• Demographic and genetic attributes of dispersing and resident individuals of an enclosed Microtus pennsylvanicus population

      Ross, Howard Alfred.; Department of Biological Sciences (Brock University, 1975-10-02)
      A dispersal polymorphism may exist in emigrants from cyclic populations of Microtus '~nnsylvanicus biasing trap-revealed movements of unenclosed animals in favour of sedentary or colonizing individuals. The dispersal tendency of emigrants from an enclosed population was investigated by releasing animals via tubes into one of two adjacent enclosures, one vacant and one inhabited. Individuals from the enclosed population were monitored for age, sex, weight and electrophoretically detectable serum transferrin genotype in an intensive live-trapping program. In 1973 the minimum number alive in the introduced enclosed study population reached approximately l67/ha when breeding stopped in October. In 1974 intensive breeding increased the population density to 333/ha by mid-July when a long decline in numbers and breeding intensity began without an intervening plateau. An adjacent unenclosed area had a much lower density and longer breeding season in 1974. The growth rate of young males in the enclosed population tended to be lowest during the decline period in 1974. Survival of the enclosed population was high throughout but was lowest during the decline phase in both sexes, especially males. Low transferrin heterozygote survival during the decline coincided with a significant heterozygote deficiency in females whereas in males genotype frequencies did not depart from Hardy-Weinberg equilibrium values throughout th.e study. Twenty-nine suitable ani.mals were released during the decline in five periods from July to November 1974. The proportions of males and transferrin heterozygotes in the released graun were generally greater than in the source population~ In the test enclosures 21% of the released animals continued their movement through the vacant area while 41% (no significant difference) moved through the inhabited enclosure. In the vacant test area, females had a greater tendency than males to continue dispersal whereas no difference was noted in the inhabited area. Low frequency of captures in the tubes, predator disturbances and cold weather forced the termination of the study. The role of dispersal as a population regulating mechanism was further substantiated. The genetic differences between emigrant and resident animals lend support to Howard's hypothesis that a genetic polymorphism influences the tendency to disperse. Support is also given to Myers' and Krebs' contention that among dispersers an additional density dependent polymorphism influences the distance dispersed.
    • Demography and molecular ecology of the solitary halictid Lasioglossum zonulum: with observations on Lasioglossum leucozonium

      Proulx, Alex; Department of Biological Sciences
      Halictid bees are excellent models for questions of both evolutionary biology and molecular ecology. While the majority of Halictid species are solitary and many are native to North America, neither solitary nor native bees have been extensively studied in terms of their population genetics. This thesis studies the social behaviour, demographic patterns and molecular ecology of the solitary Holarctic sweat bee Lasioglossum zonulum, with comparisons to its well-studied sister species Lasioglossum leucozonium. I show that L. zonulum is bivoltine in the Niagara region of southern Ontario but is univoltine in a more northern region of southern Alberta. Measurements of size, wear and ovarian development of collected females revealed that Brood 1 offspring are not altruistic workers and L. zonulum is solitary. A large proportion of foundresses were also found foraging with well-developed ovaries along with their daughters, meaning L. zonulum is solitary and partially-bivoltine in the Niagara region. L. zonulum being solitary and univoltine in Calgary suggests that it is a demographically polymorphic and not socially polymorphic. Thus, L. zonulum represents a transitional evolutionary state between solitary and eusocial behaviour in bees. I demonstrate that Lasioglossum zonulum was introduced to North America at least once from Europe in the last 500 years, with multiple introductions probable. Most North American specimens share the same mitochondrial DNA haplotype as those in Europe, with a small portion from western North America possessing distinct sequences. Investigations using microsatellite markers found North American populations to have a deficit of heterozygosity, and Bayesian analysis suggests that there are 3-4 lineages of L. zonulum in North. It is theorized that introductions could also be from Europe, Asia, or could even represent a native population which arrived via the Bering Land Bridge. I suggest that the plasticity found in L. zonulum may have a genetic cause and exists in North America due to the multiple introductions and potentially diverse geographic origins of this species. The outcome of my studies highlight why Lasioglossum zonulum is a model organism for the study of how eusociality evolved and why it warrants further and more in-depth study.
    • Development of a bovine adenovirus type 2-based gene delivery vector /

      Ojkic, Davor.; Department of Biological Sciences (Brock University, 1997-05-21)
      ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.
    • The development of a new genetic test for grapevine cultivars using a computational genomics approach

