• METHOXYPYRAZINES AND LADYBUG TAINT IN WINES

      Botezatu, Andreea Ioana; Department of Biological Sciences (Brock University, 2013-11-05)
      Methoxypyrazines are aroma active compounds found in many wine varietals. These compounds can be of either grape-derived nature or can be introduced into wines via Coccinellidae beetles. Regardless of their origin, methoxypyrazines can have either a beneficial role for wine quality, contributing to the specificity of certain wine varietals (Cabernet sauvignon, Cabernet franc, Sauvignon blanc) or a detrimental role, particularly at higher concentrations, resulting in overpowering green, unripe and herbaceous notes. When methoxypyrazines of exogenous nature are responsible for these unpleasant characteristics, wines are considered to be affected by what is generally known as Ladybug taint (LBT). This is work is a collection of studies seeking to create a sensitive analytical method for the detection and quantification of methoxypyrazines in wines; to investigate the role of different Coccinellidae species in the tainting of wines with LBT and identify the main compounds in ladybug tainted wines responsible for the typical green herbaceous characteristics; to determine the human detection threshold of 2,5-dimethyl-3-methoxypyrazine in wines as well as investigate its contribution to the aroma of wines; and finally to survey methoxypyrazine concentrations in a large set of wines from around the world. In the first study, an analytical method for the detection and quantitation of methoxypyrazines in wines was created and validated. The method employs multidimensional Gas Chromatography coupled with Mass Spectrometry to detect four different methoxypyrazines (2,5-dimethyl-3-methoxypyrazine, isobutyl methoxypyrazine, secbutyl methoxypyrazine and isopropyl methoxypyrazines) in wine. The low limits of detection for the compounds of interest, improved separation and isolation capabilities, good validation data, as well as the ease of use recommend this method as a good alternative to the existing analytical methods for methoxypyrazine detection in wine. In the second study the capacity of two Coccinellidae species, found in many wine regions – Harmonia axyridis and Coccinella septempunctata - to taint wines is evaluated. Coccinella septempunctata is shown to be as capable as causing LBT in wines as Harmonia axyridis. Dimethyl methoxypyrazine, previously thought to be of exogenous nature only (from Coccinellidae haemolymph), is also detected in control (untainted) wines. The main odor active compounds in LBT wines are investigated through Aroma Extract Dilution Assay. These compounds are identified as isopropyl methoxypyrazine, sec- and iso- butyl methoxypyrazine. In the third study, the human detection threshold for dimethyl methoxypyrazine in wine is established to be 31 ng/L in the orthonasal modality and 70 ng/L retronasally. After wines spiked with various amounts of dimethyl methoxypyrazine are evaluated sensorally, dimethyl methoxypyrazine causes significant detrimental effects to wine aroma at a concentration of 120 ng/L. The final study examines methoxypyrazine (dimethyl methoxypyrazine, isopropyl methoxypyrazine, secbutyl methoxypyrazine and isobutyl methoxypyrazine) concentrations in 187 wines from around the world. Dimethyl methoxypyrazine is detected in the majority of the red wines tested. Data are interpreted through statistical analyses. A new measure for predicting greenness/herbaceousness in wines - methoxypyrazine “total impact factor” is proposed.
    • Molecular ecology and social evolution of the eastern carpenter bee, Xylocopa virginica

