineering Pyruvate Synthesis in the Chloroplasts of Arabidopsis thaliana

Phosphoenolpyruvate (PEP) is a high energy hub metabolite which supplies carbon for aromatic amino acid biosynthesis, phenylpropanoid biosynthesis, cytoplasmic and plastidic isoprenoid biosynthesis, fatty acid biosynthesis, and the shikimate pathway. PEP is supplied by glycolytic enzymes enolase (ENO) and phosphoglyceromutase (PGM). ENO and PGM are downregulated in green tissues, presumably to avoid conflicts with the Calvin-Benson cycle, necessitating their PEP import from the cytosol. To better understand PEP/pyruvate trafficking, we chose the cue1 mutant, which is deficient in the plastid PEP transporter, as a visual reporter to monitor attempts to engineer endogenous PEP/pyruvate synthesis in the chloroplast. This was achieved by constitutively expressing PGM or ENO in chloroplasts of transgenic plants in combination with separate herbicide resistance genes (basta or kanamycin). Genetic crosses of PGM+/+ and ENO+/+ singly transformed lines generated double transgenic lines expressing both activities in the chloroplast, a combination which we predicted would enable endogenous PEP/pyruvate production in this compartment. Double transformants resulting from genetic crosses were confirmed by growth of F1 progeny on double selection media containing basta and kanamycin. Furthermore, crosses of single or double transgenics with the cue1 mutant have been implemented and will provide the basis for complementation of the cue1 phenotype and evaluation of the effects of endogenous PEP/pyruvate production in the chloroplast.

BIO481 – April 5, 2019 – Abstracts and Presentations

Session I – 9:00 am to 10:05 am – Moderator: Robert Gerlai

9:00 am Agatha Tymczak (Voula Kanelis) Spectroscopically characterizing the regulatory region of yeast cadmium factor 1

The yeast cadmium factor, Ycf1p, transports glutathione-conjugate toxic metal ions such as cadmium from the cytosol into the vacuole. Ycf1p is a member of the ABC (ATP-binding cassette) superfamily of transporters and, as such, contains the core ABC structure of two membrane-spanning domains (MSD1, MSD2) and two nucleotide binding domains (NBD1, NBD2). Ycf1p also contains an additional membrane-spanning domain (MSD0) that is connected to the core ABC structure by a cytoplasmic L0 linker.

ATP binding and hydrolysis at the NBDs drives transports of solutes across biological membranes. In addition, ABC transporters are also regulated by phosphorylation. In the case of Ycf1p, phosphorylation of one site in the L0 linker inhibits transport. In contrast, phosphorylation of the intrinsically disordered regulatory region (RR), which connects NBD1 to MSD2, increases transport. This presentation shows the in vitro characterization of the RR by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies. These tools were used to determine whether or not phosphorylation alters the conformation of RR and its interactions with NBD1. Future studies with involve RR/NBD2 binding studies and involve obtaining NMR resonance assignments for RR so that phosphorylation-dependent changes in structure and binding can be analyzed at residue-level resolution. These studies promise to address how intrinsically disordered regions in ABC transporters regulate transport.

9:15 am Mario Filice (Tim Westwood) The Role of Drosophila Heat Shock Factor in the Regulation of Key Developmental Genes

Heat shock factor (HSF) acts as a master regulator of the heat shock response (HSR) during times of acute cellular stress, a mechanism that is conserved across all organisms. During heat shock, studies have shown HSF binds to developmentally regulated genes that are not involved in the HSR such as the early 20-hydroxyecdysone (20E) inducible genes (Eip74, Eip75, and Broad) that are responsible for metamorphic changes in arthropods.

Some of these HSF binding sites overlap with ecdysone receptor (EcR) binding sites, where EcRs are responsible for 20E activation. HSF binding to these sites during HS and/or non-shock conditions may alter EcR binding and/or affect early 20E inducible gene transcription.

In this project, HSF binding sites within early 20E inducible genes were identified using in silico analysis. The most biologically significant sites were selected based upon their proximity to 20E enhancers and EcR binding sites. Electrophoretic Mobility Shift Assays are being done to detect whether HSF binds to the predicted sites within 20E inducible genes. To determine the role of HSF binding in the regulation of this class of genes in vivo, HSF’s binding sites in the EiP74 gene will be mutated via CRISPR/Cas9 gene editing. The transcription of the Eip74 gene in the mutated Drosophila will be studied under HS and non-shock using quantitative reverse transcriptase PCR.