      Martin, Robert; Department of Biological Sciences
      Due to the sale and consumption of wine and table grapes, the grapevine is an important crop for many countries, including Canada. One of the main issues in viticulture is the identification of cultivars. Many of the over 6000 different types of grape cultivars look similar in colour or shape but may have very different taste profiles and require different growing conditions, while some have the same name but are genetically different (homonym) or having different names but are genetically identical (synonyms). Genetic tests based on the use of simple single repeat (SSR), or short tandem repeats (STR) markers have been developed to determine the genetic identity of different grapevine cultivars. However, the markers used in existing tests were identified more than 2 decades ago without optimization, and with the service limited to a few places around the world imposing many hurdles for international users. This research aims to develop a new grapevine genetic test by selecting the best STR markers in taking advantage of recently available rich grapevine genomic resources. Using a computational genomics approach, a total of 13 top performing STR markers were selected based on their discrimination power for 304 grapevine cultivars. A Polymerase Chain Reaction (PCR) based test was designed to group these 13 STR markers into 5 multiplex PCR groups for assaying using the QIAGEN QIAxcel Advanced System™ for its speedy and cost-efficient DNA fragment analysis. As a way of evaluating the performance of the designed test, in silico genotyping was performed for 304 grapevine cultivars and 37 Chardonnay clones based on available whole genome sequencing data. The results showed that the test was able to distinguish all these grape cultivars and Chardonnay clones, and furthermore, the number of STR markers used in the test can be reduced to a minimum of 6 for distinguishing these cultivars and clones. Genotype-based phylogeny analysis of these cultivars and clones showed meaningful clustering patterns matching their known or assumed relationships, indicating the validity of the test. In conclusion, despite not being able to perform evaluations of the STR markers in the laboratory, the preliminary in silico results demonstrate the high efficiency of the computation genomic approach in finding top performing STR markers and predicts an excellent performance of the designed grapevine genetic test.
    • Development of packaging cell lines for rescuing BAV2 viral vectors

      Salamé, Mohamad S.; Department of Biological Sciences (Brock University, 1999-07-09)
      The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.
    • Development of techniques for testing the effect of the E1 region on adenovirus integration /

      Geng, Songsong.; Department of Biological Sciences (Brock University, 2007-06-04)
      Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.
    • Developmental variations in the peripheral erythrocytic system of the rainbow trout, Salmo gairdneri

      Keen, John Ellis.; Department of Biological Sciences (Brock University, 1986-07-09)
      The peripheral circulating erythrocytic system of the rainbow trout, l3 almo gairdner , was examined in vitro in relation differences in the morphology and multiple hemoglobin system organization of adult and juvenile red cells. Cells were separated by velocity sedimentation under unit gravity, a procedure requiring red cell exposure to an incubation medium for periods of at least three hours. Therefore , this must provide an environment in which red cells remain in a condition approximaing normalcy. Previous studies having demonstrated commonly employed media to be ineffective in this regard , a medium was developed through modification of Cortl and saline. One of the principal additions to this me dium , norepinephrine, altered cell regulation of intracellular calcium, magnesium and chloride concentrations. Catecholamine involvement was also suggeste d in the synthes is of hemoglobin . The procedure was found to separtate cells primarily by density and, to a lesser extent, by shape. Characterization of red cells revealed two subpopulations to exist . The first comprised the bulk of the cell population, and were of greater l ength, width, volume and major:minor axis ratio than the smaller population; these were adult cells. The later, juvenile cells were of smaller overall size and were more spherical in shape . Juvenile cells also possessed fewer electrophore tpically distinguishable isomorphs than did adults with only eight of eleven hemoglobin component s typically found With maturation,hemoglobin complement with the development of three more bands. The total complement of the adult cell contained 7 cathodal bands and four anodal hemoglobin isomorphs. Bands acquired with maturation comprised the smallest percentage of the cells hemoglobin. each averaging less than one-percent of the total. Whether these additional bands are derived through degradation and reaggregation of existing components or are the product of pe gQy2 synthesis is not yet known.
    • Diatoms as indicators of lake trophic status

