Species recognition is an integral component of mate selection and must occur in all sexually reproducing organisms to avoid costly hybridisation. Species recognition abilities may be comprised of both innate components and experience during ontogeny through the learning of visual, acoustic, and other sensory species-specific cues. But how greatly is the ability to recognise one‟s own species (conspecifics) over others (heterospecifics) dependent on the phylogeographic relationship of the array of potential species as social partners and to what extent is the discriminatory behaviour modulated by subject ontogeny versus species identity? Using a model system, which is widely studied in all disciplines of avian research, the zebra finch (Taeniopygia guttata castanotis), I aimed to investigate the visual and acoustic cues involved in conspecific recognition by both female and male individuals of this species. I used an array of previously untested phylogeographically relevant estrildid heterospecifics as my stimuli and tested subjects of diverse experimental ontogenetic treatments. By scoring a wide-selection of measured behavioural responses my research indicates that female and male zebra finches prefer live conspecifics over live phylogeographically relevant heterospecific stimuli and this preference is more consistent by females than males. Female zebra finches rely on both visual and acoustic features of potential social partners for accurate species discrimination; in this regard video playbacks or the diverse colour morphs of domesticated zebra finches may be useful tools for further experimentation. Additionally, females display significant individuality in their behavioural responses which may be relevant for pair bonding decisions made by both sexes. I further documented that normally-reared zebra finches will prefer song playbacks of their own species but that both rearing in an indoor restricted acoustic environment of conspecifics or cross-fostering to another species will reduce discrimination preferences, although the results may depend on the behavioural metrics analysed. This dissertation is presented as a general overview with details of my specific contributions towards the work included in this thesis, followed by discrete review and data chapters, and a final general discussion.

The Podocarpaceae is an essentially southern hemisphere family of conifers which includes seven genera: viz. Podocarpus, Dacrydium, Phyllocladus, Microstrobos, Microcachrys, Acmopyle, and Saxegothaea. The first of these is represented in every major southern continent except Antarctica, includes some 90 species, and is sub-divided into eight sections (Buchholz & Gray, 1948). Dacrydium and Phyllocladus are both fairly widely distributed around the southern borders of the Pacific Basin and include 20 and six species respectively. All three of these genera are represented in New Zealand. The remaining four genera are all of very limited distribution, again around the borders of the South Pacific, and include only one or two species each. None is represented in New Zealand.

Natural vanilla (Vanilla planifolia, V. tahitensis) is economically important for a number of producing countries, but viral diseases are prejudicial to its successful cultivation. Techniques are required to detect viruses in vanilla plants in order to establish quarantine procedures and sources of virus-free planting material. This study contributed to the progress in managing viral diseases of vanilla by firstly identifying Cucumber mosaic virus (CMV, cucumovirus) as causing severe distortion, stunting, sterility and sometimes death of vanilla plants. Vanilla CMV isolates from French Polynesia (Pacific Ocean) and Réunion Island (Indian Ocean) were classified in CMV subgroup IB, adding to the short list of subgroup IB isolates detected outside of Asia. Two isolates were putatively classified in subgroup IA, suggesting that CMV in vanilla is still evolving and/or that vanilla is infected from different sources. Subgroup II CMV from New Zealand was also experimentally infectious to V. planifolia, showing that vanilla crops should be protected from all potential sources of CMV inoculum. Existing serological and molecular detection rests were performant for the detection of CMV directly from vanilla tissue. Secondly, this study provided the first coat protein sequence information for Vanilla mosaic virus (VanMV, Potyvirus). A Cook Islands isolate (VanMV-CI) and a French Polynesia isolate (VanMV-FP) had distinctly different coat proteins. The VanMV-FP CP N-terminus contained a stretch of amino-acid repeats (GTN) typical of natively unfolded proteins. This GTN stretch was located downstream of a DVG motif (which replaced the more common aphid transmission DAG motif), suggesting a role in improving aphid transmission, or regulating formation of the HC-virus complex. CP core nucleotide sequence identities indicated VanMV-CI and VanMV-FP were strains of Dasheen mosaic virus (DsMV). In contrast, CP amino-acid sequence homologies between VanMV- CI and DsMV were intermediate between strains and species, and CP amino-acid homologies between VanMV-FP and DsMV were typical of distinct species. In addition, VanMv-CI and VanMv-FP had characteristic 3'NTR sequences and NIb/CP cleavage sites, and only infected vanilla. Hence, it is proposed that VanMV-CI and VanMV-FP are considered new Potyvirus species and named Vanilla mosaic Cook Islands virus and Vanilla mosaic French Polynesia virus. Alternatively, the two isolates may be grouped under the name Dasheen mosaic virus-Vanilla (DsMV-V) and distinguished from Dasheen mosaic virus-Dasheen(DsMV-D). Primers to VanMV-CI and VanMV-FP were designed and permitted RT-PCR detection of the viruses directly from vanilla tissue. VanMV-Cl and VanMV-FP could be differentiated from DsMV and Watermelon mosaic virus (WMV-Tonga), and differentiated from each other by comparison of amplicon size. Long-term specific potyvirus diagnosis is however expected to be difficult due to potyviral variability in vanilla. Future research should concentrate on techniques such as microarrays to permit simultaneous detection combined with specific identification of potyvirus species. Such techniques would be beneficial to viral disease management in vanilla and many other crops.