9:30 am Khadija Lakdawala (Joshua Milstein/Emilius Visser) Tethered Particle Motion as a tool for understanding transcription at the single molecule level

Tethered particle motion (TPM) is a single molecule technique used to examine conformational dynamics and interactions between biological molecules with a high degree of accuracy as compared to bulk experiments. It involves the tethering of one end of a DNA molecule to a glass slide, the other end to a polystyrene bead, and the subsequent measurement of the amplitude of the Brownian motion using bright-field microscopy. In this study, TPM was used to observe the transcriptional activity of T7 RNA polymerase. A calibration curve was constructed as a tool for verifying the tether lengths in the system by comparing the amplitude of motion to different DNA tether lengths. It was discovered that with increasing tether lengths, the amplitude of motion also increased. To observe transcriptional activity using the TPM system, DNA

tethers with the EGFP gene and the corresponding T7 promoter were constructed. The polymerase was immobilized on a glass surface, and the tethers were added to study the interaction between the polymerase and tethers. Due to an absence of bound tethers observed, an agarose gel electrophoresis method was developed to confirm and quantify enzymatic activity of T7 RNA polymerase. This study establishes a foundation for studying transcription kinetics at the single molecule level.

9:45 am Ishti Paul, Zelaikha Najmi and Amira Abozaid (Robert Gerlai) Developmental stage dependent effects of embryonic alcohol exposure on the behavior and morphology of zebrafish larvae: A strain comparison study.

Despite the known deleterious effects of alcohol, Fetal Alcohol Spectrum Disorder (FASD) remains prevalent worldwide because of alcohol consumption by pregnant women. The severity and symptoms of FASD show high variability across affected children. One possibility for this variance is consumption of alcohol at different stages of pregnancy. Another is the genotype of the fetus. Zebrafish allows precise dosing and timing of alcohol delivery during embryonic development. Here we investigated how embryonic alcohol administration affected behavior and anatomy in two different zebrafish strains, AB and WT. To examine the possible differential effects of timing of alcohol exposure during embryonic development, we immersed zebrafish embryos into 1% ethanol solution or freshwater (control) for 2 hours at 8, 16, 24, 32, or 40 hours post fertilization, respectively. Subsequently, we allowed the embryos to develop normally. At 6-8 days post-fertilization, we measured the behavior of each fish singly in a Petridish during a 30-minute session, quantifying their swim path parameters. We also measured basic anatomy features of their body and eyes. Data analysis is ongoing, but we already detected developmental stage and strain dependent alcohol effects in swim speed, turn angle and other swim path parameters, which we will report in our presentation.

Session II – 10:15 am to 12:00 pm – Moderator: Bryan Stewart

10:15 am Eun-Kyung Jung (Jumi Shin) Increasing stringency of ME47 by reducing gIII expression in vitro

Over 50% of all cancers are associated with deregulation of expression of the c-Myc gene. c-Myc’s activation domain confers the ability to upregulate gene expression toward regulation of cell metabolism and proliferation. Myc and Max are basic helix-loop-helix-zipper (bHLHZ) transcription factors that heterodimerize to bind the Enhancer sequence (E-box, 5’-CACGTG-3’). Deregulation of Myc/Max results in aberrant cell proliferation. Thus, blocking Myc/Max from binding to the E-box motif is an important target in human cancer cell treatment: we have used our designed protein ME47 to inhibit Myc/Max binding to the E-box. ME47 is a minimalist hybrid protein comprising the Max basic region with the E-47 HLH region. ME47 competitively inhibits c-Myc/Max binding to the E-box DNA, preventing abnormal regulation of downstream genes. ME47 can be evolved by directed evolution toward higher fitness under a specific selective pressure. We use PACE (phage assisted continuous evolution) for directed evolution. The goal of my project was to increase the efficiency of ME47 by lowering the gIII gene expression.

10:30 am Arif Sarowar (Bryan Stewart) Effect on neural development by specific Type Is motor neuron expression patterns in Drosophila melanogaster

The Drosophila melanogaster neuromuscular junction (NMJ) is an important structure for studying neurobiology and synaptic function. Drosophila contains two glumatergic motor neurons (MN), type Ib and Is that innervate at an NMJ. Using UAS-GAL4 techniques, we have utilized GMR27E09-GAL4, which expresses GAL4 under the control of FMR1 regulatory regions, to selectively drive in type Is MN, altering expression in one of the MNs but not the other. This novel approach allows us to examine the response of Ib MNs when Is MNs are manipulated through gene expression. In the presence of apoptotic expression, via UAS-hid, NMJs show Ib boutons/synapses without Is bouton presence in Is specific expression, while observed lethal in all neuron expression, via positive control elav-GAL4/C155 line. Using an overgrowth reporter line, via UAS-NSF2 E/Q, type 1s specific expression does not express overgrowth phenotype, characterized by long abnormal axons, showing suppression of overgrowth phenotype. In type Is overexpression in potassium channels, via UAS-KCN2, NMJs showed reduced branching, NMJ length, and lower levels of Is and Ib boutons, with no significant change in Ib bouton morphology and count. Overall this research project explores how neuron development is affected by manipulating in one of two motor neurons that innervate a common target muscle

