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Bottlenose dolphins (Tursiops truncatus) are frequently brought into contact with humans, due to their coastal habitat use. Over the last decade there has been considerable growth in the commercial dolphin-based tourism industry. This growth exposes coastal populations to boats and swimmers. New Zealand has allowed commercial operations offering swimming with wild dolphins since 1989. Little is known about the effects of boats and swimmers on dolphin behaviour. The present study examines a population of bottlenose dolphins that utilise the Bay of Islands as part of their range along the northeastern coast of New Zealand, and their behavioural response to commercial swim-with-dolphin operations. Data were collected from an independent research boat and the commercial dolphinwatching boats from December 1996 to February 2000. Photo-identification showed a closed population of 446 (95% C.I. 418-487) dolphins, with an 81% resight rate of the 378 catalogued individuals. Group size ranged from 2 – 50 dolphins, with 80% of groups (n = 160) containing 2 - 20 dolphins. The number of groups with calves present increased from 32% (n = 17) in 1997, to 63% (n = 47) in 1999. This could be explained by the increased number of neonates born; from four in 1997, to nine in 1999. Photo-identification in areas outside the Bay of Islands showed that individuals ranged minimum distances of 82 km north, and 388 km south of the Bay of Islands. All photographs taken outside the Bay of Islands were matched to known animals, suggesting that the majority of the northeastern coastal population is seen in the Bay. With the next nearest population of bottlenose dolphins approximately 1,000 km south, these dolphins probably form an isolated population ranging along the east coast of the northeastern North Island. Habitat use by the dolphins was examined using Geographic Positioning System (GPS) points simultaneously linked to behavioural states. In total, 31 groups were followed, and 847 GPS points were recorded. Position of initial encounter was recorded for 138 groups of dolphins. There was a significant correlation between water depth and sea-surface temperature, with dolphins found in deeper waters in summer when water temperature was highest, and in shallow waters in winter when the water temperature was lowest. Only one behavioural state, slow travel, was significantly different by season. Although all behaviours were observed in all zones, dolphins had preferential habitat use throughout the four zones within the Bay. To determine the level of impact on dolphin behaviour due to boat traffic, focal-group follows using a scan sample methodology were conducted from an independent research boat. A CATMOD analysis was used to determine dolphin behaviour by season, number of boats, and group size. Dolphin behaviour differed by boat number; in particular, resting behaviour decreased as boat number increased and milling behaviour increased as boat number increased. Behaviour differed when the research boat was the only boat present. Closer examination of boat type, i.e., permitted dolphin watching boats, non-permitted tour boats, and recreational boats, found a difference in dolphin behaviour in the presence of the permitted boats when compared to the other types. Dolphins rested less and engaged in more milling behaviour when the permitted boats were present. Responses to swim attempts from the commercial boats were compared from 1994 - 1995 to 1997 - 1998. In total, 255 groups were encountered of which 36% (n = 93) were exposed to at least one swim attempt. Successful swims (i.e., where at least one dolphin remained within 5 m of at least one swimmer) decreased from 48% in 1994 - 1995 to 34% in 1997 - 1998 and avoidance responses increased from 22% to 31%. Dolphin response varied according to swimmer placement from the boats. The ‘in path’ placement had the highest level of avoidance. Only ‘line abreast’ placement resulted in a decrease in avoidance. Based on the photo-identification sighting records, it was estimated that an average dolphin was exposed to 31 swim attempts per year. This suggests that individual dolphins have, with cumulative experience, become sensitised to swim attempts. If a swim attempt was successful, on average, it involved 19% of the group. Juveniles were more likely to interact with swimmers than adults. The present study suggests that the wide-ranging population of bottlenose dolphins that frequents the Bay of Islands is exposed to levels of tourism which can affect the dolphins behaviour. In particular, the permitted boats’ presence results in decreased resting behaviour by the dolphins. The apparent sensitisation of this population to swim attempts, and the relatively small number of individual dolphins that interact with swimmers, are grounds for concern about the long-term impact of interactions. Ongoing monitoring of this populations’ response to swimmers and boats, combined with a precautionary approach to management of boats and swimmers, are recommended.