The work in this thesis concerns the analysis of essential replication genes of the F plasmid of Escherichia coli. The techniques principally employed were the cloning of restriction fragments, transposition mutagenesis, maxicell analysis of proteins, and DNA sequencing. This work led to the following findings. 1. A 1.15kb region of f5 was shown to contain all the functions required for initiation of F replication. Insertions of a transposon within this 1.15kb region abolished mini-F replication. 2. A mini-F plasmid was constructed which constitutes the smallest F derivative so far reported. This plasmid has an elevated copy number, as a result of deletion of the incC region. 3. At least two proteins specified by the essential F replication region were identified in maxicells. The promoter fragments of two mini-F genes were fused to a β-qalactosidase gene and demonstrated to have low but significant in vivo activities.

The Majority of Rotorua Lakes are rapidly becoming eutrophic and/or dominated by exotic hydrophytes. Lake Rotoma was studied to provide essential information on native aquatic hydrophytes within an oligotrophic lake, and to record the initial transition towards an exotic dominated community. The lake is of volcanic origin and has a markedly fluctuating water level because of the absence of a surface outlet. An underwater SCUBA investigation was made in 1972, followed by intensive sampling from 50 systematically located one transects in 1973. A suitable quadrat size and shape was selected and the sampling error was assessed. The species frequencies were calculated from presence or absence recordings. Quadrat cover, depth, gradient and substrate were also determined. Three characean species, Chara fibrosa f. acanthopitys. (A. Br.) R.D.W., Nitella leptostachys var. leonhardii (R.D.W.) R.D.W. and N.pseudoflabellata var. mucosa (Nordst.) Bailey, dominated the vegetation. The next most common pattern contained the same three species plus Lagarosiphon major (Ridley) Moss. The initial stages of L.major invasion in to a characean community was recorded from 1973 up to 1976. The charophytes ranged in depth from 0 - 17.5m and were located upon a wide variety of substrates and gradients. The native vascular plants were absent from many transects. The plants had a depth range from 0 - 4.5m with most occurring above 3.5m. The Low Mixed Community was primarily located in water less than 1.25ma at the N.E. end of the lake, which resulted in this area having a high species diversity. The exotic hydrophytes were more widespread and abundant than the native vascular phanerogams. The distribution of L.major and Elodea canadensis Michx. appeared to coincide with boating access, recreational usage and fallen submerged trees. The depth range was 0 - 6.0m, although the available habitats within this range have not yet been fully exploited. Emergent species were abundant within the S.W. inlet and also in the lagoons around the lake shoreline where sheltered conditions and shallow gradients prevail. An experimental study on the effect of erosion and sedimentation by removing the shallow water plant cover, showed that the presence of plants moderated the extent of erosion and also encouraged sedimentation. Recolonisation of cleared areas was discussed. Annual frequency changes in the vascular hydrophytes were largely influenced by water level fluctuations and by the extension of exotic species. Water-level fluctuations were observed to influence (a) the bottom limit of plant growth (probably indirectly through light penetration), (b) the upper limit of plant growth by determining available growing space, and (c) the rate of spread of exotic species by erosion and redistribution of existing stands. The 1973 raw data was processed and standardised in the form of species presence or absence, absolute frequency, and relative frequency within each transect. Distance matrices were constructed using non-Euclidean distance functions. A non-linear multidimensional scaling technique was used to construct transect and species ordinations. The advantages and problems associated with this technique are discussed and compared to alternative methods. That adopted was successfully tested for its ability to reconstruct a place map of the North Island of New Zealand. The configuration error was calculated for each ordination and was found to be less than for the alternative methods tested. From the first species ordination a concept of species prosperity was developed which involved the number of transects and quadrats within which each species was recorded, and the maximum depth recording for each species. Non-linear analysis indicated that average gradient might be a significant influence. From the first transect ordination, four vegetation groups were recognised and discussed. Depth, substrate, species dominance and gradient were significant within the ordinations. Non-linear analysis confirmed many of the previous interpretations of the ordinations but gave no further information.