10:45 am Ifeoluwa Egbeniran (Tim Westwood) The Role of Heat Shock Proteins in Aging and Lifespan Duration in Drosophila melanogaster Heat Shock Proteins (HSPs) have been extensively studied in Drosophila, mainly for their protective functions during and after stress and in their ability to make the organism thermotolerant. Using both segmental deletions and RNAi induced gene expression knockdowns, this present study examines the effects of reducing HSP copy number in adult fruit flies. Contrary to what has been observed in the past, we found that a reduction in levels of HSP70, 23 and 22 resulted in a longer lifespan under thermal stress conditions at 33˚C compared to controls. Similarly, chromosomal deletions containing HSP70 results in an increased survival at 25˚C. The reasons why reduction in HSP levels lead to longer lifespan under normal and constant stress conditions are unclear but likely include shorter than expected lifespan in the one of the parental lines. Further experiments will need to be done to investigate this unexpected result.

11:00 am Anuj Kaushal (Bryan Stewart) Determining Genetic Interactions with NSF in Drosophila melanogaster

N-ethylmaleimide sensitive factor (NSF) is one of the most important molecules involved in molecular signaling. It allows for the reuse of SNARE proteins by disassembling the cis-SNARE complex that is formed after vesicle fusion. Understanding the genetic interactions between NSF and various other genes may provide a deeper understanding of some important signaling pathways, such as Wingless and Notch. In Drosophila melanogaster, disruption of the dNSF2 gene leads to a notched-wing phenotype. In this study, I conducted a screen to find suppressors of the wing phenotype. Through deletion interactions and gene mapping I found that mutants in the gene Lethal (2) giant larvae suppress the dNSF2 wing phenotype. This result reveals a novel genetic interaction and provides insights into dNSF2 function.

K L

11:15 am Zaleena Akheralie (Ted Erclik) Characterizing Embryonic Optic Neuroblasts in the Developing Drosophila melanogaster Brain

The optic lobe is the visual processing center of the Drosophila melanogaster brain and is comprised of four compartments; the lamina, medulla, lobula, and lobula plate. These compartments are derived from a group of embryonic epithelial cells called the optic placode. During larval stages, these cells undergo expansive symmetrical divisions before converting into asymmetrically diving stem cells, termed neuroblasts, in the 3rd instar larval stage. While it has been widely accepted that optic lobe neuroblasts are first generated in the 3rd instar larva, recent research has identified an earlier window of neuroblast formation. Embryonic Optic Neuroblasts (EONs) have been shown to develop in the late embryo, two days earlier than previously anticipated. However, the neurons generated by EON’s have not yet been characterized. I have used a combination of Drosophila genetics, immunohistochemistry, and confocal microscopy to identify the neural types generated by EONs. Preliminary data suggest that EONs contribute to two populations of neurons in the adult, one that arborizes in the lobula and another that arborizes in the lobula plate and medulla. I will further determine the developmental stage in which EONs generate these neurons. My findings suggest that an early wave of neurogenesis serves to increase neural diversity in the visual system by generating unique classes of neurons. These early born neurons may play a role in the initial assembly of the visual neural circuit.

11:30 am Denia Hurst (Tim Westwood) Localization of Heat Shock Factor (HSF) Activity During Drosophila melanogaster Embryogenesis

Heat Shock Factor (HSF) is a transcriptional activator and during stress, it upregulates the transcription of the heat shock (HS) genes that make the Heat Shock Proteins (HSPs). HSPs code for protein chaperones that help cells repair the protein damage caused by stresses such as heat shock. During stress, HSF transitions from a monomer to a trimer, allowing it to bind to the Heat Shock Elements (HSEs) in the HS gene promoters. Although it is known that HSF has a role in stress conditions, it was also found that HSF plays a role in embryogenesis under non-stress conditions. To study when and where HSF is active during normal embryogenesis, we obtained transgenic fly lines that have reporter gene constructs containing HS gene promoters linked to fluorescent proteins (e.g. Hsp70 promoter-Green Fluorescent Protein and Hsp22 promoter-DsRed fluorescent protein). Using immunohistochemistry and confocal microscopy, we are investigating when and where these reporter genes are being expressed thereby determining HSFs spatial and temporal activity in embryogenesis. These results will be combined with the results of experiments that are examining where HSF is binding in the genome during embryogenesis to get an in depth understanding of the role HSF plays in Drosophila development.

