Molecular probes for the fish-killing Raphidophytes and the phylogeny of the group

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dc.contributor.author Tyrrell, John V en
dc.date.accessioned 2007-06-28T22:46:02Z en
dc.date.available 2007-06-28T22:46:02Z en
dc.date.issued 1999 en
dc.identifier THESIS 00-236 en
dc.identifier.citation Thesis (PhD--Biological Sciences)--University of Auckland, 1999 en
dc.identifier.uri http://hdl.handle.net/2292/579 en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract Effective management of fish aquaculture stocks to avoid or mitigate the impact of fish-killing phytoplankton requires intensive spatial and temporal sampling to identify and quantify potentially harmful species so that adequate warning of a harmful bloom may be given. The current methodology based on light and electron microscopy is inadequate as a daily monitoring tool. This thesis examines the utility of oligonucleotide probes to provide a simple, cost-effective and rapid means to identify and enumerate harmful algal bloom species. The Raphidophyceae were chosen for the work because of their impact on salmon farmed in seapens in New Zealand and globally. Regions of the small subunit and the large subunit ribosomal RNA genes (SSU rRNA & LSU rRNA) were sequenced to provide variable regions for the design of oligonucleotide probes, and to provide characters to establish the phylogenetic placement of the Raphidophyceae within the heterokont algae. The'V4' domain small-subunit rDNA gene ('V4' domain SSU rDNA gene) was sequenced for the taxa Heterosigma akashiwo (Hada) ex Sournia and Chattonella antiqua (Hada) Ono to provide characters for phylogenetic analyses. These analyses suggest a relationship between the Raphidophyceae and Eustigamatophyceae. A close systematic relationship for these two classes has not been shown before. Transition frequency analysis showed that there was a major loss of phylogenetic signal within the chromophyte algae 'V4'domain SSU rDNA gene sequences. Partial large subunit ribosomal DNA sequences for the raphidophytes Heterosigma akashiwo, Chattonella antiqua, Chattonella marina (Subrahmanyan) Hara & Chihara, Chattonella ovata Hara & Chihara, Chattonella subsalsa Biecheler, Fibrocapsa japonica Toriumi & Takano, Olisthodiscus luteus Carter and the eustigamatophycean Nannochlorpsis oculata (Droop) Hibberd were used to define systematic relationships within the Raphidophyceae. Sequence analysis of the Chattonella species revealed only two ribotypes (gene sequences), ribotype I: C. subsalsa and ribotype II: C. antiqua; C. marina; C. ovata. The discovery of only two ribotypes indicates extensive morphological plasticity for this genus. Maximum parsimony and maximum likelihood analyses both produce the same phylogenetic tree and, together with tree statistics, these analyses indicate that the freshwater and marine raphidophytes form a monophyletic assemblage and that Olisthodiscus luteus is not a member of the Raphidophyceae. To determine the phylogenetic affinities of O. luteus, the entire SSU rRNA gene was sequenced and aligned with species from the heterokont algae. Phylogenetic analyses revealed no close relationship of O. luteus with any class within the heterokont algae. Previous studies also have had difficulty in defining the systematic placement of O.luteus. Accordingly, the species O. luteus is transferred to the new class Olisthophyceae. Due to its delicate structure the identification and enumeration of Heterosigma akashiwo is relatively difficult and tedious when large numbers of samples need to be screened. In this study we have examined the use of in situ hybridisation based on small subunit ribosomal RNA (SSU rRNA) -targeted fluorescent oligonucleotide probes to identify H. akashiwo specifically. Two probes Haka1-F and Haka2-F specifically labelled all H. akashiwo isolates. Haka1-F and Haka2-F were reconstructed with a biotin label instead of fluorescein and detected by a secondary labelling reaction. This procedure increased the fluorescent signal by approximately 3-4 times and was particularly useful for detecting cells in late-stationary phase growth. Both methods proved extremely effective in specifically identifying cultured H. akashiwo. The large-subunit rRNA (LSU rRNA) gene was also examined to determine its utility to provide group and species-specific probes for the fish-killing species Heterosigma akashiwo and Fibrocapsa japonica. Oligonucleotide probes were evaluated using whole cell fluorescent in situ hybridisation (FISH) to determine which regions of the LSU rRNA molecule were reactive towards the probes and to assess their specificity. Probes that reacted strongly towards the target organisms and displayed adequate or promising specificity were then incorporated into a sandwich hybridisation assay (SHA). Species-specific SHA's were successfully developed for both H. akashiwo and F. japonica, and the sensitivity of these tests is such that resource managers could utilise the assay to detect these species at concentrations well below those that result in fish mortality. The SHA, applied using an automated robotic processor, appears to be a faster, cheaper, and simpler method than other available cell detection and quantification strategies. en
dc.language.iso en en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA9990779814002091 en
dc.rights Restricted Item. Available to authenticated members of The University of Auckland. en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Molecular probes for the fish-killing Raphidophytes and the phylogeny of the group en
dc.type Thesis en
thesis.degree.discipline Biological Sciences en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.subject.marsden Fields of Research::270000 Biological Sciences::270700 Ecology and Evolution::270702 Marine and estuarine ecology (incl. marine ichthyology) en
dc.rights.holder Copyright: The author en


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