Sulfate removal from sulfomucin is believed to be a rate-limiting step in sulfomucin degradation by bacteria from the digestive tract. A novel sulfomucin-desulfating enzyme has been discovered in the anaerobic bacterium, Prevotella strain RS2, which can grow on colonic mucin as its sole energy source. The enzyme, located in the periplasm, was assayed by measuring p-nitrophenol removal from the model substrate sulfate-6-N-acetylglucosamine-1-p-nitrophenol, sulfate-6-N-acetylglucosamine being the other product. This activity differs from that of sulfatases which remove the sulfate ester group from sulfate-6-N-acetylglucosamine and its analogues substituted at the Cl position. The enzyme has been termed a sulfate-6-N-acetylglucosaminidase or sulfoglycosidase (SGL). The SGL was purified to a single protein band of 100 kDa as analyzed by SDS-PAGE. The purified SGL protein was trypsin-digested and peptide fragments were sequenced. PCR and inverse PCR were then used to amplify the entire sg/ gene from Prevotella strain RS2 genomic DNA. After inserting the gene into a suitable plasmid, active recombinant SGL was expressed using an Escherichia coli expression system. The SGL was characterized using a selection of model substrates, and shown to be an exo-enzyme that removes non-reducing terminal sulfate-6-N-acetylglucosamine residues by glycosidic bond cleavage. When tested against its putative physiological substrate, sulfomucin, the only small molecular size product detected corresponded to a sulfate-6-N-acetylglucosamine residue. Thus the SGL can catalyze a reaction, formerly thought to be performed in bacteria by the combined actions of a N-acetylglucosamine-6-sulfatase and a N-acetylglucosaminidase. Inhibition studies on the SGL were carried out. Inhibitors of the SGL and those of the sulfatases were used to confirm the presence or absence of SGL-like activity in other bacteria that inhabit environments containing sulfomucin. Four isolates, including Prevotella strain RS2, of the thirteen strains tested, appeared to have SGL-like activity. This research on the SGL with its novel catalytic activity, suggests a new mechanism by which sulfomucin desulfation can occur. The physiological importance of the enzyme is postulated to be (i) to provide energy in the form of sulfate-6-N-acetylglucosamine, for the bacterium, (ii) to remove sulfate-6-N-acetylglucosamine groups present on mucin chains, thus creating or removing sites for different adhesins, and (iii) removal of inhibitory sulfate-6-N-acetylglucosamine groups from mucin chains that limit degradation of the chain by exoglycosidases and neuraminidases.

Whole document restricted at the request of the author. / The pathways by which nutritional status is signalled to the mammary glands and the metabolic sites targeted by these pathways have not been identified. Understanding of these pathways is of particular importance in species such as rodents and ruminants in which mammary metabolism is extremely sensitive to food availability. The studies in this thesis investigated mechanisms by which nutritional state was signalled to the mammary glands using the lactating rat as an experimental model. An in vivo preparation for analysis of the effects of altered nutritional state on substrate supply to, and uptake by, the inguinal mammary glands was established, as this had not been accurately performed before in rats. This preparation was used to record a large fall in mammary glucose uptake with food deprivation and a rapid restoration of uptake after refeeding. Results demonstrated that mammary glucose uptake, and hence mammary metabolism, was not closely linked to glucose supply in lactating rats. Glucose supply is unlikely to be a key factor signalling nutritional state to the mammary glands. Experiments in which the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands was severed showed that these neural pathways did not contribute to the maintenance of mammary metabolic activity in the fed and refed states or to the suppressed activity in food deprived rats. Neural signalling is unlikely to have a direct role in controlling mammary metabolism in rats. An in vitro method for measuring the uptake of glucose by rat mammary acini was developed. Insulin administration increased glucose uptake in acini from both fed and food deprived rats. Treatment with a crude gut extract enhanced uptake of glucose in acini from food deprived, lactating rats, but not in acini from fed rats. It was concluded that insulin and/or a factor from the gut may be involved in signalling the mammary gland of the restoration of nutrient supply when food deprived rats are refed. Proteomic studies were performed to investigate the effect of food deprivation and insulin on the abundance of intracellular proteins in acini from fed and food deprived, lactating rats. Analysis of over 800 protein spots detected 7 that were regulated by food deprivation, 26 that were regulated by insulin, and 9 in which the regulation was different in acini from fed and food deprived rats. None were regulated by both food deprivation and insulin. This suggests that decreased blood insulin concentration during food deprivation is unlikely to be the only signal that results in decreased mammary metabolism. Identification of proteins affected by food deprivation and insulin has led to new insights into some of the intracellular mechanisms regulated by these factors.