11:45 am Gill Onate (Ted Erclik) Characterization of an early born population of neurons in the developing Drosophila optic lobe

The optic lobe is the primary visual processing center in the fruit fly brain and is an excellent research model in which to study the mechanisms that regulate neural diversity. The optic lobe is comprised of 3 major neuropils: the lamina, the medulla, and the lobula complex. The largest neuropil, the medulla, is comprised of over 100 distinct neural cell types. Medulla neurogenesis begins during the late larval stages from a monolayer neuroepithelium known as the Outer Proliferation Center (OPC). At the late 2nd instar larval stage, OPC neuroepithelial cells differentiate into stem cells termed neuroblasts (NBs), which subsequently divide to generate neural progenitors and medulla neurons. We have recently discovered a cluster of neurons generated by the OPC, born earlier than the majority of neurons born in the mid-late larva. Based on the location of this neuronal cluster

and the combination of transcription factors that are expressed in its neurons, I show here that these neurons are born from the central region of the OPC in the mid 2nd instar larva. Using a combination of genetic lineage tracing, immunohistochemistry and confocal microscopy, I further demonstrate that these early born neurons develop into a previously unidentified medulla tangential neuron, which arborizes in the medulla and projects to the central brain. The identification of this unique medulla neuron suggests that an unidentified patterning mechanism marks a sub-population of OPC epithelial cells for early conversion into neuroblasts.

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Session III – 1:00 pm to 3:00pm – Moderator: Baohua Liu

1:00 pm Fagun Kaushik (Joel Levine) Social Interaction Networks between D. pseudoobscura and D. persimilis

Social organization exists across the animal kingdom, and can be seen in animals, humans and even Drosophila. Social Interaction Networks (SINs) are quantitative tools of analyzing social organization in the fruit fly. Previously, SINs were found to differ between two strains of D. melanogaster, and these were shown to be structured in a non-random manner. This showcases that social organisation may be heritable under natural selection. A recent finding by the Levine lab suggests that there are greater differences in SINs between closely related species. This may indicate that social networks respond to reinforcement, which is a selection mechanism that favours the evolution of species boundaries. It was hypothesized that if SINs function to enforce species boundaries then they may differ mixed species groups, compared to isolated species groups. More specifically, each species may segregate due to differences in their social organization, despite occupying a shared environment. This was tested using two closely related species: D. pseudoobscura and D. persimilis. These species coexist in sympatry and produce hybrid offspring in the wild. Here, I acquired SINs from each species isolated in a homogenous social group, and from a mixed species social group and characterized patterns of pair-wise interactions between each social group.

1:15 pm Celine Bailleul and Samuel Nguyen (Robert Gerlai) Neurochemical and behavioural effects of stage-dependent ethanol exposure on novel tank response in juvenile zebrafish

Zebrafish have become a popular model organism for the analysis of the behavioural and biological mechanisms underlying fetal alcohol spectrum disorders (FASD). Several studies have investigated the long-term behavioural and neurochemical effects of acute embryonic alcohol exposure in adult zebrafish, however, little is known about the effects of embryonic ethanol exposure in juvenile zebrafish. Furthermore, potential developmental-stage dependent effects of embryonic alcohol exposure have not been systematically analyzed. Here, we expose zebrafish embryos to ethanol at different developmental stages (6hpf, 10 hpf, 16 hpf, 24, hpf, 36 hpf, 48 hpf) and measure the resulting behavioural and neurochemical changes in one-month old AB zebrafish. We employ the open field task an anxiety paradigm and use HPLC to quantify dopamine, serotonin and their metabolites from whole brain extract. Our results demonstrate significant developmental stage dependent effects of alcohol on certain behavioural responses. We are currently completing our HPLC analyses. We anticipate a correlation between behavioural and neurochemical effects of ethanol. We hope these findings will further our understanding of the behavioural and neural deficits commonly associated with FASD.

1:35 pm Omar Alhalabi (Baohua Liu) Characterization of the binocular ocular motor behaviour in rodents.