      Yang, Ching-jung.; Department of Biological Sciences (Brock University, 1990-07-09)
      Relationships between surface sediment diatom assemblages and lake trophic status were studied in 50 Canadian Precambrian Shield lakes in the Muskoka-Haliburton and southern Ontario regions. The purpose of this study was to develop mathematical regression models to infer lake trophic status from diatom assemblage data. To achieve this goal, however, additional investigations dealing with the evaluation of lake trophic status and the autecological features of key diatom species were carried out. Because a unifying index and classification for lake trophic status was not available, a new multiple index was developed in this study, by the computation of the physical, chemical and biological data from 85 south Ontario lakes. By using the new trophic parameter, the lake trophic level (TL) was determined: TL = 1.37 In[1 +(TP x Chl-a / SD)], where, TP=total phosphorus, Chl-a=chlorophyll-a and SD=Secchi depth. The boundaries between 7 lake trophic categories (Ultra-oligotrophic lakes: 0-0.24; Oligotrophic lakes: 0.241-1.8; Oligomesotrophic lakes: 1.813.0; Mesotrophic lakes: 3.01-4.20; Mesoeutrophic lakes: 4.21-5.4; Eutrophic lakes: 5.41-10 and Hyper-eutrophic lakes: above 10) were established. The new trophic parameter was more convenient for management of water quality, communication to the public and comparison with other lake trophic status indices than many of the previously published indices because the TL index attempts to Increase understanding of the characteristics of lakes and their comprehensive trophic states. It is more reasonable and clear for a unifying determination of true trophic states of lakes. Diatom specIes autecology analysis was central to this thesis. However, the autecological relationship of diatom species and lake trophic status had not previously been well documented. Based on the investigation of the diatom composition and variety of species abundance in 30 study lakes, the distribution optima of diatom species were determined. These determinations were based on a quantitative method called "weighted average" (Charles 1985). On this basis, the diatom species were classified into five trophic categories (oligotrophic, oligomesotrophic, mesotrophic, mesoeutrophic and eutrophic species groups). The resulting diatom trophic status autecological features were used in the regressIon analysis between diatom assemblages and lake trophic status. When the TL trophic level values of the 30 lakes were regressed against their fi ve corresponding diatom trophic groups, the two mathematical equations for expressing the assumed linear relationship between the diatom assemblages composition were determined by (1) uSIng a single regression technique: Trophic level of lake (TL) = 2.643 - 7.575 log (Index D) (r = 0.88 r2 = 0.77 P = 0.0001; n = 30) Where, Index D = (0% + OM% + M%)/(E% + ME% + M%); 4 (2) uSIng a' multiple regressIon technique: TL=4.285-0.076 0%- 0.055 OM% - 0.026 M% + 0.033 ME% + 0.065 E% (r=0.89, r2=0.792, P=O.OOOl, n=30) There was a significant correlation between measured and diatom inferred trophic levels both by single and multiple regressIon methods (P < 0.0001, n=20), when both models were applied to another 20 test lakes. Their correlation coefficients (r2 ) were also statistically significant (r2 >0.68, n=20). As such, the two transfer function models between diatoms and lake trophic status were validated. The two models obtained as noted above were developed using one group of lakes and then tested using an entirely different group of lakes. This study indicated that diatom assemblages are sensitive to lake trophic status. As indicators of lake trophic status, diatoms are especially useful in situations where no local trophic information is available and in studies of the paleotrophic history of lakes. Diatom autecological information was used to develop a theory assessing water quality and lake trophic status.
    • Differentiation of morphology and reproductive traits in the house finch (Carpodacus mexicanus): a comparison between native and introduced populations