This thesis uses molecular genetics as a tool to uncover information about the population structure and genetic variation in Hector’s dolphin (Cephalorhynchus hectori), to track population declines and to assess the evolutionary origins and taxonomic status of this species. A high-resolution genetic analysis of population structure was considered important for the determination of population boundaries and delimitation of conservation management units due to potentially unsustainable fisheries-related mortality. Population structure and dispersal rates were assessed using 281 samples collected from individual Hector’s dolphins of ten population groups representing the known geographic range of this species. Variation among mitochondrial DNA sequences (ΦST = 0.545) and microsatellite allele frequencies at six loci (RST = 0.252) indicated the presence of four genetically isolated regional populations, North Island (n = 29), East Coast South Island (n = 110), West Coast South Island (n = 122) and South Coast South Island (n = 19). Significant levels of genetic differentiation were not detected within local sub-populations of the East Coast and West Coast regional populations. However, the estimated geneflow between these sub-populations fitted a one-dimensional stepping-stone model (r2 = 0.6225) suggesting a vulnerability of local populations to fragmentation. A measure of expected mtDNA diversity (Tajima’s D statistic) suggested decline in eight of the ten populations. Microsatellite heterozygosity was also lower than expected in the East Coast and North Island regions, suggesting either further regional sub-structuring (Wahlund effect), loss of diversity due to population decline or the presence of null alleles. Examination of all Hector’s dolphin museum specimens of known origin (n = 55) enabled comparison of historic (1870 - 1987) genetic diversity to contemporary (1988 – 1999) diversity in two regional populations to assess the possibility that these populations have undergone recent declines. Over the last 20 years the North Island population has been reduced from at least three lineages (h = 0.41) to a single lineage (h = 0, p < 0.05). The diversity of the East Coast, South Island population has declined significantly from h = 0.65 to h = 0.35 (p < 0.05). These results suggest that the low abundance currently observed is due to recent population declines and that the North Island population is threatened with extinction in the near future. Based on a trend analysis of the mtDNA, it can be predicted that the East Coast South Island population may lose all mtDNA diversity within the next 20 years. Alternatively, detection of a one dimensional dispersal pattern may indicate that some populations are at risk of extirpation while others may not be in decline. If this is the case then the East Coast regional population is at risk of fragmentation. On a wider evolutionary scale, Hector’s dolphin is one of four species of the genus Cephalorhynchus, all of which suffer fisheries–related mortality. To describe the origin and radiation of these species, 485 bp of the mitochondrial DNA control region was sequenced from 320 individuals (including previously sequenced 200 Hector’s dolphins) representing nine of the ten species in the sub-family Lissodelphininae. The hypotheses that either Cephalorhynchus is a monophyletic genus or that the four species have arisen separately from pelagic Lissodelphine species and have converged morphologically were tested. The mtDNA phylogeny supported the monophyly of the genus and suggested that the genus Cephalorhynchus originated in the waters of South Africa and, following the West Wind Drift, colonised New Zealand and then South America. Secondary radiations resulting in two genetically isolated populations were found for the Kerguelen Island Commerson’s dolphin and the North Island Hector’s dolphin. A comparison of the genetic differentiation between the Commerson’s dolphins of the Kerguelen Islands (n = 11) and the coast of South America (n = 35), and between the North Island (n = 14) and South Island (n = 185) Hector’s dolphins, was conducted in order to assess the conservation and taxonomic status of these populations. A single fixed substitution in the mtDNA control region was diagnostic for the Kerguelen Island compared to South America (FST = 0.306, ΦST = 0.602) and the North Island compared to the South Island (FST = 0.442, ΦST = 0.495). Population differentiation of four microsatellite alleles (including unique alleles in each of the four populations) between the Kerguelen Island and South American Commerson’s dolphin (FST = 0.036, RST = 0.0493) and between the North and South Island Hector’s dolphins (FST = 0.391, RST = 0.3197) indicated restricted nuclear as well as maternal geneflow. These data, combined with