The pullulanase gene from Caldocellum saccharolyticum, an obligate thermophilic anaerobe, was sequenced and expressed in E. coli. Expression and substrate induction studies in E. coli showed that while gene expression was substrate inducible and the enzyme was exported into the growth medium in C. saccharolyticum, expression was non-inducible in E. coli and the enzyme remained in the cytoplasm. The nucleotide sequence of the pulA gene was shown to be 2478 basepairs (bp) in length, coding for a protein of 96 kDa. The proposed promoter sequences showed homology to both the standard E. coli sequences and the consensus sequences obtained from other C. saccharolyticum genes. The enzyme from the native organism was purified from the growth medium and shown to have a molecular mass of approximately 120 kDa. Periodic acid-Schiffs staining showed that this enzyme was glycosylated and substrate characterisation revealed that the enzyme debranched pullulan to produce only maltotriose, but hydrolysed amylopectin, amylose and β-limit dextran to produce a number of smaller oligosaccharides. The enzyme was expressed in E. coli from its own promoters and was purified from the cytoplasmic fraction. Substrate characterisation revealed that the enzyme debranched pullulan to produce only maltotriose, but had only limited activity on β-limit dextran and amylopectin, and no activity on amylose. The pullulanase gene was also expressed under the control of a heat-inducible overexpression system in E. coli and a copper-inducible expression system in yeast. Amino acid homology comparisons of the pullulanase to other pullulanase sequences and related enzymes revealed a high degree of homology, particularly around three highly conserved regions. In α-amylases amino acids in these regions are involved in catalytic activity, substrate binding and metal ion binding.