      Kozlovic, Daniel R.; Department of Biological Sciences (Brock University, 1986-07-09)
      House Finches (CarpQdacqs mexiCAnuS) were introduced to Long Island, New York from southern'California in 1940. Apparently, an initial sample of less than 100 birds has given rise to a population that now occupies much of the eastern United States. This study was to determine if morphological and reproductive changes have taken place in introduced eastern birds, which have colonized a novel environment. A study area in Goleta, California (CAL) represented the parental population whereas for comparison, House Finches in St. Catharines, Ontario (ONT) represented the introduced population. Interlocality variation in 25 morphometric characters of 100 adult House Finches was examined statistically. Singleclassification analysis of variance revealed significant interlocality differentiation in seven characters of males and nine of females. Females showed differentiation in more limb elements than males. Analysis of character variation using discriminant and principal component analysis distinguished samples on the basis of variation in shape. Compared to CAL, aNT birds (especially females) had smaller extremities relative to certain core parts and weight. Females showed similar patterns of character covariation in each locality on the second principal component, which suggests that differentiation of the ONT population may not be solely environmentally induced. Sexual dimorphism was evident in four charaoters in aNT and five in CAL. Disoriminant analysis distinguished sex on the basis of variation in shape. Males possessed a relatively larger flying apparatus and small.er hind limbs than females. The dearee of sexual dimorphism did not vary sicnifioantly between looalities. 3 Data on reproduotive parameters were oolleoted in 1983 and 1984 in ONT, and 1984 in CAL. In 1984, Bouse Finohes began breedina approximately three months earlier in CAL than in ONT. In ONT, there was no sianifioant differenoe in mean olutoh initiation date between 1983 and 1984. In both looalities most nests oontained either four or five ea",s, and olutoh size differenoes between looalites were not signifioant. Seasonal deolines in olutch size were evident in ONT but not in CAL. Intralooality variation in e.g weight and size was not related to clutch size. E",g weiaht showed no seasonal trend in ONT, but inoreased sianifioantly with breed ina season in OAL. In both looalities e8'''' weiaht increased sipifioantly with order of layina in olutohes of four but not in clutohes of five. Eag's in ONT in 1983 and 1984 were sip.ificantly larser than in CAL in 1984. The modal inoubation period was 13 days and did not vary sip.ifioantly between localites. In both looalities nestling weiaht on the day of hatohing was oorrelated to fresh ega welaht. For muoh of the period between hatohing and 14 days post-hatoh, ONT nestlinas were signifioantly laraer than CAL nestlings in terms of weiaht. bill length, bill depth, and manus length.
    • Distribution of excitation energy in photosynthesis: a study of heat loss, photo chemical activity and fluorescence emission in intact calls of cyanobacterium.

      Salehian, Omid.; Department of Biological Sciences (Brock University, 1991-10-02)
      Photosynthetic state transitions were investigated in the cyanobacterium Synechococcus sp. PCC 6301 by studying fluorescence emission, heat loss, and PS I activity in intact cells brought to state 1 and state 2. 77K fluorescence emission spectra were modelled with a sum of 6 components corresponding to PBS, PS II, and PS I emissions. The modelled data showed a large decrease in PS II fluorescence accompanied with a small increase in the PS I fluorescence upon transition to state 2 for excitation wavelengths absorbed by both PBS and ChI ll.. The fluorescence changes seen with ChI .a. excitations do not support the predictions of the mobile PBS model of state transition in PBS-containing organisms. Measurements of heat loss from intact cells in the two states were similar for both ChI it. and PBS excitations over three orders of magnitude of laser flash intensity. This suggests that the PBS does not become decoupled from PS II in state 2 as proposed by the PBS detachment model of state transition in PBS-containing organisms. PS I activity measurements done on intact cells showed no difference in the two states, in contrast with the predictions of all of the existing models of state transitions. Based on these results a model for state transition In PBScontaining organisms is proposed, with a PS II photoprotectory function.
    • Diversity among bacteria causing blotch disease on the commercial mushroom, agaricus bisporus /

      Sivanesan, Durga.; Department of Biological Sciences (Brock University, 2003-07-14)
      A total of 251 bacterial isolates were isolated from blotched mushroom samples obtained from various mushroom farms in Canada. Out of 251 stored isolates, 170 isolates were tested for pathogenicity on Agaricus bisporus through mushroom rapid pitting test with three distinct pathotypes observed: dark brown, brovm and yellow/yellow-brown blotch. Phenotypic analysis of 83 isolates showed two distinct proteinase K resistant peptide profiles. Profile group A isolates exhibited peptides with masses of 45, 18, 16 and 14 kDa and fiirther biochemical tests identified them as Pseudomonasfluorescens III and V. Profile group B isolates lacked the 16-kDa peptide and the blotch causing bacterial isolates of this group was identified as Serratia liquefaciens and Cedecea davisae. Comparative genetic analysis using Amplified Fragment Length Polymorphism (AFLP) on 50 Pseudomonas sp. isolates (Group A) showed that various blotch symptoms were caused by isolates distributed throughout the Pseudomonas sp. clusters with the exception of the Pseudomonas tolaasii group and one non-pathogenic Pseudomonas fluorescens cluster. These results show that seven distinct Pseudomonas sp. genotypes (genetic clusters) have the ability to cause various symptoms of blotch and that AFLP can discriminate blotch causing from non-blotch causing Pseudomonasfluorescens. Therefore, a complex of diverse bacterial organisms causes bacterial blotch disease
    • DNA polymerase activity in trophoblast giant cells in the mouse /

      Dean, Wendy L.; Department of Biological Sciences (Brock University, 1985-06-01)
      In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.
    • Does Gamma-aminobutyric acid function as a plant resistance mechanism against phytophagous insect activity? /