additional evidence of morphological and geographic isolation, indicated that the Kerguelen Island Commerson’s dolphin and the North Island Hector’s dolphin are likely to be reproductively isolated from their alternate con-specific populations. Examination of various species concepts and definitions of conservation units leads to the conclusion that these four populations should each be considered unique at the subspecies level for the purposes of management, protection and evolutionary potential. These results lead to the conclusion that the Hector’s dolphin consists of highly subdivided populations. As a result of this and a low reproductive potential, Hector’s dolphin populations are vulnerable to extirpation through even low levels of human induced mortality. To manage such populations, it is appropriate to consider each of the two islands as separate sub-species. Within the South Island, the populations may be further subdivided into three demographically independent Management Units – the East, West and South Coasts. The South Coast management unit is vulnerable due to its low abundance and isolation and requires further investigation. Population modelling will need to reflect the fact that the local populations within the East and West coast regions share only limited dispersal with immediately adjacent populations and are thus susceptible to fragmentation. These results also show that the population declines of the East Coast South Island and the North Island populations are of recent origin thus implicating fisheries-related mortality as the principal threat to Hector’s dolphin. To prevent further decline or fragmentation of South Island populations more stringent control of inshore gillnet fisheries is required. By contrast, current decline of the North Island population may be a result of inbreeding depression. Given the low abundance and rapid decline of the North Island population, it is imperative to evaluate the potential for inbreeding depression while continuing to mitigate all human-related threats.

Broadcast spawning marine invertebrates have been widely used as model organisms to study processes of evolution. One of these is the study of various life history stages associated with reproduction. Fertilization ecology in broadcast spawning marine organisms, i.e. the process by which sperm and egg fusion occurs once released into the ocean, has been the subject of intensive study for roughly the last 20 years, and represents thus a recent field in ecological sciences. This growth in interest was sparked by studies that showed spawning events may take place predominantly under sperm limiting conditions. More recent findings however, suggest that the occurrence of sperm competition, and the risk of polyspermy (multiple sperm entry in to the egg, which is lethal in echinoderms) can also occur during reproductive events in marine invertebrates. This has also been predicted on theoretical grounds. Even though polyspermy has been observed previously, particularly during aquaculture studies, evidence to assess the occurrence of polyspermy in situ, and the conditions under which it occurs is lacking. Simulated field studies in Evechinus chloroticus as well as laboratory studies in E. chloroticus and Coscinasterias muricata found high levels of polyspermy, even under the sperm limiting conditions that are naturally found in the field in other marine broadcast spawners. Furthermore, laboratory results in both Evechinus and Coscinasterias showed that polyspermy is most likely to increase when sperm concentrations are increased. In addition, even though increasing sperm concentrations increases monospermy to a certain extent, an increase in gamete contact times generates similar levels of monospermy. Results from these studies confirmed the mathematical model predicting some of these events. The timing of gamete release to achieve maximum fertilization success (monospermy) in a broadcast spawner is thought to be under strong selective pressures, because reproductive success is directly related to fitness. The spawning patterns in the sea urchin E. chloroticus observed in this study demonstrated that E. chloroticus spawns under varying environmental and ecological conditions. Interestingly, it appeared that highly synchronous and widespread spawning was found to occur when highly turbulent conditions existed. This may reduce the potential for polyspermy that may exist during mass spawnings under shallow and calm conditions. The demonstration of polyspermy and the confirmation of the mathematical model, suggest that polyspermy is a common and frequent occurrence during broadcast spawning events. Thus polyspermy represents an evolutionary force that may shape the evolution of reproductive phenomena at levels ranging from the gamete to the population.