      Vickruck, Jessica L; Department of Biological Sciences
      Bees are extremely valuable models in both ecology and evolutionary biology. Their link to agriculture and sensitivity to climate change make them an excellent group to examine how anthropogenic disturbance can affect how genes flow through populations. In addition, many bees demonstrate behavioural flexibility, making certain species valuable models with which to study the evolution of social groups. This thesis studies the molecular ecology and social evolution of one such bee, the eastern carpenter bee, Xylocopa virginica. As a generalist native pollinator that nests almost exclusively in milled lumber, anthropogenic disturbance and climate change have the power to drastically alter how genes flow through eastern carpenter bee populations. In addition, X. virginica is facultatively social and is an excellent organism to examine how species evolve from solitary to group living. Across their range of eastern North America, X. virginica appears to be structured into three main subpopulations: a northern group, a western group and a core group. Population genetic analyses suggest that the northern and potentially the western group represent recent range expansions. Climate data also suggest that summer and winter temperatures describe a significant amount of the genetic differentiation seen across their range. Taken together, this suggests that climate warming may have allowed eastern carpenter bees to expand their range northward. Despite nesting predominantly in disturbed areas, eastern carpenter bees have adapted to newly available habitat and appear to be thriving. This is in marked contrast to many other bee species, particularly in the genus Bombus, who appear unable to shift their ranges along with climate change. Facultatively social organisms are interesting species to study the evolution of social groups, and the remaining chapters address questions of sociality in X. virginica. I used observation nests and genetic relatedness to examined how females behave towards one another in the spring prior to the establishment of dominance hierarchies in social nests. In spring, females directed fewer aggressive behaviours and more cooperative behaviours towards familiar rather than related individuals, indicating that females use nestmate recognition rather that kin recognition when interacting with conspecifics. Overwintering groups often contain both related and unrelated individuals, indicating that many bees interacting with one another in the fall prior to overwintering may be unrelated, emphasizing the importance of recognizing nestmates. Within social carpenter bee nests three different types of female have been described: primary, secondary and tertiary. Primary females are the dominant foragers and egg layers in the nest while secondary and tertiary females appear to join a reproductive queue behind the primary. To understand the nature and flexibility of this reproductive queue I performed removal experiments across three different years. This study showed that secondary females always assumed the role of replacement primary, while tertiary females rarely opted to forage and reproduce even if they were the only female in the nest. Removal experiments demonstrated that social groups in X. virginica are complex and comprise two different reproductive strategies (breed in the current year or delay reproduction) as well as form dominance hierarchies among primary and secondary females. Several tertiary females were able to become primary or solitary females in their second summer, providing evidence for how each type of female may have evolved in social nests. Finally, I examined how competition influences the evolution and maintenance of social groups in eastern carpenter bees. In conditions of high population density significantly more social nests were present in the population, indicating that competition for limiting nesting resources drives individuals together into social groups. Within social groups relatedness was low, and siblings actually dispersed away from one another to other nests in the population, reducing competition among kin. Eastern carpenter bees appear to demonstrate an interesting evolutionary route to sociality, where very high levels of competition among kin lead to dispersal, while limited nesting substrate forces individuals back into unrelated social groups. While predicted by kin selection, social groups of this nature are previously undescribed in the Hymenoptera, and further study of eastern carpenter bees can provide novel insights into alternate routes to sociality.
    • Phage-mediated biological control of Erwinia amylovora: The role of CRISPRs and exopolysaccharide

      Yagubi, Abdelbaset; Department of Biological Sciences
      Fire blight, caused by bacterium Erwinia amylovora, is a very serious disease affecting apple, pear and other fruit plants. The development of phage-based biopesticides is currently in progress in our lab. Emergence of phage-resistant bacteria is a valid concern. Two attributes of the bacterial host that may contribute to the development of resistance were studied, the Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated (CRISPR/Cas) system and exopolysaccharide (EPS) interaction with phages. The structure of E. amylovora CRISPR/Cas system was determined in 8 E. amylovora isolates from different geographical regions. Three CRISPR-array sets named CR1, CR2 and CR3 were detected in 4 isolates, and only 2 arrays were determined in the rest of the isolates. No significant similarity was found between spacers in any of these systems to phage DNA sequenced in this study or from GenBank. Also the Cas level of expression was not stimulated during phage infection. Introduction of extra copies of Cas genes to enhance expression did not result in phage resistance. Nevertheless, E. amylovora CRISPR/Cas system was found to be efficient in blocking the transformation of plasmids carrying protospacers matched spacers in CRR1 and CRR2. Among phages that have been sequenced in this study are ΦEa9-2 and ΦEa35-70. ΦEa9-2 (Podoviridae) genome is 75,568 bp, and found to be related to coliphage N4. ΦEa35-70 (Myoviridae) genome is 271,084 bp, and found to carry a potential EPS depolymerase gene. Activity of ΦEa35-70 EPS depolymerase was only detected when cloned and expressed in E. coli, but His-tagged purified protein did not exhibit any EPS-depolymerase activities. This study offers critical information for the design of novel and effective phage-based biopesticides for the control of E. amylovora. It provides a new knowledge on the molecular structure and function of CRISPR/Cas system and EPS-phage interaction.
    • Proximate influence on eusocial caste behaviour

      Awde, David N; Department of Biological Sciences
      Queens and workers of eusocial sweat bee species are morphologically and developmentally similar, which means that each female is capable of behaving as a queen or a worker. However, few females lay eggs and behave as queens, while the majority of females provision the queen’s offspring, rarely lay eggs, and behave as workers. This makes eusocial sweat bee species, such as Lasioglossum laevissimum, excellent models to study the underling environmental (social) and genetic factors that contribute to variation in caste behaviours. My research focused on describing some of the proximate mechanisms that influence caste behaviours in L. laevissimum females. The social environment of a sweat bee colony, specifically the behaviour of a queen, can have a dramatic impact on worker behaviour. Queens suppress worker reproduction by physically bullying their workers. In a nesting aggregation at Brock University, almost half of L. laevissimum nests became queenless, which provided me with a natural experiment to assess the direct influence by queens on worker behaviour. Dissection data showed that a small proportion (17%) of workers developed their ovaries in both queenright and queenless nests. This suggests that L. laevissimum queens exert an early, negative, and strong influence on worker egg-laying behaviour, which lasts after she is gone. Next I assessed the relationship between gene expression and L. laeivsismum caste behaviours. I predicited that queens would express a gene associated with egg-laying, vitellogenin, more than workers, and that workers would express genes associated with foraging, the foraging gene, more than queens. Lasioglossum laevissimum queens had higher vitellogenin expression levels than workers, and females with high ovarian development had high vitellogenin expression, regardless of caste. On the other hand, queens and workers had similar foraging expression levels. Gene expression comparisons between queens and workers highlight two important behavioural characteristics of sweat bee castes. First, in eusocial sweat bees, both queens and workers actively provision brood at some point during the breeding season, which is reflected in their similar foraging expression levels. Secondly, queens lay eggs while a small proportion of workers have queen-like ovarian development, reflected in vitellogenin expression differences between castes.
    • Responses to Reflection in Two Invertebrate Species