      MacGregor, Kennaway Byron.; Department of Biological Sciences (Brock University, 2000-05-21)
      Gamma-aminobutyric acid (GAB A) is a ubiquitous non-protein amino acid synthesized via the decarboxylation of L-glutamate in a reaction catalyzed by the cytosolic enzyme L-glutamate decarboxylase (GAD). In animals it functions as an inhibitory neurotransmitter. In plants it accumulates rapidly in response to various stresses, but its function remains unclear. The hypothesis that GABA accumulation in leaf tissue may function as a plant resistance mechanism against phytophagous insect activity was investigated. GABA accumulation in response to mechanical stimulation, mechanical damage and insect activity was demonstrated. In wt tobacco (Nicotiana tabacum cv Samsun), mechanical stimulation or damage caused GABA to accumulate within 2 min from mean levels of 14 to 37 and 1~9 nmol g-l fresh weight (FW), respectively. In the transgenic tobacco strain CaMVGAD27c overexpressing Petunia GAD, the same treatments caused GABA to accumulate from 12 to 59 and 279 nmol g-l FW, respectively. In the transgenic tobacco strain CaMVGADilC 11 overexpressing Petunia GAD lacking an autoinhibitory domain, mechanical stimulation or damage caused GABA to accumulate from 180 to 309 and 630 nmol g-l FW, respectively. Ambulatory activity by tobacco budworm (TBW) larvae (Heliothis virescens) on leaves of CaMVGAD27c tobacco caused GABA to accumulate from 28 to 80 nmol g-l FW within 5 min. Ambulatory and leaf-rolling activity by oblique banded leaf roller (OBLR) larvae (Choristoneura rosaceana cv Harris) on wt soybean leaves (Glycine max cv Harovinton) caused GABA to accumulate from 60 to 1123 nmol g-l FW within 20 min. Increased GABA levels in leaf tissue were shown to affect phytophagous preference in TBW larvae presented with wt and transgenic tobacco leaves. When presented with leaves of Samsun wt and CaMVGAD27c plants, TBW larvae consumed more wt leaf tissue (640 ± 501 S.D. mm2 ) than transgenic leaf tissue (278 ± 338 S.D. mm2 ) nine times out of ten. When presented with leaves of Samsun wt and CaMVGAD~C11 plants, TBW larvae consumed more transgenic leaf tissue (1219 ± 1009 S.D. mm2 ) than wt leaf tissue (28 ± 31 S.D. mm2 ) ten times out of ten. These results indicate that: (1) ambulatory activity of insect larvae on leaves results in increased GABA levels, (2) transgenic tobacco leaves with increased capacity for GABA synthesis deter feeding, and (3) transgenic tobacco leaves with constitutively higher GABA levels stimulate feeding.
    • The ecological and physiological consequences of sun vs. shade nesting for the small carpenter bee, Ceratina calcarata, and their offspring

      de Haan, Jessie; Department of Biological Sciences
      Ceratina calcarata mothers choose their nesting location and that choice can have developmental consequences for their offspring. Nests in the sun experience higher developmental temperatures, reducing self-feeding insect body size through a phenomenon called the Temperature-Size Rule. How does developmental temperature affect body size in insects whose mothers’ feed them; for example, in mass-provisioning bees where all the food an offspring needs to complete development is provided by the mother upfront? What are the physiological advantages or disadvantages conveyed to offspring in sunny nests? In this thesis I used C. calcarata to investigate the ecological and behavioural consequences of nest location choice (sun or shade) on mothers, as well as the physiological consequences of developmental temperature on their offspring. Nests randomly allocated to the shade treatment were more likely to be empty when opened, indicating that shaded nesting locations were not preferred. Mothers nesting in the sun foraged more often for nectar than shade mothers, but provisioned similar sized pollen masses. Sunny nests were hotter than shaded nests, even more so if they were oriented on an angle. Offspring from sunny nests were smaller than shade bees in agreement with the Temperature Size Rule. Sunny offspring also had higher thermal tolerance than offspring from the shade but less frequent DGE bursts at 25°C compared to shade bees, suggesting a lower metabolism at a moderate temperature. As a result, I conclude offspring from sunny nests displayed irreversible developmental thermal plasticity. Offspring from sunny nests experienced a thermal trade-off as a result of their developmental conditions; they had a capacity for higher thermal tolerance as evidenced by a shifted thermal breadth, but at the expense of a smaller adult body size. Such trade-offs may enhance their response to climate change and contribute to speciation.
    • Ecological interactions between zooplankton and photosynthetic bacteria in Crawford Lake, Ontario

