 Abstract | 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
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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. |