      May, Holly; Department of Biological Sciences
      The present thesis investigates the responses to reflection in both the crayfish Procambarus clarkii and the fruit fly Drosophila melanogaster. Responses to reflection in crayfish depend on social status and the current work suggests that learning and memory consolidation are required for these responses to be altered. Crayfish were treated to either massed or spaced training fights prior to reflection testing. The results show that subordinate crayfish treated to spaced training display a response typical of subordinate crayfish but subordinate crayfish treated to massed training exhibit a response typical of dominant crayfish. Fruit flies are shown to be attracted to reflection and responses to reflection are described here for the first time. Responses in fruit flies are shown to be dependent on social status. The frequency of behaviours were altered in isolated flies but not socialized flies. The addition of pheromones cVA and 7,11-HD were used to investigate how the addition of chemical cues altered responses to reflection in fruit flies. Socialized fruit flies treated with cVA exhibited an increase in the frequency of behaviours on both mirrored and clear glass walls, while isolated flies exhibited a decrease. Socialized flies treated with 7,11-HD spent more time on mirrored walls compared to glass walls, whereas the frequency of all behaviours were decreased in isolated flies treated with 7,11-HD.
    • The role of microRNAs and retinoid signaling during spinal cord regeneration in the adult newt.

      Lepp, Amanda; Department of Biological Sciences
      The molecular events after spinal cord injury that lead to the establishment of a permissive environment and epimorphic regeneration remain unclear. Two molecular pathway regulators that may converge to create a spinal cord regeneration-permissive environment in the urodele are retinoic acid (RA) and microRNAs (miRNAs). Recent evidence suggests that RARβ-mediated signaling is necessary for tail and caudal spinal cord regeneration in the adult newt. MicroRNAs are attractive candidates as mediators of retinoid signaling during regeneration, as their pleiotropic effects are vital in situations where global changes in gene expression are required. Thus, the overall aim of this thesis was to determine if miRNAs are involved in tail and caudal spinal cord regeneration in the adult newt, and if they act as regulators and/or effectors of retinoid signaling during this process. I have demonstrated here, for the first time, that multiple miRNAs are dysregulated in response to spinal cord injury in the adult newt, as well as in response to inhibition of retinoid signaling. Two of these miRNAs, miR-133a and miR-1, appear to target RARβ2 transcripts both in vivo and in vitro. Inhibition of RA signaling via RARβ with a selective antagonist, LE135, alters the pattern of expression of these miRNAs, which leads to an inhibition of tail regeneration. These data are indicative of a negative feed back loop, albeit potentially an indirect one. I also aimed to examine which miRNAs are affected by inhibiting RA synthesis during regeneration, and provided a long list of miRNAs that are dysregulated. These data provide the foundation for future studies on the putative roles of these miRNAs, as well as their function in retinoid signaling. Overall, these studies provide the first evidence for a role for miRNAs as mediators of retinoid signaling during caudal spinal cord regeneration in any system.
    • The role of mobile elements in recent primate genomes