      Mazumder, A.; Department of Biological Sciences (Brock University, 1983-07-09)
      The present study was carried out to test the hypothesis that photosynthetic bacteria contribute a large portion of the food of filter feeding zooplankton populations in Crawford Lake, Ontario. The temporal and spatial variations of both groups of organisms are strongly dependent on one another. 14 By using C-Iabelled photosynthetic bacteria. the ingestion and clearance rates of Daphnia pulex, ~. rosea, and Keratella spp were estimated during summer and fall of 1982. These quantitative estimations of zooplankton ingestion and clearence rates on photosynthetic bacteria comprised an original addition to the literature. Photosynthetic bacteria comprised a substantial portion of the diet of all four dominant zooplankton species. The evidence for this is based on the ingestion and clearance rates of the dominant zooplankton species. Ingestion rates of D. pulex and D. rosea ranged 5 5 -1 -1 - -- 5 - -- 5 from 8.3X10 -1 to 14.6XlO -1 cells.ind. hr and 8.1X10 to 13.9X10 cells.ind. hr • Their clearance rates ranged from 0.400 to 1.000 -1 -1 -1 -1 ml.ind. hr. and 0.380 to 0.930 ml.ind. hr • The ingestion and clearance -1 -1 -1 -1 rates of Keratella spp were 600 cell.ind. hr and 0.40 ul.ind. hr respectively. Clearance rates were inversely proportional to the concentration of food cells and directly proportional to the body size of the animals. It is believed that despite the very short reg~neration times of photosynthetic bacteria (3-8 hours) their population densities were controlled in part by the feeding rates of the dominant zooplankton in Crawford Lake. By considering the regeneration times of photosynthetic bacteria and the population clearance rates of zooplankton, it was estimated that between 16 to 52% and 11 to 35% of the PHotosynthetic bacteria were' consumed· by Daphnia· pulex. and Q.. rosea per day. The temporal and spatial distribution of Daphnia pulex, !.. rosea, Keratella quadrata, K. coChlearis and photosynthetic bacteria in Crawford Lake were also investigated during the period of October, 1981 to December, 1982. The photosynthetic bacteria in the lake, constituted a major food source for only those zooplankton Which tolerate anaerobic conditions. Changes in temperature and food appeared to correlate with the seasonal changes in zooplankton density. All four dominant species of zooplankton were abundant at the lake's surface (O-4m) during winter and spring and moved downwards with the thermocline as summer stratification proceeded. Photosynthetic bacteria formed a 2 m thick layer at the chemocline. The position of this photosynthetic bacterial J-ayer changed seasonally. In the summer, the bacterial plate moved upwards and following fall mixing it moved downwards. A vertical shift of O.8m (14.5 to 15.3m) was recorded during the period of June to December. The upper limit of the photosynthetic bacteria in the water column was controlled by dissolved oxygen, and sulfide concentrations While their lower limit was controlled by light intensity. A maximum bacterio- 1 chlorophyll concentration of 81 mg Bchl.l was recorded on August 9, 1981. The seasonal distribution of photosynthetic bacteria was controlledinpart' by ·theg.-"z1ai'_.Q;~.zoopl. ank:tCm;-.Qther -ciactors associated with zooplankton grazing were oxygen and sulfide concentrations.
    • Effect of age on female choice in the field cricket, Gryllus interger