Prumnopitys taxifolia (Banks et Sol. ex D. Don) de Laub. (matai) and P. ferruginea (D. Don) de Laub. (miro) are two New Zealand podocarps with morphologically very different reproductive structures. In P. taxifolia the pollen cones are sessile on spicate fertile branches, whereas in P. ferruginea they are solitary on short scaly peduncles. Likewise, in P. taxifolia the ovulate strobilus is an attenuated spicate structure bearing globose seeds, whereas in P. ferruginea the reduced ovulate strobilus bears a dorsi-ventrally flattened, usually solitary seed on a slender scaly peduncle. Such differences throw doubt on the validity of the current delimitations of the genus Prumnopitys. A comparative embryological study of these two species was done in parallel with a morphological and anatomical study of the reproductive structures to fill gaps in their known reproductive life cycles, and to determine whether there are any differences in the male and female cones or in their embryogeny that are of taxonomic significance at the generic level. In addition the hydrodynamic properties of their saccate pollen grains were compared with that of the pollen of several other conifers and the spores of three ferns and one lycopod. Morphology and Embryology Embryologically these two species are shown to be more similar to each other and to the Chilean Prumnopitys andina than to other podocarp genera. For instance, in both species: the long tapered archegonia have prominent neck cells with a thickened cap overlying them; five free nuclear mitoses occur before cell formation in the proembryo; meiosis can result in both linear and non-linear tetrads of megaspores; the microgametophyte typically forms 7 or 8 prothallial cells before dispersal. However, new morphological and anatomical differences have emerged from this study. In the pollen cones of P. taxifolia, the microsporangia are free with a transverse stomium located close to the cone axis; in P. ferruginea they are fused with the transverse stomium located basally. In P. taxifolia ovules, a tanniniferous layer develops in the epimatium and not in the integument, and fusion of epimatium/integument/nucellus is greatest dorsi-ventrally; in P. ferruginea ovules a prominent tanniniferous layer develops only in the integument, and fusion of epimatium/integument/nucellus is greatest laterally. Furthermore, observations of insect predation point to biochemical differences between P. taxifolia and P. ferruginea. An undescribed species of gall midge appears to be specialized exclusively for the P. taxifolia life cycle, and several other insect larvae prey on the developing and mature seeds, whereas the ovules/seeds of P. ferruginea appear to have almost no insect predators. Morphologically, anatomically and biochemically, P. taxifolia and P. ferruginea may therefore be sufficiently different to warrant being two separate genera; comparable differences have been used to distinguish other genera in the Podocarpaceae. Since embryogeny is likely to be under less evolutionary pressure than morphology and anatomy, and since P. taxifolia (along with P. andina) is regarded by some as basal in the conifers, with P. ferruginea less so, their embryological similarity does not necessarily indicate close relationship, only that they are closer to each other than to the other podocarps. Although their morphological and anatomical differences may be of taxonomic significance, a better understanding of the other species currently in Prumnopitys is necessary before the relationships within the genus can be clarified. Pollen Hydrodynamics Despite the morphological differences in their ovulate cones, P. taxifolia and P. ferruginea have very similar pollination mechanisms involving an inverted micropyle, a pollination drop and saccate pollen. Saccate grains have sometimes been referred to as 'non-wettable' due to their buoyant properties, while non-saccate pollen grains have been described as 'wettable'. The hydrodynamic properties of saccate pollen grains of seven podocarp species in five genera, Dacrydium Sol. ex G. Forst., Dacrycarpus (Endl.) de Laub., Manoao Molloy, Podocarpus L'Hér. ex pers. and Prumnopitys Phil. have been tested in water, together with saccate and non-saccate pollen of four other conifer genera, Cedrus Trew (Pinaceae), Cephalotaxus Siebold & Zucc. ex Endl. (Cephalotaxaceae), Cupressus L. (Cupressaceae) and Phyllocladus Rich. ex Mirb. (Phyllocladaceae), and spores of three fern species and one lycopod species. All four spore types studied were non-wettable, whereas the bisaccate and trisaccate pollen types, like all the other conifer pollen types, were wettable, enabling the grains to cross the surface tension barrier of water. Once past this barrier, grain behaviour was governed by presence or absence of sacci. Non-saccate and vestigially saccate grains sank, whereas saccate grains behaved like air bubbles, floating up to the highest point. In addition, the grains were observed to float in water with sacci uppermost, consistent with the suggestion that distally-placed sacci serve to orientate the germinal furrow of the pollen grain towards the nucellus of an inverted ovule. Observations of pollen grains in the pollen chambers of naturally pollinated Prumnopitys ovules confirmed this. The combination of buoyancy and wettability in saccate pollen has implications for the efficiency of the typical podocarp pollination mechanism. Since saccate pollen, inverted ovules and pollination drops are also characteristic of the Pinaceae, the similarities in the pollination mechanisms and the pollen grains of P. taxifolia and P. ferruginea have no taxonomic significance at the generic level.

Potyvirus is the largest and one of the most economically important of the virus genera infecting plants. The complexities of potyvirus identification resulting from many different species, mixed infections, emerging new viruses, new hosts, and new vectors, etc., often requires the use of multiple detection methods which is time consumable and costly. Therefore an assay that can test for a range of potyviruses simultaneously, with good specificity and sensitivity, is desirable. This study looked at the feasibility of producing an oligonucleotide microarray for detection and identification of potyviruses at both species and strain level. Thirty plant samples with suspected potyvirus infections were collected from field and research laboratories in New Zealand and partial NIb gene, complete CP gene and 3΄UTR were sequenced. Twelve definitive potyviruses, one tentative potyvirus, one non-potyvirus, and one novel potyviruslike sequence were identified, six of which were first records for New Zealand. Sequence analysis showed that NIb and CP genes and the 3΄UTR contained both conserved and variable sequences which were used to design both species and strain level specific probes. Four Potyvirus species were chosen for a “proof of concept” study and probes were designed using two different software programs (ROSO and CAG software). A total of eighty five probes including 33 perfect-match and 52 mismatch probes were selected to represent the four targeted potyviruses. Each probe was synthesized with spacers of either 6 or 12 poly-cytosine or poly-thymine at the 5΄ terminus. Arrays showed high specificity to the targets when tested using nineteen different geographically diverse potyvirus isolates representing the four target species, four distinct but closely related New Zealand potyviruses, and four healthy plant species. Factors affecting the hybridization efficiency, e.g. the size of the target fragments, secondary structure of probes and targets, label type, strandedness, Tm and GC content of probes, were also investigated. The approaches and protocols developed in this study should form a useful basis for developing other potyviruses arrays and the results also provide useful insights into issues of generic interest for the development of arrays for detecting other pathogens.