      Tang, Wanxiangfu; Department of Biological Sciences
      Mobile elements (MEs), which constitute ~50% of the primate genomes, have contributed to both genome evolution and gene function as demonstrated by ample evidence discovered over the last few decades. The three studies in this thesis aims to provide a better understanding of the evolutionary profile and function of MEs in the primate genomes by taking a computational comparative genomics approach. The first study represents a comprehensive analysis of the differential ME transposition among primates via identification of species-specific MEs (SS-MEs) in eight primate genomes from the families of Hominidae and Cercopithecidae using a comparative genomics approach. In total, 230,855 SS-MEs are identified, which reveal striking differences in retrotransposition level in the eight primate genomes. The second study represents a more focused analysis for the identification of a new type of MEs, which we term “retro-DNA” for non-LTR retrotransposons derived from DNA transposons, in the recent primate genomes. By investigating biallelic DNA transposons that have both the insertion and pre-integration alleles in ten primate genomes, a total of 1,750 retro-DNA elements representing 750 unique insertion events are reported for the first time. The third study provides an analysis of the mechanism underlying the differential SINE transposition in the primate genomes. In this study, Alu profiles are compared and the Alu master copies are identified in six primate genomes in the Hominidae and Cercopithecidae groups. The results show that each lineage of the primates and each species owns a unique Alu profile exclusively defined by the AluY transposition activity, which is determined by the number of Alu master copies and their relative activity. Overall, work in this thesis provides new insights about MEs and their impact on the recent primate genomes by revealing differential ME transposition as an important mechanism in generating genome diversity among primate lineages and species through discovering a new type of MEs and preliminary analysis of the mechanism underlying the differential ME transposition among primates. Furthermore, taking advantage of the recently available primate genomes and transcriptomes data, the work in this thesis demonstrates the great potential of the comparative genomic approach in studying MEs in primate genomes.
    • The role of retinoic acid in long-term memory formation and synaptic plasticity in the mollusc Lymnaea stagnalis

      Rothwell, Cailin; Department of Biological Sciences (Brock University, 2015-02-04)
      The active metabolite of vitamin A, retinoic acid (RA), is involved in memory formation and hippocampal plasticity in vertebrates. A similar role for retinoid signaling in learning and memory formation has not previously been examined in an invertebrate species. However, the conservation of retinoid signaling between vertebrates and invertebrates is supported by the presence of retinoid signaling machinery in invertebrates. For example, in the mollusc Lymnaea stagnalis the metabolic enzymes and retinoid receptors have been cloned from the CNS. In this study I demonstrated that impairing retinoid signaling in Lymnaea by either inhibiting RALDH activity or using retinoid receptor antagonists, prevented the formation of long-term memory (LTM). However, learning and intermediate-term memory were not affected. An additional finding was that exposure to constant darkness (due to the light-sensitive nature of RA) itself enhanced memory formation. This memory-promoting effect of darkness was sufficient to overcome the inhibitory effects of RALDH inhibition, but not that of a retinoid receptor antagonist, suggesting that environmental light conditions may influence retinoid signaling. Since RA also influences synaptic plasticity underlying hippocampal-dependent memory formation, I also examined whether RA would act in a trophic manner to influence synapse formation and/or synaptic transmission between invertebrate neurons. However, I found no evidence to support an effect of RA on post-tetanic potentiation of a chemical synapse. Retinoic acid did, however, reduce transmission at electrical synapses in a cell-specific manner. Overall, these studies provide the first evidence for a role of RA in the formation of implicit long-term memories in an invertebrate species and suggest that the role of retinoid signaling in memory formation has an ancient origin.
    • STRATEGIES FOR PREVENTION AND TREATMENT OF HEPATITIS C VIRUS INFECTION

      Mansour, Hayam; Department of Biological Sciences (Brock University, 2012-06-04)
      Hepatitis C virus (HCV) is the causative agent of Hepatitis C, a serious global health problem which results in liver cirrhosis and hepatocellular carcinoma. Currently there is no effective treatment or vaccine against the virus. Therefore, development of a therapeutic vaccine is of paramount importance. In this project, three alternative approaches were used to control HCV including a DNA vaccine, a recombinant viral vaccine and RNA interference. The first approach was to test the effect of different promoters on the efficacy of a DNA vaccine against HCV. Plasmids encoding HCV-NS3 and E1 antigens were designed under three different promoters, adenoviral E1A, MLP, and CMV ie. The promoter effect on the antigen expression in 293 cells, as well as on the antibody level in immunized BALB/c mice, was evaluated. The results showed that the antigens were successfully expressed from all vectors. The CMV ie promoter induced the highest antigen expression and the highest antibody level. Second, the efficiency of a recombinant adenovirus vaccine encoding HCV-NS3 was compared to that of a HCV-NS3 plasmid vaccine. The results showed that the recombinant adenovirus vaccine induced higher antibody levels as compared to the plasmid vaccine. The relationship between the immune response and miRNA was also evaluated. The levels of mir-181, mir-155, mir-21 and mir-296 were quantified in the sera of immunized animals. mir-181 and mir-21 were found to be upregulated in animals injected with adenoviral vectors. Third, two recombinant adenoviruses encoding siRNAs targeting both the helicase and protease parts of the NS3 region were tested for their ability to inhibit NS3 expression. The results showed that the siRNA against protease was more effective in silencing the HCV-NS3 gene in a HCV replicon cell line. This result confirmed the efficiency of adenovirus for siRNA delivery. These results confirmed that CMV ie is optimum promoter for immune response induction. Adenovirus was shown to be an effective delivery vector for antigens or siRNAs. In addition, miRNAs were proved to be involved in the regulation of immune response.