      Prosser, Melaine Renee.; Department of Biological Sciences (Brock University, 1994-07-09)
      Female choice is an important element of sexual selection that may vary among females of the same species. Few researchers have investigated the causes of variation in selectivity with respect to potential mates and overall level of motivation toward a stimulus source representative of a mate. This study demonstrates that female age may be one cause of variation in female choice. Females of different ages may have different mate preferences. As females age, they have less time left to reproduce, and their residual reproductive value decreases. This should correspond to a higher reproductive effort which may be represented as increased motivation and/or decreased selectivity. The effect of age on mate choice in Gryllus integer was investigated by using a non-compensating treadmill, called the Kugel, to measure female phonotaxis. Artificially generated male calling songs of varying pulse rates were broadcast in either a singlestimulus or a three-stimulus experimental design. The pulse rates used in the calling song stimuli were 70, 64, 76, 55 and 85 pulses per second. These corresponded to the documented mean pulse rate for the species at the experimental temperature, one standard deviation below and above the mean, and 2.5 standard deviations below and above the mean, respectively. Test females were either 11-14 days or 25-28 days post-ecdysis. Trials usually were conducted two to seven hours into the scotophase. In the single-stimulus experiment, females were presented with stimuli with only one pulse rate. Older females achieved higher vector scores than younger females, indicating that older females are more motivated to mate. Both groups showed little phonotactic response towards 55 or 85 pIs, both of which lie outside the natural range of G. integer calling song at the experimental temperature. Neither group discriminated among the three pulse rates that fell within the natural range of calling song. In the three-stimulus experiment, females were presented with stimuli with one of three pulse rates, 64, 70 or 76 pIs, In alternation. Both age groups had reduced responsiveness in this experiment, perhaps due to an increase in perceived male density. Additionally, younger females responded significantly more to 64 and 70 pIs than to the higher pulse rate, indicating that they are selective with respect to mate choice. Older females did not discriminate among the three pulse rates. Therefore, it was concluded that selectivity decreases with age. A further study was conducted to determine that these effects were due to age and not due to the differing periods without a mating between the two age groups. Again, stimuli were presented in a three-stimulus experimental design. Age was held constant at 28 days and time since last mating varied from 11 to 25 days. Females varyIng in time since last mating did not differ in their responses to the calling song pulse rates. This indicated that the increased motivation and decreased selectivity exhibited In the initial experiments were due to age and not to time without a mating. Neither time of trial nor female weight had an effect upon female phonotaxis. Data are discussed in terms of mate choice, residual reproductive value, and costs of choice.
    • The effect of age on the structure and compostition of the cell walls of Choanephora cucurbitarum

      Campbell, Craig D.; Department of Biological Sciences (Brock University, 1980-07-09)
      The effect of age on the structure and composition of isolated and purified cell walls from cultures of Choanephora cucurbitarum was investigated by microchemical analyses, visible and infrared spectrophotometry, x-ray diffractometry and electron microscopy. Qualitative evaluation revealed the presence of lipids, proteins, neutral sugars, strong alkali soluble sugars, chitin, chitosan and uronic acids in the cell walls of both the 1 and 7 day old cultures. As the mycelium aged, there was a slight but statistically significant increase in the protein content, and a pronounced rise in the chitin and neutral sugar constituents of the cell walls. Conversely, the decrease in the chitosan content during this period had the net effect of altering the chitin: chitosan ratio from near unity in the younger cultures, to a 2:1 ratio in the 7 day old cell wall samples. Glutaraldehyde-osmium fixed thin sections of the 1 day old vegetative hyphae of £. curbitarum revealed the presence of a monolayered cell wall, which upon aging became bilayered. Replicas of acid hydrolysed cell walls demonstrated that both the 1 and 7 day old samples possessed an outer layer which was composed of finely granular amorphous material and randomly distributed microfibrils. The deposition of an inner secondary layer composed of parallel oriented microfibrils in the older hypha was correlated with an increase in the chitin content in the cell wall. The significance of these results with respect to the intimate relationship between composition and structure is discussed.
    • Effect of citrate on the redox properties of cytochrome C and its kinetic and binding behaviour with cytochrome oxidase /

      Brooks, Stephen P. J.; Department of Biological Sciences (Brock University, 1981-06-09)
      Increasing citrate concentration, at constant ionic strength (30 mM) decreases the rate of cytochrome ~ reduction by ascorbate. This effect is also seen at both high (600 mM) and low (19 mM) ionic strengths, and the Kapp for citrate increases with increasing ionic strength. Citrate binds d both ferri -and ferrocytochrome ~, but with a lower affinity for the latter form (Kox . .red d = 2 mM, Kd = 8 mM) as shown by an equilibrium assay with N,N,N',N', Tetramethyl E- phenylenediamine. The reaction of ferricytochrome ~with cyanide is also altered in the presence of citrate: citrate increases the K~PP for cyanide. Column chromatography of cytochrome ~-cytochrome oxidase mixtures shows citrate increases the dissociation constant of the complex. These results are confirmed in kinetic assays for the "loose"site (Km = 20 pM) only. The effect of increasing citrate observable at the "tight" site (Km = 0.25 pM) is on the turnover number and not on the K . These results suggest a mechanism m where anion binding to cytochrome £ at the tight site affects the equilibrium between two forms of cytochrome c bound cytochrome oxidase: an active and an inactive one.
    • The effect of diet and parasite load on female phonotaxis in the Texas field cricket, Gryllus integer