Various aspects of metabolism and physiology were investigated during the ontogeny of yellowtail kingfish (Seriola lalandi), a fish of growing aquaculture importance in both New Zealand and other countries. Incubation experiments between 18-24°C showed that developing eggs and larvae were heavily influenced by temperature. It appeared that at warmer temperatures larvae hatched smaller but grew on the yolk sac, whereas at cooler temperatures larvae grew inside the chorion. Oxygen consumption data supported this, with a negative correlation found between total embryonic oxygen consumption and temperature. A mechanism was proposed to explain the differential effect of temperature on ontogeny and growth. Like other marine fish with pelagic eggs, yellowtail kingfish were found to be heavily reliant on free amino acids as a source of energy. At 23°C the pattern of substrate utilisation in eggs was considerably different from that at 17-21°C, indicating that 23°C exceeded the tolerance for normal development. Inter-individual aggression by large individuals was associated with the development of size heterogeneity in juveniles. Although this aggression also affected the survival of smaller juveniles, it was not the primary agent of much of the mortality that occurs during this phase, as many of these individuals were on a degenerate developmental trajectory. Yellowtail kingfish fingerlings used for ongrowing were robust to the stressors imposed by live transport. The ontogenetic development of metabolic rate from 0.6 mg-2.2 kg did not follow the same scaling exponent as that observed for mammalian models of allometry, and has implications for interspecific studies of mass-dependent metabolism.

The Chilean oyster, Tiostrea chilensis, is a commercially important species that is native to New Zealand and the Pacific Coast of South America. The description of the variability in life history characteristics among populations of the Chilean oyster is fundamental to understanding its biology and may help in solving some of the problems encountered in culturing this species. Research presented in this thesis describes some aspects of the reproductive biology of the Chilean oyster from four populations, mainly in northern New Zealand, and compares the results with previous studies from elsewhere. Similar patterns of gametogenesis were found among three study populations at Manukau Harbour, Hauraki Gulf and Foveaux Strait. Oysters were protandrous, maturing firstly as males and later also producing ova. In all three populations the majority of the contents of all the gonads were male reproductive products. There was no evidence that the sexuality of oysters alternated rhythmically as has been reported in other species of larviparous oysters. For spawning females, the often concurrent release of ripe sperm indicated the possible presence of self fertilisation. In samples of oysters examined from Foveaux strait the sexuality of oysters was found to change markedly with size. This suggested that the infection of oysters by the parasite Bonamia may not be related to their sexuality as was previously thought. At two northern populations (Hauraki Gulf and Manukau Harbour) larvae were produced from young, small oysters, and a much larger proportion of the population was brooding larvae each year than has been reported elsewhere. In both populations, larvae were being brooded, released and were settling at all times of the year, unlike other populations. The mean fertility of the Manukau Harbour oysters was the highest so far reported for any population of this species. The size of larvae in all three northern populations were smaller than has been reported for all other locations in New Zealand and chile and is thought to be related to differences in water temperatures. Overall, the results highlight the importance of investigations into populations of molluscs across their geographical range for revealing variation in life history characteristics which may be of benefit for aquaculture. / Whole document restricted, but available by request, use the feedback form to request access.

The tellinid bivalve, Macomona liliana, is important to the structure and organisation of intertidal sandflat communities in Manukau Harbour. This thesis reports on features of the reproductive, larval supply and recruitment biology of Macomona liliana in relation to environmental factors in the harbour. Macomona liliana is gonochoric. In a mid-tide population sexual maturity occurred in animals ≥ 22 mm (shell length). Spawning is asynchronous and protracted from late November to March throughout the harbour and timing of the reproductive cycle is associated with seasonal changes in environmental variables: temperature, salinity and seasonal production of phytoplankton and benthic microalgae. A visual gonad index was useful for establishing the main periods of spawning activity. Favourable habitats are at mid-tide on the large exposed sandflats characterised by well sorted fine sands. Less favourable habitats are in sheltered bays characterised by moderate-poorly sorted sands. A delay in the onset of gametogenesis in small compared to large adults (July-August of. May-June) indicates size and/or age dependent resource allocation to growth and reproduction. Adult size classes are evenly represented in favourable habitats whereas in less favourable habitats size classes are dominated by small adults. Favourable habitats have an equal sex ratio or are biased towards females whereas in less favourable habitats the converse is true. Early life-history stages are described and techniques employed to facilitate their identification in plankton and sediment samples. A spatial Taylor's Power Plot (slope, b = 1.31) demonstrates thorough mixing of larvae in the main channels. Spatial autocorrelation models confirm that densities of larvae are higher to the south of Karore Bank than to the north. The recruitment period is December to early March but varies between successive years. Physical transport processes, post-settlement migration and high levels of mortality in Boccardia syrtis tube-mats dominate recruitment patterns in the high energy environments. Larval supply and active habitat selection by settling larvae may, however, be significant determinants of population structure in sheltered bays. Generalisations are made on the effect of habitat quality on the reproduction and recruitment biology of the Manukau Harbour metapopulation.