Optokinetic reflex (OKR) is an eye reflex which serves to stabilize moving objects onto the retina. It is a behavioral response that requires no specific training. Saccadic eye movement is another reflex which results in rapid eye movement to capture different objects in the environment. This study aims to characterize the binocular oculomotor behaviour in mice eye tracking. Using video captured at 100 frames/s, both eyes in CD1 crossed with C57BL/6 mice were analyzed under head restrained conditions. An oscillatory sinusoidal grating was used as the stimulus at varying parameters. Mice exhibited robust eye tracking at a spatial frequency of 0.16 c/d and 0.32 c/d. Data suggests that mice cannot control each individually and likely maintain binocular vision.

1:50 pm Ramsha Awan (Baohua Liu) Mapping the Circuits Underlying the Cortical Control of the Optokinetic Reflex; Neuronal Populations Projecting from the Visual Cortex to the NOT-DTN in the Mouse Model

Reflexes in mammals are usually mediated by the brainstem, but it has been found that the cortex can also play a role in modulation of these reflexes. The optokinetic reflex, optokinetic nystagmus, is an example of an innate reflex which is modulated by the cortex. To investigate the cortical control of the optokinetic reflex, the anatomical circuitry of the corticofugal projections were found. The aim of this experiment is to determine the areas of the visual cortex which project to the NOT-DTN. This was done by viral stereotaxic injection on CD1xAi14 tdTomato flexed mice. These mice were injected with retroAAV-Ef1a-mCherry-IRES-Cre virus into the NOT-DTN via iontophoresis for 20 minutes. After a 2-week waiting period, histology and confocal imaging of the brains was performed. It was found that the origin of the corticofugal projections have some area specificity. Areas in the ventral stream of the brain show the highest density of retrograde labeled neurons. In contrast, areas in the dorsal stream show very little contribution to the corticofugal projection innervating the NOT-DTN. This finding suggests that the ventral stream in mice may be the primary player in modulating the optokinetic reflex behaviour. This anatomical map will allow for functional experiments investigating the exact extent to which the cortex modulates the optokinetic reflex.

2:05 pm Kyshona Edwards (Baohua Liu) Mapping the Circuits Underlying the Cortical Control of Innate Reflexes

Innate reflexes are inflexible and instinctive behaviours that are unaffected by environmental conditions. In mammals, the brainstem was believed to be the driver of innate reflexes. Recent work has shown the cerebral cortex modulates basic visual reflexes, in addition to its role in higher cognitive functioning. To understand how the cortex modulates these reflexes, we chose the optokinetic reflex (OKR) in mice as a model system. The OKR is an oculomotor reflex driven by brainstem nuclei, that stabilizes moving images on the retina. The proposed mechanism underlying cortical modulation of the OKR is a neural circuit containing projections between the visual cortex, brainstem nuclei and other downstream targets. Previous work has shown the visual cortex sends projections to brainstem nuclei. However, the projection patterns between the brainstem and its downstream targets are still unknown. To determine the nature of these connections, we chose to map projections between the brainstem and three downstream targets using retrograde/ anterograde viral tracing. We attempted to determine if these downstream targets are independent populations and whether they receive cortical innervation. We expected these downstream targets to be distinct neural populations as projections between these targets are not known to overlap. Our data suggests connections between the brainstem and these downstream targets form both single target projections and overlapping projection patterns.

2:20 pm Sigi Maho (Robert Resiz/Diane Scott) Description of a new varanopid from Richards Spur and the evolutionary stasis of varanopids

Varanopids are a basal clade of small- to medium-sized synapsids, the ancestral group of mammals, spanning the Late Pennsylvanian to late Middle Permian environments in North America, Russia, Europe, and South Africa. The greatest varanopid diversity is observed at the fossiliferous deposits in the Richards Spur locality of Oklahoma, representative of an Early Permian upland ecosystem. Two previously described varanopid species are known only from limited post-cranial material. This study reports on a third varanopid taxon, represented by substantially more complete material, including three complete skulls. Such comprehensive cranial material allows for a detailed study of the taxon and its relationship to others varanopids. This new varanopid bears great morphological similarity to Mesenosaurus romerii from the Middle Permian Mesen River Basin in Russia. A phylogenetic analysis recovers a sister relationship between this taxon and M. romeri. This relationship, in conjunction with a detailed morphological comparison, supports the placement of this taxon within Mesenosaurus. These results reveal an unexpected extension of the geographic and temporal range of Mesenosaurus, contributing to our understanding of varanopid dispersal. The persistence of varanopids, exemplified by the evolutionary stasis of Mesenosaurus, is unusual among Paleozoic tetrapods. This phenomenon implies an exceptionally high degree of extended ecological resilience across major faunal and environmental transitions.