      MacDougall, Donald Allen.; Department of Biological Sciences (Brock University, 1996-07-09)
      Female crickets respond selectively to variations in species-specific male calling songs. This selectivity has been shown to be age-dependent; older females are less choosy. However, female quality should also affect female selectivity. The effect of female quality on mate choice was examined in Gryllus integer by comparing the phonotactic responses of females on different diets and with different parasite loads to various synthetic models of conspecific calling song. Test females were virgin, 11-14 days old, and had been maintained on one of five diets varying in protein and fat content. Phonotaxis was quantified using a non-compensating Kugel treadmill which generates vector scores incorporating the speed and direction of movement of each female. Test females were presented with four calling song models which differed in pulse rate, but were still within the natural range of the species for the experimental temperature. After testing, females were dissected and the number of gregarine parasites within the digestive tract counted. There were no significant effects of either diet or parasitism on female motivation to mate although the combined effects of these variables seem to have an effect with no apparent trend. Control females did not discriminate among song types, but there was a trend of female preferences for lower pulse rates which are closest to the mean pulse rate for the species. Heavily parasitized females did not discriminate among pulse rates altho~gh there was a similar trend of high vector scores for low pulse rates. Diet, however, affected selectivity with poorly-fed females showing significantly high vector scores for pulse rates near the species mean. Such findings raise interesting questions about energy allocation and costs and risks of phonotaxis and mate choice in acoustic Orthoptera. These results are discussed in terms of sexual selection and female mate choice.
    • Effect of diurnal cycling temperatures on cardiovascular- ventilatory function in statically-acclimated rainbow trout, Salmo gairdneri

      Henry, James Arthur Charles.; Department of Biological Sciences (Brock University, 1980-07-09)
      Four groups of rainbow trout, Salmo gairdneri, were acclimated to 2°, 10°, and 18°e, and to a diurnal temperature cycle (100 ± 4°C). To evaluate the influence of cycling temperatures in terms of an immediate as opposed to acclimatory response various ventilatory-cardiovascular rate functions were observed for trout, either acclimated to cycling temperatures or acclimated to constant temperatures and exposed to a diurnal temperature cycle for the first time (10° ± 4°C for trout acclimated to 10°C; 18°+ 4°C for trout acclimated to l8°e). Gill resistance and the cardiac to ventilatory rate ratio were then calculated. Following a post preparatory recovery period of 36 hr, measurements were made over a 48 hour period with the first 24 hours being at constant temperature in the case of statically-acclimated fish followed by 24 hours under cyclic temperature conditions. Trout exhibited marked changes in oxygen consumption (Vo ) with temp- 2 erature both between acclimation groups, and in response to the diurnal temperature cycle. This increase in oxygen uptake appears to have been achieved by adjustment of ventilatory and, to some extent, cardiovascular activity. Trout exhibited significant changes in ventilatory rate (VR), stroke volume (Vsv), and flow (VG) in response to temperature. Marked changes in cardiac rate were also observed. These findings are discussed in relation to their importance in convective oxygen transport via water and blood at the gills and tissues. Trout also exhibited marked changes in pressure waveforms associated with the action of the resp; ratory pumps with temperature. Mean differenti a 1 pressure increased with temperature as did gill resistance and utilization. This data is discussed in relation to its importance in diffusive oxygen transport and the conditions for gas exchange at the gills. With one exception, rainbow trout were able to respond to changes in oxygen demand and availability associated with changes in temperature by means of adjustments in ventilation, and possibly pafusion, and the conditions for gas exchange at the gills. Trout acclimated to 18°C, however, and exposed to high cyclic temperatures, showed signs of the ventilatory and cardiovascular distress problems commonly associated with low circulating levels of oxygen in the blood. It appears these trout were unable to fully meet the oxygen requirements associated with c~ling temperatures above 18°C. These findings were discussed in relation to possible limitations in the cardiovascular-ventilatory response at high temperatures. The response of trout acclimated to cycling temperatures was generally similar to that for trout acclimated to constant temperatures and exposed to cycling temperatures for the first time. This result suggested that both groups of fish may have been acclimated to a similar thermal range, regardless of the acclimation regime employed. Such a phenomenon would allow trout of either acclimation group to respond equally well to the imposed temperature cycle. Rainbow trout showed no evidence of significant diurnal rhythm in any parameters observed at constant temperatures (2°, 10°, and 18° C), and under a 12/12 light-dark photoperiod regime. This was not taken to indicate an absence of circadian rhythms in these trout, but rather a deficiency in the recording methods used in the study.