Previous studies indicated the marine pyridoacridone alkaloid ascididemin possessed a unique biological profile. Investigation of synthetic routes to ascididemin led to the discovery that ascididemin precursors possessed a wide range of biological activities. One precursor possessed hollow fiber in vivo antitumoral activity and continuing in vivo studies at the NCI, using subcutaneous xenograft assays, are in progress. A crystal structure of a precursor indicates that these tetracyclic heterocycles are planar, suggesting intercalation as a mechanism of antitumoral action. Ascididemin was synthesized by two novel ring E forming reactions. The superior methodology was also useful in the preparation of analogues, such as kuanoniamine A. Ascididemin possessed promising hollow fiber in vivo antitumoral activity but was poorly active in a subcutaneous xenograft study. Ascididemin was incapable of exerting antitumoral activity at a distance and further analogues were prepared to address this problem. Ten analogues were prepared, with all the non-bromine containing analogues selected for in vivo evaluation at the NCI. Two ring A analogues were prepared by a thennew synthetic route, including the antiviral natural product 11-hydroxyascididemin. 11-Methoxyascididemin was selected for hollow fiber evaluation. Two bromine containing ring D analogues were prepared, including the antifungal natural product 2-bromoleptoclinidinone. A crystal structure on 2-bromoleptoclinidinone was the first determined on a pyridoacridone alkaloid and the molecule was planar, further supporting an intercalative mechanism of action. Due to non-selective antitumoral cytotoxicity, ring D analogues are not useful antitumoral agents. Six carbon-based ring E analogues were prepared by novel methodology. All 6-substituted analogues assessed were selected for in vivo antitumoral evaluation. Hollow fiber antitumoral activity decreased with bulk of the substituent. 6-Methylascididemin has been selected for subcutaneous xenograft studies. The 5-substituted analogue prepared gave the best in vitro antitumoral profile of all alkaloids in this study and has been selected for in vivo evaluation. Ring E substituted N-8-deaza-ascididemin analogues possessed no antitumoral activity, highlighting the importance of the 1,10-phenanthroline-like bay region of ascididemin in antitumoral activity. Another four structurally novel, quinoid containing alkaloids have been selected tor in vivo evaluation. While ascididemin was the only compound capable of topoisomerase IIα cleavable complex stabilization, related alkaloids possessed a similar level of inhibitory action against this enzyme. This further supports intercalation as the dominant mechanism of action for pyridoacridone alkaloids. Ecological roles of four natural pyridoacridone alkaloids were assessed. Alkaloids were species specific antifeedant agents against important consumers. These alkaloids may have a long term detrimental effect on predator physiology, due to the well established ability of these alkaloids to interfere with cell proliferation. Ascididemin elicited avoidance responses in numerous marine species. Ascididemin has no antifouling activity against macrofoulers. Microbiological assessment of ascididemin, 11-hydroxyascididemin and 2-bromoleptoclinidinone indicated that modification of the ascididemin chromophore leads to the directing of antimicrobial activity towards a different phyla of parasites. Pyridoacridone alkaloids may be part of a non-antibody based immune system. All studies point to pyridoacridone alkaloids enhancing the eventual reproductive success of the organism. Biological and chemical evaluation of 29 New Zealand ascidians has been performed. Significant biological activity was detected in ten ascidians. Novel metabolites isolated were 2-(3'-bromo-4'-hydroxyphenol)ethanamine (Cnemidocarpa bicomuta) and 1,3-dimethylguanine (unidentified ascidian). Known metabolites isolated were 1,3-dimethylisoguanine (Cnemidocarpa bicomuta) and rubrolides A, B and C (unidentified ascidian). The survey highlighted the importance of overexpressed purine bases in ascidian metabolism. No physiological roles for these overexpressed purines are as yet apparent. Our study of NZ ascidians has led to the isolation of many compounds previously isolated from sponges. The widespread distribution of such metabolites gives credence to the theory that common metabolite-generating genes are present in both phyla, due to the evolutionary success of these genes. Two optically active 9-(5-S-methyl-5-sulfinyl-lyxofuranosyl)-9H-purin-6-amine (lyxosyl-MTAS) nucleosides were isolated from the nudibranch Doriopsis flabellifera. This is the first report of any lyxosyl-MTAS nucleoside as either a natural or synthetic product.

