Author: Patrick William Laffy
Laffy, Patrick William, 2011 Evolution, gene expression and enzymatic production of Tyrian purple: A molecular study of the Australian muricid Dicathais orbita (Neogastropoda: Muricidae), Flinders University, School of Biological Sciences
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Tyrian purple is the traditional source of purple pigmentation used in the textile industry since ancient times, sourced from the Muricidae family of neogastropod molluscs. Brominated indole derivatives of tryptophan, the precursors to Tyrian purple are potent anticancer and antibacterial compounds which may have potential for pharmaceutical development. In addition to their production within the hypobranchial gland, some members of the Muricidae invest Tyrian purple precursors within their egg capsules. The first aim of this thesis was to investigate the evolution of Tyrian purple precursor investment within the egg masses of muricid molluscs using a molecular phylogenetic analysis of 18s and 28s ribosomal RNA sequences. The second aim of this thesis was to investigate the gene expression of the hypobranchial gland of the Australian muricid Dicathais orbita, in an effort to uncover the enzymes involved in the production of Tyrian purple. The investigation into the evolution of Tyrian purple precursor investment within the egg capsules of muricid molluscs identified that the capacity for adults to invest these compounds in their egg capsules is a trait that was not ancestral and has arisen at least twice in the evolution of the Muricidae. Molecular analysis confirmed the monophyly of the Rapaninae and Ocenebrinae muricid subfamilies members and supports Tan's 2003 classification of a new muricid subfamily, the Haustrinae. These findings also support the use of D. orbita as a representative of the Rapaninae in which to study Tyrian purple synthesis and investment. Suppressive subtractive hybridization (SSH) was used to identify genes that were up-regulated or uniquely expressed in the hypobranchial gland of D. orbita. A total of 438 sequences were identified to be differentially expressed in the hypobranchial gland, including an arylsulfatase gene. Arylsulfatase activity is known to be involved in the formation of Tyrian purple from precursors in muricid molluscs. The full length arylsulfatase sequence was amplified and recombinantly expressed in a mammalian expression system. No active enzyme was produced from these experiments suggesting an incompatibility between molluscan arylsulfatase and mammalian expression systems. Initial manual sequence analysis indicated that over 65% of sequences expressed in the hypobranchial gland showed no homology to known database sequences. The subset of genes V that did show sequence matches to genes in the database showed homology to a wide variety of taxa, including chordate, molluscan and ciliate sequences. Our investigation into the gene expression of the hypobranchial gland of D. orbita enabled the functional assignment of 110 sequences using BLAST2GO automated sequence annotation. The hypobranchial gland plays a key role in muricid biology as a site of chemical interaction and biosynthesis. Manual sequence annotation also identified a number of sequences within our cDNA library that would only be functional if translated using an alternate codon translation system used by ciliate protozoans. Histological analysis of the hypobranchial gland identified intracellular ciliate protozoans present within the gland. Ciliate abundance varied in accordance to the reproductive condition of the host snail and 57 ciliate protein coding genes were identified within our cDNA library. Analysis of ribosomal RNA sequences from our expression library confirmed the presence of ciliate protozoans within the hypobranchial gland of D. orbita belonging the ciliate class Phyllopharyngea and possibly from another unidentified ciliate class. A novel use of SSH is proposed for the investigation of symbiont gene expression in other biological systems. In summary, this thesis uses molecular techniques to explore the synthesis and evolution of Tyrian purple and hypobranchial gland gene expression in the muricid mollusc D. orbita. This thesis is the first study to investigate the evolution of Tyrian purple precursor investment within the egg capsules of muricid molluscs and has revealed that this is a derived trait that has arisen at least twice since the muricids diverged from other Muricoidean species. In addition, this is the first study to investigate gene expression within the hypobranchial gland of any mollusc. This study also identified one of the gene sequences involved in the enzymatic production of these bioactive compounds. Further investigations are required in order to produce active recombinant molluscan arylsulfatase enzymes. Additional investigations are also required in order to identify the other enzymes involved in the production of Tyrian purple precursors, which would then facilitate the in vitro synthesis of these compounds in the future. If sustainable synthesis of these compounds is established, these bioactive compounds may find use in pharmaceutical or nutraceutical treatments.
Keywords: Mollusc,Tyrian purple,Muricidae,Dicathais orbita,Ciliate protozoan,transcriptomics
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Associate Professor Catherine Abbott