The use of enterococci as a microbiological indicator of water quality requires an understanding of the sources, persistence, and ecology of this group of bacteria in the environment. This research describes a series of investigations undertaken to describe the abundance, occurrence, and diversity of enterococci from aquatic and terrestrial environments. A screening protocol for environmentally sourced enterococci was developed to describe species and sub-species variability. This protocol combined classical microbiological methods of selective culture and biochemical characterisation, with molecular techniques including gene probe screening for identity and RAPD-PCR for genotypic diversity. Preliminary studies on the occurrence of enterococci in terrestrial and aquatic environments suggested that these organisms are ubiquitous. However, abundance varied between individual samples taken from terrestrial (e.g. leaf litter, sand, seaweed, animal faeces) or aquatic (marine or freshwater) environments, complicating the ability to predict the enterococci load from these sources. Enumeration of enterococci from bathing beach environments indicated occasionally high levels from seaweed and sand, where levels of up to 660 CFU/100 g (wet weight) were recorded from aged and degrading seaweed but not from fresh seaweed samples. Restriction enzyme analysis (REA) of isolates from degraded seaweed indicated a dominance of clonal populations and supported the notion of replication or survival of strains. Laboratory studies conducted to investigate enterococci persistence and growth on seaweed were not conclusive, although there was some evidence to suggest enterococci replication was occurring. This was indicated by molecular fingerprinting (REA analysis), which showed that the inoculated strain persisted for the full duration of experiments (up to 28 days). The isolation of non-inoculum strains from seaweed treatments, combined with increased abundance of these strains with incubation, suggested the persistence or replication of enterococci that were naturally occurring on seaweed. To investigate the occurrence of enterococci in bathing environments a statistical analysis of Auckland Regional Council (ARC) bathing water quality data was undertaken. This analysis indicated a strong positive correlation between enterococci and turbidity, and hence turbidity may serve as a useful physical measure to indicate deteriorating water quality. Surveys of three marine bathing beaches on Auckland's North Shore (Long Bay, Mairangi Bay, and Rothesay Bay) indicated the abundance of enterococci in all bathing beach environments surveyed. These included marine and fresh water, sand, seaweed, and stream sediment, and a significant association between enterococci levels found in the sand and in the seaweed. Enterococci screening protocols were evaluated for use in describing enterococci species and sub-species diversity in bathing beach environments. This investigation showed a diversity of enterococci from all beach environmental sources, with highest levels of species diversity from marine and stream water. Enterococci diversity did not provide clues as to the sources in marine water samples. RAPD-PCR analysis and phenotypic screening of enterococci isolates did not indicate a pattern of niche-specific associations of enterococci strains, and there was no strong evidence from this study that enterococci sub-species associate with specific environments. However, the presence of identical genotypes indicated that enterococci can persist and possibly replicate in environments such as sand and seaweed. Calculation of similarity coefficients from Ent. faecalis and Ent. casseliflavus sub-species groupings indicated a greater level of sub-specific similarity between selected environments, for example, seaweed:sand, marine water:stream water, seaweed:marine water, although this was not a guarantee that environments were biologically or ecologically associated. Where an elevated level of enterococci is measured in the absence in identifiable pollution source the separation of pigmented from non-pigmented enterococci was proposed as an indicator of the environmental or faecal nature of the enterococci within the sample. Although not tested under controlled conditions, this concept was found to have good utility for discriminating sources from elevated marine bathing water samples. Enterococci from one of 13 elevated ARC marine water samples examined was shown to be environmentally derived, with 5 of the 13 samples attributed to putative human or animal faecal sources. With further validation, this concept may be a useful means of determining source.